Leishmaniasis is an important Public Health problem in Brazil and in many parts of the world. Since treatments presently available are not very effective and pose serious side effects, finding alternative ways of controlling the disease is a necessity. Leishmania, like other trypanosomatids, present unique biological features as compared to higher eukaryotes, that can be exploited with the intent of finding new chemotherapeutical/vaccine targets. The present projects proposes to study mechanisms of cellular sorting in these parasites, as such potential targets. Lysosomal targeting of amastigote-specific cysteine proteinases (Lpcys1 and Lpcys2) in Leishmania: -Examination of the role of glycosylation in lysosomal targeting. There are two potential N-glycosylation sites in the abundant Lpcys2 proteinase. The effect of the substitution of these asparagines by site-directed mutagenesis will be investigated. -Characterization of the specific signals in the prepro region of Lpcys2 responsible for lysosomal sorting. Previous results indicate a role in targeting for this region. -Search for targeting sequences in the prepro region of Lpcys1. -Determination of the leishmanial receptors responsible for lysosomal targeting recognizing the prepro region of Lpcys2. -Determination of a role for the C-terminal extension of Lpcys2. This unique sequence contains many conserved cysteines when compared to proteinases of trypanosomes, suggesting a conserved function. When fused to the C-terminus of green fluorescent protein (GFP) and expressed in Leishmania, was shown to enhance fluorescence, indicating a possible role in protection from degradation. Flagellar targeting in Leishmania: -Examination of signals responsible for targeting of the flagellum-specific protein Flag-1.
