Metacaspases, autophagins and metallocarboxypeptidases: potential new targets for chemotherapy of the trypanosomiases.

During the last decade, de novo drug discovery approaches have come into focus due to the increased number of parasite pathogen genomes sequenced and the subsequent availability of genome-scale functional datasets. In order to prioritize target proteins, these approaches consider traits commonly thought to be desirable in a drug target, including essentiality, druggability (whether drug-like molecules are likely to interact with the target), assayability, importance in lifecycle stages of the pathogen relevant to human health, and specificity (i.e. the target is absent from, or substantially different in, the host). Proteases from protozoan parasites have become popular drug targets since these enzymes accomplish both housekeeping tasks common to many eukaryotes as well as functions highly specific to the parasite life style. Trypanosoma cruzi, the parasitic flagellate, agent of Chagas Disease, contains several cysteine, serine, threonine and metallo proteinases. This review will deal with peculiar families described in this parasite. Among them, two eukaryote homologues of the carboxypeptidases Taq are promising targets due to their particular phylogenetic distribution. Also absent in metazoans, metacaspases are essential peptidases playing important roles in cell growth, death and differentiation of trypanosomatids. Finally, autophagins are involved in the regulation of a conserved degradative pathway, the autophagy pathway, and result important for parasite survival under nutritional stress conditions and differentiation. Although so far there are no specific inhibitors for these families, the increasing knowledge of their biochemical properties, including substrate specificity, crystal structure, and biological functions, is an essential step towards the development of inhibitors.

Cysteine proteinases of Trypanosoma cruzi: from digestive enzymes to programmed cell death mediators

Trypanosoma cruzi, the parasite causing Chagas disease, contains a number of proteolytic enzymes. The recent completion of the genome sequence of the T. cruzi CL Brener clone suggests the presence of 70 cysteine peptidases, 40 serine peptidases (none of them from the chymotrypsin family), about 250 metallopeptidases (most leishmanolysin homologues), 25 threonine peptidases, and only two aspartyl peptidases, none of them from the pepsin family. The cysteine peptidases belong to 7 families of Clan CA, 3 families of Clan CD, and one each of Clans CE and CF In Clan CA, the C1 family is represented by cruzipains 1 and 2, biochemically well characterized, as well as cathepsin B and two other cathepsins. There are a number of homologues to calpains (family C2), probably non-functional, lacking the Ca-binding domain. Family C54 includes the Atg4 proteinases (autophagins), which seem to be involved in the autophagic process. Clan CD includes family C14, the metacaspases. We have expressed the metacaspases TcMCA3 and TcMCA5, and obtained indirect evidence of their participation in programmed cell death induced by fresh human serum in the parasite. More experiments are required to better define their role in apoptosis.

The high stability of cruzipain against pH-induced inactivation is not dependent on its C-terminal domain.

Unlike mammalian lysosomal cysteine proteases, the trypanosomal cysteine protease cruzipain contains a 130-amino acid residue C-terminal domain, in addition to the catalytic domain, and it is stable at neutral pH. The endogenous (with C-terminal domain) and recombinant (without C-terminal domain) cruzipains exhibit similar stabilities at both acid (k(inac)=3.1x10(-3) s(-1) and 4.4x10(-3) s(-1) at pH 2.75 for endogenous and recombinant cruzipain, respectively) and alkaline pH (k(inac)=3.0x10(-3) s(-1) and 3. 7x10(-3) s(-1) at pH 9.15 for endogenous and recombinant cruzipain, respectively). The pH-induced inactivation, which is a highly pH dependent first order process, is irreversible and accompanied by significant changes of secondary and tertiary structure as revealed by circular dichroism measurements. The different stability of cruzipain as compared to related proteases, is therefore due mainly to the different number, nature and distribution of charged residues within the catalytic domain and not due to addition of the C-terminal domain.

Purification and some properties of serine hydroxymethyltransferase from Trypanosoma cruzi.

A single form of serine hydroxymethyltransferase (SHMT) was detected in epimastigotes of Trypanosoma cruzi, in contrast to the three isoforms of the enzyme characterized from another trypanosomatid, Crithidia fasciculata [Capelluto D.G.S., Hellman U., Cazzulo J.J. & Cannata J.J.B. (1999) Mol. Biochem. Parasitol. 98, 187-201]. The T. cruzi SHMT was found to be highly unstable in crude extracts. In the presence of the cysteine proteinase inhibitors N-alpha-p-tosyl-L-lysine chloromethyl ketone and Ltrans-3-carboxyoxiran-2-carbonyl-L-leucylagmatine, however, the enzyme could be purified to homogeneity. Digitonin treatment of intact cells suggested that the enzyme is cytosolic. T. cruzi SHMT presents a monomeric structure shown by the apparent molecular masses of 69 kDa (native) and 55 kDa (subunit) determined by Sephadex G-200 gel filtration and SDS/PAGE, respectively. This is in contrast to the tetrameric SHMTs described in C. fasciculata and other eukaryotes. The enzyme was pyridoxal phosphate-dependent after L-cysteine and hydroxylamine treatments and it was strongly inhibited by the substrate analog folate, which was competitive towards tetrahydrofolate and noncompetitive towards L-serine. Partial sequencing of tryptic internal peptides of the enzyme indicate considerable similarity with other SHMTs, particularly from those of plant origin.

Cathepsin S and cruzipain are inhibited by equistatin from Actinia equina.

Cathepsin S has been isolated for the first time from human tissue. It has a molecular mass of 24 kDa and an isoelectric point in the range of 8.2 to 8.6. The enzyme is inhibited by equistatin, which belongs to the thyropins, a new family of protein inhibitors, with an inhibition constant of Ki = 0.40 +/- 0.07 nM. Cruzipain, a cathepsin L-like enzyme sharing a 130 amino acid long C-terminal extension, is also strongly inhibited by equistatin (Ki = 0.028 +/- 0.006 nM). Together with previously reported data, these results further indicate that a functional heterogeneity exists among thyropin inhibitors, as demonstrated by their interaction with cathepsin S and cruzipain.

Trypanosoma cruzi calreticulin is a lectin that binds monoglucosylated oligosaccharides but not protein moieties of glycoproteins.

Trypanosoma cruzi is a protozoan parasite that belongs to an early branch in evolution. Although it lacks several features of the pathway of protein N-glycosylation and oligosaccharide processing present in the endoplasmic reticulum of higher eukaryotes, it displays UDP-Glc:glycoprotein glucosyltransferase and glucosidase II activities. It is herewith reported that this protozoan also expresses a calreticulin-like molecule, the third component of the quality control of glycoprotein folding. No calnexin-encoding gene was detected. Recombinant T. cruzi calreticulin specifically recognized free monoglucosylated high-mannose-type oligosaccharides. Addition of anti-calreticulin serum to extracts obtained from cells pulse-chased with [35S]Met plus [35S]Cys immunoprecipitated two proteins that were identified as calreticulin and the lysosomal proteinase cruzipain (a major soluble glycoprotein). The latter but not the former protein disappeared from immunoprecipitates upon chasing cells. Contrary to what happens in mammalian cells, addition of the glucosidase II inhibitor 1-deoxynojirimycin promoted calreticulin-cruzipain interaction. This result is consistent with the known pathway of protein N-glycosylation and oligosaccharide processing occurring in T. cruzi. A treatment of the calreticulin-cruzipain complexes with endo-beta-N-acetylglucosaminidase H either before or after addition of anti-calreticulin serum completely disrupted calreticulin-cruzipain interaction. In addition, mature monoglucosylated but not unglucosylated cruzipain isolated from lysosomes was found to interact with recombinant calreticulin. It was concluded that the quality control of glycoprotein folding appeared early in evolution, and that T. cruzi calreticulin binds monoglucosylated oligosaccharides but not the protein moiety of cruzipain. Furthermore, evidence is presented indicating that glucosyltransferase glucosylated cruzipain at its last folding stages.

Purification and partial characterization of three isoforms of serine hydroxymethyltransferase from Crithidia fasciculata.

Three molecular forms of serine hydroxymethyltransferase (SHMT) have been detected in choanomastigotes of Crithidia fasciculata by DEAE-cellulose chromatography. The three isoforms (named SHMT I, II, and III) presented small differences in charge and molecular weight. Digitonin treatment of intact cells suggested that SHMT III is cytosolic, whereas the other two isoforms are particle bound, one being mitochondrial (SHMT I) and the other one very likely glycosomal (SHMT II). The three SHMT isoforms were purified to homogeneity, and their physicochemical and kinetic properties studied. Determination of their native and subunit molecular masses revealed that all of them have a tetrameric structure. The three isoforms were shown to be PLP-dependent enzymes after L-cysteine and hydroxylamine hydrochloride treatments. They showed similar pH optima, bimodal kinetics for L-serine and Michaelis-Menten kinetics for THF.

[Cruzipain, major cysteine proteinase of Trypanosoma cruzi: sequence and genomic organization of the codifying genes]. / La cruzipaína, cisteína proteinasa principal del Trypanosoma cruzi. Secuencia y organización genómica de los genes que la codifican.

Cruzipain is the major cysteine proteinase present in Trypanosoma cruzi, the parasite causing the American Trypanosomiasis, Chagas disease. The enzyme is encoded by a high number of genes (up to 130 in the Tul 2 stock) placed in head-to-tail tandems, and located in two to four chromosomes. The simultaneous expression of several different genes results in the production of a complex mixture of isoforms. Those known as a group as "cruzipain 1" differ essentially at the level of the C-terminal domain, in their amino-acid sequence and in their type of N-glycosylation. The existence of a more distantly related cysteine proteinase, "cruzipain 2", has been demonstrated; it differs markedly, particularly at the level of the catalytic moiety.

Substrate inhibition of cruzipain is not affected by the C-terminal domain.

Endogenous and recombinant cruzipain, the major cysteine proteinase from the protozoan parasite Trypanosoma cruzi, exhibit differences in the protein and circular dichroism spectra probably attributed to the absence of the C-terminal domain in the recombinant enzyme. Substrate hydrolysis of both molecules at 25 degrees C and neutral pH obeyed Michaelis-Menten kinetics whereas significant substrate inhibition was observed above neutral pH. The results suggest that substrate inhibition of cruzipain is pH-dependent, and that the C-terminal domain does not play an essential role in this process.

Membrane-bound cysteine proteinase isoforms in different developmental stages of Trypanosoma cruzi.

Cysteine proteinase isoforms, immunologically cross-reactive with cruzipain and with a similar apparent molecular mass, have been identified in epimastigotes of Trypanosoma cruzi by extraction and phase partition using the detergent Triton X-114. These isoforms are concentrated in the microsomal fraction obtained after differential centrifugation, which is known to consist essentially of plasma membrane, can be labelled by incubation of live parasites with sulfo-NHS-biotin, and bind to cystatin-sepharose affinity columns. They are present, albeit with a different electrophoretic pattern, in the epimastigote, amastigote and trypomastigote stages of the parasite.

Polymorphisms of the genes encoding cruzipain, the major cysteine proteinase of Trypanosoma cruzi, in the region encoding the C-terminal domain.

Forty-eight cDNA clones obtained from different developmental stages of Trypanosoma cruzi and all encoding the C-terminal domain of the major cysteine proteinase (cruzipain) have been sequenced. A number of polymorphisms were detected, seven of them resulting in amino acid replacements. The predicted pI values of the corresponding gene products varied between 7.05 and 8.12. These changes in amino acid sequence, together with previously reported variations in carbohydrate composition at the only N-glycosylation site in the C-terminal domain, may account for most of the heterogeneities found in the mature enzyme.

A fragment of the major histocompatibility complex class II-associated p41 invariant chain inhibits cruzipain, the major cysteine proteinase from Trypanosoma cruzi.

A peptide fragment derived from the p41 form of the invariant chain (Ii) associated with the major histocompatibility complex (MHC) class II molecule has been shown to inhibit the mammalian lysosomal cysteine proteinase, cathepsin L, and to be a novel cysteine proteinase inhibitor, distinct from cystatins. Here we report that this same fragment also binds to and inhibits cruzipain, the cathepsin L-like enzyme from the protozoan parasite Trypanosoma cruzi. The binding of the Ii fragment to cruzipain is fast (k(ass) = 2.4 x 10(7) M(-1) s(-1) and tight (Ki = 5.8 x 10(-11) M). The inhibition is competitive. These results suggest the possibility of using the invariant chain as a model for the specific inhibition of cruzipain in vivo, i.e. as a potential drug to combat Chagas' disease.

Cruzipain, the major cysteine proteinase from the protozoan parasite Trypanosoma cruzi.

Trypanosoma cruzi, the parasitic protozoan which causes the American Trypanosomiasis, Chagas disease, contains a major cysteine proteinase (CP), cruzipain. The enzyme belongs to the papain family, but contains, as other CPs from Trypanosomatids, an unusual C-terminal extension. This C-terminal domain contains a number of post-translational modifications and is responsible for the immunodominant antigenic character of cruzipain in natural human infections. In addition, this domain is probably the cause of most of the microheterogeneities found in natural cruzipain. Irreversible inhibitors of CPs are able to block the parasite's life cycle at the differentiation steps, suggesting an essential role for CPs for parasite survival, and opening up possibilities of developing new chemotherapeutic agents against Chagas disease based on specific cruzipain inhibitors.

High-molecular-weight kininogen binds two molecules of cysteine proteinases with different rate constants.

Fluorescence titrations showed that high-molecular-weight kininogen binds two molecules of papain, cruzipain and cathepsin S with high affinity. The 2:1 binding stoichiometry was confirmed by stopped-flow kinetic measurements of papain binding, which also revealed that the two sites bind the enzyme with different association rate constants (kass,1 = 23.0 x 10(6) M-1 s-1 and kass,2 = 3.4 x 10(6) M-1 s-1). As for low-molecular-weight kininogen, comparison of these kinetic constants with previous data for intact low- and high-molecular-weight kininogen and the separated domains indicated that the faster-binding site is also the tighter-binding site and is that of domain 3, whereas the slower-binding, lower-affinity site is on domain 2. The results further demonstrate that there is no appreciable steric interference between the two domains or by the kininogen light chain in the binding of proteinases. Similarly, the binding of kininogen via its light chain to a surface, as indicated by the binding to the model surface, heparin, did not affect the inhibitory properties of kininogen. The M(r) of high-molecular-weight kininogen was determined to be 83,500 by sedimentation equilibrium measurements, in agreement with the value calculated from amino acid sequence and carbohydrate analysis.

The use of UDP-Glc:glycoprotein glucosyltransferase for radiolabeling protein-linked high mannose-type oligosaccharides.

A simple method for specifically radiolabeling high mannose-type oligosaccharides linked to protein backbones has been developed. The method is based on the fact that incubation of rat liver UDP-Glc:glycoprotein glucosyltransferase, glucose-labelled UDP-Glc and a denatured high mannose-type glycoprotein target leads to the glucosylation of the oligosaccharide. In the case described here it allowed to follow easily the purification, by HPLC and affinity chromatography, of labelled glycopeptides obtained by controlled proteolysis of cruzipain, a cysteine proteinase isolated from the human pathogen Trypanosoma cruzi. It was thus determined that the N-glycosylation site in Asn33 of cruzipain is occupied by high mannose-type oligosaccharides.

Hydrolysis of synthetic peptides by cruzipain, the major cysteine proteinase from Trypanosoma cruzi, provides evidence for self-processing and the possibility of more specific substrates for the enzyme.

Two synthetic peptides, peptide G, with the sequence KEEASSAVVGGPG, consisting of the last 10 amino acid residues of the catalytic domain, plus the first 3 at the C-terminal domain, of cruzipain, and peptide R, with the sequence KEEASSAVVRGPG, were hydrolyzed by the enzyme, as shown by reversed-phase HPLC. Peptide R was the best substrate, with a Vmax/K(m) ratio 6-fold higher as compared with peptide G, in good agreement with previous studies indicating that, in small peptides, cruzipain prefers R or K at P1. The optimal pH values for hydrolysis of peptides G and R were 6.8 and 8.0, respectively. A p-nitroanilide derivative containing the P1-P3 residues, Z-VVR-pNA, was an excellent substrate for cruzipain, with a K(m) value (33 microM at pH 9.0) lower than that for Bz-PFR-pNA (66 microM). These results open the possibility of synthesizing more specific substrates and inhibitors of cruzipain.

Effects of divalent cations and nucleotides on the 14CO2-oxaloacetate exchange catalyzed by the phosphoenol pyruvate carboxykinase from the moderate halophile, Vibrio costicola.

The phosphoenolpyruvate carboxykinase (PEPCK) from Vibrio costicola catalyzed a 14CO2-oxaloacetate exchange reaction with an unusual nucleotide specificity. ATP gave the higher apparent catalytic efficiency (Vmax/Km, 6.78), followed by GTP (1.30), CTP (0.87) and ITP (0.66). Maximal activity required a divalent cation; CdCl2 and MgCl2 synergistically activated the enzyme, when added in the presence of MnCl2. The sigmoidal saturation curve for MnCl2 (apparent n 2.11) was converted into a hyperbola by 0.01 mM CdCl2 (apparent n 1). The results suggest a double role of the divalent cation in the reaction mechanism, namely as part of the MeATP2- substrate and as free Me2+. Mn2+ would be the best for the first, and Cd2+ for the second role. Preincubation with 0.01 mM CdCl2 increased the activity of the enzyme assayed with MgATP2- through an increase in Vmax; addition of CdCl2 to the reaction mixture elicited further activation, through a 17-fold decrease in the apparent Km for MgATP2-. These results, together with the biphasic curve of activation by CdCl2 when used alone, suggest the existence of two different sites for free Cd2+ on the enzyme.

Retention of glucose units added by the UDP-GLC:glycoprotein glucosyltransferase delays exit of glycoproteins from the endoplasmic reticulum.

It has been proposed that the UDP-Glc:glycoprotein glucosyltransferase, an endoplasmic reticulum enzyme that only glucosylates improperly folded glycoproteins forming protein-linked Glc1Man7-9-GlcNAc2 from the corresponding unglucosylated species, participates together with lectin-like chaperones that recognize monoglucosylated oligosaccharides in the control mechanism by which cells only allow passage of properly folded glycoproteins to the Golgi apparatus. Trypanosoma cruzi cells were used to test this model as in trypanosomatids addition of glucosidase inhibitors leads to the accumulation of only monoglucosylated oligosaccharides, their formation being catalyzed by the UDP-Glc:glycoprotein glucosyltransferase. In all other eukaryotic cells the inhibitors produce underglycosylation of proteins and/or accumulation of oliogosaccharides containing two or three glucose units. Cruzipain, a lysosomal proteinase having three potential N-glycosylation sites, two at the catalytic domain and one at the COOH-terminal domain, was isolated in a glucosylated form from cells grown in the presence of the glucosidase II inhibitor 1-deoxynojirimycin. The oligosaccharides present at the single glycosylation site of the COOH-terminal domain were glucosylated in some cruzipain molecules but not in others, this result being consistent with an asynchronous folding of glycoproteins in the endoplasmic reticulum. In spite of not affecting cell growth rate or the cellular general metabolism in short and long term incubations, 1-deoxynojirimycin caused a marked delay in the arrival of cruzipain to lysosomes. These results are compatible with the model proposed by which monoglucosylated glycoproteins may be transiently retained in the endoplasmic reticulum by lectin-like anchors recognizing monoglucosylated oligosaccharides.

Inhibition of cruzipain, the major cysteine proteinase of the protozoan parasite, Trypanosoma cruzi, by proteinase inhibitors of the cystatin superfamily.

Cruzipain, the major cysteine proteinase from Trypanosoma cruzi epimastigotes, purified to a sequentially pure form, exists in multiple forms with pI values between 3.7 and 5.1, and an apparent molecular mass of 41 kDa. The enzyme is stable between pH 4.5-9.5. Cruzipain was found to be rapidly and tightly inhibited by various protein inhibitors of the cystatin superfamily (kass = 1.7-79 x 10(6) M-1s-1, Kd = 1.4-72 pM). These results suggest a possible defensive role for the host's cystatins after parasite infection, and may be of use for the design of new therapeutic drugs.