ATP-Dependent Protein Unfolding and Translocation by the Eukaryotic Proteasome

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Project Summary Protein degradation is tightly regulated by ATP-dependent compartmental proteases of the AAA+ family. The major AAA+ protease in eukaryotic cells is the 26S proteasome, a 35-subunit complex that degrades proteins marked with poly-ubiquitin chains and controls protein homeostasis as well as numerous vital processes. Despite the proteasome’s great importance for cell viability, its detailed mechanisms for substrate selection and processing, and in particular its regulation and fine-tuning, for instance by substrate-attached ubiquitin chains, remain largely elusive. During the past granting period, we were able to significantly advance our understanding of proteasome structure and function. We solved high-resolution structures of the substrate- engaged proteasome at different stages of the ATP-hydrolysis cycle, established the first complete kinetic picture of substrate degradation, revealed how major conformational changes of the proteasome are coupled to individual steps of substrate processing, and uncovered how these conformational transitions are in part regulated by interactions between proteasomal subcomplexes. Our biochemical tools, recombinant expression systems, and site-specific fluorescence-labeling strategies put us into a unique position to tackle the numerous outstanding questions about ubiquitin-mediated protein turnover, the molecular mechanisms of the 26S proteasome and other AAA+ motors, and the regulation of pathways connected to the ubiquitin-proteasome system. Especially our newly established single-molecule FRET-based assays allow unprecedented studies of substrate interactions and progression through the proteasome regulatory particle, as well as the conformational dynamics of the proteasome. Exciting preliminary data indicate that substrate-attached ubiquitin chains affect the conformational switching, the kinetics of substrate engagement and degradation, and the unfolding power of the proteasome depending on the chain length and linkage type. A primary goal is to investigate how the proteasome utilizes its three main ubiquitin receptors and allosteric networks between proteasomal subcomplexes to read out this “ubiquitin code” and fine-tune its activities. We will employ a multidisciplinary approach that includes in-vitro biochemical, single-molecule, and atomic-resolution structural studies. A pathway upstream of the 26S proteasome is the AAA+ protein unfoldase Cdc48 (p97/VCP in human). In a new research direction, we will use fluorescence- and FRET-based assays combined with a series of differentially ubiquitinated and labeled model proteins to investigate how Cdc48 in complex with its adaptor Ufd1/Npl4 engages and unfolds its substrates, and how the dynamics of Cdc48-adaptor interactions determine substrate delivery, unfolding, and deubiquitination. Besides advancing our general understanding of ubiquitin-dependent protein unfolding and degradation, our research also has substantial medical relevance and offers great potential for the development of new small-molecule drugs, as both the 26S proteasome and p97 fulfill numerous regulatory functions in all cells and play important roles in various human diseases.