Role of Mechanisms of Efflux in Manganese Homeostasis and Detoxification

外排机制在锰稳态和解毒中的作用

• 批准号: 8609168
• 负责人: Somshuvra Mukhopadhyay
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-16 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8609168
• 关键词: Adult; Advisory Committees; American; Area; Award; Behavioral; Beryllium; Binding; Biomedical Research; Cells; Cellular biology; Child; Cognitive; Collaborations; Cytosol; Data; Defect; Development; Development Plans; Disease; Drug Metabolic Detoxication; Elements; Endosomes; Ensure; Environment; Excision; Exposure to; Faculty; Fluorescence; Foundations; Future; Genetic; Goals; Golgi Apparatus; Hepatolenticular Degeneration; Homeostasis; Human; Hybrids; International; Ion Pumps; Ions; Life; Lysosomes; Mammalian Cell; Mammals; Manganese; Maps; Mediating; Medical; Mentors; Metals; Microscopy; Minor; Molecular; Mutate; Nature; Nerve Degeneration; Neurologic; Occupational; Parkinson Disease; Pathway interactions; Pharmaceutical Preparations; Play; Population; Positioning Attribute; Postdoctoral Fellow; Process; Proteins; Publications; Pump; RNA Interference; Regulation; Research; Research Personnel; Research Project Grants; Research Training; Risk; Role; Route; Societies; Structure; Students; Syndrome; Testing; Time; Toxic effect; Toxin; Training; Work; Yeasts; abstracting; base; career; career development; cell fixing; clinically relevant; cytotoxic; domain mapping; experience; gel electrophoresis; gene replacement; genome-wide; human disease; improved; innovation; meetings; metal poisoning; mutant; neurotoxic; neurotoxicity; novel; planetary Atmosphere; post-doctoral training; pulmonary arterial hypertension; response; screening; sensor; skills; structural biology; success; symposium; therapy development; trafficking; uptake

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.

项目总结/摘要 研究项目： Mn是一种必需元素，在升高的细胞水平下变得有毒，从而导致 无法治愈的神经毒性综合征迄今为止，对哺乳动物锰稳态和中毒的研究已成为焦点 锰内流的机制，但确定的流入转运蛋白既不具体的锰，也不受管制 通过细胞锰水平。相比之下，尽管外排在细胞内起着重要作用，但胞质锰外排的作用研究不足。 在维持其他金属的体内平衡中起重要作用。我们现在已经确定了一个重要的作用， Mn通过摄取到高尔基体，然后分泌到维持细胞Mn水平和保护 在高暴露期间防止过量的Mn积累。在此基础上，我们的目标是阐明 调节机制管理锰流出的高尔基体，以便更好地了解这一作用， 锰稳态和锰诱导的神经毒性的发展的基本过程。 我们的研究结果表明，锰进入高尔基体的吸收需要SPCA 1，高尔基体定位的Ca/Mn泵， SPCA 1在高尔基体和核内体之间快速运输。由于离子泵的亚细胞运输调节 为了了解它们的活性，我们的首要目标是阐明SPCA 1贩运在调节锰外排中的作用。 我们还发现，增加的高尔基体内Mn诱导高尔基体蛋白GPP 130的快速降解 并且GPP 130水平影响锰的外排和毒性。因此，我们的第二个目标是阐明机制 GPP 130通过其调节Mn的流出和毒性。 最后，由于调节锰外排的其他因素仍有待确定，我们的第三个目标是进行一项研究。 全基因组RNAi筛选改变高尔基体对Mn的控制的蛋白质。在屏幕上，修改后的版本 GPP 130的表达可作为高尔基体内Mn的一种新型传感器。 拟议的研究将提供一个关键的机制，但尚未探索的锰方面的理解 内稳态，这是直接相关的锰诱导的神经毒性的病理生物学。 候选人/职业发展计划/环境： 我的目标是成为一名独立的研究者，在金属的亚细胞调节领域广泛工作。 离子稳态作为一种手段，以了解病理生物学，并开发治疗，疾病发生 因为金属中毒我的学术道路非常适合这个目标。我开始从事生物医学 完成我的医学培训后，我进行了一项研究，目的是为进一步了解我们的知识做出贡献。 无法治愈的人类疾病。我的毕业论文是关于肺动脉的分子机制 高血压，给了我研究临床相关细胞生物学问题所需的技能。我有 在我的博士后培训期间扩展了这些技能。K99奖现在对我的成功至关重要，因为 这将给我必要的保护时间来完成我在亚当·林斯泰特博士手下的训练， 两年后开始我的独立职业生涯。 具体的职业发展活动，以帮助我从实习生到教师的过渡如下。我已经 我将独立完成我的工作，这将在未来两年继续下去。我目前正在指导一个博士 并将继续这样做，以获得指导学生的经验。我将进一步 在此期间提高了我的技术技能，并熟练掌握了酵母双杂交，二维荧光凝胶 电泳和全基因组RNAi筛选。这将增加我已经在后获得的技能- 博士阶段（基因敲除后的基因替换，蛋白质的结构功能分析和3-和4-D生命 和固定细胞显微术）。由于我工作的跨学科性质，我们建立了富有成效的 与唐纳德史密斯博士，锰毒性专家，马克麦克白博士，结构生物学和博士。 乔纳森明登，一个生物化学家。在接下来的两年里，我将继续定期与这些教师互动。 对于该奖项，他们将作为一个咨询委员会的一部分，由林斯泰特博士担任主席，该委员会将会见每一个 3-4这几个月来，我一直在努力，以确保我的进步。为了进一步提高我的科学技能，我还将 参加了几次国家和国际会议（例如，2011年的金属细胞生物学GRC;年度 美国细胞生物学协会（American Society for Cell Biology）在颁奖期间举行的会议上发表了演讲。 Linstedt博士作为导师有着出色的记录，并在他的指导下为接下来的两个项目工作。 多年的经验将使我具备在独立环境中取得成功所需的技能。创新和 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 在两年后申请教职的时候提高我的资历期间开展的工作 R 00阶段将提出多个问题，这将为我职业生涯中的扩展研究开辟令人兴奋的途径。 因此，K99/R 00奖将直接帮助我从博士后实习生过渡到初级教师。