Affinity Purification Strategy to Capture Human Endogenous Proteasome Complexes Diversity and to Identify Proteasome-interacting Proteins

Bousquet, Marie‐Pierre; Baudelet, Emilie; Guèrin, Frédéric; Matondo, Mariette; Uttenweiler-Joseph, Sandrine; Burlet‐Schiltz, Odile; Monsarrat, Bernard

doi:10.1074/mcp.m800193-mcp200

Cited by 67 publications

(75 citation statements)

References 76 publications

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“…How the p97 complex delivers the ubiquitinated IB␣ to the 26S proteasome is another important question in IB␣ proteolysis. p97 and several p97 cofactors associate with the 26S proteasome (53,54). However, it is still unclear if the p97 complex contacts the proteasome directly or via additional polyubiquitin receptors.…”

Section: Discussionmentioning

confidence: 99%

The p97-UFD1L-NPL4 Protein Complex Mediates Cytokine-Induced IκBα Proteolysis

Zhang

et al. 2014

Molecular and Cellular Biology

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IB␣ is an inhibitor of NF-B, a family of transcription factors that transactivate genes related to inflammation. Upon inflammatory stimuli, IB␣ is rapidly degraded via the ubiquitin-proteasome pathway. While it is very clear that the SCF ␤-TRCP ubiquitin ligase ubiquitinates IB␣ upon stimulation, little is known about the postubiquitinational events of IB␣ proteolysis. Here, we report that p97, a valosin-containing protein (also called VCP), plays an essential role in the postubiquitinational regulation of IB␣ turnover after tumor necrosis factor alpha (TNF-␣) or interleukin-1␤ (IL-1␤) treatment. The ATPase activity of p97 is essential for its role in IB␣ proteolysis. Moreover, we found that UFD1L and NPL4, two cofactors of p97, assist p97 to control the postubiquitinational regulation of IB␣. The p97-UFD1L-NPL4 protein complex specifically associates with ubiquitinated IB␣ via the interactions between p97 and the SCF ␤-TRCP ubiquitin ligase and between the polyubiquitin binding domain of UFD1L and polyubiquitinated IB␣. Furthermore, we observed that the postubiquitinational regulation of IB␣ by the p97-UFD1L-NPL4 complex is important for NF-B activation under stimuli.

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Section: Discussionmentioning

confidence: 99%

The p97-UFD1L-NPL4 Protein Complex Mediates Cytokine-Induced IκBα Proteolysis

Zhang

et al. 2014

Molecular and Cellular Biology

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“…Apart from the HB tag (46 -48, 80, 82, 84), antibodies (78,81,87) and epitope-based tags such as Myc (79) and hemagglutinin-FLAG (83) have been used for similar applications. Unlike HB tag-based affinity purification, antibody-based affinity purification must be carried out under native conditions, which will result in higher nonspecific background and thus interfere with subsequent analyses.…”

Section: Qtax Methods (In Vivo Cross-linking-ap-qms)mentioning

confidence: 99%

Profiling of Protein Interaction Networks of Protein Complexes Using Affinity Purification and Quantitative Mass Spectrometry

Kaake

Wang

Huang

2010

Molecular & Cellular Proteomics

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Protein-protein interactions are important for nearly all biological processes, and it is known that aberrant protein-protein interactions can lead to human disease and cancer. Recent evidence has suggested that protein interaction interfaces describe a new class of attractive targets for drug development. Full characterization of protein interaction networks of protein complexes and their dynamics in response to various cellular cues will provide essential information for us to understand how protein complexes work together in cells to maintain cell viability and normal homeostasis. Affinity purification coupled with quantitative mass spectrometry has become the primary method for studying in vivo protein interactions of protein complexes and whole organism proteomes. Recent developments in sample preparation and affinity purification strategies allow the capture, identification, and quantification of protein interactions of protein complexes that are stable, dynamic, transient, and/or weak. Essential biological processes in cells such as DNA replication, transcription, translation, protein degradation, and cell cycle control are carried out by an assembly of protein molecules that interact and form multisubunit protein complexes (1-5). These macromolecular complexes are important cellular machineries that work hand in hand to maintain normal cell homeostasis. Although the function of each individual subunit is important, the function of the complex as a whole is not simply the sum of its individual components (6 -8). Traditional biochemical methods focus on characterizing a single protein.Although still valid and useful, these methods cannot account for the complexity of macromolecular protein machines. Recently, technological developments in mass spectrometrybased proteomics approaches have made comprehensive characterization of protein complexes possible by enabling the determination of dynamic protein complex compositions, stoichiometries, posttranslational modifications, assemblies, structures, and protein interaction networks (8 -17). This review will report the advances in the study of protein-protein interactions by mass spectrometry.Protein-protein interactions are important for nearly all biological processes, and it is known that aberrant protein-protein interactions can lead to human disease and cancer (18 -20). Sufficient evidence has demonstrated that modulation of protein-protein binding represents an emerging therapeutic paradigm, and protein interaction interfaces describe a new class of attractive targets for drug development. For example, inactivation of tumor suppressor p53 has been implicated in various cancers, and one mechanism of inactivation is the inappropriate interaction with the E3 ubiquitin ligase MDM2, leading to excessive degradation of p53 (21). Disruption of this interaction by small molecule inhibitors has shown promise as a potential tool for cancer treatment (21-25). Current work has illustrated how generating protein interaction networks of protein complexes and profiling t...

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“…In the second dimension, proteins were separated in a 0.5-mm-thick SDS-PAGE gel (20 3 20 cm) and stained with colloidal blue. For identification, protein spots were excised from the gel, digested with trypsin, and analyzed by nano-liquid chromatography coupled to tandem mass spectrometry (MS/MS) on an ESI-LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific) operating in positive mode, as recently described (18).…”

Section: Two-dimensional Gel Electrophoresis and Liquid Chromatographmentioning

confidence: 99%

Analysis of the Processing of Seven Human Tumor Antigens by Intermediate Proteasomes

Guillaume

Stroobant

Bousquet

et al. 2012

The Journal of Immunology

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We recently described two proteasome subtypes that are intermediate between the standard proteasome and the immunoproteasome. They contain only one (β5i) or two (β1i and β5i) of the three inducible catalytic subunits of the immunoproteasome. They are present in tumor cells and abundant in normal human tissues. We described two tumor antigenic peptides that are uniquely produced by these intermediate proteasomes. In this work, we studied the production by intermediate proteasomes of tumor antigenic peptides known to be produced exclusively by the immunoproteasome (MAGE-A3114–122, MAGE-C242–50, MAGE-C2336–344) or the standard proteasome (Melan-A26–35, tyrosinase369–377, gp100209–217). We observed that intermediate proteasomes efficiently produced the former peptides, but not the latter. Two peptides from the first group were equally produced by both intermediate proteasomes, whereas MAGE-C2336–344 was only produced by intermediate proteasome β1i-β5i. Those results explain the recognition of tumor cells devoid of immunoproteasome by CTL recognizing peptides not produced by the standard proteasome. We also describe a third antigenic peptide that is produced exclusively by an intermediate proteasome: peptide MAGE-C2191–200 is produced only by intermediate proteasome β1i-β5i. Analyzing in vitro digests, we observed that the lack of production by a given proteasome usually results from destruction of the antigenic peptide by internal cleavage. Interestingly, we observed that the immunoproteasome and the intermediate proteasomes fail to cleave between hydrophobic residues, despite a higher chymotrypsin-like activity measured on fluorogenic substrates. Altogether, our results indicate that the repertoire of peptides produced by intermediate proteasomes largely matches the repertoire produced by the immunoproteasome, but also contains additional peptides.

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Affinity Purification Strategy to Capture Human Endogenous Proteasome Complexes Diversity and to Identify Proteasome-interacting Proteins

Cited by 67 publications

References 76 publications

The p97-UFD1L-NPL4 Protein Complex Mediates Cytokine-Induced IκBα Proteolysis

The p97-UFD1L-NPL4 Protein Complex Mediates Cytokine-Induced IκBα Proteolysis

Profiling of Protein Interaction Networks of Protein Complexes Using Affinity Purification and Quantitative Mass Spectrometry

Analysis of the Processing of Seven Human Tumor Antigens by Intermediate Proteasomes

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