Lipid Specific Membrane Interaction of Aptamers and Cytotoxicity

We aim to discover diagnostic tools to detect phosphatidylserine (PS) externalization on apoptotic cell surface using PS binding aptamers, AAAGAC and TAAAGA, and hence to understand chemotherapy drug efficacy when inducing apoptosis into cancer cells. The entropic fragment-based approach designed aptamers have been investigated to inspect three aspects: lipid specificity in aptamers’ membrane binding and bilayer physical properties-induced regulation of binding mechanisms, the apoptosis-induced cancer cell surface binding of aptamers, and the aptamer-induced cytotoxicity. The liposome binding assays show preferred membrane binding of aptamers due to presence of PS in predominantly phosphatidylcholine-contained liposomes. Two membrane stiffness reducing amphiphiles triton X-100 and capsaicin were found to enhance membrane’s aptamer adsorption suggesting that bilayer physical properties influence membrane’s adsorption of drugs. Microscopic images of fluorescence-tagged aptamer treated LoVo cells show strong fluorescence intensity only if apoptosis is induced. Aptamers find enhanced PS molecules to bind with on the surface of apoptotic over nonapoptotic cells. In cytotoxicity experiments, TAAAGA (over poor PS binding aptamer CAGAAAAAAAC) was found cytotoxic towards RBL cells due to perhaps binding with nonapoptotic externalized PS randomly and thus slowly breaching plasma membrane integrity. In these three experimental investigations, we found aptamers to act on membranes at comparable concentrations and specifically with PS binding manner. Earlier, we reported the origins of actions through molecular mechanism studies—aptamers interact with lipids using mainly charge-based interactions. Lipids and aptamers hold distinguishable charge properties, and hence, lipid–aptamer association follows distinguishable energetics due to electrostatic and van der Waals interactions. We discover that our PS binding aptamers, due to lipid-specific interactions, appear as diagnostic tools capable of detecting drug-induced apoptosis in cancer cells.

Structural, functional and computational studies of membrane recognition by Plasmodium Perforin-Like Proteins 1 and 2.

Perforin-like proteins (PLPs) play key roles in the mechanisms associated with parasitic disease caused by apicomplexans such as Plasmodium (malaria) and Toxoplasma. The T. gondii PLP1 (TgPLP1) mediates tachyzoite egress from cells, while the five Plasmodium PLPs carry out various roles in the life cycle of the parasite and with respect to the molecular basis of disease. Here we focus on Plasmodium vivax PLP1 and PLP2 (PvPLP1 and PvPLP2) compared to TgPLP1; PvPLP1 is important for invasion of mammalian hosts by the parasite and establishment of a chronic infection, PvPLP2 is important during the symptomatic blood stage of the parasite life cycle. Determination of the crystal structure of the membrane-binding APCβ domain of PvPLP1 reveals notable differences with that of TgPLP1, which are reflected in its inability to bind lipid bilayers in the way that TgPLP1 and PvPLP2 can be shown to. Molecular dynamics simulations combined with site-directed mutagenesis and functional assays allow a dissection of the binding interactions of TgPLP1 and PvPLP2 on lipid bilayers, and reveal a similar tropism for lipids found enriched in the inner leaflet of the mammalian plasma membrane. In addition to this shared mode of membrane binding PvPLP2 displays a secondary synergistic interaction side-on from its principal bilayer interface. This study underlines the substantial differences between the biophysical properties of the APCβ domains of Apicomplexan PLPs, which reflect their significant sequence diversity. Such differences will be important factors in determining the cell targeting and membrane-binding activity of the different proteins, in their different developmental roles within parasite life cycles.

Structural dissection of the first events following membrane binding of the islet amyloid polypeptide

Amyloid forming proteins are involved in many pathologies and often belong to the class of intrinsically disordered proteins. One of these proteins is the islet amyloid polypeptide (IAPP), which is the main constituent of the amyloid fibrils found in the pancreas of type 2 diabetes patients. The molecular mechanism of IAPP-induced cell death is not yet understood, however it is known that the cell membrane plays a dual role, being a catalyst of IAPP aggregation and the target of IAPP toxicity. Using FTIR spectroscopy, transmission electron microscopy, and molecular dynamics simulations we investigate the very first molecular steps following IAPP binding to a lipid membrane. In particular, we assess the combined effects of the charge state of amino-acid residue 18 and the IAPP-membrane interactions on the structures of monomeric and aggregated IAPP. Both our experiments and simulations reveal distinct IAPP-membrane interaction modes for the various IAPP variants. Membrane binding causes IAPP to fold into an amphipathic helix, which in the case of H18K- and H18R-IAPP can easily insert below the lipid headgroups. For all IAPP variants but H18E-IAPP, the membrane-bound α-helical structure is an intermediate on the way to IAPP amyloid aggregation, while H18E-IAPP remains in a stable helical conformation. The fibrillar aggregates of wild-type IAPP and H18K-IAPP are dominated by an antiparallel β-sheet conformation, while H18R- and H18A-IAPP exhibit both antiparallel and parallel β-sheets as well as amorphous aggregates. In summary, our results emphasize the importance of residue 18 for the structure and membrane interaction of IAPP. This residue is thus a good target for destabilizing amyloid fibrils of IAPP and inhibit its toxic actions by possible therapeutic molecules.

Implications of Membrane Binding by the Fe-S Cluster-Containing N-Terminal Domain in the Drosophila Mitochondrial Replicative DNA Helicase

Recent evidence suggests that iron-sulfur clusters (ISCs) in DNA replicative proteins sense DNA-mediated charge transfer to modulate nuclear DNA replication. In the mitochondrial DNA replisome, only the replicative DNA helicase (mtDNA helicase) from Drosophila melanogaster (Dm) has been shown to contain an ISC in its N-terminal, primase-like domain (NTD). In this report, we confirm the presence of the ISC and demonstrate the importance of a metal cofactor in the structural stability of the Dm mtDNA helicase. Further, we show that the NTD also serves a role in membrane binding. We demonstrate that the NTD binds to asolectin liposomes, which mimic phospholipid membranes, through electrostatic interactions. Notably, membrane binding is more specific with increasing cardiolipin content, which is characteristically high in the mitochondrial inner membrane (MIM). We suggest that the N-terminal domain of the mtDNA helicase interacts with the MIM to recruit mtDNA and initiate mtDNA replication. Furthermore, Dm NUBPL, the known ISC donor for respiratory complex I and a putative donor for Dm mtDNA helicase, was identified as a peripheral membrane protein that is likely to execute membrane-mediated ISC delivery to its target proteins.

Synaptotagmin-1 C2B domains cooperatively stabilize the fusion stalk via a master-servant mechanism

Synaptotagmin-1 is a low-affinity Ca2+ sensor that triggers synchronous vesicle fusion. It contains two similar C2 domains (C2A and C2B) that cooperate in membrane binding, being the C2B domain the main responsible for the membrane fusion process due to its polybasic patch KRLKKKKTTIKK (321-332). In this work, a master-servant mechanism between two identical C2B domains is shown to control the formation of the fusion stalk. Two regions in C2B are essential for the process, the well-known polybasic patch and a recently described pair of arginines (398,399). The master domain shows strong PIP2 interactions with its polybasic patch and its pair of arginines. At the same time, the servant analogously cooperates with the master to reduce the total work to form the fusion stalk. The strategic mutation (T328E,T329E) in both master and servant domains disrupts the cooperative mechanism, drastically increasing the free energy needed to induce the fusion stalk, however with negligible effects on the master domain interactions with PIP2. These data point to a difference in the behavior of the servant domain, which is unable to sustain its PIP2 interactions neither through its polybasic patch nor through its pair of arginines, in the end losing its ability to assist the master in the formation of the fusion stalk.

The sperm protein Spaca6 is essential for fertilization in zebrafish

Fertilization is a key process in all sexually reproducing species, yet the molecular mechanisms that underlie this event remain unclear. To date, only a few proteins have been shown to be essential for sperm-egg binding and fusion in mice, and only some are conserved across vertebrates. One of these conserved, testis-expressed factors is SPACA6, yet its function has not been investigated outside of mammals. Here we show that zebrafish spaca6 encodes for a sperm membrane protein which is essential for fertilization. Zebrafish spaca6 knockout males are sterile. Furthermore, Spaca6-deficient sperm have normal morphology, are motile, and can approach the egg, but fail to bind to the egg and therefore cannot complete fertilization. Interestingly, sperm lacking Spaca6 have decreased levels of another essential and conserved sperm fertility factor, Dcst2, revealing a previously unknown dependence of Dcst2 expression on Spaca6. Together, our results show that zebrafish Spaca6 regulates Dcst2 levels and is required for binding between the sperm membrane and the oolemma. This is in contrast to murine SPACA6, which was reported not to be required for sperm-egg membrane binding but necessary for fusion. These findings demonstrate that Spaca6 is essential for zebrafish fertilization and is a conserved sperm factor in vertebrate reproduction.