Human plasma plasminogen internalization route in Plasmodium falciparum-infected erythrocytes.

BACKGROUND: The intra-erythrocytic development of the malaria parasite Plasmodium falciparum depends on the uptake of a number of essential nutrients from the host cell and blood plasma. It is widely recognized that the parasite imports low molecular weight solutes from the plasma and the consumption of these nutrients by P. falciparum has been extensively analysed. However, although it was already shown that the parasite also imports functional proteins from the vertebrate host, the internalization route through the different infected erythrocyte membranes has not yet been elucidated. In order to further understand the uptake mechanism, the study examined the trafficking of human plasminogen from the extracellular medium into P. falciparum-infected red blood cells. METHODS: Plasmodium falciparum clone 3D7 was cultured in standard HEPES-buffered RPMI 1640 medium supplemented with 0.5% AlbuMAX. Exogenous human plasminogen was added to the P. falciparum culture and the uptake of this protein by the parasites was analysed by electron microscopy and Western blotting. Immunoprecipitation and mass spectrometry were performed to investigate possible protein interactions that may assist plasminogen import into infected erythrocytes. The effect of pharmacological inhibitors of different cellular physiological processes in plasminogen uptake was also tested. RESULTS: It was observed that plasminogen was selectively internalized by P. falciparum-infected erythrocytes, with localization in plasma membrane erythrocyte and parasite's cytosol. The protein was not detected in parasitic food vacuole and haemoglobin-containing vesicles. Furthermore, in erythrocyte cytoplasm, plasminogen was associated with the parasite-derived membranous structures tubovesicular network (TVN) and Maurer's clefts. Several proteins were identified in immunoprecipitation assay and may be involved in the delivery of plasminogen across the P. falciparum multiple compartments. CONCLUSION: The findings here reported reveal new features regarding the acquisition of plasma proteins of the host by P. falciparum-infected erythrocytes, a mechanism that involves the exomembrane system, which is distinct from the haemoglobin uptake, clarifying a route that may be potentially targeted for inhibition studies.

Evaluación de la proteólisis en células aisladas vivas de Plasmodium falciparum aisladas durante los estadios asexuales sanguíneos / Proteolysis assessment in isolated Plasmodium falciparum living cells at the asexual blood stages

It has been demonstrated that proteases play crucial roles in Plasmodium falciparum infection and therefore have been considered as targets for the development of new therapeutic drugs. The aim of this study was to describe the specific proteolytic activity profile in all blood stages of P. falciparum isolated parasites in order to explore new antimalarial options. For this purpose, we used the fluorogenic substrate Z-Phe-Arg-MCA (Z: carbobenzoxy, MCA: 7-amino-4-methyl coumarine) and classic inhibitors for the different classes of proteolytic enzymes, such as phenylmethylsulfonyl fluoride (PMSF), 1.10-phenantroline, pepstatin A and E64 to study the inhibition profiles. As expected, due to the high metabolic activity in mature stages, the substrate was mostly degraded in the trophozoite and schizont, with specific activities ~ 20 times higher than in early stages (merozoite/rings). The major actors in substrate hydrolysis were cysteine proteases, as confirmed by the complete hydrolysis inhibition with E64 addition. Proteolytic activity was also inhibited in the presence of PMSF in all but the schizont stage. However, PMSF inhibition was the result of unspecific interaction with cysteine proteases as demonstrated by reversion of inhibition by dithiotreitol (DTT), indicating that serine protease activity is very low or null. To our knowledge, this is the first report aiming to describe the proteolytic profile of P. falciparum isolated parasites at all the erythrocytic cycle stages. The results and protocol described herein can be useful in the elucidation of stage specific action of proteolysis-inhibiting drugs and aid in the development of antimalarial compounds with protease inhibitory activity(AU)

e ha demostrado que las proteasas desempeñan funciones vitales en la infección por Plasmodium falciparum, y por lo tanto se consideran dianas en la elaboración de nuevos medicamentos terapéuticos. El objetivo del estudio era describir el perfil de actividad proteolítica específica de todas las etapas sanguíneas de parásitos aislados de P. falciparum con vistas a explorar nuevas opciones antimaláricas. Con ese propósito, utilizamos el sustrato fluorogénico Z-Phe-Arg-AMC (Z: carbobenzoxi, AMC: 7-amino-4-metilcumarina) e inhibidores clásicos para las diferentes clases de enzimas proteolíticas, tales como el fluoruro de fenilmetilsulfonilo (PMSF), 1,10-fenantrolina, pepstatina A y E64 para estudiar los perfiles de inhibición. Como se esperaba, debido a la elevada actividad metabólica de las etapas de madurez, el sustrato fue degradado mayormente en el trofozoíto y el esquizonte, con actividad específica ~ 20 veces superior a la de las etapas tempranas (merozoíto/ anillos). Los principales actores en la hidrólisis del sustrato fueron las cisteínas proteasas, lo que fue confirmado por la inhibición completa de la hidrólisis con la adición de E64. La actividad proteolítica también fue inhibida en presencia de PMSF en todas las etapas excepto el esquizonte. Sin embargo, la inhibición del PMSF fue resultado de una interacción inespecífica con las cisteínas proteasas, según lo demuestra la reversión de la inhibición con el ditiotreitol (DTT), lo que indica que la actividad de la serina proteasa es muy baja o inexistente. Que sepamos, este es el primer informe dirigido a describir el perfil proteolítico de parásitos aislados de P. falciparum en todas las etapas del ciclo eritrocítico. Los resultados y el protocolo que aquí se describen pueden ser útiles para dilucidar la acción específica de los medicamentos inhibidores de proteólisis en cada etapa, así como contribuir al desarrollo de compuestos antimaláricos con actividad inhibidora de la proteasa(AU)

Plasmodium falciparum histidine triad protein and calmodulin modulates calcium homeostasis and intracellular proteolysis.

Calcium signaling has an essential role in fundamental processes of Plasmodium life cycle, including migration, cell invasion and parasite development. Two important players in calcium homeostasis, the Histidine Triad (HIT) protein that is implicated in calcium signaling in mammalian cells and calmodulin, which is a classic calcium sensor in eukaryotes are present in Plasmodium falciparum, however theirs function is unknown in the parasite. Here, we investigated the involvement of the P. falciparum Histidine Triad protein (PfHint-1) and calmodulin (PfCaM) in calcium signaling and intracellular proteolysis. For this, we targeted PfHint-1 with a hemagglutinin tail and overexpressed both proteins. We observed that PfHint-1 is expressed throughout the erythrocytic stages and partially colocalizes to the endoplasmic reticulum. Parasites overexpressing PfHint-1 displayed lower ER Ca2+ content and a higher [Ca2+]cyt rise in the parasite cytosol upon Ca2+ addition to the extracellular medium after depletion of ER calcium store. PfCaM-overexpressing parasites exhibit a higher [Ca2+]cyt rise after challenge with the calmodulin inhibitor, calmidazolium. The calcium-dependent proteolytic activity in PfCaM- and PfHint-1-overexpressing parasites was increased and correlated to alterations in calcium homeostasis. Taken together, our results indicate the participation of these proteins in P. falciparum fundamental cellular processes and highlights promising targets for the development of antimalarial drugs.

Inhibition of Plasmodium falciparum cysteine proteases by the sugarcane cystatin CaneCPI-4.

Malaria is a disease caused by Plasmodium parasites that affects hundreds of millions of people. Plasmodium proteases are involved in invasion, erythrocyte egress and degradation of host proteins. Falcipains are well-studied cysteine peptidases located in P. falciparum food vacuoles that participate in hemoglobin degradation. Cystatins are natural cysteine protease inhibitors that are implicated in a wide range of regulatory processes. Here, we report that a cystatin from sugarcane, CaneCPI-4, is selectively internalized into P. falciparum infected erythrocytes and is not processed by the parasite proteolytic machinery. Furthermore, we demonstrated the inhibition of P. falciparum cysteine proteases by CaneCPI-4, suggesting that it can exert inhibitory functions inside the parasites. The inhibition of the proteolytic activity of parasite cells is specific to this cystatin, as the addition of an anti-CaneCPI-4 antibody completely abolished the inhibition. We extended the studies to recombinant falcipain-2 and falcipain-3 and demonstrated that CaneCPI-4 strongly inhibits these enzymes, with IC50 values of 12nM and 42nM, respectively. We also demonstrated that CaneCPI-4 decreased the hemozoin formation in the parasites, affecting the parasitemia. Taken together, this study identified a natural molecule as a potential antimalarial that specifically targets falcipains and also contributes to a better understanding of macromolecule acquisition by Plasmodium falciparum infected RBCs.

Calmidazolium evokes high calcium fluctuations in Plasmodium falciparum.

Calcium and calmodulin (CaM) are important players in eukaryote cell signaling. In the present study, by using a knockin approach, we demonstrated the expression and localization of CaM in all erythrocytic stages of Plasmodium falciparum. Under extracellular Ca(2+)-free conditions, calmidazolium (CZ), a potent CaM inhibitor, promoted a transient cytosolic calcium ([Ca(2+)]cyt) increase in isolated trophozoites, indicating that CZ mobilizes intracellular sources of calcium. In the same extracellular Ca(2+)-free conditions, the [Ca(2+)]cyt rise elicited by CZ treatment was ~3.5 fold higher when the endoplasmic reticulum (ER) calcium store was previously depleted ruling out the mobilization of calcium from the ER by CZ. The effects of the Ca(2+)/H(+) ionophore ionomycin (ION) and the Na(+)/H(+) ionophore monensin (MON) suggest that the [Ca(2+)]cyt-increasing effect of CZ is driven by the removal of Ca(2+) from at least one Ca(2+)-CaM-related (CaMR) protein as well as by the mobilization of Ca(2+) from intracellular acidic calcium stores. Moreover, we showed that the mitochondrion participates in the sequestration of the cytosolic Ca(2+) elicited by CZ. Finally, the modulation of membrane Ca(2+) channels by CZ and thapsigargin (THG) was demonstrated. The opened channels were blocked by the unspecific calcium channel blocker Co(2+) but not by 2-APB (capacitative calcium entry inhibitor) or nifedipine (L-type Ca(2+) channel inhibitor). Taken together, the results suggested that one CaMR protein is an important modulator of calcium signaling and homeostasis during the Plasmodium intraerythrocytic cell cycle, working as a relevant intracellular Ca(2+) reservoir in the parasite.

Hypervalent organotellurium compounds as inhibitors of P. falciparum calcium-dependent cysteine proteases.

Hypervalent organotellurium compounds (organotelluranes) have shown several promising applications, including their use as potent and selective cysteine protease inhibitors and antiprotozoal agents. Here, we report the antimalarial activities of three organotellurane derivatives (RF05, RF07 and RF19) in two Plasmodium falciparum strains (CQS 3D7 and CQR W2), which demonstrated significant decreases in parasitemia in vitro. The inhibition of intracellular P. falciparum proteases by RF05, RF07 and RF19 was determined and the IC50 values were 3.7±1.0µM, 1.1±0.2µM and 0.2±0.01µM, respectively. Using an assay performed in the presence of the ER Ca(2+)-ATPase inhibitor we showed that the main enzymatic targets were cysteine proteases stimulated by calcium (calpains). None of the compounds tested caused haemolysis or a significant decrease in endothelial cell viability in the concentration range used for the inhibition assay. Taken together, the results suggest promising compounds for the development of antimalarial drugs.

Theoretical insight into the mechanism for the inhibition of the cysteine protease cathepsin B by 1,2,4-thiadiazole derivatives.

Several cellular disorders have been related to the overexpression of the cysteine protease cathepsin B (CatB), such as rheumatic arthritis, muscular dystrophy, osteoporosis, Alzheimer's disease, and tumor metastasis. Therefore, inhibiting CatB may be a way to control unregulated cellular functions and prevent tissue malformations. The inhibitory action of 1,2,4-thiadiazole (TDZ) derivatives has been associated in the literature with their ability to form disulfide bridges with the catalytic cysteine of CatB. In this work, we present molecular modeling and docking studies of a series of eight 1,2,4-thiadiazole compounds. Substitutions at two positions (3 and 5) on the 1,2,4-thiadiazole ring were analyzed, and the docking scores were correlated to experimental data. A correlation was found with the sequence of scores of four related compounds with different substituents at position 5. No correlation was observed for changes at position 3. In addition, quantum chemistry calculations were performed on smaller molecular models to study the mechanism of inhibition of TDZ at the active site of CatB. All possible protonation states of the ligand and the active site residues were assessed. The tautomeric form in which the proton is located on N2 was identified as the species that has the structural and energetic characteristics that would allow the ring opening of 1,2,4-thiadiazole.