RESUMEN
Leishmania ISPs are ecotin-like natural peptide inhibitors of trypsin-family serine peptidases, enzymes that are absent from the Leishmania genome. This led to the proposal that ISPs inhibit host serine peptidases and we have recently shown that ISP2 inhibits neutrophil elastase, thereby enhancing parasite survival in murine macrophages. In this study we show that ISP1 has less serine peptidase inhibitory activity than ISP2, and in promastigotes both are generally located in the cytosol and along the flagellum. However, in haptomonad promastigotes there is a prominent accumulation of ISP1 and ISP2 in the hemidesmosome and for ISP2 on the cell surface. An L. major mutant deficient in all three ISP genes (Δisp1/2/3) was generated and compared with Δisp2/3 mutants to elucidate the physiological role of ISP1. In in vitro cultures, the Δisp1/2/3 mutant contained more haptomonad, nectomonad and leptomonad promastigotes with elongated flagella and reduced motility compared with Δisp2/3 populations, moreover it was characterized by very high levels of release of exosome-like vesicles from the flagellar pocket. These data suggest that ISP1 has a primary role in flagellar homeostasis, disruption of which affects differentiation and flagellar pocket dynamics.
Asunto(s)
Leishmania major/fisiología , Inhibidores de Proteasas/metabolismo , Proteínas Inhibidoras de Proteinasas Secretoras/metabolismo , Proteínas Protozoarias/metabolismo , Animales , Células Cultivadas , Flagelos/metabolismo , Flagelos/ultraestructura , Técnicas de Inactivación de Genes , Interacciones Huésped-Parásitos , Leishmania major/genética , Leishmania major/metabolismo , Leishmania major/ultraestructura , Macrófagos Peritoneales/parasitología , Ratones , Ratones Endogámicos BALB C , Inhibidores de Proteasas/química , Transporte de Proteínas , Proteínas Inhibidoras de Proteinasas Secretoras/química , Proteínas Inhibidoras de Proteinasas Secretoras/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Vesículas Secretoras/metabolismo , Vesículas Secretoras/ultraestructura , Serina Proteasas/químicaRESUMEN
The role of protein phosphorylation in the life cycle of malaria parasites is slowly emerging. Here we combine global phospho-proteomic analysis with kinome-wide reverse genetics to assess the importance of protein phosphorylation in Plasmodium falciparum asexual proliferation. We identify 1177 phosphorylation sites on 650 parasite proteins that are involved in a wide range of general cellular activities such as DNA synthesis, transcription and metabolism as well as key parasite processes such as invasion and cyto-adherence. Several parasite protein kinases are themselves phosphorylated on putative regulatory residues, including tyrosines in the activation loop of PfGSK3 and PfCLK3; we show that phosphorylation of PfCLK3 Y526 is essential for full kinase activity. A kinome-wide reverse genetics strategy identified 36 parasite kinases as likely essential for erythrocytic schizogony. These studies not only reveal processes that are regulated by protein phosphorylation, but also define potential anti-malarial drug targets within the parasite kinome.
Asunto(s)
Malaria Falciparum/metabolismo , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidad , Proteómica/métodos , Proteínas Protozoarias/metabolismo , Animales , Humanos , FosforilaciónRESUMEN
Over the last decade, several protein kinases inhibitors have reached the market for cancer chemotherapy. The kinomes of pathogens represent potentially attractive targets in infectious diseases. The functions of the majority of protein kinases of Plasmodium falciparum, the parasitic protist responsible for the most virulent form of human malaria, remain unknown. Here we present a thorough characterisation of PfTKL3 (PF13_0258), an enzyme that belongs to the tyrosine kinase-like kinase (TKL) group. We demonstrate by reverse genetics that PfTKL3 is essential for asexual parasite proliferation in human erythrocytes. PfTKL3 is expressed in both asexual and gametocytes stages, and in the latter the protein co-localises with cytoskeleton microtubules. Recombinant PfTKL3 displays in vitro autophosphorylation activity and is able to phosphorylate exogenous substrates, and both activities are dramatically dependent on the presence of an N-terminal "sterile alpha-motif" domain. This study identifies PfTKL3 as a validated drug target amenable to high-throughput screening.
Asunto(s)
Plasmodium falciparum/enzimología , Plasmodium falciparum/fisiología , Proteínas Tirosina Quinasas/metabolismo , Secuencias de Aminoácidos , Animales , Eritrocitos/metabolismo , Eritrocitos/parasitología , Eucariontes , Humanos , Malaria/enzimología , Malaria Falciparum , Parásitos/metabolismo , Fosforilación , Plasmodium falciparum/metabolismo , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , S-Adenosilmetionina/metabolismoRESUMEN
Malaria still remains one of the deadliest infectious diseases, and has a tremendous morbidity and mortality impact in the developing world. The propensity of the parasites to develop drug resistance, and the relative reluctance of the pharmaceutical industry to invest massively in the developments of drugs that would offer only limited marketing prospects, are major issues in antimalarial drug discovery. Protein kinases (PKs) have become a major family of targets for drug discovery research in a number of disease contexts, which has generated considerable resources such as kinase-directed libraries and high throughput kinase inhibition assays. The phylogenetic distance between malaria parasites and their human host translates into important divergences in their respective kinomes, and most Plasmodium kinases display atypical properties (as compared to mammalian PKs) that can be exploited towards selective inhibition. Here, we discuss the taxon-specific kinases possessed by malaria parasites, and give an overview of target PKs that have been validated by reverse genetics, either in the human malaria parasite Plasmodium falciparum or in the rodent model Plasmodium berghei. We also briefly allude to the possibility of attacking Plasmodium through the inhibition of human PKs that are required for survival of this obligatory intracellular parasite, and which are targets for other human diseases.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Malaria/tratamiento farmacológico , Plasmodium berghei/enzimología , Plasmodium falciparum/enzimología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Quinasas , Proteínas Protozoarias/antagonistas & inhibidores , Animales , Humanos , Malaria/enzimología , Inhibidores de Proteínas Quinasas/químicaRESUMEN
Ecotin is a potent inhibitor of family S1A serine peptidases, enzymes lacking in the protozoan parasite Leishmania major. Nevertheless, L. major has three ecotin-like genes, termed inhibitor of serine peptidase (ISP). ISP1 is expressed in vector-borne procyclic and metacyclic promastigotes, whereas ISP2 is also expressed in the mammalian amastigote stage. Recombinant ISP2 inhibited neutrophil elastase, trypsin and chymotrypsin with K(i)s between 7.7 and 83 nM. L. major ISP2-ISP3 double null mutants (Deltaisp2/3) were created. These grew normally as promastigotes, but were internalized by macrophages more efficiently than wild-type parasites due to the upregulation of phagocytosis by a mechanism dependent on serine peptidase activity. Deltaisp2/3 promastigotes transformed to amastigotes, but failed to divide for 48 h. Intracellular multiplication of Deltaisp2/3 was similar to wild-type parasites when serine peptidase inhibitors were present, suggesting that defective intracellular growth results from the lack of serine peptidase inhibition during promastigote uptake. Deltaisp2/3 mutants were more infective than wild-type parasites to BALB/c mice at the early stages of infection, but became equivalent as the infection progressed. These data support the hypothesis that ISPs of L. major target host serine peptidases and influence the early stages of infection of the mammalian host.
Asunto(s)
Leishmania major/inmunología , Leishmania major/patogenicidad , Macrófagos/parasitología , Proteínas Protozoarias/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Secuencia de Aminoácidos , Animales , Quimotripsina/antagonistas & inhibidores , Eliminación de Gen , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/parasitología , Elastasa de Leucocito/antagonistas & inhibidores , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Fagocitosis/inmunología , Proteínas Protozoarias/genética , Alineación de Secuencia , Inhibidores de Serina Proteinasa/genética , Tripsina/metabolismoRESUMEN
The FK506-binding proteins (FKBs) represent ubiquitous enzymes that catalyze the rate-limiting peptidyl prolyl cis-trans isomerization step in protein folding. The nematode Caenorhabditis elegans has eight FKBs, three of which (FKB-3, -4, and -5) have dual peptidyl prolyl cis-trans isomerase (PPIase) domains, signal peptides and ER retention signals. PPIase activity has been detected for recombinant FKB-3. Both FKB-3 and -5 are expressed in the exoskeleton-synthesizing hypodermis with transcript peaks that correspond to the molting and collagen synthesis cycles. FKB-4 is expressed at a low level throughout development. No phenotypes were observed in deletion mutants in each of the secretory pathway FKBs. Combined triple and fkb-4, -5 double deletion mutants were however found to arrest at 12 degrees C, but developed normally at 15-25 degrees C. This cold-sensitive larval lethal effect was not maternally derived, occurred during embryogenesis, and could be rescued following the transgenic introduction of a wild type copy of either fkb-4 or fkb-5. The temperature-sensitive defects also affected molting, cuticle collagen expression, hypodermal seam cell morphology, and the structural integrity of the cuticular extracellular matrix. This study establishes that the secretory pathway FK506-binding PPIase enzymes are essential for normal nematode development, collagen biogenesis, and the formation of an intact exoskeleton under adverse physiological conditions.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Isomerasa de Peptidilprolil/deficiencia , Proteínas de Unión a Tacrolimus/deficiencia , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Frío , Matriz Extracelular/patología , Eliminación de Gen , Muda/genéticaRESUMEN
Pyrococcus furiosus protease I (PFPI) is a multimeric cysteine peptidase from P. furiosus. Genome analyses indicate that orthologues are present in rather few other organisms, including Dictyostelium discoideum and several bacteria, Archaea and plants. An open reading frame (ORF) coding for a PFPI-like protein (PFP1) was identified in Leishmania major and Leishmania mexicana and full-length spliced and polyadenylated PFP1 mRNA detected for both species. Vestiges of a PFPI-like gene could also be identified in Leishmania braziliensis and Leishmania infantum, but no ORF remains owing to the presence of frame-shifts and stop codons. No evidence for a PFPI-like gene could be found in the syntenic region of Trypanosoma brucei or Trypanosoma cruzi, raising the possibility that the PFPI-like genes were acquired by a lateral gene transfer event after the divergence of trypanosomes and Leishmania. The gene may have subsequently degenerated into a pseudogene in some Leishmania species, owing to the loss of relevant biological function. However, antibodies raised against L. mexicana recombinant protein detected PFP1 in promastigote extracts of L. major, but not in L. mexicana promastigote or amastigote extracts. The expression of PFP1 in L. major suggests that PFP1 might contribute to the disease tropism that distinguishes this Leishmania species from others.
Asunto(s)
Proteínas Arqueales/genética , Genes Protozoarios , Leishmania major/genética , Leishmania/genética , Péptido Hidrolasas/genética , Seudogenes , Proteínas Recombinantes de Fusión/genética , Secuencia de Aminoácidos , Animales , Proteínas Arqueales/química , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Péptido Hidrolasas/química , Proteínas Recombinantes de Fusión/químicaRESUMEN
An NAD(+)-dependent glutamate dehydrogenase (GDH; EC 1.4.1.24) was cloned from the ruminal ciliate protozoan, Entodinium caudatum. The gene had high sequence similarity to GDH genes from the Bacteroides (class)--a class of bacteria which is highly represented in the rumen. When expressed in Escherichia coli the enzyme had a high affinity for ammonia and alpha-ketoglutarate (apparent K(m) of 2.33 and 0.71 mM, respectively) and a low affinity for glutamate (apparent K(m) of 98 mM). GDH activity and GDH mRNA concentration were increased by incubating washed E. caudatum cells with ammonia and antibiotics. These results suggest that the GDH is an anabolic enzyme catalysing the assimilation of ammonia by E. caudatum in the rumen and that the gene was probably acquired by lateral gene transfer from a ruminal bacterium.
Asunto(s)
Cilióforos/enzimología , Genes Protozoarios , Glutamato Deshidrogenasa/genética , Glutamato Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Amoníaco/metabolismo , Animales , Bacteroides/genética , Secuencia de Bases , Clonación Molecular , ADN Protozoario/química , ADN Protozoario/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica , Transferencia de Gen Horizontal , Glutamato Deshidrogenasa/aislamiento & purificación , Ácido Glutámico/metabolismo , Ácidos Cetoglutáricos/metabolismo , Datos de Secuencia Molecular , Filogenia , ARN Mensajero/análisis , ARN Protozoario/análisis , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
A novel protein disulfide isomerase gene, pdi-3, was isolated from the nematode Caenorhabditis elegans. This gene encodes an enzyme related to the ERp60 class of thioredoxin proteins and was found to exhibit unusual enzymatic properties. Recombinant protein displayed both disulfide bond isomerase activity and calcium-dependent transglutaminase-like cross-linking activity. The pdi-3 transcript was developmentally constitutively expressed, and the encoded protein is present in many tissues including the gut and the hypodermis. The nematode hypodermis synthesizes the essential collagenous extracellular matrix (ECM) called the cuticle. Transcript disruption via double-stranded RNA interference resulted in dramatic and specific synthetic phenotypes in several C. elegans mutant alleles with weakened cuticles: sqt-3(e2117), dpy-18(e364, ok162, and bx26). These nematodes displayed severe dumpy phenotypes and disrupted lateral alae, a destabilized cuticle and abnormal male and hermaphrodite tail morphologies. These defects were confirmed to be consistent with hypodermal seam cell abnormalities and corresponded with the severe disruption of a cuticle collagen. Wild type nematodes did not exhibit observable morphological defects; however, cuticle collagen localization was mildly disrupted following pdi-3 RNA interference. The unusual thioredoxin enzyme, protein disulfide isomerase-3, may therefore play a role in ECM assembly. This enzyme is required for the proper maintenance of post-embryonic body shape in strains with a weakened cuticle, perhaps through ECM stabilization via cross-linking activity, disulfide isomerase protein folding activity, protein disulfide isomerase chaperone activity, or via multifunctional events.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Transglutaminasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Cartilla de ADN , Inmunohistoquímica , Datos de Secuencia Molecular , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/genética , ARN/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
The free-living nematode Caenorhabditis elegans expresses 18 cyclophilin isoforms, eight of which are conserved single domain forms, comprising two closely related secreted or type B forms (CYP-5 and CYP-6). Recombinant CYP-5 has been purified, crystallised and the X-ray structure solved to a resolution of 1.75A. The detailed molecular architecture most strongly resembles the structure of human cyclophilin B with conserved changes in loop structure and N and C-terminal extensions. Interestingly, the active site pocket is occupied by a molecule of dithiothreitol though this has little effect on the geometry of the active site which is similar to other cyclophilin structures. The peptidyl-prolyl isomerase activity of CYP-5 has been characterised against the substrate N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and gives a k(cat)/K(m) value of 3.6x10(6)M(-1)s(-1) that compares with a value of 6.3x10(6)M(-1)s(-1) for human cyclophilin B. The immunosuppressive drug cyclosporin A binds and inhibits CYP-5 with an IC(50) value of 50nM, which is comparable to the value of 84nM found for human cyclophilin B. CYP-6 has 67% sequence identity with CYP-5 and a molecular model was built based on the CYP-5 crystal structure. The model shows that CYP-5 and CYP-6 are likely to have very similar structures, but with a markedly increased number of negative charges distributed around the surface of CYP-6. The spatial expression patterns of the cyclophilin B isoforms were examined using transgenic animals carrying a LacZ reporter fusion to these genes, and both cyp-5 and cyp-6 are found to be expressed in an overlapping fashion in the nematode gut. The temporal expression pattern of cyp-5 was further determined and revealed a constitutive expression pattern, with highest abundance levels being found in the embryo.