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Structural Insights Into Substrate Recognition and Processing by the 20S Proteasome

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Structural Insights Into Substrate Recognition and Processing by the 20S Proteasome biomolecules Review Structural Insights into Substrate Recognition and Processing by the 20S Proteasome Indrajit Sahu * and Michael H. Glickman * Faculty of Biology, Technion-Israel Institute of Technology, 32000 Haifa, Israel * Correspondence: [email protected] (I.S.); [email protected] (M.H.G.); Tel.: +972-0586747499 (I.S.); +972-4-829-4552 (M.H.G.) Abstract: Four decades of proteasome research have yielded extensive information on ubiquitin- dependent proteolysis. The archetype of proteasomes is a 20S barrel-shaped complex that does not rely on ubiquitin as a degradation signal but can degrade substrates with a considerable unstructured stretch. Since roughly half of all proteasomes in most eukaryotic cells are free 20S complexes, ubiquitin-independent protein degradation may coexist with ubiquitin-dependent degradation by the highly regulated 26S proteasome. This article reviews recent advances in our understanding of the biochemical and structural features that underlie the proteolytic mechanism of 20S proteasomes. The two outer α-rings of 20S proteasomes provide a number of potential docking sites for loosely folded polypeptides. The binding of a substrate can induce asymmetric conformational changes, trigger gate opening, and initiate its own degradation through a protease-driven translocation mechanism. Consequently, the substrate translocates through two additional narrow apertures augmented by the β-catalytic active sites. The overall pulling force through the two annuli results in a protease-like unfolding of the substrate and subsequent proteolysis in the catalytic chamber. Although both proteasomes contain identical β-catalytic active sites, the differential translocation mechanisms yield distinct peptide products. Nonoverlapping substrate repertoires and product outcomes rationalize cohabitation of both proteasome complexes in cells. Citation: Sahu, I.; Glickman, M.H. Structural Insights into Substrate Keywords: 20S proteasome; protein degradation; intrinsically disordered proteins; enzyme func- Recognition and Processing by the tional cycle; peptides; peptidome; proteome; oxidative stress 20S Proteasome. Biomolecules 2021, 11, 148. https://doi.org/10.3390/ biom11020148 1. Introduction Academic Editor: Paolo Cascio Two major species of proteasome coexist in most cells: the 20S proteasome as a stan- Received: 3 January 2021 dalone complex, and the 20S complex as a core particle that is associated with regulatory Accepted: 21 January 2021 Published: 24 January 2021 particles or activators [1–3]. The 20S is a self-compartmentalized protease complex [4] that carefully selects substrates having substantiate disordered or misfolded stretches [5–7] Publisher’s Note: MDPI stays neutral and proteolyses them once they enter into the inner chamber. Under in vitro conditions with regard to jurisdictional claims in without any activators or associated unfoldase activities, the 20S proteasome proteolyses published maps and institutional affil- unstructured proteins or extended polypeptides [8–14]. Interestingly, archaea and some iations. bacteria have 20S proteasomes alongside other ATP-dependent proteases, which supports the idea of the 20S complex being a primordial protein-degrading machine [15]. Usually, these simple 20S proteasomes are made up of 14 copies of α and β subunits each [16,17], intermittently aided by a loosely associated homomeric ring of ATPases [15,18,19]. All β subunits in archaeal proteasomes are catalytically active proteases arranged in two con- Copyright: © 2021 by the authors. α Licensee MDPI, Basel, Switzerland. certed rings around an enclosed catalytic chamber, with the 14 subunits forming two This article is an open access article outer rings through which substrate enters. In contrast to archaea, 20S proteasomes in distributed under the terms and eukaryotes display greater complexity, with seven different types of α and β subunits each, conditions of the Creative Commons stacked in a four-ringed 20S complex. The symmetry mismatch between seven different Attribution (CC BY) license (https:// α subunits in each outer ring enables a gating mechanism, which provides a regulatory creativecommons.org/licenses/by/ system for substrate entry [20–23]. Regarding the β subunits, out of seven only three retain 4.0/). proteolytic activity in eukaryotes influencing the peptide product outcome [24]. Biomolecules 2021, 11, 148. https://doi.org/10.3390/biom11020148 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, x 2 of 16 ferent α subunits in each outer ring enables a gating mechanism, which provides a regu‐ latory system for substrate entry [20–23]. Regarding the β subunits, out of seven only three Biomolecules 2021, 11, 148 retain proteolytic activity in eukaryotes influencing the peptide product outcome2 of 15 [24]. In eukaryotic cells, 20S proteasomes function independently and act directly on dis‐ ordered proteins [25–29] or oxidized proteins [6,14,26,30,31], substantiating its potential role as a Instand eukaryotic‐alone cells, protease. 20S proteasomes Nevertheless, function its independentlyactivity is augmented and act directly by various on disor- activa‐ tors deredthat attach proteins to [25 the–29 ]20S or oxidized proteasome, proteins aiding [6,14,26 ,30substrate,31], substantiating recruitment its potential and processing, role therebyas a broadening stand-alone protease. its substrate Nevertheless, repertoire. its activity The is19S augmented regulatory by various particle activators (RP) is that the major activatorattach that to the enables 20S proteasome, the 20S aidingproteasome substrate to recruitment degrade virtually and processing, any protein thereby broad-tagged with ening its substrate repertoire. The 19S regulatory particle (RP) is the major activator that the small protein modifier ubiquitin [32,33]. Either one or two 19S RPs can attach to a enables the 20S proteasome to degrade virtually any protein tagged with the small protein singlemodifier 20S catalytic ubiquitin core [32 particle,33]. Either (CP) one to or form two 19Sthe RPs singly can capped attach to 26S, a single or the 20S doubly catalytic capped 30S, respectivelycore particle (CP) (Figure to form 1) the[34]. singly We cappedrefer readers 26S, or theto doublya number capped of recent 30S, respectively reviews on 26S proteasomes(Figure1)[ [35–40].34]. We refer Additional, readers to simpler, a number non of recent‐ATPase reviews activators on 26S proteasomes can also associate [35–40]. with 20S CPsAdditional, (e.g., PA200, simpler, PA28; non-ATPase [3,18,41–48]), activators although can also associatetheir mode with of 20S action CPs (e.g., in substrate PA200, deg‐ radationPA28; is [ 3vague,18,41– 48and]), althoughwill not theirbe detailed mode of in action this inreview. substrate degradation is vague and will not be detailed in this review. Figure 1.Figure Cellular 1. Cellular distribution distribution of different of different proteasome proteasome complexes complexes in mammalian cells. cells. The The 20S 20S model model figure figure was generated was generated by ChimeraXby ChimeraX using the using 20S the cryo 20S cryo-EM‐EM structure structure [49] [49].. The The averageaverage percentages percentages are calculatedare calculated based onbased published on published reports [3]. reports [3]. The sizeThe sizeof each of each proteasome proteasome species corresponds corresponds to the to averagethe average value ofvalue the range of the denoted range to denoted visually illustrate to visually their illustrate relative their relative abundanceabundance in in cell cell lines lines that that have have been been quantified. quantified. Although the 20S complex as a core particle is an integral part of all species of largerAlthough complex the 20S proteasomes complex in as eukaryotes a core particle [10,50 ],is itan is integral quite abundant part of asall a species free com- of larger complexplex inproteasomes many cell types, in eukaryotes estimated in [10,50], some studies it is quite at ~50% abundant of all proteasome as a free complex species [3 in] many cell types,(Figure estimated1). It has been in some suggested studies that at free ~50% 20S of complexes all proteasome may be species proteasome [3] (Figure assembly 1). It has been intermediates,suggested that 26S free breakdown 20S complexes products (duemay to be disassembly), proteasome or assembly stand-alone intermediates, proteolytic 26S enzymes [1,5–7,14,21,51–57]. Since the ratio of 20S to 26S proteasome varies across different breakdowncellular conditions,products a (due dynamic to equilibriumdisassembly), between or thestand two‐ speciesalone mayproteolytic be part of enzymes an adap- [1,5– 7,14,21,51–57].tive response Since to cellular the ratio needs of [20S58]. Althoughto 26S proteasome its functionality varies as across an independently different cellular active con‐ ditions,enzyme a dynamic under cellular equilibrium conditions between is a matter the of two debate, species recent may advances be part have of highlighted an adaptive re‐ sponsea role to forcellular 20S as needs a functional [58]. proteasomeAlthough inits cells. functionality Various reports as an suggest independently a role of 20S active as en‐ zymean under emergency cellular proteasome conditions under is a cellular matter stress, of debate, for example recent oxidative advances stress have or hypoxic highlighted a role forstress 20S [25 as,49 a, 56functional,59–61], to proteasome provide survival in cells. benefits Various under proteotoxicity reports
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