- Email: [email protected]
Proteasome: from structure to function
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Proteasome: from structure to function Daniela Stock*, Petra M Nederloft, Erika Seem011ert, Wolfgang Baumeistert, Robert Hubert and Jan LSwe During the past two years, significant progress has been made in understanding the structure and function of the proteasome. Recent work has revealed the three-dimensional structure of the 700 kDa proteolytic complex at atomic resolution and elucidated its novel catalytic mechanism. Close relationships to a number of other amino-terminal hydrolases have emerged, making the proteasomal subunits the prototype of this newly discovered structural superfamily.
recognized by subunits localized in the 19S caps [12-14]. These caps also contain subunits with ATP-binding sites [15,16], which may serve to unfold the protein [17,18]. 20S proteasomes without 19S caps only degrade unfolded proteins independently of ATP [19] (Fig. 1), It is now well established that the ubiquitin-proteasome pathway is involved in a muhitude of cellular functions such as cell growth and division, and degradation of short-lived regulatory proteins such as oncogene products [20-22], transcription factors [23-25], and cyclins [26,27].
Addresses
*$Abteilung Strukturforschung, Max-Planck-lnstitutfor Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany t Abteilung fiir Strukturelle Molekularbiologie, Max-Planck-lnstitutfLir Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany * e-mail: [email protected] Current Opinion in Biotechnology 1996, 7:3?6-385
© Current Biology Ltd ISSN 0958-1669 Abbreviations AGA aspartylglucosamidase cNLS complementary nuclear localization signal endoplasmic reticulum ER GAT glutamine PRPP amidotransferase LMP low molecular weight protein nuclear localization signal NLS Ntn amino-terminal nucleophile penicillin acylase PA PRPP 5-phosphoribosyl-1 -pyrophosphate transporter associated with antigen presentation TAP
Introduction Although 20S proteasomes were first described almost 30 years ago [1], their widespread distribution and cellular roles have only recently been defined. T h e 700kDa multicatalytic protease complex is ubiquitous in eukaryotic cells and is also found in a number of archaebacteria and eubacteria [2,3",4*]. It is the central cytosolic protease and is responsible for intracellular protein turnover. It is also critically involved in many regulatory processes and, in higher eukaryotes, in antigen processing. Intracellular protein turnover is carried out by a nonlysosomal, ATP- and ubiquitin-dependent pathway that controls the lifespan of most cellular proteins (for reviews, see [5-10]). T h e 26S proteasome, an approximately 2000 kDa muhiprotein complex, is the proteolytic enzyme responsible for the ATP-dependent degradation of proteins. T h e 20S proteasome forms the catalytic core of the 26S complex, whereas 19S caps, which associate with the termini of this core have multiple nonproteolytic functions [11]. Proteins are marked for degradation by conjugation with ubiquitin. T h e ubiquitinated protein is
There is strong evidence that the proteasome is involved in antigen processing in the M H C I pathway of antigen presentation in higher eukaryotes (for reviews, see [28-31]). T h e proteasome contributes to this pathway by degrading cellular proteins into peptides suitable as antigenic peptides. These are then transferred to the endoplasmic reticulum via transporters associated with antigen presentation (TAPs) 1 and 2 [32]. Here, the peptides assemble with the M H C I heavy chain and 182 microglobulin into the trimolecular MHC complex. This complex is subsequently exported to the plasma membrane where it is presenmd to cytotoxic T lymphocytes (Fig, 2). During the past two years, much progress has been made in elucidating the structure and catalytic mechanism of this important enzyme using both biochemical and biophysical approaches. On the basis of studies with small synthetic peptides and protease inhibitors, at least five distinct proteolytic activities have been identified in the eukaryotic proteasome [33]. T h e proteasome of the archaeon Thermoplasma acidophilum has oOy a single, chymotrypsin-like activity [34]. Experiments with denatured substrates present a somewhat different picture, showing that the enzyme hydrolyzes virtually any peptide bond [35-38]. Nevertheless, the degradation products fall into a rather narrow size range, suggesting that some kind of molecular ruler exists [37]. X-ray structural studies [39°,40], as well as site-directed mutagenesis experiments with the archaebacterial enzyme [41,42°], and localization of the covalently bound inhibitor, lactacystin, in a eukaryotic proteasome subunit [43 °] led to the identification of the proteasome's active site nucleophile and showed that the proteasome represents the first example of a novel class of proteases. Structural comparisons revealed surprising similarity between a growing number of enzymes with amino-terminal hydrolase activity [44,45°]. T h e recently discovered proteasomes from a methanogenic archaeon [3 °] and from a eubacterium [4"] indicate that the proteasome is more widely distributed
Proteasome: from structure to function Stock
et aL
377
Figure 1
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/ The ubiquitin-proteasome pathway. Native proteins are ubiquitinated by a three-enzyme cascade (involving E l - E 3 ) [74,75] that generates a thioester at the carboxyl terminus of the protein. The thioester is then used to form an isopeptide bond to the e-amino group of a lysine residue in the protein. Further ubiquitin (Ub) molecules are linked to the first one, forming chains of ubiquitin on the protein. These chains are recognized by the 26S proteasome, the thioester bonds are hydrolyzed by an isopeptidase, and the substrate is unfolded by an ATPase, all located in the 1 gS caps of the 26S proteasome. The 20S proteasome degrades unfolded or denatured proteins into peptides of defined length (7-14 amino acids) which, together with peptides generated from proteolysis by the 26S proteasome, are either degraded into their constituent amino acids by intracellular exopeptidases or transported to the cell surface via the endoplasmic reticulum (ER) and the Golgi to act as antigenic peptides presented by MHC I molecules.
throughout all three Urkingdoms than was previously assumed. T h e scope of this review follows major advances of the past years, which were mainly obtained from structural studies, using electron microscopy and X-ray crystallography, as well as from using site-directed mutagenesis experiments.
Primary and quaternary structures According to electron microscopic investigations, the quaternary structure of the 20S proteasome is conserved between archaea and humans [2]. T h e cylindrical complex, approximately 150/~ in height and 110,~. in diameter, shows a characteristic pattern of four rings of seven subunits stacked along the cylinder axis ([46]; Fig. 3). T h e archaebacterial proteasome contains only two different subunits, 0~ and 13, whereas proteasomes from eukaryotes have a more complex subunit composition, consisting of multiple or-type and 13-type subunits which are related to the respective subunits of the archaebacterial proteasome [47]. As determined by immunoelectronmicroscopy [48], o~ subunits form the outer rings of the complex, whereas the two inner rings consist of 13 subunits. This relative simplicity made the archaebacterial enzyme a suitable model system for unraveling the structure and catalytic mechanism of the 20S proteasome.
The completion of the primary structure determination of the 14 subunits of the yeast proteasome revealed a high sequence identity of all the subunits [49]. Sequence comparison of all known eukaryotic proteasomal subunits implies the presence of a total of 14 subgroups, which can be divided into seven (~-type and seven p-type groups. These considerations, as well as studies using monoclonal antibodies against individual subunits [50,51], suggest that each of the 14 eukaryotic subunits has a distinct place in the complex. Immunoelectronmicroscopic studies using an antibody against subunit HsN3 from human proteasomes [52] and the structure of the 26S proteasome [53] are in agreement with this concept and reveal the existence of a local dyad of the complex.
Mechanistic and functional studies Much effort has been spent determining the nature and location of the active site of the proteasome. T h e cloning and expression of the Thermoplasma subunits in Escherichia coil [54] was a prerequisite for site-directed mutagenesis studies as well as for the X-ray structure determination. Inhibitor studies had suggested that the 20S proteasome might be an unusual type of cysteine or serine protease [33,34]. After mutating the only cysteine, both histidines, and all the serines of the 13 subunit as well as highly conserved aspartates, it became clear that the proteasome's catalytic mechanism had to be a new one [41].
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Figure 2
(a) Humoral immune response
Cellular immune response CD8 (killer) T lymphocyte
lymphokines
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The role of the proteasome in the immune response. The present model divides the immune system into three interacting pathways attacking antigens (a) in the extracellular space (humoral immune system), (b) in the cytosol, and (¢) during endocytosis, whereby lymphokines act as mediators. (a) Antigens in the extracellular space can be attacked unspecifically by the complement system (not shown) and specifically by antibody molecules, produced by plasma cells derived from B lymphocytes. (b) Protein antigens in the cytosol of most cell types are degraded by the proteasome into small peptides, which are then transported into the ER via TAP and presented on the cell surface by MHC class I molecules. (c) Protein antigens being taken up by endocytosis, mainly by macrophages, are degraded in the lysosome. Peptides are transported into the ER and are presented by MHC class il molecules. Ag, antigen; B, B lymphocyte; P, antibody-presenting cell; T, cytotoxic T lymphocyte.
Another part of the functional puzzle was solved by the electron microscopy of gold-labeled insulin B chains
incubated with Thermoplasma20S proteasomes [18]. T h e 1.5 nm gold particles were clearly visible at the entrance
Proteasome: from structure to function Stock et aL
Figure 3
379
The 'immuno-proteasome'
T h e efficiency of peptide production for this purpose seems to be further optimized in proteasomes containing the three M H C li encoded proteasomal subunits, low molecular weight protein (LMP)2, LMP7, and LMP10, which can replace their constitutive counterparts upon interferon-y stimulation. Rock and coworkers [55] had shown previously that inhibitors of the proteasome are able to block antigen presentation in vivo. Meanwhile, these findings have been corroborated by investigations using an LMP2-deficient cell line [56 °] and LMP7 knock-out mice [57], which were both shown to have reduced MHC I-restricted antigen presentation. T h e latter studies, as well as the investigations done by Kuckelkorn and coworkers [58], showed that the incorporation of a single LMP subunit or of both LMP2 and LMP7 into the proteasome alters its cleavage-site preference and thus the quality of the generated peptides. These results are consistent with the findings of Nandi and coworkers [59°], who recently discovered a third interferon-y inducible subunit, LMP10; they suggcsted that the three interferon-y induced proteasomal subunits can replace the constitutive 13-type subunits and increase the repertoire of potentially antigenic peptides by increasing the populations of proteasomes with different cleavage-site preferences in the cell.
The tertiary structure
The structure of the 20S proteasome. (a) Averaged electron micrograph of negatively stained Thermoplasma20S proteasomes. (b) Subunit stoichiometry of the complex (s-type subunits are shown lightly shaded and [[]-type subunits are darkly shaded).
to the putative channel of the proteasome, thereby inactivating the complex. This experiment suggested that the site of proteolytic cleavage is located in an inner compartment of the proteasome that is not accessible to large substrates or folded proteins. Further clues regarding the function of the proteasome were obtained by investigating the cleavage products: it could be shown that the incubation of the proteolytic complex with either oxidized human hemoglobin or with the insulin B chain resulted in degradation products of a rather narrow size range, peaking around hepta- to nonapeptides [36-38]. This observation led to the proposal of a molecular ruler determining the length of the cleavage products [37]. Taking into account that M H C I molecules preferentially bind peptides of 9 (+1) residues, the proteasome appears to be capable of providing well tailored peptide fragments for the cellular immune response.
In order to obtain more detailed insights into the proteolytic machinery, we have analyzed the crystal structure of the Thermoplasma 20S proteasome. T h e recombinant protein could be crystallized [60], and the crystal structure was determined to a resolution of 3.4~, using single isomorphous replacement and cyclic averaging techniques, which were facilitated by the high internal symmetry of the archaebacterial complex [39*]. T h e overall features of the crystal structure were in good agreement with the information obtained by electron microscopy at lower resolution. T h e core of the proteasome revealed a number of previously unknown features: the four rings of the ~ and 13 subunits encase a channel that penetrates the complex along the sevenfold axis and that widens into three large cavities that are limited by narrow constrictions (Figs 4 and 5). T h e two outermost constrictions form a passage from the cytosol to the inside of the cylinder and are only 13A in diameter. T h e crystal structure of the proteasome in complex with a small peptide inhibitor localized the active sites within the central cavity of the complex. Thus, the structure of the proteasome seems to be well designed to protect cytosolic proteins from being arbitrarily degraded. T h e fact that the proteasome buries its proteolytic compartment in its interior allows it to control access of the substrates. Hence the proteasome could be substituted for an organelle with complex membrane and targeting systems, which appears to be the reason for the unusual size of the proteinase.
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Figure 4 The crystal structure of the Thermoplasma20S proteasome. A ribbon drawing of a cross section through the proteasome, cut open along the sevenfold axis. The penetrating channel is widened by three large cavities that are limited by narrow constrictions (see also Fig. 5). The outer constriction is only 13 ~ in diameter and prevents folded proteins from being degraded. The internal twofold symmetry of the complex is clearly visible at the center of the molecule.
Figure 5 The molecular ruler mechanism of the 20S proteasome. The orientation of the surface representation is identical to that in Figure 4. The 14 active sites are located in two antiparallel rings around the central cavity and are highlighted by showing the amino-terminal threonines as dark van der Waals surfaces. The distance between two neighbouring active sites in one ring corresponds to a peptide length of about seven amino acids in extended conformation. The same is true for twofold related active sites, but these have opposite orientations and are separated by a small constriction between the rings. Some of the possible distances between the successively cleaving active sites that are thought to determine the length of the cleavage products are indicated by black bars. The molecular ruler mechanism is thought to act on a statistical basis that determines the average size of the resulting products to be in the range of heptapeptides to nonapeptides.
Another surprising phenomenon highlighted by the crystal structure was the strong conservation of the fold of the two subunits, ot and 13, in spite of their low sequence identity (25%). T h e molecular structure consists of a central five-stranded 13 sandwich, flanked by two bundles of
ot helices, one above and one below. T h e amino-terminal 35 amino acid extension of the ~ subunit forms an additional c~ helix in front of the l-sheet cleft (Fig. 6). Because eukaryotic m-type and [3-type subunits display even higher sequence identities to their corresponding ar-
Proteasome: from structure to function Stock et aL
chaebacterial prototypes, and because secondary structure predictions suggest a strong conservation of the fold of all proteasomal subunits, it can be expected that the tertiary structure is also highly conserved (J L6we, unpublished data). Proteasomal o~-type subunits contain sequences resembling nuclear localization signals (NLSs) that are also found in the Thermoplasma proteasome [61]. Meanwhile, it has been demonstrated that the putative NLS sequences are functional in vitro [62°]. Upon transplanting them to large non-nuclear reporter molecules, the NLS sequences direct the reporter molecules into the nuclei of HeLa or 3T3 cells. It remains to be established whether the NLS and the complementary NLS (cNLS) sequences, which are in close proximity to each other in the structure, do indeed interact. A model suggesting the masking and unmasking of the NLS by the cNLS, whereby phosphorylation acts as a switch, had been put forward originally by Tanaka and coworkers [63]. Figure 6
381
have similar catalytic mechanisms: they all use the aminoterminal amino acid as a nucleophile [44,45°]. Presently known members of this family are the glutamine PRPP (5phosphoribosyl-l-pyrophosphate) amidotransferase (GAT) [64], the penicillin acylase (PA) [65], the proteasome, and the aspartylglucosamidase (AGA)[66]. GAT uses an amino-terminal cysteine as a nucleophile, PA uses a serine, and AGA and the proteasome 13 subunit use a threonine. T h e architecture of the Ntn fold, characterized by a core of two stacked antiparallel 13 sheets flanked on both sides by ~ helices, is most simply exemplified by the proteasome subunits, whereas GAT, PA, and AGA have several extra 13 strands and o~ helices (Fig. 7). Moreover, all these hydrolascs are post-translationally processed [45°]. It can be assumed that the Ntn fold not only provides the capacity for nucleophilic attack but also for autocatalytic cleavage. In addition, the four enzymes show significant differences in their tertiary structures and have completely different quaternary structures. Because there is no obvious sequence homology between the enzymes and there are differences in the topology (i.e. in the connectivities between secondary structure elements), particularly between GAT and the other members of the family, the evolutionary relationship remains unclear. The catalytic mechanism T h e long enigmatic catalytic nucleophile of the 20S proteasome was identified independently and almost simultaneously by three different approaches, described below.
The structure of the ~ subunit of the Thermoplasma20S proteasome. The ribbon drawing is in standard orientation. The ~ subunits have a nearly identical fold but lack the amino-terminal ec helix on the left side. The highly homologous eukaryotic subunits are predicted to also have the same fold. Thermoplasmaand eukaryotic c~ subunits contain NLSs, and sequences complementary to them (cNLSs), which are thought to be recognized by subunits of the nuclear pore complex. The side chains of the corresponding sequences in the Thermoplasma o~subunit (NLS, residues 49-56; cNLS, residues 201-206) are shown in ball-and-stick representation. Within' the complex, these residues form the top of the cylinder and are easily accessible from the solvent.
T h e p r o t e a s o m e fold: i n h e r i t e d or r e i n v e n t e d ? What was initially assumed to be a new fold unique to the proteasomal subunits turned out to be common to a superfamily of amino-terminal (Ntn) hydrolases that all
Firstly, the X-ray structure analysis of a proteasome crystal in complex with the small peptide aldehyde inhibitor Ac-Suc-Leu-Leu-norleucinal (commercially available as calpain inhibitor I), an inhibitor of the proteasome's chymotryptic activity [39",40], identified the binding site and mode of binding, and suggested a catalytic mechanism. T h e O1 of T h r l of the 13 subunit is the nucleophile and the amino-terminal amino group the proton acceptor. Since Glu1317 and Lys1333 are the only polar residues close to the active site, they are probably also players in the catalytic reaction. T h e oligopeptidase activity (the molecular ruler) may be explained by the fact that neighbouring active sites are about 28A apart from each other, a distance spanned by an octapeptide in extended conformation (Fig. 5). Well-defined substrate binding is only observed in the P-direction, suggesting oligopeptide formation from the carboxyl terminus and thus carboxypeptidase activity of the 20S proteasome. Secondly, cysteine, serine and histidine residues were ruled out of being involved in the catalytic mechanism [41] by site-directed mutagenesis. Another well-conserved amino acid is the amino-terminal threonine, after removal of the prosequence of the Thermoplasma 13 subunit. Substitution of T h r l to alanine yielded a completely inactive protein, whereas substitution with serine turned out to have no obvious ~ffect on proteolysis [42"[.
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Figure 7 A stereoview of the Ntn fold. Superposition of the Ntn fold of the 13subunits of penicillin acylase (lightly shaded; Protein Data Bank [PDB] code: 1PNK) [65] and the Thermoplasma proteasome (darkly shaded; PDB code: 1 PMA) with a root mean square deviation of 1.8 A over 88 Coc atoms. The positions of the amino-terminal amino acids Thrl of the proteasoma113 subunit and Cysl of the PA ]3 subunit, which in both cases act as the catalytic nucleophile, are indicated by showing their side chains (Stick diagram).
Mutagenesis of Glu1817 and Lys1833 as suggested by the X-ray structure also led to inactivation of the proteasome, but this does not imply direct participation in the proteolytic reaction, as conformational changes caused by disruption of the salt bridge formed by these residues may OCCUr.
The third approach involved labeling with lactacystin, a Streptomvces metabolite that was discovered because of its ability to induce neurite outgrowth in the murine neuroblastoma cell line, Neuro-2a, and which also inhibits proliferation of other cells [67]. The target could be identified using 3H-lactacystine which was shown to bind to the amino-terminal threonine of a proteasomal 13-type subunit [43*]. Subsequent kinetic studies proved lactacystin to be the first specific inhibitor of the proteasome. Three distinct proteolytic activities (trypsin-like, chymotrypsinlike and peptidyl-glutamyl-peptide hydrolyzing) were inhibited by lactacystin at different rates, the first two activities irreversibly. The binding of lactacystin to Thrl of the 18subunit from the Thermoplasma proteasome could be verified by X-ray structure analysis of proteasome crystals incubated with lactacystin (J L6we, D Stock, unpublished data). These data, as well as the studies carried out by Dick and coworkers [68], show that a hydrolyzed form of lactacystin is responsible for the inhibition.
New proteasomes The fact that Thermoplasma had long been the only archaeon in which 20S proteasomes had been found led to the proposal that these organisms might have been the precursors of eukaryotic cytoplasm. Maupin-Furlow and Ferry [3°] were recently able to isolate a 20S proteasome from the distantly related methanogenic archaeon Methanosarcina thermophila. Therefore, proteasomes appear to be more widespread amongst archaea than anticipated. The sequence of the two subunits of the methanogenic proteasome revealed a high degree of
sequence similarity to the corresponding subunits of the Thermoplasma proteasome. Much effort has been spent finding eubacterial proteasomes. Sequences related to the proteasomal 13-type subunits were found in a number of eubacteria, including E. co/i, Mvcobacterium /eprae, and Bacillus subtilis [69]. Meanwhile, the first eubacterial 20S proteasome has been discovered and isolated from the nocardioform actinomycete, Rhodococcus sp. [4"]. The isolation and sequencing revealed four types of subunits termed ~1, ~2, 181, and ]32. The topology of the subunits within the complex remains to be established.
Conclusions Biochemical and biophysical approaches have elucidated many facets of the proteasome's structure and mechanism. The availability of the archaebacterial enzyme, having been functionally expressed in E. co/i, has opened up the possibility of characterizing the protein by site-directed mutagenesis [41,42°,54]. This work, as well as inhibitor and X-ray studies, allowed the identification of the active site nucleophile and elucidated the catalytic mechanism of the Thermoplasma enzyme. Because sequence comparisons to eukaryotic subunits revealed a high degree of similarity [47,49], these enzymes are expected to be structurally very similar and to function by the same mechanism. Remaining questions are related to the positions of the 14 different subunits in the complex and to the assignment of the different catalytic activities, although recent kinetic studies on the chymotryptic activity revealed a cooperativity of the active sites [70°]. The first attempts have been made to localize the subunits by means of monoclonal antibodies [52]. A meaningful nomenclature of all proteasomal subunits will have to await the completion of these studies or the elucidation of a three-dimensional structure of a eukaryotic proteasome at atomic resolution.
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The first steps in this direction have already been taken [71].
6.
Hochstrasser M: Ubiquitin, proteasomes, and the regulation of intracellular protein degradation. Curr Opin Ceil Bio/1995, 7:215-223.
The important roles of the proteasome, both in cellular protein turnover and in antigen presentation, have been demonstrated in many experiments. Functional studies were hitherto hampered by the fact that the available inhibitors were not specific enough to distinguish between the proteasome and other cellular proteases. Lactacystin is rather specific, although it inhibits the different activities of the proteasome at different rates [43°]. The search for an inhibitor that simply blocks the narrow entrance to the proteasome's interior appears to be the most promising option.
7.
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Hochstrasser M: Protein degradation or regulation: Ub the judge. Ce//1996, 84:813-815.
1t.
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12.
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13.
Ferrell K, Deveraux Q, Van Nocker S, Rechsteiner M: Molecular cloning and expression of a multiubiquiUn chain binding subunit of the human 26S protease. FEBS Lett 1996, 381:143-~148.
14.
Van Nocker S, Deveraux CI, Rechsteiner M, Vierstra RD: Arabidopsis MBP1 gene encodes a conserved ubiquitin recognition component of the 26S proteasome. Proc Nat/Acad Sci USA 1996, 93:856-860.
15.
Akiyama K, Yokota K, Kagawa S, Shimbara N, DeMartino GN, Slaughter CA, Noda C, Tanaka K: cDNA cloning of a new putative ATPase subunit p45 of the human 26S proteasome, a homolog of yeast transcriptional factor sugl p. FEBS Lett 1995, 363:151-156.
16.
Rubin DM, Coux O, Wefes I, Hengartner C, Young RA, Goldberg AL, Finley D: Identification of the gal4 suppressor sugl as a subunit of the yeast 26S proteasome. Nature 1996, 379:655-657.
17.
Lupas A, Koster A J, Baumeister W: Structural features of 26S and 20S proteasomes. Enzym Protein 1993, 47:252-273.
18.
Wenzel T, Baumeister W: Conformational constraints in proteindegradation by the 20S proteasome. Nat Struct Bio/1995, 2:199-204.
19.
Wenzel T, Baumeister W: Thermoplesma acidophilum proteasomes degrade partially unfolded and ubiquitinassociated proteins. FEBS Lett 1993, 326:215-218.
20.
JarieI-Encontre I, Pariat M, Martin F, Carillo S, Salvat C, Piechaczyk M: Ubiquitinylation is not an absolute requirement for degradation of c-Jun protein by the 26S proteasome. J Biol Chem 1995, 270:11623-11627.
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Stancovski I, Gonen H, Orian A, Schwartz AL, Ciechanover A: Degradation of the proto-oncogene product c-fos by the ubiquitin proteolytic system in vivo and in vitro - identification and characterization of the conjugating enzymes. Mo/Cell Bio/ 1995, 15:7106-7116.
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Tsurumi C, Ishida N, Tamura T, Kakizuka A, Nishida E, Okumura E, Kishimoto T, Inagaki M, Okazaki K, Sagata N e t aL: Degradation of c-fos by the 26S proteasome is accelerated by c-jun and multiple protein-kinases. Mo/Cell Bio11995, 15:5682-5687.
23.
Chen ZJ, Hagler J, Palombella VJ, Melandri F, Scherer D, Ballard D, Maniatis T: Signal-induced site-specific phosphorylation targets IKB(x to the ubiquitin-proteasome pathway. Genes Dev 1995, 9:1586-1597.
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The mechanism of oligopeptide generation by the 20S proteasome is still hypothetical. Until now, the assumptions are merely based on conclusions from X-ray crystallographic [39•,40] and degradation studies [36-38]. The most challenging task for the future is to achieve a detailed understanding of very high molecular weight aggregates such as the 26S proteasome or the complexes of the 20S proteasome with PA28 or other activators and inhibitors. This might not be easy to accomplish in view of the labile nature of some of these complexes. Recent advances include sequencing and expression of the proteasomal activator PA28 subunits [72] as well as characterization of some of the 19S subunits such as the multiubiquitin binding protein [13,14], the ubiquitin isopeptidase subunit [73], and of the ATPases [15,16,70°].
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Maupin-Fudow JA, Ferry JG: A proteasome from the methanogenic archaeon Methanosarcina thermophila. J B/o/ Chem 1995, 270:28617-28622. The isolation and molecular cloning of the 20S proteasome from the archeaon M. thermophila is described. Sequences of the two different subunits are closely related to those of the Thermop/asma proteasome and indicate a wider distribution of the proteasome among archaea than was previously assumed. 4. •
TamuraT, Nagy I, Lupas A, Lottspeich F, Cejka Z, Schools G, Tanaka K, Demot R, Baumeister W: The first characterization of a eubacterial proteasome - the 20S complex of Rhodococcus. Curt Bio/1995, 5:766-??4. Isolation and characterization of the 20S proteasome from Rhodococcus sp. reveals a close relationship between the eubacterial, the archaebacterial and eukaryotic enzymes. The quartemary structure, as determined by electron microscopy, and the catalytic mechanism, as deduced from the sequence, seem to be conserved throughout the three Urkingdoms. 5.
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DahlmannB, Kuehn L, Grziwa A, Zwickl P, Baumeister W: Biochemical properties of the proteasome from Thermoplasma acidophilum. Eur J Biochem 1992, 208:789-797. Dick LR, Moomaw CR, DeMartino GN, Slaughter CA: Degradation of oxidized insulin B chain by the multiproteinase complex macropain (proteasome). Biochemistry 1991, 30:2725-2734. Dick LR, Aldrich C, Jameson SC, Moomaw CR, Pramanik BC, Doyle CK, DeMartino GN, Bevan MJ, Forman JM, Slaughter CA: Proteolytic processing of ovalbumin and ~-galactosidase by the proteasome to yield antigenic peptides. J Immune/1994, 152:3884-3894.
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EhringB, Meyer TH, Eckerskorn C, Lottspeich F, Tampe R: Effects of major-histocompatibility-complex-encoded subunits on the peptidase and proteolytic activities of human 20S proteasomes - cleavage of proteins and antigenic peptides. Eur J Biochem 1996, 235:404-415.
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L~we J, Stock D, Jap R, Zwickl P, Baumeister W, Huber R: Crystal-structure of th.e 20S proteasome from the archaeon T. acidophilum at 3.4 A resolution. Science 1995, 268:533-539. The X-ray structure determination, structure description and the first characterization of the active site of the archaebacterial 20S proteasome. •
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Stock D, Ditzel L, Baumeister W, Huber R, Lbwe J: The catalytic mechanism of the 20S proteasome of Thermoplasma acidophilum revealed by X-ray crystallography. Co/d Spring Harb Symp Quant Bio/1996, 60:525-532. SeemLillerE, Lupas A, Z(.ihl F, Zwickl P, Baumeister W: The proteasome from Thermoplasma acidophilum is neither a cysteine- nor a serine-protease. FEBS Lett 1995, 359:173-178.
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Seem011erE, Lupas A, Stock D, LSwe J, Huber R, Baumeister W: Proteasome from Thermoplasma acidophilum - a threonine protease. Science 1995, 268:579-582. Site-directed mutagenesis studies of the active site of the 20S proteaseme from T. acidophi/um and a discussion on eukaryotic active sites. 43. •
FenteanyG, Standaert RF, Lane WS, Choi S, Corey F_I, Schreiber SL: Inhibition of proteasome activities and subunitspecific amino-terminal threonine modification by lactacystin. Science 1995, 268:726-731. This paper describes the discovery of lactacystin as a specific proteasome inhibitor. 44. 45. •
56. •
Sibille C, Gould KG, Willard-Gallo K, Thomson S, Rivett AJ, Powis S, Butcher GW, Debaetselier P: LMP2(+) proteasomes are required for the presentation of specific antigens to cytotoxic T-lymphocytes. Curr Bio11995, 5:923-930. The first in vivo study of the effect of LMP2 on antigen presentation. 57.
Stohwasser R, Kuckelkorn U, Kraft R, Kostka S, Kloetzel PM: 20S proteasome from LMP7 knock out mica reveals altered proteolytic activities and cleavage site preferences. FEBS Lett 1996, 383:109-113.
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Kuckelkorn U, Frentzel S, Kraft R, Kostka S, Groettrup M, Kloetzel PM: Incorporation of major histocompatibility complexencoded subunits LMP2 and LMP7 changes the quality of the 20S proteasome polypeptide processing products independent of interferon-•. Eur J Immunol 1995, 25:2605-2611.
59. •
Nandi D, Jiang H, Monaco JJ: Identification of MECL-1 (LMP10) as the third IFN-y-inducible proteasome subunit. J Immune/ 1996, 156:2361-2364. The authors present the idea that the replacement of subunits alters the cleavage specificity of the 20S proteasome by increasing the number of proteasome subpopulations in a statistical manner. 60.
Jap B, PLihler G, L, icke H, Typke D, Lbwe J, Stock D, Huber R, Baumeister W: Preliminary X-ray crystallographic study on the proteasome from Thermoplasma acidophilum. J Mo/ Biol 1993, 234:681-884.
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Grziwa A, Dahlmann B, Cejka Z, Santarius U, Baumeister W: Localisation of a sequence motif complementary to the nuclear Iocalisation signal in proteasomes from Thermoplasma acidophilum by immunoelectron microscopy. J Struct Biol 1992, 109:168-175.
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Nederlof PM, Wang HR, Baumeister W: Nuclear-localization signals of human and Thermoplasma proteasomal c~subunits are functional in-vitro. Proc Nat/Acad Sci USA 1995, 92:12060-12064.
ArtymiukPJ: A sting in the (N-terminal) tail. Nat Struct Bio/1995, 2:1035-1037.
BranniganJA, Dodson G, Duggleby HJ, Moody PCE, Smith JL, TomchickDR, Murzin AG: A protein catalytic framework with an N-terminal nucleophile is capable of self-activation. Nature 1995, 378:416-419. This paper describes the first classification of the Ntn-hydrolase fold.
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Interestingly, upon transplantation of the NLS signal from the Thermoplasma 20S proteasome into proteins, the proteins are directed to the nucleus of HeLa or 3T3 cells even though Thermoplasrna lacks a nucleus. 63.
TanakaK, Yoshimura T, Tamura T, Fujiwara T, Kumatori A, Ichihara A: Possible mechanism of nuclear translocation of proteasomes. FEBS Lett 1990, 271:41-46.
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Duggleby HJ, Tolley SP, Hill CP, Dodson EJ, Dodson G, Moody PCE: Penicillin acylase has a single-amino-acid catalytic centre. Nature 1995, 373:264-268.
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FenteanyG, Standaert RF, Reichard GA, Corey EJ, Schreiber SL: A ~-Iactone related to lactacystin induces neurite outgrowth in a neuroblastoma cell line and inhibits cell cycle progression in an osteosarcoma cell line. Proc Nat/Acad Sci USA 1994, 91:3358-3362.
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Dick LR, Cruikshank AA, Grenier L, Melandri FD, Nunes SL, Stein RL: Mechanistic studies on the inactivation of the proteasome by lactacystin. J Biol Chem 1996, 271 :?273-72?6.
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Stein RL, Melandri F, Dick L: Kinetic characterization of the" chymotryptic activity of the 20S proteasome. Biochemistry 1996, 35:3899-3908. The authors present kinetic evidence for cooperativity between active sites in the 20S proteasome and a detailed kinetic analysis. 71.
Morimoto Y, Mizushima 1", Yagi A, Tanahashi N, Tanaka K, Ichihara A, Tsukihara T: Ordered structure of the crystallized bovine 20S proteasome. J Biochern (Tokyo) 1995, 117:471-474.
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Ahn JY, Tanahashi N, Akiyama KY, Hisamatsu H, Noda C, Tanaka K, Chung CH, Shibmara N, Willy PJ, Mott JD et a/.: Primary structures of two homologous subunits of PA28, a y-interferoninducible protein activator of the 20S proteasome. FEBS Left 1995, 366:37-42.
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EytanE, Armon T, Heller H, Beck S, Hershko A: Ubiquitin Cterminal hydrolase activity associated with the 26S protease complex..J Bio/Chem 1993, 268:4668-4674.
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Scheffner M, Nuber U, Huibregtse JM: Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade. Nature 1995, 373:81-83.
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Proteasome: from structure to function Daniela Stock*, Petra M Nederloft, Erika Seem011ert, Wolfgang Baumeistert, Robert Hubert and Jan LSwe During the past two years, significant progress has been made in understanding the structure and function of the proteasome. Recent work has revealed the three-dimensional structure of the 700 kDa proteolytic complex at atomic resolution and elucidated its novel catalytic mechanism. Close relationships to a number of other amino-terminal hydrolases have emerged, making the proteasomal subunits the prototype of this newly discovered structural superfamily.
recognized by subunits localized in the 19S caps [12-14]. These caps also contain subunits with ATP-binding sites [15,16], which may serve to unfold the protein [17,18]. 20S proteasomes without 19S caps only degrade unfolded proteins independently of ATP [19] (Fig. 1), It is now well established that the ubiquitin-proteasome pathway is involved in a muhitude of cellular functions such as cell growth and division, and degradation of short-lived regulatory proteins such as oncogene products [20-22], transcription factors [23-25], and cyclins [26,27].
Addresses
*$Abteilung Strukturforschung, Max-Planck-lnstitutfor Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany t Abteilung fiir Strukturelle Molekularbiologie, Max-Planck-lnstitutfLir Biochemie, Am Klopferspitz 18a, 82152 Martinsried, Germany * e-mail: [email protected] Current Opinion in Biotechnology 1996, 7:3?6-385
© Current Biology Ltd ISSN 0958-1669 Abbreviations AGA aspartylglucosamidase cNLS complementary nuclear localization signal endoplasmic reticulum ER GAT glutamine PRPP amidotransferase LMP low molecular weight protein nuclear localization signal NLS Ntn amino-terminal nucleophile penicillin acylase PA PRPP 5-phosphoribosyl-1 -pyrophosphate transporter associated with antigen presentation TAP
Introduction Although 20S proteasomes were first described almost 30 years ago [1], their widespread distribution and cellular roles have only recently been defined. T h e 700kDa multicatalytic protease complex is ubiquitous in eukaryotic cells and is also found in a number of archaebacteria and eubacteria [2,3",4*]. It is the central cytosolic protease and is responsible for intracellular protein turnover. It is also critically involved in many regulatory processes and, in higher eukaryotes, in antigen processing. Intracellular protein turnover is carried out by a nonlysosomal, ATP- and ubiquitin-dependent pathway that controls the lifespan of most cellular proteins (for reviews, see [5-10]). T h e 26S proteasome, an approximately 2000 kDa muhiprotein complex, is the proteolytic enzyme responsible for the ATP-dependent degradation of proteins. T h e 20S proteasome forms the catalytic core of the 26S complex, whereas 19S caps, which associate with the termini of this core have multiple nonproteolytic functions [11]. Proteins are marked for degradation by conjugation with ubiquitin. T h e ubiquitinated protein is
There is strong evidence that the proteasome is involved in antigen processing in the M H C I pathway of antigen presentation in higher eukaryotes (for reviews, see [28-31]). T h e proteasome contributes to this pathway by degrading cellular proteins into peptides suitable as antigenic peptides. These are then transferred to the endoplasmic reticulum via transporters associated with antigen presentation (TAPs) 1 and 2 [32]. Here, the peptides assemble with the M H C I heavy chain and 182 microglobulin into the trimolecular MHC complex. This complex is subsequently exported to the plasma membrane where it is presenmd to cytotoxic T lymphocytes (Fig, 2). During the past two years, much progress has been made in elucidating the structure and catalytic mechanism of this important enzyme using both biochemical and biophysical approaches. On the basis of studies with small synthetic peptides and protease inhibitors, at least five distinct proteolytic activities have been identified in the eukaryotic proteasome [33]. T h e proteasome of the archaeon Thermoplasma acidophilum has oOy a single, chymotrypsin-like activity [34]. Experiments with denatured substrates present a somewhat different picture, showing that the enzyme hydrolyzes virtually any peptide bond [35-38]. Nevertheless, the degradation products fall into a rather narrow size range, suggesting that some kind of molecular ruler exists [37]. X-ray structural studies [39°,40], as well as site-directed mutagenesis experiments with the archaebacterial enzyme [41,42°], and localization of the covalently bound inhibitor, lactacystin, in a eukaryotic proteasome subunit [43 °] led to the identification of the proteasome's active site nucleophile and showed that the proteasome represents the first example of a novel class of proteases. Structural comparisons revealed surprising similarity between a growing number of enzymes with amino-terminal hydrolase activity [44,45°]. T h e recently discovered proteasomes from a methanogenic archaeon [3 °] and from a eubacterium [4"] indicate that the proteasome is more widely distributed
Proteasome: from structure to function Stock
et aL
377
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/ The ubiquitin-proteasome pathway. Native proteins are ubiquitinated by a three-enzyme cascade (involving E l - E 3 ) [74,75] that generates a thioester at the carboxyl terminus of the protein. The thioester is then used to form an isopeptide bond to the e-amino group of a lysine residue in the protein. Further ubiquitin (Ub) molecules are linked to the first one, forming chains of ubiquitin on the protein. These chains are recognized by the 26S proteasome, the thioester bonds are hydrolyzed by an isopeptidase, and the substrate is unfolded by an ATPase, all located in the 1 gS caps of the 26S proteasome. The 20S proteasome degrades unfolded or denatured proteins into peptides of defined length (7-14 amino acids) which, together with peptides generated from proteolysis by the 26S proteasome, are either degraded into their constituent amino acids by intracellular exopeptidases or transported to the cell surface via the endoplasmic reticulum (ER) and the Golgi to act as antigenic peptides presented by MHC I molecules.
throughout all three Urkingdoms than was previously assumed. T h e scope of this review follows major advances of the past years, which were mainly obtained from structural studies, using electron microscopy and X-ray crystallography, as well as from using site-directed mutagenesis experiments.
Primary and quaternary structures According to electron microscopic investigations, the quaternary structure of the 20S proteasome is conserved between archaea and humans [2]. T h e cylindrical complex, approximately 150/~ in height and 110,~. in diameter, shows a characteristic pattern of four rings of seven subunits stacked along the cylinder axis ([46]; Fig. 3). T h e archaebacterial proteasome contains only two different subunits, 0~ and 13, whereas proteasomes from eukaryotes have a more complex subunit composition, consisting of multiple or-type and 13-type subunits which are related to the respective subunits of the archaebacterial proteasome [47]. As determined by immunoelectronmicroscopy [48], o~ subunits form the outer rings of the complex, whereas the two inner rings consist of 13 subunits. This relative simplicity made the archaebacterial enzyme a suitable model system for unraveling the structure and catalytic mechanism of the 20S proteasome.
The completion of the primary structure determination of the 14 subunits of the yeast proteasome revealed a high sequence identity of all the subunits [49]. Sequence comparison of all known eukaryotic proteasomal subunits implies the presence of a total of 14 subgroups, which can be divided into seven (~-type and seven p-type groups. These considerations, as well as studies using monoclonal antibodies against individual subunits [50,51], suggest that each of the 14 eukaryotic subunits has a distinct place in the complex. Immunoelectronmicroscopic studies using an antibody against subunit HsN3 from human proteasomes [52] and the structure of the 26S proteasome [53] are in agreement with this concept and reveal the existence of a local dyad of the complex.
Mechanistic and functional studies Much effort has been spent determining the nature and location of the active site of the proteasome. T h e cloning and expression of the Thermoplasma subunits in Escherichia coil [54] was a prerequisite for site-directed mutagenesis studies as well as for the X-ray structure determination. Inhibitor studies had suggested that the 20S proteasome might be an unusual type of cysteine or serine protease [33,34]. After mutating the only cysteine, both histidines, and all the serines of the 13 subunit as well as highly conserved aspartates, it became clear that the proteasome's catalytic mechanism had to be a new one [41].
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Proteinengineering
Figure 2
(a) Humoral immune response
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The role of the proteasome in the immune response. The present model divides the immune system into three interacting pathways attacking antigens (a) in the extracellular space (humoral immune system), (b) in the cytosol, and (¢) during endocytosis, whereby lymphokines act as mediators. (a) Antigens in the extracellular space can be attacked unspecifically by the complement system (not shown) and specifically by antibody molecules, produced by plasma cells derived from B lymphocytes. (b) Protein antigens in the cytosol of most cell types are degraded by the proteasome into small peptides, which are then transported into the ER via TAP and presented on the cell surface by MHC class I molecules. (c) Protein antigens being taken up by endocytosis, mainly by macrophages, are degraded in the lysosome. Peptides are transported into the ER and are presented by MHC class il molecules. Ag, antigen; B, B lymphocyte; P, antibody-presenting cell; T, cytotoxic T lymphocyte.
Another part of the functional puzzle was solved by the electron microscopy of gold-labeled insulin B chains
incubated with Thermoplasma20S proteasomes [18]. T h e 1.5 nm gold particles were clearly visible at the entrance
Proteasome: from structure to function Stock et aL
Figure 3
379
The 'immuno-proteasome'
T h e efficiency of peptide production for this purpose seems to be further optimized in proteasomes containing the three M H C li encoded proteasomal subunits, low molecular weight protein (LMP)2, LMP7, and LMP10, which can replace their constitutive counterparts upon interferon-y stimulation. Rock and coworkers [55] had shown previously that inhibitors of the proteasome are able to block antigen presentation in vivo. Meanwhile, these findings have been corroborated by investigations using an LMP2-deficient cell line [56 °] and LMP7 knock-out mice [57], which were both shown to have reduced MHC I-restricted antigen presentation. T h e latter studies, as well as the investigations done by Kuckelkorn and coworkers [58], showed that the incorporation of a single LMP subunit or of both LMP2 and LMP7 into the proteasome alters its cleavage-site preference and thus the quality of the generated peptides. These results are consistent with the findings of Nandi and coworkers [59°], who recently discovered a third interferon-y inducible subunit, LMP10; they suggcsted that the three interferon-y induced proteasomal subunits can replace the constitutive 13-type subunits and increase the repertoire of potentially antigenic peptides by increasing the populations of proteasomes with different cleavage-site preferences in the cell.
The tertiary structure
The structure of the 20S proteasome. (a) Averaged electron micrograph of negatively stained Thermoplasma20S proteasomes. (b) Subunit stoichiometry of the complex (s-type subunits are shown lightly shaded and [[]-type subunits are darkly shaded).
to the putative channel of the proteasome, thereby inactivating the complex. This experiment suggested that the site of proteolytic cleavage is located in an inner compartment of the proteasome that is not accessible to large substrates or folded proteins. Further clues regarding the function of the proteasome were obtained by investigating the cleavage products: it could be shown that the incubation of the proteolytic complex with either oxidized human hemoglobin or with the insulin B chain resulted in degradation products of a rather narrow size range, peaking around hepta- to nonapeptides [36-38]. This observation led to the proposal of a molecular ruler determining the length of the cleavage products [37]. Taking into account that M H C I molecules preferentially bind peptides of 9 (+1) residues, the proteasome appears to be capable of providing well tailored peptide fragments for the cellular immune response.
In order to obtain more detailed insights into the proteolytic machinery, we have analyzed the crystal structure of the Thermoplasma 20S proteasome. T h e recombinant protein could be crystallized [60], and the crystal structure was determined to a resolution of 3.4~, using single isomorphous replacement and cyclic averaging techniques, which were facilitated by the high internal symmetry of the archaebacterial complex [39*]. T h e overall features of the crystal structure were in good agreement with the information obtained by electron microscopy at lower resolution. T h e core of the proteasome revealed a number of previously unknown features: the four rings of the ~ and 13 subunits encase a channel that penetrates the complex along the sevenfold axis and that widens into three large cavities that are limited by narrow constrictions (Figs 4 and 5). T h e two outermost constrictions form a passage from the cytosol to the inside of the cylinder and are only 13A in diameter. T h e crystal structure of the proteasome in complex with a small peptide inhibitor localized the active sites within the central cavity of the complex. Thus, the structure of the proteasome seems to be well designed to protect cytosolic proteins from being arbitrarily degraded. T h e fact that the proteasome buries its proteolytic compartment in its interior allows it to control access of the substrates. Hence the proteasome could be substituted for an organelle with complex membrane and targeting systems, which appears to be the reason for the unusual size of the proteinase.
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Figure 4 The crystal structure of the Thermoplasma20S proteasome. A ribbon drawing of a cross section through the proteasome, cut open along the sevenfold axis. The penetrating channel is widened by three large cavities that are limited by narrow constrictions (see also Fig. 5). The outer constriction is only 13 ~ in diameter and prevents folded proteins from being degraded. The internal twofold symmetry of the complex is clearly visible at the center of the molecule.
Figure 5 The molecular ruler mechanism of the 20S proteasome. The orientation of the surface representation is identical to that in Figure 4. The 14 active sites are located in two antiparallel rings around the central cavity and are highlighted by showing the amino-terminal threonines as dark van der Waals surfaces. The distance between two neighbouring active sites in one ring corresponds to a peptide length of about seven amino acids in extended conformation. The same is true for twofold related active sites, but these have opposite orientations and are separated by a small constriction between the rings. Some of the possible distances between the successively cleaving active sites that are thought to determine the length of the cleavage products are indicated by black bars. The molecular ruler mechanism is thought to act on a statistical basis that determines the average size of the resulting products to be in the range of heptapeptides to nonapeptides.
Another surprising phenomenon highlighted by the crystal structure was the strong conservation of the fold of the two subunits, ot and 13, in spite of their low sequence identity (25%). T h e molecular structure consists of a central five-stranded 13 sandwich, flanked by two bundles of
ot helices, one above and one below. T h e amino-terminal 35 amino acid extension of the ~ subunit forms an additional c~ helix in front of the l-sheet cleft (Fig. 6). Because eukaryotic m-type and [3-type subunits display even higher sequence identities to their corresponding ar-
Proteasome: from structure to function Stock et aL
chaebacterial prototypes, and because secondary structure predictions suggest a strong conservation of the fold of all proteasomal subunits, it can be expected that the tertiary structure is also highly conserved (J L6we, unpublished data). Proteasomal o~-type subunits contain sequences resembling nuclear localization signals (NLSs) that are also found in the Thermoplasma proteasome [61]. Meanwhile, it has been demonstrated that the putative NLS sequences are functional in vitro [62°]. Upon transplanting them to large non-nuclear reporter molecules, the NLS sequences direct the reporter molecules into the nuclei of HeLa or 3T3 cells. It remains to be established whether the NLS and the complementary NLS (cNLS) sequences, which are in close proximity to each other in the structure, do indeed interact. A model suggesting the masking and unmasking of the NLS by the cNLS, whereby phosphorylation acts as a switch, had been put forward originally by Tanaka and coworkers [63]. Figure 6
381
have similar catalytic mechanisms: they all use the aminoterminal amino acid as a nucleophile [44,45°]. Presently known members of this family are the glutamine PRPP (5phosphoribosyl-l-pyrophosphate) amidotransferase (GAT) [64], the penicillin acylase (PA) [65], the proteasome, and the aspartylglucosamidase (AGA)[66]. GAT uses an amino-terminal cysteine as a nucleophile, PA uses a serine, and AGA and the proteasome 13 subunit use a threonine. T h e architecture of the Ntn fold, characterized by a core of two stacked antiparallel 13 sheets flanked on both sides by ~ helices, is most simply exemplified by the proteasome subunits, whereas GAT, PA, and AGA have several extra 13 strands and o~ helices (Fig. 7). Moreover, all these hydrolascs are post-translationally processed [45°]. It can be assumed that the Ntn fold not only provides the capacity for nucleophilic attack but also for autocatalytic cleavage. In addition, the four enzymes show significant differences in their tertiary structures and have completely different quaternary structures. Because there is no obvious sequence homology between the enzymes and there are differences in the topology (i.e. in the connectivities between secondary structure elements), particularly between GAT and the other members of the family, the evolutionary relationship remains unclear. The catalytic mechanism T h e long enigmatic catalytic nucleophile of the 20S proteasome was identified independently and almost simultaneously by three different approaches, described below.
The structure of the ~ subunit of the Thermoplasma20S proteasome. The ribbon drawing is in standard orientation. The ~ subunits have a nearly identical fold but lack the amino-terminal ec helix on the left side. The highly homologous eukaryotic subunits are predicted to also have the same fold. Thermoplasmaand eukaryotic c~ subunits contain NLSs, and sequences complementary to them (cNLSs), which are thought to be recognized by subunits of the nuclear pore complex. The side chains of the corresponding sequences in the Thermoplasma o~subunit (NLS, residues 49-56; cNLS, residues 201-206) are shown in ball-and-stick representation. Within' the complex, these residues form the top of the cylinder and are easily accessible from the solvent.
T h e p r o t e a s o m e fold: i n h e r i t e d or r e i n v e n t e d ? What was initially assumed to be a new fold unique to the proteasomal subunits turned out to be common to a superfamily of amino-terminal (Ntn) hydrolases that all
Firstly, the X-ray structure analysis of a proteasome crystal in complex with the small peptide aldehyde inhibitor Ac-Suc-Leu-Leu-norleucinal (commercially available as calpain inhibitor I), an inhibitor of the proteasome's chymotryptic activity [39",40], identified the binding site and mode of binding, and suggested a catalytic mechanism. T h e O1 of T h r l of the 13 subunit is the nucleophile and the amino-terminal amino group the proton acceptor. Since Glu1317 and Lys1333 are the only polar residues close to the active site, they are probably also players in the catalytic reaction. T h e oligopeptidase activity (the molecular ruler) may be explained by the fact that neighbouring active sites are about 28A apart from each other, a distance spanned by an octapeptide in extended conformation (Fig. 5). Well-defined substrate binding is only observed in the P-direction, suggesting oligopeptide formation from the carboxyl terminus and thus carboxypeptidase activity of the 20S proteasome. Secondly, cysteine, serine and histidine residues were ruled out of being involved in the catalytic mechanism [41] by site-directed mutagenesis. Another well-conserved amino acid is the amino-terminal threonine, after removal of the prosequence of the Thermoplasma 13 subunit. Substitution of T h r l to alanine yielded a completely inactive protein, whereas substitution with serine turned out to have no obvious ~ffect on proteolysis [42"[.
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Protein engineering
Figure 7 A stereoview of the Ntn fold. Superposition of the Ntn fold of the 13subunits of penicillin acylase (lightly shaded; Protein Data Bank [PDB] code: 1PNK) [65] and the Thermoplasma proteasome (darkly shaded; PDB code: 1 PMA) with a root mean square deviation of 1.8 A over 88 Coc atoms. The positions of the amino-terminal amino acids Thrl of the proteasoma113 subunit and Cysl of the PA ]3 subunit, which in both cases act as the catalytic nucleophile, are indicated by showing their side chains (Stick diagram).
Mutagenesis of Glu1817 and Lys1833 as suggested by the X-ray structure also led to inactivation of the proteasome, but this does not imply direct participation in the proteolytic reaction, as conformational changes caused by disruption of the salt bridge formed by these residues may OCCUr.
The third approach involved labeling with lactacystin, a Streptomvces metabolite that was discovered because of its ability to induce neurite outgrowth in the murine neuroblastoma cell line, Neuro-2a, and which also inhibits proliferation of other cells [67]. The target could be identified using 3H-lactacystine which was shown to bind to the amino-terminal threonine of a proteasomal 13-type subunit [43*]. Subsequent kinetic studies proved lactacystin to be the first specific inhibitor of the proteasome. Three distinct proteolytic activities (trypsin-like, chymotrypsinlike and peptidyl-glutamyl-peptide hydrolyzing) were inhibited by lactacystin at different rates, the first two activities irreversibly. The binding of lactacystin to Thrl of the 18subunit from the Thermoplasma proteasome could be verified by X-ray structure analysis of proteasome crystals incubated with lactacystin (J L6we, D Stock, unpublished data). These data, as well as the studies carried out by Dick and coworkers [68], show that a hydrolyzed form of lactacystin is responsible for the inhibition.
New proteasomes The fact that Thermoplasma had long been the only archaeon in which 20S proteasomes had been found led to the proposal that these organisms might have been the precursors of eukaryotic cytoplasm. Maupin-Furlow and Ferry [3°] were recently able to isolate a 20S proteasome from the distantly related methanogenic archaeon Methanosarcina thermophila. Therefore, proteasomes appear to be more widespread amongst archaea than anticipated. The sequence of the two subunits of the methanogenic proteasome revealed a high degree of
sequence similarity to the corresponding subunits of the Thermoplasma proteasome. Much effort has been spent finding eubacterial proteasomes. Sequences related to the proteasomal 13-type subunits were found in a number of eubacteria, including E. co/i, Mvcobacterium /eprae, and Bacillus subtilis [69]. Meanwhile, the first eubacterial 20S proteasome has been discovered and isolated from the nocardioform actinomycete, Rhodococcus sp. [4"]. The isolation and sequencing revealed four types of subunits termed ~1, ~2, 181, and ]32. The topology of the subunits within the complex remains to be established.
Conclusions Biochemical and biophysical approaches have elucidated many facets of the proteasome's structure and mechanism. The availability of the archaebacterial enzyme, having been functionally expressed in E. co/i, has opened up the possibility of characterizing the protein by site-directed mutagenesis [41,42°,54]. This work, as well as inhibitor and X-ray studies, allowed the identification of the active site nucleophile and elucidated the catalytic mechanism of the Thermoplasma enzyme. Because sequence comparisons to eukaryotic subunits revealed a high degree of similarity [47,49], these enzymes are expected to be structurally very similar and to function by the same mechanism. Remaining questions are related to the positions of the 14 different subunits in the complex and to the assignment of the different catalytic activities, although recent kinetic studies on the chymotryptic activity revealed a cooperativity of the active sites [70°]. The first attempts have been made to localize the subunits by means of monoclonal antibodies [52]. A meaningful nomenclature of all proteasomal subunits will have to await the completion of these studies or the elucidation of a three-dimensional structure of a eukaryotic proteasome at atomic resolution.
Proteasome: from structure to function Stock eta/.
383
The first steps in this direction have already been taken [71].
6.
Hochstrasser M: Ubiquitin, proteasomes, and the regulation of intracellular protein degradation. Curr Opin Ceil Bio/1995, 7:215-223.
The important roles of the proteasome, both in cellular protein turnover and in antigen presentation, have been demonstrated in many experiments. Functional studies were hitherto hampered by the fact that the available inhibitors were not specific enough to distinguish between the proteasome and other cellular proteases. Lactacystin is rather specific, although it inhibits the different activities of the proteasome at different rates [43°]. The search for an inhibitor that simply blocks the narrow entrance to the proteasome's interior appears to be the most promising option.
7.
Jentsch S, Schlenker S: Selective protein-degradation - a journey's-end within the proteasome. Ceil 1995, 82:881-884.
8.
Rubin DM, Finley D: Proteolysis. The proteasome: a proteindegrading organelle. Curt Bio/1995, 5:854-858.
9.
Hilt W, Wolf HD: Proteasomes: destruction as a programme. Trends Biochem Sci 1996, 21:96-102.
10.
Hochstrasser M: Protein degradation or regulation: Ub the judge. Ce//1996, 84:813-815.
1t.
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12.
Baboshina OV, Haas AL: Novel multiubiquitin chain linkages catalyzed by the conjugating enzymes E2EpF and RAD6 are recognized by 26S proteasome subunit 5. J Bio/Chem 1996, 271:2823-2831.
13.
Ferrell K, Deveraux Q, Van Nocker S, Rechsteiner M: Molecular cloning and expression of a multiubiquiUn chain binding subunit of the human 26S protease. FEBS Lett 1996, 381:143-~148.
14.
Van Nocker S, Deveraux CI, Rechsteiner M, Vierstra RD: Arabidopsis MBP1 gene encodes a conserved ubiquitin recognition component of the 26S proteasome. Proc Nat/Acad Sci USA 1996, 93:856-860.
15.
Akiyama K, Yokota K, Kagawa S, Shimbara N, DeMartino GN, Slaughter CA, Noda C, Tanaka K: cDNA cloning of a new putative ATPase subunit p45 of the human 26S proteasome, a homolog of yeast transcriptional factor sugl p. FEBS Lett 1995, 363:151-156.
16.
Rubin DM, Coux O, Wefes I, Hengartner C, Young RA, Goldberg AL, Finley D: Identification of the gal4 suppressor sugl as a subunit of the yeast 26S proteasome. Nature 1996, 379:655-657.
17.
Lupas A, Koster A J, Baumeister W: Structural features of 26S and 20S proteasomes. Enzym Protein 1993, 47:252-273.
18.
Wenzel T, Baumeister W: Conformational constraints in proteindegradation by the 20S proteasome. Nat Struct Bio/1995, 2:199-204.
19.
Wenzel T, Baumeister W: Thermoplesma acidophilum proteasomes degrade partially unfolded and ubiquitinassociated proteins. FEBS Lett 1993, 326:215-218.
20.
JarieI-Encontre I, Pariat M, Martin F, Carillo S, Salvat C, Piechaczyk M: Ubiquitinylation is not an absolute requirement for degradation of c-Jun protein by the 26S proteasome. J Biol Chem 1995, 270:11623-11627.
21.
Stancovski I, Gonen H, Orian A, Schwartz AL, Ciechanover A: Degradation of the proto-oncogene product c-fos by the ubiquitin proteolytic system in vivo and in vitro - identification and characterization of the conjugating enzymes. Mo/Cell Bio/ 1995, 15:7106-7116.
22.
Tsurumi C, Ishida N, Tamura T, Kakizuka A, Nishida E, Okumura E, Kishimoto T, Inagaki M, Okazaki K, Sagata N e t aL: Degradation of c-fos by the 26S proteasome is accelerated by c-jun and multiple protein-kinases. Mo/Cell Bio11995, 15:5682-5687.
23.
Chen ZJ, Hagler J, Palombella VJ, Melandri F, Scherer D, Ballard D, Maniatis T: Signal-induced site-specific phosphorylation targets IKB(x to the ubiquitin-proteasome pathway. Genes Dev 1995, 9:1586-1597.
24.
Finco TS, Baldwin AS: Mechanistic aspects of NF~B regulation - the emerging role of phosphorylation and proteolysis. /mmunity 1995, 3:263-272.
25.
Traenckner EBM, Baeuerle PA: Appearance of apparently ubiquitin-conjugated IKBcxduring its phosphorylation-induced degradation in intact cells. J Cell Sci 1995, suppl 19:79-84.
26.
Ichihara A, Tanaka K: Roles of proteasomes in cell-growth. Mol B/o/ReD 1995. 21:49-52.
The mechanism of oligopeptide generation by the 20S proteasome is still hypothetical. Until now, the assumptions are merely based on conclusions from X-ray crystallographic [39•,40] and degradation studies [36-38]. The most challenging task for the future is to achieve a detailed understanding of very high molecular weight aggregates such as the 26S proteasome or the complexes of the 20S proteasome with PA28 or other activators and inhibitors. This might not be easy to accomplish in view of the labile nature of some of these complexes. Recent advances include sequencing and expression of the proteasomal activator PA28 subunits [72] as well as characterization of some of the 19S subunits such as the multiubiquitin binding protein [13,14], the ubiquitin isopeptidase subunit [73], and of the ATPases [15,16,70°].
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