Illuminating degradation: The coordination and timing of substrate processing by the 26S proteasome
Selective protein degradation is a constant and critical process in cells. It is essential for maintaining tight control over protein abundance, which allows for finely tuned regulatory responses in a variety of cellular pathways. The degradation of certain proteins is also an important step in the regulation of many physiologically important cellular processes, such as cell cycle progression and stress responses. In addition, misfolded proteins accumulated both spontaneously and as a result of cellular stress must be degraded to prevent the formation of cytotoxic protein aggregates, which have been implicated as the cause of several neurodegenerative diseases. The 26S proteasome is the principal macromolecular machine responsible for protein degradation in eukaryotes. To reliably process all the proteins presented to it in the complex cellular environment, the proteasome must combine high promiscuity with exceptional substrate selectivity. Recent structural and biochemical studies have shed light on some of the steps involved in proteasomal substrate processing but have been mostly limited to static views of the process with little information about the dynamics of substrate processing. Hence, much remains to be learned about the detailed kinetics and coordination of the underlying substrate-processing steps of the proteasome, and how they correlate with observed conformational states.
The goal of my thesis work described here was to develop assays which would measure the kinetics of proteasomal degradation and reveal important details of the processing mechanism. I began by developing a method for the targeted incorporation and labeling of unnatural amino-acids at specific residues in recombinantly expressed sub-complexes of the 26S proteasome. I then designed a series of FRET and anisotropy-based assays to probe substrate-proteasome interactions, the individual steps of the processing pathway, and the conformational state of the proteasome itself. Using these assays, I developed a complete kinetic picture of proteasomal degradation, which reveals that the engagement steps prior to substrate commitment are fast relative to subsequent deubiquitination, translocation and unfolding. Furthermore, by modulating the architecture of the substrate and then investigating the kinetics of its processing, I found that non-ideal substrates are rapidly rejected by the proteasome, which thus employs a kinetic proofreading mechanism to ensure degradation fidelity and substrate prioritization.