Function and Mechanism of CUL4 E3 Ligases in Human Diseases

描述(申请人提供)：大多数，如果不是全部，细胞过程通过泛素系统调节调节蛋白的及时降解。特定的底物蛋白是通过E3泛素连接酶的活性而被降解的。我们先前发现，两个小的环指蛋白ROC1和ROC2是最大的E3连接酶复合体--cullin环E3连接酶(CRL)家族的重要催化成分。我们还发现，cullin 3和cullin 4可以分别组装多达200个和100个不同的CRL3和CRL4 E3复合体。在这一应用中，我们提出了两条研究路线，旨在了解两个特定的CRL4 E3泛素连接酶复合体CRL4G？2和CRL4WDR5的功能和机制，以及这些CRL4复合体的变化如何导致两种主要的人类疾病，心力衰竭和X连锁智力低下。在第一个目标中，我们将研究CRL4G？2的分子功能及其在心血管疾病中的作用。我们提供的数据表明，WD40蛋白，G蛋白？亚基2(G？2)，与DDB1-CUL4A结合，靶向G蛋白偶联受体激酶(GRK2)，进行泛素化。GRK2升高以前与心肌梗死、心力衰竭、门脉高压、胰岛素抵抗和阿尔茨海默病有关。我们建议首先通过确定CRL4与其他G和GRK蛋白的相互作用来探索CRL4在G蛋白偶联受体(GPCR)信号调节中的广泛参与。然后，我们将集中在一个特征良好的GPCR信号通路-肾上腺素能受体(AR)信号，以确定CRL4G？2介导的GRK2泛素化的机制。我们还将确定Cul4a基因的缺失是否会损害小鼠的心功能。在第二个目的中，我们提供的数据表明，CUL4B是X-连锁智力低下(XLMR)患者中最常见的突变基因之一，而不是CUL4A，是WDR5的核E3连接酶，WD40蛋白是组蛋白H3K4三甲基化的重要组成部分。我们建议确定CUL4B介导的WDR5泛素化在神经细胞增殖、存活和分化中的作用。我们还建议开发和鉴定一种新的小鼠模型--条件性Cul4b突变小鼠品系，以确定Cul4b在脑中的功能。最后，我们建议广泛地确定CUL4B在染色质修饰和基因表达调节中的功能。 公共卫生相关性：基于在上一个资助周期中的发现，我们建议确定两个不同的基于cullin的泛素连接酶复合体的机制和功能，每个复合体都与一种特定的人类疾病有关：CRL4GNB2与心血管疾病有关，CRL4WDR5与X连锁精神发育迟滞有关。

DESCRIPTION (provided by applicant): Most, if not all, cellular processes are regulated by timely degradation of regulatory proteins through the ubiquitin system. Specific substrate proteins are targeted for degradation through the activity of E3 ubiquitin ligases. We previously discovered that two small RING finger proteins, ROC1 and ROC2, function as the essential catalytic components of the largest family of E3 ligase complexes, the cullin-RING E3 ligases (CRLs). We also discovered that cullin 3 and cullin 4 could assemble as many as 200 and 100 distinct CRL3 and CRL4 E3 complexes, respectively. In this application, we propose two lines of research aimed at understanding the function and mechanism of two specific CRL4 E3 ubiquitin ligase complexes, CRL4G¿2, and CRL4WDR5, and how alterations of these CRL4 complexes contribute to two major human diseases, heart failure and X-linked mental retardation. In the first Aim, we will examine the molecular function of CRL4 G¿2 and its role in cardiovascular disease. We present data demonstrating that a WD40 protein, G-protein ¿ subunit 2 (G¿2), associates with DDB1- CUL4A to target G protein-coupled receptor kinases (GRK2), for ubiquitylation. Elevated GRK2 has previously been associated with myocardial infarction, heart failure, portal hypertension, insulin resistance, and Alzheimer's disease. We propose first to explore how broadly CRL4 is involved in the regulation of G- protein coupled receptor (GPCR) signaling by determining the interaction of CRL4 with other G¿ and GRK proteins. We will then focus on one well-characterized GPCR signaling pathway, ¿-adrenergic receptors (¿ - AR) signaling, to determine the mechanism of CRL4G¿2 mediated GRK2 ubiquitylation. We will also determine whether loss of Cul4a impairs heart function in mouse. In the second Aim, we present data demonstrating that CUL4B, one of the most frequently mutated genes in X-linked mental retardation (XLMR) patients, but not CUL4A, is a nuclear E3 ligase of WDR5, a WD40 protein and an essential component for histone H3K4 trimethylation. We propose to determine the function of CUL4B-mediated WDR5 ubiquitylation in neuronal cell proliferation, survival and differentiation. We also propose to develop and characterize a novel mouse model, conditional Cul4b mutant mouse strain, to determine the function of Cul4b in brain. Finally, we propose to determine broadly the function of CUL4B in the regulation of chromatin modification and gene expression. PUBLIC HEALTH RELEVANCE: Based on the discoveries made during the last funding cycle, we propose to determine the mechanism and function of two distinct cullin-based ubiquitin ligase complexes, each linked with a specific human disease: CRL4GNB2 in cardiovascular disease and CRL4WDR5 in X-linked mental retardation.