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

基本信息

批准号：
8829855
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
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-04-16 至 2018-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8829855
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 post-doctoral training pulmonary arterial hypertension response scientific atmosphere screening sensor skills structural biology success symposium therapy development trafficking uptake

项目摘要

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.

项目摘要/摘要研究项目： MN是必不可少的元素，在升高的细胞水平上变得有毒无法治愈的神经毒性综合征。迄今为止，对哺乳动物的MN稳态和毒理症的研究已集中关于MN涌入的机制，但已确定的涌入转运蛋白既不针对MN也不是受调节的通过细胞MN水平。相反，胞质Mn的外排的作用被研究了，尽管外排起来在维持其他金属的稳态中的重要作用。我们现在已经确定了外排的重要作用 Mn通过摄取对高尔基体的吸收，然后分泌在维持细胞MN水平和保护中在升高的暴露期间反对MN积累过多。基于此，我们的目标是阐明高尔基人管理MN外排的监管机制，以便更好地了解这一点的作用 MN稳态的基本过程以及MN诱导的神经毒性的发展。我们的结果表明，Mn对高尔基体的摄取需要SPCA1，Golgi定位的CA/MN泵，并且 SPCA1在高尔基体和内体之间迅速流动。作为离子泵的亚细胞贩运调节他们的活动，我们的第一个目的是阐明SPCA1运输在调节MN外排的作用。我们还发现，高尔基体内MN的增加会诱导高尔基蛋白GPP130的快速降解 GPP130水平会影响Mn外排和毒性。因此，我们的第二个目标是阐明机制 GPP130调节MN外排和毒性。最后，由于调节MN外排的其他因素仍有待确定，我们的第三个目标是执行基因组宽的RNAi筛选，用于改变高尔基体对MN的控制。在屏幕上，修改版本 GPP130的GPP130将充当Golgi Lumenal Mn的新传感器。拟议的研究将提供对MN关键但未开发的方面的机械理解与MN诱导的神经毒性的病理生物学直接相关的稳态。候选人/职业发展计划/环境：我的目标是成为在金属亚细胞调节领域广泛工作的独立研究者离子体内平衡是了解发生的疾病的病理生物学和发展疗法的一种手段由于金属毒性。我的学术道路非常适合这一目标。我从事生物医学的职业完成我的医疗培训后的研究旨在为促进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，为我提供了研究临床相关的细胞生物学问题所需的技能。我有在我的博士后培训期间，扩大了这些技能。 K99奖对于我的成功至关重要，因为在亚当·林斯泰特（Adam Linstedt）博士的领导下完成我的培训并帮助我，这将为我提供保护的时间启动我的独立职业两年。以下特定的职业发展活动，以帮助我从实习生到教师的过渡。我已经独立执行我的工作，这将在未来两年内持续。我目前正在指导博士学位在她的研究中，学生将继续这样做，以获得指导学生的经验。我会进一步在此期间提高我的技术技能，并提高酵母2杂交的熟练度，2 d荧光凝胶电泳和全基因组RNAi筛选。这将增加我在帖子中已经获得的技能 - 博士期（敲低后的基因置换，蛋白质的结构功能分析以及3-和4-D Live 和固定细胞显微镜）。由于我的工作的跨学科性质，我们已经建立了富有成果的与MN毒性专家Donald Smith博士合作； Mark Macbeth博士，结构生物学和博士乔纳森·明登（Jonathan Minden），生物化学家。在接下来的两年中，我将继续与这些教师进行定期互动。为了获得该奖项，他们将担任由Linstedt博士主持的咨询委员会的一部分，该委员会将符合每一个 3-4个月以确保我正在迅速进步。为了进一步完善我的科学技能，我也将参加几次国家和国际会议（例如，2011年关于金属细胞生物学的GRC；美国细胞生物学学会会议）在奖项期间。 Linstedt博士作为导师有着出色的往绩，并在接下来的两个指导下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期间执行的工作将帮助我生成更多数据，添加到我的出版物中并提高我的证书，同时申请教师职位两年。在 R00时期将提出多个问题，这将为我职业生涯的扩展研究打开令人兴奋的途径。因此，K99/R00奖将直接帮助我从博士后学员到初级教师的过渡。