Nuclear Function of a Membrane Ubiquitin Ligase

智力上的优点。大多数细胞活动是由蛋白质表达的精确调控控制的。蛋白质表达的终止通常是通过将蛋白质转移到细胞循环中心实现的，在那里它们被分解成氨基酸，可以在新的蛋白质合成中重新利用。进入这些回收中心受到严格的监管。在蛋白酶体内翻转的蛋白质需要一种特殊的修饰，作为进入的钥匙或信号。这个“关键”是一小段名为泛素的氨基酸序列，它通过一类被称为泛素连接酶的调节蛋白与蛋白质连接或连接。因此，泛素连接酶通过识别和标记要破坏的其他蛋白质来控制蛋白质的表达。泛素连接酶具有多种底物，因此通常影响多个关键的细胞通路，包括调节细胞周期、细胞程序性死亡和发育过程中信号通路的整合。揭示泛素连接酶的功能对于我们理解所有的细胞过程是至关重要的。大多数泛素连接酶是使细胞质蛋白泛素化的可溶性蛋白质。这项研究将集中于识别特定泛素连接酶标记的周转蛋白质，泛素连接酶是一种内体膜蛋白，当外膜下的细胞信号通路被激活时，它会稳定并移动到内核膜。为了鉴定这种连接酶标记的蛋白质，实验将比较连接酶激活时和表达下调时纯化的内核膜的蛋白质组成。当连接酶的表达被下调时，逃脱泛素介导的降解并因此在内核膜部分增加数量的蛋白质将通过质谱学进行鉴定。识别与特定核功能相关的连接酶底物将表明内体泛素连接酶在核调节中的作用。广泛影响：这些研究将作为教育本科生、研究生和博士后研究员的工具，包括代表不足的群体的成员。该项目是基于一名研究生最初获得的观察结果。这些研究得到了一位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专业的本科生，这些学生要么选择课程学分进行研究，要么在REU的资助下在实验室度过夏天。本科生参加实验室会议，有自己的项目，并被列入出版物。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展示数据。指导本科生是在实验室对研究生和博士后进行的极好的培训，教他们如何教学，如何组织和安排，以及鼓励他人。

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.