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Allosteric Effects in the Regulation of 26S Proteasome Activities
Perspective
Allosteric Effects in the Regulation of 26S Proteasome Activities
Paweł Śledź, Friedrich Förster and Wolfgang Baumeister Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
Correspondence to Wolfgang Baumeister: [email protected] http://dx.doi.org/10.1016/j.jmb.2013.01.036 Edited by C. Kalodimos
Abstract The 26S proteasome is the executive arm of the ubiquitin-proteasome system. This 2.5-MDa complex comprising the 20S core particle (CP) and the 19S regulatory particle (RP) is able to effectively execute its function due to a tightly regulated network of allosteric interactions. From this perspective, we summarize the current state of knowledge on these regulatory interdependencies. We classify them into the three functional layers—within the CP, within the RP, and at the CP–RP interface. In the CP, allosteric effects are thought to couple the gate opening and substrate proteolysis. Gate opening depends on events occurring in the RP— ATP hydrolysis and substrate binding. Finally, a number of processes occurring solely in the RP, like ATP hydrolysis or substrate deubiquitylation, are also proposed to be allosterically regulated. Recent advances in structural studies of 26S proteasome open up new avenues for dissecting and rationalizing the molecular basis of these regulatory networks. © 2013 Elsevier Ltd. All rights reserved.
Introduction The 26S proteasome is a molecular machine of 2.5-MDa, which is the executive arm of the ubiquitinproteasome system. It eliminates proteins no longer needed at certain stages of a cell's life cycle and those misfolded or damaged. Such proteins are marked for destruction by the covalent attachment of multiple ubiquitins—polyubiquitin chains—via the ubiquitin ligation cascades. 1–3 The 26S proteasome comprises two major subcomplexes: the 20S core particle (CP) harboring the proteolytically active sites and the 19S regulatory particle (RP) responsible for substrate recognition and recruitment, as well as for its ATP-dependent processing (Fig. 1a). While the CP has been studied in great detail and high-resolution atomic structures became available almost two decades ago, 6,7 the structure of the 26S holocomplex remained elusive. Hitherto, all attempts to obtain crystals of the holocomplex suitable for X-ray crystallography were met with frustration. Only recently, the subunit architecture of the entire 26S assembly has been established: two independent approaches yielded essentially the same result. 8,9 A combination of cryo-
electron microscopy (EM), biochemistry, and integrative modeling approaches has resolved the architecture of the RP. Very recently, an atomic model of the RP has been obtained using cryo-EM in conjunction with molecular-dynamics-based flexible fitting (Fig. 1a). 4 These structural studies have paved the way for a deeper mechanistic understanding of this intriguing molecular machine. The challenge now is to elucidate the network of interactions between proteasomal subunits responsible for control of the protein degradation process. Here, we review our current understanding of this regulatory network and highlight some key events from the initial recognition of proteins marked for degradation to their proteolytic cleavage. Several hypotheses implying allosteric effects have been put forward, but structural evidence for them remains scarce (Table 1). The 26S proteasome exhibits a modular architecture The CP is built of four stacked seven-membered rings, two α-subunit rings flanking the two central β-
0022-2836/$ - see front matter © 2013 Elsevier Ltd. All rights reserved.
J. Mol. Biol. (2013) 425, 1415–1423
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Perspective: Regulation of 26S Proteasome Activities
Fig. 1. (a) Architecture of 26S proteasome. Left: cross-section of 26S proteasome shows compartmentalization of the CP and its active sites. Right: pseudoatomic models of yeast 26S proteasome indicating positions of different types of subunits. 4 Rpn1 (a PC subunit) has been omitted as the structural information is limited due to its flexibility. (b) Domain composition of RP subunits. 5 The RP is assembled of two distinct subcomplexes, the “base” and the “lid”. The AAAATPases (Rpt1–Rpt6) consist of a disordered region, a coiled coil, an oligosaccharide binding (OB) domain, and the canonical AAA-module (from N- to C-terminus). The two largest subunits, Rpn1 and Rpn2, consist of a domain with a tetratricopeptide-like repeats (TPR-like), a torus that is conserved in proteasomal and cyclosome subunits (PC-torus), and a β-sandwich (β-sw) C-terminal domain. Rpn13 is a pleckstrin-like receptor for ubiquitin (PRU). The lid subunits Rpn3, Rpn5, Rpn6, Rpn7, Rpn9, and Rpn12 all exhibit a conserved architecture: the N-terminal domain is TPR-like, followed by a domain that is conserved in proteasome, COP9, and initiation factor 3 (PCI), and C-terminal helices that contribute to a helical bundle (HB), which is essential for structural integrity of the lid. Both Rpn8 and Rpn11 possess a conserved MPN domain that is followed by HB helices. In the ubiquitin receptor Rpn10, a von Willebrand factor domain (vWFa) precedes ubiquitin binding motifs (UB).
subunit rings. Of the seven β-subunits (β1–β7), β1, β2, and β5 are proteolytically active. 10 These active sites, including the N-terminal threonines, are positioned near the interfaces of neighboring βsubunits. 6,7 The proteolytic sites are sequestered from the cellular environment through the selfcompartmentalizing barrel-like structure of the CP. This architectural principle offers protection of the intracellular proteins from unwanted proteolysis. 11 The CP alone can only degrade unfolded proteins and peptides. 12 The two α-rings together with their
neighboring β-rings form two smaller compartments, referred to as the “antechambers” (Fig. 1a). They provide the microenvironment that maintains proteins translocated into the CP in an unfolded state in preparation for their degradation. 13,14 The passage through the center of α-ring (the “α-annulus”) is controlled by the conformational changes of the loops. 15 The RP is an essential control element of the proteolytic activity of the CP. The RP comprises six ATPase subunits (Rpt1–Rpt6), two MPN (Mpr1 and
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Table 1. Summary of proposed allosteric effects observed within the regulatory network of the 26S proteasome Location
Effect
Details
Bite–chew mechanism Active sites of β1, β2, and β5 subunits cross-regulate their activity Coupling between the gate and active sites Transition state at the active sites may be coupled with the gate opening CP–RP interface Gate opening by ATPases C-termini of AAA-ATPases interact with α-ring inducing gate opening Gate opening by the substrate Substrate binding stimulates gate-opening-dependent activities of the proteasome RP ATP hydrolysis ATPase subunits regulate each other's activity hydrolyzing ATP in a concerted manner Rpn11 Incorporation of Rpn11 into the complex switches on its proteolytic activity Uch37 Transient association of Uch37 with 26S proteasomes activates it CP
Pad1 N-terminal) subunits (Rpn8 and Rpn11), two PC (conserved in proteasome and cyclosome) subunits (Rpn1 and Rpn2), six PCI (conserved in proteasome, COP9 and initiation factor 3) subunits (Rpn3, Rpn5, Rpn6, Rpn7, Rpn9, and Rpn12), and two ubiquitin receptors (Rpn10 and Rpn13) 5 (Fig. 1b). The ATPases are responsible for ATPdependent substrate unfolding and its translocation into the antechambers. 16,17 Rpn11 forms a heterodimer with Rpn8 and is a metalloprotease with deubiquitylating activity. 18,19 It is responsible for the removal of polyubiquitin chains prior to degradation, and its active site is positioned near the mouth of the AAA-ATPase module. The Ub receptors are located at the distal end of RP and recognize polyubiquitin, providing the initial affinity of the proteasome for its substrates. 20–22 The role of the PC and PCI subunits is less well understood. They appear to participate in control of RP activities through the appropriate positioning of other subunits and by acting as a scaffold. For example, Rpn1 is known to be a docking site for a number of proteasome-interacting proteins, such as the shuttling Ub receptors Rad23, Dsk2, and Ddi1, as well as the deubiquitylating enzyme Ubp6. 3 In addition, some E3 ligases seem to bind to the RP, but their precise interaction sites are still unknown. 23,24 Allosteric effects upon binding to the active sites in the CP Kisselev et al. studied the proteolytic cleavage step in some detail using biochemical assays and described the network of interactions between the distinct types of active sites within the CP—“chymotrypsin-like”, “trypsin-like”, and “caspase-like”. 25 They have shown that proteolysis at the chymotrypsin-like subunit β5 significantly stimulates the activity of the caspase-like β1 subunit, while proteolysis at caspase-like site is inhibitory to chymotrypsin-like activity of β2. The authors concluded that a cyclic “bite–chew mechanism” of protein degradation is in place with the chymotrypsin-like site performing initial cleavage (“bite”) followed by the series of caspase-like proteolysis events (“chewing” of the protein substrate).
The CP gate may possibly also exercise allosteric regulation controlling the substrate cleavage inside the CP of 26S proteasome. The gate opening has been proposed to be coupled with the transition state in proteolysis. Atomic force microscopy experiments and observations of repetitive transitions between the open-gated and closed-gated proteasomes were taken as evidence that the open-gated form is stabilized upon exposure to substrates or inhibitors. 26 However, the low resolution of the atomic force microscopy experiments does not allow visualizing such details with confidence and further biophysical and structural experiments are needed to validate this model. In another experiment, it was observed that the interaction between CP and RP is stabilized in the presence of a CP active-site inhibitor implying an allosteric effect, but the mechanism underlying stabilization remains unclear. 27 However, both experiments suggest that conformational changes of the active sites may propagate to the gate and even to the RP. The CP gate may be under allosteric control The CP and RP interfaces have different symmetries (pseudo-C7 for the CP versus pseudo-C6 for the AAA-ATPases; see Fig. 2a and b), making a perfect fit between them impossible and limiting contacts to a few sites. 28,30 Therefore, the CP–RP interaction is relatively weak, which allows the RP to undergo extensive conformational changes. The network of regulatory interactions within the proteasome naturally follows the intersubunit contacts. The CP contacts the RP through the proteasomal AAA-ATPase modules (their C-terminal “small” domains; Fig. 2c) and the α-ring, as well as through the Rpn5 and Rpn6 subunits. 8,9 The AAAATPases of the RP have essential roles in the regulation of substrate processing. They are responsible for association of the RP with the CP, activation of the CP proteolytic activity through controlling of gate opening, and unfolding of the substrate in preparation for degradation. All these activities are likely to be controlled by the nucleotide state of the ATPases, as it has been shown for the archaeal counterpart of the 26S proteasome consisting of the
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Perspective: Regulation of 26S Proteasome Activities
Fig. 2. The AAA-ATPases of 26S proteasome. (a) The asymmetric interface between CP and RP (pockets on the surface of CP are shown with circles) 28; (b) top view of hexameric ring of AAA-ATPase; (c) structure of PAN monomer with ADP bound, large and small domains indicated with gray and white, respectively. 29
homohexameric AAA-ATPase PAN (proteasomeactivating nucleotidase) and the CP. 31 Upon binding of nucleotides, the AAA-ATPases assume conformations that promote interactions with the CP. These structural rearrangements propagate to other subunits in the RP and CP. In the 26S proteasome, every other ATPase subunit contains conserved hydrophobic tyrosine-X motif (HbYX motif) at its Cterminus (Fig. 2a). This HbYX motif can open the CP gate for substrate entry, as shown using sevenresidue peptides containing this motif. 32 Structural insights into the underlying molecular mechanism of the PAN–CP system were obtained with the help of these seven-residue peptides mimicking the C-terminal tail of PAN. 33 The peptides were complexed with the CP to induce gate opening and examined by cryo-EM. The three-dimensional reconstruction revealed additional densities spanning the pockets on the surface of the α-ring of 20S proteasome. The peptides appeared to interact with residues of both subunits forming the pocket, including Gly34 and Leu81 residues of the Thermoplasma acidophilum α-subunit and Arg28 residue of its neighbor. This interaction causes the entire α-ring of the proteasome to rotate by 4° with respect to the β-ring, resulting in an opening of the gate through a displacement of the loops forming the α-annulus. Similar effects have been observed for proteasome activation by 11S particle (PA26); however, in this case, the mechanism appears to be different. PA26, a homoheptameric non-ATPase, contacts the CP
through its C-termini, and the displacement of the pore-forming loops is achieved through their direct interactions with activation loops of PA26. A hypothesis that a similar mechanism might be in place for gate opening in PAN–20S complex as well as 26S proteasome (based on the experiments with PA26 hybrid constructs featuring C-terminus from PAN) has also been put forward. 34,35 However, Yu et al. have engineered PA26 particle in a similar way introducing the HbYX motif and mutating the activation loop and they observed a HbYX-dependent gate opening coupled to the α-ring rotation in both X-ray and EM structures. 36 This discrepancy of results despite similar experimental methodology will have to be addressed. The mechanism of the α-ring rotation observed by Rabl et al. is not fully understood. 33 However, it appears to be regulated through long-range conformational changes induced upon binding of the Ctermini of selected ATPase subunits. In the recently determined cryo-EM structures of the 26S proteasome in the presence of ATP, the gate appeared to be much more closed than observed for the CP– HbYX peptide complex and an α-ring rotation was not observed. 4,8,9 However, these maps were averaged over an ensemble of different nucleotide states. A preliminary computational dissection of coexisting dynamic states of yeast 26S proteasome suggests that twisting of the α-ring and changes in the gate density resembling those described by Rabl et al. may indeed also be observed with 26S proteasome (unpublished data).
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Gate opening has been also proposed to be induced upon binding of polyubiquitin conjugates. 37,38 Peth et al. have demonstrated that polyubiquitylated species associate with Ubp6 (deubiquitylating enzyme interacting with the Rpn1) and stimulate gateopening-dependent proteolytic activities of the proteasome. 39 This effect required the presence of Ubp6 or its catalytically inactive mutant, suggesting an indirect mechanism of gate regulation. The approximate location of Ubp6 is at least 5 nm away from the CP gate 9 hinting at an allosteric mechanism. A mechanism through which the proteasome may be inhibited in neurodegenerative diseases has been recently proposed. It has been shown that gate may be kept closed upon interaction of aggregated Prp with the 20S proteasome. 40 It has been shown that this effect is due to binding of aggregated Prp to the side of the proteasome rather than directly obstructing the gate. Nucleotide-dependent conformational changes of AAA-ATPases support substrate processing Proteasomal ATPases have been proposed to fulfill additional ATP-dependent roles. They unfold protein substrates by exerting the required mechanical force through movements of the AAA-ATPase pore loops, threading them through the pores. The pore loops are highly conserved and contain a hydrophobic aromatic residue (Phe or Tyr) that provides the handle for interaction with substrate proteins. 15 Hydrolysis of ATP coincides with relative movements of the large and small ATPase domain, forming a nucleotide-binding cleft at their interface (Fig. 2c). Two models of ATP hydrolysis and its effect on the proteasome's protein unfolding power have been proposed. Studies of Smith et al. with PAN suggested that the AAA-ATPase subunits hydrolyze ATP in pairs in a tightly regulated manner and that 26S ATPases may act similarly. 41 The nucleotide state of each subunit is thought to affect the nucleotide binding capacities of its neighbors. Smith et al. explain the observed enzymatic activity and nucleotide binding experiments by three possible nucleotide-binding states of the ATPases—tight binding, loose binding, and nonbinding. During the ATPase cycle, two subunits, for symmetry reasons presumably located at the opposite faces of the ring, hydrolyze ATP and change their conformation to become ADP-binding subunits. At the same time, ADP is released from their neighbors, which become nonbinding subunits, and the pair of subunits that was previously nonbinding changes its conformation to bind ATP. An alternative model of ATP hydrolysis has been proposed by Lander et al. 9 In the 26S proteasome reconstruction in the presence of saturated ATP, they observed that the pore loops of the AAAmodules are arranged in a staircase-like topologi-
cally open conformation, 9 similar to lockwasher-like helicase structures. 42 They interpret this conformation as a low-energy state and stochastic ATP hydrolysis of the different subunits may trigger translocation and unfolding, similar to a model previously proposed for the AAA-ATPase ClpX. 15,43 Since the stoichiometry studies are performed in a way detecting only tightly bound nucleotide and only one structural snapshot of proteasomal ATPase is available, none of the hypotheses can be definitely proven based on collected experimental evidence. Given the number of contacts between the ATPase subunits, it is expected that, at least, some regulation between them exists and indeed staircase-like conformations have also been hypothesized to imply an ordered mechanism of reaction in the case of helicases. 42,44 While the ATPase hexameric ring seems to be topologically open in the structure of ATP-exposed proteasome, we have also observed other conformations, dependent on the nucleotide state (unpublished results). Further structural studies will allow deconstructing the different stages of nucleotide cycle and elucidate the mechanism of ATP hydrolysis. Glynn et al. used a combination of biochemical cross-linking experiments and X-ray crystallography to study the nucleotide-dependent conformational changes in AAA-ATPases of ClpX protease, closely related to proteasomal ATPases. 45 It has been proposed that the small domain forms a rigid body with the large domain of neighboring subunits and stays as such during all the ATPase movements. While data for 26S proteasome are still limited, we have preliminary results suggesting that such intersubunit dimers exist also in the case of the 26S ATPases. Deubiquitylation activity may be regulated allosterically The structure of the 26S proteasome suggests that ubiquitin moieties are removed from the substrates concomitantly with translocation into the AAAATPase pore. Rpn11 has been proposed to be allosterically regulated by the AAA-ATPase module. 3,9 The metalloprotease neither exhibits proteolytic activity in its monomeric form nor does it as part of the isolated lid. However, upon assembly of the full RP, Rpn11 is activated. 18,19 Substantial structural rearrangements of the Rpn8/Rpn11 dimer have been observed when the lid is integrated into the RP by low-resolution negative-stain EM also hinting at an allosteric activation mechanism. 9 Based on the atomic model of the Rpn8/Rpn11 dimer in the RP, a possible mechanism of this allosteric activation was proposed: the C-termini are part a helical bundle, which was hypothesized to block the active site of the isolated lid and to free the active site upon integration into the RP. 4
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Fig. 3. The mobility of the 19S RP. (a) Regions of highest mobility (marker with warm colors using the variance analysis) are situated in the RP; (b) an example of wagging motions made by the 19S RP; one conformation shown in solid and another shown in cross-hatched.
The proteasome-associated deubiquitylating enzyme Ubp6 and its mammalian homolog Usp14 also get activated upon binding to the 26S proteasome 46; the activity of Ubp6 is increased approximately 300fold due to a long-range interaction with the RP. 47 Another deubiquitylating enzyme, Uch37, was found to associate with proteasomes in most species other than Saccharomyces cerevisiae has also been shown to be allosterically regulated. 48–50 In cytoplasm, Uch37 is self-inhibited by its C-terminus and unable to hydrolyze diubiquitin. When it is recruited to the 26S proteasome through binding to the human ortholog of Rpn13 (Adrm1), the self-inhibition is relieved and deubiquitylating activity is significantly increased. Interestingly, the formation of isolated Uch37–Adrm1 complexes does not lead to activation hinting at a more complex mechanism. The non-ATPases undergo a “wagging” or “wobbling” motion The structure of the 26S proteasome suggests that movements of the ATPase propagate to the nonATPases. The horseshoe scaffold formed by PCI subunits of the lid contacts the CP via two subunits— Rpn5 and in particular Rpn6. 51 Interestingly, upregulation of Rpn6 has been shown to be responsible for increased proteasome-dependent activities in stem cells from different organisms. 52,53 However, it remains to be shown whether this effect is primarily due to stoichiometric limitations in 26S proteasome
assembly or this relates to allosteric effects in the regulatory network. Rpn5 and Rpn6 are also the major contact sites between the ATPases and the lid. Conformational changes in the ATPase ring resulting from a change in the nucleotide state, for example, triggered by ATP hydrolysis, may result in movements of the whole lid. In this case, the Rpn5/6-CP contact is preserved and the ATPase pushing these PCI subunits makes them act as a lever and change the positioning of the apex of the RP. As a result, structural features such as the OB (oligonucleotide binding) domain of the ATPases may move affecting trafficking of the substrate through its central pore or may also change the positions of Ub receptors with respect to the ATPases. Our preliminary EM data have indicated that such kinds of movements do occur (Fig. 3). In principle, they are similar to the whole RP “wagging movements” seen in the low-resolution studies of the 26S proteasome long time ago. 54 Improvements in data acquisition and therefore resolution of the data set allowed now not only for more detailed insights into motions of the RP but also for a dissection of the intersubunit interactions responsible for them. 4,8 The dependence of the lid conformation on the nucleotide state of the ATPase ring explains some previous observations that binding of ATP positively regulates binding and degradation of protein substrates. 41,55 We expect that further studies will provide deeper insight into the regulatory role of RP and help to
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Perspective: Regulation of 26S Proteasome Activities
explain previous functional observations such as the role of ATP binding in substrate hydrolysis.
Summary
2. 3.
26S proteasome is regulated at different levels, which allows for a controlled execution of its proteolytic roles in the cell. Many of its functions, such as substrate recognition, deubiquitylation, unfolding, and degradation, appear to be controlled allosterically. The challenge ahead of us is now to elucidate their structural basis. Recent developments in methodology including the ability to dissect coexisting conformations in highly dynamic assemblies by cryo-EM and advanced image classification approaches will allow in-depth studies to be performed on these effects. We expect that a better understanding of the proteasome's regulation will allow for the development of more selective therapeutic agents targeting steps upstream of the final proteolytic cleavage event.
4.
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7.
8.
Acknowledgements
9.
Our research is supported by funding from European Union Seventh Framework Programme PROSPECTS (Proteomics Specification in Space and Time Grant HEALTH-F4-2008-201648), Deutsche Forschungsgemeinschaft (SFB-1035 to W.B.), Human Frontiers Science Program (Career Development Award to F.F.), and European Molecular Biology Organization and Marie Curie Actions (EMBO LongTerm Fellowship to P.Ś.). We thank Antje Aufderheide and Florian Beck for help with Fig. 3. Received 30 November 2012; Received in revised form 30 January 2013; Accepted 30 January 2013 Available online 8 February 2013
10.
11. 12. 13.
14. Keywords: allostery; 26S proteasome; proteostasis; cryo-electron microscopy Abbreviations used: CP, core particle; RP, regulatory particle, EM, electron microscopy.
References 1. Voges, D., Zwickl, P. & Baumeister, W. (1999). The 26S proteasome: a molecular machine designed for
15.
16.
17.
controlled proteolysis. Annu. Rev. Biochem. 68, 1015–1068. Tanaka, K. (2009). The proteasome: overview of structure and functions. Proc. Jpn. Acad., Ser. B, 85, 12–36. Finley, D. (2009). Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu. Rev. Biochem. 78, 477–513. Beck, F., Unverdorben, P., Bohn, S., Schweitzer, A., Pfeifer, G., Sakata, E. et al. (2012). Near-atomic resolution structural model of the yeast 26S proteasome. Proc. Natl Acad. Sci. USA, 109, 14870–14875. Förster, F., Lasker, K., Nickell, S., Sali, A. & Baumeister, W. (2010). Toward an integrated structural model of the 26S proteasome. Mol. Cell. Proteomics, 9, 1666–1677. Löwe, J., Stock, D., Jap, B., Zwickl, P., Baumeister, W. & Huber, R. (1995). Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 Ǻ resolution. Science, 268, 533–539. Groll, M., Ditzel, L., Löwe, J., Stock, D., Bochtler, M., Bartunik, H. D. & Huber, R. (1997). Structure of 20S proteasome from yeast at 2.4 Ǻ resolution. Nature, 386, 463–471. Lasker, K., Förster, F., Bohn, S., Walzthoeni, T., Villa, E., Unverdorben, P. et al. (2012). Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach. Proc. Natl Acad. Sci. USA, 109, 1380–1387. Lander, G. C., Estrin, E., Matyskiela, M. E., Bashore, C., Nogales, E. & Martin, A. (2012). Complete subunit architecture of the proteasome regulatory particle. Nature, 482, 186–191. Seemüller, E., Lupas, A., Stock, D., Löwe, J., Huber, R. & Baumeister, W. (1995). Proteasome from Thermoplasma acidophilum: a threonine protease. Science, 268, 579–582. Baumeister, W., Walz, J., Zühl, F. & Seemüller, E. (1998). The proteasome: paradigm of a self-compartmentalizing protease. Cell, 92, 367–380. Wenzel, T. & Baumeister, W. (1995). Conformational constraints in protein degradation by the 20S proteasome. Nat. Struct. Biol. 2, 199–204. Ruschak, A. M., Religa, T. L., Breuer, S., Witt, S. & Kay, L. E. (2010). The proteasome antechamber maintains substrates in an unfolded state. Nature, 467, 868–871. Sharon, M., Witt, S., Felderer, K., Rockel, B., Baumeister, W. & Robinson, C. V. (2006). 20S proteasomes have the potential to keep substrates in store for continual degradation. J. Biol. Chem. 281, 9569–9575. Martin, A., Baker, T. A. & Sauer, R. T. (2008). Pore loops of the AAA + ClpX machine grip substrates to drive translocation and unfolding. Nat. Struct. Mol. Biol. 15, 1147–1151. Förster, F., Lasker, K., Beck, F., Nickell, S., Sali, A. & Baumeister, W. (2009). An atomic model AAAATPase/20S core particle sub-complex of the 26S proteasome. Biochem. Biophys. Res. Commun. 388, 228–233. Sauer, R. T. & Baker, T. A. (2011). AAA + proteases: ATP-fueled machines of protein destruction. Annu. Rev. Biochem. 80, 587–612.
1422 18. Verma, R., Aravind, L., Oania, R., McDonald, W. H., Yates, J. R., 3rd, Koonin, E. V. & Deshaies, R. J. (2002). Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science, 298, 611–615. 19. Yao, T. & Cohen, R. E. (2002). A cryptic protease couples deubiquitination and degradation by the proteasome. Nature, 419, 403–407. 20. Sakata, E., Bohn, S., Mihalache, O., Kiss, P., Beck, F., Nagy, I. et al. (2012). Localization of the proteasomal ubiquitin receptors Rpn10 and Rpn13 by electron cryomicroscopy. Proc. Natl Acad. Sci. USA, 109, 1479–1484. 21. Deveraux, Q., Ustrell, V., Pickart, C. & Rechsteiner, M. (1994). A 26S protease subunit that binds ubiquitin conjugates. J. Biol. Chem. 269, 7059–7061. 22. van Nocker, S., Sadis, S., Rubin, D. M., Glickman, M., Fu, H., Coux, O. et al. (1996). The multiubiquitinchain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover. Mol. Cell. Biol. 16, 6020–6028. 23. Xie, Y. & Varshavsky, A. (2000). Physical association of ubiquitin ligases and the 26S proteasome. Proc. Natl Acad. Sci. USA, 97, 2497–2502. 24. Crosas, B., Hanna, J., Kirkpatrick, D. S., Zhang, D. P., Tone, Y., Hathaway, N. A. et al. (2006). Ubiquitin chains are remodeled at the proteasome by opposing ubiquitin ligase and deubiquitinating activities. Cell, 127, 1401–1413. 25. Kisselev, A. F., Akopian, T. N., Castillo, V. & Goldberg, A. L. (1999). Proteasome active sites allosterically regulate each other, suggesting a cyclical bite–chew mechanism for protein breakdown. Mol. Cell, 4, 395–402. 26. Osmulski, P. A., Hochstrasser, M. & Gaczynska, M. (2009). A tetrahedral transition state at the active sites of the 20S proteasome is coupled to opening of the αring channel. Structure, 17, 1137–1147. 27. Kleijnen, M. F., Roelofs, J., Park, S., Hathaway, N. A., Glickman, M., King, R. W. & Finley, D. (2007). Stability of the proteasome can be regulated allosterically through engagement of its proteolytic active sites. Nat. Struct. Mol. Biol. 14, 1180–1188. 28. Tian, G., Park, S., Lee, M. J., Huck, B., McAllister, F., Hill, C. P. et al. (2011). An asymmetric interface between the regulatory and core particles of the proteasome. Nat. Struct. Mol. Biol. 18, 1259–1267. 29. Zhang, F., Hu, M., Tian, G., Zhang, P., Finley, D., Jeffrey, P. D. & Shi, Y. (2009). Structural insights into the regulatory particle of the proteasome from Methanocaldococcus jannaschii. Mol. Cell, 34, 473–484. 30. Bohn, S., Beck, F., Sakata, E., Walzthoeni, T., Beck, M., Aebersold, R. et al. (2010). Structure of the 26S proteasome from Schizosaccharomyces pombe at subnanometer resolution. Proc. Natl Acad. Sci. USA, 107, 20992–20997. 31. Smith, D. M., Kafri, G., Cheng, Y., Ng, D., Walz, T. & Goldberg, A. L. (2005). ATP binding to PAN or the 26S ATPases causes association with the 20S proteasome, gate opening, and translocation of unfolded proteins. Mol. Cell, 20, 687–698. 32. Smith, D. M., Chang, S. C., Park, S., Finley, D., Cheng, Y. & Goldberg, A. L. (2007). Docking of the
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33.
34.
35. 36.
37. 38.
39.
40.
41.
42. 43. 44. 45.
46.
47.
48.
proteasomal ATPases' carboxyl termini in the 20S proteasome's α ring opens the gate for substrate entry. Mol. Cell, 27, 731–744. Rabl, J., Smith, D. M., Yu, Y., Chang, S. C., Goldberg, A. L. & Cheng, Y. (2008). Mechanism of gate opening in the 20S proteasome by the proteasomal ATPases. Mol. Cell, 30, 360–368. Stadtmueller, B. M., Ferrell, K., Whitby, F. G., Heroux, A., Robinson, H., Myszka, D. G. & Hill, C. P. (2010). Structural models for interactions between the 20S proteasome and its PAN/19S activators. J. Biol. Chem. 285, 13–17. Stadtmueller, B. M. & Hill, C. P. (2011). Proteasome activators. Mol. Cell, 41, 8–19. Yu, Y., Smith, D. M., Kim, H. M., Rodriguez, V., Goldberg, A. L. & Cheng, Y. (2010). Interactions of PAN's C-termini with archaeal 20S proteasome and implications for the eukaryotic proteasome–ATPase interactions. EMBO J. 29, 692–702. Li, X. & DeMartino, G. N. (2009). Variably modulated gating of the 26S proteasome by ATP and polyubiquitin. Biochem. J. 421, 397–404. Bech-Otschir, D., Helfrich, A., Enenkel, C., Consiglieri, G., Seeger, M., Holzhütter, H. G. et al. (2009). Polyubiquitin substrates allosterically activate their own degradation by the 26S proteasome. Nat. Struct. Mol. Biol. 16, 219–225. Peth, A., Besche, H. C. & Goldberg, A. L. (2009). Ubiquitinated proteins activate the proteasome by binding to Usp14/Ubp6, which causes 20S gate opening. Mol. Cell, 36, 794–804. Deriziotis, P., André, R., Smith, D. M., Goold, R., Kinghorn, K. J., Kristiansen, M. et al. (2011). Misfolded PrP impairs the UPS by interaction with the 20S proteasome and inhibition of substrate entry. EMBO J. 30, 3065–3077. Smith, D. M., Fraga, H., Reis, C., Kafri, G. & Goldberg, A. L. (2011). ATP binds to proteasomal ATPases in pairs with distinct functional effects, implying an ordered reaction cycle. Cell, 144, 526–538. Enemark, E. J. & Joshua-Tor, L. (2006). Mechanism of DNA translocation in a replicative hexameric helicase. Nature, 442, 270–275. Martin, A., Baker, T. A. & Sauer, R. T. (2005). Rebuilt AAA + motors reveal operating principles for ATPfuelled machines. Nature, 437, 1115–1120. Thomsen, N. D. & Berger, J. M. (2009). Running in reverse: the structural basis for translocation polarity in hexameric helicases. Cell, 139, 523–534. Glynn, S. E., Nager, A. R., Baker, T. A. & Sauer, R. T. (2012). Dynamic and static components power unfolding in topologically closed rings of a AAA + proteolytic machine. Nat. Struct. Mol. Biol. 19, 616–622. Borodovsky, A., Kessler, B. M., Casagrande, R., Overkleeft, H. S., Wilkinson, K. D. & Ploegh, H. L. (2001). A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14. EMBO J. 20, 5187–5196. Leggett, D. S., Hanna, J., Borodovsky, A., Crosas, B., Schmidt, M., Baker, R. T. et al. (2002). Multiple associated proteins regulate proteasome structure and function. Mol. Cell, 10, 495–507. Yao, T., Song, L., Xu, W., DeMartino, G. N., Florens, L., Swanson, S. K. et al. (2006). Proteasome
Perspective: Regulation of 26S Proteasome Activities
recruitment and activation of the Uch37 deubiquitinating enzyme by Adrm1. Nat. Cell Biol. 8, 994–1002. 49. Hamazaki, J., Iemura, S., Natsume, T., Yashiroda, H., Tanaka, K. & Murata, S. (2006). A novel proteasome interacting protein recruits the deubiquitinating enzyme UCH37 to 26S proteasomes. EMBO J. 25, 4524–4536. 50. Qiu, X.-B., Ouyang, S. Y., Li, C. J., Miao, S., Wang, L. & Goldberg, A. L. (2006). hRpn13/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37. EMBO J. 25, 5742–5753. 51. Pathare, G. R., Nagy, I., Bohn, S., Unverdorben, P., Hubert, A., Körner, R. et al. (2012). The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together. Proc. Natl Acad. Sci. USA, 109, 149–154.
1423 52. Vilchez, D., Boyer, L., Morantte, I., Lutz, M., Merkwirth, C., Joyce, D. et al. (2012). Increased proteasome activity in human embryonic stem cells is regulated by PSMD11. Nature, 489, 304–308. 53. Vilchez, D., Morantte, I., Liu, Z., Douglas, P. M., Merkwirth, C., Rodrigues, A. P. et al. (2012). RPN-6 determines C. elegans longevity under proteotoxic stress conditions. Nature, 489, 263–268. 54. Walz, J., Erdmann, A., Kania, M., Typke, D., Koster, A. J. & Baumeister, W. (1998). 26S proteasome structure revealed by three-dimensional electron microscopy. J. Struct. Biol. 121, 19–29. 55. Peth, A., Uchiki, T. & Goldberg, A. L. (2010). ATPdependent steps in the binding of ubiquitin conjugates to the 26S proteasome that commit to degradation. Mol. Cell, 40, 671–681.
Allosteric Effects in the Regulation of 26S Proteasome Activities
Paweł Śledź, Friedrich Förster and Wolfgang Baumeister Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany
Correspondence to Wolfgang Baumeister: [email protected] http://dx.doi.org/10.1016/j.jmb.2013.01.036 Edited by C. Kalodimos
Abstract The 26S proteasome is the executive arm of the ubiquitin-proteasome system. This 2.5-MDa complex comprising the 20S core particle (CP) and the 19S regulatory particle (RP) is able to effectively execute its function due to a tightly regulated network of allosteric interactions. From this perspective, we summarize the current state of knowledge on these regulatory interdependencies. We classify them into the three functional layers—within the CP, within the RP, and at the CP–RP interface. In the CP, allosteric effects are thought to couple the gate opening and substrate proteolysis. Gate opening depends on events occurring in the RP— ATP hydrolysis and substrate binding. Finally, a number of processes occurring solely in the RP, like ATP hydrolysis or substrate deubiquitylation, are also proposed to be allosterically regulated. Recent advances in structural studies of 26S proteasome open up new avenues for dissecting and rationalizing the molecular basis of these regulatory networks. © 2013 Elsevier Ltd. All rights reserved.
Introduction The 26S proteasome is a molecular machine of 2.5-MDa, which is the executive arm of the ubiquitinproteasome system. It eliminates proteins no longer needed at certain stages of a cell's life cycle and those misfolded or damaged. Such proteins are marked for destruction by the covalent attachment of multiple ubiquitins—polyubiquitin chains—via the ubiquitin ligation cascades. 1–3 The 26S proteasome comprises two major subcomplexes: the 20S core particle (CP) harboring the proteolytically active sites and the 19S regulatory particle (RP) responsible for substrate recognition and recruitment, as well as for its ATP-dependent processing (Fig. 1a). While the CP has been studied in great detail and high-resolution atomic structures became available almost two decades ago, 6,7 the structure of the 26S holocomplex remained elusive. Hitherto, all attempts to obtain crystals of the holocomplex suitable for X-ray crystallography were met with frustration. Only recently, the subunit architecture of the entire 26S assembly has been established: two independent approaches yielded essentially the same result. 8,9 A combination of cryo-
electron microscopy (EM), biochemistry, and integrative modeling approaches has resolved the architecture of the RP. Very recently, an atomic model of the RP has been obtained using cryo-EM in conjunction with molecular-dynamics-based flexible fitting (Fig. 1a). 4 These structural studies have paved the way for a deeper mechanistic understanding of this intriguing molecular machine. The challenge now is to elucidate the network of interactions between proteasomal subunits responsible for control of the protein degradation process. Here, we review our current understanding of this regulatory network and highlight some key events from the initial recognition of proteins marked for degradation to their proteolytic cleavage. Several hypotheses implying allosteric effects have been put forward, but structural evidence for them remains scarce (Table 1). The 26S proteasome exhibits a modular architecture The CP is built of four stacked seven-membered rings, two α-subunit rings flanking the two central β-
0022-2836/$ - see front matter © 2013 Elsevier Ltd. All rights reserved.
J. Mol. Biol. (2013) 425, 1415–1423
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Perspective: Regulation of 26S Proteasome Activities
Fig. 1. (a) Architecture of 26S proteasome. Left: cross-section of 26S proteasome shows compartmentalization of the CP and its active sites. Right: pseudoatomic models of yeast 26S proteasome indicating positions of different types of subunits. 4 Rpn1 (a PC subunit) has been omitted as the structural information is limited due to its flexibility. (b) Domain composition of RP subunits. 5 The RP is assembled of two distinct subcomplexes, the “base” and the “lid”. The AAAATPases (Rpt1–Rpt6) consist of a disordered region, a coiled coil, an oligosaccharide binding (OB) domain, and the canonical AAA-module (from N- to C-terminus). The two largest subunits, Rpn1 and Rpn2, consist of a domain with a tetratricopeptide-like repeats (TPR-like), a torus that is conserved in proteasomal and cyclosome subunits (PC-torus), and a β-sandwich (β-sw) C-terminal domain. Rpn13 is a pleckstrin-like receptor for ubiquitin (PRU). The lid subunits Rpn3, Rpn5, Rpn6, Rpn7, Rpn9, and Rpn12 all exhibit a conserved architecture: the N-terminal domain is TPR-like, followed by a domain that is conserved in proteasome, COP9, and initiation factor 3 (PCI), and C-terminal helices that contribute to a helical bundle (HB), which is essential for structural integrity of the lid. Both Rpn8 and Rpn11 possess a conserved MPN domain that is followed by HB helices. In the ubiquitin receptor Rpn10, a von Willebrand factor domain (vWFa) precedes ubiquitin binding motifs (UB).
subunit rings. Of the seven β-subunits (β1–β7), β1, β2, and β5 are proteolytically active. 10 These active sites, including the N-terminal threonines, are positioned near the interfaces of neighboring βsubunits. 6,7 The proteolytic sites are sequestered from the cellular environment through the selfcompartmentalizing barrel-like structure of the CP. This architectural principle offers protection of the intracellular proteins from unwanted proteolysis. 11 The CP alone can only degrade unfolded proteins and peptides. 12 The two α-rings together with their
neighboring β-rings form two smaller compartments, referred to as the “antechambers” (Fig. 1a). They provide the microenvironment that maintains proteins translocated into the CP in an unfolded state in preparation for their degradation. 13,14 The passage through the center of α-ring (the “α-annulus”) is controlled by the conformational changes of the loops. 15 The RP is an essential control element of the proteolytic activity of the CP. The RP comprises six ATPase subunits (Rpt1–Rpt6), two MPN (Mpr1 and
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Perspective: Regulation of 26S Proteasome Activities
Table 1. Summary of proposed allosteric effects observed within the regulatory network of the 26S proteasome Location
Effect
Details
Bite–chew mechanism Active sites of β1, β2, and β5 subunits cross-regulate their activity Coupling between the gate and active sites Transition state at the active sites may be coupled with the gate opening CP–RP interface Gate opening by ATPases C-termini of AAA-ATPases interact with α-ring inducing gate opening Gate opening by the substrate Substrate binding stimulates gate-opening-dependent activities of the proteasome RP ATP hydrolysis ATPase subunits regulate each other's activity hydrolyzing ATP in a concerted manner Rpn11 Incorporation of Rpn11 into the complex switches on its proteolytic activity Uch37 Transient association of Uch37 with 26S proteasomes activates it CP
Pad1 N-terminal) subunits (Rpn8 and Rpn11), two PC (conserved in proteasome and cyclosome) subunits (Rpn1 and Rpn2), six PCI (conserved in proteasome, COP9 and initiation factor 3) subunits (Rpn3, Rpn5, Rpn6, Rpn7, Rpn9, and Rpn12), and two ubiquitin receptors (Rpn10 and Rpn13) 5 (Fig. 1b). The ATPases are responsible for ATPdependent substrate unfolding and its translocation into the antechambers. 16,17 Rpn11 forms a heterodimer with Rpn8 and is a metalloprotease with deubiquitylating activity. 18,19 It is responsible for the removal of polyubiquitin chains prior to degradation, and its active site is positioned near the mouth of the AAA-ATPase module. The Ub receptors are located at the distal end of RP and recognize polyubiquitin, providing the initial affinity of the proteasome for its substrates. 20–22 The role of the PC and PCI subunits is less well understood. They appear to participate in control of RP activities through the appropriate positioning of other subunits and by acting as a scaffold. For example, Rpn1 is known to be a docking site for a number of proteasome-interacting proteins, such as the shuttling Ub receptors Rad23, Dsk2, and Ddi1, as well as the deubiquitylating enzyme Ubp6. 3 In addition, some E3 ligases seem to bind to the RP, but their precise interaction sites are still unknown. 23,24 Allosteric effects upon binding to the active sites in the CP Kisselev et al. studied the proteolytic cleavage step in some detail using biochemical assays and described the network of interactions between the distinct types of active sites within the CP—“chymotrypsin-like”, “trypsin-like”, and “caspase-like”. 25 They have shown that proteolysis at the chymotrypsin-like subunit β5 significantly stimulates the activity of the caspase-like β1 subunit, while proteolysis at caspase-like site is inhibitory to chymotrypsin-like activity of β2. The authors concluded that a cyclic “bite–chew mechanism” of protein degradation is in place with the chymotrypsin-like site performing initial cleavage (“bite”) followed by the series of caspase-like proteolysis events (“chewing” of the protein substrate).
The CP gate may possibly also exercise allosteric regulation controlling the substrate cleavage inside the CP of 26S proteasome. The gate opening has been proposed to be coupled with the transition state in proteolysis. Atomic force microscopy experiments and observations of repetitive transitions between the open-gated and closed-gated proteasomes were taken as evidence that the open-gated form is stabilized upon exposure to substrates or inhibitors. 26 However, the low resolution of the atomic force microscopy experiments does not allow visualizing such details with confidence and further biophysical and structural experiments are needed to validate this model. In another experiment, it was observed that the interaction between CP and RP is stabilized in the presence of a CP active-site inhibitor implying an allosteric effect, but the mechanism underlying stabilization remains unclear. 27 However, both experiments suggest that conformational changes of the active sites may propagate to the gate and even to the RP. The CP gate may be under allosteric control The CP and RP interfaces have different symmetries (pseudo-C7 for the CP versus pseudo-C6 for the AAA-ATPases; see Fig. 2a and b), making a perfect fit between them impossible and limiting contacts to a few sites. 28,30 Therefore, the CP–RP interaction is relatively weak, which allows the RP to undergo extensive conformational changes. The network of regulatory interactions within the proteasome naturally follows the intersubunit contacts. The CP contacts the RP through the proteasomal AAA-ATPase modules (their C-terminal “small” domains; Fig. 2c) and the α-ring, as well as through the Rpn5 and Rpn6 subunits. 8,9 The AAAATPases of the RP have essential roles in the regulation of substrate processing. They are responsible for association of the RP with the CP, activation of the CP proteolytic activity through controlling of gate opening, and unfolding of the substrate in preparation for degradation. All these activities are likely to be controlled by the nucleotide state of the ATPases, as it has been shown for the archaeal counterpart of the 26S proteasome consisting of the
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Perspective: Regulation of 26S Proteasome Activities
Fig. 2. The AAA-ATPases of 26S proteasome. (a) The asymmetric interface between CP and RP (pockets on the surface of CP are shown with circles) 28; (b) top view of hexameric ring of AAA-ATPase; (c) structure of PAN monomer with ADP bound, large and small domains indicated with gray and white, respectively. 29
homohexameric AAA-ATPase PAN (proteasomeactivating nucleotidase) and the CP. 31 Upon binding of nucleotides, the AAA-ATPases assume conformations that promote interactions with the CP. These structural rearrangements propagate to other subunits in the RP and CP. In the 26S proteasome, every other ATPase subunit contains conserved hydrophobic tyrosine-X motif (HbYX motif) at its Cterminus (Fig. 2a). This HbYX motif can open the CP gate for substrate entry, as shown using sevenresidue peptides containing this motif. 32 Structural insights into the underlying molecular mechanism of the PAN–CP system were obtained with the help of these seven-residue peptides mimicking the C-terminal tail of PAN. 33 The peptides were complexed with the CP to induce gate opening and examined by cryo-EM. The three-dimensional reconstruction revealed additional densities spanning the pockets on the surface of the α-ring of 20S proteasome. The peptides appeared to interact with residues of both subunits forming the pocket, including Gly34 and Leu81 residues of the Thermoplasma acidophilum α-subunit and Arg28 residue of its neighbor. This interaction causes the entire α-ring of the proteasome to rotate by 4° with respect to the β-ring, resulting in an opening of the gate through a displacement of the loops forming the α-annulus. Similar effects have been observed for proteasome activation by 11S particle (PA26); however, in this case, the mechanism appears to be different. PA26, a homoheptameric non-ATPase, contacts the CP
through its C-termini, and the displacement of the pore-forming loops is achieved through their direct interactions with activation loops of PA26. A hypothesis that a similar mechanism might be in place for gate opening in PAN–20S complex as well as 26S proteasome (based on the experiments with PA26 hybrid constructs featuring C-terminus from PAN) has also been put forward. 34,35 However, Yu et al. have engineered PA26 particle in a similar way introducing the HbYX motif and mutating the activation loop and they observed a HbYX-dependent gate opening coupled to the α-ring rotation in both X-ray and EM structures. 36 This discrepancy of results despite similar experimental methodology will have to be addressed. The mechanism of the α-ring rotation observed by Rabl et al. is not fully understood. 33 However, it appears to be regulated through long-range conformational changes induced upon binding of the Ctermini of selected ATPase subunits. In the recently determined cryo-EM structures of the 26S proteasome in the presence of ATP, the gate appeared to be much more closed than observed for the CP– HbYX peptide complex and an α-ring rotation was not observed. 4,8,9 However, these maps were averaged over an ensemble of different nucleotide states. A preliminary computational dissection of coexisting dynamic states of yeast 26S proteasome suggests that twisting of the α-ring and changes in the gate density resembling those described by Rabl et al. may indeed also be observed with 26S proteasome (unpublished data).
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Perspective: Regulation of 26S Proteasome Activities
Gate opening has been also proposed to be induced upon binding of polyubiquitin conjugates. 37,38 Peth et al. have demonstrated that polyubiquitylated species associate with Ubp6 (deubiquitylating enzyme interacting with the Rpn1) and stimulate gateopening-dependent proteolytic activities of the proteasome. 39 This effect required the presence of Ubp6 or its catalytically inactive mutant, suggesting an indirect mechanism of gate regulation. The approximate location of Ubp6 is at least 5 nm away from the CP gate 9 hinting at an allosteric mechanism. A mechanism through which the proteasome may be inhibited in neurodegenerative diseases has been recently proposed. It has been shown that gate may be kept closed upon interaction of aggregated Prp with the 20S proteasome. 40 It has been shown that this effect is due to binding of aggregated Prp to the side of the proteasome rather than directly obstructing the gate. Nucleotide-dependent conformational changes of AAA-ATPases support substrate processing Proteasomal ATPases have been proposed to fulfill additional ATP-dependent roles. They unfold protein substrates by exerting the required mechanical force through movements of the AAA-ATPase pore loops, threading them through the pores. The pore loops are highly conserved and contain a hydrophobic aromatic residue (Phe or Tyr) that provides the handle for interaction with substrate proteins. 15 Hydrolysis of ATP coincides with relative movements of the large and small ATPase domain, forming a nucleotide-binding cleft at their interface (Fig. 2c). Two models of ATP hydrolysis and its effect on the proteasome's protein unfolding power have been proposed. Studies of Smith et al. with PAN suggested that the AAA-ATPase subunits hydrolyze ATP in pairs in a tightly regulated manner and that 26S ATPases may act similarly. 41 The nucleotide state of each subunit is thought to affect the nucleotide binding capacities of its neighbors. Smith et al. explain the observed enzymatic activity and nucleotide binding experiments by three possible nucleotide-binding states of the ATPases—tight binding, loose binding, and nonbinding. During the ATPase cycle, two subunits, for symmetry reasons presumably located at the opposite faces of the ring, hydrolyze ATP and change their conformation to become ADP-binding subunits. At the same time, ADP is released from their neighbors, which become nonbinding subunits, and the pair of subunits that was previously nonbinding changes its conformation to bind ATP. An alternative model of ATP hydrolysis has been proposed by Lander et al. 9 In the 26S proteasome reconstruction in the presence of saturated ATP, they observed that the pore loops of the AAAmodules are arranged in a staircase-like topologi-
cally open conformation, 9 similar to lockwasher-like helicase structures. 42 They interpret this conformation as a low-energy state and stochastic ATP hydrolysis of the different subunits may trigger translocation and unfolding, similar to a model previously proposed for the AAA-ATPase ClpX. 15,43 Since the stoichiometry studies are performed in a way detecting only tightly bound nucleotide and only one structural snapshot of proteasomal ATPase is available, none of the hypotheses can be definitely proven based on collected experimental evidence. Given the number of contacts between the ATPase subunits, it is expected that, at least, some regulation between them exists and indeed staircase-like conformations have also been hypothesized to imply an ordered mechanism of reaction in the case of helicases. 42,44 While the ATPase hexameric ring seems to be topologically open in the structure of ATP-exposed proteasome, we have also observed other conformations, dependent on the nucleotide state (unpublished results). Further structural studies will allow deconstructing the different stages of nucleotide cycle and elucidate the mechanism of ATP hydrolysis. Glynn et al. used a combination of biochemical cross-linking experiments and X-ray crystallography to study the nucleotide-dependent conformational changes in AAA-ATPases of ClpX protease, closely related to proteasomal ATPases. 45 It has been proposed that the small domain forms a rigid body with the large domain of neighboring subunits and stays as such during all the ATPase movements. While data for 26S proteasome are still limited, we have preliminary results suggesting that such intersubunit dimers exist also in the case of the 26S ATPases. Deubiquitylation activity may be regulated allosterically The structure of the 26S proteasome suggests that ubiquitin moieties are removed from the substrates concomitantly with translocation into the AAAATPase pore. Rpn11 has been proposed to be allosterically regulated by the AAA-ATPase module. 3,9 The metalloprotease neither exhibits proteolytic activity in its monomeric form nor does it as part of the isolated lid. However, upon assembly of the full RP, Rpn11 is activated. 18,19 Substantial structural rearrangements of the Rpn8/Rpn11 dimer have been observed when the lid is integrated into the RP by low-resolution negative-stain EM also hinting at an allosteric activation mechanism. 9 Based on the atomic model of the Rpn8/Rpn11 dimer in the RP, a possible mechanism of this allosteric activation was proposed: the C-termini are part a helical bundle, which was hypothesized to block the active site of the isolated lid and to free the active site upon integration into the RP. 4
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Perspective: Regulation of 26S Proteasome Activities
Fig. 3. The mobility of the 19S RP. (a) Regions of highest mobility (marker with warm colors using the variance analysis) are situated in the RP; (b) an example of wagging motions made by the 19S RP; one conformation shown in solid and another shown in cross-hatched.
The proteasome-associated deubiquitylating enzyme Ubp6 and its mammalian homolog Usp14 also get activated upon binding to the 26S proteasome 46; the activity of Ubp6 is increased approximately 300fold due to a long-range interaction with the RP. 47 Another deubiquitylating enzyme, Uch37, was found to associate with proteasomes in most species other than Saccharomyces cerevisiae has also been shown to be allosterically regulated. 48–50 In cytoplasm, Uch37 is self-inhibited by its C-terminus and unable to hydrolyze diubiquitin. When it is recruited to the 26S proteasome through binding to the human ortholog of Rpn13 (Adrm1), the self-inhibition is relieved and deubiquitylating activity is significantly increased. Interestingly, the formation of isolated Uch37–Adrm1 complexes does not lead to activation hinting at a more complex mechanism. The non-ATPases undergo a “wagging” or “wobbling” motion The structure of the 26S proteasome suggests that movements of the ATPase propagate to the nonATPases. The horseshoe scaffold formed by PCI subunits of the lid contacts the CP via two subunits— Rpn5 and in particular Rpn6. 51 Interestingly, upregulation of Rpn6 has been shown to be responsible for increased proteasome-dependent activities in stem cells from different organisms. 52,53 However, it remains to be shown whether this effect is primarily due to stoichiometric limitations in 26S proteasome
assembly or this relates to allosteric effects in the regulatory network. Rpn5 and Rpn6 are also the major contact sites between the ATPases and the lid. Conformational changes in the ATPase ring resulting from a change in the nucleotide state, for example, triggered by ATP hydrolysis, may result in movements of the whole lid. In this case, the Rpn5/6-CP contact is preserved and the ATPase pushing these PCI subunits makes them act as a lever and change the positioning of the apex of the RP. As a result, structural features such as the OB (oligonucleotide binding) domain of the ATPases may move affecting trafficking of the substrate through its central pore or may also change the positions of Ub receptors with respect to the ATPases. Our preliminary EM data have indicated that such kinds of movements do occur (Fig. 3). In principle, they are similar to the whole RP “wagging movements” seen in the low-resolution studies of the 26S proteasome long time ago. 54 Improvements in data acquisition and therefore resolution of the data set allowed now not only for more detailed insights into motions of the RP but also for a dissection of the intersubunit interactions responsible for them. 4,8 The dependence of the lid conformation on the nucleotide state of the ATPase ring explains some previous observations that binding of ATP positively regulates binding and degradation of protein substrates. 41,55 We expect that further studies will provide deeper insight into the regulatory role of RP and help to
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Perspective: Regulation of 26S Proteasome Activities
explain previous functional observations such as the role of ATP binding in substrate hydrolysis.
Summary
2. 3.
26S proteasome is regulated at different levels, which allows for a controlled execution of its proteolytic roles in the cell. Many of its functions, such as substrate recognition, deubiquitylation, unfolding, and degradation, appear to be controlled allosterically. The challenge ahead of us is now to elucidate their structural basis. Recent developments in methodology including the ability to dissect coexisting conformations in highly dynamic assemblies by cryo-EM and advanced image classification approaches will allow in-depth studies to be performed on these effects. We expect that a better understanding of the proteasome's regulation will allow for the development of more selective therapeutic agents targeting steps upstream of the final proteolytic cleavage event.
4.
5.
6.
7.
8.
Acknowledgements
9.
Our research is supported by funding from European Union Seventh Framework Programme PROSPECTS (Proteomics Specification in Space and Time Grant HEALTH-F4-2008-201648), Deutsche Forschungsgemeinschaft (SFB-1035 to W.B.), Human Frontiers Science Program (Career Development Award to F.F.), and European Molecular Biology Organization and Marie Curie Actions (EMBO LongTerm Fellowship to P.Ś.). We thank Antje Aufderheide and Florian Beck for help with Fig. 3. Received 30 November 2012; Received in revised form 30 January 2013; Accepted 30 January 2013 Available online 8 February 2013
10.
11. 12. 13.
14. Keywords: allostery; 26S proteasome; proteostasis; cryo-electron microscopy Abbreviations used: CP, core particle; RP, regulatory particle, EM, electron microscopy.
References 1. Voges, D., Zwickl, P. & Baumeister, W. (1999). The 26S proteasome: a molecular machine designed for
15.
16.
17.
controlled proteolysis. Annu. Rev. Biochem. 68, 1015–1068. Tanaka, K. (2009). The proteasome: overview of structure and functions. Proc. Jpn. Acad., Ser. B, 85, 12–36. Finley, D. (2009). Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu. Rev. Biochem. 78, 477–513. Beck, F., Unverdorben, P., Bohn, S., Schweitzer, A., Pfeifer, G., Sakata, E. et al. (2012). Near-atomic resolution structural model of the yeast 26S proteasome. Proc. Natl Acad. Sci. USA, 109, 14870–14875. Förster, F., Lasker, K., Nickell, S., Sali, A. & Baumeister, W. (2010). Toward an integrated structural model of the 26S proteasome. Mol. Cell. Proteomics, 9, 1666–1677. Löwe, J., Stock, D., Jap, B., Zwickl, P., Baumeister, W. & Huber, R. (1995). Crystal structure of the 20S proteasome from the archaeon T. acidophilum at 3.4 Ǻ resolution. Science, 268, 533–539. Groll, M., Ditzel, L., Löwe, J., Stock, D., Bochtler, M., Bartunik, H. D. & Huber, R. (1997). Structure of 20S proteasome from yeast at 2.4 Ǻ resolution. Nature, 386, 463–471. Lasker, K., Förster, F., Bohn, S., Walzthoeni, T., Villa, E., Unverdorben, P. et al. (2012). Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach. Proc. Natl Acad. Sci. USA, 109, 1380–1387. Lander, G. C., Estrin, E., Matyskiela, M. E., Bashore, C., Nogales, E. & Martin, A. (2012). Complete subunit architecture of the proteasome regulatory particle. Nature, 482, 186–191. Seemüller, E., Lupas, A., Stock, D., Löwe, J., Huber, R. & Baumeister, W. (1995). Proteasome from Thermoplasma acidophilum: a threonine protease. Science, 268, 579–582. Baumeister, W., Walz, J., Zühl, F. & Seemüller, E. (1998). The proteasome: paradigm of a self-compartmentalizing protease. Cell, 92, 367–380. Wenzel, T. & Baumeister, W. (1995). Conformational constraints in protein degradation by the 20S proteasome. Nat. Struct. Biol. 2, 199–204. Ruschak, A. M., Religa, T. L., Breuer, S., Witt, S. & Kay, L. E. (2010). The proteasome antechamber maintains substrates in an unfolded state. Nature, 467, 868–871. Sharon, M., Witt, S., Felderer, K., Rockel, B., Baumeister, W. & Robinson, C. V. (2006). 20S proteasomes have the potential to keep substrates in store for continual degradation. J. Biol. Chem. 281, 9569–9575. Martin, A., Baker, T. A. & Sauer, R. T. (2008). Pore loops of the AAA + ClpX machine grip substrates to drive translocation and unfolding. Nat. Struct. Mol. Biol. 15, 1147–1151. Förster, F., Lasker, K., Beck, F., Nickell, S., Sali, A. & Baumeister, W. (2009). An atomic model AAAATPase/20S core particle sub-complex of the 26S proteasome. Biochem. Biophys. Res. Commun. 388, 228–233. Sauer, R. T. & Baker, T. A. (2011). AAA + proteases: ATP-fueled machines of protein destruction. Annu. Rev. Biochem. 80, 587–612.
1422 18. Verma, R., Aravind, L., Oania, R., McDonald, W. H., Yates, J. R., 3rd, Koonin, E. V. & Deshaies, R. J. (2002). Role of Rpn11 metalloprotease in deubiquitination and degradation by the 26S proteasome. Science, 298, 611–615. 19. Yao, T. & Cohen, R. E. (2002). A cryptic protease couples deubiquitination and degradation by the proteasome. Nature, 419, 403–407. 20. Sakata, E., Bohn, S., Mihalache, O., Kiss, P., Beck, F., Nagy, I. et al. (2012). Localization of the proteasomal ubiquitin receptors Rpn10 and Rpn13 by electron cryomicroscopy. Proc. Natl Acad. Sci. USA, 109, 1479–1484. 21. Deveraux, Q., Ustrell, V., Pickart, C. & Rechsteiner, M. (1994). A 26S protease subunit that binds ubiquitin conjugates. J. Biol. Chem. 269, 7059–7061. 22. van Nocker, S., Sadis, S., Rubin, D. M., Glickman, M., Fu, H., Coux, O. et al. (1996). The multiubiquitinchain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a nonessential, substrate-specific role in protein turnover. Mol. Cell. Biol. 16, 6020–6028. 23. Xie, Y. & Varshavsky, A. (2000). Physical association of ubiquitin ligases and the 26S proteasome. Proc. Natl Acad. Sci. USA, 97, 2497–2502. 24. Crosas, B., Hanna, J., Kirkpatrick, D. S., Zhang, D. P., Tone, Y., Hathaway, N. A. et al. (2006). Ubiquitin chains are remodeled at the proteasome by opposing ubiquitin ligase and deubiquitinating activities. Cell, 127, 1401–1413. 25. Kisselev, A. F., Akopian, T. N., Castillo, V. & Goldberg, A. L. (1999). Proteasome active sites allosterically regulate each other, suggesting a cyclical bite–chew mechanism for protein breakdown. Mol. Cell, 4, 395–402. 26. Osmulski, P. A., Hochstrasser, M. & Gaczynska, M. (2009). A tetrahedral transition state at the active sites of the 20S proteasome is coupled to opening of the αring channel. Structure, 17, 1137–1147. 27. Kleijnen, M. F., Roelofs, J., Park, S., Hathaway, N. A., Glickman, M., King, R. W. & Finley, D. (2007). Stability of the proteasome can be regulated allosterically through engagement of its proteolytic active sites. Nat. Struct. Mol. Biol. 14, 1180–1188. 28. Tian, G., Park, S., Lee, M. J., Huck, B., McAllister, F., Hill, C. P. et al. (2011). An asymmetric interface between the regulatory and core particles of the proteasome. Nat. Struct. Mol. Biol. 18, 1259–1267. 29. Zhang, F., Hu, M., Tian, G., Zhang, P., Finley, D., Jeffrey, P. D. & Shi, Y. (2009). Structural insights into the regulatory particle of the proteasome from Methanocaldococcus jannaschii. Mol. Cell, 34, 473–484. 30. Bohn, S., Beck, F., Sakata, E., Walzthoeni, T., Beck, M., Aebersold, R. et al. (2010). Structure of the 26S proteasome from Schizosaccharomyces pombe at subnanometer resolution. Proc. Natl Acad. Sci. USA, 107, 20992–20997. 31. Smith, D. M., Kafri, G., Cheng, Y., Ng, D., Walz, T. & Goldberg, A. L. (2005). ATP binding to PAN or the 26S ATPases causes association with the 20S proteasome, gate opening, and translocation of unfolded proteins. Mol. Cell, 20, 687–698. 32. Smith, D. M., Chang, S. C., Park, S., Finley, D., Cheng, Y. & Goldberg, A. L. (2007). Docking of the
Perspective: Regulation of 26S Proteasome Activities
33.
34.
35. 36.
37. 38.
39.
40.
41.
42. 43. 44. 45.
46.
47.
48.
proteasomal ATPases' carboxyl termini in the 20S proteasome's α ring opens the gate for substrate entry. Mol. Cell, 27, 731–744. Rabl, J., Smith, D. M., Yu, Y., Chang, S. C., Goldberg, A. L. & Cheng, Y. (2008). Mechanism of gate opening in the 20S proteasome by the proteasomal ATPases. Mol. Cell, 30, 360–368. Stadtmueller, B. M., Ferrell, K., Whitby, F. G., Heroux, A., Robinson, H., Myszka, D. G. & Hill, C. P. (2010). Structural models for interactions between the 20S proteasome and its PAN/19S activators. J. Biol. Chem. 285, 13–17. Stadtmueller, B. M. & Hill, C. P. (2011). Proteasome activators. Mol. Cell, 41, 8–19. Yu, Y., Smith, D. M., Kim, H. M., Rodriguez, V., Goldberg, A. L. & Cheng, Y. (2010). Interactions of PAN's C-termini with archaeal 20S proteasome and implications for the eukaryotic proteasome–ATPase interactions. EMBO J. 29, 692–702. Li, X. & DeMartino, G. N. (2009). Variably modulated gating of the 26S proteasome by ATP and polyubiquitin. Biochem. J. 421, 397–404. Bech-Otschir, D., Helfrich, A., Enenkel, C., Consiglieri, G., Seeger, M., Holzhütter, H. G. et al. (2009). Polyubiquitin substrates allosterically activate their own degradation by the 26S proteasome. Nat. Struct. Mol. Biol. 16, 219–225. Peth, A., Besche, H. C. & Goldberg, A. L. (2009). Ubiquitinated proteins activate the proteasome by binding to Usp14/Ubp6, which causes 20S gate opening. Mol. Cell, 36, 794–804. Deriziotis, P., André, R., Smith, D. M., Goold, R., Kinghorn, K. J., Kristiansen, M. et al. (2011). Misfolded PrP impairs the UPS by interaction with the 20S proteasome and inhibition of substrate entry. EMBO J. 30, 3065–3077. Smith, D. M., Fraga, H., Reis, C., Kafri, G. & Goldberg, A. L. (2011). ATP binds to proteasomal ATPases in pairs with distinct functional effects, implying an ordered reaction cycle. Cell, 144, 526–538. Enemark, E. J. & Joshua-Tor, L. (2006). Mechanism of DNA translocation in a replicative hexameric helicase. Nature, 442, 270–275. Martin, A., Baker, T. A. & Sauer, R. T. (2005). Rebuilt AAA + motors reveal operating principles for ATPfuelled machines. Nature, 437, 1115–1120. Thomsen, N. D. & Berger, J. M. (2009). Running in reverse: the structural basis for translocation polarity in hexameric helicases. Cell, 139, 523–534. Glynn, S. E., Nager, A. R., Baker, T. A. & Sauer, R. T. (2012). Dynamic and static components power unfolding in topologically closed rings of a AAA + proteolytic machine. Nat. Struct. Mol. Biol. 19, 616–622. Borodovsky, A., Kessler, B. M., Casagrande, R., Overkleeft, H. S., Wilkinson, K. D. & Ploegh, H. L. (2001). A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14. EMBO J. 20, 5187–5196. Leggett, D. S., Hanna, J., Borodovsky, A., Crosas, B., Schmidt, M., Baker, R. T. et al. (2002). Multiple associated proteins regulate proteasome structure and function. Mol. Cell, 10, 495–507. Yao, T., Song, L., Xu, W., DeMartino, G. N., Florens, L., Swanson, S. K. et al. (2006). Proteasome
Perspective: Regulation of 26S Proteasome Activities
recruitment and activation of the Uch37 deubiquitinating enzyme by Adrm1. Nat. Cell Biol. 8, 994–1002. 49. Hamazaki, J., Iemura, S., Natsume, T., Yashiroda, H., Tanaka, K. & Murata, S. (2006). A novel proteasome interacting protein recruits the deubiquitinating enzyme UCH37 to 26S proteasomes. EMBO J. 25, 4524–4536. 50. Qiu, X.-B., Ouyang, S. Y., Li, C. J., Miao, S., Wang, L. & Goldberg, A. L. (2006). hRpn13/ADRM1/GP110 is a novel proteasome subunit that binds the deubiquitinating enzyme, UCH37. EMBO J. 25, 5742–5753. 51. Pathare, G. R., Nagy, I., Bohn, S., Unverdorben, P., Hubert, A., Körner, R. et al. (2012). The proteasomal subunit Rpn6 is a molecular clamp holding the core and regulatory subcomplexes together. Proc. Natl Acad. Sci. USA, 109, 149–154.
1423 52. Vilchez, D., Boyer, L., Morantte, I., Lutz, M., Merkwirth, C., Joyce, D. et al. (2012). Increased proteasome activity in human embryonic stem cells is regulated by PSMD11. Nature, 489, 304–308. 53. Vilchez, D., Morantte, I., Liu, Z., Douglas, P. M., Merkwirth, C., Rodrigues, A. P. et al. (2012). RPN-6 determines C. elegans longevity under proteotoxic stress conditions. Nature, 489, 263–268. 54. Walz, J., Erdmann, A., Kania, M., Typke, D., Koster, A. J. & Baumeister, W. (1998). 26S proteasome structure revealed by three-dimensional electron microscopy. J. Struct. Biol. 121, 19–29. 55. Peth, A., Uchiki, T. & Goldberg, A. L. (2010). ATPdependent steps in the binding of ubiquitin conjugates to the 26S proteasome that commit to degradation. Mol. Cell, 40, 671–681.