biomolecules Review PA28γ: New Insights on an Ancient Proteasome Activator Paolo Cascio Department of Veterinary Sciences, University of Turin, Largo P. Braccini 2, 10095 Grugliasco, Italy; [email protected] Abstract: PA28 (also known as 11S, REG or PSME) is a family of proteasome regulators whose members are widely present in many of the eukaryotic supergroups. In jawed vertebrates they are represented by three paralogs, PA28α, PA28β, and PA28γ, which assemble as heptameric hetero (PA28αβ) or homo (PA28γ) rings on one or both extremities of the 20S proteasome cylindrical structure. While they share high sequence and structural similarities, the three isoforms significantly differ in terms of their biochemical and biological properties. In fact, PA28α and PA28β seem to have appeared more recently and to have evolved very rapidly to perform new functions that are specifically aimed at optimizing the process of MHC class I antigen presentation. In line with this, PA28αβ favors release of peptide products by proteasomes and is particularly suited to support adaptive immune responses without, however, affecting hydrolysis rates of protein substrates. On the contrary, PA28γ seems to be a slow-evolving gene that is most similar to the common ancestor of the PA28 activators family, and very likely retains its original functions. Notably, PA28γ has a prevalent nuclear localization and is involved in the regulation of several essential cellular processes including cell growth and proliferation, apoptosis, chromatin structure and organization, and response to DNA damage. In striking contrast with the activity of PA28αβ, most of these diverse biological functions of PA28γ seem to depend on its ability to markedly enhance degradation rates of regulatory protein by 20S proteasome. The present review will focus on the molecular mechanisms and biochemical properties of PA28γ, which are likely to account for its various and complex biological functions and highlight the common features with the PA28αβ paralog. Citation: Cascio, P. PA28γ: New Insights on an Ancient Proteasome Keywords: PA28γ; PA28αβ; proteasome; proteasome activator; proteasome gate; protein degrada- Activator. Biomolecules 2021, 11, 228. tion; ATP-independent proteolysis; ubiquitin–proteasome system (UPS); intrinsically disordered https://doi.org/10.3390/ proteins (IDPs); proteostasis biom11020228 Academic Editor: Shigeo Murata Received: 2 January 2021 1. The Ubiquitin–Proteasome System (UPS) Accepted: 3 February 2021 Published: 5 February 2021 In living organisms, proteins are constantly subject to synthesis and degradation processes that are aimed at rapidly adapting the proteome of the cell to any change Publisher’s Note: MDPI stays neutral in its metabolic and physiological needs, and to guarantee the maintenance of cellular with regard to jurisdictional claims in homeostasis challenged by exogenous and endogenous stimuli and stresses. In particular, published maps and institutional affil- each cell needs to remove proteins whose function is no longer required at a specific time iations. (e.g., regulatory proteins or transcription factors), damaged proteins, covalently modified (e.g., oxidized) or presenting any other alteration or modification that could be potentially dangerous or toxic, propeptides from inactive precursors, and proteins from which to obtain amino acids to be used as an energy source in case of insufficient caloric intake [1]. In eukaryotic cells, the large majority of intracellular proteins are hydrolyzed by the 26S Copyright: © 2021 by the author. Licensee MDPI, Basel, Switzerland. proteasome, a large (2.4 MDa) multimeric protease abundantly expressed in the nucleus This article is an open access article and cytosol [2]. The central core of the 26S proteasome consists of the relatively latent distributed under the terms and 20S proteolytic particle, at the two free ends of which may associate various regulatory conditions of the Creative Commons complexes that perform the task of controlling and modulating the functions of the protease Attribution (CC BY) license (https:// in different ways [3]. The 20S proteasome, whose internal cavity harbors proteolytic sites, creativecommons.org/licenses/by/ is the central core of this proteolytic macromolecular machine: it has a cylindrical structure 4.0/). with a molecular weight of ~700 kDa and is formed by four overlapping rings of seven Biomolecules 2021, 11, 228. https://doi.org/10.3390/biom11020228 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, x FOR PEER REVIEW 2 of 23 Biomolecules 2021, 11, 228 2 of 23 a molecular weight of ~700 kDa and is formed by four overlapping rings of seven subunits easubunitsch. The two each. outer The tworings outer are constituted rings are constituted of α subunits, of α andsubunits, the two and inner the twoones innerof β sub- ones unitsof β subunits[4]. The active [4]. The catalytic active catalyticsites of the sites constitutive of the constitutive 20S proteasomes 20S proteasomes are located are locatedon the β1,on β2, the andβ1, ββ52, subuni and βts5 (Figure subunits 1). (Figure In principle,1). In proteasomes principle, proteasomes can hydrolyze can the hydrolyze C-terminal the amideC-terminal bond amideof any amino bond of acid any except amino proline. acid except However, proline. proteolytic However, activities proteolytic assessed activities by shortassessed fluorogenic by short peptides fluorogenic have peptides identified have three identified well-defined three well-definedhydrolyzing preferences: hydrolyzing trpreferences:ypsin-like (i.e. trypsin-like, hydrolysis (i.e., at the hydrolysis C-terminus at the of C-terminus basic residues, of basic performed residues, by performed β2 subunit), by chymotrypsinβ2 subunit), chymotrypsin-like-like (i.e., hydrolysis (i.e., at hydrolysisthe C-terminus at the of C-terminus hydrophobic of hydrophobicresidues, performed residues, byperformed β5 subunit), by andβ5 subunit), caspase-like and (i.e. caspase-like, hydrolysis (i.e., at the hydrolysis C-terminus at the of C-terminusacidic residues, of acidic per- formedresidues, by performedβ1 subunit) by [5]β. 1 subunit) [5]. x 90 α5 α4 20S Proteasome α6 α3 (Side view) α7 α2 Heptameric α-ring α1 β5 x 90 β6 CT-L β4 β7 β3 C-L T-L Heptameric β-ring β1 β2 FigureFigure 1. 1. SchematicSchematic structure structure of of 20S 20S proteasome. proteasome. Proteolytic Proteolytic active active sites sites of of β1,β1, β2β,2, and and β5β 5subunits subunits are are depicted depicted as as scissors. scissors. CC-L:-L: caspase caspase-like;-like; T T-L:-L: trypsin trypsin-like;-like; CT CT-L:-L: chymotrypsin chymotrypsin-like.-like. UnderUnder the the stimulus stimulus of of γγ-interferon-interferon (INF (INF--γ)γ )or or other other pro pro-inflammatory-inflammatory cytokines, cytokines, new new catalyticcatalytic subunits subunits are are synthesized synthesized (β1i, (β1i, β2i,β2i, and and β5i)β5i) that that replace replace the the constitutive constitutive one one to to formform newly newly assembled assembled 20S 20S immunoproteasomes immunoproteasomes [6] [6.]. Experiments Experiments with with small small fluorogenic fluorogenic peptidespeptides demonstrated demonstrated that that immunoproteasomes immunoproteasomes possess possess a agreater greater ability ability to to hydrolyze hydrolyze afterafter hydrophobic hydrophobic amino acidsacids andand a a reduced reduced capacity capacity to cleaveto cleave after after acidic acidic residues, residue whiles, whileconflicting conflicting results results are reported are reported concerning concerning its capacity its capacity to cleave to cleave after after basic basic residues residues [7,8]. Consequently, peptides produced by immunoproteasomes are expected to have a greater [7,8]. Consequently, peptides produced by immunoproteasomes are expected to have a amount of hydrophobic and a lower amount of negatively charged C-termini, which fa- greater amount of hydrophobic and a lower amount of negatively charged C-termini, vors uptake by TAP transporters and tight binding to MHC class I molecules [9]. Crystal which favors uptake by TAP transporters and tight binding to MHC class I molecules [9]. structures of the mouse constitutive and immuno 20S proteasomes revealed differences in Crystal structures of the mouse constitutive and immuno 20S proteasomes revealed dif- the substrate-specificity pockets between the catalytic constitutive and immuno β subunits, ferences in the substrate-specificity pockets between the catalytic constitutive and im- which largely provide an explanation for the observed differences in cleavage specifici- muno β subunits, which largely provide an explanation for the observed differences in ties [10]. In fact, whereas the substrate pocket of β1 accommodates an acidic P1 residue, cleavage specificities [10]. In fact, whereas the substrate pocket of β1 accommodates an that of β1i interacts with a small, hydrophobic P1 residue. Moreover, the active sites of both acidic P1 residue, that of β1i interacts with a small, hydrophobic P1 residue. Moreover, β5 and β5i are surrounded by nonpolar environments, but the pocket of β5i is significantly the active sites of both β5 and β5i are surrounded by nonpolar environments, but the larger than that of β5, thus enabling accommodation of a bulky, hydrophobic P1 residue. pocket of β5i is significantly larger than that of β5, thus enabling accommodation of a Finally, the substrate-binding pockets of the mouse β2 and β2i subunits are essentially bulky,identical, hydrophobic