The function and regulation of the cullin family E3 ubiquitin ligases

描述（由申请人提供）：消除不需要的和有害的蛋白质对细胞和生物体的功能至关重要。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物，称为cullin- RING E3泛素连接酶。这些E3复合物通过用死亡标签链（即泛素）标记目标蛋白质，从而指导它们的破坏，从而发挥“分子死亡触发器”的作用。这些途径的缺陷调控在包括癌症在内的人类疾病中是明显的。我们的长期目标是了解cullin-RING E3型“分子死亡触发器”的确切功能，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CUL7 E3如何消除IRS-1蛋白靶标，从而促进肿瘤抑制。具体来说，我们将进行生化实验，以确定IRS-1中“内置”的代码，该代码指导其与CUL7 E3的分子相互作用，从而触发泛素标记并因此降解。我们将定义那些修改IRS-1降解“代码”的细胞成分，将其设置为“ON”或“OFF”。此外，我们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，即通过cul7e3消除IRS-1有助于细胞通过灭活主要的促生长活性PI3K来抑制癌症。细胞转化试验将被开发来解决这个问题。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。鉴于其在理解癌症发生和预防方面的重要分支，我们将剖析这种新型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起作用的。公共卫生相关性：该项目旨在探索最初在该实验室发现的CUL7 E3泛素连接酶复合体执行的两种抗癌活性。实验概述了CUL7 E3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，深入的分子和细胞生物学方法将用于探索IRS-1诱导永久性细胞停滞（衰老）的新功能，这已经成为包括人类在内的多细胞生物的重要抗癌机制。

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.