Intellectual merit. The protein content inside a cell is constantly changing. Depending on the cell's needs, certain proteins are made while others are removed or degraded. Protein degradation must be highly specific and is therefore tightly regulated by compartmental proteases, large barrel-shaped complexes that utilize the energy from ATP hydrolysis to mechanically unravel and then translocate protein substrates into a sequestered internal chamber for degradation. The major ATP-dependent protease in eukaryotic cells is the 26S proteasome, which controls protein turnover and numerous vital processes by specifically degrading regulatory proteins involved for instance in cell division, signal transduction, and programmed cell death. Most substrates are marked for degradation by the reversible attachment of another small protein, ubiquitin, which can be consecutively linked together to form poly-ubiquitin chains that act as a targeting signal to the proteasome. The goal of this research is to elucidate the detailed mechanisms of ubiquitin-dependent protein degradation by using novel approaches for the structural, quantitative biochemical, and biophysical characterization of the 26S proteasome. Specific aims are 1) to understand the intricate coupling between substrate degradation and the preceding removal of ubiquitin chains, 2) to gain detailed structural and functional insight into the architecture of the proteasome molecular machine and its mechanisms for substrate recognition, unfolding, and ubiquitin removal, and 3) to use unprecedented single-molecule measurements to provide a glimpse into the mechanochemistry of substrate recognition and degradation. It is expected that elucidating these molecular mechanisms will help to define the principles that control the degradation of highly diverse proteins in vivo, and thus further the understanding of the various regulatory functions of preoteasomes in eukaryotic cells. Due to the ubiquitous functions of the proteasome in protein turnover and maintenance of crucial pathways, the results and novel tools generated through this research will strongly impact other cell-biological studies and approaches.
Broader impacts. A detailed knowledge of the molecular mechanisms for substrate processing will allow the development of dedicated proteasomes for the temporally controlled elimination of specific enzymes and other proteins in the cell, which is highly desired for applications in bioengineering and systems biology, and may aid the synthesis of drug precursors or biofuels. The proposed research will be integrated with teaching and outreach to strengthen education in the science, technology, engineering, and mathematics (STEM) fields. Due to their strongly collaborative nature, the research projects provide intense interdisciplinary training for undergraduates, postdocs, and graduate students at the intersection of biology, physics, and chemistry. Furthermore, in order to start science education and outreach already at a young age, a program will be developed that includes summer research internships for high school students, a Laboratory Boot Camp program with courses for undergraduates, community college students, and high school teachers, and the organization of science fairs at local public charter schools with high percentage of underrepresented minorities.
