Báo cáo khoa học: The propeptide of cruzipain ) a potent selective inhibitor of the trypanosomal enzymes cruzipain and brucipain, and of the human enzyme cathepsin F ppt

The propeptide of cruzipain ) a potent selective inhibitor of the trypanosomal enzymes cruzipain and brucipain, and of the human enzyme cathepsin F Flavia C G Reis1, Tatiana F R Costa1, Traian Sulea2, Alessandra Mezzetti2, Julio Scharfstein1, ´ Dieter Bromme3, Robert Menard2 and Ana Paula C A Lima1 ă ´ Instituto de Biofısica Carlos Chagas Filho, CCS, Universidade Federal Rio de Janeiro, RJ, Brazil Biotechnology Research Institute, National Research Council of Canada, Montreal, Canada Department of Oral and Biological Sciences, University of British Columbia, Vancouver, Canada Keywords brucipain; cathepsin F; cruzipain; inhibition; propeptide Correspondence ´ A P C A Lima, Instituto de Biofısica Carlos Chagas Filho, Bloco G, Centro de ˆ ´ Ciencias da Saude, Universidade Federal ´ria, Rio de Janeiro, Cidade Universita 21949-900, Rio de Janeiro, RJ, Brazil Fax: +55 21 2280 8193 Tel: +55 21 2209 6591 E-mail: anapaula@biof.ufrj.br (Received 25 September 2006, revised 30 November 2006, accepted 22 December 2006) Papain-like cysteine proteases of pathogenic protozoa play important roles in parasite growth, differentiation and host cell invasion The main cysteine proteases of Trypanosoma cruzi (cruzipain) and of Trypanosoma brucei (brucipain) are validated targets for the development of new chemotherapies These proteases are synthesized as precursors and activated upon removal of the N-terminal prodomain Here we report potent and selective inhibition of cruzipain and brucipain by the recombinant full-length prodomain of cruzipain The propeptide did not inhibit human cathepsins S, K or B or papain at the tested concentrations, and moderately inhibited human cathepsin V Human cathepsin F was very efficiently inhibited (Ki of 32 pm), an interesting finding indicating that cruzipain propeptide is able to discriminate cathepsin F from other cathepsin L-like enzymes Comparative structural modeling and analysis identified the interaction between the b1p–a3p loop of the propeptide and the propeptide-binding loop of mature enzymes as a plausible cause of the observed inhibitory selectivity doi:10.1111/j.1742-4658.2007.05666.x Pathogenic trypanosomes are the cause of major parasitic diseases, which pose a threat to public health in many developing countries [1] Trypanosoma cruzi is the etiologic agent of Chagas’ disease, a chronic illness endemic in Central and South America, associated with heart failure and ⁄ or denervation of the digestive system Trypanosoma brucei causes sleeping sickness, a widespread disease in sub-Saharan Africa that is characterized by neurologic degeneration, psychiatric disorders, and inevitable death if left untreated Present chemotherapy relies on compounds presenting low efficacy and ⁄ or high toxicity, and there is an urgent need for the development of alternative drugs In many pathogenic parasites, the activity of papainlike cysteine proteases (CPs) seems to be crucial for growth, development and tissue ⁄ host cell penetration [2] The use of synthetic irreversible CP inhibitors in animals experimentally infected with T cruzi [3] or with T brucei [4] has significantly reduced parasitemia and mortality, validating these enzymes as promising targets for the development of new drugs The main targets of these compounds are the well-characterized major CPs of T cruzi, cruzipain (or cruzain) [5–7], and the CP of T brucei, brucipain (or rhodesain) [8,9] The resolution of the X-ray structure of cruzain in complex with diazomethane peptidyl inhibitors provided the basis for structure-based inhibitor design [10] Although several studies have explored the inhibition of cruzipain by peptidyl epoxy ketones [11], aldehydes [12], vinyl sulfones [13], thiosemicarbazones [14] and alpha-keto-based compounds [15], there are still ongoing efforts to find new mechanism-based small-molecule Abbreviations CP, cysteine protease; PBL, proregion-binding loop; PCZ, recombinant propeptide of cruzipain 1224 FEBS Journal 274 (2007) 1224–1234 ª 2007 The Authors Journal compilation ª 2007 FEBS F C G Reis et al inhibitors and structural information that could lead to the development of efficient and specific inhibitors for both cruzipain and brucipain CPs are synthesized as inactive precursors that possess a signal peptide, a prodomain, and a mature domain that retains catalytic activity In trypanosomal CPs, the mature enzyme is composed of a central domain that corresponds to the mature domain of mammalian CPs and an additional 130-residue C-terminal extension with unknown function [2] The C-terminal extension is not essential for enzyme activity Activation of papain-like CPs is achieved by proteolytic excision of the prodomain, an event believed to occur in vivo by a multistep process that may involve multiple endosomal ⁄ lysosomal peptidases or even proteases present in the extracellular environment [16,17] Diverse functions have been attributed to the prodomain present in CP precursors (proenzyme): (a) inhibition of enzyme activity through its interaction with the active site; (b) folding assistance [18]; and (c) targeting of the precursors to the endosomal–lysosomal system [19] Determination of the three-dimensional structure of zymogens revealed that enzyme inhibition by the propeptide is accomplished by its binding to the enzyme’s active site with the backbone in the reverse direction, thus protecting it from hydrolysis [20] The discovery that the propeptides of cathepsins B [21], L [22], K and S [23–25] are potent inhibitors that display a certain degree of selectivity for their parent enzymes has paved the way for the design of new selective inhibitory compounds Indeed, this approach was successful in the production of short peptidyl noncovalent inhibitors based on the mode of inhibition of cathepsin L by its prodomain These synthetic inhibitors present 300-fold selectivity for cathepsin L over cathepsin K, whereas the full-length propeptide of cathepsin L presented only two-fold selectivity for the parental enzyme [26] Although CPs are widely expressed in parasitic organisms, very little information has been reported on the inhibitory properties of their prodomains The propeptide of the main cathepsin L-like CP from the worm Fasciola hepatica was described as highly selective for the parasite’s enzymes, being practically incapable of inactivating mammalian cathepsins L, K and B and papain [27] In another study, the inhibitory potential of 23 overlapping synthetic peptides spanning the propeptide of a CP from the cattle parasite Trypanosoma congolense (congopain) has also been described [28] This study identified a few peptides presenting a certain degree of selectivity for the inhibition of congopain and cruzipain relative to cathepsins B and L [28] Cathepsin F inhibition by propeptide of cruzipain In this study, the propeptide of cruzipain was produced and tested as an inhibitor of trypanosomal CPs Selectivity with regard to human lysosomal CPs was also investigated We show that the recombinant propeptide of cruzipain is a potent inhibitor of the mature enzyme, as well as brucipain, and further demonstrate that the propeptide of cruzipain selectively inactivates human cathepsin F with high potency The molecular basis for selective inhibition and the implications of these findings are discussed Results and Discussion Expression and purification of the propeptide of cruzipain The predicted full-length propeptide of cruzipain (Cys19–Gly122) was cloned by PCR and expressed in Escherichia coli as a fusion protein with a 6xHis tag at the N-terminus This type of N-terminal tag was previously used for the expression of the propeptide of human cathepsin K, and it did not interfere with the inhibitory activity of the protein [24] Although some of the recombinant propeptide of cruzipain (PCZ) was produced in its soluble form, a great amount was found in inclusion bodies In order to maximize production and to optimize purification, the fusion protein was denatured in a buffer containing urea, and was subsequently refolded and used in the inhibition assays The purity of refolded PCZ was checked by SDS ⁄ PAGE (Fig 1A), and its chemical integrity was verified by MS Analysis by MALDI-TOF MS showed a major single peak with a molecular mass of 14 019 Da, and a minor peak with a molecular mass of 14 161 Da (Fig 1B) The major peak corresponded to the full-length construct containing: (a) residues MRGS (introduced by the plasmid multiple cloning site immediately before the His-tag); (b) the six histidines of the tag; (c) the full-length propeptide of cruzipain (Cys19–Gly122); and (d) residues RGVDLQPSLIS at the C-terminus, which resulted from the fusion of the propeptide with a small stretch of coding sequence within the multiple cloning site of the pQE30 plasmid before the stop codon The predicted molecular mass of the fusion protein is 14 075 Da (14 kDa), which is within 1% error of the mass corresponding to the major peak This indicates that the great majority of the purified propeptide was homogeneous and had no detectable truncations Inhibitory activity of the propeptide Next, we tested the inhibitory properties of PCZ towards the parent enzyme, cruzipain, purified from FEBS Journal 274 (2007) 1224–1234 ª 2007 The Authors Journal compilation ª 2007 FEBS 1225 Cathepsin F inhibition by propeptide of cruzipain A 600 MW KDa Fluorescence units A F C G Reis et al 26.6 Control 0.02 nM 400 0.03 nM 0.06 nM 200 20 0 B 1/v (M–11.s–1) 14.3 200 400 600 seconds 800 1000 1.5 µM 0.8 0.6 3.0 µM 0.4 0.2 B 100 14015.00 878.2 90 –0.02 % Intensity 80 70 0.02 0.04 0.06 0.08 0.1 [I] nM 14063.56 60 Fig Inhibition of cruzipain by its propeptide (A) Progress curves for the inhibition of cruzipain by its full-length recombinant propeptide The figure shows the generation of product with time in the absence or in the presence of PCZ at the concentrations indicated for each curve (B) Plots of ⁄ v versus [I] showing competitive inhibition The substrate concentrations used in the kinetic measurements are indicated in the graph 50 40 30 20 10 12400 16600 20800 Mass (m/z) 25000 Fig Expression of the full-length propeptide of cruzipain (A) A DNA fragment encoding the full-length propeptide of cruzipain (Cys19–Gly122) was cloned in fusion with a polyhistidine tag in pQE30 and expressed in E coli Denatured fusion protein (PCZ) was purified in an Ni–nitrilotriacetic acid column and subsequently renaturated, filtered and dialyzed SDS ⁄ PAGE of the purification of PCZ: bacterial homogenate after induction (lane 1), denaturated PCZ eluted from Ni–nitrilotriacetic acid (lane 2), PCZ after renaturation and dialysis (lane 3) (B) MS analysis by MALDI-TOF MS Approximately nmol of the purified propeptide was precipitated with trichloroacetic acid and analyzed by MS The measured mass for His-tagged PCZ was 14 019.01 Da, which is within 56 Da of the average calculated mass T cruzi epimastigotes We observed time-dependent inhibition of cruzipain (Fig 2A) by PCZ, as noted previously for the inhibition of mammalian CPs by their 1226 propeptides, which follows slow-binding kinetics, in the pH 5–6 range [21–25] The linear portions of the progress curves where steady state was reached were used to calculate the rate constant of substrate hydrolysis in the presence of the inhibitor (vi) and to determine Ki The Dixon plots (1 ⁄ v versus [I]) [29] resulted in intersecting lines, showing competitive inhibition (Fig 2B) At pH 6.5, PCZ inactivated cruzipain competitively, with an inhibition constant (Ki) of 0.018 nm Remarkably, this was the lowest Ki value described so far for the inhibition of a papain-like CP by its cognate propeptide The propeptide was also found to be a very good inhibitor of brucipain, with a Ki value of 0.0163 nm at pH 6.5 This finding is not surprising, as cruzipain and brucipain share a high degree of sequence similarity, both in the mature part and in their propeptides Along these lines, a previous study using 15-mer synthetic FEBS Journal 274 (2007) 1224–1234 ª 2007 The Authors Journal compilation ª 2007 FEBS F C G Reis et al Cathepsin F inhibition by propeptide of cruzipain Table Dissociation constants for the inhibition of cruzipain by its propeptide Cruzipain purified from T cruzi epimastigotes was assayed for inhibition by the propeptide as described in Experimental procedures The steady-state velocities were calculated by linear regression, and the results were used to plot ⁄ v versus [I] to calculate the Ki values [36] Ki (nM) 5.0 5.5 6.0 6.5 7.0 0.2637 0.0565 0.027 0.018 0.034 Vo x 10–11 (M/S) pH ± ± ± ± ± 0.0001 0.0005 0.007 0.002 0.007 peptides based on the prodomain of the CP congopain from the cattle trypanosome T congolense showed cross-inhibition of congopain and cruzipain [28] However, the Ki values of the inhibitory peptides were in the micromolar range The potency of PCZ as an inhibitor of cruzipain and brucipain decreased moderately at the more acidic pH value of (Table 1), but remained in the subnanomolar range The very low Ki values at pH values ranging from to suggest that if full-length propeptide was generated upon procruzipain maturation, it would probably inhibit the mature enzyme very efficiently Notably, it was suggested that in T cruzi, the autocatalytic processing of cruzipain zymogens occurs at the Golgi complex [30], a compartment that usually presents only a mildly acidic environment In this context, it is possible that the liberated full-length propeptide exerts a crucial regulatory function in keeping cruzipain inactive until it reaches lysosome-like organelles (reservosomes) The propeptide of cruzipain is a potent inhibitor of the cognate enzyme, and it could be expected that PCZ would likewise efficiently inactivate the CPs from other trypanosomatids that share high sequence similarity with cruzipain Indeed, a preliminary test using the homogenates of trypanosomatids such as T rangeli, T brucei and Leishmania donovani revealed that PCZ inhibited approximately 90% of the overall peptidase activity detected in L donovani and 50–70% of the activity detected in the homogenates from the other protozoa (Fig 3) These results suggest that PCZ would be a good candidate as a lead for the design of potent peptidyl inhibitors aimed at the inactivation of CPs from several pathogenic protozoa The propeptide of cruzipain is a potent inhibitor of cathepsin F L donovani 100 T cruzi T brucei T rangeli 10 0,1 Fig The propeptide of cruzipain inactivates endogenous CPs of trypanosomatids Parasites were cultivated as described in Experimental procedures, and lysates were incubated with control buffer (clear bars), with nM propeptide (gray bars), or with 30 lM E-64 (dark bars) The peptidase activity was measured by addition of 10 lM Z-Phe-Arg-MCA and monitored continuously The graphs represent initial velocities calculated by linear regression of the progress curves and F (in the subnanomolar range), and the residual activity of the peptidases was measured using a synthetic fluorogenic substrate This analysis revealed relatively weak inhibition of cathepsins L and V, whereas neither papain nor cathepsins B, S and K were inactivated by PCZ when incubated with up to 25 nm of the propeptide (Table 2) We observed degradation of PCZ by cathepsins S, L and V, but not by cathepsins B or K after 60 of incubation (data not shown) During the invasion of mammalian cells by T cruzi, the host cell lysosomes are recruited to the point of contact between host cell and parasites, and this is followed by the delivery of the lysosomal content to Table Inhibition of papain-like enzymes by the propeptide of cruzipain The enzymes were assayed for inhibition by the propeptide of cruzipain as described in Experimental procedures The steadystate velocities were calculated by linear regression and the results were used to plot ⁄ v versus [I] to calculate the Ki values [29] ND, not detected 19 Enzyme Cruzipain Brucipain Cathepsin Cathepsin Cathepsin Cathepsin Cathepsin Cathepsin Ki (nM) at pH 6.5 B F K La S V 0.018 0.0163 ND 0.032 ND 2.05 ND 5.18 ± 0.002 ± 0.0001 ± 0.003 ± 0.25 ± 0.54 a To evaluate the selectivity of PCZ as an inhibitor of papain-like enzymes, the propeptide was incubated for 30 with papain or human cathepsins B, L, S, K, V Ki determination was performed at pH 5, due to enzyme instability at higher pH values Degradation of PCZ was observed when it was incubated with cathepsins S, K, L and V at [E] > 500 nM for over 60 FEBS Journal 274 (2007) 1224–1234 ª 2007 The Authors Journal compilation ª 2007 FEBS 1227 F C G Reis et al the extracellular environment [31] In view of our observations, if cathepsins S or L are present at high concentrations at the parasite–host cell point of contact, they could mediate degradation of the propeptide present therein Interestingly, significant inhibition of cathepsin F by PCZ was observed The inactivation constants for inhibition of cathepsin F were subsequently determined (Table 2), and showed that PCZ exhibits very high potency towards cathepsin F, and is 150-fold less effective in inhibiting cathepsin V Interestingly, the Ki for cathepsin F was in the same range as that observed with the cognate enzyme (cruzipain) and with brucipain, although mature cathepsin F shares considerably less sequence identity with mature cruzipain (49%) than does mature brucipain (71%) The activity of cathepsin L was minimally inhibited at the maximal concentration of PCZ used (25 nm) The Ki could not be determined at pH 6.5, because cathepsin L inactivates over time at this pH However, when determined at pH 5.0, the Ki for inhibition of cathepsin L by PCZ was in the same range as observed for the inactivation of cathepsin V This was expected, as cathepsin L is very similar to cathepsin V, and its sequence identity to cruzipain (43%) is also comparable to that of cathepsin V (41%) In order to be able to inhibit endosomal ⁄ lysosomal CPs in living cells, PCZ would have to be, at least to some extent, resistant to degradation by peptidases that are present in these acidic compartments We determined whether PCZ would be able to inhibit CPs present in the pool of endogenous peptidases of mammalian cell homogenates The tissue distribution of cathepsin L is quite broad, whereas cathepsins F and V present a much more restricted expression pattern It has been reported that both cathepsin V and cathepsin F are expressed in human monocyte-derived macrophages [32,33] and that the latter is also expressed in smooth muscle cells Therefore, we tested the inactivation of endogenous CPs of human cells with diverse origins by PCZ (Fig 4) The incubation of whole cell homogenates with PCZ led to a 20–30% drop in the total peptidase activity of monocytes ⁄ macrophages (U937 cell line), smooth muscle cells and Hela cells, whereas it reduced the peptidase activity of prostate epithelial cells (DU145) by 4% Substrate hydrolysis by the cell homogenates was fully inhibited by E-64 (data not shown), confirming that we were measuring mainly the activity of papain-like CPs in our assays Cathepsin F is a key enzyme responsible for the degradation of the invariant chain (Ii) in macrophages, playing a fundamental role in antigen presentation 1228 % inhibition of peptidase activity Cathepsin F inhibition by propeptide of cruzipain 30 25 20 15 10 Prostate epithelium Hela Smooth Monocytes/ muscle macrophages Fig The propeptide of cruzipain inactivates endogenous CPs of mammalian cells Human cell lines were cultivated as described in Experimental procedures, and cell lysates (10 lgỈmL)1) were incubated with nM propeptide or with control buffer, for 15 at room temperature The residual peptidase activity was measured by the hydrolysis of lM Z-Phe-Arg-MCA The peptidase activity of all lysates was fully inhibited by 10 lM E-64 (not shown) The initial velocities were calculated by linear regression of the substrate hydrolysis curves, and the graph represents the percentage of inhibition, considering the peptidase activity of untreated lysates as 100% The graph is representative of three independent experiments [34] Cathepsin F also modifies low-density lipoprotein particles in vitro, is present in human atherosclerotic lesions [35], and partially degrades lipid free apoA-1 and high-density lipoprotein (HDL3) in macrophages, thus blocking cholesterol efflux from these cells [32] It would be interesting to know whether T cruzi can secrete the cruzipain propeptide in its intact form If so, it is possible that antigen-presenting cells such as macrophages could internalize the propeptide, which, once in the endosomal ⁄ lysosomal compartments, would be able to inhibit cathepsin F As cathepsin F participates in the degradation of Ii, indirectly modulating the levels of class II MHC molecules available for binding of antigenic peptides, one can speculate that inhibition of this peptidase by the propeptide could affect antigen presentation by such cells Comparative structural analysis of papain-like CPs Given the remarkable selectivity of PCZ as an inhibitor of cathepsin F and of the two trypanosomal CPs tested, we set out to investigate the possible molecular basis of selectivity The sequence alignment of the propeptide domains of several papain-like enzymes (Fig 5A) revealed that the prodomains of cruzipain and of brucipain are highly similar The high sequence identity observed between the prodomains of the trypanosomal enzymes (60%) could account for the FEBS Journal 274 (2007) 1224–1234 ª 2007 The Authors Journal compilation ª 2007 FEBS F C G Reis et al potent cross-inhibition of brucipain by PCZ The prodomain of cruzipain shares the highest identity with the C-terminal portion (residues Pro147–Leu251) of the prodomain of cathepsin F (32%) and that of cathepsin W (33%) among the mammalian CPs, albeit at a significantly lower level than with brucipain The PCZ similarity to the prodomains of human capthepsins L, V, S and K is significantly lower (18–25%), showing that sequence conservation qualitatively correlates to a certain degree with the overall inhibition pattern shown in Table PCZ is significantly different from the prodomain of cathepsin B (not shown) and is not expected to inhibit this carboxypeptidase, due to the occluding loop in the primed side of the binding site groove Although the prodomains of cathepsins F and W share the same degree of similarity to the prodomain of cruzipain, the similarity of the mature domain of cathepsin F to that of cruzipain is higher (48%) than that between the mature domains of cruzipain and cathepsin W (32%) In order to understand the structural basis for the PCZ inhibitory selectivity among endopeptidases, we modeled the three-dimensional structure of cruzipain propeptide in complex with the crystal structure of mature cruzipain The modeled PCZ–cruzipain complex was then compared with the experimental structural data available for the other enzymes, particularly surveying the residues and structural features at the PCZ–cruzipain contact interface that are conserved in brucipain and cathepsin F but not in cathepsins V, L, S and K The most significant difference is located in the loop connecting strand b1p to helix a3p of the proregion, and occurs concertedly with a structural change in the interacting portion of the mature enzyme known as the proregion-binding loop (PBL) As seen in Fig 5A, the b1p–a3p loop of the cruzipain proregion, which is two residues shorter than those of the cathepsin L, K, V or S proregions (see also Fig 5A), would result in a steric conflict with the PBL side chain Phe145 of mature cathepsin L, as well as with the corresponding side chains at this position in mature cathepsins K (Phe144), V (Phe146) and S (Leu147) The interfering side chain appears to be conformationally restricted by the PBL main chain in all these enzymes The collision with PCZ is, however, circumvented in the case of mature cruzipain and cathepsin F, due to a two-residue deletion in the PBL sequence, which changes the main chain local conformation relative to the other cathepsins, and in turn moves the corresponding interfering side chain (the smaller Thr147 of cruzipain, and Phe141 of cathepsin F) out of the way On the basis of homology-based sequence alignments of mature enzymes, the same Cathepsin F inhibition by propeptide of cruzipain would hold true for PCZ binding to brucipain and cathepsin W, which are, however, omitted from Fig 5B, due to the unavailability of experimental structures for these enzymes It remains to be tested whether PCZ is indeed a potent inhibitor of cathepsin W, as predicted by this analysis However, active cathepsin W has not been successfully expressed to date Conversely, the two-residue insertion Asn55p-(Ala ⁄ His)56p in the proregion loop b1p–a3p of cathepsins L, K, V and S relative to the cruzipain, brucipain and cathepsin F and W proregions (Fig 5A), allows good steric complementarity with the PBL side chain at position 145 (cathepsin L numbering) of the corresponding mature enzymes (Fig 5B), in addition to a hydrogen bond observed between the conserved Asn55p side chain and the PBL main chain at position 144 (not shown) The predicted structural determinant of PCZ specificity for cathepsin F can be verified experimentally by mutagenesis in several ways, either on the b1–a3 loops of propeptides or in the interacting PBL segments of the mature enzymes For example, a recombinant PCZ variant with a two-residue-longer b1–a3 loop, as in the prosegments of cathepsins L, K, V and S, should exhibit a tendency to level off or even reverse the cathepsin inhibition pattern of the wildtype recombinant PCZ Other structural features may be invoked to further explain the observed inhibitory selectivity of cruzipain propeptide against cathepsins V, L, K and S (Table 2) In general, it is expected that the fine modulation of binding affinity and specificity between mature enzymes and the  100-residue propeptides results from the cumulative effect of small contributions scattered along the extensive binding interface For example, the shorter b1p–a3p loop of PCZ appears to force a small displacement of its helix a3p and to allow the accommodation of an additional residue (His81p) at the end of this helix, close to the active site, in comparison to the structures of procathepsins L and K These more subtle structural changes may have a differential impact on the affinity of binding to mature cathepsins V, L, K and S A movement of helix a3p perpendicular to its axis has been documented between the proregions of cathepsins L and K, and is speculated to affect the proregion inhibitory selectivity [36] Also, residues Asn82p and Gly83p from the putative active sitespanning segment YHNGAA of PCZ are conserved in the proregions of cathepsins L and K, where they bind in the reverse substrate-binding mode into the S1¢ and S1 subsites, respectively [20] Their flanking residues, however, are more conserved in brucipain FEBS Journal 274 (2007) 1224–1234 ª 2007 The Authors Journal compilation ª 2007 FEBS 1229 Cathepsin F inhibition by propeptide of cruzipain F C G Reis et al A B Fig Structural basis of PCZ inhibitory selectivity (A) Sequence alignment of propeptides from papain-like CPs Residues identical in at least half of the sequences are highlighted on a black background; those similar in at least half of the sequences are highlighted on a gray background Secondary structure elements, as observed in the crystal structure of procathepsin L (Protein Data Bank accession number: 1CS8), are indicated below the alignment Amino acid identities (%) of the cruzipain proregion with other CP proregions are given on the right (B) Interactions with the PBLs of the mature enzymes as a basis for the PCZ inhibitory selectivity The PBL segments of mature cruzipain (Protein Data Bank accession number: 1ME3, modeled in complex with its propeptide) and mature cathepsins F, L, K, V and S (Protein Data Bank accession numbers: 1MD6, 1CS8, 1BY8, 1FH0 and 1NQC, respectively), displayed as Ca traces, correspond to the sequences aligned in the upper right corner The PBL segments of brucipain and cathepsin W, whose propeptides are aligned in (A), are not shown in (B), due to the unavailability of experimental structures for these mature enzymes The PBL-interacting propeptide segments of cruzipain (model) and procathepsins L and K (Protein Data Bank accession numbers: 1CS8 and 1BY8, respectively), rendered by secondary structure, correspond to the sequences aligned in the lower left corner The last four displayed residues (underlined) of these propeptide segments extend into subsites S1¢ to S3 of the mature enzymes Cruzipain is shown in green, cathepsin F in magenta, and the other cathepsins in white An arrow indicates the two-residue insertions (highlighted) in the b1p–a3p loops of cathepsin L and K propeptides relative to the cruzipain propeptide This length variability in the propeptide sequences correlates with the steric requirements for interaction imposed by the conformation adopted by a PBL residue, displayed as a ball-and-stick model and highlighted in the sequence alignment and cathepsin F than in the cathepsin V, L, K and S proregions, and thus they may represent another source of selectivity among these enzymes, also depending on the physicochemical properties of their substrate-binding site groove Given that CP proregions adopt folded structures upon binding to the mature enzymes, we cannot exclude that even the PCZ regions located remotely from the interface and active site could be implicated in the fine-tuning of inhibitory selectivity 1230 The selectivity of PCZ for cathepsin F over other mammalian cathepsins is a remarkable observation indicating that it could serve as a lead for the development of potent selective inhibitors of cathepsin F The knowledge generated by our study could contribute to the design and production of potent small synthetic inhibitors selective for either cruzipain or cathepsin F Such selective inhibitors would be of great interest for the study of the biological functions and pathologic states in which these enzymes are implicated FEBS Journal 274 (2007) 1224–1234 ª 2007 The Authors Journal compilation ª 2007 FEBS F C G Reis et al Experimental procedures Expression of the propeptide of cruzipain Cathepsin F inhibition by propeptide of cruzipain (pH 6.5), 2.5 mm dithiothreitol, and 5% dimethylsulfoxide 14 Substrate hydrolysis was monitored in a Hitachi F-4500 spectrofluorimeter (Tokyo, Japan) at 380 nm excitation and 440 nm emission, in continuous assays The initial velocities were calculated by linear regression of the hydrolysis curves The propeptide titer was calculated by linear regression of the titration curves as being equal to the X-value at which V0 ¼ The titrations were performed with the propeptide at concentrations at least five-fold above the Ki Complementary oligonucleotides directed to residues Cys19 (5¢-GCGGATCCTGCCTCGTCCCCGCGGCG-3¢) and Gly122 (5¢-CGCCCGGGGCCAACAACCTCCACGTC-3¢), which span the full-length predicted prodomain of the cruzipain gene of T cruzi Dm28c, were used as primers in a PCR using the cruzipain gene as a template and Pfx polymerase (Invitrogen, Grand Island, NY, USA) The Proteolytic enzymes amplification conditions were as follows: 30 cycles of denaturation (94 °C, min), annealing (55 °C, min), and extenCruzipain was purified from T cruzi Dm28c epimastigotes sion (68 °C, min) The resulting fragment was excised from as previously described [37] Human cathepsin B and a 0.8% agarose gel, digested with BamHI and SmaI, cloned human cathepsin S were purchased from Sigma and Calbiointo the respective sites of pQE30 (Qiagen, Valencia, CA, chem (La Jolla, CA, USA), respectively Human cathepsins USA), and sequenced in an automated Applied Biosystems L, V, K and F were expressed in Pichia pastoris as previ9 (Foster City, CA, USA) sequencer E coli M15 (pREP4) ously described [38,39] The detailed cloning and produc(Qiagen) was used as a host for the expression of the fusion tion of recombinant brucipain will be described elsewhere protein containing a 6xHis N-terminal tag and the full-length (T F R Costa, F C G Reis, L Juliano, J Scharfstein, & predicted propeptide of cruzipain The cells were grown in A P C A Lima, unpublished results) Briefly, a DNA LB medium supplemented with 100 lgỈmL)1 ampicillin and fragment spanning the predicted first residue of the prodo25 lgỈmL)1 kanamycin overnight at 37 °C, diluted 20 times main (Cys22) and the last residue of the mature domain 10 in fresh medium, and cultivated until a D600 nm of 0.8 was (Val339) of brucipain was cloned in the BamHI and KpnI reached Recombinant protein expression was induced by sites of pQE30 for expression in E coli M15 (pREP4) The addition of mm isopropyl thio-b-d-galactoside at 30 °C, for purification of His-tagged recombinant probrucipain and 11 h The cells were harvested by centrifugation at 5000 g in vitro refolding were performed as described above for the using a Sorvall Centrifuge RC28S (Wilmington, DE, USA) propeptide of cruzipain, with the exception of a 6-day incuwith GSA rotor, washed in 20 mm phosphate buffer (pH 7.2) bation at °C for complete refolding of the proenzyme and 150 mm NaCl (NaCl ⁄ Pi), and lysed in 100 mm The proenzyme was activated by diluting in 50 mm NaOAc NaH2PO4, 10 mm Tris, and m urea (pH 8), under agitation, (pH 5.5), 200 mm NaCl, mm EDTA, and mm dithiofor h at room temperature The soluble material was recovthreitol Mature enzyme was subsequently affinity-purified 12 ered by centrifugation at 10 000 g for 20 using a Sorvall in thiopropyl sepharose 6B (Pharmacia-Amersham, Upsala, Centrifuge RC28S with F28 ⁄ 36 rotor, and the recombinant Sweden), according to the manufacturer’s instructions The propeptide was purified in an Ni–nitrilotriacetic acid–agarose purified enzyme was stable at °C for month The resin according to the manufacturer’s instructions The eluted enzyme concentration was determined by active site titrasamples (1 mgỈmL)1) were pooled and submitted to in vitro tion with E-64, as previously described [40] refolding by incubation with mm dithiothreitol at 37 °C for 45 min, followed by 100-fold dilution in ice-cold 100 mm MS analysis Tris ⁄ HCl (pH 8), mm EDTA, m KCl, and 20% glycerol, and further incubation at °C for 24 h The solution was The refolded propeptide (5.4 nmol) was precipitated with filtered through 0.2 lm cellulose acetate filters (Millipore, trichloroacetic acid and resupended in de-ionized water 13 Bedford, MA, USA), and centrifuged at 10 000 g for 20 The protein was mixed at : v ⁄ v with the matrix solution using a Sorvall Centrifuge RC28S with F28 ⁄ 36 rotor, and the (a-cyano-4-hydroxycinnamic acid), applied on the steel soluble fraction was concentrated and dialyzed by successive plate appropriate for the introduction of samples in the dilutions in ice-cold Milli-Q water by ultrafiltration in kDa Voyager DE Pro equipment, and allowed to dry at room cut-off DIAFLO membranes (Amicon, Bedford, MA, USA) temperature The mass profile was subsequently obtained The titer of the propeptide was determined by active site titrawith MALDI-TOF MS (Voyager DE-Pro ⁄ Applied Biosystion of cruzipain The activated enzyme (previously titrated tem, Foster City, CA, USA) with E-64) was incubated with different volumes of the propeptide in 50 mm Na2HPO4, 200 mm NaCl, mm EDTA Kinetic measurements (pH 6.5), and 2.5 mm dithiothreitol, for 20 at room temperature The residual activity of the enzyme was determined Kinetic measurements were carried out in continuous assays by the hydrolysis of lm Z-Phe-Arg-AMC (Sigma, St Louis, 15 using a Hitachi F-4500 spectrofluorimeter at 28 °C All the MO, USA) in 50 mm Na2HPO4, 200 mm NaCl, mm EDTA FEBS Journal 274 (2007) 1224–1234 ª 2007 The Authors Journal compilation ª 2007 FEBS 1231 Cathepsin F inhibition by propeptide of cruzipain F C G Reis et al enzymes were stable at the different pH values during the assays The kinetics of inhibition of cruzipain were measured using the Z-Arg-Arg-MCA substrate at 1.5 lm and lm ([S]