Abstract
The 26S proteasome is the most complex ATP-dependent protease machinery, of ~2.5MDa mass, ubiquitously found in all eukaryotes. It selectively degrades ubiquitin-conjugated proteins and plays fundamentally indispensable roles in regulating almost all major aspects of cellular activities. To serve as the sole terminal "processor" for myriad ubiquitylation pathways, the proteasome evolved exceptional adaptability in dynamically organizing a large network of proteins, including ubiquitin receptors, shuttle factors, deubiquitinases, AAA-ATPase unfoldases, and ubiquitin ligases, to enable substrate selectivity and processing efficiency and to achieve regulation precision of a vast diversity of substrates. The inner working of the 26S proteasome is among the most sophisticated, enigmatic mechanisms of enzyme machinery in eukaryotic cells. Recent breakthroughs in three-dimensional atomic-level visualization of the 26S proteasome dynamics during polyubiquitylated substrate degradation elucidated an extensively detailed picture of its functional mechanisms, owing to progressive methodological advances associated with cryogenic electron microscopy (cryo-EM). Multiple sites of ubiquitin binding in the proteasome revealed a canonical mode of ubiquitin-dependent substrate engagement. The proteasome conformation in the act of substrate deubiquitylation provided insights into how the deubiquitylating activity of RPN11 is enhanced in the holoenzyme and is coupled to substrate translocation. Intriguingly, three principal modes of coordinated ATP hydrolysis in the heterohexameric AAA-ATPase motor were discovered to regulate intermediate functional steps of the proteasome, including ubiquitin-substrate engagement, deubiquitylation, initiation of substrate translocation and processive substrate degradation. The atomic dissection of the innermost working of the 26S proteasome opens up a new era in our understanding of the ubiquitin-proteasome system and has far-reaching implications in health and disease.
Highlights
Human cells express more than 20,000 genes, which direct synthesis of a comparable number of proteins by the ribosome
Complex dynamics of the proteasome in the act of substrate processing at the atomic level are compared in detail with structural characterization of proteasome dynamics at the near-atomic or pseudo-atomic level under various in vitro biochemical conditions
It becomes increasingly apparent that the lid interactions with the base and Core Particle (CP) favor two sets of ATPase conformations characterized in two sets of comparable states [ED1, 5D/5T, C3-b, s6] and [ED2, SD2, 4D, C3-a, s4]. (See below: section Operating Principles of Proteasomal Associated with diverse cellular Activities (AAA)-ATPase Motor, for more details)
Summary
Human cells express more than 20,000 genes, which direct synthesis of a comparable number of proteins by the ribosome. RP-CP interface relationship, and CP gate, but RP-CP interface different ATPase and CP gate) conformers) Due to their higher resolution, better map quality and relative completeness in defining the energy landscape of the proteasome, the states of the substrate-bound human proteasome in a buffer containing 1 mM ATP and 1 mM ATPγS (Dong et al 2019) are used as a canonical reference for comparisons with the available states of proteasome under other four distinct categories of biochemical conditions: (1) the substrate-free human proteasome in the presence of 1 mM ATP or ATPγS (Chen et al 2016a; Zhu et al 2018), (2) the substrate-bound yeast proteasome with inactivated RPN11 in the presence of 1 mM ATP (de la Pena et al 2018), (3) the Lys48-linked tetraubiquitin-bound yeast proteasome in the presence of 1 mM ATP (Ding et al 2019), and (4) the substrate-free yeast proteasome with one of the nucleotides or nucleotide analogs, including the cases of 1 mM ATP, 2 or 4 mM ATPγS, 1 mM ADP, ADP-AlFx, ADP-BeFx, ATP/BeFx, or AMP-PNP (Wehmer et al 2017; Eisele et al 2018; Ding et al 2017). The interaction of RPT6 with the α2 subunit is regulated by phosphorylation on RPT6, which stimulates proteasome activity (Satoh et al 2001; Asai et al 2009)
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