Nuclear Function of a Membrane Ubiquitin Ligase

膜泛素连接酶的核功能

• 批准号: 0938796
• 负责人: Ann Erickson
• 金额: $ 12万
• 依托单位: University of North Carolina at Chapel Hill
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-15 至 2013-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0938796&HistoricalAwards=false
• 关键词: Nuclear; Function; Membrane; Ubiquitin; Ligase

Intellectual merit. Most cellular events are controlled by precise regulation of protein expression. Termination of protein expression is commonly achieved by moving proteins into cellular recycling centers, where they are broken down into amino acids that can be reutilized in new protein synthesis. Entry into these recycling centers is tightly regulated. Proteins that are turned-over inside proteasomes, barrel-shaped recycling centers in the cytoplasm, need a special modification that serves as a key or signal for entry. This "key" is a small sequence of amino acids, named ubiquitin, which is linked or ligated to proteins by a class of regulatory proteins known as ubiquitin ligases. Ubiquitin ligases thus control protein expression by their ability to recognize and tag other proteins for destruction. Ubiquitin ligases have multiple substrates and thus commonly impact multiple key cellular pathways, including regulation of the cell cycle, programmed cell death, and the integration of signaling pathways during development. Uncovering the function of ubiquitin ligases is essential to our understanding of all cellular processes.Most ubiquitin ligases are soluble proteins that ubiquitinate cytoplasmic proteins. This research will focus on identifying the proteins tagged for turnover by a specific ubiquitin ligase, an endosomal membrane protein that is stabilized and moves to the inner nuclear membrane when cell signaling pathways under the outer cell membrane are activated. To identify proteins tagged by this ligase, experiments will compare the protein composition of purified inner nuclear membrane when the ligase is active and when its expression is knocked down. Proteins that escape ubiquitin-mediated degradation and thus increase in quantity in the inner nuclear membrane fraction when expression of the ligase is knocked down will be identified by mass spectrometry. Identification of ligase substrates correlated with specific nuclear functions will indicate a role for the endosomal ubiquitin ligase in nuclear regulation.Broader impacts: These studies will serve as a vehicle for educating undergraduates, graduate students, and postdoctoral fellows, including members of under-represented groups. The project is based on observations initially obtained by a graduate student. The studies were confirmed and expanded by a postdoctoral fellow. The principal investigator has and will continue to train undergraduate chemistry and biology majors, who either elect research for course credit or spend the summer in the lab with REU grant support. The undergraduates attend lab meetings, have their own projects, and are included on publications. All trainees not only master new techniques but, more importantly, learn to analyze data, to propose creative solutions to problems, and to summarize and present data in a professional format. Mentoring undergraduates is excellent training for the graduate students and postdocs in the lab, teaching them to teach, to organize and schedule, and to encourage others.

智力上的优点。 大多数细胞事件是通过精确调节蛋白质表达来控制的。 蛋白质表达的终止通常是通过将蛋白质转移到细胞回收中心来实现的，在那里它们被分解成可以在新的蛋白质合成中重新利用的氨基酸。 进入这些回收中心受到严格监管。在蛋白酶体（细胞质中的桶形回收中心）内翻转的蛋白质需要特殊的修饰，作为进入的关键或信号。 这个“钥匙”是一小段氨基酸序列，称为泛素，它通过一类称为泛素连接酶的调节蛋白与蛋白质连接或连接。 因此，泛素连接酶通过其识别和标记其他蛋白质以进行破坏的能力来控制蛋白质表达。 泛素连接酶具有多种底物，因此通常会影响多种关键细胞途径，包括细胞周期的调节、程序性细胞死亡以及发育过程中信号传导途径的整合。揭示泛素连接酶的功能对于我们理解所有细胞过程至关重要。大多数泛素连接酶是泛素化细胞质蛋白的可溶性蛋白质。 这项研究将重点鉴定由特定泛素连接酶标记的蛋白质，泛素连接酶是一种内体膜蛋白，当外细胞膜下的细胞信号传导途径被激活时，它会稳定并移动到内核膜。为了鉴定该连接酶标记的蛋白质，实验将比较连接酶激活时和其表达被抑制时纯化的内核膜的蛋白质组成。 当连接酶的表达被敲低时，逃脱泛素介导的降解并因此在内核膜部分中数量增加的蛋白质将通过质谱法来鉴定。 与特定核功能相关的连接酶底物的鉴定将表明内体泛素连接酶在核调节中的作用。更广泛的影响：这些研究将作为教育本科生、研究生和博士后研究员（包括代表性不足群体的成员）的工具。 该项目基于研究生最初获得的观察结果。 该研究得到了一名博士后研究员的证实和扩展。 首席研究员已经并将继续培训化学和生物学专业的本科生，他们要么选择研究作为课程学分，要么在 REU 资助的支持下在实验室度过暑假。 本科生参加实验室会议，拥有自己的项目，并发表在出版物上。所有学员不仅掌握新技术，更重要的是学会分析数据，提出创造性的问题解决方案，并以专业的格式总结和呈现数据。指导本科生对于实验室里的研究生和博士后来说是很好的培训，教他们如何教学、如何组织和安排时间以及如何鼓励他人。