Analysis of NGAL Metabolism Identifies a Therapy for Iron Overload

描述（由申请人提供）：铁的运输造成了一个显著的问题，因为游离三价铁在生理pH的需氧溶液中是不溶的（< 10-18 M），而在被一些螯合剂溶解时，产生了一种反应形式的铁，其可以产生有毒的氧物质。因此需要专门的机制来运输铁，并且这些专门的机制在利用保守基序直接结合铁（转铁蛋白和铁蛋白）或利用嵌入的辅因子的蛋白质中发现。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显示出令人惊讶的有限表型（Barasch，Developmental Cell，2009）。我们发现称为Ngal的脂质运载蛋白超家族的成员在结合称为儿茶酚或由儿茶酚构建的相关细菌铁载体的新型辅因子家族时充当高亲和力铁载体（Barasch，Molecular Cell，2002）。在铁的存在下，Ngal：铁载体：FeIII复合物的形成以亚纳摩尔亲和力发生（Barasch，《自然化学生物学》，2010年），形成亮红色蛋白质，该蛋白质在溶液中稳定多日，并且在体内稳定运输其紧密结合的铁。Ngal在体内表达，但许多“损伤”刺激物使其浓度提高几个数量级。此后，Ngal在血清中运输，并被认为是由肾受体巨蛋白捕获，其中Ngal清除铁载体：Fe复合物。虽然我们已经了解了大量关于Ngal的尿液形式的代谢（它从远端肾单位表达并作为全长蛋白质在尿液中排泄），但我们对这种清除系统和巨蛋白受体的作用知之甚少，巨蛋白受体是Ngal唯一确认的受体。为了深入研究这一过程，我们将研究巨蛋白的条件突变体，对于野生型小鼠的研究，我们正在创建一系列Ngal突变体，其中一些绕过巨蛋白所在的近端小管，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以高亲和力与铁载体FeIII结合（它们是红色蛋白质），并且它们肯定可以排泄铁，我们推测它们是以氧化还原非活性的方式。事实上，Ngal：铁载体：Fe复合物不是将铁捐赠给微生物，这是小分子螯合剂的主要关注点，而是从细菌中螯合铁。总之，在这个提议中，我们测试了巨蛋白是Ngal的关键再循环受体的假设，并且由于这个想法，我们提出当巨蛋白-Ngal复合物被抑制时，Ngal可以在尿液中携带紧密结合的铁，因此作为铁过载疾病的常见综合征的安全的新型治疗剂。 公共卫生相关性：铁超载疾病是临床医学中常见的疾病，其治疗已被证明对许多细胞系具有毒性以及诱导细菌生长。铁超负荷是输血的常见后果，但它在肝炎、慢性肾病以及常见遗传性疾病如血色病中是众所周知的。我们已经发现了一种基于Ngal蛋白的新型铁转运途径，该蛋白在人类不同类型的组织损伤中大量表达。我们对Ngal代谢的研究已经证明了Ngal可以用作安全的治疗性铁螯合剂的概念。

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. PUBLIC HEALTH RELEVANCE: Iron overload diseases are common occurrences in clinical medicine, and their therapies have proved toxic to many cell lineages as well as inductive of bacterial growth. Iron overload is a common sequel of blood transfusions, but it is well known in hepatitis, chronic kidney disease as well as in common hereditary diseases such as hemachromatosis. We have discovered a novel iron trafficking pathway based on the protein Ngal, which is massively expressed in the human in different types of tissue damage. Our studies in Ngal metabolism have led to a proof of concept that Ngal can be used as a safe, therapeutic iron chelator.