Gates, Channels, and Switches: Elements of the Proteasome Machine

Finley, Daniel; Chen, Xiang; Walters, Kylie J.

doi:10.1016/j.tibs.2015.10.009

Cited by 236 publications

(236 citation statements)

References 158 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).…”

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confidence: 99%

Structural basis for dynamic regulation of the human 26S proteasome

Chen

et al. 2016

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

159

310

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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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Structural basis for dynamic regulation of the human 26S proteasome

Chen

et al. 2016

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

159

310

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“…T he 26S proteasome is an ATP-dependent multisubunit protease degrading polyubiquitylated proteins (1,2). It operates at the executive end of the Ubiquitin-Proteasome System (UPS) and has a key role in cellular proteostasis.…”

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Structure of the human 26S proteasome at a resolution of 3.9 Å

Schweitzer

Aufderheide

Rudack

et al. 2016

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

187

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Protein degradation in eukaryotic cells is performed by the UbiquitinProteasome System (UPS). The 26S proteasome holocomplex consists of a core particle (CP) that proteolytically degrades polyubiquitylated proteins, and a regulatory particle (RP) containing the AAA-ATPase module. This module controls access to the proteolytic chamber inside the CP and is surrounded by non-ATPase subunits (Rpns) that recognize substrates and deubiquitylate them before unfolding and degradation. The architecture of the 26S holocomplex is highly conserved between yeast and humans. The structure of the human 26S holocomplex described here reveals previously unidentified features of the AAA-ATPase heterohexamer. One subunit, Rpt6, has ADP bound, whereas the other five have ATP in their binding pockets. Rpt6 is structurally distinct from the other five Rpt subunits, most notably in its pore loop region. For Rpns, the map reveals two main, previously undetected, features: the C terminus of Rpn3 protrudes into the mouth of the ATPase ring; and Rpn1 and Rpn2, the largest proteasome subunits, are linked by an extended connection. The structural features of the 26S proteasome observed in this study are likely to be important for coordinating the proteasomal subunits during substrate processing.proteostasis | cryo-electron microscopy | AAA-ATPase | integrative modeling T he 26S proteasome is an ATP-dependent multisubunit protease degrading polyubiquitylated proteins (1, 2). It operates at the executive end of the Ubiquitin-Proteasome System (UPS) and has a key role in cellular proteostasis. The 26S proteasome selectively removes misfolded proteins or proteins no longer needed and it is critically involved in numerous cellular processes such as protein quality control, regulation of metabolism, cell cycle control, or antigen presentation. Malfunctions of the UPS are associated with various pathologies, including neurodegenerative diseases and cancer. Therefore, the proteasome is an important pharmaceutical target, and a high-resolution structure is a prerequisite for structure-based drug design (3).The 26S proteasome comprises the 20S cylindrical core particle (CP), where proteolysis takes place, and 19S regulatory particles (RPs). In cellular environments, 26S holocomplexes with either one or two RPs bound to the ends of the cylindershaped CP coexist (4). The role of the RPs is to recruit ubiquitylated substrates, to cleave off their polyubiquitin tags, and to unfold and translocate them into the CP for degradation into short peptides. Whereas X-ray crystallography has revealed the atomic structures first of archaeal 20S proteasome (5) and subsequently of the yeast (6) and mammalian proteasome (7), only lower-resolution structures were available for the 26S holocomplex. Given the compositional and conformational heterogeneity of the RP, single-particle cryo-electron microscopy (cryo-EM) has been the most successful approach to determining the structure of the 26S holocomplex (8). At this point, the most detailed insights have been obtained ...

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“…Delivery of ubiquitinated substrates to the 26S proteasome is an important regulatory step in the ubiquitin proteasome system (UPS) (2,3). It is generally believed that the initial recognition of substrates by the proteasome is mediated by the substrate-attached polyubiquitin chain (1)(2)(3).…”

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confidence: 99%

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confidence: 99%

“…It is generally believed that the initial recognition of substrates by the proteasome is mediated by the substrate-attached polyubiquitin chain (1)(2)(3). Ubiquitin receptors (UbRs) are a group of proteins that can recognize and bind polyubiquitin chains (2,3,12). Five types of UbRs have been well recognized to be associated with proteasomal degradation, including the two proteasome subunits Rpn10 (13) and Rpn13/ADRM1 (14), and three "shuttling factors," Rad23, Dsk2, and Ddi1 (2, 3) (Table I).…”

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Characterization of Dynamic UbR-Proteasome Subcomplexes by In vivo Cross-linking (X) Assisted Bimolecular Tandem Affinity Purification (XBAP) and Label-free Quantitation

Yang

Wang

et al. 2016

Molecular & Cellular Proteomics

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Proteasomes are protein degradation machines that exist in cells as heterogeneous and dynamic populations. A group of proteins function as ubiquitin receptors (UbRs) that can recognize and deliver ubiquitinated substrates to proteasome complexes for degradation. Defining composition of proteasome complexes engaged with UbRs is critical to understand proteasome function. However, because of the dynamic nature of UbR interactions with the proteasome, it remains technically challenging to capture and isolate UbR-proteasome subcomplexes using conventional purification strategies. As a result, distinguishing the molecular differences among these subcomplexes remains elusive. We have developed a novel affinity purification strategy, in vivo cross-linking (X) assisted bimolecular tandem affinity purification strategy (XBAP), to effectively isolate dynamic UbR-proteasome subcomplexes and define their subunit compositions using label-free quantitative mass spectrometry. In this work, we have analyzed seven distinctive UbR-proteasome complexes and found that all of them contain the same type of the 26S holocomplex. However, selected UbRs interact with a group of proteasome interacting proteins that may link each UbR to specific cellular pathways. The compositional similarities and differences among the seven UbRproteasome subcomplexes have provided new insights on functional entities of proteasomal degradation machineries. The strategy described here represents a gen- Proteasomes are multisubunit protein complexes that are responsible for the degradation of ubiquitinated substrates to maintain cell viability and homeostasis. The 26S proteasome is composed of at least 33 subunits (1-3), which can be divided into two subcomplexes: the 20S catalytic core particle (CP) 1 and the 19S regulatory particle (RP). The 20S CP is responsible for various proteolytic activities, and has a highly conserved "barrel"-like structure consisting of two copies each of 14 nonidentical subunits (␣1-7, ␤1-7), which are arranged into four heptameric rings stacked in the order of ␣ 7 ␤ 7 ␤ 7 ␣ 7 (4, 5). The 19S RP intimately interacts with the 20S CP, regulating its activity. In addition, the 19S RP carries diverse functions including substrate recognition and deubiquitination, protein unfolding, and substrate translocation to the 20S CP for degradation (2, 3, 6 -8). In contrast to the highly ordered and stable structure of the 20S CP, the 19S RP appears to be much more flexible and dynamic (3, 9 -11). Current structural analyses have revealed that six Rpt subunits of the 19S RP form a hexameric AAA-ATPase ring to associate with the cylinder ends of the 20S CP, and are surrounded by a shell of Rpn subunits (9 -11). Apart from the

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Gates, Channels, and Switches: Elements of the Proteasome Machine

Cited by 236 publications

References 158 publications

Structural basis for dynamic regulation of the human 26S proteasome

Structural basis for dynamic regulation of the human 26S proteasome

Structure of the human 26S proteasome at a resolution of 3.9 Å

Characterization of Dynamic UbR-Proteasome Subcomplexes by In vivo Cross-linking (X) Assisted Bimolecular Tandem Affinity Purification (XBAP) and Label-free Quantitation

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