Mechanisms of Iron-Dependent Regulation of ZIP-14

 描述(由申请人提供)：该项目旨在确定一个新发现的铁进口体ZIP14的调节机制，并研究其在体内建立铁稳态中的作用。阐明ZIP14的S调控机制为我们更好地了解铁的正常代谢和铁超载障碍的发病机制提供了机会。因此，这项拟议的研究直接关系到公共卫生和美国国立卫生研究院的使命。在提交这份申请时，我有三年半的博士后研究经验。我的长期职业目标是成为一家主要学术机构的独立研究员，为生物医学知识和一般健康改善做出贡献。我决心致力于研究铁代谢紊乱的分子基础，并将这些知识用于提高这些疾病患者的生活质量。我博士后研究的中心主题是调查铁代谢的潜在机制，并确定治疗基因靶点，以便在个性化的基础上应用基因特异性治疗。在可预见的未来，这仍将是我的首要目标。在这个K99/R00奖项的五年时间里，我的中心目标是实现研究独立。该奖项的指导阶段提供了接受必要的培训和开发验证和进一步描述ZIP14所需的工具的完美机会。我会利用这个机会 为期两年的指导阶段，以获得额外的方法培训，提高我的沟通技能，并增强我的管理和教学技能。我将在K99阶段的第一年结束时开始寻找大学教员的职位，以确保及时和顺利地过渡到R00阶段。我将继续为建立研究独立性而努力，在第二到第三年的过渡期间准备R01支持的申请 独立R00阶段。到目前为止，我已经完成的项目以及我在这个主题上的未来目标表明，一条适合K99/R00资助机制的职业道路。在我的初步研究中，我已经确定了一种氨基酸残基，它参与调节ZIP14的铁敏感性。这个残基与最近在脑铁超载患者中发现的一种突变氨基酸紧密相连，这表明该区域附近存在一个潜在的铁感应基序。我还发现了一种新的蛋白酶体介导的ZIP14降解途径。这一途径涉及ZIP14从细胞膜到细胞质的错位。值得注意的是，铁阻止了从膜上提取ZIP14进行蛋白酶体加工。此外，我发现ZIP14泛素化受到P53的正向调节。然而，决定ZIP14的S铁敏感性的关键区域， 目前尚不清楚ZIP14的S膜错位机制，以及P53在ZIP14铁依赖性调控中的作用。因此，需要对铁的调节和ZIP14的降解过程进行更全面的机制研究。重要的是，进一步探索ZIP14在铁代谢紊乱发病机制中的作用是启动独立研究事业的理想项目。在对ZIP14进行全面分析之前，需要在指导阶段创建和执行一些初步实验、分析和试剂。在完成这些步骤后，授标的独立阶段将允许对指导阶段的数据所产生的假设进行全面的后续分析。此外，这个K99/R00奖将使我能够建立一个结构化的系统，用于验证和表征ZIP14在体外和体内的作用，我最终的实验室可以用它来探索铁生物学的新课题。

 DESCRIPTION (provided by applicant): This project aims to determine the mechanisms underlying the regulation of a newly identified iron importer, ZIP14, and to examine its function in establishing iron homeostasis in the body. Elucidating the mechanisms of ZIP14's regulation offers opportunities to enhance our understanding of both the normal iron metabolism and the pathogenesis of iron-overload disorders. Therefore, this proposed research is directly relevant to public health and the NIH's mission. I have three and a half years of postdoctoral research experience when submitting this application. My long term career goal is to become an independent investigator at a major academic institution and to contribute to biomedical knowledge and general health improvement. I am determined to devote my career to examine the molecular basis of iron metabolism disorders and put this knowledge to use for improving the life quality of patients with these diseases. The central theme of my postdoctoral research has been the investigation of the mechanisms underlying iron-metabolism and the identification of therapeutic gene targets such that gene- specific therapies can be applied on a personalized basis. This will remain my primary objective for the foreseeable future. My central goal during the five-year span of this K99/R00 award is to achieve research independence. The mentored phase of this award offers the perfect opportunity to receive the necessary training and develop the tools needed for validation and further characterization of ZIP14. I will take advantage of the two-year mentored phase to gain additional methodological training, improve my communication skills, and enhance my management and teaching skills. I will start looking for university faculty positions towards the end of the first year of the K99 stage to ensure timely and smooth transition to the R00 phase. I will continue to work toward establishing research independence by preparing an application for R01 support during the second to the third year's transition of the independent R00 phase. The projects that I have accomplished to this point as well as my future goals regarding this subject suggest a career path that is ideal for the K99/R00 funding mechanism. In my preliminary studies, I have identified an amino acid residue that is involved in regulating the iron sensitivity of ZIP14. This residue is closely adjacent to a mutated amino acid that was recently identified in patients with brain iron overload, suggesting the existence of a potential iron sensing motif near this region. I have also found a novel proteasome-mediated pathway for the degradation of ZIP14. This pathway involves dislocation of ZIP14 from the membrane into the cell cytosol. Of note, iron prevents the extraction of ZIP14 from the membrane for proteasomal processing. Further, I found that ZIP14 ubiquitination is positively regulated by p53. However, the region that is critical in determining ZIP14's iron sensitivity, the mechanism of ZIP14's membrane dislocation, and the role of p53 in the iron- dependent regulation of ZIP14 are not known. Therefore, a more complete examination of the mechanisms underlying the iron regulation and the degradation process of ZIP14 is required. Importantly, further exploration of the contribution of ZIP14 to the pathogenesis of disorders in iron metabolism represents an ideal project from which to launch an independent research career. There are a number of preliminary experiments, assays, and reagents that need to be created and performed during the mentored phase before a full analysis of ZIP14 can be undertaken. Upon completion of these steps, the independent phase of the award will allow for complete follow-up analysis of the hypotheses generated by data during the mentored phase. Moreover, this K99/R00 award will enable me to establish a structured system for validation and characterization of the in vitro and in vivo roles of ZIP14 with which my eventual laboratory can explore new topics in iron biology.