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

描述(申请人提供)：虽然铁是许多蛋白质的基本辅因子，但其良好的化学性质也会促进破坏大分子的有毒副反应。未能保持适当的铁平衡可能会导致贫血或铁超载紊乱，并增加感染的易感性。细胞铁的稳态是通过对负责铁的摄取、释放、利用和储存的基因产物的转录后调节来维持的。当细胞内游离铁利用率低时，铁调节蛋白1和2(Irp1和2)与这些mRNA5‘或3’非翻译区内的铁反应元件(IRES)结合，从而影响它们随后的翻译或稳定性。当细胞内游离铁可获得性高时，IRP1组装一个铁-硫簇，导致蛋白质失去与IRES的亲和力，而IRP2优先泛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，已被证明是极具争议的。为了解决与细胞铁感应和IRP调节相关的悬而未决的问题，基于细胞的siRNA筛选被用来鉴定调节IRP2稳定性的E3泛素连接酶。已经从该屏幕中选出了排名靠前的候选人进行进一步的表征。初步研究表明，当细胞内游离铁利用率较高时，含有FBXL5蛋白的E3泛素连接酶复合体SCFFBXL5直接针对IRP2降解蛋白酶体。FBXL5本身的稳定性是受调节的，在铁和氧气充足的条件下积累，并在铁耗尽时被降解。FBXL5似乎含有一个铁和氧结合的杂氰菊酯结构域，该结构域作为配体结合的调节开关调节FBXL5‘S的分化稳定性。这些观察结果表明，通过氢氰菊酯结构域感知铁、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。本研究的主要目的是验证SCFFBXL5在IRP2调控中的作用，研究FBXL5的S功能和调控的分子机制(S)，并探讨FBXL5在维持哺乳动物体内铁稳态中的重要性。具体地说，这项建议的目的是(1)利用培养细胞和体外重组分析来定位和表征FBXL5的功能和调节域，(2)使用各种生物物理技术来研究杂氰菊酯传感器的配体结合特性，(3)识别调控杂氰菊酯结构域的铁依赖稳定性的额外因素(S)，以及(4)建立和鉴定缺乏FBXL5表达的小鼠。综上所述，这些研究将极大地促进我们对哺乳动物铁稳态的理解，并可能为相关人类疾病的治疗提供新的见解。

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.