Mechanisms and Intracellular Components in Exocytosis, Membrane Assembly and Repair, and Parasite Replication

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Living cells are open non-equilibrium systems. To exist, a cell requires precisely controlled maintenance of gradients in the chemical potential, between the extracellular environment, the cytoplasm, and the lumen of organelles, of many constituents. The amphiphilic nature of lipid molecules, self-assembling into lipid bilayers, provides an extremely low permeability barrier to both electrolytes and large nonelectrolytes. For the processes of life, a continuous exchange of matter must occur across all membranes. For example, uptake of large molecules and compounds from the outside occurs via endocytosis, phagocytosis, and macro and micro pinocytosis, secretion occurs via exocytosis, and intracellular protein trafficking via transport vesicles between endoplasmic reticulum, Golgi apparatus, endosomes, and lysosomes. The movement of such membrane-bound cargo dictates membrane recycling, or cells and organelles would be incapable of maintaining their volumes and shapes. These ubiquitous and multifarious events, plus the accommodation of lipid bilayers to membrane proteins and transient pores, all require the lipid bilayer to change its topology. Physical models of these topological changes, essential to life, must take into account the energy of membrane deformations within the framework of an adequate theory of elasticity. 1. For pore formation, to avoid solvent and tension effects, giant unilamellar vesicles (GUVs) were used, and transport kinetics were measured by the entry of the impermeant fluorescent dye calcein. Because the timescale of electrical pulses needed to restructure bilayers into pores is much shorter than the time resolution of current techniques for. membrane transport kinetics measurements, the lifetimes of lipid bilayer electropores were measured using systematic variation of the initial MD simulation conditions, whereas GUV transport kinetics were detected in response to a nanosecond timescale variation in the applied electric pulse lifetimes and interpulse intervals. Molecular transport after GUV permeabilization induced by multiple pulses is additive for interpulse intervals as short as 50 ns but not 5-ns intervals, consistent with the 1050- ns lifetimes of electropores in MD simulations. Although the results were mostly consistent between GUV and MD simulations, the kinetics of ultrashort, electric-field-induced permeabilization of GUVs were significantly different from published results in cells exposed to ultrashort (6 and 2 ns) electric fields, suggesting that cellular electroporation involves additional structures and processes. The experimental data are consistent with the hypothesis that calcein-permeable lipid electropores in GUVs are created within a few nanoseconds and that most are annihilated within a few tens of nanoseconds, consistent with molecular simulations, but in contrast with the typical persistent electropermeabilization (many seconds to minutes) observed in living cells. Furthermore, the magnitudes of the increases in intravesicular dye concentration are much smaller with nanosecond-pulsed electric fields than those observed in the presence of the pore-forming peptide melittin or exposure to influenza virus at a low pH. Nanosecond bipolar pulse cancellation, a phenomenon recently described in cells, was not observed in GUVs. The absence of persistent electropermeabilization and nanosecond bipolar cancellation of GUVs suggests that the electropermeabilization of cells involves structures and processes that go beyond transport through lipid pores and that models of electroporation must be modified accordingly. 2. Plasmodium falciparum VAR2CSA binds to chondroitin sulfate A (CSA) on the surface of the syncytiotrophoblast during placental malaria. This interaction facilitates placental sequestration of malaria parasites resulting in severe health outcomes for both the mother and her offspring. Furthermore, CSA is presented by diverse cancer cells and specific targeting of cells by VAR2CSA may become a viable approach for cancer treatment. Here, we determined the Cryo-EM structures of the full-length ectodomain of VAR2CSA from P. falciparum strain NF54 in complex with CSA, and VAR2CSA from a second P. falciparum strain FCR3. The architecture of VAR2CSA is composed of a stable core flanked by a flexible arm. CSA traverses the core domain by binding within two channels and CSA binding does not induce major conformational changes in VAR2CSA. The CSA-binding elements are conserved across VAR2CSA variants and are flanked by polymorphic segments, suggesting immune selection outside the CSA-binding sites. This work provides paths for developing interventions against placental. malaria and cancer. 3. The search for new antimalarial drugs will be a perpetual task until multitargeted drug mixtures are joined by vigorous anti-mosquito efforts and efficient, affordable, and heat-resistant vaccines identified by the World Health Organization to eliminate malaria. In recently published work, a potential leap forward in a large and persistent effort to develop a drug against a crucial but as yet undrugged stage of the parasites replication that our section has studied for years and for which we determined the pathway of morphological stages matched with specific enzymatic progression via proteolysis: egress of daughter parasites from infected erythrocytes. We have developed an outline of the work that is now needed to test this new hypothesis on drug design for parasite egress. In brief, first, inhibitor specificity and toxicity remains to be carefully explored, as serine proteases are a common group of enzymes in parasite and host. Fortunately, there are structural differences in the parasite SUB1 catalytic center and the catalytic centers of mammalian serine proteases. Compound 3j requires much higher concentrations to affect tested mammalian serine proteases only mildly. Second, the effect of mechanical stress has not yet been considered. Mature parasites, enclosed in two membranes, can be experimentally released by mechanical disruption of membranes. Can the shear stress created by blood circulation do the same job and halt the effect of inhibitors? To address this concern and to evaluate inhibitors potency, off target effects, and toxicity, a humanized mouse model of malaria would be a good choice to employ.