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Affinity Purification Strategy to Capture Human Endogenous Proteasome Complexes Diversity and to Identify Proteasome-interacting Proteins
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Edité par CCSD ; American Society for Biochemistry and Molecular Biology -
International audience. An affinity purification strategy was developed to characterize human proteasome complexes diversity as well as endogenous proteasome-interacting proteins (PIPs). This single step procedure, initially used for 20 S proteasome purification, was adapted to purify all existing physiological proteasome complexes associated to their various regulatory complexes and to their interacting partners. The method was applied to the purification of proteasome complexes and their PIPs from human erythrocytes but can be used to purify proteasomes from any human sample as starting material. The benefit of in vivo formaldehyde cross-linking as a stabilizer of protein-protein interactions was studied by comparing the status of purified proteasomes and the identified proteins in both protocols (with or without formaldehyde cross-linking). Subsequent proteomics analyses identified all proteasomal subunits, known regulators, and recently assigned partners. Moreover other proteins implicated at different levels of the ubiquitin-proteasome system were also identified for the first time as PIPs. One of them, the ubiquitin-specific protease USP7, also known as HAUSP, is an important player in the p53-HDM2 pathway. The specificity of the interaction was further confirmed using a complementary approach that consisted of the reverse immunoprecipitation with HAUSP as a bait. Altogether we provide a valuable tool that should contribute, through the identification of partners likely to affect proteasomal function, to a better understanding of this complex proteolytic machinery in any living human cell and/or organ/tissue and in different cell physiological states.Proteasome-mediated and lysosomal degradations are the two main mechanisms involved in turnover of intracellular proteins. The 26 S proteasome is the proteolytic machine of the ubiquitin-proteasome pathway (UPP)1 (1, 2). In most cases, the degradation is processed by two successive steps: (i) polyubiquitination of the substrate and (ii) proteolysis of the tagged protein by the 26 S proteasome (2). The proteasome degrades abnormal and non-functional proteins generated under normal and stress conditions but also tightly regulates major cellular processes (cell cycle progression, transcription, apoptosis, DNA repair, epitope generation, etc.) by controlling the cellular pool of key regulatory proteins (3). Therefore a dysregulation of this machinery can lead to various pathologies such as neurodegenerative diseases (4) or cancers (5). The proteasome has recently been identified as a therapeutic target for cancer treatment (6).The eukaryotic 26 S proteasome can be divided into subcomplexes, one 670-kDa 20 S core particle where proteolysis occurs and one or two axially positioned 900-kDa 19 S regulatory particles responsible for polyubiquitinated substrates recognition, ATP-dependent substrate unfolding, and ubiquitin recycling (7). The eukaryotic 20 S proteasome is a stable complex (8) composed of 28 subunits, arranged in four stacked rings with seven unique α subunits in the two outer rings and seven unique β subunits in the two inner rings (9). Six catalytic proteolytic active sites are located on the proteasome subunits β1, β2, and β5. Upon interferon-γ-induced immune response in mammals, these catalytic subunits are replaced by the immunosubunits β1i, β2i, and β5i, respectively, which induce some changes in the proteolytic activities of the complex (10).Eukaryotic 19 S regulatory particle, also called PA700, is connected to the 20 S catalytic core through the α ring. It is composed of ∼16 electrophoretically distinct subunits with molecular masses ranging from 25 to 112 kDa (11) corresponding to at least 23 proteins at the present knowledge. The 19 S regulatory particle has a base comprising six proteasomal ATPases (Rpt1–Rpt6 in yeast), three additional non-ATPase subunits (Rpn1, Rpn2, and Rpn13), and a lid structure composed of at least 14 non-ATPase subunits and that is assumed to be connected to the base by the Rpn10 subunit. Although some subunits have been identified as key components for substrate recognition (Rpn10 and Rpt5) (12, 13), for opening the core particle gate (Rpt2) (14), and for deubiquitination (Rpn11) (15), the precise function of most subunits still remains to be elucidated. Functional characterization of 19 S subunits is difficult because the structural organization of the complex is not well defined on account of labile and dynamic interactions of several subunits (16).In addition to PA700, the two outer α rings of the 20 S proteasome can associate to other regulatory caps, PA28αβ, PA28γ, and PA200; the main role of these regulators is to open the gate into and out of the catalytic chamber. This leads to the formation of several different subpopulations of highly dynamic proteasome complexes because one 20 S core particle can interact at its two sides with either two identical regulators or two different ones, thus forming hybrid proteasomes (17). These hybrid proteasomes have been implicated in major cellular processes such as immune surveillance, regulation of cell size and growth, apoptosis, or DNA repair, but the exact proteolytic function of each of these different complexes is mainly unresolved and constitutes today a challenge in proteasome biology (18). Moreover several transient interacting proteins of human 26 S proteasome have recently been identified, but their functional impact remains, for most of them, to be elucidated (18, 19). They are, however, of particular importance because proteasome dynamic association to various proteins is likely to regulate its stability and activity upon diverse stimuli.Multidimensional chromatography has been used to purify the 26 S proteasome from various species to homogeneity (8, 20–24). These methods have improved our knowledge on proteasome structure but failed to catch labile interactors due to the use of high salt concentrations. Affinity purification or co-immunoprecipitation methods (25–28) as well as genome-wide two-hybrid surveys (29) have proven to be much more efficient and led to the identification of numerous additional proteasome subunits and associated proteins from yeast.The few works dealing with the characterization of the human proteasome-interacting protein network have been published very recently and were conducted by proteomics approaches (17, 30–32). A strategy relying on the tagging of a 19 S subunit and mass spectrometric analyses of co-immunoprecipitated complexes identified Adrm1, the human orthologue of the yeast Rpn13 subunit (30, 31). A similar approach combined with a SILAC (stable isotope labeling with amino acids in cell culture) strategy enabled distinguishing proteins that transiently interact with the proteasome from stable proteasome-interacting proteins (PIPs) (32).These strategies, involving mass spectrometry-based identification associated to biochemical approaches such as immunoprecipitation or affinity purification, have been successfully applied to the study of PIPs and support the idea that recent developments in proteomics are powerful tools for the study of protein networks (33). Protein-protein interactions inside protein complexes are difficult to maintain during the purification procedure, and the use of chemical cross-linkers can be useful for transient interactor recovery (34) and have been successfully used for 26 S proteasomes structural studies (35).However, all the affinity-based methods described to date to purify proteasome complexes and PIPs rely on overexpression or tagging strategies. Moreover most of them are based on the use of a 19 S subunit as a bait, which implies that only proteasome containing at least one 19 S regulatory particle can be purified. Proteasome pools involving only PA28 or PA200 regulators might be associated with specific partners. Therefore, methods to efficiently purify all proteasome complexes would be of great interest.We developed a single step affinity purification protocol to characterize all physiological proteasome complexes and their PIPs in human cells. It is based on the high affinity binding of a subunit of the 20 S core particle to a monoclonal antibody. A differential proteomics strategy with a control antibody was used to distinguish specific PIPs from nonspecific interactors. Moreover the benefit of in vivo formaldehyde cross-linking on overall proteasome complexes recovery was also assessed. This strategy led to the identification of new PIPs of the human proteasome, including the deubiquitinase HAUSP for which specific interaction was further confirmed
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