An atomic model AAA-ATPase/20S core particle sub-complex of the 26S proteasome

The proteasome is the major engine of protein degradation in all eukaryotic cells. At the heart of this machine is a heterohexameric ring of AAA (ATPases associated with diverse cellular activities) proteins that unfolds ubiquitylated target proteins that are concurrently translocated into a proteolytic chamber and degraded into peptides. Using cryoelectron microscopy, we determined a near–atomic-resolution structure of the 2.5-MDa human proteasome in its ground state, as well as subnanometer-resolution structures of the holoenzyme in three alternative conformational states. The substrate-unfolding AAA-ATPase channel is narrowed by 10 inward-facing pore loops arranged into two helices that run in parallel with each other, one hydrophobic in character and the other highly charged. The gate of the core particle was unexpectedly found closed in the ground state and open in only one of the alternative states. Coordinated, stepwise conformational changes of the regulatory particle couple ATP hydrolysis to substrate translocation and regulate gating of the core particle, leading to processive degradation.

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Nucleotide-Driven Triple-State Remodeling of the AAA-ATPase Channel in the Activated Human 26S Proteasome

10.1101/132613 ◽

2017 ◽

Cited By ~ 1

Author(s):

Yanan Zhu ◽

Wei Li Wang ◽

Daqi Yu ◽

Qi Ouyang ◽

Ying Lu ◽

...

Keyword(s):

Conformational Changes ◽

26S Proteasome ◽

Eukaryotic Cells ◽

Alternative States ◽

Nucleotide Exchange ◽

Core Particle ◽

Aaa Atpase ◽

The Core ◽

Gate Opening ◽

Proteolytic Chamber

SUMMARYThe proteasome is a sophisticated ATP-dependent molecular machine responsible for protein degradation in all eukaryotic cells. It remains elusive how conformational changes of the AAA-ATPase unfoldase in the regulatory particle (RP) control the gating of substrate-translocation channel to the proteolytic chamber of the core particle (CP). Here we report three alternative states of the ATP-γS-bound human proteasome, in which the CP gate is asymmetrically open, visualized by cryo-EM at near-atomic resolutions. Only four nucleotides are stably bound to the AAA-ATPase ring in the open-gate states. Concerted nucleotide exchange gives rise to a back-and-forth wobbling motion of the AAA-ATPase channel, coincident with remarkable transitions of their pore loops between the spiral staircase and saddle-shaped circle topologies. Gate opening in the CP is thus controlled with nucleotide-driven remodeling of the AAA-ATPase unfoldase. These findings demonstrate an elegant mechanism of allosteric coordination among sub-machines within the holoenzyme that is crucial for substrate translocation.

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A VCP inhibitor substrate trapping approach (VISTA) enables proteomic profiling of endogenous ERAD substrates

Molecular Biology of the Cell ◽

10.1091/mbc.e17-08-0514 ◽

2018 ◽

Vol 29 (9) ◽

pp. 1021-1030 ◽

Cited By ~ 17

Author(s):

Edmond Y. Huang ◽

Milton To ◽

Erica Tran ◽

Lorraine T. Ador Dionisio ◽

Hyejin J. Cho ◽

...

Keyword(s):

Secretory Pathway ◽

Carcinoma Cells ◽

26S Proteasome ◽

Proteomic Profiling ◽

Aaa Atpase ◽

Early Secretory Pathway ◽

Lysine Residues ◽

Discovery Method ◽

Established Role ◽

Human Hepatocellular Carcinoma Cells

Endoplasmic reticulum (ER)–associated degradation (ERAD) mediates the proteasomal clearance of proteins from the early secretory pathway. In this process, ubiquitinated substrates are extracted from membrane-embedded dislocation complexes by the AAA ATPase VCP and targeted to the cytosolic 26S proteasome. In addition to its well-established role in the degradation of misfolded proteins, ERAD also regulates the abundance of key proteins such as enzymes involved in cholesterol synthesis. However, due to the lack of generalizable methods, our understanding of the scope of proteins targeted by ERAD remains limited. To overcome this obstacle, we developed a VCP inhibitor substrate trapping approach (VISTA) to identify endogenous ERAD substrates. VISTA exploits the small-molecule VCP inhibitor CB5083 to trap ERAD substrates in a membrane-associated, ubiquitinated form. This strategy, coupled with quantitative ubiquitin proteomics, identified previously validated (e.g., ApoB100, Insig2, and DHCR7) and novel (e.g., SCD1 and RNF5) ERAD substrates in cultured human hepatocellular carcinoma cells. Moreover, our results indicate that RNF5 autoubiquitination on multiple lysine residues targets it for ubiquitin and VCP-dependent clearance. Thus, VISTA provides a generalizable discovery method that expands the available toolbox of strategies to elucidate the ERAD substrate landscape.

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Tagging the proteasome active site β5 causes tag specific phenotypes in yeast

Scientific Reports ◽

10.1038/s41598-020-75126-1 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Kenrick A. Waite ◽

Alicia Burris ◽

Jeroen Roelofs

Keyword(s):

Nitrogen Starvation ◽

Critical Role ◽

26S Proteasome ◽

Core Particle ◽

Poor Growth ◽

Physiological Conditions ◽

Cellular Processes ◽

Storage Granules ◽

Fluorescent Tags ◽

Gfp Tag

Abstract The efficient and timely degradation of proteins is crucial for many cellular processes and to maintain general proteostasis. The proteasome, a complex multisubunit protease, plays a critical role in protein degradation. Therefore, it is important to understand the assembly, regulation, and localization of proteasome complexes in the cell under different conditions. Fluorescent tags are often utilized to study proteasomes. A GFP-tag on the β5 subunit, one of the core particle (CP) subunits with catalytic activity, has been shown to be incorporated into proteasomes and commonly used by the field. We report here that a tag on this subunit results in aberrant phenotypes that are not observed when several other CP subunits are tagged. These phenotypes appear in combination with other proteasome mutations and include poor growth, and, more significantly, altered 26S proteasome localization. In strains defective for autophagy, β5-GFP tagged proteasomes, unlike other CP tags, localize to granules upon nitrogen starvation. These granules are reflective of previously described proteasome storage granules but display unique properties. This suggests proteasomes with a β5-GFP tag are specifically recognized and sequestered depending on physiological conditions. In all, our data indicate the intricacy of tagging proteasomes, and possibly, large complexes in general.

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New insights into the human 26S proteasome function and regulation

10.1101/2021.10.03.462214 ◽

2021 ◽

Author(s):

Miglė Kišonaitė ◽

Pavel Afanasyev ◽

Jonida Tafilaku ◽

Ana Toste Rêgo ◽

Paula C. A. da Fonseca

Keyword(s):

High Resolution ◽

Structural Data ◽

26S Proteasome ◽

Aaa Atpase ◽

Physiological Processes ◽

Allosteric Communication ◽

Atpase Subunits ◽

Strict Regulation ◽

Major Conformation

SummaryThe 26S proteasome is a protease complex essential for proteostasis and strict regulation of diverse critical physiological processes, the mechanisms of which are still not fully described. The human 26S proteasome purification was optimized without exogenous nucleotides, to preserve the endogenous nucleotide occupancy and conformation of its AAA-ATPase subunits. This unveiled important effects on the proteasome function and structure resulting from exposure to Ca2+ or Mg2+, with important physiological implications. This sample, with an added model degron designed to mimic the minimum canonical ubiquitin signal for proteasomal recognition, was analysed by high-resolution cryo-EM. Two proteasome conformations were resolved, with only one capable of degron binding. The structural data show that this occurs without major conformation rearrangements and allows to infer into the allosteric communication between ubiquitin degron binding and the peptidase activities. These results revise existing concepts on the 26S proteasome function and regulation, opening important opportunities for further research.

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Structural insights into the functional cycle of the ATPase module of the 26S proteasome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1621129114 ◽

2017 ◽

Vol 114 (6) ◽

pp. 1305-1310 ◽

Cited By ~ 88

Author(s):

Marc Wehmer ◽

Till Rudack ◽

Florian Beck ◽

Antje Aufderheide ◽

Günter Pfeifer ◽

...

Keyword(s):

Atp Hydrolysis ◽

Ubiquitin Proteasome System ◽

26S Proteasome ◽

Core Particle ◽

Nucleotide Analogs ◽

The Core ◽

Cryo Electron Microscopy ◽

Intracellular Proteins ◽

Ubiquitin Proteasome ◽

Structural Insights

In eukaryotic cells, the ubiquitin–proteasome system (UPS) is responsible for the regulated degradation of intracellular proteins. The 26S holocomplex comprises the core particle (CP), where proteolysis takes place, and one or two regulatory particles (RPs). The base of the RP is formed by a heterohexameric AAA+ ATPase module, which unfolds and translocates substrates into the CP. Applying single-particle cryo-electron microscopy (cryo-EM) and image classification to samples in the presence of different nucleotides and nucleotide analogs, we were able to observe four distinct conformational states (s1 to s4). The resolution of the four conformers allowed for the construction of atomic models of the AAA+ ATPase module as it progresses through the functional cycle. In a hitherto unobserved state (s4), the gate controlling access to the CP is open. The structures described in this study allow us to put forward a model for the 26S functional cycle driven by ATP hydrolysis.

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Chaperone-driven proteasome assembly

Biochemical Society Transactions ◽

10.1042/bst0360807 ◽

2008 ◽

Vol 36 (5) ◽

pp. 807-812 ◽

Cited By ~ 28

Author(s):

Rina Rosenzweig ◽

Michael H. Glickman

Keyword(s):

26S Proteasome ◽

Biologically Active ◽

Core Particle ◽

Regulatory Particle ◽

Particle Attachment

Assembly of the 34-subunit, 2.5 MDa 26S proteasome is a carefully choreographed intricate process. It starts with formation of a seven-membered α-ring that serves as a template for assembly of the complementary β-ring-forming ‘half-proteasomes’. Dimerization results in a latent 20S core particle that can serve further as a platform for 19S regulatory particle attachment and formation of the biologically active 26S proteasome for ubiquitin-dependent proteolysis. Both general and dedicated proteasome assembly chaperones regulate the efficiency and outcome of critical steps in proteasome biogenesis, and in complex association.

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The proteasome cap RPT5/Rpt5p subunit prevents aggregation of unfolded ricin A chain

Biochemical Journal ◽

10.1042/bj20130133 ◽

2013 ◽

Vol 453 (3) ◽

pp. 435-445 ◽

Cited By ~ 12

Author(s):

Paola Pietroni ◽

Nishi Vasisht ◽

Jonathan P. Cook ◽

David M. Roberts ◽

J. Michael Lord ◽

...

Keyword(s):

Mammalian Cells ◽

Proteasome Inhibitors ◽

Retrograde Transport ◽

26S Proteasome ◽

Atpase Subunit ◽

Aaa Atpase ◽

A Chain ◽

Ricin A

The plant cytotoxin ricin enters mammalian cells by receptor-mediated endocytosis, undergoing retrograde transport to the ER (endoplasmic reticulum) where its catalytic A chain (RTA) is reductively separated from the holotoxin to enter the cytosol and inactivate ribosomes. The currently accepted model is that the bulk of ER-dislocated RTA is degraded by proteasomes. We show in the present study that the proteasome has a more complex role in ricin intoxication than previously recognized, that the previously reported increase in sensitivity of mammalian cells to ricin in the presence of proteasome inhibitors simply reflects toxicity of the inhibitors themselves, and that RTA is a very poor substrate for proteasomal degradation. Denatured RTA and casein compete for a binding site on the regulatory particle of the 26S proteasome, but their fates differ. Casein is degraded, but the mammalian 26S proteasome AAA (ATPase associated with various cellular activities)-ATPase subunit RPT5 acts as a chaperone that prevents aggregation of denatured RTA and stimulates recovery of catalytic RTA activity in vitro. Furthermore, in vivo, the ATPase activity of Rpt5p is required for maximal toxicity of RTA dislocated from the Saccharomyces cerevisiae ER. The results of the present study implicate RPT5/Rpt5p in the triage of substrates in which either activation (folding) or inactivation (degradation) pathways may be initiated.

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Chaperone-assisted assembly of the proteasome core particle

Biochemical Society Transactions ◽

10.1042/bst0380029 ◽

2010 ◽

Vol 38 (1) ◽

pp. 29-33 ◽

Cited By ~ 21

Author(s):

Ana C. Matias ◽

Paula C. Ramos ◽

R. Jürgen Dohmen

Keyword(s):

Active Sites ◽

26S Proteasome ◽

Eukaryotic Cells ◽

Main Function ◽

Core Particle ◽

Lysosomal Protease ◽

Assembly Pathway ◽

Multimeric Complex ◽

Autocatalytic Processing ◽

Α Subunits

The 26S proteasome is a non-lysosomal protease in the cytosol and nucleus of eukaryotic cells. Its main function is to mediate ubiquitin-dependent proteolysis. The 26S proteasome is a multimeric complex composed by the 20S proteasome CP (core particle) and the 19S RPs (regulatory particles). Although the atomic structure of the 26S proteasome has not yet been determined, high-resolution structures are available for its CP. Studies on the complicated assembly pathway of the proteasome have revealed that it involves an unprecedented number of dedicated chaperones. Assembly of the CP alone involves three conserved proteasome-assembly chaperones [PAC1–PAC2, PAC3–PAC4 and UMP1 (ubiquitin-mediated proteolysis 1)]. Whereas the two heterodimeric PACs have been implicated in the formation of rings of the seven distinct α subunits, UMP1 is important for the formation and dimerization of proteasome precursor complexes containing β subunits. Dimerization coincides with the incorporation of the last β subunit (β7). Additional modules important for the assembly of precursor complexes and their dimerization reside in the β subunits themselves, either as transient or as permanent extensions. Particularly important domains are the propeptide of β5 and the C-terminal extensions of β2 and β7. Upon maturation of the active sites by autocatalytic processing, UMP1 is degraded by the native proteasome.

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Structure of an endogenous yeast 26S proteasome reveals two major conformational states

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1601561113 ◽

2016 ◽

Vol 113 (10) ◽

pp. 2642-2647 ◽

Cited By ~ 51

Author(s):

Bai Luan ◽

Xiuliang Huang ◽

Jianping Wu ◽

Ziqing Mei ◽

Yiwei Wang ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Enzyme Activity ◽

Molecular Basis ◽

Biochemical Analysis ◽

26S Proteasome ◽

Core Particle ◽

Conformational States ◽

The Core ◽

Substrate Structure ◽

Exogenous Substrate

The eukaryotic proteasome mediates degradation of polyubiquitinated proteins. Here we report the single-particle cryoelectron microscopy (cryo-EM) structures of the endogenous 26S proteasome from Saccharomyces cerevisiae at 4.6- to 6.3-Å resolution. The fine features of the cryo-EM maps allow modeling of 18 subunits in the regulatory particle and 28 in the core particle. The proteasome exhibits two distinct conformational states, designated M1 and M2, which correspond to those reported previously for the proteasome purified in the presence of ATP-γS and ATP, respectively. These conformations also correspond to those of the proteasome in the presence and absence of exogenous substrate. Structure-guided biochemical analysis reveals enhanced deubiquitylating enzyme activity of Rpn11 upon assembly of the lid. Our structures serve as a molecular basis for mechanistic understanding of proteasome function.

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