Plasmodium falciparum causes the most severe form of human malaria. During its 48-hour life cycle inside human red blood cells (RBCs) the parasite replicates into infective merozoites, which must then exit the host cell to invade new erythrocytes. Mounting evidence suggests that proteases are involved in host cell rupture and parasite release, although the molecular mechanisms underlying this process remain largely uncharacterized. Recently, we identified a novel P. falciparum cysteine protease named falcipain-2 (FP-2) that cleaves host erythrocyte membrane ankyrin, protein 4.1, adducin, and dematin with maximum proteolytic activity at late stages of parasite development. Based on our findings we propose that FP-2 mediates the cleavage of erythrocyte membrane skeletal proteins that are vital to the stability of red cell membrane, thus modulating the parasite release in vivo. The aim of this proposal is to define the functional role of FP-2 in vivo. Specifically, the following issues will be addressed: (1) What are the FP-2-mediated cleavage sites of erythrocyte ankyrin, adducin, and dematin? We have recently identified the site of protein 4.1 cleavage by FP-2, and have isolated an inhibitory peptide (Pi) that blocks all known functions of FP-2 in vitro. Similar strategy will be used to determine the precise sequence of the cleavage sites of ankyrin, adducin, and dematin. Also, we will determine the specificity of FP-2 by using a series of peptides derived from Pi in which critical residues are varied based on substrate specificities of other papain family enzymes. The potent peptide(s) will be selected to determine it's (their) effect on the intraerythrocytic growth and ultimate release of P. falciparum parasite. (2) Is parasite-derived cytosolic falcipain-2 accessible to the erythrocyte membrane skeleton in vivo? This will be achieved by immunodetection of FP-2 within parasite- infected erythrocytes, and in subcellular fractions, and by studying biosynthesis and maturation of FP-2. Furthermore, we will use a cell-permeable cysteine protease inhibitor in P. falciparum cultures as well as in the mouse in vivo model to elucidate the role of cysteine proteases in parasite release. (3) What is the role of falcipain-2 in th intraerythrocytic life cycle of P. falciparum? To directly confirm the function of FP-2 in vivo and to determine whether FP-2 alone is sufficient for parasite release, a model will be developed by disrupting the FP- 2 gene using gene targeting technology. Together, the proposed studies on FP-2 may begin to reveal molecular mechanisms underlying the release of malaria parasite from the host erythrocyte.