Function and Mechanism of CUL4 E3 Ligases in Human Diseases

描述（由申请人提供）：大多数（如果不是全部）细胞过程通过泛素系统及时降解调节蛋白来调节。特异性底物蛋白通过E3泛素连接酶的活性靶向降解。我们以前发现，两个小的RING指蛋白，ROC1和ROC2，作为E3连接酶复合物的最大家族，cullin-RING E3连接酶（CRL）的基本催化组分。我们还发现cullin 3和cullin 4分别可以组装多达200个和100个不同的CRL 3和CRL 4 E3复合物。在本申请中，我们提出了两条研究路线，旨在了解两种特定的CRL 4 E3泛素连接酶复合物，CRL 4G <$2和CRL 4WDR 5的功能和机制，以及这些CRL 4复合物的改变如何导致两种主要的人类疾病，心力衰竭和X连锁精神发育迟滞。在第一个目标中，我们将研究CRL 4 G <$2的分子功能及其在心血管疾病中的作用。我们目前的数据表明，WD 40蛋白，G蛋白亚基2（G <$2），与DDB1-CUL4A的目标G蛋白偶联受体激酶（GRK2），泛素化。GRK2升高以前与心肌梗死、心力衰竭、门脉高压、胰岛素抵抗和阿尔茨海默病有关。我们建议首先探索如何广泛的CRL 4参与G蛋白偶联受体（GPCR）信号的调节，通过确定CRL 4与其他G蛋白和GRK蛋白的相互作用。然后，我们将集中在一个充分表征的GPCR信号通路，<$-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的缺失是否损害小鼠的心脏功能。在第二个目标，我们目前的数据表明，CUL4B，最常见的突变基因之一，在X连锁精神发育迟滞（XLMR）患者，但不是CUL4A，是一个核E3连接酶的WDR5，WD 40蛋白和组蛋白H3K4三甲基化的必要组成部分。我们建议确定CUL4B介导的WDR5泛素化在神经细胞增殖，存活和分化中的功能。我们还建议开发和表征一种新的小鼠模型，条件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控中的功能。

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.