Deciphering preferential interactions within supramolecular protein complexes: the proteasome caseby B. Fabre, T. Lambour, L. Garrigues, F. Amalric, N. Vigneron, T. Menneteau, A. Stella, B. Monsarrat, B. Van den Eynde, O. Burlet-Schiltz, M.-P. Bousquet-Dubouch

Molecular Systems Biology



Deciphering preferential interactions within supramolecular protein complexes: the proteasome case

Bertrand Fabre1,2, Thomas Lambour1,2, Luc Garrigues1,2, François Amalric1,2, Nathalie Vigneron3,4,5,

Thomas Menneteau1,2, Alexandre Stella1,2, Bernard Monsarrat1,2, Benoît Van den Eynde3,4,5,

Odile Burlet-Schiltz1,2,* & Marie-Pierre Bousquet-Dubouch1,2,**


In eukaryotic cells, intracellular protein breakdown is mainly performed by the ubiquitin–proteasome system. Proteasomes are supramolecular protein complexes formed by the association of multiple sub-complexes and interacting proteins. Therefore, they exhibit a very high heterogeneity whose function is still not well understood. Here, using a newly developed method based on the combination of affinity purification and protein correlation profiling associated with high-resolution mass spectrometry, we comprehensively characterized proteasome heterogeneity and identified previously unknown preferential associations within proteasome sub-complexes. In particular, we showed for the first time that the two main proteasome subtypes, standard proteasome and immunoproteasome, 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-c. The new method developed here constitutes an innovative and powerful strategy that could be broadly applied for unraveling the dynamic and heterogeneous nature of other biologically relevant supramolecular protein complexes.

Keywords affinity purification; correlation profiling; label-free quantitative proteomics; mass spectrometry

Subject Categories Post-translational Modifications, Proteolysis &


DOI 10.15252/msb.20145497 | Received 12 June 2014 | Revised 21 November 2014 | Accepted 3 December 2014

Mol Syst Biol. (2015) 11: 771


The proteasome is a supramolecular protein machinery that is central to protein homeostasis. In all eukaryotic cells, it is involved in the selective degradation of most short-lived intracellular proteins (Hershko & Ciechanover, 1998; Glickman & Ciechanover, 2002), ensuring subtle modulation of gene expression, but also the removal of misfolded, aberrant (i.e. oxidized or mutated), or otherwise damaged proteins, avoiding cytotoxicity. In higher eukaryotes, it is critical to the immune response because it generates antigenic peptide precursors that can be recognized by cytotoxic T lymphocytes (CTLs) on MHC class I molecules.

Proteasome particles are formed by the dynamic association of several sub-complexes, a 20S core particle (20S CP), either single or associated with one or two regulatory particles (RPs) of identical or different protein composition. Proteasome complexes thus display a high degree of heterogeneity. The 20S CP presents a a7b7b7a7 barrel-like structure and was shown to exist in the eukaryotic cell as four different subtypes, depending on the subsets of incorporated catalytic beta subunits. In the standard proteasome (sP20S), the two b rings each contain three standard catalytic subunits, b1, b2, and b5, which are replaced by distinct immunosubunits, b1i, b2i, and b5i, in the immunoproteasome (iP20S), respectively. Two intermediate 20S CP subtypes, b5i 20S proteasome (b5i P20S) and b1ib5i 20S proteasome (b1ib5i P20S), bearing a mixed incorporation of standard and immunosubunits, b1, b2, b5i and b1i, b2, b5i, respectively, have also been identified in a wide range of cell types and tissues (Guillaume et al, 2010). The catalytic subunits are responsible for the three proteasome proteolytic activities (trypsin like, chymotrypsin like, and caspase like), which can be modulated by the replacement of standard subunits by immunosubunits (Orlowski & Wilk, 2000;

Basler et al, 2013). The iP20S is induced during the immune response in mammals but also exists in various amounts as 1 CNRS, IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France 2 Université de Toulouse, UPS, IPBS, Toulouse, France 3 Ludwig Institute for Cancer Research, Brussels, Belgium 4 WELBIO (Walloon Excellence in Life Sciences and Biotechnology), Brussels, Belgium 5 de Duve Institute, Université catholique de Louvain, Brussels, Belgium *Corresponding author. Tel: +33 561175547; E-mail: **Corresponding author. Tel: +33 561175544; E-mail: ª 2015 The Authors. Published under the terms of the CC BY 4.0 license Molecular Systems Biology 11: 771 | 2015 1

Published online: January 5, 2015 constitutive proteasome complexes, depending on tissues or cell type (Dahlmann et al, 2000; Zoeger et al, 2006; Klare et al, 2007;

Bousquet-Dubouch et al, 2008; Guillaume et al, 2010, 2012). The two a rings are located at the opposite ends of the proteolytic cavity and regulate the access of substrates to catalytic sites through gated pores of 13 A˚ in diameter. In mammals, gate opening can be efficiently triggered through the association of the 20S CP with four main different RPs, the 19S regulatory particle (19S RP), PA28ab, PA28c (otherwise known as 11S RPs), and PA200. One 20S CP can interact at its two sides with either two identical regulators or two different ones, thus forming hybrid proteasomes (Tanahashi et al, 2000). The most studied regulator, the 19S RP, is involved in the recognition, the unfolding, and the translocation of poly-ubiquitinated substrates into the 20S CP for degradation. In addition to the 19S RP, PA28ab and

PA28c RPs are abundant 20S proteasome-associated regulators present in the cytosol and the nucleus, respectively (Drews et al, 2007;

Fabre et al, 2013). They catalyze protein degradation through an ubiquitin-independent pathway, which still needs to be completely clarified (Stadtmueller & Hill, 2011; Kish-Trier & Hill, 2013).

Although the binding constants between the different 20S subtypes and its different RPs are not known, the binding mode between the a-ring and RPs has been established precisely by numerous structural studies and was found to be shared among species (Stadtmueller & Hill, 2011; Beck et al, 2012; da Fonseca et al, 2012; Lander et al, 2012; Lasker et al, 2012; Kish-Trier & Hill, 2013). In all cases, it involves the C-termini of RP subunits and a pocket at the interface between a-subunits. Recently, in-solution