Nuclear Function of a Membrane Ubiquitin Ligase

智力上的优点。 大多数细胞事件都是由蛋白质表达的精确调节控制的。 蛋白质表达的终止通常通过将蛋白质移动到细胞回收中心来实现，在那里它们被分解成可以在新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管制。蛋白酶体是细胞质中的桶形回收中心，在蛋白酶体内翻转的蛋白质需要特殊的修饰，作为进入的钥匙或信号。 这个“钥匙”是一个小的氨基酸序列，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、程序性细胞死亡和发育期间信号传导途径的整合。揭示泛素连接酶的功能对我们理解所有细胞过程至关重要。大多数泛素连接酶是可溶性蛋白质，它们泛素化细胞质蛋白。 这项研究将集中在识别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通路被激活时，内体膜蛋白被稳定并移动到内核膜。为了鉴定由该连接酶标记的蛋白质，实验将比较当连接酶有活性时和当其表达被敲低时纯化的内核膜的蛋白质组成。 逃避泛素介导的降解并因此在连接酶的表达被敲低时在内核膜部分中数量增加的蛋白质将通过质谱法鉴定。 识别与特定的核功能相关的连接酶底物将表明核regulation.broader影响的内体泛素连接酶的作用：这些研究将作为一种工具，为教育本科生，研究生和博士后研究员，包括代表性不足的群体的成员。 该项目是基于最初由一名研究生获得的观察。 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本科化学和生物学专业的学生，他们要么选择课程学分的研究，要么在REU资助的实验室度过夏天。 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，但更重要的是，学会分析数据，提出创造性的解决方案的问题，并总结和呈现数据在专业格式。指导本科生是对研究生和博士后在实验室的良好培训，教他们教学，组织和安排，并鼓励他人。

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.