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DAVID M. RUBIN AND DANIEL FINLEY The : a -degrading ? The crystal structure of the proteasome suggests that degradation of -protein conjugates is achieved by unfolding the protein and translocating it through a channel into a peptidase-containing chamber.

The specificity pockets of intestinal such as Proteasome active sites have long guided thinking about selectivity in Proteasome subunits are arranged into four heptameric proteolysis. Using the polypeptide ubiquitin to tag pro- rings, which are stacked together to form a hollow teins for degradation has, by contrast, seemed a rather cylinder (Fig. 1). In the proteasome from Thermoplasma baroque approach to the problem of substrate discrimina- acidophilum, an archaebacterium, the subunits are of two tion. We now know that ubiquitination accounts for the types, at and 3, located in the outer and inner rings, turnover of , -dependent inhibitors, respectively (Fig. 1) [3]. The eukaryotic forms of these , c-Jun and other oncoproteins [1,2]. However, it has subunits are surprisingly similar to those of T acidophilum, remained unclear why, of the many known proteolytic although in the a and 13 genes are differenti- mechanisms, only this one has evolved a tagging step. ated into families. In the Saccharomyces cerevisiae, for Recent studies of the 26S , a 2 megadalton com- example, there are seven at subunit genes and seven 3 plex that degrades ubiquitinated , have pointed subunit genes. The yeast proteasome presumably contains to an elegant explanation [3]. two copies of each subunit, resulting in a structure with two-fold (C-2) symmetry [10]. The ability of the 26S protease to degrade proteins gen- erally depends on both the ubiquitination of the substrate Specific, -penetrating inhibitors of the proteasome and the presence of ATP. There are, however, exceptions. have been discovered recently [11,12]. These inhibitors Studies of showed that these are active-site-directed agents that covalently bind to 3 requirements can be uncoupled: its degradation requires subunits of the proteasome, identifying them as the ATP but not ubiquitin [4]. Moreover, oligopeptide degra- proteolytically active subunits [3,12]. Remarkably, the co- dation requires neither ubiquitin nor ATP. Based on the valently modified residue in both eukaryotic and archae- crystal structure of the catalytic core of the 26S protease bacterial is an amino-terminal [3], these disparate observations can be reconciled by (Thrl) [3,12]; the proteasome therefore exemplifies a new postulating that ubiquitin and ATP act in different ways class of proteases that have threonine as the active-site to promote the passage of substrates through the 'mol- nucleophile. In support of this view, substitution of Thrl ecular sieve'. At the distal end of the sieve are the pro- with serine in the archaebacterial 13 subunit results in a teolytic active sites that degrade the protein. It is the fully active protease that has acquired sensitivity to serine ability of a protein to pass through the sieve, therefore, protease inhibitors [13]. Surprisingly, a serine residue has that may qualify it as a substrate. never been found at position 1 in either archaebacterial or eukaryotic proteasomes. Perhaps threonine serves more The catalytic core of the 26S protease is a 20S particle effectively than serine in amino-terminal propeptide known as the proteasome (Fig. 1). Although the chem- removal, which has not been assayed using the mutant istry of degradation occurs in this complex, isolated pro- proteasomes [13]. Propeptide removal may be essential for teasomes are inactive on physiological substrates and activity because of the extraordinary placement of the their activity is not stimulated by either ATP or ubiqui- at the amino terminus of the mature protein. tin [5]. The ability of the proteasome to degrade physio- The amino group of Thrl might actually participate in logical substrates is restored by a complex known as the , substituting for histidine as a general base in the 19S particle (Fig. 1) [5-8]. A specific subunit of the 19S of serine proteases [3,13]. particle appears to mediate recognition of ubiquitin-pro- tein conjugates [9]; this subunit (S5) binds polymers of In the archaebacterial proteasome, all of the 3 subunits are ubiquitin even when they have not been linked to any identical and therefore presumably active. In eukaryotes, substrate. Such binding events, which presumably initiate by contrast, three of the seven 3 subunits may be proteo- the degradative process, appear to be ATP-independent, lytically active, based on the presence of a Thrl residue as is the final event of -bond cleavage. The ATP- [13]. This is consistent with the existence of at least three dependence of 26S function may therefore reflect an distinct peptidase specificities in the eukaryotic protea- intermediate step in the mechanism. As ubiquitin bind- some, as suggested previously by experiments with chro- ing and peptide-bond cleavage occur within different mogenic peptide substrates [5,10,14]. Thus, the protea- parts of the 26S protease, the intermediate step may some appears to contain separate active sites for involve translocation of the substrate from the 19S bearing basic, acidic and hydrophobic residues on the particle to the proteasome. amino-terminal side of the scissile bond (the P1 position).

854 © Current Biology 1995, Vol 5 No 8 DISPATCH 855

Fig. 1. Structural features of the 26S protease. Left, contour plot of the 26S protease derived from electron micro- scopy and image analysis [24]. Top right, schematic cross-section of the proteasome, showing the location of a subunits (red), subunits (blue) and peptidase active sites (yellow). Bottom right, schematic view of subunit arrangement in the proteasome.

Interestingly, defects in the -like activity of multiple influenza is defective in the the yeast proteasome can result from substitutions in absence of LMP-2 expression [17,18]. either of two 3 subunits, Prel or Pre2 [15]. Whereas Pre2 contains Thrl and is therefore presumably active, One explanation for the phenotype of the LMP-2 and Prel lacks the Thrl residue. These observations, LMP-7 mutants is that replacing active subunits may together with the apparent inactivity of isolated P3sub- bias the spectrum of oligopeptide degradation products. units, have suggested the existence of cooperative inter- As peptides produced by the proteasome seem to serve as actions between 3 subunits. By analogy to the structure raw material for class I presentation [11,16], an of the archaebacterial proteasome, it is now apparent that altered cleavage pattern could enhance the ability of the the pre2-2 results in the replacement of an immune system to respond to foreign antigens. In this active-site residue. Moreover, the prel-1 mutation can model, the inducible '' generates apparently inhibit Pre2 activity specifically, because the peptides with a high affinity for downstream components substituted residue lies within an c helix involved in of the antigen-presentation pathway, such as the TAP V3-3 subunit interactions. Similarly, substitutions in peptide transporter, which shuttles peptides from the another two 3 subunits (Pre3 and Pre4) can inhibit the to the , or the class I ability of proteasomes to cleave peptides with acidic P1 MHC molecule itself. The influence of inducible [3 sub- residues [10]. units on the pattern of cleavage sites has yet to be demonstrated for physiological substrates. However, the In , each of the three active 3 subunits is found immunoproteasome has an enhanced ability to cleave in distinct basal and inducible forms [13,14]. Induction is chromogenic peptide substrates with hydrophobic mediated by y-interferon-dependent transcriptional reg- residues at the P1 position [14]. It might therefore prefer- ulation in response to viral or other stresses. entially generate degradation products with hydrophobic That inducible [3 subunits play a role in class I antigen carboxyl termini, which are high-affinity substrates for presentation is well-documented [16-18]. For example, the TAP transporter [19,20]. mice with a targeted deletion of the gene encoding the inducible LMP-7 subunit present certain antigens ineffi- Proteasome structure ciently. These mice also have reduced cell-surface expres- The crystal structure of the T acidophilum proteasome [3] sion of MHC class I molecules, which can be restored by shows that its active sites face the central cavity (Fig. 1). the addition of exogenous peptide [16]. This implies that The significance of this observation follows from the LMP-7 is required for peptide supply to MHC class I inaccessible nature of the cavity as a whole. The side molecules. Similar observations are reported for LMP-2 walls of the proteasomal cylinder contain no major open- on pages 923-930 of this issue [18]. The presentation of ings, whereas the faces of the cylinder contain channels 856 Current Biology 1995, Vol 5 No 8

that are remarkably narrow - no more than 13 A in unfolding by interacting directly with the substrate protein diameter (Fig. 1) [3]. In comparison, a helices have and having a higher affinity for its unfolded form. diameters of approximately 11 A. Thus, folded proteins would not be expected to pass through this channel, as In this hypothetical model, the bind peptide confirmed in a recent study [21]. Even a 14-residue pep- segments that are buried in the folded structure of the tide such as somatostatin cannot be degraded by the pro- protein. Substrate sequences that can interact with the teasome without prior reduction of its bond. ATPase would then be expected to be required for Moreover, when 20 A nanogold particles are conjugated degradation. However, many degradation signals have to substrate peptides, they accumulate at the orifice of been characterized and shown to function by triggering the proteasome as if they are too large to enter the cavity. ubiquitination rather than a later step in the pathway [1]. In such complexes, the nanogold-linked peptide cannot Perhaps the recognition sites for 26S ATPases are suffi- be degraded, nor are free peptides degraded when added ciently degenerate that a typical protein would have subsequently. This indicates that substrate traffic is redundant target sites. It is also possible that the ATPases restricted to the orifices at the ends of the cylinder [21]. have different binding specificities, further increasing the Another implication of these data is that peptides may range of substrate sequences that can be bound. These have a significant affinity for the proteasome channel. considerations may help to explain the ability of the 26S protease to degrade a wide variety of multiubiquitinated The need to sequester major proteases from the cyto- proteins, including proteins that are not normally plasm was suggested decades ago by the discovery of the degraded but have been engineered to carry a ubiquiti- , and subsequent findings that most cytoplasmic nation signal [1]. The ability of the 26S protease to do so proteins are not degraded in only reinforced implies that it has a remarkably robust ability to unfold the problem. It now appears that the tight shell of the proteins of different kinds. proteasome substitutes for the phospholipid barrier in forming a compartmentalized space that is analogous to Presumably, the ATPases act preferentially on ubiquiti- the lumen of a cytoplasmic organelle. Another similarity nated proteins (see below). According to this view, sub- to protein import into is in the suggested strates follow an ordered pathway through the 26S requirement for proteins to be unfolded before entering protease, interacting in a stepwise fashion with a ubiqui- the hydrolytic chamber of the proteasome [21,22]. tin-binding site on subunit S5 [9], an unfolding site that Unfolding is clearly required for passage through the pro- includes the ATPases, the protein-translocating channel tein-conducting channels of the endoplasmic reticulum and finally the peptidase sites of the J3 subunits. This and [23]. In the case of the proteasome, putative cycle is depicted in Figure 2. the channel has a hydrophobic character. Each a subunit contributes a tyrosine residue to form the 13 A sieve; Translocation tyrosines (as well as methionines) are also present within The translocation of substrate into the proteasome poses an internal, 22 A constriction in the cavity of the protea- an interesting set of problems. Which portion of the sub- some (Fig. 1). The hydrophobicity of these inner surfaces strate leads its migration into the proteasome? Is it simply may stabilize the unfolded forms of substrate proteins as the structural domain that is most susceptible to unfold- they migrate toward the active sites [3]. ing? Another possibility is that the ubiquitination site of the substrate, being anchored to the 19S particle by the The channels leading into the hydrolytic chamber of the ubiquitin chain, is prevented from leading translocation. proteasome open on the outside directly into the 19S Can only free ends lead translocation of the substrate, or particle (Fig. 1) [24]. Therefore, one may assume that is the channel large enough that extended polypeptide the 19S particle forms a sister passage which is continu- chains can pass through as loops? ous with the proteasome channel. Remarkably, the 19S particle contains at least four distinct ATPase subunits The driving force for translocation remains uncertain, [6-8]. Because interaction of the 19S particle with the even in many cases of protein translocation across mem- proteasome is ATP-dependent [25], the ATPases possibly branes [26]. Recent studies suggest that the unidirection- reside at the base of the 19S, contacting the proteasome ality of protein transport into mitochondria is imparted directly and lining the substrate's pathway through the by the sequential binding of molecules in the 19S particle. matrix space [27]. Bound Hsp70 may act as a molecular ratchet, sterically interfering with retrograde motion. In Unfolding the case of the proteasome, it is plausible that the forward As described above, studies of the proteasome imply that progress of translocation results from of the some factor must act upstream in the pathway to unfold substrate at the distal end of the channel. This mechanism proteolytic substrates [3,21]. The 19S particle could medi- would differ from that of mitochondrial protein import in ate the unfolding because it directly contacts the protea- that the process driving translocation (peptide-bond some channel. This model is further supported by the cleavage) does not do so by preventing retrograde motion presence of multiple ATPases in the 19S particle - of the body of the substrate. However, because the pepti- ATPases are known to modulate in other dase sites are deep within the cavity of the proteasome, systems [23]. The 26S ATPases might mediate protein substantial retrograde motion could occur after a cleavage DISPATCH 857

Fig. 2. The putative proteasome cycle. The proteolytic substrate is shown in red, ubiquitin in green. The peptidase sites are shown as blue scissors. For simplicity, the 26S protease is shown with a single 19S particle; the 19S parti- cle is shown without the protein mass that occupies the mouth of the wedge (see Fig. 1). Degradation of the substrate to oligopeptides is accompanied by the regeneration of free ubiquitin mol- ecules. Ubiquitin is activated at its car- boxyl terminus by an El , then passed to an E2 enzyme, and finally to an E3 enzyme (ubiquitin-protein ligase) [32]. All three form thiolester bonds to ubiquitin, although in some cases the E2 enzyme might directly donate ubiquitin to target proteins [32]. The proteolytic substrate is linked to ubiquitin by an isopeptide bond. Multi- ple ubiquitin groups are added to the substrate sequentially to form a multi- ubiquitin chain. The steps of the protea- some cycle are discussed in the text.

event without aborting translocation. Energetically favor- the GroE, protein folding is inefficient in that able interactions, between the substrate and the substrates must undergo multiple cycles of GroE-binding hydrophobic surfaces of the channel and the internal, and ATP-driven release in order to fold [30]. Both pro- 22 A constriction, might also serve to minimize abortive tein unfolding and translocation by the 26S protease translocation. The same interactions could facilitate initial might similarly require multiple trials to reach com- 'threading' of the channel as well. pletion. However, because the conjugate is anchored by its ubiquitin tag, the trials might follow one another Ubiquitin tagging without conjugate dissociation from the 26S complex. A major difference between the 26S protease and the Stable binding of the chain during unfolding and translo- simple, ATP-dependent proteases of cation motions of the substrate would require flexibility [28,29], is in the use of a tagging mechanism by the 26S in the conjugate. For example, the rotational freedom in protease. ATPases of the prokaryotic proteases must the ubiquitin-substrate linkage might allow the substrate apparently perform both tasks of substrate selection and to swivel and thus encounter the ATPases in a range of substrate unfolding. By holding the substrate in place, orientations. Also, because the chain is a repeating struc- the ubiquitin tag may serve to increase the local concen- ture, possible jittering in the register of chain-binding tration of substrate in the vicinity of the 26S ATPases, may allow translational freedom along the pathway of and thus to drive the unfolding reaction. In the case of substrate translocation. 858 Current Biology 1995, Vol 5 No 8

Upon degradation of substrate, de-ubiquitinating enzymes most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 1994, 78:761-771. regenerate free ubiquitin from the conjugate [1]. This 12. Fenteany G, Standaert RF, Lane WS, Choi S, Corey EJ,Schreiber SL: suggests that ubiquitin is not threaded into the protea- Inhibition of proteasome activities and subunit-specific amino- some along with substrate (Fig. 2). Perhaps this is a result terminal threonine modification by lactacystin. Science 1995, 268:726-731. of the extreme resistance of ubiquitin to unfolding. 13. Seemuller E, Lupas A, Stock D, Lowe J, Huber R, Baumeister W: Pro- Given that ubiquitin cannot enter the proteasome, its teasome from Thermoplasma acidophilum: a . cleavage from the substrate may be necessary for substrate Science 1995, 268:579-582. 14. Gaczynska M, Rock KL, Goldberg AL: Role of proteasomes in antigen translocation to be completed. If ubiquitin groups were presentation. 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