The major research focus in my laboratory is the investigation of protein ubiquitination and degradation by the proteasome. We discovered that Rad23 is a shuttle-factor that can bind ubiquitinated proteins and deliver them to the proteasome, to initiate degradation. The domains in Rad23 that bind ubiquitinated proteins and the proteasome were identified. We are using molecular, biochemical and genetic methods (in both yeast and cell-culture based systems), to understand the mechanism of intracellular proteolysis and its significance in DNA repair, stress-response and human neurodegenerative diseases.
DNA repair: Current studies are exploring the role of Rad23 in controlling the stability of the DNA repair protein Rad4. Mutations in the human homolog of Rad4 lead to Xerodermapigmentosum, a debilitating disease in which patients are extremely sensitive to sunlight. Rad23 is extensively phosphorylated in vivo. The significance of this post-translation modification and its significance in DNA repair is under investigation.
Stress-response: We used genetic and biochemical methods to establish a link between Rad23 and the proteasome. Loss of both Rad23 and a specific proteasome subunit results in diverse proteolytic and growth defects. Current studies are focused on characterizing a number of dosage suppressors that can alleviate the defects of this double mutant strain. One of these suppressors is Sts1. and our studies suggest that it is a new regulatory factor in the Ub/proteasome proteolytic system.
Neurodegenerative diseases: We recently described the first biochemical function for the ataxin-3 protein. Mutations in this protein cause Machado-Joseph disease, a fatal neurodegenerative condition. Ataxin-3 interacts with the shuttle-factor Rad23, as well as the chaperone VCP/p97. Our studies suggest that ataxin-3 is a novel component of the proteasome that can bind ubiquitinated substrates. In other studies we are characterizing alpha-synuclein and Parkin, which are causative factors in Parkinson's disease.
Proteomics: We developed affinity matrices to rapidly purify cellular ubiquitinated proteins and the intact proteasome. We have begun to identify targets of ubiquitin-mediated proteolysis, and novel components of the proteasome, by high-resolution two-dimensional gel electrophoresis and mass spectrometry. We have also implemented this approach for characterizing heart disease, breast cancer and colorectal cancer.
