Biomedical Research and Research Training

项目摘要 肿瘤坏死因子（TNF）是一种重要的细胞因子，其协调细胞因子的产生、炎症、细胞凋亡和细胞凋亡。 存活和细胞死亡。生死决定是如何对TNF作出反应的，目前还不完全清楚。 几种分子事件决定了TNF刺激细胞是否导致转录反应， 促进细胞存活或是否会导致细胞死亡。TNF受体的下游信号传导严重 受翻译后修饰（包括遍在化）的调节。LUBAC，一种泛素（Ub）E3连接酶， Sharpin，HOIL和HOIP的，是迄今为止描述的唯一可以修饰具有线性Ub链的蛋白质的Ub连接酶 （Metl-Ub链）。LUBAC活性对于TNF-α诱导的NF-kB依赖性促存活基因转录是必需的 刺激.来源于LUBAC组分中具有生殖系突变的患者的细胞引起缺陷性NF-κ B-κ B。 依赖的基因转录和细胞死亡途径的异常激活。类似地， 有缺陷的LUBAC组分引起慢性炎症和多个器官和组织中的细胞凋亡增加。 我们发表的研究表明，细胞死亡，而不是在NF-κ B依赖基因转录的缺陷， 在LUBAC缺陷（sharpinpdm）动物中， FADD是死亡诱导信号的重要组成部分， 复杂.这一认识使我们假设LUBAC活性是预防细胞凋亡的直接必要条件。 死亡在目标1中，我们将讨论LUBAC修饰Met 1-Ub蛋白靶点的分子基础 店我们将讨论LUBAC如何使用Met 1-Ub链修改FADD。在目标2中，我们将描述 FADD修饰在细胞中的功能相关性，并使用新的基于Ub的工具来富集Met 1-Ub链。在 目的3，我们将研究LUBAC自身泛素化的功能以及磷酸化如何调节LUBAC自身泛素化， LUBAC的线性Ub链形成活性。我们的研究将为原发性高血压的病因学提供机制性的见解。 与LUBAC组分中生殖系突变患者相关的免疫缺陷。

PROJECT SUMMARY Tumor necrosis factor (TNF) is an important cytokine that coordinates cytokine production, inflammation, cell survival, and cell death. How life and death decisions are made in response to TNF is not completely understood. Several molecular events determine whether TNF stimulation of cells leads to a transcriptional response that promotes cell survival or whether it can lead to cell death. Signaling downstream of the TNF receptor is heavily regulated by post-translation modifications including ubiquitination. LUBAC, a ubiquitin (Ub) E3 ligase consisting of Sharpin, HOIL and HOIP, is the only Ub ligase described to date that can modify proteins with linear Ub chains (Met1-Ub chains). LUBAC activity is essential for NF-kB dependent transcription of prosurvival genes upon TNF stimulation. Cells derived from patients with germline mutations in LUBAC components elicit defective NF-kB- dependent gene transcription and aberrant activation of cell death pathways. Similarly, murine models with defective LUBAC components elicit chronic inflammation and increased apoptosis in multiple organs and tissues. Our published studies indicate that cell death, rather than a deficiency in NF-kB dependent gene transcription, in LUBAC-deficient (sharpincpdm) animals is driving the TNF-dependent chronic inflammation as this phenotype is reversed by a compound deficiency in FADD—an important component of the death inducing signaling complex. This insight led us to the hypothesis that LUBAC activity is directly required for the prevention of cell death. In aim 1, we will address the molecular basis for LUBAC modification of protein targets with Met1-Ub chains. We will address how LUBAC can modify FADD with Met1-Ub chains. In aim 2, we will characterize the functional relevance of FADD modification in cells and use a new Ub-based tool to enrich for Met1-Ub chains. In aim 3, we will investigate the function of LUBAC auto-ubiquitination and how phosphorylation can regulate LUBAC’s linear Ub chain forming activity. Our studies will provide mechanistic insight into the etiology of primary immune deficiencies associated with patients with germline mutations in LUBAC components.