Intracellular superoxide (O 32 ) was manipulated in M14 melanoma cells by overexpression or repression of Cu/Zn SOD using a tetracycline-inducible expression system. Scavenging intracellular O 3 2 increased tumor cell sensitivity to daunorubicin, etoposide, and pMC540, whereas expression of the antisense SOD mRNA significantly decreased cell sensitivity to drug treatment. Whereas Cu/Zn SOD overexpressing cells exhibited higher activation of the executioner caspase 3 upon drug exposure, caspase 3 activation was significantly lower when Cu/Zn SOD was repressed by antisense expression. These data show that intracellular O 3 2 regulates tumor cell response to drug-induced cell death via a direct or indirect effect on the caspase activation pathway.z 1999 Federation of European Biochemical Societies.
The mechanism by which a malaria merozoite recognizes a suitable host cell is mediated by a cascade of receptor-ligand interactions. In addition to the availability of the appropriate receptors, intracellular ATP plays an important role in determining whether erythrocytes are suitable for merozoite invasion. Recent work has shown that ATP secreted from erythrocytes signals a number of cellular processes. To determine whether ATP signaling might be involved in merozoite invasion, we investigated whether known plasmodium invasion proteins contain nucleotide binding motifs. Malaria is caused by unicellular protozoan parasites and is considered to be one of the most important infectious diseases still affecting mankind today. The complex life cycle of the parasite is characterized by three invasive forms: the sporozoite and merozoite that invade hepatocytes and erythrocytes in the vertebrate host, respectively, and the ookinetes inside the insect vector that penetrates the mosquito midgut epithelium (1-5). In the case of merozoites, specific organelles (rhoptries, micronemes, and dense granules) at the apical end of the parasite contain proteins that play an important role in the recognition and invasion of the host cell (6). Recognition of specific erythrocyte receptors by the merozoite is mediated by at least two gene families, the reticulocyte-binding protein homologues (RBL) 4 and the erythrocyte binding ligands (7,8). Like the erythrocyte binding ligands, the RBLs are also found in varying numbers in all plasmodium species with each member believed to play a role in recognizing a different receptor (7-16). In general, the members of RBL are large transmembrane proteins with molecular masses above 200 kDa that get proteolytically cleaved during the invasion process (14,17). In Plasmodium yoelii, members of the RBL termed Py235 (P. yoelii 235-kDa rhoptry protein) have been shown to be potential virulence factors that enable the parasite to invade a wider range of host erythrocytes (18 -20). In addition, Py235 is also involved in clonal phenotypic variation of merozoites (21) enabling the parasite to evade immune responses and adapt to changes in the host environment during the invasion step (22). Recently it has been demonstrated that variation in the amount of Py235 expressed in merozoites defines the host cell repertoire that a parasite can invade (20). Studies carried out on the RBLs of Plasmodium falciparum (PfRH1, (10, 14 -16, 23, 24) have provided additional evidence that different RBL members interact with different receptors on the erythrocyte and that these interactions are crucial for invasion. Previously work in Plasmodium vivax has indicated that RBLs may have an initial sensing role preceding and possibly enabling the subsequent interaction of the erythrocyte binding ligand member with its corresponding receptor (25). In a more recent study the erythrocyte binding region of PfRH1 and PfRH4 has been identified (26,71), showing that only a relatively small region of these proteins is actually invol...
Invasion of the host cell by the malaria parasite is a key step for parasite survival and the only stage of its life cycle where the parasite is extracellular, and it is therefore a target for an antimalaria intervention strategy. Multiple members of the reticulocyte binding protein homologues (RH) family are found in all plasmodia and have been shown to bind to host red blood cells directly. In the study described here, we delineated the erythrocyte binding domain (EBD) of one member of the RH family, termed Py235, from Plasmodium yoelii. Moreover, we have obtained the low-resolution structure of the EBD using small-angle X-ray scattering. Comparison of the EDB structure to other characterized Plasmodium receptor binding domains suggests that there may be an overall structural conservation. These findings may help in developing new approaches to target receptor ligand interactions mediated by parasite proteins.Differences in the ability of the invasive form of the malaria parasite, the merozoite, to recognize and invade red blood cells (RBC) have a direct impact on disease severity. In the case of the human parasite Plasmodium vivax, invasion is restricted to reticulocytes, leading to a lower parasite burden than that of Plasmodium falciparum, which is able to invade RBC of all ages. Differences in the repertoire and expression of parasite ligands on the merozoite are responsible in defining the invasion characteristics of the different parasite species.Two protein families, called erythrocyte binding-like proteins (EBL) and reticulocyte binding protein homologues (RH), are found in all Plasmodium species and have been shown to be key ligands that enable the parasite to recognize different receptors on the RBC surface (reviewed in references 22 and 34). The total number of EBL varies between different parasite species, with P. falciparum having five members while P. vivax has only a single member (1,11,20). All members of the EBL proteins are defined by the presence of the cysteinerich Duffy binding-like (DBL) domain, with each DBL domain mediating binding to a single receptor on the RBC (1, 2, 26, 40-42). Both in P. falciparum and in P. vivax the RBC receptors recognized by the different members of the EBL family are known. The receptor recognized by each EBL directly correlates with the binding specificity of its DBL domain. As with the EBL, the number of RH varies between different parasite species, ranging from as few as 6 members in P. falciparum to as many as 14 in the rodent malaria parasite Plasmodium yoelii (12,13,20). In P. falciparum, different members of the RH are able to recognize different receptors on the RBC, and the combination of RH and EBL expressed in the merozoite defines unique invasion pathways (3, 6-8, 16, 17, 19, 21, 26, 31, 35, 39, 42, 46, 47, 49, 50, 56, 62, 65). Unlike the case of the EBL, there is no easily identifiable domain structure in RH, making it difficult to identify functional domains within these large proteins. Recently, the erythrocyte binding regions of PfRH1, PfRH4, ...
Invasion of the erythrocyte by the invasive form of the malaria parasite, the merozoite, is a complex process involving numerous parasite proteins. The reticulocyte-binding protein homologues (RH) family of merozoite proteins has been previously shown to play an important role in the invasion process. Previously, it has been shown that the RH proteins of Plasmodium yoelii, Py235, play a role as an ATP/ADP sensor. Binding of Py235 to the erythrocyte surface is increased in the presence of ATP, while ADP has an inhibitory effect. The sensor domain of Py235 is called NBD94 and the segment that has been shown to covalently bind the adenine nucleotide is made up by the residues (483) FNEIKEKLKHYNFDDFVKEE(502) . Here, we report on the solution nuclear magnetic resonance structure of this peptide (NBD94(483-502) ) showing the presence of an α-helix between amino acid residues 485 and 491. The N- and C-terminal segments of the structure bend at tyrosine 493, a residue important for ATP binding. Importantly, erythrocyte-binding assays demonstrate that NBD94(483-502) can directly interfere with the binding of native Py235 to erythrocytes, suggesting a direct role of this region in erythrocyte binding. The data will provide the foundation for future studies to identify new compounds that directly interfere with the invasion process.
Alternative invasion pathways 54 1.10 The present investigation 57 CHAPTER 2 MATERIAL AND METHODS 60 2.1 Materials (Chemicals, reagents and kits) 61 2.2 Bioinformatics 63
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