Analysis of NGAL Metabolism Identifies a Therapy for Iron Overload

描述（由申请人提供）：铁的运输带来了一个重大问题，因为在生理pH值下，游离铁在有氧溶液中不溶（< 10-18 M），而在一些螯合剂的增溶作用下，会产生一种反应形式的铁，可以产生有毒的氧。因此，转运铁需要专门的机制，而这些专门的机制存在于利用保守基序直接结合铁（转铁蛋白和铁蛋白）或利用嵌入辅因子的蛋白质中。虽然细胞外铁转运主要由转铁蛋白介导，但携带这些基因缺失的小鼠表现出令人惊讶的有限表型（Barasch, Developmental Cell, 2009）。我们发现，脂质钙蛋白超家族的成员Ngal在结合名为儿茶酚的新型辅助因子家族或由儿茶酚构建的相关细菌铁载体时，充当了高亲和力的铁载体（Barasch, Molecular Cell, 2002）。在铁存在的情况下，Ngal：铁载体：FeIII复合物的形成以亚纳摩尔亲和力发生（Barasch, Nature Chemical Biology, 2010），形成一种鲜红色的蛋白质，该蛋白质在溶液中稳定了许多天，并且在体内稳定地运输其紧密结合的铁。Ngal在体内表达，但一些“损伤”刺激会使其浓度提高几个数量级。此后，Ngal在血清中运输，被认为被肾受体巨噬蛋白捕获，在那里Ngal清除铁载体：铁复合物。虽然我们已经了解了大量关于Ngal尿液形式的代谢（它从远端肾元表达，并作为全长蛋白在尿液中排泄），但我们对这种清除系统和meggalin受体的作用知之甚少，meggalin受体是唯一确认的Ngal受体。为了深入研究这一过程，我们将检查meggalin的条件突变体，对于野生型小鼠的研究，我们正在创建一系列Ngal突变体，其中一些绕过meggalin所在的近端小管，导致它们存在于尿液中。在这些研究中，我们意识到突变体仍然可以高亲和力地与铁载体：FeIII结合（它们是红色的蛋白质），并且它们肯定可以以氧化还原非活性的方式排出铁。事实上，Ngal：铁载体：铁复合物并没有将铁捐献给微生物，这是小分子螯合剂的主要关注点，而是从细菌中隔离铁。总之，在本提案中，我们验证了meggalin是Ngal的关键再循环受体的假设，并且由于这一想法，我们提出当meggalin -Ngal复合物被抑制时，Ngal可以在尿液中携带紧密结合的铁，因此可以作为一种安全的新型治疗铁超载疾病常见综合征的药物。

DESCRIPTION (provided by applicant): The transport of iron poses a significant problem because free ferric iron is insoluble (< 10-18 M) in aerobic solutions at physiologic pH, while upon solubilization by some chelators, a reactive form of iron is created that can produce toxic oxygen species. Specialized mechanisms are consequently required to traffic iron and these specialized mechanisms are found in proteins which utilize conserved motifs to directly bind iron (transferrin and ferritin) or utilize embedded cofactors. While extracellular iron transport is largely mediated by transferrin, mice carrying deletions of these genes displayed surprisingly limited phenotypes (Barasch, Developmental Cell, 2009). We found that a member of the lipocalin superfamily called Ngal acted as a high affinity iron carrier (Barasch, Molecular Cell, 2002) when binding a family of novel cofactors called the catechols or else the related bacterial siderophores constructed from catechol. In the presence of iron, formation of the Ngal:siderophore:FeIII complex occurred at subnanomolar affinity (Barasch, Nature Chemical Biology, 2010) forming a bright red protein, which was stable for many days in solution and stable in vivo for transport of its tightly bound iron. Ngal is expressed in vivo, but a number of "damage" stimuli raise its concentration by orders of magnitude. Thereafter, Ngal traffics in the serum and is thought to be captured by the kidney receptor megalin, where Ngal clears the siderophore:Fe complex. While we have learned a great deal about the metabolism of the urinary form of Ngal (it is expressed from the distal nephron and is excreted in the urine as a full length protein), we know much less about this clearance system and the role of the megalin receptor, which is the only confirmed receptor for Ngal. To study this process in depth we will examine a conditional mutant of megalin, and for studies in wild type mice we are creating a series of Ngal mutants, some of which bypass the proximal tubule where megalin is located, resulting in their presence in the urine. During these studies we realized that the mutants could still bind to siderophore:FeIII at high affinity (they were red colored proteins), and that they could definitely excrete iron, we speculate in a redox inactive manner. Indeed, rather than donate iron to micro-organisms, which is a major concern for small molecule chelators, the Ngal:siderophore:Fe complexes sequester iron from bacteria. In sum, in this proposal, we test the hypothesis that megalin is the key recycling receptor for Ngal and as a result of this idea, we propose that when the megalin-Ngal complex is inhibited, Ngal can carry tightly bound iron in the urine, hence serving as a safe, novel therapeutic for the common syndromes of iron overload diseases.