Tuning the proteasome to brighten the end of the journey.

Am J Physiol Cell Physiol 311: C793–C804, 2016. First published September 7, 2016; doi:10.1152/ajpcell.00198.2016.

Themes

Tuning the proteasome to brighten the end of the journey Thibault Mayor,1* Michal Sharon,2 and Michael H. Glickman3 1

Department of Biochemistry and Molecular Biology, Michael Smith Laboratories, University of British Columbia, Vancouver, Canada; 2Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot, Israel; and 3Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel Submitted 5 July 2016; accepted in final form 4 September 2016

proteasome; proteolysis; ubiquitin; UPS

PROTEIN HOMEOSTASIS, also called proteostasis, is maintained by coordinating protein production, folding, and degradation to keep the proteome in a functional state. The ubiquitin proteasome system (UPS) is the primary proteolytic path for shortlived proteins in the nucleus and cytosol of eukaryotic cells. Regulation of the UPS relies on complex protein networks that typically lead to the ubiquitination of the targeted polypeptides that are then recognized and hydrolyzed by the proteasome. Ubiquitination involves the formation of an isopeptide bond between a lysine residue of the substrate (in most cases) and the C-terminal end of ubiquitin, a short 76 amino acid protein highly conserved in eukaryotes. The substrate modification hinges on the ATP-dependent E1 activating enzyme that transfers an activated ubiquitin to one of the E2 conjugating enzymes, which then mediates ubiquitination with the help of an E3 ligase (59, 88, 113, 134, 144). The modification can be reversed or altered by deubiquitinases, which are ubiquitinspecific proteases. Encoded in the human genome, there are two ubiquitin E1 enzymes, an estimated 35 E2s, over 600 different E3s, and 80 deubiquitinases. Since ubiquitination is a highly selective as well as a dynamic and tightly regulated process, it is often thought to exert most of the control over the UPS. However, a number of recent studies have illuminated how regulation of the proteasome, such as by post translation modifications (PTMs), is important for proteostasis. In addition, inhibition of the proteasome can be effective at selectively impairing cancer cells. As such, there are currently three

* This review article is part of a Theme series: The Control of the Proteostasis Network in Health and Diseases. Address for reprint requests and other correspondence: T. Mayor, Dept. of Biochemistry and Molecular Biology, Michael Smith Laboratories, Univ. of British Columbia, Vancouver, BC, Canada (e-mail: [email protected]). http://www.ajpcell.org

approved small chemicals (bortezombid, carfilzombid, and ixazomib) to treat relapsed multiple myeloma and mantle cell lymphoma. Therefore, modulation of the proteasome activity could be used in therapeutics to remedy or further precipitate an imbalanced protein homeostasis. The 26S Proteasome: A Formidable Molecular Machinery That “Grinds” Targeted Proteins The proteasome is invariably composed of one barrel-shaped core particle (CP), also known as the 20S complex, in which resides the proteolytic activity (80). The 20S CP is composed of four stacked heptameric rings with a central cavity. In eukaryotic cells, the two outer rings contain seven different but related ␣-subunits, whereas the two inner rings contain seven related ␤-subunits. Three of the ␤-subunits contain active protease sites with different peptidase activities: caspase-like post acidic (␤1), trypsin-like post basic (␤2), and chymotrypsin-like post hydrophobic (␤5). These active sites are located inside the cavity of the 20S CP. The N-terminal ends of the ␣-subunits form a gate that precludes access to the proteolytic active sites within the proteolytic chamber in the closed state. Opening of the gate, which permits the entry of unfolded polypeptides through the narrow channel, is promoted by the docking of proteasome activators over the ␣-ring. The main activator is the 19S regulatory particle (RP, also known as PA700) that binds to the 20S CP to form the 26S proteasome. The holoenzyme can be either singly or doubly capped, if one or both sides of the CP are occupied, respectively. The 19S RP is composed of 19 different subunits that can dissociate into two subcomplexes: the base and the lid (see also Fig. 1B). We witnessed a formidable advancement of our understanding of the RP modus operandi ever since a few years

0363-6143/16 Copyright © 2016 the American Physiological Society

C793

Downloaded from http://ajpcell.physiology.org/ by 10.220.33.2 on April 6, 2017

Mayor T, Sharon M, Glickman MH. Tuning the proteasome to brighten the end of the journey. Am J Physiol Cell Physiol 311: C793–C804, 2016. First published September 7, 2016; doi:10.1152/ajpcell.00198.2016.—Degradation by the proteasome is the fate for a large portion of cellular proteins, and it plays a major role in maintaining protein homeostasis, as well as in regulating many cellular processes like cell cycle progression. A decrease in proteasome activity has been linked to aging and several age-related neurodegenerative pathologies and highlights the importance of the ubiquitin proteasome system regulation. While the proteasome has been traditionally viewed as a constitutive element of proteolysis, major studies have highlighted how different regulatory mechanisms can impact its activity. Importantly, alterations of proteasomal activity may have major impacts for its function and in therapeutics. On one hand, increasing proteasome activity could be beneficial to prevent the age-related downfall of protein homeostasis, whereas inhibiting or reducing its activity can prevent the proliferation of cancer cells.

Themes C794

PROTEASOME REGULATION AND PROTEOLYSIS

A PSGs

Starvation

Oxidative Stress

Disassembly

Blm10

lid

S-glutathion. α-subunit (Cys)

base

DJ1

20S CP

Nrf2 NQO1

Degradation Disassembly 19S RP ubiquitin-dependent degradation

B

ubiquitin-independent degradation

DYRK2 (Kin) CaMKII (Kin) Rpt3(T25) Rpt6(S120) Rsp5 (E3) Rpn10(K84)

Ube3C (E3) Rpn13(K21/34?)

Base

Rpn11 Rpn

Lid

Rpn1 ATPases PKA (Kin) Rpn6(S14)

20S

ago when the resolution of cryoelectron microscopy improved to allow the determination of the RP architecture and subunit arrangement (20, 63, 65). The base of the RP consists of a hexameric complex composed of six AAA⫹ ATPases (Rpt1-6) that form two superimposed rings to establish a pore. Since substrates can only pass in an extended polypeptide state, the six ATPase subunits coordinate ATP-dependent conformational changes to unfold the protein and translocate it through the pore (7). In addition to the hexameric ATPase complex, the base of the RP also contains the Rpn1, Rpn2, and Rpn13 scaffolding subunits. Notably, both Rpn1 and Rpn13 (as well as Rpn10 lid subunit) contain domains that bind to ubiquitin or ubiquitin shuttling receptors, and thereby participate in substrate recognition and anchoring during the unfolding process (48, 115). The lid structure of the RP is closely related to the COP9 signalosome (CSN) and eukaryotic initiation factor 3 (eIF3) complexes. The lid is laterally bound to partially cover the base, as well as contact the side of the CP. Six of the lid subunits (Rpn3, 5, 6, 7, 9, and 12) assemble into a scaffold that is shaped as a horseshoe with radiating protrusions (35), and holds the Rpn8-Rpn11 core catalytic heterodimer on its inner side. Rpn11 is a metalloprotease that serves as a deubiquitinase that cleaves ubiquitin chains from proteasome substrates (130, 143). At the periphery of the scaffold, the Rpn10/S5a ubiquitin binding receptor is associated to Rpn9. There are also numerous proteasome-associated proteins, many binding to the RP like the shuttling ubiquitin receptors Rad23, Dsk2, and Ddi1,

c-Abl (Kin) α4(Y151)

as well as Ubp6/Usp14 and mammalian Uch37 deubiquitinases that assist with the targeting of substrates and modulate proteasome activity. Binding, unfolding, translocation, and deubiquitination of proteasome targets by the RP are tightly connected activities that ensure an efficient processing of the substrates. The substrate has to be deubiquitinated to facilitate its threading into the channel formed by the ATPase subunits of the base, and then into the CP. One current model is that an incoming ubiquitinated substrate is held by one of the ubiquitin-binding receptors (Rpn1, 10, or 13) so that the proximal ubiquitin can be cleaved by Rpn11. The distance between the Rpn11 active site and the ubiquitin receptors likely explains why substrates either conjugated to a chain containing at least four ubiquitin moieties or multiubiquitinated (i.e., conjugated on multiple sites) are more efficiently degraded by the proteasome (58, 118). The challenge is that once a substrate is deubiquitinated, it loses its anchorage to the proteasome. Therefore, Rpn11 action is timed to prevent the premature substrate release and acts late in the catalytic cycle, after the ATPase engagement and alignment of the substrate in the catalytic pore (21, 82, 98, 140). The presence of an unstructured domain on the substrate near the site conjugated to the ubiquitin chain also greatly increases substrate processivity (51). As a consequence of unfolding, the unstructured substrate domains presumably form additional contact sites with the aromatic hydrophobic loops that protrude from the ATPase domains in the RP central

AJP-Cell Physiol • doi:10.1152/ajpcell.00198.2016 • www.ajpcell.org


Fig. 1. A: schematic representation of the 26S disassembly prior to the formation of proteasome storage granules (PSGs) and upon oxidative stress. Dotted arrows represent hypothetical paths and gray arrows regulatory mechanisms. B: schematic representation of a singly capped 26S proteasome and major regulatory mechanisms reviewed here that are mediated by kinases (kin) or E3-ubiquitin ligase (E3).

Themes PROTEASOME REGULATION AND PROTEOLYSIS

Aging and the Downfall of Proteostasis A lower proteolytic capacity of the proteasome has been linked to cellular aging and several age-related neurodegenerative diseases. Reduced proteasome activity has been observed in numerous mammalian tissues (e.g., rat liver, heart, and brain cortex) and human cell types (epidermal and lymphocytes) derived from older organisms (12, 13, 15, 43, 55). Notably, a decrease in proteasome activity has been associated with oxidation, which occurs to a higher degree on 20S subunits upon aging, leading to both glycation and lipid peroxidation (15). In contrast, increased proteasome activity has been observed in human centenarians (18). In addition to these observations, genetic studies also support a causal link between proteolysis and aging. Reduction of proteasome activity by either replacing one of the CP peptidases with a less active variant or knocking out the REG␥ (also known as the S11 regulatory particle that in addition to the 19S also regulates the CP) induced a decrease of lifespan in mouse (72, 120). Increasing expression of proteasome subunits leads to a longer replicative lifespan in yeast (61). Remarkably, upregulation of the Rpn6

lid subunit in Caenorhabditis elegans was sufficient to cause a higher proteasome activity and extend lifespan in conditions that promote proteotoxic stresses (e.g., heat shock or elevated oxidation) (133). Taken together, these studies strongly indicate that cellular proteolytic activity is a key limiting factor to lifespan. Therefore, it is important to determine how regulation of the proteasome may impact protein homeostasis. In this review, we will focus on recent studies that illustrate how proteasome levels are brought upon and how its activity is regulated by posttranslational mechanisms. The Proteasome Life Cycle Proteasome biogenesis: a concerted effort. The ability of cells to promptly enhance proteasome activity is vital to handle conditions that require rapid turnover of proteins. Indeed, rapid elevation of proteasomes is observed in response to proteotoxic stress (such as heat shock, oxidative, unfolded protein response) or upon proteasome inhibition. The concerted expression of all proteasome subunits requires the presence of master transcription factors. In yeast, the stress-induced transcription factor Rpn4 regulates the combined expression of proteasome subunits by recognizing a nonamer proteasome-associated control element (PACE) on the promoter regions (141). It is now evident that proteasome chaperones and all components needed to build proteasome complexes de novo are also upregulated by Rpn4 responding to a similar element to PACE (116). Rpn4 is also a proteasome substrate thus its levels are under tight regulation and self-limiting; when the cellular proteolytic capacity is sufficient, Rpn4 levels are low. Consequently, stabilization of Rpn4 feeds-forward to elevated expression of proteasome-encoding genes. In multicellular organisms, nuclear respiratory factor 1 (Nrf1) and nuclear factor (erythroid-derived)-like 2 (Nrf2) appear to be Rpn4 counterparts in mammalian cells that activate their target genes through a common cis-acting element named ARE (antioxidant response element). Similar to Rpn4, Nrf1 induces proteasome expression under conditions where proteolysis is insufficient (104, 105). Nrf2 is a general regulator of the oxidative stress response that induces expression of genes encoding both antioxidant enzymes and stress response proteins, including proteasome subunits, providing cells with a strategy to overcome bouts of oxidation assaults (62, 147). However, recent work suggests that Nrf2 may preferably induce the expression of 20S subunits (see also the section below, Dining Without Ubiquitin: The Attention Is on the 20S) (102, 103). Response to proteotoxicity is also highly regulated in plants, with the proteasome partaking in alleviating the stress. A heterodimeric transcription factor in Arabidopsis, NAC53-NAC78, induces expression of proteasome subunits alongside ubiquitination enzymes, chaperones, autophagy components, and oxidation detoxification enzymes, through a consensus cis-element in their promotors (37). Activation of these transcription factors may be a strategy to overcome the proteolysis defects related to proteotoxic-related diseases. Indeed, overexpression of the SKN-1 transcription factor, the Nrf orthologue in worm, leads to an increased lifespan through coordinated induction of proteasome subunits (17, 74). Remarkably, in one case, induction of a single proteasomal subunit, Rpn6, also led to higher proteasome activity (132, 133), a somewhat baffling phenomenon that may allude to a



channel to both increase substrate affinity and initiate translocation, similar to what was observed with the prokaryotic ClpA and ClpX AAA⫹ proteolytic unfoldases (45, 85). The presence of multiple RP subunits that can bind to either ubiquitin and ubiquitin shuttling receptors could allow the substrate to be arranged in different conformations until it is favorably presented for translocation. As Rpn11 activity is coupled to the RP ATPase activity (130), the deubiquitinase only cleaves off ubiquitin chains once unfolding and translocation is initiated by the ATPases, which can then hold the substrate immobilized to the proteasome. Structural analyses by cryoelectron microscopy offered additional clues on how these two activities are coordinated, based on major conformational rearrangements of the RP in the presence of substrates. When proteasomes are incubated with substrates or ATP-␥-S that hydrolyzes more slowly, the two rings formed by the six ATPases are aligned with the axis formed by the CP cylinder (87, 117). In contrast, in an alternative state (referred as the S1, apo or ground state), which is obtained in the presence of ATP but in the absence of substrates, the two rings are not aligned with the CP, preventing access to the 20S cavity. Importantly, in the presence of substrates or ATP-␥-S, the position of Rpn11 is shifted in the active state so that its catalytic site and ubiquitin binding interface are accessible and ideally positioned near the entrance of the pore (87, 127). These studies show how structural rearrangement could help coordinate the different events during substrate processing. Presence of these different structural states enabled Baumeister and colleagues to analyze in situ the distribution of the different proteasome populations in neuronal cells (4). Their work indicates that three quarters of the assembled 26S were in the ground state and therefore not engaged with substrates, suggesting that proteasomes are supernumerary and thereby not limiting the degradation of substrates in these conditions. However, it should be noted that these analyses cannot yet distinguish between proteasomes that are “competent” versus proteasomes that are potentially less active or perhaps compromised due to PTMs and presence of additional factors that could either promote or inhibit its proteolytic activity.

C795

Themes C796


narrative [the reader is referred to detailed reviews and references to original findings therein (39, 111, 122)], four chaperones accompany three pairs of ATPase subunits to assemble the hexameric ring. Specifically, Hsm3 associates with Rpt1Rpt2 (and Rpn1), Nas2 with Rpt4-Rpt5, while both Rpn14 and Nas6 chaperones associate with Rpt3-Rpt6. By covering the C-termini of the ATPase subunits, these chaperones not only aid pairwise assembly, but also inhibit premature attachment of partially assembled bases to the 20S (96). Active dissociation of the four chaperones is triggered by nucleotide hydrolysis in the newly formed ATPase ring (56, 96). The last step is probably expedited by the attachment of the lid, giving precedence for a fully assembled 19S over only the base. Under stress conditions, Adc17 is an additional upregulated chaperone protein that assists the formation of the Rpt6-Rpt3 ATPase pair in yeast (42). Remarkably, the Mpk1 Map kinase, which is negatively regulated by mammalian target of rapamycin (mTOR), is required to increase levels of Adc17 and four other RP base assembly chaperones (a.k.a. Nas2, Nas6, Hsm3, and Rpn14) in a posttranscriptional manner (109). In this case, inhibition of target of rapamycin complex 1 (TORC1) is sufficient to induce higher levels of 26S proteasome activity. This evolutionarily conserved pathway (from yeast to mammals) links the regulation of proteasome abundance to the mTOR kinase that thereby controls both the two major proteolytic systems in the cell (the proteasome and autophagy). In general terms, the RP-lid is congealed from two subcomplexes, module 1 (Rpn5, Rpn6, Rpn8, Rpn9, and Rpn11) and module 2 (Rpn3, Rpn7, and Rpn12). Module 1 self-assembles piecemeal, is competent to attach to the previously assembled base-CP in vitro, and has been identified bound to base-CP in vivo (145), or associates first to module 2 subunit. In contrast to module 1, for which no chaperone has been identified, Sem1/DSS1 tethers Rpn3 with Rpn7, which can associate with module 1 (121). Lastly, attachment of Rpn12 alters conformation of Rpn6 rendering it more fit to fasten the lid and base together, and eventually tether 19S RP to 20S CP (123). Rpn6 proteasome subunit was shown by cryoelectron microscopy to span the lid, base, and 20S CP (35, 97, 127) and likely plays a key role in regulating proteasome activity levels in the cell (see also later section related to the work from Duff and Goldberg labs). It remains unclear whether chaperones are needed to bring proteasome subcomplexes together, especially following proteasome disassembly. At least in vitro, the 20S and 19S readily snap together without exogenous factors (76, 78). This property is physiologically relevant, since the 19S RP and 20S CP are also generated from disassembled 26S proteasomes. The dynamic equilibrium between 26S holoenzymes, and stable 19S and 20S entities, apparently responds to cellular proteolytic needs, as examined in more detail in the following section. Proteasome disassembly and storage, for possible disposal. Proteasomal localization is dynamic and recent studies show how congregation of proteasomes may be regulated to control cell proteolytic activity. A large portion of proteasomes are nuclear (particularly prominent in yeast but common to many eukaryotic cell types), although they are present throughout the cytosol and next to membranes (ER, mitochondria, etc.) (33). Shifting populations between the cytosol and nucleus may respond to local proteolytic needs. For instance, N-myristoyl-



pool of excess free proteasome subunits harnessed for rapid deployment. In the reverse direction, a precedent observation demonstrated that proteasome levels can be subject to endogenous regulation through miR-101, which decreases proteasome biogenesis (148). Chaperone-assisted assembly of the proteasome puzzle. With a machine as intricate as the 26S proteasome, great care must be taken to ensure proper timing of subunit incorporation. The task is especially delicate as many subunits are close paralogs of each other and have to be properly inserted despite their structural and sequence similarities. At least 10 different chaperones aid in 26S proteasome assembly (39, 111, 122). Nevertheless, none of the 10 proteasome chaperones documented so far are essential in yeast grown under typical laboratory conditions (“log phase”), indicating that their role is likely to facilitate or direct self-assembly rather than to serve as mandatory assembly factors. For this reason, there are likely alternate routes that lead to the assembly of the 26S proteasome holoenzyme. Five chaperones aid in the assembly and maturation of the 20S barrel. PAC1-PAC2 forge an ␣-ring out of seven different ␣-subunits, while PAC3-PAC4 and Ump1 order the seven ␤-subunits onto the ␣-ring template (chaperone nomenclature is somewhat muddled, with PAC1-4 also known as Poc1-4, Pba1-4, or Dmp1-4, whereas Ump1 is named POMP in humans). PAC3-PAC4 are released prior to incorporation of the last ␤-subunit (pro-␤7) and, only then, two half-CPs (␣7-␤7) can snap together. Ump1 remains trapped inside the proteolytic chamber to be then degraded after the activation of the proteases, which occurs after the cleavage of the N-terminal domains of the ␤1-, ␤2-, and ␤5-subunits (which then reveals the threonine nucleophile). Maturation of these ␤-subunits sites also induces a conformational change in the ␣-ring, reducing the affinity of PAC1-PAC2. The ␣-ring surface is now available for 19S RP to bind (137). During the chaperone-mediated assembly of the CP, a different composition of ␣-subunits can lead to alternate 20S CP with a second ␣4 (PSMA7) instead of a missing ␣3 (PSMA4) (95). That composition change, which results in a less tightened gate, could possibly generate a wider range of peptide products, providing a potential evolutionary advantage. An interesting observation is that iRhom, an endoplasmic reticulum (ER)-resident rhomboid protease induced by unfolded protein response (UPR) stress, was shown to physically associate with 20S CP chaperones to stimulate proteasome assembly in the vicinity of the ER (69). The 19S RP, being asymmetric and more diverse in its makeup than the 20S CP, poses greater challenges to de novo assembly. It is unclear whether there is a unique or preferred assembly path for the 19S RP. The assembly process appears to be incredibly efficient with few free subunits or intermediate subcomplexes detected (at least in dividing yeast cells) (94). To infer the assembly order, most studies relied on mutations to isolate transitory intermediates, although illuminating, these somewhat artificial bottlenecks could themselves influence the preferred assembly path. One current model is that the 19S RP assembles to completion and then attaches to the 20S CP as an intact unit. Alternatively, there could be a bottom-up assembly of 19S (base to lid) onto the 20S ␣-ring surface. So far, five chaperones dedicated to the RP assembly have been identified: Nas2/p27, Nas6/p28, Hsm3/S5b, and Rpn14/PAAF and Sem1/ DSS1 (yeast/mammals, respectively). In a severely abridged


Dining Without Ubiquitin: The Attention Is On the 20S Most of the proteins are selectively targeted for degradation through ubiquitin tagging. However, there are multiple examples of ubiquitin-independent proteolysis, which are not exceptions but reflect that ubiquitination is not an exclusive prerequisite for degradation by proteasomes (9, 34). For instance, the presence of the 19S regulatory particle is not mandatory for the degradation of partially unfolded proteins that can be mediated by the 20S proteasome complex alone. In fact, the two alternative proteasomal degradation mechanisms, ubiquitin-dependent and ubiquitin-independent, are not mutually exclusive, and different populations of the same protein can be sent to degradation via either pathway (5). Substrates of the 20S proteasome are made up of proteins that have become partially or completely unfolded due to aging, mutations, or oxidation (2, 9, 101). Native proteins containing large unstructured segments (⬎30 amino acids in length), or with entirely disordered sequences, are also susceptible to degradation by the 20S proteasome (9). This latter group of substrates is dominated by key regulatory and signaling proteins with essential roles in cell cycle progression, oncogenesis, and age-related pathologies. For example, proteins such as tumor suppressor p53 (5), cell cycle regulator p21 (73, 114), and the neurodegenerative disease-related proteins tau (14, 22) and ␣-synuclein (119) have been shown to be substrates of the 20S proteasome. Considering that 44% of human protein-coding genes contain intrinsically disordered regions (128), it is reasonable to assume that the number of proteins recognized as substrates of the ubiquitin-independent pathway will continue to increase in the future. The biological relevance of this degradation pathway has been ascertained by several recent findings. Firstly, a large portion of proteasomes in mammalian tissue culture cells were found to be uncapped 20S proteasomes in a study in which cross-linking was employed prior to the biochemical purification and mass spectrometry analysis of proteasomes (36). Secondly, at least 20% of cellular proteins were shown to undergo 20S proteasomal cleavage (8), and the number of specific proteins known to undergo in vivo degradation by the 20S proteasome is increasing (49). Moreover, under oxidizing conditions, the 20S proteasome was identified as the major degradation machinery, while ubiquitination enzymes may be more sensitive redox conditions (101). Under these conditions, the capacity of the 20S degradation route can be enhanced by multiple processes. The first leads to a 20S proteasome level increase through disassembly of the 26S proteasome into its 20S and 19S components (Fig. 1A) (38, 78). In the mammalian system, the chaperone Hsp70 is involved in this process by stabilizing the 19S particle after its dissociation from the 20S proteasome, and enhancing the reassembly of functional 26S proteasomes once the oxidative stress is eliminated (38). In yeast, H2O2-induced disassembly of 26S proteasomes is assisted by both the proteasome-associated protein Ecm29 (136) and the Hsp90 chaperone (50). In addition, for the 20S to be remarkably resilient, as a protein complex, to oxidative environments (78), oxidation may actually facilitate access to its active sites resulting in enhanced proteolytic capacity (24). S-glutathionylation of cysteines on ␣-subunits may protect the proteasome from oxidative damage, simultaneously opening a gate into the internal proteolytic chamber thereby increasing



ation of Rpt2 has been shown to increase the abundance of 26S proteasomes in nuclei (57). In quiescent cells, proteasomes tend to congregate into proteasome storage granules (PSGs), found primarily next to ER/nuclear membranes (64). Entry of proteasome components into PSGs appears to be reversible, sequestering reservoirs for future use. Young cells are more efficient at relocalizing proteasomes into PSGs than older cells, suggesting a defensive role for PSGs in long-term survivable (129). Disassembly of 26S proteasomes into 20S CP and 19S RP tends to correlate with entry into PSGs (135, 138) (Fig. 1A). Instead of 19S RP, an alternative cap, Blm10 (similar to PA200 in mammals) facilitates the localization of 20S CP into PSGs (138). Importantly, observations under different conditions could lead to PSGs with variable functions or properties. Despite some similarities, PSGs are distinct from deposits of insoluble proteins (100). In some cases, misfolded or damaged proteasome subunits are localized to insoluble protein deposits aided by chaperones such as heat shock protein 42 (Hsp42) (99), which might be distinct from PSGs. Even so, persistent nutrient deprivation or prolonged proteasome inactivity does seem to culminate with selective proteasome autophagy (“proteaphagy”; see below) (83, 135); how this elimination relates to PSGs is unclear and should be further investigated. Long-term starvation induces dissociation of 26S proteasomes into 20S CP and 19S RP, which generally appear as intact complexes, though their long-term fate—stable or eventually removed— can be dependent on environment and health of cells (6, 129, 135). Recent work from the Vierstra lab showed that, in plants, polyubiquitination of proteasomes stimulated by inactivity and nutrient starvation may trigger removal by autophagy (83, 84). In this case, selected autophagy is mediated by none other than Rpn10, one of the ubiquitin receptors resident in the proteasome itself. Independently, 26S proteasome holoenzymes also tend to dissociate into intact and stable 20S CP and 19S RP subcomplexes when exposed to short periods of oxidation (78). The two processes may be linked through mitochondria health and alterations in metabolic activity. Indicative of a reversible equilibrium of 26S holocomplexes with 19S RP and 20S CP, 26S proteasomes rapidly reform from its subcomplexes in vitro, or in vivo upon resumption of standard growth conditions (6, 78). As PSGs have been studied primarily in yeast, expanding these studies to mammalian cells, tissues, or animal models would require transposing conditions accordingly to illuminate specific conditions and their relative importance for survival during starvation-induced quiescence. Different signals for sequestering 20S CP and 19S RP in PSGs or sending them to autophagy may serve not just to downregulate overall proteolytic activity, but to also alter the ratio of 19S RP to 20S CP, thereby increasing cellular levels of free 20S CP or even those of asymmetric singly capped proteasomes over the symmetric doubly capped proteasomes. It is often suggested that 20S CP, with or without ATPaseindependent regulator caps such as PA200/Blm10 and S11 (a heptameric structure also known as PA28 or REG), may participate in ubiquitin-independent proteolysis (106) (see next section). Therefore, 26S proteasome dissociation may attenuate ubiquitin-dependent turnover, yet at the same time, increase ubiquitin-independent proteolysis of misfolded or damaged proteins by the ATP-frugal 20S CP.

C797

Themes C798


Tweaking Proteasome Activity Numerous PTMs on the proteasome have been reported, including nearly 450 phosphorylation sites on subunits of the human 26S proteasome that are listed in the PhosphoSite.org database at the time of writing this review. In addition, numerous other modifications on proteasomal subunits, such as ubiquitination and acetylation, have been reported and compiled in recent reviews (19, 46). A major challenge is to infer which of these modifications impact the activity or function of the proteasome and thus may be implicated in diseases. Fortunately, several recent studies have begun to shed some light on how the proteasome machinery is regulated by PTMs. In

this section of the review, we will summarize recent studies that have deepened our understanding of the different regulation mechanisms that control the activity of the proteasome and impact overall degradation rates. We will emphasize how both the increase or decrease of proteasome activity adapts to cellular needs and how the inability to do so is relevant to different diseases. Regulation of the traffic of substrates to the proteasome. Docking of the ubiquitinated proteins at the proteasome may be regulated to control the flux of substrates at the proteasome. As indicated above, there are various proteasome subunits that can bind to ubiquitin to mediate substrate recognition. In addition, there are several so-called shuttling receptors that transiently associate with ubiquitinated substrates and with the proteasome. Despite their discovery over 10 years ago (32, 110, 131), it remains largely unclear how the triage of proteasome substrates is governed. A recent striking study indicates that one of these shuttling receptor proteins, ubiquilin-2 (homologous to yeast Dsk2), is specifically required to promote the proteasomal degradation of misfolded proteins that aggregate (47). Interestingly, mutations of ubiquilin-2 that impair its function cause amyotrophic lateral sclerosis (26). Regulation of proteasome receptors and the proteasome-associated pool of shuttling proteins may therefore have a major impact on the degradation of distinct subsets of substrates. Ubiquitination of both Rpn10 and Rpn13 proteasome substrate receptors have been shown to impact their function (Fig. 1B) (10, 52). While ubiquitination of proteasome subunits has been reported before, it was mostly viewed as a possible collateral product of the UPS activity (several E3 ligases have been found associated to the proteasome) or as a possible targeting of nonassembled and misfolded proteasome subunits. However, it is now becoming clearer that, in some cases, ubiquitination of proteasome subunits is not inadvertent. Ubiquitinated Rpn13 was shown to be associated to the proteasome and Rpn13 conjugation levels on the 26S were increased upon chemical inhibition of the proteasome (10). Rpn13 ubiquitination was found to be dependent on Hul5/Ube3c, a E3 ubiquitin ligase that can be found associated with the proteasome (70). Proteasomes conjugated in vitro, in which Rpn13 became fully modified, displayed a marked reduced ability to both bind and degrade ubiquitinated substrates. Goldberg and colleagues proposed a prospective model in which ubiquitination of Rpn13 by Hul5/Ube3c could avert binding of ubiquitinated substrates to proteasomes that are inactive (10). Likewise, the Rpn10 subunit can be found conjugated in vivo, and proteasomes incubated with recombinant Rpn10 fused to a ubiquitin moiety failed to degrade a ubiquitinated substrate in vitro (52). There are two pools of cellular Rpn10 that are either associated or not with the proteasome, and proteasome-free Rpn10 was shown to retain the Dsk2 shuttling receptor (86). As ubiquitinated Rpn10 displays a reduced binding to both the proteasome and Dsk2, conjugation of Rpn10 could promote recruitment of Dsk2 at the proteasome (150). Another recent study also showed that Rad23, another proteasome shuttling receptor, can be found phosphorylated in vivo and that Rad23 with a phosphomimetic mutation displayed reduced affinity to the proteasome (75). These studies show that multiple mechanisms are likely regulating the association to the proteasome of both substrate and shuttling receptors, as well in some cases their ability to bind to ubiquitinated proteins. It will be impor-



ability to degrade oxidized proteins (23). Under these conditions, it has also been shown that there is specifically more de novo synthesis of 20S proteasomes subunits mediated by the Nrf2 master regulator of antioxidant transcriptional responses in metazoan cells (102, 103). In summary, these findings suggest that 20S-mediated proteolysis is not limited to rare cases, but rather represents a complementary degradation route that is critical, especially under oxidative stress, for removing damaged, unfolded proteins, and for maintaining normal levels of proteins containing intrinsically disordered regions. To date, relatively little is known about the mechanisms that control the 20S proteasome degradation route. However, the recent identification of two related proteins that coordinate the 20S proteasome function, NQO1 (5, 90) and DJ-1 (89), has begun to reveal some details on the regulatory principles of this degradation system. Interestingly, NQO1 and DJ-1 share a wide spectrum of common features (reviewed in refs. 29, 139). Both proteins are homodimers consisting of a flavodoxin-like fold and known to be involved in the cellular defense mechanism against oxidative stress. In addition, the expression of DJ-1 and NQO1 is increased in several types of cancer (reviewed in refs. 108, 139) and mutations in both are associated with neurodegenerative diseases. In particular, mutations in NQO1 lead to an increased risk of developing Alzheimer’s disease (125), while mutations in DJ-1 are linked to familial Parkinson’s disease (54, 71, 139). Recently, these two proteins where shown to be involved in the regulation of the 20S proteasome in response to oxidative stress (89) (Fig. 1A). Both DJ-1 and NQO1 can physically bind to the 20S proteasome and prevent proteolysis. In addition, DJ-1 was shown to stabilize Nrf2, which in turn leads to the expression of NQO1. Nrf2, on the other hand, also promotes the expression of 20S proteasome subunits, in accordance with the cell’s need to rapidly eliminate oxidatively damaged proteins. This regulatory circuit probably sustains the balance between the need to efficiently eliminate oxidatively damaged proteins, ensure proper proteasomal function, and maintain the abundance of the naturally unfolded proteins. Overall, under basal conditions, proteolysis by the 26S proteasome likely constitutes the dominant route for protein turnover. Under stress conditions however, like oxidative conditions, when rapid elimination of proteins is required for maintaining cellular viability, contribution of the 20S CP to overall proteolysis seems to become essential. In this case, degradation by the 20S proteasome overcomes the enzymatic cascade required for ubiquitination to not only enhance degradation rate but also spare the energy costs associated with it.


recombinant PKA kinase in vitro, and when purified from a variety of tissue culture cells in which PKA was activated chemically (79, 146). Importantly, the in vitro activation of the isolated proteasomes by PKA indicates that the mechanism does not require alteration of proteasome levels or additional factors, i.e., the increased activity was intrinsic to the proteasome. Alongside the increased peptidase activity, purified 26S proteasomes displayed an increase of both ATP hydrolysis activity (mediated by the base AAA⫹ ATPases) and turnover rate of ubiquitinated substrates (79). While previous work indicated that the base Rpt6 subunit was the main PKA target (146), the more recent analysis pointed to the S14 of the mammalian Rpn6 lid subunit (79). Remarkably, a phosphomimetic mutation of the Rpn6 serine (i.e., S14D) led to higher proteasome activity, while the mutation to alanine led to lower proteasome activity (79). While the N-terminal sequence of Rpn6 is not conserved between higher and lower eukaryotes, that region is located at one extremity of the 19S RP structure of the yeast proteasome that was resolved and lies on the lateral side of the CP (63, 65). One possibility is that phosphorylation of this region could tighten the assembly of the RP to the CP. Previous studies have pointed to a similar assembly function for phosphorylation (3, 40, 149). However, one experiment from Duff and colleagues suggests a possible alternative mechanism. While levels of singled capped and doubly capped proteasomes separated in a native gel were not altered, the peptidase activity was markedly higher for singly capped proteasomes in PKA-activated cells. One possibility is that Rpn6 phosphorylation causes a conformational change of the CP that could for instance facilitate gate opening to permit the entry of the substrates into the core cavity. Note that the standard assay employed to measure proteasome peptidase activity is both influenced by the CP protease activities, as well as the opening of the gate, as the employed short fluorogenic peptides cannot properly diffuse inside the CP. Additional structural analyses will be required to further explore these possibilities. Regardless of the exact activation mechanisms, these studies highlight the potential for therapeutics targeting kinases that regulate or impact proteasome activity to treat pathologies in which there is a decline in proteostasis. Turn it off or down! While an increase in proteasome activity is coveted in some cases, abolition of its activity is beneficial with others. Utilization of two different classes of proteasome inhibitors to treat multiple myeloma (the bortezombid and ixazomib boronates and the irreversible carfilzombid epoxyketone) perhaps best exemplifies this paradigm. How the inhibition of the proteasome leads to the clearance of cancer cells still remains to be determined. On one hand, inhibition of the proteasome may alter the activation of specific pathways such as NF-␬B that mediates an inflammation response and is critical for many tumors including in multiple myeloma (25). Alternatively, proteasome inhibition may also exacerbate cytotoxic stresses due to the accumulation of nondegraded misfolded proteins in the ER and cytosol (28, 44, 93). ER-stress response can induce apoptosis above a certain threshold and may be particularly prevalent within the antibody producing plasma cells affected in multiple myeloma. Aneuploidy, a hallmark of cancer cells that is also prevalent in multiple myeloma (16), also causes an increased load of misfolded polypeptides, especially multimeric proteins expressed from supernumerary chromosomes (27). In agreement with the link



tant to determine how substrate specificity at the proteasome may be impacted by these PTMs, and whether proteasomes at distinct cell localizations display different substrate preferences. A quest for beefed-up proteasomes. Since a decrease in proteostasis has been associated with aging and numerous age-related diseases, treatments that promote activity of the UPS could be exploited to reestablish the correct proteostatic balance in affected cells. One explored avenue is the regulation of auxiliary proteins associated with the proteasome-like deubiquitinases that could control proteostasis by impacting the flux of proteasome substrates. In addition to Rpn11, Usp14 (or Ubp6 in yeast) is one of the two main additional deubiquitinases associated with the human proteasome (70). Finley and colleagues recently showed that Usp14 primarily cleaves off supernumerary ubiquitin chains on proteasome substrates when more than one ubiquitin chain are conjugated to different sites (67). Previously, it was shown that the addition of a small chemical or RNA heptamers that specifically inhibit Usp14 deubiquitinase activity leads to a better clearance of several ectopically expressed aggregation prone proteins in tissue culture cells such as TDP43 and tau (66, 68). In contrast, a recent study showed that phosphorylation of S432 (serine at position 432) of Usp14 by Akt kinase can activate the deubiquitinase both in vitro and in vivo, and thereby reduce turnover of a model proteasome substrate (142). These studies suggest that regulation of Usp14 could control global proteostasis and that its inhibition could be employed to potentially accelerate the degradation of proteins that accumulate in cells with lower proteostatic capacity. A key matter to delineate is to determine which endogenous proteasome substrates are processed by Usp14. The activity of the proteasome can also be directly modulated by posttranslation modifications. For instance, phosphorylation of the proteasome by protein kinase A (PKA), also known as cAMP-dependent kinase, leads to increased proteasome activity (Fig. 1B)(3, 79, 91, 146, 149). Remarkably, Duff and colleagues recently showed very promising results using a mouse model for tauopathy, in which proteasome activity was reestablished following a pharmacological treatment that targets PKA (91). The authors of the study used the Tg4510 model mice, in which the expression of the human tau protein with a pathogenic mutation (P301L) leads to the progressive development of neurofibrillary tangles, neuronal loss, and behavioral impairments (112). In these mice, there is decreased proteasome activity and an accumulation of ubiquitin proteins in brain tissues concomitant to the accumulation of insoluble tau in five-month-old individuals. Notably, administration of the PKA activator rolipram led to increased proteasome activity (concurrent to higher phosphorylation of the 26S), as well as lower levels of insoluble tau, and improved cognition in the treated mice (91). Interestingly, the treatment in Tg4510 mice was only effective if rolipram was administrated early on, suggesting that there was a point of no return upon development of the pathology. One question raised by this study is whether the PKA activation directly leads to higher proteasome activity. Work from a second related study, which also corroborated previous analyses, indicates that it is likely the case. Indeed, 26S proteasomes displayed higher peptidase activities, measured using small fluorescent peptides, following incubation with the

C799

Themes C800


T25 (41). Knockdown of DYRK2 led to lower proteasome peptidase activity. Similarly, overexpression of the catalytically inactive kinase mutant led to lower proteasome activity and lower turnover of a model proteasome substrate in HEK293T cells that expressed the wild-type but not Rpt3 mutated on T25. As well, proteasomes incubated with the active kinase in vitro displayed higher activity. Remarkably, either expression of the Rpt3-T25A mutant or knockdown of DYRK2 impaired xenograft tumor growth in nude mice. In this case, higher proteasome activity seems to be required for progression of cell cycle during the S and G2 phases. Interestingly, lower activity of the proteasome may be required for the earlier G1/S transition. It was shown that the c-Abl tyrosine kinase can interact and phosphorylate the ␣-4 20S subunit (on Y151) (77). In this case, phosphorylation led to lower proteasomal activity. Another intriguing point is that DYRK2 was previously suggested to be a negative regulator of cell cycle progression (92); its inhibition may exert distinctive outcomes in different cell types or conditions. One important point will be to determine how phosphorylation of T25 can activate the proteasome. The T25 of Rpt3 is located next to a region that forms a coiled coil with Rpt6 and extends away from the ATPase domain, in a location near the pinnacle of the 19S RP structure that is distant from the 20S CP. Interestingly, in the presence of a substrate, the 26S ATPase activity was further enhanced following proteasome phosphorylation by active DYRK2 in vitro (41), suggesting that Rpt3 phosphorylation may promote or stabilize a change of conformation of the lid that is favorable to substrate translocation and degradation. Structural analyses will be required to further assess this possibility. A pivotal role of this coiled coil region formed by Rpt3 and Rpt6 is supported by the presence of a downstream residue on S120 of Rpt6 that is also regulated by phosphorylation. Phosphorylation of Rpt6 S120 by calcium/calmodulin-dependent protein kinase II (CaMKII) has been shown to upregulate proteasome activity at synapses where proteasome is also recruited by binding to CaMKII (11, 31). Correct tuning of the proteasome activity is key to controlling synaptic strength and neuronal activity. In cultured rat hippocampal neurons, expression of the Rpt6 S120D phosphomimetic mutant caused a significant decrease in amplitude of excitatory postsynaptic currents (30). This UPS function may be crucial in controlling neuronal plasticity. For instance, phosphorylation of the proteasome by CaMKII in amygdala has been shown to be important for long-term memories in rats upon fear conditioning (53). It will be important to further elucidate how distinct regulatory kinases and phosphorylation sites on the proteasome specifically impact proteasome function at the cellular and organism levels, as one could potentially control both proteasome and proteostasis in a very targeted manner. Conclusion There have been major leaps in the proteasome field in the recent years. The high-resolution structural analyses aided by cryoelectron microscopy now provide an unprecedented glimpse of the organization of the 19S RP. In addition, the combination of careful biochemical investigations together with in vivo analyses using specific proteasome mutants (e.g., phosphomimetic) has revealed multiple novel mechanisms to regulate protein degradation at the proteasome level. Joint efforts using



between cytotoxic stress and proteasome activity in aneuploidy, loss of function of the yeast Ubp6 deubiquitinase (homologous to human Usp14) was shown to improve fitness of disomic cells, presumably due to enhanced proteasome activity in these cells (124). Two independent and recent studies also provide additional evidence for a role of global proteostasis in multiple myeloma therapeutics (1, 126). To better understand resistance mechanisms to proteasome inhibition treatment that can lead to relapse, two different screens were undertaken using RNAi and retroviral gene-trap insertion approaches. In both cases it was found that a reduction of 19S subunit levels leads to a higher survival of cancer cells after proteasome inhibition, while lower levels of 20S subunits increased sensitivity to drug treatment (1, 126). Remarkably, levels of 19S subunits in cells isolated from patients and in different cancer cell lines can be correlated to the response to the treatment and their sensitivity to proteasome inhibition, respectively (lower levels being associated with lower efficacy). Further analysis of cells with lower Rpn1 levels showed that protein synthesis was slightly but significantly lower in these cells, which could reduce the load of misfolded proteins (126). As well, downregulation of several genes participating in protein synthesis also increased cell survival upon proteasome inhibition (1). A lower translation rate could reduce the number of proteins misfolded after synthesis, as well as increase the folding capacity of the cells by “freeing up” chaperones. Consistent with a lower basal level of proteotoxic stress, transcriptome analysis of cells with lower Rpn1 levels revealed that expression of many heat shock proteins (HSPs) was reduced (126). These results suggest that a rewiring of the proteostatic network could lower the dependence on the proteasome and therefore reduce the efficacy of the treatment in these cells. How the proteostatic network is modulated by changes in the 26S/20S ratios remain to be determined. These studies further stress that combinatory treatment will likely contribute to better prognosis. For instance, ixazomib is currently prescribed together with lenalidomide, which is a novel class of drug that binds to an E3 ligase (cereblon) to alter its substrate specificity (60, 81). Intriguingly, both drugs have somewhat opposing activities by inhibiting and promoting proteolysis. As downregulation of chaperone proteins also increased cell death upon proteasome inhibition (1), the combination of both actions could cause a synergetic effect to specifically target cancer cells. Indeed, previous clinical trials in which both Hsp90 and the proteasome were inhibited showed encouraging results (107). In addition to chemical inhibition of the CP proteases, inhibition of proteasome phosphorylation could also be employed in order specifically affect cancer cells. Phosphorylation of Rpt3 T25 was recently shown to be cell cycle regulated by the Dixon lab (41). Phosphorylation of this ATPase lid subunit appeared in S phase and was maintained until G2/M phases. Remarkably, in cells in which T25 was mutated to an alanine residue in both copies of Rpt3 gene using CRISPR/ Cas9 technology, proliferation was dampened with a marked delay of S phase progression in the synchronized cells. Proteasomes purified from these cells were less active when assessed with small substrate peptides. In contrast, ectopic expression of the phosphomimetic mutant (T25D) led to higher proteasome activity. A kinome-wide screen revealed that DYRK2 kinase was likely the kinase that phosphorylate Rpt3


both approaches will hopefully, in the near future, unveil how PTMs and regulatory proteins may exert specific conformational changes to regulate the 26S proteasome. A major view has been thus far that the proteasome works similarly to a funnel, which is fed substrates by a large network of E3 ubiquitin ligases that received much of our attention. It is now apparent that the “proteasome-funnel” is malleable by being able to adapt to the needs of different cells or cellular compartments, and that the regulation of the proteasome activity can have a major impact on the cellular proteolytic capacity. ACKNOWLEDGMENTS We apologize in advance to colleagues in the field whose many excellent and important studies may have not been cited here. We thank Amalia Rose for comments on the manuscript.

This work was supported by a grant from the Canada Institutes of Health Research (CIHR), a Michael Smith Foundation for Health Research Career Award, a Feinberg Foundation Visiting Faculty Fellowship from the Weizmann Institute and Lady Davis Visiting Fellowship from the Technion. M. Sharon is grateful for the support of a Starting Grant from the European Research Council (ERC) (Horizon 2020)/ERC Grant Agreement no. 636752. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS T.M. prepared figures; T.M., M.S., and M.H.G. drafted manuscript; T.M., M.S., and M.H.G. edited and revised manuscript; T.M., M.S., and M.H.G. approved final version of manuscript. REFERENCES 1. Acosta-Alvear D, Cho MY, Wild T, Buchholz TJ, Lerner AG, Simakova O, Hahn J, Korde N, Landgren O, Maric I, Choudhary C, Walter P, Weissman JS, Kampmann M. Paradoxical resistance of multiple myeloma to proteasome inhibitors by decreased levels of 19S proteasomal subunits. Elife 4: e08153, 2015. 2. Aiken CT, Kaake RM, Wang X, Huang L. Oxidative stress-mediated regulation of proteasome complexes. Mol Cell Proteomics 10: R110 006924. 2011year⬎. 3. Asai M, Tsukamoto O, Minamino T, Asanuma H, Fujita M, Asano Y, Takahama H, Sasaki H, Higo S, Asakura M, Takashima S, Hori M, Kitakaze M. PKA rapidly enhances proteasome assembly and activity in in vivo canine hearts. J Mol Cell Cardiol 46: 452–462, 2009. 4. Asano S, Fukuda Y, Beck F, Aufderheide A, Forster F, Danev R, Baumeister Proteasomes W. A molecular census of 26S proteasomes in intact neurons. Science 347: 439 –442, 2015. 5. Asher G, Tsvetkov P, Kahana C, Shaul Y. A mechanism of ubiquitinindependent proteasomal degradation of the tumor suppressors p53 and p73. Genes Dev 19: 316 –321, 2005. 6. Bajorek M, Finley D, Glickman MH. Proteasome disassembly and downregulation is correlated with viability during stationary phase. Curr Biol 13: 1140 –1144, 2003. 7. Bar-Nun S, Glickman MH. Proteasomal AAA-ATPases: structure and function. Biochim Biophys Acta 1823: 67–82, 2012. 8. Baugh JM, Viktorova EG, Pilipenko EV. Proteasomes can degrade a significant proportion of cellular proteins independent of ubiquitination. J Mol Biol 386: 814 –827, 2009. 9. Ben-Nissan G, Sharon M. Regulating the 20S proteasome ubiquitinindependent degradation pathway. Biomolecules 4: 862–884, 2014. 10. Besche HC, Sha Z, Kukushkin NV, Peth A, Hock EM, Kim W, Gygi S, Gutierrez JA, Liao H, Dick L, Goldberg AL. Autoubiquitination of the 26S proteasome on Rpn13 regulates breakdown of ubiquitin conjugates. EMBO J 33: 1159 –1176, 2014. 11. Bingol B, Wang CF, Arnott D, Cheng D, Peng J, Sheng M. Autophosphorylated CaMKIIalpha acts as a scaffold to recruit proteasomes to dendritic spines. Cell 140: 567–578, 2010.

12. Bulteau AL, Petropoulos I, Friguet B. Age-related alterations of proteasome structure and function in aging epidermis. Exp Gerontol 35: 767–777, 2000. 13. Bulteau AL, Szweda LI, Friguet B. Age-dependent declines in proteasome activity in the heart. Arch Biochem Biophys 397: 298 –304, 2002. 14. Cardozo C, Michaud C. Proteasome-mediated degradation of tau proteins occurs independently of the chymotrypsin-like activity by a nonprocessive pathway. Arch Biochem Biophys 408: 103–110, 2002. 15. Carrard G, Dieu M, Raes M, Toussaint O, Friguet B. Impact of ageing on proteasome structure and function in human lymphocytes. Int J Biochem Cell Biol 35: 728 –739, 2003. 16. Chesi M, Bergsagel PL. Advances in the pathogenesis and diagnosis of multiple myeloma. Int J Lab Hematol 37, Suppl 1: 108 –114, 2015. 17. Chondrogianni N, Georgila K, Kourtis N, Tavernarakis N, Gonos ES. 20S proteasome activation promotes life span extension and resistance to proteotoxicity in Caenorhabditis elegans. FASEB J 29: 611–622, 2015. 18. Chondrogianni N, Petropoulos I, Franceschi C, Friguet B, Gonos ES. Fibroblast cultures from healthy centenarians have an active proteasome. Exp Gerontol 35: 721–728, 2000. 19. Cui Z, Scruggs SB, Gilda JE, Ping P, Gomes AV. Regulation of cardiac proteasomes by ubiquitination, SUMOylation, and beyond. J Mol Cell Cardiol 71: 32–42, 2014. 20. da Fonseca PC, He J, Morris EP. Molecular model of the human 26S proteasome. Mol Cell 46: 54 –66, 2012. 21. Dambacher CM, Worden EJ, Herzik MA, Martin A, Lander GC. Atomic structure of the 26S proteasome lid reveals the mechanism of deubiquitinase inhibition. Elife 5: e13027, 2016. 22. David DC, Layfield R, Serpell L, Narain Y, Goedert M, Spillantini MG. Proteasomal degradation of tau protein. J Neurochem 83: 176 –185, 2002. 23. Demasi M, Hand A, Ohara E, Oliveira CL, Bicev RN, Bertoncini CA, Netto LE. 20S proteasome activity is modified via S-glutathionylation based on intracellular redox status of the yeast Saccharomyces cerevisiae: implications for the degradation of oxidized proteins. Arch Biochem Biophys 557: 65–71, 2014. 24. Demasi M, Netto LE, Silva GM, Hand A, de Oliveira CL, Bicev RN, Gozzo F, Barros MH, Leme JM, Ohara E. Redox regulation of the proteasome via S-glutathionylation. Redox Biol 2: 44 –51, 2014. 25. Demchenko YN, Kuehl WM. A critical role for the NFkB pathway in multiple myeloma. Oncotarget 1: 59 –68, 2010. 26. Deng HX, Chen W, Hong ST, Boycott KM, Gorrie GH, Siddique N, Yang Y, Fecto F, Shi Y, Zhai H, Jiang H, Hirano M, Rampersaud E, Jansen GH, Donkervoort S, Bigio EH, Brooks BR, Ajroud K, Sufit RL, Haines JL, Mugnaini E, Pericak-Vance MA, Siddique T. Mutations in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/dementia. Nature 477: 211–215, 2011. 27. Dephoure N, Hwang S, O’Sullivan C, Dodgson SE, Gygi SP, Amon A, Torres EM. Quantitative proteomic analysis reveals posttranslational responses to aneuploidy in yeast. Elife 3: e03023, 2014. 28. Deshaies RJ. Proteotoxic crisis, the ubiquitin-proteasome system, and cancer therapy. BMC Biol 12: 94, 2014. 29. Dinkova-Kostova AT, Talalay P. NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), a multifunctional antioxidant enzyme and exceptionally versatile cytoprotector. Arch Biochem Biophys 501: 116 –123, 2010. 30. Djakovic SN, Marquez-Lona EM, Jakawich SK, Wright R, Chu C, Sutton MA, Patrick GN. Phosphorylation of Rpt6 regulates synaptic strength in hippocampal neurons. J Neurosci 32: 5126 –5131, 2012. 31. Djakovic SN, Schwarz LA, Barylko B, DeMartino GN, Patrick GN. Regulation of the proteasome by neuronal activity and calcium/calmodulin-dependent protein kinase II. J Biol Chem 284: 26655–26665, 2009. 32. Elsasser S, Chandler-Militello D, Muller B, Hanna J, Finley D. Rad23 and Rpn10 serve as alternative ubiquitin receptors for the proteasome. J Biol Chem 279: 26817–26822, 2004. 33. Enenkel C. Nuclear transport of yeast proteasomes. Biomolecules 4: 940 –955, 2014. 34. Erales J, Coffino P. Ubiquitin-independent proteasomal degradation. Biochim Biophys Acta 1843: 216 –221, 2014. 35. Estrin E, Lopez-Blanco JR, Chacon P, Martin A. Formation of an intricate helical bundle dictates the assembly of the 26S proteasome lid. Structure 21: 1624 –1635, 2013. 36. Fabre B, Lambour T, Delobel J, Amalric F, Monsarrat B, BurletSchiltz O, Bousquet-Dubouch MP. Subcellular distribution and dynam-



GRANTS

C801

Themes C802

37. 38.

39. 40. 41.

43. 44.

45. 46. 47.

48. 49. 50. 51. 52.

53.

54. 55. 56. 57.

58.

ics of active proteasome complexes unraveled by a workflow combining in vivo complex cross-linking and quantitative proteomics. Mol Cell Proteomics 12: 687–699, 2013. Gladman NP, Marshall RS, Lee KH, Vierstra RD. The proteasome stress regulon is controlled by a pair of NAC transcription factors in Arabidopsis. Plant Cell 28: 1279 –1296, 2016. Grune T, Catalgol B, Licht A, Ermak G, Pickering AM, Ngo JK, Davies KJ. HSP70 mediates dissociation and reassociation of the 26S proteasome during adaptation to oxidative stress. Free Radic Biol Med 51: 1355–1364, 2011. Gu ZC, Enenkel C. Proteasome assembly. Cell Mol Life Sci 71: 4729 –4745, 2014. Guo X, Engel JL, Xiao J, Tagliabracci VS, Wang X, Huang L, Dixon JE. UBLCP1 is a 26S proteasome phosphatase that regulates nuclear proteasome activity. Proc Natl Acad Sci USA 108: 18649 –18654, 2011. Guo X, Wang X, Wang Z, Banerjee S, Yang J, Huang L, Dixon JE. Site-specific proteasome phosphorylation controls cell proliferation and tumorigenesis. Nat Cell Biol 18: 202–212, 2016. Hanssum A, Zhong Z, Rousseau A, Krzyzosiak A, Sigurdardottir A, Bertolotti A. An inducible chaperone adapts proteasome assembly to stress. Mol Cell 55: 566 –577, 2014. Hayashi T, Goto S. Age-related changes in the 20S and 26S proteasome activities in the liver of male F344 rats. Mech Ageing Dev 102: 55–66, 1998. Hideshima T, Bradner JE, Wong J, Chauhan D, Richardson P, Schreiber SL, Anderson KC. Small-molecule inhibition of proteasome and aggresome function induces synergistic antitumor activity in multiple myeloma. Proc Natl Acad Sci USA 102: 8567–8572, 2005. Hinnerwisch J, Fenton WA, Furtak KJ, Farr GW, Horwich AL. Loops in the central channel of ClpA chaperone mediate protein binding, unfolding, and translocation. Cell 121: 1029 –1041, 2005. Hirano H, Kimura Y, Kimura A. Biological significance of co- and post-translational modifications of the yeast 26S proteasome. J Proteomics 134: 37–46, 2016. Hjerpe R, Bett JS, Keuss MJ, Solovyova A, McWilliams TG, Johnson C, Sahu I, Varghese J, Wood N, Wightman M, Osborne G, Bates GP, Glickman MH, Trost M, Knebel A, Marchesi F, Kurz T. UBQLN2 mediates autophagy-independent protein aggregate clearance by the proteasome. Cell 166: 935–949, 2016. Husnjak K, Elsasser S, Zhang N, Chen X, Randles L, Shi Y, Hofmann K, Walters KJ, Finley D, Dikic I. Proteasome subunit Rpn13 is a novel ubiquitin receptor. Nature 453: 481–488, 2008. Hwang J, Winkler L, Kalejta RF. Ubiquitin-independent proteasomal degradation during oncogenic viral infections. Biochim Biophys Acta 1816: 147–157, 2011. Imai J, Maruya M, Yashiroda H, Yahara I, Tanaka K. The molecular chaperone Hsp90 plays a role in the assembly and maintenance of the 26S proteasome. EMBO J 22: 3557–3567, 2003. Inobe T, Fishbain S, Prakash S, Matouschek A. Defining the geometry of the two-component proteasome degron. Nat Chem Biol 7: 161–167, 2011. Isasa M, Katz EJ, Kim W, Yugo V, Gonzalez S, Kirkpatrick DS, Thomson TM, Finley D, Gygi SP, Crosas B. Monoubiquitination of RPN10 regulates substrate recruitment to the proteasome. Mol Cell 38: 733–745, 2010. Jarome TJ, Kwapis JL, Ruenzel WL, Helmstetter FJ. CaMKII, but not protein kinase A, regulates Rpt6 phosphorylation and proteasome activity during the formation of long-term memories. Front Behav Neurosci 7: 115, 2013. Kahle PJ, Waak J, Gasser T. DJ-1 and prevention of oxidative stress in Parkinson’s disease and other age-related disorders. Free Radic Biol Med 47: 1354 –1361, 2009. Keller JN, Hanni KB, Markesbery WR. Possible involvement of proteasome inhibition in aging: implications for oxidative stress. Mech Ageing Dev 113: 61–70, 2000. Kim YC, Li X, Thompson D, DeMartino GN. ATP binding by proteasomal ATPases regulates cellular assembly and substrate-induced functions of the 26 S proteasome. J Biol Chem 288: 3334 –3345, 2013. Kimura A, Kurata Y, Nakabayashi J, Kagawa H, Hirano H. N-myristoylation of the Rpt2 subunit of the yeast 26S proteasome is implicated in the subcellular compartment-specific protein quality control system. J Proteomics 130: 33–41, 2016. Kirkpatrick DS, Hathaway NA, Hanna J, Elsasser S, Rush J, Finley D, King RW, Gygi SP. Quantitative analysis of in vitro ubiquitinated

59. 60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71. 72.

73.

74.

75.

76.

77.

78.

cyclin B1 reveals complex chain topology. Nat Cell Biol 8: 700 –710, 2006. Kleiger G, Mayor T. Perilous journey: a tour of the ubiquitin-proteasome system. Trends Cell Biol 24: 352–359, 2014. Kronke J, Udeshi ND, Narla A, Grauman P, Hurst SN, McConkey M, Svinkina T, Heckl D, Comer E, Li X, Ciarlo C, Hartman E, Munshi N, Schenone M, Schreiber SL, Carr SA, Ebert BL. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343: 301–305, 2014. Kruegel U, Robison B, Dange T, Kahlert G, Delaney JR, Kotireddy S, Tsuchiya M, Tsuchiyama S, Murakami CJ, Schleit J, Sutphin G, Carr D, Tar K, Dittmar G, Kaeberlein M, Kennedy BK, Schmidt M. Elevated proteasome capacity extends replicative lifespan in Saccharomyces cerevisiae. PLoS Genet 7: e1002253, 2011. Kwak MK, Wakabayashi N, Greenlaw JL, Yamamoto M, Kensler TW. Antioxidants enhance mammalian proteasome expression through the Keap1-Nrf2 signaling pathway. Mol Cell Biol 23: 8786 –8794, 2003. Lander GC, Estrin E, Matyskiela ME, Bashore C, Nogales E, Martin A. Complete subunit architecture of the proteasome regulatory particle. Nature 482: 186 –191, 2012. Laporte D, Salin B, Daignan-Fornier B, Sagot I. Reversible cytoplasmic localization of the proteasome in quiescent yeast cells. J Cell Biol 181: 737–745, 2008. Lasker K, Forster F, Bohn S, Walzthoeni T, Villa E, Unverdorben P, Beck F, Aebersold R, Sali A, Baumeister W. Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach. Proc Natl Acad Sci USA 109: 1380 –1387, 2012. Lee BH, Lee MJ, Park S, Oh DC, Elsasser S, Chen PC, Gartner C, Dimova N, Hanna J, Gygi SP, Wilson SM, King RW, Finley D. Enhancement of proteasome activity by a small-molecule inhibitor of USP14. Nature 467: 179 –184, 2010. Lee BH, Lu Y, Prado MA, Shi Y, Tian G, Sun S, Elsasser S, Gygi SP, King RW, Finley D. USP14 deubiquitinates proteasome-bound substrates that are ubiquitinated at multiple sites. Nature 532: 398 –401, 2016. Lee JH, Shin SK, Jiang Y, Choi WH, Hong C, Kim DE, Lee MJ. Facilitated tau degradation by USP14 aptamers via enhanced proteasome activity. Sci Rep 5: 10757, 2015. Lee W, Kim Y, Park J, Shim S, Lee J, Hong SH, Ahn HH, Lee H, Jung YK. iRhom1 regulates proteasome activity via PAC1/2 under ER stress. Sci Rep 5: 11559, 2015. Leggett DS, Hanna J, Borodovsky A, Crosas B, Schmidt M, Baker RT, Walz T, Ploegh H, Finley D. Multiple associated proteins regulate proteasome structure and function. Mol Cell 10: 495–507, 2002. Lev N, Roncevic D, Ickowicz D, Melamed E, Offen D. Role of DJ-1 in Parkinson’s disease. J Mol Neurosci 29: 215–225, 2006. Li L, Zhao D, Wei H, Yao L, Dang Y, Amjad A, Xu J, Liu J, Guo L, Li D, Li Z, Zuo D, Zhang Y, Liu J, Huang S, Jia C, Wang L, Wang Y, Xie Y, Luo J, Zhang B, Luo H, Donehower LA, Moses RE, Xiao J, O’Malley BW, Li X. REGgamma deficiency promotes premature aging via the casein kinase 1 pathway. Proc Natl Acad Sci USA 110: 11005–11010, 2013. Li X, Amazit L, Long W, Lonard DM, Monaco JJ, O’Malley BW. Ubiquitin- and ATP-independent proteolytic turnover of p21 by the REGgamma-proteasome pathway. Mol Cell 26: 831–842, 2007. Li X, Matilainen O, Jin C, Glover-Cutter KM, Holmberg CI, Blackwell TK. Specific SKN-1/Nrf stress responses to perturbations in translation elongation and proteasome activity. PLoS Genet 7: e1002119, 2011. Liang RY, Chen L, Ko BT, Shen YH, Li YT, Chen BR, Lin KT, Madura K, Chuang SM. Rad23 interaction with the proteasome is regulated by phosphorylation of its ubiquitin-like (UbL) domain. J Mol Biol 426: 4049 –4060, 2014. Liu CW, Li X, Thompson D, Wooding K, Chang TL, Tang Z, Yu H, Thomas PJ, DeMartino GN. ATP binding and ATP hydrolysis play distinct roles in the function of 26S proteasome. Mol Cell 24: 39 –50, 2006. Liu X, Huang W, Li C, Li P, Yuan J, Li X, Qiu XB, Ma Q, Cao C. Interaction between c-Abl and Arg tyrosine kinases and proteasome subunit PSMA7 regulates proteasome degradation. Mol Cell 22: 317– 327, 2006. Livnat-Levanon N, Kevei E, Kleifeld O, Krutauz D, Segref A, Rinaldi T, Erpapazoglou Z, Cohen M, Reis N, Hoppe T, Glickman



42.



79.

80. 81.

82.

84. 85. 86.

87. 88. 89. 90.

91.

92. 93. 94.

95. 96.

97.

98.

99. Peters LZ, Karmon O, David-Kadoch G, Hazan R, Yu T, Glickman MH, Ben-Aroya S. The protein quality control machinery regulates its misassembled proteasome subunits. PLoS Genet 11: e1005178, 2015. 100. Peters LZ, Karmon O, Miodownik S, Ben-Aroya S. Proteasome storage granules are transiently associated with the insoluble protein deposit (IPOD). J Cell Sci 2016. 101. Pickering AM, Davies KJ. Degradation of damaged proteins: the main function of the 20S proteasome. Prog Mol Biol Transl Sci 109: 227–248, 2012. 102. Pickering AM, Linder RA, Zhang H, Forman HJ, Davies KJ. Nrf2dependent induction of proteasome and Pa28alphabeta regulator are required for adaptation to oxidative stress. J Biol Chem 287: 10021– 10031, 2012. 103. Pickering AM, Staab TA, Tower J, Sieburth D, Davies KJ. A conserved role for the 20S proteasome and Nrf2 transcription factor in oxidative stress adaptation in mammals, Caenorhabditis elegans and Drosophila melanogaster. J Exp Biol 216: 543–553, 2013. 104. Radhakrishnan SK, den Besten W, Deshaies RJ. p97-dependent retrotranslocation and proteolytic processing govern formation of active Nrf1 upon proteasome inhibition. Elife 3: e01856, 2014. 105. Radhakrishnan SK, Lee CS, Young P, Beskow A, Chan JY, Deshaies RJ. Transcription factor Nrf1 mediates the proteasome recovery pathway after proteasome inhibition in mammalian cells. Mol Cell 38: 17–28, 2010. 106. Raynes R, Pomatto LC, Davies KJ. Degradation of oxidized proteins by the proteasome: distinguishing between the 20S, 26S, and immunoproteasome proteolytic pathways. Mol Aspects Med 50: 41–55, 2016. 107. Richardson PG, Mitsiades CS, Laubach JP, Lonial S, Chanan-Khan AA, Anderson KC. Inhibition of heat shock protein 90 (HSP90) as a therapeutic strategy for the treatment of myeloma and other cancers. Br J Haematol 152: 367–379, 2011. 108. Ross D, Siegel D. NAD(P)H:quinone oxidoreductase 1 (NQO1, DTdiaphorase), functions and pharmacogenetics. Methods Enzymol 382: 115–144, 2004. 109. Rousseau A, Bertolotti A. An evolutionarily conserved pathway controls proteasome homeostasis. Nature 536: 184 –189, 2016. 110. Saeki Y, Saitoh A, Toh-e A, Yokosawa H. Ubiquitin-like proteins and Rpn10 play cooperative roles in ubiquitin-dependent proteolysis. Biochem Biophys Res Commun 293: 986 –992, 2002. 111. Sahara K, Kogleck L, Yashiroda H, Murata S. The mechanism for molecular assembly of the proteasome. Adv Biol Regul 54: 51–58, 2014. 112. Santacruz K, Lewis J, Spires T, Paulson J, Kotilinek L, Ingelsson M, Guimaraes A, DeTure M, Ramsden M, McGowan E, Forster C, Yue M, Orne J, Janus C, Mariash A, Kuskowski M, Hyman B, Hutton M, Ashe KH. Tau suppression in a neurodegenerative mouse model improves memory function. Science 309: 476 –481, 2005. 113. Schulman BA, Harper JW. Ubiquitin-like protein activation by E1 enzymes: the apex for downstream signalling pathways. Nat Rev Mol Cell Biol 10: 319 –331, 2009. 114. Sheaff RJ, Singer JD, Swanger J, Smitherman M, Roberts JM, Clurman BE. Proteasomal turnover of p21Cip1 does not require p21Cip1 ubiquitination. Mol Cell 5: 403–410, 2000. 115. Shi Y, Chen X, Elsasser S, Stocks BB, Tian G, Lee BH, Shi Y, Zhang N, de Poot SA, Tuebing F, Sun S, Vannoy J, Tarasov SG, Engen JR, Finley D, Walters KJ. Rpn1 provides adjacent receptor sites for substrate binding and deubiquitination by the proteasome. Science 351: 2016. 116. Shirozu R, Yashiroda H, Murata S. Identification of minimum Rpn4responsive elements in genes related to proteasome functions. FEBS Lett 589: 933–940, 2015. 117. Sledz P, Unverdorben P, Beck F, Pfeifer G, Schweitzer A, Forster F, Baumeister W. Structure of the 26S proteasome with ATP-gammaS bound provides insights into the mechanism of nucleotide-dependent substrate translocation. Proc Natl Acad Sci USA 110: 7264 –7269, 2013. 118. Thrower JS, Hoffman L, Rechsteiner M, Pickart CM. Recognition of the polyubiquitin proteolytic signal. EMBO J 19: 94 –102, 2000. 119. Tofaris GK, Layfield R, Spillantini MG. ␣-Synuclein metabolism and aggregation is linked to ubiquitin-independent degradation by the proteasome. FEBS Lett 509: 22–26, 2001. 120. Tomaru U, Takahashi S, Ishizu A, Miyatake Y, Gohda A, Suzuki S, Ono A, Ohara J, Baba T, Murata S, Tanaka K, Kasahara M. Decreased proteasomal activity causes age-related phenotypes and promotes the development of metabolic abnormalities. Am J Pathol 180: 963–972, 2012.



83.

MH. Reversible 26S proteasome disassembly upon mitochondrial stress. Cell Rep 7: 1371–1380, 2014. Lokireddy S, Kukushkin NV, Goldberg AL. cAMP-induced phosphorylation of 26S proteasomes on Rpn6/PSMD11 enhances their activity and the degradation of misfolded proteins. Proc Natl Acad Sci USA 112: E7176-7185, 2015. Lowe J, Stock D, Jap B, Zwickl P, Baumeister W, Huber R. Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 34 A resolution. Science 268: 533–539, 1995. Lu G, Middleton RE, Sun H, Naniong M, Ott CJ, Mitsiades CS, Wong KK, Bradner JE, Kaelin WG Jr. The myeloma drug lenalidomide promotes the cereblon-dependent destruction of Ikaros proteins. Science 343: 305–309, 2014. Mansour W, Nakasone MA, von Delbruck M, Yu Z, Krutauz D, Reis N, Kleifeld O, Sommer T, Fushman D, Glickman MH. Disassembly of Lys11 and mixed linkage polyubiquitin conjugates provides insights into function of proteasomal deubiquitinases Rpn11 and Ubp6. J Biol Chem 290: 4688 –4704, 2015. Marshall RS, Li F, Gemperline DC, Book AJ, Vierstra RD. Autophagic degradation of the 26S proteasome is mediated by the dual ATG8/ubiquitin receptor RPN10 in Arabidopsis. Mol Cell 58: 1053– 1066, 2015. Marshall RS, Vierstra RD. Eat or be eaten: the autophagic plight of inactive 26S proteasomes. Autophagy 11: 1927–1928, 2015. Martin A, Baker TA, Sauer RT. Pore loops of the AAA⫹ ClpX machine grip substrates to drive translocation and unfolding. Nat Struct Mol Biol 15: 1147–1151, 2008. Matiuhin Y, Kirkpatrick DS, Ziv I, Kim W, Dakshinamurthy A, Kleifeld O, Gygi SP, Reis N, Glickman MH. Extraproteasomal Rpn10 restricts access of the polyubiquitin-binding protein Dsk2 to proteasome. Mol Cell 32: 415–425, 2008. Matyskiela ME, Lander GC, Martin A. Conformational switching of the 26S proteasome enables substrate degradation. Nat Struct Mol Biol 20: 781–788, 2013. Metzger MB, Hristova VA, Weissman AM. HECT and RING finger families of E3 ubiquitin ligases at a glance. J Cell Sci 125: 531–537, 2012. Moscovitz O, Ben-Nissan G, Fainer I, Pollack D, Mizrachi L, Sharon M. The Parkinson’s-associated protein DJ-1 regulates the 20S proteasome. Nat Commun 6: 6609, 2015. Moscovitz O, Tsvetkov P, Hazan N, Michaelevski I, Keisar H, Ben-Nissan G, Shaul Y, Sharon M. A mutually inhibitory feedback loop between the 20S proteasome and its regulator, NQO1. Mol Cell 47: 76 –86, 2012. Myeku N, Clelland CL, Emrani S, Kukushkin NV, Yu WH, Goldberg AL, Duff KE. Tau-driven 26S proteasome impairment and cognitive dysfunction can be prevented early in disease by activating cAMPPKA signaling. Nat Med 22: 46 –53, 2016. Nihira NT, Yoshida K. Engagement of DYRK2 in proper control for cell division. Cell Cycle 14: 802–807, 2015. Obeng EA, Carlson LM, Gutman DM, Harrington WJ Jr, Lee KP, andBoise LH. Proteasome inhibitors induce a terminal unfolded protein response in multiple myeloma cells. Blood 107: 4907–4916, 2006. Pack CG, Yukii H, Toh-e A, Kudo T, Tsuchiya H, Kaiho A, Sakata E, Murata S, Yokosawa H, Sako Y, Baumeister W, Tanaka K, Saeki Y. Quantitative live-cell imaging reveals spatio-temporal dynamics and cytoplasmic assembly of the 26S proteasome. Nat Commun 5: 3396, 2014. Padmanabhan A, Vuong SA, Hochstrasser M. Assembly of an evolutionarily conserved alternative proteasome isoform in human cells. Cell Rep 14: 2962–2974, 2016. Park S, Li X, Kim HM, Singh CR, Tian G, Hoyt MA, Lovell S, Battaile KP, Zolkiewski M, Coffino P, Roelofs J, Cheng Y, Finley D. Reconfiguration of the proteasome during chaperone-mediated assembly. Nature 497: 512–516, 2013. Pathare GR, Nagy I, Bohn S, Unverdorben P, Hubert A, Korner R, Nickell S, Lasker K, Sali A, Tamura T, Nishioka T, Forster F, Baumeister W, Bracher A. The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together. Proc Natl Acad Sci USA 109: 149 –154, 2012. Pathare GR, Nagy I, Sledz P, Anderson DJ, Zhou HJ, Pardon E, Steyaert J, Forster F, Bracher A, Baumeister W. Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11. Proc Natl Acad Sci USA 111: 2984 –2989, 2014.

C803

Themes C804

PROTEASOME REGULATION AND PROTEOLYSIS 135. Waite KA, De-La Mota-Peynado A, Vontz G, Roelofs J. starvation induces proteasome autophagy with different pathways for core and regulatory particles. J Biol Chem 291: 3239 –3253, 2016. 136. Wang X, Yen J, Kaiser P, Huang L. Regulation of the 26S proteasome complex during oxidative stress. Sci Signal 3: ra88, 2010. 137. Wani PS, Rowland MA, Ondracek A, Deeds EJ, Roelofs J. Maturation of the proteasome core particle induces an affinity switch that controls regulatory particle association. Nat Commun 6: 6384, 2015. 138. Weberruss MH, Savulescu AF, Jando J, Bissinger T, Harel A, Glickman MH, Enenkel C. Blm10 facilitates nuclear import of proteasome core particles. EMBO J 32: 2697–2707, 2013. 139. Wilson MA. The role of cysteine oxidation in DJ-1 function and dysfunction. Antioxid Redox Signal 15: 111–122, 2011. 140. Worden EJ, Padovani C, Martin A. Structure of the Rpn11-Rpn8 dimer reveals mechanisms of substrate deubiquitination during proteasomal degradation. Nat Struct Mol Biol 21: 220 –227, 2014. 141. Xie Y, Varshavsky A. RPN4 is a ligand, substrate, and transcriptional regulator of the 26S proteasome: A negative feedback circuit. Proc Natl Acad Sci USA 98: 3056 –3061, 2001. 142. Xu D, Shan B, Lee BH, Zhu K, Zhang T, Sun H, Liu M, Shi L, Liang W, Qian L, Xiao J, Wang L, Pan L, Finley D, Yuan J. Phosphorylation and activation of ubiquitin-specific protease-14 by Akt regulates the ubiquitin-proteasome system. Elife 4: e10510, 2015. 143. Yao T, Cohen RE. A cryptic protease couples deubiquitination and degradation by the proteasome. Nature 419: 403–407, 2002. 144. Ye Y, Rape M. Building ubiquitin chains: E2 enzymes at work. Nat Rev Mol Cell Biol 10: 755–764, 2009. 145. Yu Z, Livnat-Levanon N, Kleifeld O, Mansour W, Nakasone MA, Castaneda CA, Dixon EK, Fushman D, Reis N, Pick E, Glickman MH. Base-CP proteasome can serve as a platform for stepwise lid formation. Biosci Rep 35: 2015. 146. Zhang F, Hu Y, Huang P, Toleman CA, Paterson AJ, Kudlow JE. Proteasome function is regulated by cyclic AMP-dependent protein kinase through phosphorylation of Rpt6. J Biol Chem 282: 22460 – 22471, 2007. 147. Zhang H, Davies KJ, Forman HJ. Oxidative stress response and Nrf2 signaling in aging. Free Radic Biol Med 88: 314 –336, 2015. 148. Zhang X, Schulz R, Edmunds S, Kruger E, Markert E, Gaedcke J, Cormet-Boyaka E, Ghadimi M, Beissbarth T, Levine AJ, Moll UM, Dobbelstein M. MicroRNA-101 suppresses tumor cell proliferation by acting as an endogenous proteasome inhibitor via targeting the proteasome assembly factor POMP. Mol Cell 59: 243–257, 2015. 149. Zong C, Gomes AV, Drews O, Li X, Young GW, Berhane B, Qiao X, French SW, Bardag-Gorce F, Ping P. Regulation of murine cardiac 20S proteasomes: role of associating partners. Circ Res 99: 372–380, 2006. 150. Zuin A, Bichmann A, Isasa M, Puig-Sarries P, Diaz LM, Crosas B. Rpn10 monoubiquitination orchestrates the association of the ubiquilintype DSK2 receptor with the proteasome. Biochem J 472: 353–365, 2015.



121. Tomko RJ Jr, Hochstrasser M. The intrinsically disordered Sem1 protein functions as a molecular tether during proteasome lid biogenesis. Mol Cell 53: 433–443, 2014. 122. Tomko RJ Jr, Hochstrasser M. Molecular architecture and assembly of the eukaryotic proteasome. Annu Rev Biochem 82: 415–445, 2013. 123. Tomko RJ Jr, Taylor DW, Chen ZA, Wang HW, Rappsilber J, Hochstrasser M. A single alpha helix drives extensive remodeling of the proteasome lid and completion of regulatory particle assembly. Cell 163: 432–444, 2015. 124. Torres EM, Dephoure N, Panneerselvam A, Tucker CM, Whittaker CA, Gygi SP, Dunham MJ, Amon A. Identification of aneuploidytolerating mutations. Cell 143: 71–83, 2010. 125. Tsvetkov P, Adamovich Y, Elliott E, Shaul Y. E3 ligase STUB1/CHIP regulates NAD(P)H:quinone oxidoreductase 1 (NQO1) accumulation in aged brain, a process impaired in certain Alzheimer disease patients. J Biol Chem 286: 8839 –8845, 2011. 126. Tsvetkov P, Mendillo ML, Zhao J, Carette JE, Merrill PH, Cikes D, Varadarajan M, van Diemen FR, Penninger JM, Goldberg AL, Brummelkamp TR, Santagata S, Lindquist S. Compromising the 19S proteasome complex protects cells from reduced flux through the proteasome. Elife 4: 2015. 127. Unverdorben P, Beck F, Sledz P, Schweitzer A, Pfeifer G, Plitzko JM, Baumeister W, Forster F. Deep classification of a large cryo-EM dataset defines the conformational landscape of the 26S proteasome. Proc Natl Acad Sci USA 111: 5544 –5549, 2014. 128. van der Lee R, Buljan M, Lang B, Weatheritt RJ, Daughdrill GW, Dunker AK, Fuxreiter M, Gough J, Gsponer J, Jones DT, Kim PM, Kriwacki RW, Oldfield CJ, Pappu RV, Tompa P, Uversky VN, Wright PE, Babu MM. Classification of intrinsically disordered regions and proteins. Chem Rev 114: 6589 –6631, 2014. 129. van Deventer S, Menendez-Benito V, van Leeuwen F, Neefjes J. N-terminal acetylation and replicative age affect proteasome localization and cell fitness during aging. J Cell Sci 128: 109 –117, 2015. 130. Verma R, Aravind L, Oania R, McDonald WH, Yates JR, 3rd Koonin EV, Deshaies RJ. Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science 298: 611–615, 2002. 131. Verma R, Oania R, Graumann J, Deshaies RJ. Multiubiquitin chain receptors define a layer of substrate selectivity in the ubiquitin-proteasome system. Cell 118: 99 –110, 2004. 132. Vilchez D, Boyer L, Morantte I, Lutz M, Merkwirth C, Joyce D, Spencer B, Page L, Masliah E, Berggren WT, Gage FH, Dillin A. Increased proteasome activity in human embryonic stem cells is regulated by PSMD11. Nature 489: 304 –308, 2012. 133. Vilchez D, Morantte I, Liu Z, Douglas PM, Merkwirth C, Rodrigues AP, Manning G, Dillin A. RPN-6 determines C. elegans longevity under proteotoxic stress conditions. Nature 489: 263–268, 2012. 134. Vittal V, Stewart MD, Brzovic PS, Klevit RE. Regulating the regulators: recent revelations in the control of E3 ubiquitin ligases. J Biol Chem 290: 21244 –21251, 2015.

A journey to the end of the message.

Accompanying our patients at the end of their journey.

A journey into insidious world of MALT lymphoma of the ileum: from the beginning to the end.

Journey to the center of the centrosome.

End-Stopping Predicts Curvature Tuning along the Ventral Stream.

Treatment of dyslipidemia and cardiovascular outcomes: the journey so far--is this the end for statins?

Fine-Tuning of FACT by the Ubiquitin Proteasome System in Regulation of Transcriptional Elongation.

The Journey.

The Journey.

The role of medical culture in the journey to resilience.

Chromatin assembly: Journey to the CENter of the chromosome.

The journey from proton to gamma knife.

The journey to independent nurse practitioner practice.

Tuning to the right signal.

The journey from the past to the future.

The Journey to Adult Congenital Heart Disease.

The journey from stem cell to macrophage.

Creatine kinase-MB: the journey to obsolescence.

End of the road: confounding results of the CORE trial terminate the arduous journey of cilengitide for glioblastoma.

Documenting the cancer journey.

From the Editor: The Journey to Open Access.

The Journey to High Reliability in the NICU.

A Haemodialysis Journey from the West to the East.

The road to virtual: the Sauls Memorial Virtual Library journey.