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的重要辅因子，但其有利的化学性质也会促进损害大分子的毒性副反应。如果不能维持适当的铁稳态，可能会导致贫血或铁超载疾病，以及增加感染的易感性。细胞内铁稳态是由负责铁的摄取、释放、利用和储存的基因产物的协调转录后调节来维持的。当细胞游离铁可用性低时，铁调节蛋白1和2（IRP1和2）结合这些mRNA的5'或3'非翻译区内的铁反应元件（IRE）以影响其随后的翻译或稳定性。当细胞游离铁的可用性高时，IRP1组装铁硫簇，导致蛋白质失去对IRES的亲和力，而IRP2优先被蛋白酶体泛素化和降解。然而，细胞如何感知铁水平并随后调节IRP2降解的潜在机制知之甚少，并且已被证明是极具争议的。为了解决与细胞铁传感和IRP调节相关的突出问题，进行基于细胞的siRNA筛选以鉴定调节IRP 2稳定性的E3泛素连接酶。从该筛选中选择最佳候选物用于进一步表征。初步研究表明，E3泛素连接酶复合物含有FBXL5蛋白，SCFFBXL5，直接靶向IRP2蛋白酶体降解时，细胞游离铁的可用性高。FBXL5本身的稳定性受到调节，在铁和氧充足的条件下积累，并在铁耗尽时降解。FBXL5似乎含有铁和氧结合的血红蛋白结构域，其充当介导FBXL5的差异稳定性的配体结合调节开关。这些观察结果表明，铁传感通过血红素域，FBXL5积累，IRP2调节，和细胞反应，以维持哺乳动物细胞铁稳态之间的直接机制联系。该提案的广泛目标是验证SCFFBXL5在IRP2调节中的作用，表征负责FBXL5功能和调节的分子机制，并研究FBXL5对维持哺乳动物体内铁稳态的重要性。具体地，该提议旨在（1）使用培养的细胞和体外重建测定来绘制和表征FBXL5的功能和调节结构域，（2）使用多种生物物理技术来研究血红蛋白传感器的配体结合特性，（3）鉴定调节血红蛋白结构域的铁依赖性稳定性的额外因子，和（4）产生和表征缺乏FBXL5表达的小鼠。总之，这些研究将极大地告知我们的哺乳动物铁稳态的理解，并可能为相关的人类疾病的治疗提供新的见解。 公共卫生相关性：未能维持适当的铁稳态可导致影响全世界数百万人的各种疾病状态，包括贫血、铁超负荷紊乱和感染易感性增加。对负责感知和响应铁可用性变化的细胞通路的更好理解可能为此类情况下的治疗干预提供新途径。为此，该提案描述了哺乳动物铁稳态的候选传感器和调节器FBXL5的表征。

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.