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USP14-regulated allostery of the human proteasome by time-resolved cryo-EM.

<div class='caption-title'>Time-resolved cryo-EM analysis of the conformational landscape of USP14–proteasome complexes in the act of substrate degradation.</div>
<div class='caption-title'>a, Side-chain interactions between the USP14 UBL domain and the RPN1 T2 site in the proteasome state ED2.1USP14. b, Structural comparison of the blocking loops by superimposing the USP14 structure in state ED2.1USP14 with two crystal structures of USP14 in its isolated form (PDB ID: 2AYN) and in complex with ubiquitin aldehyde5 (UbAl) (PDB ID: 2AYO). c, Magnified view of the ubiquitin–USP–OB sandwich architecture in state ED2.1USP14. d, Local cryo-EM density of the BL1 motif in state ED2.1USP14 in mesh representation superimposed with its atomic model in cartoon representation from two opposite orientations, showing its β-hairpin conformation. e–g, Magnified views of the interfaces between the catalytic Cys114 of USP14 and the C-terminal Gly76 of ubiquitin (e), between the USP14 BL1 motif and the RPT1 OB domain (f), and between the USP14 BL3 motif and the RPT2 OB domain (g). Key residues mediating the inter-molecular interactions are shown in stick representation in a–g. h, In vitro degradation of Ubn–Sic1PY by the human proteasome assembled with USP14 variants at 37 °C, analysed by SDS–PAGE and western blot using anti-T7 antibody to visualize the fusion protein T7–Sic1PY. See Supplementary Fig. 1 for gel source data. These experiments were repeated independently three times with consistent results. The proteasome without USP14 (no USP14; labelled W/O above the leftmost lanes) is used as a negative control. Lanes labelled WT correspond to the proteasome bound to wild-type USP14. i, Ubiquitin–AMC hydrolysis by the USP14 mutants dictates their DUB activity in the proteasome. RFU, relative fluorescence units. RFU values at 60 min are shown. All labelled P values were computed against the wild-type USP14 using a two-tailed unpaired t-test. Data are mean ± s.d. from three independent experiments. Each experiment includes three replicates. The quantification of wild-type USP14 was used as a denominator to normalize all measurements.</div>
<div class='caption-title'>a, b, Side-by-side comparison of the USP14–ATPase subcomplex structures aligned against the CP in six substrate-engaged states (a) and four substrate-inhibited states (b). c, d, Plots of distance from the pore-1 loop of each ATPase to the CP (c) and to the substrate (d) in distinct states. e, The AAA domain structures of the ATPase motor in six substrate-engaged states. f, Varying architecture of the pore-1 loop staircase interacting with the substrate in distinct states. The distances from disengaged pore-1 loops to the substrate are marked. The side chains of the pore-1 loop residues, featuring a consensus sequence of K/M-Y/F-V/L/I, are shown in stick representation, with the aromatic residues highlighted in transparent sphere representation. g, Electrostatic surface representation of the full-length USP14, coloured according to electrostatic potential from red (−5.0 kT e−1, negatively charged) to blue (5.0 kT e−1, positively charged). h, Atomic model of the USP14–RPT1 subcomplex in state ED4USP14 in cartoon representation. i, j, Magnified views of the USP-AAA interface I (i) and II (j) in state ED4USP14, with the interacting pairs of residues in stick representation. k, Changes of the USP-AAA interface in distinct states, characterized by measuring the shortest distance between four USP14 residues (Y285, R371, K375 and N383) and the main chains of RPT1 AAA domain. l, ATPase activity was quantified by measuring the release of phosphate from ATP hydrolysis of the proteasome. All labelled P values were computed by comparison with wild-type USP14 using a two-tailed unpaired t-test. Data are mean ± s.d. from three independent experiments, each with three replicates. The quantification of USP14-free proteasome was used as a denominator to normalize all measurements in each experiment.</div>
Zhang S, Zou S, Yin D, Zhao L, Finley D, Wu Z, Mao Y
OpenAccess logo Nature 605 567-574 (2022)
Related entries: 7w37, 7w38, 7w39, 7w3a, 7w3b, 7w3c, 7w3f, 7w3g, 7w3h, 7w3i, 7w3j, 7w3k

Cited: 16 times
EuropePMC logo PMID: 35477760

Abstract

Proteasomal degradation of ubiquitylated proteins is tightly regulated at multiple levels1-3. A primary regulatory checkpoint is the removal of ubiquitin chains from substrates by the deubiquitylating enzyme ubiquitin-specific protease 14 (USP14), which reversibly binds the proteasome and confers the ability to edit and reject substrates. How USP14 is activated and regulates proteasome function remain unknown4-7. Here we present high-resolution cryo-electron microscopy structures of human USP14 in complex with the 26S proteasome in 13 distinct conformational states captured during degradation of polyubiquitylated proteins. Time-resolved cryo-electron microscopy analysis of the conformational continuum revealed two parallel pathways of proteasome state transitions induced by USP14, and captured transient conversion of substrate-engaged intermediates into substrate-inhibited intermediates. On the substrate-engaged pathway, ubiquitin-dependent activation of USP14 allosterically reprograms the conformational landscape of the AAA-ATPase motor and stimulates opening of the core particle gate8-10, enabling observation of a near-complete cycle of asymmetric ATP hydrolysis around the ATPase ring during processive substrate unfolding. Dynamic USP14-ATPase interactions decouple the ATPase activity from RPN11-catalysed deubiquitylation11-13 and kinetically introduce three regulatory checkpoints on the proteasome, at the steps of ubiquitin recognition, substrate translocation initiation and ubiquitin chain recycling. These findings provide insights into the complete functional cycle of the USP14-regulated proteasome and establish mechanistic foundations for the discovery of USP14-targeted therapies.

Reviews citing this publication (8)

  1. AlphaFold, allosteric, and orthosteric drug discovery: Ways forward. Nussinov R, Zhang M, Liu Y, Jang H. Drug Discov Today 28 103551 (2023)
  2. Frozen in time: analyzing molecular dynamics with time-resolved cryo-EM. Amann SJ, Keihsler D, Bodrug T, Brown NG, Haselbach D. Structure 31 4-19 (2023)
  3. Ubiquitin-specific peptidases: Players in bone metabolism. Shen J, Lin X, Dai F, Chen G, Lin H, Fang B, Liu H. Cell Prolif 56 e13444 (2023)
  4. Allostery Modulates Interactions between Proteasome Core Particles and Regulatory Particles. Coffino P, Cheng Y. Biomolecules 12 764 (2022)
  5. Deubiquitinases in cancer. Dewson G, Eichhorn PJA, Komander D. Nat Rev Cancer 23 842-862 (2023)
  6. Intersections of Ubiquitin-Proteosome System and Autophagy in Promoting Growth of Glioblastoma Multiforme: Challenges and Opportunities. Visintin R, Ray SK. Cells 11 4063 (2022)
  7. Proteasome substrate receptors and their therapeutic potential. Osei-Amponsa V, Walters KJ. Trends Biochem Sci 47 950-964 (2022)
  8. Ubiquitin-Dependent and Independent Proteasomal Degradation in Host-Pathogen Interactions. Bialek W, Collawn JF, Bartoszewski R. Molecules 28 6740 (2023)

Articles citing this publication (8)

  1. A closed translocation channel in the substrate-free AAA+ ClpXP protease diminishes rogue degradation. Ghanbarpour A, Cohen SE, Fei X, Kinman LF, Bell TA, Zhang JJ, Baker TA, Davis JH, Sauer RT. Nat Commun 14 7281 (2023)
  2. ATP-binding and hydrolysis of human NLRP3. Brinkschulte R, Fußhöller DM, Hoss F, Rodríguez-Alcázar JF, Lauterbach MA, Kolbe CC, Rauen M, Ince S, Herrmann C, Latz E, Geyer M. Commun Biol 5 1176 (2022)
  3. An Unsupervised Classification Algorithm for Heterogeneous Cryo-EM Projection Images Based on Autoencoders. Wang X, Lu Y, Lin X, Li J, Zhang Z. Int J Mol Sci 24 8380 (2023)
  4. Choreographing atomic motions of macromolecular machines in action: Towards dynamics-based drug discovery. Zou S, Mao Y. Clin Transl Med 12 e977 (2022)
  5. Inhibition of USP14 promotes TNFα-induced cell death in head and neck squamous cell carcinoma (HNSCC). Morgan EL, Toni T, Viswanathan R, Robbins Y, Yang X, Cheng H, Gunti S, Huynh A, Sowers AL, Mitchell JB, Allen CT, Chen Z, Van Waes C. Cell Death Differ 30 1382-1396 (2023)
  6. Transient Structural Dynamics of Glycogen Phosphorylase from Nonequilibrium Hydrogen/Deuterium-Exchange Mass Spectrometry. Kish M, Ivory DP, Phillips JJ. J Am Chem Soc 146 298-307 (2024)
  7. USP14 governs CYP2E1 to promote nonalcoholic fatty liver disease through deubiquitination and stabilization of HSP90AA1. Wei D, Tian X, Zhu L, Wang H, Sun C. Cell Death Dis 14 566 (2023)
  8. Visualizing Conformational Space of Functional Biomolecular Complexes by Deep Manifold Learning. Wu Z, Chen E, Zhang S, Ma Y, Mao Y. Int J Mol Sci 23 8872 (2022)


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