26th Annual Student Research Conference
Purification of proteasomal substrates from mouse (Mus musculus) tissue using proteasomal polyubiquitin-binding domains
Jared D. James
Dr. Dorota Skowyra (Saint Louis University Department of Biochemistry) and Dr. Cynthia Cooper, Faculty Mentors
The proteasome, an organelle, is responsible for the degradation of 80% of cellular proteins. Defects in this degradation pathway contribute to many diseases including Alzheimer's, liver anti-trypsin deficiency, and diabetes. We propose that a key insight into the development and treatment of these diseases could be provided by the identification of changes in the queue of proteasomal substrates. Since ubiquitin is used to tag proteins for degradation, changes could be monitored by utilizing the specificity of various polyubiquitin binding receptors in the ubiquitin-mediated proteolytic pathway. As proof of concept, we show that the Mus musculus Psmd4 proteasomal ubiquitin-binding domain can be used as an affinity reagent to co-purify ubiquitin-conjugated proteins in complexes with the 26S proteasome from mouse liver extracts, and that in vitro incubation of these co-purified complexes at 37 °C is sufficient to generate proteasomal product peptides in amounts and purities sufficient for identification by mass spectroscopy.
Keywords: Proteolysis, Psmd4, S5a, Proteasomal Substrates, ubiquitin-binding
Topic(s):Biology
Chemistry
Presentation Type: Oral Paper
Session: 310-1
Location: MG 2001
Time: 1:00