Seema Dalal

Roles for Two Aminopeptidases in Vacuolar Hemoglobin Catabolism in Plasmodium falciparum

During the erythrocytic stage of its life cycle, the human malaria parasite Plasmodium falciparum catabolizes large quantities of host-cell hemoglobin in an acidic organelle, the food vacuole. A current model for the catabolism of globin-derived oligopeptides invokes peptide transport out of the food vacuole followed by hydrolysis to amino acids by cytosolic aminopeptidases. To test this model, we have examined the roles of four parasite aminopeptidases during the erythrocytic cycle. Localization of tagged aminopeptidases, coupled with biochemical analysis of enriched food vacuoles, revealed the presence of amino acid-generating pathways in the food vacuole as well as the cytosol. Based on the localization data and in vitro assays, we propose a specific role for one of the plasmodial enzymes, aminopeptidase P, in the catabolism of proline-containing peptides in both the vacuole and the cytosol. We establish an apparent requirement for three of the four aminopeptidases (including the two food vacuole enzymes) for efficient parasite proliferation. To gain insight into the impact of aminopeptidase inhibition on parasite development, we examined the effect of the presence of amino acids in the culture medium of the parasite on the toxicity of the aminopeptidase inhibitor bestatin. The ability of bestatin to block parasite replication was only slightly affected when 19 of 20 amino acids were withdrawn from the medium, indicating that exogenous amino acids cannot compensate for the loss of aminopeptidase activity. Together, these results support the development of aminopeptidase inhibitors as novel chemotherapeutics directed against malaria.

Interaction of TorsinA with Its Major Binding Partners Is Impaired by the Dystonia-associated ΔGAG Deletion

Early onset (DYT1) torsion dystonia is a dominantly inherited movement disorder associated with a three-base pair (ΔGAG) deletion that removes a glutamic acid residue from the protein torsinA. TorsinA is an essential AAA+ (ATPases associated with a variety of cellular activities) ATPase found in the endoplasmic reticulum and nuclear envelope of higher eukaryotes, but what it does and how changes caused by the ΔGAG deletion lead to dystonia are not known. Here, we asked how the DYT1 mutation affects association of torsinA with interacting proteins. Using immunoprecipitation and mass spectrometry, we first established that the related transmembrane proteins LULL1 and LAP1 are prominent binding partners for torsinA in U2OS cells. Comparative analysis demonstrates that these two proteins are targeted to the endoplasmic reticulum or nuclear envelope by their divergent N-terminal domains. Binding of torsinA to their C-terminal lumenal domains is stabilized when residues in any one of three motifs implicated in ATP hydrolysis (Walker B, sensor 1, and sensor 2) are mutated. Importantly, the ΔGAG deletion does not stabilize this binding. Indeed, deleting the ΔGAG encoded glutamic acid residue from any of the three ATP hydrolysis mutants destabilizes their association with LULL1 and LAP1C, suggesting a possible basis for loss of torsinA function. Impaired interaction of torsinA with LULL1 and/or LAP1 may thus contribute to the development of dystonia.

SNARE Complex Zero Layer Residues Are Not Critical for N-Ethylmaleimide-sensitive Factor-mediated Disassembly

Membrane-anchored SNAREs assemble into SNARE complexes that bring membranes together to promote fusion. SNARE complexes are parallel four-helix bundles stabilized in part by hydrophobic interactions within their core. At the center of SNARE complexes is a distinctive zero layer that consists of one arginine and three glutamines. This zero layer is thought to play a special role in the biology of the SNARE complex. One proposal is that the polar residues of the zero layer enable N-ethylmaleimide-sensitive factor (NSF)-mediated SNARE complex disassembly. Here, we studied the effects of manipulating the zero layer of the well studied synaptic SNARE complex in vitro and in vivo. Using a fluorescence-based assay to follow SNARE complex disassembly in real time, we found that the maximal rate at which NSF disassembles complexes was unaffected by mutations in the zero layer, including single replacement of the syntaxin glutamine with arginine as well as multiple replacement of all four layer residues with non-polar amino acids. To determine whether syntaxin with arginine instead of glutamine in its zero layer can support SNARE function in vivo, we introduced it as a transgene into a Caenorhabditis elegans syntaxin-null strain. Mutant syntaxin rescued viability and locomotory defects similarly to wild-type syntaxin, demonstrating that SNARE complexes with two glutamines and two arginines in the zero layer can support neurotransmission. These findings show that residues of the zero layer do not play an essential role in NSF-mediated disassembly.

Evidence for catalytic roles for Plasmodium falciparum aminopeptidase P in the food vacuole and cytosol

The metalloenzyme aminopeptidase P catalyzes the hydrolysis of amino acids from the amino termini of peptides with a prolyl residue in the second position. The human malaria parasite Plasmodium falciparum expresses a homolog of aminopeptidase P during its asexual intraerythrocytic cycle. P. falciparum aminopeptidase P (PfAPP) shares with mammalian cytosolic aminopeptidase P a three-domain, homodimeric organization and is most active with Mn(II) as the cofactor. A distinguishing feature of PfAPP is a 120-amino acid amino-terminal extension that appears to be removed from the mature protein. PfAPP is present in the food vacuole and cytosol of the parasite, a distribution that suggests roles in vacuolar hemoglobin catabolism and cytosolic peptide turnover. To evaluate the plausibility of these putative functions, the stability and kinetic properties of recombinant PfAPP were evaluated at the acidic pH of the food vacuole and at the near-neutral pH of the cytosol. PfAPP exhibited high stability at 37 °C in the pH range 5.0–7.5. In contrast, recombinant human cytosolic APP1 was unstable and formed a high molecular weight aggregate at acidic pH. At both acidic and slightly basic pH values, PfAPP efficiently hydrolyzed the amino-terminal X-Pro bond of the nonapeptide bradykinin and of two globin pentapeptides that are potential in vivo substrates. These results provide support for roles for PfAPP in peptide catabolism in both the food vacuole and the cytosol and suggest that PfAPP has evolved a dual distribution in response to the metabolic needs of the intraerythrocytic parasite.

Evidence for a Golgi-to-endosome protein sorting pathway in Plasmodium falciparum.

During the asexual intraerythrocytic stage, the malaria parasite Plasmodium falciparum must traffic newly-synthesized proteins to a broad array of destinations within and beyond the parasites plasma membrane. In this study, we have localized two well-conserved protein components of eukaryotic endosomes, the retromer complex and the small GTPase Rab7, to define a previously-undescribed endosomal compartment in P. falciparum. Retromer and Rab7 co-localized to a small number of punctate structures within parasites. These structures, which we refer to as endosomes, lie in close proximity to the Golgi apparatus and, like the Golgi apparatus, are inherited by daughter merozoites. However, the endosome is clearly distinct from the Golgi apparatus as neither retromer nor Rab7 redistributed to the endoplasmic reticulum upon brefeldin A treatment. Nascent rhoptries (specialized secretory organelles required for invasion) developed adjacent to endosomes, an observation that suggests a role for the endosome in rhoptry biogenesis. A P. falciparum homolog of the sortilin family of protein sorting receptors (PfSortilin) was localized to the Golgi apparatus. Together, these results elaborate a putative Golgi-to-endosome protein sorting pathway in asexual blood stage parasites and suggest that one role of retromer is to mediate the retrograde transport of PfSortilin from the endosome to the Golgi apparatus.

Nanomolar Antimalarial Agents against Chloroquine-Resistant Plasmodium falciparum from Medicinal Plants and Their Structure–Activity Relationships

Inspired by the discovery of the antimalarial drug artemisinin from a traditional Chinese medicine (TCM), a natural product library of 44 lindenane-type sesquiterpenoids was assessed for activities against the Dd2 chloroquine-resistant strain of the malaria parasite Plasmodium falciparum. These compounds were mainly isolated from plants of the Chloranthus genus, many species of which are named Sikuaiwa in TCM and have long been used to treat malaria. The compounds consisted of 41 sesquiterpenoid dimers and three monomers, including the 12 new dimers 1-12 isolated from Chloranthus fortunei. The results showed that 16 dimers exhibited potent antiplasmodial activities (<100 nM); in particular, compounds 1, 14, and 19 exhibited low nanomolar activities with IC50 values ranging from 1 to 7 nM, which is comparable to the potency of artemisinin, and selectivity index values toward mammalian cells greater than 500. A comprehensive structure-activity relationship study indicated that three functional groups are essential and two motifs can be modified.

Fortunoids A–C, Three Sesquiterpenoid Dimers with Different Carbon Skeletons from Chloranthus fortunei

Three dimeric sesquiterpenoids (1-3), fortunoid A (1) possessing a new carbon skeleton of rearranged lindenane dimer and fortunoids B (2) and C (3) representing the first example of the dimers of a lindenane and a eudesmane sesquiterpene, were isolated from Chloranthus fortunei. Their structures with absolute configurations were established by spectroscopic data and electric circular dichroism analysis. Their biosynthetic origins were also proposed. Compounds 1 and 2 showed moderate antimalarial activities.

Antiplasmodial Sesquiterpenoid Lactones from Trichospira verticillata : Structure Elucidation by Spectroscopic Methods and Comparison of Experimental and Calculated ECD Data

A dichloromethane extract of Trichospira verticillata from the Natural Products Discovery Institute was discovered to have good antiplasmodial activity (IC50 ∼5 μg/mL). After purification by liquid-liquid partition and C18 reversed-phase HPLC, four new germacranolide-type sesquiterpenoid lactones named trichospirolides A-D (1-4) were isolated. The structures of the new compounds were elucidated by analysis of their 1D and 2D NMR and MS data. The relative and absolute configurations were assigned based on a comparison of calculated and experimental ECD and UV spectra, specific rotations, internuclear distances, and coupling constants for all possible diastereomers for each compound. Among these four compounds, the conjugated dienone 1 displayed the most potent antiplasmodial activity, with an IC50 value of 1.5 μM.

Antimalarial activity of the isolates from the marine sponge Hyrtios erectus against the chloroquine-resistant Dd2 strain of Plasmodium falciparum

Abstract This work reports the isolation of the three known compounds, smenotronic acid (1), ilimaquinone (2), and pelorol (3), from the 85% methanol (MeOH)-soluble fraction of the sponge Hyrtios erectus. The structures of the isolated compounds were determined with the help of modern spectroscopic techniques, and the resulting compounds were then subjected to in vitro screening to determine their antimalarial potential against the chloroquine-resistant Dd2 strain of Plasmodium falciparum. Amongst the three compounds, pelorol (3) showed significant activity against P. falciparum with an IC50 value of 0.8 μM. For smenotronic acid (1) and ilimaquinone (2), moderate activities were observed with IC50 values of 3.51 and 2.11 μM, respectively.