RESUMO
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.
Assuntos
Carboxipeptidases/antagonistas & inibidores , Caspases/química , Proteínas de Protozoários/antagonistas & inibidores , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/enzimologia , Tripanossomíase/tratamento farmacológico , Carboxipeptidases/metabolismo , Caspases/metabolismo , Biologia Computacional , Humanos , Proteínas de Protozoários/metabolismo , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/metabolismo , Tripanossomíase/parasitologiaRESUMO
Metacaspases are distant relatives of animal caspases present in plants, fungi and protozoa. At variance with caspases, metacaspases exhibit stringent specificity for basic amino-acid residues and are absolutely dependent on millimolar concentrations of calcium. In the protozoan parasite Trypanosoma cruzi, metacaspases have been suggested to be involved in an apoptosis-like phenomenon upon exposure of the parasite to fresh human serum (FHS). Nuclear relocalization of metacaspases was observed after FHS treatment and overexpression of metacaspase-5 led to enhanced sensitivity to this stimulus. Here we report some biochemical properties of T. cruzi metacaspases. Performing fluorescent-activated cell sorting (FACS) analysis of epimastigotes inducibly overexpressing metacaspase-3, we demonstrate a role for this metacaspase in cell cycle progression, protection of epimastigotes from naturally occurring cell death and differentiation to infective metacyclic trypomastigotes. We also show that regulation of metacaspase-3 activity is important for cell cycle completion inside the mammalian host. On the other hand, inducible overexpression of metacaspase-5 lacking its C-terminal domain caused an apoptotic-like response. These results suggest that the two T. cruzi metacaspases could play an important role in the life cycle and bring to light the close relationship between cell division, death and differentiation in this ancient unicellular eukaryote.
Assuntos
Caspases/metabolismo , Trypanosoma cruzi/citologia , Trypanosoma cruzi/enzimologia , Animais , Animais Geneticamente Modificados , Arginina/metabolismo , Caspases/biossíntese , Caspases/química , Caspases/genética , Morte Celular/fisiologia , Diferenciação Celular/fisiologia , Processos de Crescimento Celular/fisiologia , Pontos de Checagem da Fase G1 do Ciclo Celular , Plasmídeos/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/sangue , Fase S , Especificidade por Substrato , Transfecção , Trypanosoma cruzi/genética , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
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.(AU)
Assuntos
Humanos , Animais , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/genética , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Sequência de Aminoácidos , Apoptose , Sistema Livre de Células , Genoma de Protozoário , Estágios do Ciclo de Vida , Dados de Sequência Molecular , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , TransfecçãoRESUMO
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.
Assuntos
Humanos , Animais , Sequência de Aminoácidos , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Cisteína Endopeptidases/química , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética , Apoptose , Sistema Livre de Células , Genoma de Protozoário , Estágios do Ciclo de Vida , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/química , Alinhamento de Sequência , TransfecçãoAssuntos
Citosol/enzimologia , Escherichia coli/enzimologia , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Análise de Sequência de DNA , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , Escherichia coli/genética , Dados de Sequência Molecular , Trypanosoma brucei brucei/genéticaRESUMO
Two malate dehydrogenase isoforms, named MDH1 and MDH2, have been purified to homogeneity from Trypanosoma cruzi epimastigotes. Both enzymes consist of subunits with a molecular mass close to 33 kDa; native molecular mass determination by gel filtration, however, indicated that MDH1 is a dimer, whereas MDH2 is a tetramer. Both isoforms did not cross-react immunologically. The N-termini of both MDH isoforms and several tryptic peptides of MDH1 (amounting to about one third of the complete molecule) have been sequenced by automated Edman degradation. The tryptic digests of both enzymes have also been analysed by mass spectrometry (MALDI-TOF MS). The apparent Km values in both directions of the reaction have been determined, as well as the possible inhibition by excess of the substrate oxaloacetate. The sequence data, together with the pI values and the presence or absence of oxaloacetate inhibition indicate that the dimeric MDH1 is the mitochondrial isoenzyme, whereas the tetrameric MDH2 is the glycosomal isoenzyme. No evidence was found for the presence of a cytosolic isoform.
Assuntos
Isoenzimas , Malato Desidrogenase/química , Malato Desidrogenase/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Reações Cruzadas/imunologia , Dimerização , Ponto Isoelétrico , Malato Desidrogenase/imunologia , Malato Desidrogenase/isolamento & purificação , Dados de Sequência Molecular , Análise de Sequência de Proteína , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Glicina Hidroximetiltransferase/isolamento & purificação , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Crithidia fasciculata/enzimologia , Citosol/enzimologia , Inibidores Enzimáticos/farmacologia , Glicina Hidroximetiltransferase/genética , Glicina Hidroximetiltransferase/metabolismo , Cinética , Dados de Sequência Molecular , Peso Molecular , Estrutura Quaternária de Proteína , Especificidade da Espécie , Especificidade por Substrato , Trypanosoma cruzi/genéticaRESUMO
Trypanosoma cruzi, the protozoan parasite causing Chagas disease, contains a novel aromatic alpha-hydroxy acid dehydrogenase. This enzyme is responsible, together with tyrosine aminotransferase, for the catabolism of aromatic amino acids, which leads to the excretion of aromatic lactate derivatives into the culture medium. The gene encoding the aromatic alpha-hydroxy acid dehydrogenase has been cloned through a combined approach using screening of an expression genomic library with antibodies, peptide sequencing and PCR amplification. Its sequence shows high similarity to the cytosolic malate dehydrogenases. However, the enzyme has no malate dehydrogenase activity. The gene seems to be present in a single copy per haploid genome and is differentially expressed throughout the parasite's life cycle, the highest levels being found in the insect forms of T. cruzi. The purified recombinant enzyme, expressed in Escherichia coli, was unable to reduce oxaloacetate and had kinetic constants similar to those of the natural aromatic alpha-hydroxy acid dehydrogenase. Sequence comparisons suggest that the aromatic alpha-hydroxy acid dehydrogenase derives from a cytosolic malate dehydrogenase no longer present in the parasite, made redundant by the presence of a glycosomal malate dehydrogenase as a member of a shuttle device involving the mitochondrial isoenzyme.
Assuntos
Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Proteínas de Protozoários , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Citosol/enzimologia , Primers do DNA/genética , Escherichia coli/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Genes de Protozoários , Humanos , Cinética , Malato Desidrogenase/genética , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Lectinas/metabolismo , Oligossacarídeos/metabolismo , Proteínas de Protozoários/metabolismo , Ribonucleoproteínas/metabolismo , Trypanosoma cruzi/química , 1-Desoxinojirimicina/farmacologia , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/imunologia , Calreticulina , Sequência de Carboidratos , Clonagem Molecular , Cisteína Endopeptidases/imunologia , Cisteína Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Inibidores Enzimáticos/farmacologia , Glicoproteínas/química , Glicoproteínas/metabolismo , Inibidores de Glicosídeo Hidrolases , Glicosilação , Hexosaminidases/farmacologia , Soros Imunes , Lectinas/efeitos dos fármacos , Lectinas/genética , Dados de Sequência Molecular , Testes de Precipitina , Dobramento de Proteína , Proteínas de Protozoários/efeitos dos fármacos , Proteínas de Protozoários/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/imunologia , Frações Subcelulares , alfa-GlucosidasesRESUMO
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.