Analysis of NGAL Metabolism Identifies a Therapy for Iron Overload

描述(由申请人提供)：铁的运输是一个重要的问题，因为游离铁在生理pH下不溶于好氧溶液(&lt；10-18M)，而当被一些螯合剂溶解时，会产生一种活性形式的铁，可以产生有毒的氧物种。因此，需要专门的机制来运输铁，这些专门的机制存在于蛋白质中，这些蛋白质利用保守的基序直接结合铁(转铁蛋白和铁蛋白)或利用嵌入的辅因子。虽然细胞外铁的运输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的表型(Barasch，Development Cell，2009)。我们发现，Lipocalin超家族的一个成员NGAL在结合一族称为儿茶酚或相关的从儿茶酚构建的细菌铁载体时，扮演着一个高亲和力的铁载体的角色(Barasch，分子细胞，2002)。在铁的存在下，NGAL：铁载体：FeIII的络合物以亚纳摩尔亲和力形成(Barasch，自然化学生物学，2010)，形成一种鲜红色的蛋白质，它在溶液中稳定数天，并在体内稳定地运输其紧密结合的铁。NGAL是在体内表达的，但一些“损伤”刺激会使其浓度升高几个数量级。此后，NGAL运输血清，并被认为被肾脏受体megalin捕获，在那里NGAL清除铁载体：铁复合体。虽然我们已经了解了大量关于NGAL尿液形式的代谢(它表达于远端肾单位，并以全长蛋白的形式在尿液中排泄)，但我们对这个清除系统和megalin受体的作用知之甚少，megalin受体是唯一已证实的NGAL受体。为了深入研究这一过程，我们将研究megalin的一个条件突变体，为了在野生型小鼠中进行研究，我们正在创建一系列NGAL突变体，其中一些突变体绕过了megalin所在的近端小管，导致它们存在于尿液中。在这些研究中，我们意识到突变株仍然可以高亲和力结合铁载体：FeIII(它们是红色蛋白质)，并且它们肯定可以排泄铁，我们推测是以氧化还原失活的方式进行的。事实上，NGAL：铁载体：铁的复合体将铁从细菌中隔离出来，而不是将铁捐赠给微生物，这是小分子螯合剂的主要担忧。总之，在这项建议中，我们验证了巨蛋白是NGAL的关键循环受体的假设，并基于这一想法，我们提出当巨蛋白-NGAL复合体被抑制时，NGAL可以在尿液中携带紧密结合的铁，从而为铁超载疾病的常见症状提供一种安全、新颖的治疗方法。 公共卫生相关性：铁超负荷疾病是临床医学中常见的疾病，其治疗方法已被证明对许多细胞系有害，并诱导细菌生长。铁超载是输血的常见后遗症，但它在肝炎、慢性肾脏疾病以及常见的遗传性疾病如血色素沉着症中都是众所周知的。我们发现了一种新的基于NGAL蛋白的铁转运途径，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可以作为一种安全的、治疗性的铁络合剂。

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.