Structural Biology of the Proteasome

Kish-Trier, Erik; Hill, Christopher P.

doi:10.1146/annurev-biophys-083012-130417

Cited by 196 publications

(193 citation statements)

References 104 publications

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“…In eukaryotic cells, selective protein degradation is mostly carried out by a set of pathways of ubiquitylation that terminate in a 2.5-MDa protein proteolytic complex, called the 26S proteasome. The ubiquitin-proteasome pathways are essential parts of important biological processes, such as cell division, differentiation, innate immunity, adaptive immunity, regulation of gene expression, and the response to proteotoxic stress (1)(2)(3)(4). The proteasome is also an important therapeutic target in multiple myeloma (5,6).…”

mentioning

confidence: 99%

“…The intact proteasome has not been resolved to a level at which a reliable Cα-backbone can be traced with spatial assignment of amino acids, although major advances have been made in recent years (2,3,(7)(8)(9)(10)(11)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). Several RP subunits have been resolved at high resolution by X-ray crystallography (14-16, 18, 21-24).…”

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confidence: 99%

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Structural basis for dynamic regulation of the human 26S proteasome

Chen

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

156

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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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confidence: 99%

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Structural basis for dynamic regulation of the human 26S proteasome

Chen

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

156

310

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“…The proteolytic activity of this multicatalytic protease resides inside the 20S core particle, built of four stacked rings of seven ␣ and ␤ subunits with ␣ 7 ␤ 7 ␤ 7 ␣ 7 organization. The internal cavity of the complex, defined by the two ␤ rings, encloses three pairs of distinct active sites named according to their differential cleavage site specificity: chymotrypsin-like (CT-L), 3 trypsin-like (T-L), and caspase-like (C-L) catalytic sites (1). Due to the closed conformation of the native 20S core complex, binding of different proteasome activators is required to induce opening of the 20S entry pores and allow injection of substrates into the catalytic chamber for degradation (2,3).…”

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confidence: 99%

Inhibition of Proteasome Activity Induces Formation of Alternative Proteasome Complexes

Welk

Coux

Kleene

et al. 2016

Journal of Biological Chemistry

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The proteasome is an intracellular protease complex consisting of the 20S catalytic core and its associated regulators, including the 19S complex, PA28␣␤, PA28␥, PA200, and PI31. Inhibition of the proteasome induces autoregulatory de novo formation of 20S and 26S proteasome complexes. Formation of alternative proteasome complexes, however, has not been investigated so far. We here show that catalytic proteasome inhibition results in fast recruitment of PA28␥ and PA200 to 20S and 26S proteasomes within 2-6 h. Rapid formation of alternative proteasome complexes did not involve transcriptional activation of PA28␥ and PA200 but rather recruitment of preexisting activators to 20S and 26S proteasome complexes. Recruitment of proteasomal activators depended on the extent of active site inhibition of the proteasome with inhibition of ␤5 active sites being sufficient for inducing recruitment. Moreover, specific inhibition of 26S proteasome activity via siRNA-mediated knockdown of the 19S subunit RPN6 induced recruitment of only PA200 to 20S proteasomes, whereas PA28␥ was not mobilized. Here, formation of alternative PA200 complexes involved transcriptional activation of the activator. Alternative proteasome complexes persisted when cells had regained proteasome activity after pulse exposure to proteasome inhibitors. Knockdown of PA28␥ sensitized cells to proteasome inhibitor-mediated growth arrest. Thus, formation of alternative proteasome complexes appears to be a formerly unrecognized but integral part of the cellular response to impaired proteasome function and altered proteostasis.The proteasome, one of the main proteolytic systems of the cell, is essential for maintenance of protein homeostasis and thus of cellular functions. The proteolytic activity of this multicatalytic protease resides inside the 20S core particle, built of four stacked rings of seven ␣ and ␤ subunits with ␣ 7

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“…The α 7 β 7 β 7 α 7 ring topology of the 20S enzyme ensures that the proteolytic active sites, which reside in the β-subunits, are sequestered and can only cleave substrates that enter the chamber through narrow axial pores formed by the α-subunits (3). As a consequence, ATP-dependent unfolding of natively folded protein substrates by the AAA+ ring and subsequent polypeptide translocation through a narrow axial channel and into the 20S chamber are required for degradation (4).…”

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confidence: 99%

“…It was proposed that as ATP hydrolysis progresses in the PAN and Rpt 1-6 rings, wobbling motions arise because the HbYX tails of newly ATP-bound subunits interact with binding pockets in the 20S α-subunit, whereas HbYX motifs in posthydrolytic subunits assume a nonbinding conformation (14). However, recent EM studies have shown that the rings of Rpt [1][2][3][4][5][6] and 20S shift into more symmetric and coaxially aligned positions upon substrate engagement, questioning the functional importance of dynamic wobbling (15).…”

mentioning

confidence: 99%

Architecture and assembly of the archaeal Cdc48⋅20S proteasome

Barthelme¹,

Chen²,

Grabenstatter

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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ATP-dependent proteases maintain protein quality control and regulate diverse intracellular functions. Proteasomes are primarily responsible for these tasks in the archaeal and eukaryotic domains of life. Even the simplest of these proteases function as large complexes, consisting of the 20S peptidase, a barrel-like structure composed of four heptameric rings, and one or two AAA+ (ATPase associated with a variety of cellular activities) ring hexamers, which use cycles of ATP binding and hydrolysis to unfold and translocate substrates into the 20S proteolytic chamber. Understanding how the AAA+ and 20S components of these enzymes interact and collaborate to execute protein degradation is important, but the highly dynamic nature of prokaryotic proteasomes has hampered structural characterization. Here, we use electron microscopy to determine the architecture of an archaeal Cdc48·20S proteasome, which we stabilized by site-specific cross-linking. This complex displays coaxial alignment of Cdc48 and 20S and is enzymatically active, demonstrating that AAA+ unfoldase wobbling with respect to 20S is not required for function. In the complex, the N-terminal domain of Cdc48, which regulates ATP hydrolysis and degradation, packs against the D1 ring of Cdc48 in a coplanar fashion, constraining mechanisms by which the N-terminal domain alters 20S affinity and degradation activity.AAA+ protease | dynamic wobbling model | p97/VCP P roteasomes are large macromolecular complexes that degrade misfolded or damaged proteins to maintain cellular homeostasis and quality control in all domains of life. In addition, selective proteasomal turnover of regulatory proteins is often a critical element of signaling cascades that allow cells to respond to changing conditions and environmental stress (1, 2). Proteasomes consist of the self-compartmentalized 20S peptidase and one or two AAA+ (ATPase associated with a variety of cellular activities) family ring hexamers. The α 7 β 7 β 7 α 7 ring topology of the 20S enzyme ensures that the proteolytic active sites, which reside in the β-subunits, are sequestered and can only cleave substrates that enter the chamber through narrow axial pores formed by the α-subunits (3). As a consequence, ATP-dependent unfolding of natively folded protein substrates by the AAA+ ring and subsequent polypeptide translocation through a narrow axial channel and into the 20S chamber are required for degradation (4).Although the 20S peptidase is the degradation module in all proteasomes, the associated AAA+ unfolding machines differ. For example, homohexamers of Mpa/Arc in actinobacteria and PAN in archaea serve as proteasomal motors, whereas a heterohexameric Rpt 1-6 ring in the 19S particle is the motor of the eukaryotic 26S proteasome (2, 5). Characteristic of type-I AAA+ enzymes, Mpa/Arc, PAN, and Rpt 1-6 subunits contain a single AAA+ module for ATP binding and hydrolysis (6). By contrast, as found in type-II AAA+ enzymes, the homohexameric Cdc48 motor in the recently discovered archaeal Cdc48·20S proteasome cont...

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Structural Biology of the Proteasome

Cited by 196 publications

References 104 publications

Structural basis for dynamic regulation of the human 26S proteasome

Structural basis for dynamic regulation of the human 26S proteasome

Inhibition of Proteasome Activity Induces Formation of Alternative Proteasome Complexes

Architecture and assembly of the archaeal Cdc48⋅20S proteasome

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