The function and regulation of the cullin family E3 ubiquitin ligases

描述(由申请人提供)：清除不需要的和有害的蛋白质对细胞和有机体的功能至关重要。包括我们在内的许多实验室在过去十年的工作中发现了大量结构相关的蛋白质复合体，称为cullin-ring E3泛素连接酶。这些E3复合体的作用是通过用死亡标签链标记目标蛋白，即泛素，从而引导它们的破坏，从而发挥“分子死亡触发器”的作用。这些通路的调节缺陷在包括癌症在内的人类疾病中都很明显。我们的长期目标是了解扑杀环E3型“分子死亡触发器”的精确功能，并开始努力开发针对人类癌症的治疗策略。我们项目的短期目标是确定这种类型的CUL7E3如何发挥作用，消除一种名为IRS-1的蛋白质靶标，从而有助于抑制肿瘤。具体地说，我们将进行生化实验，以确定IRS-1中指导其与CUL7 E3分子相互作用的“内置”代码，这会触发泛素标记，从而导致降解。我们将定义那些修改IRS-1降级“代码”的蜂窝组件，将其设置为“开”或“关”。此外，我们还将进行深入的分子和细胞分析，以了解CUL7 E3是如何组装成“分子死亡触发器”的。我们将致力于探索我们的初步发现，即CUL7 E3消除IRS-1有助于细胞通过灭活一种被称为PI3K的主要生长促进活性来抑制癌症。将开发细胞转化分析来解决这一问题。此外，我们最近发现，IRS-1可以通过驱动癌细胞进入永久生长抑制状态(衰老)来预防癌症。鉴于它在理解癌症启动和预防方面的重要分支，我们将剖析这一新的衰老程序的组成部分，并利用分子和细胞生物学工具了解它是如何发挥作用的。公共卫生相关性：该项目旨在探索最初在本实验室发现的由CUL7 E3泛素连接酶复合体执行的两种抗癌活动。实验被概述以了解CUL7E3如何指导IRS-1信号蛋白的降解，以及这一过程如何通过失活PI3K生长促进功能来抑制癌症。此外，将利用深入的分子和细胞生物学方法来探索IRS-1在诱导永久性细胞停滞(衰老)方面的新功能，这已成为包括人类在内的多细胞生物中的一种突出的抗癌机制。

DESCRIPTION (provided by applicant): Elimination of unwanted and harmful proteins is of key importance for the function of a cell and an organism. Work from many laboratories including ours in the past decade have uncovered a large number of structurally related protein complexes, named cullin- RING E3 ubiquitin ligases. These E3 complexes function as "molecular death trigger' by tagging target proteins with a chain of death-tag, known as ubiquitin, thereby directing their destruction. Defective regulation of these pathways is manifest in human diseases including cancers. Our long-range goal is to understand the precise functioning of cullin-RING E3 type of "molecular death triggers," and initiate efforts to develop therapeutic strategies against human cancers. The short-term objective of our project is to determine how one such type, the CUL7 E3, functions to eliminate a protein target called IRS-1, thereby contributing to tumor suppression. Specifically, we will carry out biochemical experiments to identify the "built-in" code in IRS-1 that directs its molecular interactions with the CUL7 E3, which triggers ubiquitin tagging and hence degradation. We will define those cellular components that modify the IRS-1 degradation "code," setting it "ON" or "OFF." In addition, we will perform in- depth molecular and cellular analysis to understand how the CUL7 E3 is assembled into a "molecular death trigger." We will devote our major efforts to explore our preliminary findings that elimination of IRS-1 by the CUL7 E3 helps a cell suppress cancer by inactivating a major growth- promoting activity known as PI3K. Cell transformation assays will be developed to address this issue. In addition, we have recently discovered that IRS-1 could prevent cancer by driving cancerous cells to a permanent growth arresting state (senescence). Given its significant ramification in understanding cancer initiation and prevention, we will dissect the components of this novel senescence program and understand how it functions using molecular and cell biology tools. PUBLIC HEALTH RELEVANCE: This project intends to explore two anti-cancer activities that are executed by the CUL7 E3 ubiquitin ligase complex, initially discovered in this laboratory. Experiments are outlined to understand how the CUL7 E3 directs the degradation of the IRS-1 signaling protein, and how this process suppresses cancer by inactivating the PI3K-growth promoting function. Moreover, in-depth molecular and cell biology methods will be employed to explore the novel function by IRS-1 in inducing permanent cell arrest (senescence), which has emerged as a prominent anti-cancer mechanism in multi- cellular organisms including humans.