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Structural and functional analyses of human proteasome complexes

We are interested in understanding the structure/activity relationships of human proteasome, a large multicatalytic complex in charge of intracellular protein degradation, which is a therapeutic target for some cancers. To this aim we have developed proteomic strategies to characterize the subunit composition of the catalytic core of the complex, to determine the distribution of the various regulatory complexes associated to the catalytic core complex, to identify proteins interacting with the proteasome, and to quantify the variations in the composition of proteasome complexes in different cells and also within the cell in different subcellular compartments. This fine description of proteasome complexes gives us important information to understand better its activity in several cell localizations and under different cellular states.

The proteasome is the proteolytic machinery of the ubiquitin-proteasome system (UPS), the main pathway responsible for degradation of intracellular proteins. As the major cellular protease, the proteasome is a key player in eukaryotic protein homeostasis and dysregulation of the UPS has been involved in neurodegenerative diseases and cancers. Because of this, protea­somes have been identified as therapeutic targets, especially for some cancers. Therefore, understanding the structure/function relationship controlling proteasome activity is of major interest in biology.

 

 

Diversity of proteasome complexes from Bousquet et al. Proteomics to study the diversity and dynamics of proteasome complexes: from fundamentals to the clinic. Expert Rev Proteomics. 2011 Aug;8(4):459-81. doi: 10.1586/EPR.11.41.

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Proteasomes complexes consist of a 20S catalytic core particle (CP) comprising 14 to 17 different proteins, either single, or associated to one or two regulatory particles of identical or different protein composition. In mammals, four activators have been identified, the 19S regulatory particle (RP), PA28a/b, PA28g, and PA200. The 26S proteasome is a particular form where the 20S CP is capped by two 19S RPs, forming a 2.4 MDa complex; it is involved in the ubiquitin-dependent degradation of proteins through specialized functions of specific subunits of the 19S, ie polyubiquitinated substrate recognition, ATP-dependent substrate unfolding, and ubiquitin recycling. Hybrid proteasomes correspond to forms where the 20S CP is capped with two different regulators, mainly 19S RP and PA28. The eukaryotic 20S proteasome is assembled in four stacked ring-shape heptamers, with seven unique a subunits in the two outer rings and seven unique b subunits in the two inner rings. The two b rings contain each 3 catalytic subunits, b1, b2, and b5, which are totally or partially replaced by the so-called immuno-subunits, b1i, b2i, and b5i, in the immuno-proteasome or intermediate-type proteasomes, respectively. The catalytic subunits are responsible for three main proteasome proteolytic activities (trypsin-like, chymotrypsin-like, and caspase-like), which can be modulated by the replacement of standard subunits by immunosubunits. The immuno-proteasome is induced during the immune response in mammals but, together with intermediate-type proteasomes, also exists as constitutive proteasome complexes, depending on tissues or cell type, and can have distinct proteolytic activities. The functional roles of these different proteasome subtypes are largely unresolved. The association of the 20S CP with its different regulators is ensured through the two a rings, which also regulate the entry of substrates into the CP. 

 

Proteasome complexes therefore exhibit a high degree of heterogeneity in its overall subunit composition. Given the broad function of proteasomes, in quality control, antigenic peptide generation or short-lived protein-tuned regulation, the cell is likely to adapt proteasome plasticity and dynamics to meet specific subcellular needs or to respond to stress or other stimuli. However, the precise intracellular subunit composition and distribution of proteasome complexes remains largely undetermined. This could be explained by the highly dynamic state of proteasome complexes, their heterogeneity and instability which make them inherently difficult to study. To deal with this, efficient strategies are needed to purify and quantify fully assembled, active proteasome complexes in homogeneous cellular fractions. 

Proteomic strategies constitute complementary methods to structure determination approaches for the study of protein machines. The association of modern mass spectrometry strategies and efficient biochemical approaches, in particular affinity-purification methods, has been critical for the initial success of functional proteomics.

The purpose of our project is to carry out a comprehensive study of the proteasome complexes diversity in eukaryotic cells to gain insights into the role of the different forms of proteasome. 

Based on a previous study where we optimized an affinity purification-mass spectrometry (AP-MS) strategy to efficiently purify all endogenous human proteasome complexes (Bousquet-Dubouch et al. Mol. Cell Proteomics 2009, 8:1150), we have developed further the approach to determine the dynamics of proteasome complexes in space (subcellular localization) and time (upon IFN stimulation). To maintain the labile interactions between the 20S core complex and the regulatory complexes, we determined for the first time experimental conditions for efficient in vivo crosslinking using formaldehyde which are compatible with subsequent subcellular fractionation (nucleus, cytoplasm, microsomes). We then used AP-MS and label-free quantitative proteomics to determine the subcellular distribution of proteasome complexes (Fabre et al. Mol. Cell Proteomics 2013, 12: 687)

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Strategy used for the determination of the dynamics of proteasome complexes in space (subcellular localization) and time (upon IFN gamma stimulation).

 

 

We have thus established that the composition of the 20S catalytic core remains unchanged in the three cellular compartments of U937 leukemic human cells whereas the composition of associated regulatory complexes varies significantly. We have also shown that a high content of free 20S complex (>60%) is present in all compartments. These results suggest that the regulation of proteasome activity is rather performed by the dynamic association of the 20S core particle with proteasome activators than by changes in 20S proteasome subunits composition. Moreover, free 20S proteasome might constitute a dynamic platform allowing proteasome activity to be rapidly increased or decreased on cellular demand. Specific proteasome interacting proteins have also been identified in each cellular compartment, giving some hints into major pathways where proteasome complexes are involved in.

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Subcellular composition of 20S proteasome complexes and associated regulatory complexes in human U937 leukemic cells (Fabre et al, Mol. Cell Proteomics, 2013, 12, 687. doi: 10.1074/mcp.M112.023317).

We have extended the determination of proteasome complexes composition in a wide range of human cell lines (Fabre et al. J.Proteome Res. 2014, 13: 3027) and showed that proteasome complexes are highly dynamic protein assemblies, the activity of which being regulated at different levels by variations in the composition of 20S catalytic subunits (A), in the stoichiometry of bound regulators (B), and in the rate of the 20S catalytic core complex assembly (C).

Stoichiometry of 20S proteasome subunits, associates regulators and assembly chaperones in a wide range of human cell lines (Fabre B., Lambour T. et al. J. Proteome Res. 2014, 13:3027. doi: 10.1021/pr500193k).
The fraction of 20S proteasome in the course of assembly can be estimated using the fraction of 20S proteasome associated PAC1/PAC2 dimer. 

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We also developed a new method based on the combination of affinity purification and protein correlation profiling associated to high resolution mass spectrometry, to comprehensively characterize proteasome heterogeneity and identify preferential associations within proteasome sub-complexes. In particular, we showed for the first time that the two main proteasome sub-types, standard proteasome (sP20S) and immunoproteasome (iP20S), interact with a different subset of important regulators. This trend was observed in very diverse human cell types and was confirmed by changing the relative proportions of both 20S proteasome forms using interferon-. The new method developed here, which we called “PAC-AP-HR-MS” for Protein Abundance Correlation associated to Affinity Purification and High Resolution Mass Spectrometry, constitutes an innovative and powerful strategy that should be easily transferred to other molecular systems to facilitate the in-depth characterization of heterogeneous protein complexes.

Other studies of the group on structure-activity relationships of proteasome have also been published:

  • Bousquet-Dubouch, M. P., Baudelet, E., Guérin, F., Matondo, M., Uttenweiler-Joseph, S., Burlet-Schiltz, O. & Monsarrat, B. (2009). Affinity purification strategy to capture human endogeneous proteasome complexes diversity and to identify proteasome interacting proteins. Mol Cell Proteomics. 8: 1150 - 1164. F1000 9.0 Exceptional.
  • Bousquet-Dubouch, M. P., Nguen S., Bouyssié D., Burlet-Schiltz O., French S.W., Monsarrat B., Gorce-Bardag F. (2009). Chronic ethanol feeding affects Proteasome Interacting Proteins. Proteomics. 9: 3609-3622.
  • Matondo M, Bousquet-Dubouch MP, Gallay N, Uttenweiler-Joseph S, Recher C, Payrastre B, Manenti S, Monsarrat B, Burlet-Schiltz O. (2010). Proteasome inhibitor-induced apoptosis in acute myeloid leukemia: A correlation with the proteasome status. Leuk Res. 34(4): 498-506.
  • Guillaume B, Chapiro J, Stroobant V, Colau D, Van Holle B, Parvizi G, Bousquet-Dubouch MP, Théate I, Parmentier N, Van den Eynde BJ. (2010) Two abundant proteasome subtypes that uniquely process some antigens presented by HLA class I molecules. Proc Natl Acad Sci U S A 107(43):18599-604.
  • Uttenweiler-Joseph S, Bouyssié D, Calligaris D, Lutz PG, Monsarrat B, Burlet-Schiltz O. Quantitative proteomic analysis to decipher the differential apoptotic response of bortezomib treated APL cells before and after retinoic acid differentiation reveals involvement of protein toxicity mechanisms. Proteomics. 2013 Jan;13(1):37-47.
  • Guillaume B, Stroobant V, Bousquet-Dubouch MP, Colau D, Chapiro J, Parmentier N, Dalet A, Van den Eynde BJ. Analysis of the Processing of Seven Human Tumor Antigens by Intermediate Proteasomes. J Immunol. 2012 Aug 27. 
  • Fabre B, Lambour T, Bouyssié D, Menneteau T, Monsarrat B, Burlet-Schiltz O, Bousquet-Dubouch MP. Comparison of label-free quantification methods for the determination of protein complexes subunits stoichiometry. EuPA Open Proteomics 2014, 82-86.