RUI: Effect of Substrate Polyubiquitination on Proteasomal Unfolding Ability

底物多泛素化对蛋白酶体展开能力的影响蛋白酶体是细胞内的一种分子机器，它通过展开不需要或不需要的蛋白质，将它们送入中心腔室，然后将它们切成可循环利用的小块，从而去除它们。注定要被破坏的蛋白质被标记上一条由一种叫做泛素的小蛋白质的多个拷贝组成的链。在这个研究项目中，研究者将研究泛素链如何连接与蛋白酶体展开泛素修饰蛋白以降解的能力之间的关系。这项工作将使人们更好地理解为什么一些蛋白质会被降解，而另一些则不会，并对细胞过程的理解产生广泛的影响。许多本科生和硕士生将参与这个项目，从而提高他们的批判性思维和科学探究技能，这将为他们在医学、工业、学术界或其他领域的职业生涯提供很好的帮助。研究人员将与维拉诺瓦大学多元文化事务中心合作，增加来自代表性不足群体的学生参与这项研究和其他研究经验。他还将致力于为学生开发一种以探究为基础的实验课，这将有助于让那些不进行独立研究的学生接触到研究型环境。26S蛋白酶体负责真核生物细胞内蛋白质的大部分降解。虽然大多数底物被降解成小肽，但一些“光滑”底物仅部分降解，释放的片段承担新的细胞功能。Kraut实验室的总体研究目标是更好地了解蛋白酶体加工及其调控失效的分子基础，并研究泛素附着的特定残基、展开能力和蛋白质降解之间的关系。这个项目的假设是，多泛素链增加了蛋白酶体的展开能力，即使这些链已经从底物上移除。初步证据表明，与k63链相比，通过K48连接的泛素链导致更多的降解过程，部分降解片段的释放更少，这表明多泛素化确实影响了展开能力，并有助于解释为什么k63链通常不会导致细胞降解。该项目将通过确定蛋白酶体的哪些成分是增强加工能力所必需的，以及不同类型的泛素链如何影响蛋白酶体展开和降解底物或释放底物的速度，来确定泛素链在展开和降解过程中如何与控制底物的马达蛋白交流。研究者已经建立了一种进程性试验，测量蛋白酶体在降解开始后展开蛋白质结构域的能力，并能够分析展开和降解与释放的潜在动力学。该试验使用多种底物和蛋白酶体突变体，将用于探索在底物已经经历降解过程后k48链激活展开的机制。

Effect of Substrate Polyubiquitination on Proteasomal Unfolding Ability The proteasome is a molecular machine inside cells that removes unwanted or unneeded proteins by unfolding them, feeding them into a central chamber, and then chopping them up into small pieces that can be recycled. Proteins destined for destruction are tagged with a chain composed of multiple copies of a small protein called ubiquitin. In this research project, the investigator will examine the relationship between how ubiquitin chains are connected and the ability of the proteasome to unfold the ubiquitin-modified protein targeted for degradation. The work will lead to a better understanding of why some proteins are degraded and others are spared, and have broad impact on the understanding of cellular processes. Many undergraduate and masters students will work on this project and thereby enhance their critical thinking and scientific inquiry skills, which will serve them well as they go on to careers in medicine, industry, academia or other fields. The investigator will work with Villanova's Center for Multicultural Affairs to increase the involvement of students from underrepresented groups in both this and other research experiences. He will also work to develop an inquiry-based lab class for students, which will help to expose students who do not do independent research to a research-type setting.The 26S proteasome is responsible for the bulk of intracellular protein degradation in eukaryotes. While most substrates are degraded into small peptides, some "slippery" substrates are only partially degraded, with the released fragments taking on new cellular functions. The overall goal of research in the Kraut lab is to better understand the molecular basis of proteasomal processivity and its regulated failure, and to examine the relationship between the specific residues of ubiquitin attachment, unfolding ability and protein degradation. The hypothesis underlying this project is that polyubiquitin chains increase the unfolding ability of the proteasome, even after the chains have been removed from the substrate. Preliminary evidence suggests that chains linked through K48 of ubiquitin lead to more processive degradation with less release of partially degraded fragments than K63-linked chains, indicating that polyubiquitination does affect unfolding ability, and helping to explain why K63-linked chains normally don't lead to degradation in the cell. The project will focus on determining how ubiquitin chains communicate with the motor proteins that grip the substrate during unfolding and degradation by determining which components of the proteasome are required for enhanced processivity and how different types of ubiquitin chains affect the speed with which the proteasome unfolds and degrades its substrates or instead releases them. The investigator has established a processivity assay that measures the ability of the proteasome to unfold protein domains well after degradation is initiated, and enables analysis of the underlying kinetics of unfolding and degradation versus release. This assay, using multiple substrates and proteasome mutants, will be used to probe the mechanism by which K48-linked chains activate unfolding after a substrate is already undergoing the degradation process.