Visualizing Chaperone-mediated Multistep Assembly of the Human 20S Proteasome

Overview

Journal Nat Struct Mol Biol

Specialties Cell Biology
Molecular Biology

Date 2024 Apr 10

PMID 38600324

Authors

Frank Adolf

Jiale Du

Ellen A Goodall

Richard M Walsh Jr

Shaun Rawson

Susanne von Gronau

J Wade Harper

John Hanna

Brenda A Schulman

Affiliations

Soon will be listed here.

Abstract

Dedicated assembly factors orchestrate the stepwise production of many molecular machines, including the 28-subunit proteasome core particle (CP) that mediates protein degradation. Here we report cryo-electron microscopy reconstructions of seven recombinant human subcomplexes that visualize all five chaperones and the three active site propeptides across a wide swath of the assembly pathway. Comparison of these chaperone-bound intermediates and a matching mature CP reveals molecular mechanisms determining the order of successive subunit additions, as well as how proteasome subcomplexes and assembly factors structurally adapt upon progressive subunit incorporation to stabilize intermediates, facilitate the formation of subsequent intermediates and ultimately rearrange to coordinate proteolytic activation with gated access to active sites. This work establishes a methodologic approach for structural analysis of multiprotein complex assembly intermediates, illuminates specific functions of assembly factors and reveals conceptual principles underlying human proteasome biogenesis, thus providing an explanation for many previous biochemical and genetic observations.

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References

Lowe J, Stock D, Jap B, Zwickl P, Baumeister W, Huber R . Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution. Science. 1995; 268(5210):533-9. DOI: 10.1126/science.7725097. View

Groll M, Ditzel L, Lowe J, Stock D, Bochtler M, Bartunik H . Structure of 20S proteasome from yeast at 2.4 A resolution. Nature. 1997; 386(6624):463-71. DOI: 10.1038/386463a0. View

Arendt C, Hochstrasser M . Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation. Proc Natl Acad Sci U S A. 1997; 94(14):7156-61. PMC: 23776. DOI: 10.1073/pnas.94.14.7156. View

Unno M, Mizushima T, Morimoto Y, Tomisugi Y, Tanaka K, Yasuoka N . The structure of the mammalian 20S proteasome at 2.75 A resolution. Structure. 2002; 10(5):609-18. DOI: 10.1016/s0969-2126(02)00748-7. View

Chen X, Htet Z, Lopez-Alfonzo E, Martin A, Walters K . Proteasome interaction with ubiquitinated substrates: from mechanisms to therapies. FEBS J. 2020; 288(18):5231-5251. PMC: 8131406. DOI: 10.1111/febs.15638. View

Knowlton J, Johnston S, Whitby F, Realini C, Zhang Z, Rechsteiner M . Structure of the proteasome activator REGalpha (PA28alpha). Nature. 1997; 390(6660):639-43. DOI: 10.1038/37670. View

Chen J, Wang Y, Xu C, Chen K, Zhao Q, Wang S . Cryo-EM of mammalian PA28αβ-iCP immunoproteasome reveals a distinct mechanism of proteasome activation by PA28αβ. Nat Commun. 2021; 12(1):739. PMC: 7854634. DOI: 10.1038/s41467-021-21028-3. View

Zhao J, Makhija S, Zhou C, Zhang H, Wang Y, Muralidharan M . Structural insights into the human PA28-20S proteasome enabled by efficient tagging and purification of endogenous proteins. Proc Natl Acad Sci U S A. 2022; 119(33):e2207200119. PMC: 9388094. DOI: 10.1073/pnas.2207200119. View

Forster A, Whitby F, Hill C . The pore of activated 20S proteasomes has an ordered 7-fold symmetric conformation. EMBO J. 2003; 22(17):4356-64. PMC: 202378. DOI: 10.1093/emboj/cdg436. View

10.

Whitby F, Masters E, Kramer L, Knowlton J, Yao Y, Wang C . Structural basis for the activation of 20S proteasomes by 11S regulators. Nature. 2000; 408(6808):115-20. DOI: 10.1038/35040607. View

11.

Kohler A, Cascio P, Leggett D, Woo K, GOLDBERG A, Finley D . The axial channel of the proteasome core particle is gated by the Rpt2 ATPase and controls both substrate entry and product release. Mol Cell. 2001; 7(6):1143-52. DOI: 10.1016/s1097-2765(01)00274-x. View

12.

Smith D, Kafri G, Cheng Y, Ng D, Walz T, Goldberg A . ATP binding to PAN or the 26S ATPases causes association with the 20S proteasome, gate opening, and translocation of unfolded proteins. Mol Cell. 2005; 20(5):687-98. DOI: 10.1016/j.molcel.2005.10.019. View

13.

Smith D, Chang S, Park S, Finley D, Cheng Y, Goldberg A . Docking of the proteasomal ATPases' carboxyl termini in the 20S proteasome's alpha ring opens the gate for substrate entry. Mol Cell. 2007; 27(5):731-44. PMC: 2083707. DOI: 10.1016/j.molcel.2007.06.033. View

14.

Rabl J, Smith D, Yu Y, Chang S, Goldberg A, Cheng Y . Mechanism of gate opening in the 20S proteasome by the proteasomal ATPases. Mol Cell. 2008; 30(3):360-8. PMC: 4141531. DOI: 10.1016/j.molcel.2008.03.004. View

15.

Wehmer M, Rudack T, Beck F, Aufderheide A, Pfeifer G, Plitzko J . Structural insights into the functional cycle of the ATPase module of the 26S proteasome. Proc Natl Acad Sci U S A. 2017; 114(6):1305-1310. PMC: 5307450. DOI: 10.1073/pnas.1621129114. View

16.

H de la Pena A, Goodall E, Gates S, Lander G, Martin A . Substrate-engaged 26 proteasome structures reveal mechanisms for ATP-hydrolysis-driven translocation. Science. 2018; 362(6418). PMC: 6519459. DOI: 10.1126/science.aav0725. View

17.

DeMartino G, Slaughter C . The proteasome, a novel protease regulated by multiple mechanisms. J Biol Chem. 1999; 274(32):22123-6. DOI: 10.1074/jbc.274.32.22123. View

18.

Sadre-Bazzaz K, Whitby F, Robinson H, Formosa T, Hill C . Structure of a Blm10 complex reveals common mechanisms for proteasome binding and gate opening. Mol Cell. 2010; 37(5):728-35. PMC: 2859072. DOI: 10.1016/j.molcel.2010.02.002. View

19.

Rego A, da Fonseca P . Characterization of Fully Recombinant Human 20S and 20S-PA200 Proteasome Complexes. Mol Cell. 2019; 76(1):138-147.e5. PMC: 6863390. DOI: 10.1016/j.molcel.2019.07.014. View

20.

Guan H, Wang Y, Yu T, Huang Y, Li M, Saeed A . Cryo-EM structures of the human PA200 and PA200-20S complex reveal regulation of proteasome gate opening and two PA200 apertures. PLoS Biol. 2020; 18(3):e3000654. PMC: 7077846. DOI: 10.1371/journal.pbio.3000654. View