Analysis of NGAL Metabolism Identifies a Therapy for Iron Overload

NGAL 代谢分析确定了铁过载的治疗方法

基本信息

批准号：
8095786
负责人：
JONATHAN M. BARASCH
金额：
$ 24.58万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-01 至 2013-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8095786
关键词：
Acute Renal Failure with Renal Papillary Necrosis Aerobic Affinity Agranulocytosis Animals Bacteria Binding Biological Availability Biological Markers Biology Blood Circulation Blood Transfusion Bypass Carrier Proteins Catechols Cell Lineage Cells Chelating Agents Chemicals Chronic Kidney Failure Clinical Medicine Color Complex Data Deferoxamine Development Disease Distal Endocrine Endocytosis Engineering Enterobactin Evaluation Excretory function Family Ferritin Gelatinase A Gene Deletion Generations Genetic Growth Harvest Hepatitis Hereditary Disease Histocompatibility Testing Hour Human In Transferrin Inherited Interruption Iron Iron Chelation Iron Overload Kidney Knock-out LDL-Receptor Related Protein 2 Learning Left Length Ligands Liver Lung Lysosomes Measures Mediating Metabolism Modeling Molecular Mus Nature Nephrons Neutropenia New Agents Nutrient Organ Organism Oxidation-Reduction Oxidative Stress Oxygen Paper Pathway interactions Patients Peritoneum Phenotype Physiological Process Proteins Reaction Reactive Oxygen Species Role Series Serum Siderophores Skin Solutions Stimulus Structure Sum Surface Plasmon Resonance Syndrome System Techniques Testing Therapeutic Toxic effect Transferrin Transport Process Urine Wild Type Mouse X-Ray Crystallography base chelation cofactor extracellular genotoxicity hearing impairment in vivo member microorganism mutant non-genetic novel novel therapeutics prevent receptor receptor recycling small molecule trafficking urinary

项目摘要

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.

描述（由申请人提供）：铁的运输提出了一个重大问题，因为游离三价铁在生理 pH 值下不溶于有氧溶液（< 10-18 M），而在一些螯合剂溶解后，会产生反应形式的铁，能产生有毒的氧气。因此需要专门的机制来运输铁，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）或利用嵌入的辅助因子的蛋白质中。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，脂质运载蛋白超家族的一个成员（称为 Ngal）在与称为儿茶酚或由儿茶酚构建的相关细菌铁载体的新型辅助因子家族结合时，充当高亲和力铁载体（Barasch，分子细胞，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），形成鲜红色蛋白质，该蛋白质在溶液中稳定多天，并且在体内运输其紧密结合的铁。 Ngal 在体内表达，但许多“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，并被认为被肾脏受体巨蛋白捕获，其中 Ngal 清除铁载体：Fe 复合物。虽然我们对尿液形式 Ngal 的代谢（它从远端肾单位表达并以全长蛋白质的形式在尿液中排泄）了解很多，但我们对这种清除系统以及 Ngal 的作用知之甚少。巨蛋白受体，这是唯一被证实的 Ngal 受体。为了深入研究这个过程，我们将检查巨蛋白的条件突变体，并且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巨蛋白所在的近端小管，导致它们出现在尿液中。在这些研究中，我们意识到突变体仍然可以以高亲和力结合铁载体：FeIII（它们是红色蛋白质），并且它们肯定可以分泌铁，我们推测以氧化还原非活性方式。事实上，Ngal:铁载体:Fe复合物并没有将铁提供给微生物（这是小分子螯合剂的主要关注点），而是将铁从细菌中螯合起来。总之，在这个提案中，我们测试了巨蛋白是 Ngal 的关键回收受体的假设，并且由于这个想法，我们提出当巨蛋白-Ngal 复合物受到抑制时，Ngal 可以在尿液中携带紧密结合的铁，因此，它可以作为铁超载疾病常见综合征的安全、新颖的治疗方法。公众健康相关性：铁超载疾病在临床医学中很常见，其治疗方法已被证明对许多细胞谱系有毒并诱导细菌生长。铁超载是输血的常见后遗症，但在肝炎、慢性肾病以及血色素沉着病等常见遗传性疾病中也是众所周知的。我们发现了一种基于 Ngal 蛋白的新型铁运输途径，该蛋白在人体不同类型的组织损伤中大量表达。我们对 Ngal 代谢的研究证明了 Ngal 可用作安全的治疗性铁螯合剂。