RUI: Effect of Substrate Polyubiquitination on Proteasomal Unfolding Ability

Villanova University, Villanova PA

Investigators

Abstract

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.