Concerted structural changes in the peptidase and the propeller domains of prolyl oligopeptidase are required for substrate binding

Prolyl oligopeptidase contains a peptidase domain and its catalytic triad is covered by the central tunnel of a seven-bladed beta-propeller. This domain makes the enzyme an oligopeptidase by excluding large structured peptides from the active site. The apparently rigid crystal structure does not explain how the substrate can approach the catalytic groups. Two possibilities of substrate access were investigated: either blades 1 and 7 of the propeller domain move apart, or the peptidase and/or propeller domains move to create an entry site at the domain interface. Engineering disulfide bridges to the expected oscillating structures prevented such movements, which destroyed the catalytic activity and precluded substrate binding. This indicated that concerted movements of the propeller and the peptidase domains are essential for the enzyme action.     

Mapping sequence differences between thimet oligopeptidase and neurolysin implicates key residues in substrate recognition

The highly homologous endopeptidases thimet oligopeptidase and neurolysin are both restricted to short peptide substrates and share many of the same cleavage sites on bioactive and synthetic peptides. They sometimes target different sites on the same peptide, however, and defining the determinants of differential recognition will help us to understand how both enzymes specifically target a wide variety of cleavage site sequences. We have mapped the positions of the 224 surface residues that differ in sequence between the two enzymes onto the surface of the neurolysin crystal structure. Although the deep active site channel accounts for about one quarter of the total surface area, only 11% of the residue differences map to this region. Four isolated sequence changes (R470/E469, R491/M490, N496/H495, and T499/R498; neurolysin residues given first) are well positioned to affect recognition of substrate peptides, and differences in cleavage site specificity can be largely rationalized on the basis of these changes. We also mapped the positions of three cysteine residues believed to be responsible for multimerization of thimet oligopeptidase, a process that inactivates the enzyme. These residues are clustered on the outside of one channel wall, where multimerization via disulfide formation is unlikely to block the substrate-binding site. Finally, we mapped the regulatory phosphorylation site in thimet oligopeptidase to a location on the outside of the molecule well away from the active site, which indicates this modification has an indirect effect on activity.     

Fluorescence resonance energy transfer (FRET) peptides and cycloretro-inverso peptides derived from bradykinin as substrates and inhibitors of prolyl oligopeptidase

Prolyl oligopeptidase (POP, EC 3.4.21.26) is a member of a family of serine peptidases with post-proline cleaving activity towards peptides. It is located in the cytosol in active form but without hydrolytic activity on proteins or peptides higher than 30 amino acids. Its function is not well defined, but it is involved in central nervous system disorders. Here, we studied the substrate specificity of wild type POP (POPwt) and its C255T variant lacking the non-catalytic Cys(255). This residue is located in the seven-bladed beta-propeller domain that regulates the activity of POP. Fluorescence resonance energy transfer (FRET) peptides were used with sequences derived from bradykinin-containing region of human kininogen and flanked by Abz (ortho-aminobenzoic acid) and EDDnp [N-ethylenediamine-(2,4-dinitrophenyl)]. The peptide Abz-GFSPFRQ-EDDnp was taken as leader substrate for the synthesis of five series of peptides modified at the P(3), P(2), P'(1), P'(2) and P'(3) residues. The optimal amino acids in each position for POPwt resulted in the sequence RRPYIR that is very similar to the C-terminal sequence of neurotensin. The cyclic peptides c(G((n))FSPFR) (n=1-4) were hydrolyzed by POP; their cycloretro and cycloretro-inverso analogues were inhibitors in the micromolar range. The differences between POPwt and its C255T mutant in the hydrolysis of the series derived from Abz-GFSPFRQ-EDDnp were restricted to the non-prime site of the substrates. The kinetic data of hydrolysis and inhibition by the cyclic peptides are consistent with the structures of POP-substrate/inhibitor complexes and with the substrate specificity data obtained with linear FRET peptides. All together, these results give information about the POP-substrate/inhibitor interactions that further complete knowledge of this important oligopeptidase.     

Oligopeptidases, and the emergence of the prolyl oligopeptidase family.

Oligopeptidases are endopeptidases that are not proteinases in the strict sense, since they do not hydrolyse peptide bonds in proteins, but act only on smaller polypeptides or oligopeptides. These enzymes apparently perform important, specialized biological functions that include the modification or destruction of peptide messenger molecules. Oligopeptidases have few naturally occurring inhibitors, and their distinctive specificity prevents them from interacting with alpha 2-macroglobulin, unlike the great majority of endopeptidases. The specificity of these specialized endopeptidases doubtless depends upon the three-dimensional structure of the active site, but no crystallographic structure is yet available for an oligopeptidase. Study of the primary structure of prolyl oligopeptidase has recently shown that it is a member of a new family of serine-type peptidases most of which are exopeptidases. The alignment of the sequences leads to the identification of some catalytic triad residues that have not yet been elucidated experimentally.     

Properties of the prolyl oligopeptidase homologue from Pyrococcus furiosus

Prolyl oligopeptidase (POP), the paradigm of a serine peptidase family, hydrolyses peptides, but not proteins. The thermophilic POP from Pyrococcus furiosus (Pfu) appeared to be an exception, since it hydrolysed large proteins. Here we demonstrate that the Pfu POP does not display appreciable activity against azocasein. The autolysis observed earlier was an artefact. We have also found that the pH-rate profile is different from that of the mammalian enzyme and the low pK(a) extracted from the curve represents the ionization of the catalytic histidine. We conclude that some oligopeptidases may be true endopeptidases, cleaving at disordered segments of proteins, but with very low efficacy.     

Baicalin, a prodrug able to reach the CNS, is a prolyl oligopeptidase inhibitor

Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyzes proline-containing peptides at the carboxy terminus of proline residues. It has been associated with schizophrenia, bipolar affective disorder, and related neuropsychiatric disorders and therefore may have important clinical implications. In a previous work, we used (19)F NMR to search for new prolyl oligopeptidase inhibitors from a library of traditional Chinese medicine plant extracts, and identified several extracts as powerful inhibitors of this peptidase. Here, the flavonoid baicalin was isolated as the active component of an extract of Scutellaria baicalensis roots having prolyl oligopeptidase inhibitory activity. Baicalin inhibited prolyl oligopeptidase in a dose-dependent manner. Inhibition experiments using baicalin analogs showed that the sugar moiety was not necessary for activity. The IC(50)s of baicalin and its aglycone derivative baicalein were rather similar, showing that the sugar moiety was not involved in the interaction of baicalin with POP. These results were confirmed by saturation transfer difference NMR experiments. To further understand the absorption and transport mechanisms of baicalin and baicalein, we evaluated their transport in vitro through the gastrointestinal tract and the blood-brain barrier using a Parallel Artificial Membrane Permeability Assay. The molecule which potentially crosses both barriers was identified as baicalein, the aglycone moiety of baicalin. Our results show that baicalin is a new prodrug able to inhibit prolyl oligopeptidase. As baicalin is a natural compound with a long history of safe administration to humans, it is a highly attractive base from which to develop new treatments for schizophrenia, bipolar affective disorder, and related neuropsychiatric diseases.     

The loops facing the active site of prolyl oligopeptidase are crucial components in substrate gating and specificity

Prolyl oligopeptidase (POP) has emerged as a drug target for neurological diseases. A flexible loop structure comprising loop A (res. 189–209) and loop B (res. 577–608) at the domain interface is implicated in substrate entry to the active site. Here we determined kinetic and structural properties of POP with mutations in loop A, loop B, and in two additional flexible loops (the catalytic His loop, propeller Asp/Glu loop). POP lacking loop A proved to be an inefficient enzyme, as did POP with a mutation in loop B (T590C). Both variants displayed an altered substrate preference profile, with reduced ligand binding capacity. Conversely, the T202C mutation increased the flexibility of loop A, enhancing the catalytic efficiency beyond that of the native enzyme. The T590C mutation in loop B increased the preference for shorter peptides, indicating a role in substrate gating. Loop A and the His loop are disordered in the H680A mutant crystal structure, as seen in previous bacterial POP structures, implying coordinated structural dynamics of these loops. Unlike native POP, variants with a malfunctioning loop A were not inhibited by a 17-mer peptide that may bind non-productively to an exosite involving loop A. Biophysical studies suggest a predominantly closed resting state for POP with higher flexibility at the physiological temperature. The flexible loop A, loop B and His loop system at the active site is the main regulator of substrate gating and specificity and represents a new inhibitor target.     

Prolyl, cystyl and pyroglutamyl peptidase activities in the hippocampus and hypothalamus of streptozotocin-induced diabetic rats

Prolyl, cystyl and pyroglutamyl peptidases are emerging targets for diabetes and cognitive deficit therapies. The present study is focused on the influence of diabetes mellitus induced by streptozotocin on levels of representative hydrolytic activities of these enzymes in the rat hypothalamus and hippocampus. Streptozotocin-diabetic rats presented about 348mg glucose/dL blood, and a slightly increased hematocrit and plasma osmolality. The activities of soluble and membrane-bound dipeptidyl-peptidase IV, and soluble cystyl aminopeptidase did not differ between diabetic and control rats in both brain areas. Hippocampal soluble prolyl oligopeptidase presented similar activities between diabetic and controls. Increased activities in diabetics were observed for soluble prolyl oligopeptidase (1.78-fold) and membrane-bound cystyl aminopeptidase (2.55-fold) in the hypothalamus, and for membrane-bound cystyl aminopeptidase (5.14-fold) in the hippocampus. In both brain areas, the activities of membrane-bound and soluble pyroglutamyl aminopeptidase were slightly lower (<0.7-fold) in diabetics. All modifications (except hematocrit) observed in streptozotocin-treated rats were mitigated by the administration of insulin. Glucose and/or insulin were shown to alter in vitro the hypothalamic activities of soluble pyroglutamyl aminopeptidase and prolyl oligopeptidase, as well as membrane-bound cystyl aminopeptidase. These data provide the first evidence that diabetes mellitus generates direct and indirect effects on the activity levels of brain peptidases. The implied regional control of regulatory peptide activity by these peptidases suggests novel potential approaches to understand certain disruptions on mediator and modulatory functions in diabetes mellitus.     

Cleavage of the Lys196-Ser197 bond of prolyl oligopeptidase: enhanced catalytic activity for one of the two active enzyme forms.

Prolyl oligopeptidase, a representative of a new family of serine proteases, is remarkably sensitive to ionic strength and has two catalytically active forms, which interconvert with changing pH [Polgár, L. (1991) Eur. J. Biochem. 197, 441-447]. To reveal whether conformational changes are associated with these effects, prolyl oligopeptidase was digested with trypsin. SDS gel electrophoresis studies demonstrated that tryptic digestion of the 75-kDa native protein generated two fragments, one having a molecular mass of 51 kDa and the other of 26 kDa. The digestion was markedly dependent on the ionic strength. Specifically, the digestion proceeded more rapidly in 0.05 M Hepes buffer than in 0.05 M Hepes buffer containing 0.5 M NaCl. Moreover, the nicked enzyme formed at low ionic strength was not stable but degraded and inactivated during an extended incubation. The digestion experiments suggested that alteration in the ionic strength elicits conformational changes in native prolyl oligopeptidase, and this may account for the enhanced catalytic activity observed at higher ionic strength. The two fragments of the nicked prolyl oligopeptidase did not separate during size-exclusion chromatography under nondenaturing conditions on a Superose 12 column and eluted in place of the native enzyme, indicating that they were strongly associated. The reactive serine residues of the nicked enzyme was labeled with tritiated diisopropyl phosphofluoridate, and the fragments were separated by size-exclusion chromatography in urea.(ABSTRACT TRUNCATED AT 250 WORDS)     

Development of a novel assay for proprotein converting enzyme activity on a multiplex bead‐based array system

We describe the development of a novel, robust assay system for determining the changes in activity of proprotein converting enzymes. An assay for prolyl oligopeptidase (POP) activity was constructed using a peptide‐streptavidin substrate coupled to magnetic microspheres and cleavage was detected by loss of streptavidin on the MAGPIX reader. Test analysis of postmortem pituitary extracts from schizophrenia patients showed an increase in POP activity compared to controls. The results were validated using both fluorometric and Western blot analyses for POP activity and immunoreactivity, respectively. The assays can be multiplexed for measuring the activity of multiple proprotein cleaving enzymes simultaneously in laboratory and clinical settings and should add valuable new information for conditions such as neuropsychiatric diseases, diabetes, endocrine dysfunction, and cancer, where effects on proteolysis of biologically active peptides play a key role.     

Four day inhibition of prolyl oligopeptidase causes significant changes in the peptidome of rat brain, liver and kidney

Prolyl oligopeptidase (PREP) cleaves short peptides at the C-side of proline. Although several proline containing neuropeptides have been shown to be efficiently cleaved by PREP in vitro, the actual physiological substrates of this peptidase are still a matter of controversy. The aim of this study was to evaluate the changes in the peptidome of rat tissues caused by a repeated 4-day administration of the potent and specific PREP inhibitor KYP-2047, using our recently developed iTRAQ-based technique. We found tissue-dependent changes in the levels of specific subsets of peptides mainly derived from cytosolic proteins. Particularly in the kidney, where the levels of cytochrome c oxidase were found decreased, many of the altered peptides originated from mitochondrial proteins being involved in energy metabolism. However, in the hypothalamus, we found significant changes in peptides derived from hormone precursors. We could not confirm a role of PREP as the metabolising enzyme for β-endorphin, galanin, octadecaneuropeptide, neuropeptide–glutamic acid–isoleucine, substance P, somatostatin, enkephalin and neuropeptide Y. Furthermore, changes in the degradation patterns of some of these neuropeptides, and also most of those derived from other larger proteins, did not follow specificity to proline. After a 4-day treatment, we found a significant amount of peptides, all derived from secreted pro-proteins, being cleaved with pair of basic residue specificity. In vitro experiments indicated that PREP modifies the endogenous dibasic residue specific proteolysis, in a KYP-2047 sensitive way. These findings suggest that PREP may act indirectly within the routes leading to the specific peptide changes that we observed. The data reported here suggest a wider tissue specific physiological role of PREP rather than the mere metabolism of proline containing active peptides and hormones.     

Inhibition of Prolyl Oligopeptidase by Fmoc-Aminoacylpyrrolidine-2-Nitriles

Abstract: Prolyl oligopeptidase (EC 3.4.21.26), a widely distributed cytosolic enzyme, cleaves peptidylprolyl peptide and peptidylprolyl amino acid bonds in many neuropeptide substrates. Its action on vasopressin has been proposed as the underlying mechanism accounting for the ability of inhibitors of prolyl oligopeptidase to reverse scopolamine-induced amnesia in rats. Future behavioral studies would be facilitated by the availability of potent inhibitors readily synthesized from common intermediates. A series of Fmoc-aminoacylpyrrolidine-2-nitriles prepared by a simple two-step synthesis were found to be potent noncompetitive inhibitors of the rabbit brain enzyme. The most potent inhibitors, Fmoc-prolyl-pyrrolidine-2-nitrile and Fmoc-alanyl-pyrrolidine-2-nitrile, each have a K _i of 5 n M . The compounds are cell permeable and stable. They do not inhibit the related enzyme dipeptidyl peptidase IV (EC 3.4.14.5). When administered intraperitoneally to mice, Fmoc-alanyl-pyrrolidine-2-nitrile crosses the blood-brain barrier to inhibit brain prolyl oligopeptidase. The ease of synthesis, potency, efficacy in vivo, stability, and specificity of Fmoc-aminoacylpyrrolidine-2-nitriles may make them inhibitors of choice in studies probing the physiological significance of prolyl oligopeptidase.     

Free ATP inhibits thimet oligopeptidase (EC 3.4.24.15) activity, induces autophosphorylation in vitro, and controls oligopeptide degradation in macrophage.

The fate of the proteasome-generated peptides depends upon the cytosolic peptidases whose activities ought to be regulated. One of the most important oligopeptide-degrading and -binding proteins in the cytosol is the thimet oligopeptidase (EC 3.4.24.15), ubiquitously found in mammalian tissues. To date, there is no indication whether thimet oligopeptidase activities are physiologically regulated. Here, we present evidences suggesting that the concentration of unbound ATP in the cytosol regulates the thimet oligopeptidase activities both, in vitro and ex vivo. To perform these studies two oligopeptides were used: a quenched fluorescent peptide, which is susceptible to thimet oligopeptidase degradation, and the ovalbumin257-264 (MHC class I ovalbumin epitope), which displays high affinity to the thimet oligopeptidase without being degraded. We also showed that the thimet oligopeptidase undergoes autophosphorylation by ATP, a modification that does not affect the peptidase activity. The autophosphorylation is abolished in the presence of the thimet oligopeptidase substrates, as well as by the effect of a site directed inhibitor of this enzyme, and by the substitution of Glu474 for Asp at the metallo-peptidase motif. Altogether, the results presented here suggest that Zn2+ at the active center of the thimet oligopeptidase is the target for the ATP binding, leading to the inhibition of the enzyme activity, and inducing autophosphorylation. These effects, which depend upon the concentration of the unbound ATP, may help to explain the fate of the proteasomal-generated oligopeptides in the cytosol.     

Biochemical and pharmacological aspects of two bradykinin-potentiating peptides obtained from tryptic hydrolysis of casein

Peptides that display bradykinin-potentiating activity have been obtained from a number of distinct sources, such as snake venoms, fibrinogen, and casein. This paper describes the characterization of two new peptides generated by tryptic hydrolysis of casein. No homology was found with other known vasoactive or vasopotentiating peptides, especially by the lack of Ile-Pro-Pro motif. The peptides EMPFPK and YPVEPFTE, corresponding to the gamma casein sequence (108-113 and 114-121, respectively), displayed a selective potentiating activity on isolated guinea pig ileum for bradykinin. Besides, the octapeptide YPVEPFTE showed an in vitro competitive inhibitor effect on angiotensin-converting enzyme and thimet oligopeptidase and presented an opiate-like activity, increasing two times the latence time in the hot-plate assay. The results suggest that the isolated bioactive peptides act on conversion and/or inactivation of endogenous peptides by enzymes such as angiotensin-converting enzyme and thimet oligopeptidase by modifying several systemic responses such as blood-pressure regulation and in pain response.     

Unusual secondary specificity of prolyl oligopeptidase and the different reactivities of its two forms toward charged substrates.

Prolyl oligopeptidase belongs to a new family of serine proteases which contains both exo- and endopeptidases, and this suggests that the enzyme binds its substrate in a special manner. Its secondary specificity, i.e., its interaction with the other residues linked to the proline that accounts for the primary specificity, has been investigated by using peptide substrates of various length and charge. Elongation of the classic dipeptide substrate Z-Gly-Pro-2-naphthylamide with 1-3 residues (Gln, Ala-Gln, Ala-Ala-Gln, and Ala-Lys-Gln) resulted in decreased specificity rate constants. This indicated a limited binding site for prolyl oligopeptidase, a major difference from the finding with other serine endopeptidases. Insertion of charged residues into the substrates, such as lysine or aspartic acid, considerably affected the rates and the pH-rate profiles. The rate constants were higher with the positively charged peptides and lower with the substrates bearing a negative charge. These electrostatic effects were reduced at high ionic strength. The results can be interpreted in terms of a negatively charged active site, which exists at high pH and exerts electrostatic attraction or repulsion toward charged substrates. The pH dependencies of the rate constants with neutral substrates exhibited roughly bell-shaped curves, whereas with charged substrates the existence of two active enzyme forms was clearly demonstrated. The physiologically competent high pH form preferred positively charged substrates (Z-Lys-Pro-2-(4-methoxy)naphthylamide, Z-Ala-Lys-Gln-Gly-Pro-2-naphthylamide), whereas the low pH form reacted faster with the negatively charged substrate (Z-Asp-Gly-Pro-2-naphthylamide).(ABSTRACT TRUNCATED AT 250 WORDS)     

Oligopeptidase B: a processing peptidase involved in pathogenesis

Oligopeptidase B is a "processing peptidase" from the prolyl oligopeptidase family of serine peptidases present in Gram negative bacteria, protozoa and plants. Unlike the prototype prolyl oligopeptidase, oligopeptidase B hydrolyses peptides on the carboxyl side of pairs of basic amino acid residues. Molecular modelling and mutation studies have identified carboxyl dyads in the C-terminal catalytic domain that mediate substrate and inhibitor binding. The peptidase is efficiently inhibited by non-peptide irreversible serine peptidase inhibitors, peptidyl-chloromethylketones, -phosphonate alpha-aminoalkyl diphenyl esters with basic residues at P1, and tripeptide aldehydes, but not by proteinaceous host plasma inhibitors such as alpha2-macroglobulin and serpins. Access of these large molecular mass inhibitors and substrates larger than approximately 30 amino acid residues to the catalytic cleft is restricted by the N-terminal beta-propeller domain. The physiological role of oligopeptidase B from various sources has not yet been elucidated. However, the peptidase has been identified as an important virulence factor and therapeutic agent in animal trypanosomosis. This review highlights the structure-function properties of oligopeptidase B in context with its physiological and/or pathological roles which make the enzyme a promising drug target.     

Ascorbate is consumed stoichiometrically in the uncoupled reactions catalyzed by prolyl 4-hydroxylase and lysyl hydroxylase

The hydroxylation of proline and lysine residues by the collagen hydroxylases is coupled with a stoichiometric decarboxylation of 2-oxoglutarate. Ascorbate is virtually a specific requirement for these enzymes, but previous studies have demonstrated that it is not consumed during most catalytic cycles. Prolyl 4-hydroxylase and lysyl hydroxylase are known also to catalyze an uncoupled decarboxylation of 2-oxoglutarate in the absence of the peptide substrate. It is shown here that, unlike the complete hydroxylation reaction, the uncoupled decarboxylation reaction involves stoichiometric ascorbate consumption. This stoichiometric ascorbate consumption was also seen when the rate of the uncoupled prolyl 4-hydroxylase reaction was enhanced by the addition of poly(L-proline). Since collagen hydroxylases may catalyze occasional uncoupled reaction cycles even in the presence of the peptide substrates, the main function of ascorbate in these reactions in vivo is suggested to be that of reactivating the enzymes after such uncoupled cycles.     

Characterization of membrane-bound prolyl endopeptidase from brain

Prolyl oligopeptidase (POP) is a serine protease that cleaves small peptides at the carboxyl side of an internal proline residue. Substance P, arginine-vasopressin, thyroliberin and gonadoliberin are proposed physiological substrates of this protease. POP has been implicated in a variety of brain processes, including learning, memory, and mood regulation, as well as in pathologies such as neurodegeneration, hypertension, and psychiatric disorders. Although POP has been considered to be a soluble cytoplasmic peptidase, significant levels of activity have been detected in membranes and in extracellular fluids such as serum, cerebrospinal fluid, seminal fluid, and urine, suggesting the existence of noncytoplasmic forms. Furthermore, a closely associated membrane prolyl endopeptidase (PE) activity has been previously detected in synaptosomes and shown to be different from the cytoplasmic POP activity. Here we isolated, purified and characterized this membrane-bound PE, herein referred to as mPOP. Although, when attached to membranes, mPOP presents certain features that distinguish it from the classical POP, our results indicate that this protein has the same amino acid sequence as POP except for the possible addition of a hydrophobic membrane anchor. The kinetic properties of detergent-soluble mPOP are fully comparable to those of POP; however, when attached to the membranes in its natural conformation, mPOP is significantly less active and, moreover, it migrates anomalously in SDS/PAGE. Our results are the first to show that membrane-bound and cytoplasmic POP are encoded by variants of the same gene.     

Extremophilic proteases as novel and efficient tools in short peptide synthesis

The objective of this review is to outline the crucial role that peptides play in various sectors, including medicine. Different ways of producing these compounds are discussed with an emphasis on the benefits offered by industrial enzyme biotechnology. This paper describes mechanisms of peptide bond formation using a range of proteases with different active site structures. Importantly, these enzymes may be further improved chemically and/or genetically to make them better suited for their various applications and process conditions. The focus is on extremophilic proteases, whose potential does not seem to have been fully appreciated to date. The structure of these proteins is somewhat different from that of the common commercially available enzymes, making them effective at high salinity and high or low temperatures, which are often favorable to peptide synthesis. Examples of such enzymes include halophilic, thermophilic, and psychrophilic proteases; this paper also mentions some promising catalytic proteins which require further study in this respect.     

β-Propeller repeats and a PDZ domain in the tricorn protease: predicted self-compartmentalisation and C-terminal polypeptide-binding strategies of substrate selection

Prokaryotic proteases demonstrate a variety of substrate-selection strategies that prevent uncontrolled protein degradation. Proteasomes and ClpXP-like proteases form oligomeric structures that exclude large substrates from central solvated chambers containing their active sites. Monomeric prolyl oligopeptidases have been shown to contain beta-propeller structures that similarly reduce access to their catalytic residues. By contrast, Tsp-like enzymes contain PDZ domains that are thought to specifically target C-terminal polypeptides. We have investigated the sequence of Thermoplasma acidophilum tricorn protease using recently-developed database search methods. The tricorn protease is known to associate into a 20 hexamer capsid enclosing an extremely large cavity that is 37 nm in diameter. It is unknown, however, how this enzyme selects its small oligopeptide substrates. Our results demonstrate the presence in tricorn protease of a PDZ domain and two predicted six-bladed beta-propeller domains. We suggest that the PDZ domain is involved in targeting non-polar C-terminal peptides, similar to those generated by the T. acidophilum proteasome, whereas the beta-propeller domains serve to exclude large substrates from the tricorn protease active site in a similar manner to that previously indicated for prolyl oligopeptidase.