Mechanistic Characterization and Genetic Analysis of Systemic Iron Homeostasis

说明(申请人提供)：作为血红素的一种成分和许多铁依赖的酶的成分，铁对氧化磷酸化、红血球生成和许多其他化学反应是必不可少的，这些化学反应对几乎所有生物的生存都是至关重要的。铁缺乏困扰着世界上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。过量存在时，它也可能是有毒的，甚至是致命的。尽管人们早就意识到体内的铁含量和血浆铁水平受到严格的调控，但铁稳态的许多分子细节仍然模糊不清，甚至连铁缺乏或过剩是如何感知的基本问题也仍然难以捉摸。最近，我们利用位置克隆技术筛选出了一种在缺铁检测中具有非冗余功能的蛋白质。这项工作旨在了解MASK，一种ENU诱导的表型，其特征是进行性脱发和小细胞性贫血，以及小鼠缺铁。MASK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“低铁”感受器的重要组成部分。具体来说：1.TMPRSS6的正常功能是抑制铁稳态的中枢调节因子--海普西丁的表达；2.TMPRSS6对编码海普西丁的基因HAMP的抑制在所有已知的刺激诱导途径中占据主导地位；3.TMPRSS6的抑制作用是由HAMP启动子的近端元件(S)介导的；4.TMPRSS6的蛋白水解性对HAMP的抑制是必不可少的；5.TMPRSS6的胞浆结构域能够自主地驱动抑制信号转导。此外，我们还研究了TMPRSS6高表达的肝癌细胞株HepG2细胞在基础条件下和在高效表达上调因子BMP2刺激下的转录谱。一些基因被发现受到TMPRSS6过表达的差异调节，这为分析信号转导提供了可能的联系。在这项提案中，我们计划继续我们的工作，以充分表征TMPRSS6发出的响应铁衰减的信号级联。将进行三个实验课程。我们将：1.研究TMPRSS6的结构特征，希望了解TMPRSS6在细胞膜上启动的信号转导的近端事件；2.进行基于亲和力的蛋白质相互作用研究，目的是分离与TMPRSS6在信号通路中相关的蛋白质(S)；3.进行正向遗传筛选，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，而且能够创造有效的策略来对抗各种铁吸收障碍。与公共健康相关：这项建议的目的是描述一种信号通路，允许检测体内的铁缺乏，并探索通过遗传方法找到铁稳态的全套调节蛋白的可能性。

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.