Role of Mechanisms of Efflux in Manganese Homeostasis and Detoxification

项目摘要/摘要 研究项目： 锰是一种基本元素，在细胞水平升高时会变得有毒，从而导致 无法治愈的神经毒性综合症。到目前为止，哺乳动物体内锰的动态平衡和中毒的研究一直是重点。 关于锰内流机制的探讨但已鉴定的内流转运蛋白既不是锰的特异性转运体，也不是受调控的转运体 通过细胞内的锰水平。相反，细胞内锰外流的作用还没有被充分研究，尽管外流在细胞内起着重要的作用。 在维持其他金属的动态平衡方面发挥重要作用。我们现在已经确定了外流的重要作用。 在维持细胞内锰水平和保护细胞的过程中，通过摄取高尔基体和分泌锰而获得的锰 防止在增加暴露期间过量积累锰。在此基础上，我们的目标是阐明 调节高尔基体锰外流的机制，以更好地理解这一作用 锰动态平衡和锰致神经毒性发展的基本过程。 我们的结果表明，锰的摄取到高尔基体需要SPCA1，一个高尔基体局部的钙/锰泵和 SPCA1在高尔基体和内体之间快速运输。随着离子泵亚细胞运输的调节 我们的第一个目的是阐明SPCA1转运在调节锰外流中的作用。 我们还发现，高尔基体内锰的增加会导致高尔基体蛋白GPP130的快速降解 GPP130水平影响锰的外流和毒性。因此，我们的第二个目标是阐明这一机制 GPP130通过其调节锰的外流和毒性。 最后，由于调节锰外流的其他因素仍有待确定，我们的第三个目标是进行 全基因组RNAi筛选改变高尔基体对锰的控制的蛋白质。在屏幕上，修改后的版本 作为一种新型的高尔基体腔锰传感器。 拟议的研究将提供对MN一个关键但未被探索的方面的机械性理解 动态平衡与锰诱导的神经毒性的病理生物学直接相关。 应聘者/职业发展计划/环境： 我的目标是成为一名独立的研究员，在金属亚细胞调控领域广泛工作。 离子动态平衡是了解疾病的病理生物学和开发治疗方法的一种手段 由于金属毒性。我的学术道路非常适合这个目标。我开始了生物医学的职业生涯 完成医学培训后的研究，目的是为加深我们的理解做出贡献 无法治愈的人类疾病。我的研究生工作，关于肺动脉的分子机制 高血压，给了我必要的技能来研究临床相关的细胞生物学问题。我有过 在我的博士后培训期间，我扩展了这些技能。K99奖现在对我的成功至关重要，因为 它将给我必要的保护时间来完成亚当·林斯泰特博士的训练，并帮助我 两年后开始我的独立事业。 帮助我从实习生过渡到教职员工的具体职业发展活动如下。我已经 独立完成我的工作，这将在接下来的两年里继续下去。我目前正在指导一名博士 她将继续从事研究，并将继续这样做，以获得指导学生的经验。我会进一步 在此期间，我提高了自己的技术技能，并熟练掌握了酵母-2-杂交、二维荧光凝胶 电泳法和全基因组RNAi筛选。这将增加我在帖子中已经获得的技能- 博士期(基因敲除后的基因替换，蛋白质的结构功能分析和三维和四维肝 和固定细胞显微镜)。由于我工作的跨学科性质，我们建立了卓有成效的 与锰毒性专家唐纳德·史密斯博士、结构生物学博士马克·麦克白博士和麦克白博士合作。 生物化学家乔纳森·明登。在接下来的两年里，我将继续定期与这些教职员工互动。 对于该奖项，他们将作为咨询委员会的一部分，该委员会由林斯泰博士担任主席，该委员会将会见每一位 3-4个月，以确保我取得快速进步。为了进一步提高我的科学技能，我还将 出席若干国内和国际会议(例如，2011年金属细胞生物学全球研究中心；年度 美国细胞生物学学会会议)。 林斯泰博士作为一名导师有着良好的业绩记录，并在他的指导下在接下来的两年里工作 多年将为我提供在独立环境中取得成功所需的技能。创新的和 CMU和大匹兹堡地区的互动科学氛围也将有助于我的培训和研究。 此外，在K99期间完成的工作将帮助我生成更多数据，添加到我的出版物中 并在两年后申请教职时提高我的资历。在此期间完成的工作 R00阶段将提出多个问题，这些问题将为我职业生涯中更广泛的研究开辟令人兴奋的道路。 因此，K99/R00奖项将直接帮助我从博士后实习生过渡到初级教师。

Project Summary/Abstract Research Project: Mn is an essential element that becomes toxic at elevated cellular levels leading to the onset of an incurable neurotoxic syndrome. To date, research on Mn homeostasis and toxicosis in mammals has focused on the mechanisms of Mn influx but the identified influx transporters are neither specific for Mn nor regulated by cellular Mn levels. In contrast, the role of efflux of cytosolic Mn is understudied although efflux plays a significant role in maintaining homeostasis of other metals. We have now identified an important role for efflux of Mn via uptake into the Golgi apparatus followed by secretion in maintaining cellular Mn levels and protecting against excess Mn accumulation during elevated exposure. Based on this, our goal is to elucidate the regulatory mechanisms governing Mn efflux by the Golgi to so as to better understand the role of this fundamental process in Mn homeostasis and in the development of Mn-induced neurotoxicity. Our results indicate that uptake of Mn into the Golgi requires SPCA1, a Golgi-localized Ca/Mn pump and that SPCA1 rapidly traffics between the Golgi and endosomes. As subcellular trafficking of ion pumps regulate their activity, our first aim is to elucidate the role of SPCA1 trafficking in regulating Mn efflux. We also discovered that increased intra-Golgi Mn induces rapid degradation of the Golgi protein GPP130 and that GPP130 levels impact Mn efflux and toxicity. Therefore, our second aim is to elucidate the mechanism by which GPP130 regulates Mn efflux and toxicity. Finally, as additional factors regulating Mn efflux remain to be identified, our third aim is to perform a genome wide RNAi screen for proteins that alter control of Mn by the Golgi. In the screen, a modified version of GPP130 will serve as a novel sensor of Golgi lumenal Mn. The proposed studies will provide mechanistic understanding of a crucial but unexplored aspect of Mn homeostasis that is directly relevant to the pathobiology of Mn-induced neurotoxicity. Candidate/ Career Development Plan/ Environment: My goal is to be an independent investigator working broadly in the area of subcellular regulation of metal ion homeostasis as a means to understand the pathobiology of, and develop therapies for, diseases that occur due to metal toxicity. My academic path is well-suited to this goal. I embarked on a career in biomedical research after completing my medical training with the aim of making contributions to further our understanding of incurable human diseases. My graduate work, on the molecular mechanisms of pulmonary arterial hypertension, gave me the skills necessary to investigate clinically relevant cell biology questions. I have expanded these skills during my post-doctoral training. The K99 award is now crucial for my success because it will give me the protected time essential to complete my training under Dr. Adam Linstedt and help me initiate my independent career two years hence. Specific career development activities to aid my transition from trainee-to-faculty are as follows. I already perform my work independently and this will continue over the next two years. I am currently guiding a PHD student in her research and will continue to do so to gain experience in mentoring students. I will further improve my technical skills during this period and gain proficiency in yeast-2-hybrid, 2 D fluorescence gel electrophoresis and genome-wide RNAi screening. This will add to the skills I have already gained in the post- doctoral period (gene replacement after knockdown, structure function analyses of proteins and 3- and 4-D live and fixed cell microscopy). Due to the interdisciplinary nature of my work, we have established fruitful collaborations with Dr. Donald Smith, an expert in Mn toxicity; Dr. Mark Macbeth, a structural biology and Dr. Jonathan Minden, a biochemist. I will continue to interact regularly with these faculty over the next two years. For the award, they will serve as part of an advisory committee, chaired by Dr. Linstedt, which will meet every 3-4 months to ensure that I am making rapid progress. To further refine my scientific skills, I will also be attending several national and international conferences (e.g. GRC on Cell Biology of Metals in 2011; annual meeting of the American Society for Cell Biology) during the award period. Dr. Linstedt has an excellent track record as a mentor and working under his guidance for the next two years will provide me with the skills necessary to succeed in an independent setting. The innovative and interactive scientific atmosphere in CMU and the greater Pittsburgh area will also aid my training and research. Further, the work performed during the K99 period will help me generate more data, add to my publications and improve my credentials while applying for faculty positions two years hence. The work performed during the R00 period will raise multiple questions that will open exciting avenues for extended research in my career. Thus, the K99/R00 award will act directly aid my transition from post-doctoral trainee to junior faculty.