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The Walters lab studies the structural and mechanistic basis of ubiquitin signaling events, proteasome function, and protein quality control. We use an interdisciplinary approach that merges functional cell-based assays with a variety of biophysical and structural biology techniques to define the mechanistic underpinnings of how protein lifetimes are regulated in cells. We have helped to identify the mechanisms of substrate recognition by the proteasome, identifying Rpn1 and Rpn13 as substrate receptors that bind to ubiquitin chains directly and to the shuttle factors that deliver ubiquitinated substrates to the proteasome. By using NMR spectroscopy, we have solved structures of these receptors complexed with ubiquitin chains and shuttle factors. We have also used cryoelectron microscopy to study ubiquitin chain binding to the 26S proteasome.

We discovered that Rpn13 is targeted in its ubiquitin-binding domain by cell permeable molecules that restrict cancer cell proliferation. We generated CRISPR cell lines that have revealed the importance of Rpn13 at the proteasome and its requirement for triggered apoptosis by these small molecules. Small molecules that inhibit the catalytic core particle of the proteasome are approved for treatment of hematological cancers and the targeting of Rpn13 is expected to function synergistically with these core particle inhibitors. We are further developing other Rpn13-targeting molecules by using structure-based drug design methods.

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In a related project, we recently identified a binding site for the E3 ubiquitin ligase E6AP/UBE3A at the proteasome in substrate receptor Rpn10. This functional domain is the first found at the proteasome to recruit ubiquitination machinery. E6AP is targeted by the human papilloma virus E6 oncoprotein leading to degradation of tumor suppressor p53 and its dysfunction is also associated with prostate cancer, Angelman syndrome, and autism spectrum disorders. We continue to study the role of E6AP at the proteasome as well as other protein-protein interactions in the ubiquitin-proteasome pathway to understand the determinants of protein targeting for degradation as well as the mechanistic features that go awry in these pathways during carcinogenesis and neurological disorders.