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Human Lactoferricin Derived Peptides Induce Apoptosis Specifically in Cancer Cells through Targeting Membranous Phosphatidylserine
Monday, February 13, 2017 912-Plat The Delta Peptide of Ebola Virus has Potent Viroporin Activity Jing He1, Lilia Melnik2, Alexander Komin3, Charles G. Starr1, Taylor Fuselier1, Gregory Wiedman3, Cameron F. Morris1, Yilin Wang1, Kalina Hristova3, William Gallaher4, Robert F. Garry2, William C. Wimley1. 1 Biochemistry, Tulane University, New Orleans, LA, USA, 2Microbiology and Immunology, Tulane University, New Orleans, LA, USA, 3Materials Science and Engineering, Johns Hopkins University, Baltimore, MD, USA, 4 Mockingbird Nature Research Group, Pearl River, LA, USA. The Delta peptide, cleaved from the soluble glycoprotein (sGP) of Ebola virus (EBOV), is a partially conserved, 40-residue nonstructural polypeptide that has no known function. This peptide shows high similarity in amino acid composition to membrane permeabilizing peptides, including a peptide in the diarrheainducing viroporin, NSP4, of Rotavirus. In addition, EBOV disease (EVD) survivors from West Africa have antibodies against the Delta peptide. Therefore, we hypothesize that the Delta peptide has an important role as a viroporin in EVD pathology. Here we show that a peptide corresponding to the serumstable, conserved C-terminal sequence of Delta peptide permeabilizes and kills nucleated mammalian cells, and potently increasing ion permeability across synthetic lipid bilayers. Importantly, this permeabilizing activity is eliminated by the reduction of the disulfide bond between conserved cysteines. We conclude that EBOV Delta peptide does not form large unregulated pores through membranes, but rather increases ionic permeation, which is similar to other viroporins. By this mechanism, it could contribute to EVD pathology in human through vascular leakage, enterotoxic effects, or other cytotoxic pathways. 913-Plat Identification of Receptor-Based Peptides to Inhibit Leukotoxin Activity Eric Krueger, Shannon Hayes, Angela C. Brown. Department of Chemical and Biomolecular Engineering, Lehigh University, Bethlehem, PA, USA. The leukotoxin (LtxA), secreted by the bacterium Aggregatibacter actinomycetemcomitans, is a member of the RTX family of toxins which targets leukocytes through several mechanisms. The toxin’s specificity for human white blood cells is driven by its recognition of the lymphocyte function-associated antigen 1 (LFA-1) integrin. This project investigates inhibition of LtxA-LFA-1 binding on living cells as an anti-virulence strategy to inhibit LtxA cytotoxicity. Specifically, we are investigating using small peptides to block (A) LFA-1 binding sites on LtxA and (B) LtxA-binding sites on LFA-1. Several LtxA binding sites on LFA-1, including a region on the aL b-propeller, have been proposed. Therefore, we have synthesized peptides corresponding to these domains and characterized their capability to inhibit LtxA binding to LFA-1 and subsequent cytotoxicity in human immune cells. We found that four of the five peptides, specifically those corresponding to sequential b-strands in the b-propeller domain, inhibited LtxA activity, demonstrating the effectiveness of this approach. The LFA-1 binding sites on LtxA are less established, but three monoclonal antibodies have previously been identified based on their inhibition of LtxA activity. We hypothesized that the epitopes to at least some of the antibodies are involved in this essential recognition process and synthesized peptides based on these epitopes to block the LtxA binding site on LFA-1. We are currently applying biophysical methods to investigate the mechanism by which these peptides inhibit LtxA binding to LFA-1 to fully characterize this proteinprotein interaction. The results of our investigations will facilitate the development of peptides to inhibit LtxA activity and can have broader implications through applying a similar approach to hinder the cytotoxic activity of other RTX toxins. 914-Plat Functional Reconstitution of V-ATPase Proteolipid Ring into a Planar Lipid Membrane Sergio Couoh-Cardel1, Yi-Ching Hsueh2, Stephan Wilkens1, Liviu Movileanu2. 1 Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA, 2Physics, Syracuse University, Syracuse, NY, USA. In this work, we show the first purification and functional characterization of the proteolipid ring (c-ring) complex of the membrane-bound Vo sector of the eukaryotic vacuolar Hþ-ATPase (V-ATPase) from the yeast Saccharomyces cerevisiae using techniques of membrane biochemistry, structural biology, and single-molecule electrophysiology. Our work was motivated by, and builds on, a large body of literature spanning more than three decades that suggested that Vo, besides its proton pumping function in holo V-ATPase, has other, so-called ‘‘non-canonical’’ functions in essential processes, such as membrane fusion and neurotransmitter release. However, direct evidence that Vo, and specifically, its core proteolipid ring (c-ring) complex, possesses the
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required properties to fulfill these non-canonical functions is still lacking. Our results presented here show that the c-ring forms a large-conductance transmembrane pore, which inserted into planar lipid bilayers with a preferred orientation. Moreover, using single-particle cryo-electron microscopy, we demonstrate that the purified c-ring forms dimers through an interaction mediated by the cytoplasmic loops of the c subunits. Taken together, these findings provide the first direct attestation in support of the proposed non-canonical functions of the V-ATPase Vo sector.1 This work was supported by NIH grants R01 GM058600 (to S.W.) and R01 GM088403 (to L.M.). 1. Couoh-Cardel, S.; Hsueh, Y. C.; Wilkens, S.; Movileanu, L., Yeast V-ATPase Proteolipid Ring Acts as a Large-conductance Transmembrane Protein Pore. Scientific reports 2016, 6, 24774. 915-Plat Human Lactoferricin Derived Peptides Induce Apoptosis Specifically in Cancer Cells through Targeting Membranous Phosphatidylserine Sabrina Riedl1, Beate Rinner2, Helmut Schaider3, Bernadette Liegl-Atzwanger4, Christina Wodlej1, Karl Lohner1, Dagmar Zweytick1. 1 Institute of Molecular Biosciences, University of Graz, Graz, Austria, 2 Biomedical Research, Medical University of Graz, Graz, Austria, 3Center for Medical Research, The University of Queensland, Brisbane, Australia, 4 Institute of Pathology, Medical University of Graz, Graz, Austria. Cancer with poor prognosis and treatability, like glioblastoma or malignant melanoma demand for new effective treatment and is therefore a focus in our studies. Recently we were able to demonstrate that the negatively charged lipid phosphatidylserine (PS) is specifically exposed by cancer cells and metastases and thus represents a uniform and novel target for cancer therapy (Riedl et al., BBA 2011). This yielded the important basis for the design of cationic antitumor peptides derived from human Lactoferricin (hLFcin). Some of these hLFcin derivatives show highly increased activity towards human malignant melanoma and glioblastoma cells compared to its parent peptide hLFcin. They can discriminate between neoplastic and non-neoplastic cells interacting specifically with negatively charged membrane components such as PS, specifically exposed on cancer cells. We have improved peptide specificity towards cancer cells by variations of peptide length, net positive charge and hydrophobicity, consequently influencing the secondary structure and the mechanism by which peptides kill the target cell. In our studies we found characteristic features of hLFcin derivatives which facilitate specific killing of cancer cells. Time dependent studies clearly revealed that a highly active but nonselective peptide acts through a direct membranolytic mechanism whereas the selective peptides presumably trigger apoptosis, which is also membrane-mediated by primary interactions with the negatively charged PS exposed by cancer cells and further intracellular targets. The role of cholesterol in triggering a specific peptide-cancer membrane interaction will also be discussed. Studies in mouse melanoma and glioblastoma xenografts confirm successful activity of the derived peptides also in vivo. Acknowledgment: FWF (P24608). 916-Plat Population Dynamics of Antimicrobial Peptide’s Activity is Governed by their Retention in Dead Bacterial Cells Mehdi Snoussi1, Paul Talledo2, Nathan-Alexander Del Rosario1, Lannah Abasi3, Federico Prokopzuk1, Bae-Yeun Ha4, Sattar Taheri-Araghi2. 1 Department of Biology, California State University, Northridge, Northridge, CA, USA, 2Department of Physics and Astronomy, California State University, Northridge, Northridge, CA, USA, 3Department of Chemistry and Biochemistry, California State University, Northridge, Northridge, CA, USA, 4 Department of Physics and Astronomy, University of Waterloo, Waterloo, ON, Canada. Antimicrobial peptides (AMPs) are broad spectrum antibiotics that utilize electrostatics to selectively target bacteria. Like every antibiotic, AMPs need a minimum concentration (known as MIC) to effectively inhibit growth of a bacterial population. Despite our molecular knowledge on AMPs, we still lack a comprehensive understanding of the AMP’s dynamics at the cellular and the population level. In this talk, I present our recent findings that MIC of AMPs is strongly dependent on the cell density, even in dilute cultures where there is no direct cell-to-cell interactions. This suggests that individual cells may adsorb a large number of AMPs that considerably reduces their concentration in the solution. To further investigate this, we used a live single-cell imaging platform to track fluorescently tagged AMPs and the time evolution of their translocation into bacteria. Our single-cell analysis show that bacteria not only adsorb a large fraction of AMPs from the culture, but dead cells also retain them almost permanently which sequesters AMPs availability for attacking more cells.
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required properties to fulfill these non-canonical functions is still lacking. Our results presented here show that the c-ring forms a large-conductance transmembrane pore, which inserted into planar lipid bilayers with a preferred orientation. Moreover, using single-particle cryo-electron microscopy, we demonstrate that the purified c-ring forms dimers through an interaction mediated by the cytoplasmic loops of the c subunits. Taken together, these findings provide the first direct attestation in support of the proposed non-canonical functions of the V-ATPase Vo sector.1 This work was supported by NIH grants R01 GM058600 (to S.W.) and R01 GM088403 (to L.M.). 1. Couoh-Cardel, S.; Hsueh, Y. C.; Wilkens, S.; Movileanu, L., Yeast V-ATPase Proteolipid Ring Acts as a Large-conductance Transmembrane Protein Pore. Scientific reports 2016, 6, 24774. 915-Plat Human Lactoferricin Derived Peptides Induce Apoptosis Specifically in Cancer Cells through Targeting Membranous Phosphatidylserine Sabrina Riedl1, Beate Rinner2, Helmut Schaider3, Bernadette Liegl-Atzwanger4, Christina Wodlej1, Karl Lohner1, Dagmar Zweytick1. 1 Institute of Molecular Biosciences, University of Graz, Graz, Austria, 2 Biomedical Research, Medical University of Graz, Graz, Austria, 3Center for Medical Research, The University of Queensland, Brisbane, Australia, 4 Institute of Pathology, Medical University of Graz, Graz, Austria. Cancer with poor prognosis and treatability, like glioblastoma or malignant melanoma demand for new effective treatment and is therefore a focus in our studies. Recently we were able to demonstrate that the negatively charged lipid phosphatidylserine (PS) is specifically exposed by cancer cells and metastases and thus represents a uniform and novel target for cancer therapy (Riedl et al., BBA 2011). This yielded the important basis for the design of cationic antitumor peptides derived from human Lactoferricin (hLFcin). Some of these hLFcin derivatives show highly increased activity towards human malignant melanoma and glioblastoma cells compared to its parent peptide hLFcin. They can discriminate between neoplastic and non-neoplastic cells interacting specifically with negatively charged membrane components such as PS, specifically exposed on cancer cells. We have improved peptide specificity towards cancer cells by variations of peptide length, net positive charge and hydrophobicity, consequently influencing the secondary structure and the mechanism by which peptides kill the target cell. In our studies we found characteristic features of hLFcin derivatives which facilitate specific killing of cancer cells. Time dependent studies clearly revealed that a highly active but nonselective peptide acts through a direct membranolytic mechanism whereas the selective peptides presumably trigger apoptosis, which is also membrane-mediated by primary interactions with the negatively charged PS exposed by cancer cells and further intracellular targets. The role of cholesterol in triggering a specific peptide-cancer membrane interaction will also be discussed. Studies in mouse melanoma and glioblastoma xenografts confirm successful activity of the derived peptides also in vivo. Acknowledgment: FWF (P24608). 916-Plat Population Dynamics of Antimicrobial Peptide’s Activity is Governed by their Retention in Dead Bacterial Cells Mehdi Snoussi1, Paul Talledo2, Nathan-Alexander Del Rosario1, Lannah Abasi3, Federico Prokopzuk1, Bae-Yeun Ha4, Sattar Taheri-Araghi2. 1 Department of Biology, California State University, Northridge, Northridge, CA, USA, 2Department of Physics and Astronomy, California State University, Northridge, Northridge, CA, USA, 3Department of Chemistry and Biochemistry, California State University, Northridge, Northridge, CA, USA, 4 Department of Physics and Astronomy, University of Waterloo, Waterloo, ON, Canada. Antimicrobial peptides (AMPs) are broad spectrum antibiotics that utilize electrostatics to selectively target bacteria. Like every antibiotic, AMPs need a minimum concentration (known as MIC) to effectively inhibit growth of a bacterial population. Despite our molecular knowledge on AMPs, we still lack a comprehensive understanding of the AMP’s dynamics at the cellular and the population level. In this talk, I present our recent findings that MIC of AMPs is strongly dependent on the cell density, even in dilute cultures where there is no direct cell-to-cell interactions. This suggests that individual cells may adsorb a large number of AMPs that considerably reduces their concentration in the solution. To further investigate this, we used a live single-cell imaging platform to track fluorescently tagged AMPs and the time evolution of their translocation into bacteria. Our single-cell analysis show that bacteria not only adsorb a large fraction of AMPs from the culture, but dead cells also retain them almost permanently which sequesters AMPs availability for attacking more cells.