Study of an iron-responsive E3 ligase regulating mammalian iron homeostasis

描述（由申请人提供）：虽然铁是许多蛋白质必不可少的辅助因子，但其有利的化学性质也可以促进毒副反应，破坏大分子。不能维持适当的铁稳态可导致贫血或铁超载紊乱，以及增加对感染的易感性。细胞铁稳态是由负责铁摄取、释放、利用和储存的基因产物的转录后协调调节维持的。当细胞游离铁可用性低时，铁调控蛋白1和2 （IRP1和2）在这些mrna的5‘或3’非翻译区结合铁反应元件（IREs），影响其后续翻译或稳定性。当细胞的游离铁可用性高时，IRP1组装铁硫簇，导致蛋白质失去对IREs的亲和力，而IRP2优先泛素化并被蛋白酶体降解。然而，细胞如何感知铁水平并随后调节IRP2降解的潜在机制尚不清楚，并且已被证明极具争议性。为了解决与细胞铁传感和IRP调节相关的悬而未决的问题，研究人员进行了基于细胞的siRNA筛选，以鉴定调节IRP2稳定性的E3泛素连接酶。从该筛选中选择最佳候选人进行进一步表征。初步研究表明，当细胞游离铁可用性高时，含有FBXL5蛋白的E3泛素连接酶复合物SCFFBXL5直接靶向IRP2进行蛋白酶体降解。FBXL5本身的稳定性受到调控，在铁和氧充满的条件下积累，在铁耗尽时靶向降解。FBXL5似乎含有铁和氧结合的氰菊酯结构域，该结构域作为配体结合调节开关介导FBXL5的差异稳定性。这些观察结果表明，通过氰菊酯结构域的铁感知、FBXL5积累、IRP2调节和维持哺乳动物细胞铁稳态的细胞反应之间存在直接的机制联系。本研究的主要目标是验证SCFFBXL5在IRP2调控中的作用，表征FBXL5功能和调控的分子机制，并研究FBXL5在维持哺乳动物体内铁稳态中的重要性。具体而言，本项目旨在(1)利用培养细胞和体外重构分析绘制和表征FBXL5的功能和调控结构域；(2)利用多种生物物理技术研究氯氰菊酯传感器的配体结合特性；(3)确定调节氯氰菊酯结构域铁依赖性稳定性的其他因素；(4)生成和表征缺乏FBXL5表达的小鼠。总之，这些研究将大大提高我们对哺乳动物铁稳态的认识，并可能为治疗相关的人类疾病提供新的见解。

DESCRIPTION (provided by applicant): While iron is an essential cofactor for many proteins, its favorable chemical properties can also promote toxic side reactions that damage macromolecules. Failure to maintain proper iron homeostasis can lead to anemia or iron overload disorders, as well as increased susceptibility to infection. Cellular iron homeostasis is maintained by the coordinate posttranscriptional regulation of gene products responsible for iron uptake, release, utilization, and storage. When cellular free iron availability is low, Iron Regulatory Proteins 1 and 2 (IRP1 and 2) bind Iron Response Elements (IREs) within the 5' or 3' untranslated regions of these mRNAs to affect their subsequent translation or stability. When cellular free iron availability is high, IRP1 assembles an iron-sulfur cluster, causing the protein to lose its affinity for IREs, while IRP2 is preferentially ubiquitinated and degraded by the proteasome. However, the underlying mechanism of how the cell senses iron levels and subsequently regulates IRP2 degradation is poorly understood and has proven to be extremely controversial. To address the outstanding questions related to cellular iron sensing and IRP regulation, a cell-based siRNA screen was performed to identify E3 ubiquitin ligases that regulate IRP2 stability. The top candidate from that screen has been selected for further characterization. Preliminary studies indicate that the E3 ubiquitin ligase complex containing the FBXL5 protein, SCFFBXL5, directly targets IRP2 for proteasomal degradation when cellular free iron availability is high. The stability of FBXL5 itself is regulated, accumulating under iron and oxygen replete conditions and targeted for degradation upon iron depletion. FBXL5 appears to contain an iron- and oxygen-binding hemerythrin domain that acts as a ligand-binding regulatory switch mediating FBXL5's differential stability. These observations suggest a direct mechanistic link between iron sensing via a hemerythrin domain, FBXL5 accumulation, IRP2 regulation, and cellular responses to maintain mammalian cellular iron homeostasis. The broad objectives of this proposal are to validate the role of SCFFBXL5 in the regulation of IRP2, characterize the molecular mechanisms responsible for FBXL5's function(s) and regulation, and investigate the importance of FBXL5 to the maintenance of mammalian iron homeostasis in vivo. Specifically, this proposal aims to (1) map and characterize the functional and regulatory domains of FBXL5 using cultured cells and in vitro reconstitution assays, (2) investigate the ligand binding properties of the hemerythrin sensor using a variety of biophysical techniques, (3) identify additional factor(s) that regulate the iron-dependent stability of the hemerythrin domain, and (4) generate and characterize mice lacking FBXL5 expression. Together these studies will greatly inform our understanding of mammalian iron homeostasis and may provide new insights for treatment of related human diseases. PUBLIC HEALTH RELEVANCE: Failure to maintain proper iron homeostasis can lead to a variety of disease states affecting millions worldwide including anemia, iron overload disorders, and increased susceptibility to infection. Improved understanding of the cellular pathways responsible for sensing and responding to changes in iron availability may provide new avenues for therapeutic intervention in such cases. To that end, this proposal describes the characterization of a candidate sensor and regulator of mammalian iron homeostasis, FBXL5.