Mechanistic Characterization and Genetic Analysis of Systemic Iron Homeostasis

描述（由申请人提供）：作为血红素和许多铁依赖性酶的组成部分，铁是氧化磷酸化、红细胞生成和许多其他化学反应所必需的，这些化学反应对几乎所有生物的生存都至关重要。缺铁困扰着世界上30%的人口，并可能导致贫血、发育迟缓、生育问题和免疫功能障碍。然而，铁是一把双刃剑。过量时，它也可能是有毒的，甚至是致命的。虽然人们早就认识到体内铁含量和血浆铁水平受到精心调节，但铁稳态的许多分子细节尚不清楚，甚至关于铁缺乏或过量是如何被感知的基本问题也仍然难以捉摸。近年来，我们利用定位克隆技术鉴定出一种在铁缺乏检测中具有非冗余功能的蛋白。本研究旨在了解enu诱导的一种以小鼠进行性脱发、小细胞性贫血和缺铁为特征的表型。掩膜表型可追溯到影响II型跨膜丝氨酸蛋白酶6 （TMPRSS6）的剪接错误。此外，我们的数据显示TMPRSS6是肝细胞中“低铁”传感装置的重要组成部分。具体:1。TMPRSS6的正常功能是抑制铁稳态中枢调节因子hepcidin的表达；2. 在所有已知的刺激诱导的hepcidin上调途径中，TMPRSS6对编码hepcidin的基因Hamp的抑制占主导地位；3. TMPRSS6的抑制作用是由Hamp启动子的近端元件介导的；4. TMPRSS6的蛋白水解活性对抑制Hamp至关重要；5. TMPRSS6的胞质结构域能够自主驱动抑制信号转导。此外，我们还研究了TMPRSS6过表达的HepG2细胞（一种肝癌细胞系）在基础条件和hepcidin表达上调因子BMP2刺激下的转录谱。少数基因被发现受TMPRSS6过表达的差异调节，为信号转导分析提供了可能的联系。在本研究中，我们计划进一步研究TMPRSS6在铁衰减下发出的信号级联。将进行三项试验。我们将：1；研究TMPRSS6的结构特征，希望了解TMPRSS6在细胞膜上启动信号转导的近端事件；2. 开展基于亲和的蛋白相互作用研究，旨在分离信号通路中与TMPRSS6相关的蛋白；3. 进行前向基因筛选，寻找破坏小鼠全身铁平衡的突变，以扩大和完善我们对铁稳态的认识。通过这些研究，我们期望我们不仅能够充分阐明铁的稳态调节，而且能够制定有效的策略来对抗各种铁吸收障碍。公共卫生相关性：本提案旨在表征一种允许检测体内铁缺乏的信号通路，并探索通过遗传方法找到一整套铁稳态调节蛋白的可能性。

DESCRIPTION (provided by applicant): As a component of heme and a constituent of many iron-dependent enzymes, iron is essential for oxidative phosphorylation, erythropoiesis, and many other chemical reactions that are vital for survival of almost all living organisms. Iron deficiency afflicts 30% of the world's population, and can cause anemia, developmental retardation, fertility problems and immune dysfunction. However, iron is a double-edged sword. It can also be toxic or even lethal when present in excess. Although it has long been appreciated that body iron content and plasma iron levels are meticulously regulated, many of the molecular details of iron homeostasis are obscure, and even the basic question about how iron deficiency or excess are sensed remains elusive. Recently we used positional cloning to identify a protein with non-redundant function in the detection of iron deficiency. This work was aimed at the understanding of mask, an ENU-induced phenotype characterized by progressive alopecia and microcytic anemia, and iron deficiency in mice. Mask phenotype was traced to a splicing error affecting the type II transmembrane serine protease 6 (TMPRSS6). Moreover, our data revealed that TMPRSS6 is an essential component of the "low iron" sensing apparatus in hepatocytes. Specifically: 1. the normal function of TMPRSS6 is to inhibit the expression of hepcidin, the central regulator of iron homeostasis; 2. the suppression of Hamp, the gene encoding hepcidin by TMPRSS6, dominates over all known stimulus-induced pathways for upregulation of hepcidin; 3. the inhibitory effect of TMPRSS6 is mediated by proximal element(s) of the Hamp promoter; 4. the proteolytic activity of TMPRSS6 is essential for Hamp suppression; 5. the cytoplasmic domain of TMPRSS6 is able to autonomously drive the suppressive signal transduction. In addition, we investigated the transcription profiles in HepG2 cells (a liver carcinoma cell line) with TMPRSS6 overexpression under basal condition and upon stimulation with the potent upregulator of hepcidin expression, BMP2. A few genes were found to be differentially modulated by TMPRSS6 overexpression, providing possible links in the analysis of signal transduction. In this proposal, we plan to carry our work forward to fully characterize the signaling cascade emanating from TMPRSS6 in response to iron attenuation. Three courses of experimentation will be pursued. We will: 1. investigate the structural characteristics of TMPRSS6, hoping to gain insight on the proximal events in signal transduction initiated by TMPRSS6 on the cell membrane; 2. perform affinity-based protein interaction studies, aiming to isolate the protein(s) that associate with TMPRSS6 in the signaling pathway; 3. conduct a forward genetic screen to search for mutations that disrupt systemic iron balance in mice, in order to expand and complete our knowledge of iron homeostasis. Through these studies, we expect that we will not only be able to fully elucidate iron homeostatic regulation, but also create effective strategies combating various disorders of iron absorption. PUBLIC HEALTH RELEVANCE: This proposal is aimed at characterization of a signaling pathway that permits the detection of iron deficiency in the body, and explores the possibility of finding the complete set of regulatory proteins for iron homeostasis by a genetic approach.