Subcellular Distribution and Dynamics of Active Proteasome Complexes Unraveled by a Workflow Combining in Vivo Complex Cross-Linking and Quantitative Proteomics

Fabre, Bertrand; Lambour, Thomas; Delobel, Julien; Amalríc, François; Monsarrat, Bernard; Burlet‐Schiltz, Odile; Bousquet, Marie‐Pierre

doi:10.1074/mcp.m112.023317

Cited by 88 publications

(137 citation statements)

References 49 publications

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“…Because of this, Ecm29 is often missed in purified mammalian proteasomes (45,46). Here, we have reproducibly captured Ecm29 after FA cross-linking, which corroborates well with previous results (48,54). In addition, dynamic proteasome interactors have displayed much higher abundances in isolated proteasome complexes after cross-linking when comparing to noncross-linked samples.…”

Section: Discussionsupporting

confidence: 91%

“…As shown, the three selected proteolytic activities were at their highest when cells were treated with 0.05% formaldehyde prior to cell lysis. These results further confirm that proteasomes are highly dynamic entities in cells and suggest that low level cross-linking can be beneficial for stabilizing proteasome structure and function during cell lysis prior to affinity purification, in good agreement with a previous report (54). To further evaluate the effect of mild cross-linking on subsequent purification and MS analysis, we purified proteasomes using HF-Rpn13/ADRM1 as the bait from 293 HF-Rpn13/ADRM1_Rpn11-TB cells.…”

Section: Developing a New Dual-bait Strategy For Effective Isolation supporting

confidence: 89%

“…Although the composition of protein complexes resulting from denaturing purification can be readily determined, subunit stoichiometry can be misrepresented because of variance in protein crosslinking efficiency, leading to varying protein absolute abundances. Previously, it has been shown that mild crosslinking can be coupled with native purification to preserve protein interactions (52)(53)(54). In order to isolate UbR-proteasome subcomplexes and determine their quantitative differences in proteasome subunit composition, we have developed a novel affinity purification strategy XBAP, i.e.…”

Section: Ubqln1 (Aka Plic-1) Ubqln2 (Aka Plic-2) Ubqln3 Ubqlnmentioning

confidence: 99%

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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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Section: Discussionsupporting

confidence: 91%

Section: Developing a New Dual-bait Strategy For Effective Isolation supporting

confidence: 89%

Section: Ubqln1 (Aka Plic-1) Ubqln2 (Aka Plic-2) Ubqln3 Ubqlnmentioning

confidence: 99%

See 1 more Smart Citation

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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“…2A). These localizations generally agree with those described in the published literature, where all subunits have been found in both the cytoplasm and the nucleus (26)(27)(28)(29)(30). The only real differences we found include the apparently exclusively cytoplasmic localization of PA28␤ and PA200 in our experiments; however, longer exposures might have revealed some fraction of these proteins within the nucleus.…”

Section: Resultssupporting

confidence: 92%

Ubiquitin-Independent Proteasomal Degradation of Tumor Suppressors by Human Cytomegalovirus pp71 Requires the 19S Regulatory Particle

Winkler

Hwang

Kalejta

2013

J Virol

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bProteasomes generally degrade substrates tagged with polyubiquitin chains. In rare cases, however, proteasomes can degrade proteins without prior ubiquitination. For example, the human cytomegalovirus (HCMV) pp71 protein induces the proteasomedependent, ubiquitin-independent degradation of the retinoblastoma (Rb) and Daxx proteins. These transcriptional corepressors and tumor suppressors inhibit the expression of cellular or viral genes that are required for efficient viral replication. Proteasomes are composed of a 20S catalytic core with or without one or two activator complexes, of which there are four different types. Here, we show that only one of these activators, the 19S regulatory particle that normally participates in ubiquitin-dependent protein degradation, is required for pp71-mediated degradation of Rb and Daxx. We report the unique use of a well-established route of substrate delivery to the proteasome by a viral protein to promote infection. The proteasome is responsible for the bulk of protein turnover within cells. Most substrates arrive at the proteasome polyubiquitinated, where the ubiquitin chains mediate proteasome binding but are then removed prior to substrate degradation by the proteasomal 20S catalytic core. The core particle can associate with activator complexes that modulate proteasome function (1). For example, the 19S regulatory particle (RP) associates with one or both ends of the 20S core to form the 26S proteasome species responsible for ubiquitin-mediated degradation events. Proteasomal activity is critical for cellular homeostasis, cell cycle progression, transcription, DNA repair, and dichotomously, both the success of viral infections and essential defenses against viral pathogens (2-6).Proteasomal degradation of viral antigens to generate peptides displayed by major histocompatibility group (MHC) molecules is a well-documented part of adaptive immunity (4). Less well appreciated is the number of cellular intrinsic defense proteins targeted for proteasomal degradation by viral factors (7,8). For example, human cytomegalovirus (HCMV) infections, which cause severe disease in immunocompromised, -suppressed, or -naive individuals, induce the degradation of several cellular transcriptional corepressors to create an environment conducive to productive, lytic infection. Within the tegument layer of its virion, HCMV packages the viral pp71 protein, which is introduced into cells immediately upon infection, traffics to the nucleus, and induces the degradation of BclAF1, Daxx, and the retinoblastoma (Rb) family members Rb, p107, and p130 (9-11). BclAF1 and Daxx degradation promotes viral immediate early (IE) gene expression. Rb family inactivation, which also occurs through phosphorylation by the virally encoded kinase UL97 (12), likely increases the efficiency of viral DNA replication. These pp71-dependent degradation events are prevented by pharmacologic inhibition of the 20S catalytic core, indicating that they are proteasomal processes. Other experimental evidence, however, indica...

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“…Enrichment of proteasomal subunits in the nuclear fraction was previously reported in rat liver and several non‐muscle cells 31. Dynamic sub‐cellular distribution of proteasomal subunits was found in cancer cell cultures, and components of the 19S were enriched in the nuclear fraction 32. The change in proteasomal activity during cell fusion could also be due to the increase in expression of specific subunits.…”

Section: Resultsmentioning

confidence: 68%

Proteasomal activity‐based probes mark protein homeostasis in muscles

Raz¹,

Raz²,

Soriano

et al. 2017

J cachexia sarcopenia muscle

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BackgroundProtein homeostasis, primarily regulated by the ubiquitin–proteasome system is crucial for proper function of cells. In tissues of post‐mitotic cells, the impaired ubiquitin–proteasome system is found in a wide range of neuromuscular disorders. Activity‐based probes (ABPs) measure proteasomal proteolytic subunits and can be used to report protein homeostasis. Despite the crucial role of the proteasome in neuromuscular pathologies, ABPs were not employed in muscle cells and tissues, and measurement of proteasomal activity was carried out in vitro using low‐throughput procedures.MethodsWe screened six ABPs for specific application in muscle cell culture using high throughput call‐based imaging procedures. We then determined an in situ proteasomal activity in myofibers of muscle cryosections.ResultsWe demonstrate that LWA300, a pan‐reactive proteasomal probe, is most suitable to report proteasomal activity in muscle cells using cell‐based bio‐imaging. We found that proteasomal activity is two‐fold and three‐fold enhanced in fused muscle cell culture compared with non‐fused cells. Moreover, we found that proteasomal activity can discriminate between muscles. Across muscles, a relative higher proteasomal activity was found in hybrid myofibers whereas fast‐twitch myofibers displayed lower activity.ConclusionsOur study demonstrates that proteasomal activity differ between muscles and between myofiber types. We suggest that ABPs can be used to report disease progression and treatment efficacy.

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Subcellular Distribution and Dynamics of Active Proteasome Complexes Unraveled by a Workflow Combining in Vivo Complex Cross-Linking and Quantitative Proteomics

Cited by 88 publications

References 49 publications

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

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

Ubiquitin-Independent Proteasomal Degradation of Tumor Suppressors by Human Cytomegalovirus pp71 Requires the 19S Regulatory Particle

Proteasomal activity‐based probes mark protein homeostasis in muscles

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