Protein Bioengineering in the Ubiquitin-Proteasome System, Poster 3
Abstract
Cdc48, an essential ring-shaped enzyme, has a critical role in eukaryotic endoplasmic reticulum-associated degradation (ERAD). In ERAD, Cdc48 associates with the Ufd1/Npl4 (UN) complex as cofactors to extract misfolded... [ view full abstract ]
Cdc48, an essential ring-shaped enzyme, has a critical role in eukaryotic endoplasmic reticulum-associated degradation (ERAD). In ERAD, Cdc48 associates with the Ufd1/Npl4 (UN) complex as cofactors to extract misfolded proteins from the ER membrane so they can be delivered the “molecular kiss of death” by a cellular trash disposal system called the proteasome. This process destroys misfolded proteins and keeps them from aggregating together into plaques which are thought to cause cell death in neurodegenerative diseases.
I worked on two projects related to the Cdc48/UN complex and its role in ERAD over five months in the Rapoport Laboratory at Harvard Medical School. In the first project, we bioengineered several protein substrates to investigate the geometry of proteasomal degradation. To degrade a substrate, the proteasome must recognize and bind a molecular marker called a ubiquitin-tag, but the geometry of this binding process, and how the proteasome initiates degradation, aren't well understood.
In the second project, we started work necessary for structural analysis of the complex. This has never been accomplished using proteins purified from Saccharomyces cerivisiae (yeast) due to structural instability of yeast proteins. Chaetomium thermophilum, a thermophilic fungus that lives at 60°C, is an ideal candidate for structural work on the Cdc48/UN complex because its proteins can survive far more trying conditions than those of yeast. I developed an in vitro deubiquitination assay using purified components from C. thermophilum to demonstrate analogous function of C. thermophilum proteins involved in ERAD to their yeast counterparts.
Authors
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Christopher Diak '18.5
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Tom Rapoport
Topic Area
Science & Technology
Session
P1 » Poster Presentations: Group 1 and Refreshments (10:30am - Friday, 21st April, MBH Great Hall, 331 and 338)