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PTEN Binding Mechanism with Compositionally Diverse Lipid Model Membranes
390a
Tuesday, February 14, 2017
into the nanoparticles the interfacial adsorption capability of the complex increased slightly depending on the palmitoylation state of the protein. We have also analyzed the interaction of the complexes with surfactant monolayers and bilayers. Fluorescently labeled nanocomplexes seem to associate to both lipid structures with no apparent lipid transfer. These nanostructures could then constitute an efficient vehicle to direct molecules such as proteins or drugs to the respiratory air-liquid interface. 1926-Pos Board B246 Lipid Bilayer Association of the Alternatively Translated Region of PTEN-Long Anne-Marie Bryant, Arne Gericke. Worcester Polytechnic Institute, Worcester, MA, USA. PTEN-Long is a 576-amino acid translational variant of phosphatase and tension homolog on chromosome ten (PTEN), a tumor suppressor gene and antagonist to phosphoinositide-3-kinase (PI3K) signaling. PTEN-Long (PTEN-L) exhibits at the N-terminal end an alternative translation region (ATR) with additional 173 N-terminal amino acids to the normal PTEN open reading frame. PTEN-L is secreted from cells, can exist outside the cell and shows activity towards PI(3,4,5)P3 after cell entry. Therefore, PTEN-L may have therapeutic uses by restoring a functional tumor suppressor protein to tumor cells. The mechanism by which PTEN-L enters cells is currently unknown. The ATR region of PTEN-L is evolutionary conserved and contains a polyarginine stretch with homology to cell permeable peptides, suggesting that PTEN-L may be able to traverse the plasma membrane passively. It has been suggested that the N-terminal ATR part of PTEN-L is intrinsically disordered, which we confirmed using CD and FTIR spectroscopy. Our FTIR studies did not reveal any observable structural change of PTEN-L ATR upon lipid binding to PC, PS, and PI(4,5)P2. We further found that the integrity of model lipid bilayers is compromised upon interaction of PTEN-L’s ATR. We are currently further delineating the lipid binding preferences of the ATR part of PTEN-L, as well as the full-length protein using surface plasmon resonance (SPR) and FRET techniques. 1927-Pos Board B247 PTEN Binding Mechanism with Compositionally Diverse Lipid Model Membranes Brittany M. Neumann1, Katrice E. McLoughlin1, Vanessa Pinderi1, Rakesh Harishchandra1, Alonzo Ross2, Arne Gericke1. 1 Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, MA, USA, 2Chemistry and Biochemistry, University of Massachusetts Medical School, Worcester, MA, USA. PTEN is one of the most frequent mutated tumor suppressor proteins in human cancer. This project investigates how PTEN interacts with lipid model membranes of different composition and morphology. We hypothesize that PTEN binds synergistically to the plasma membrane (PM) by interacting with phosphatidylinositol-(4,5)-bisphosphate(PI(4,5)P2) through its PBM domain as well as non-specifically, electrostatically through its C2 domain with anionic lipids. While the currently accepted model is that PTEN’s C2 domain interacts with phosphatidylserine (PS), we hypothesize that the C2 domain can interact with any anionic lipid, as long as this lipid has a sufficiently high local concentration. PI(4,5)P2 is present in the PM at about 1%, while PS is found at about 30%. We observed in model membrane studies that PI(4,5)P2 and PS have only a limited tendency to co-localize in a domain. In contrast, phosphatidylinositol (PI) (about 8% of PM lipids), aids in the formation of PI(4,5)P2 enriched domains through headgroup hydrogen bond formation. A PI/PI(4,5)P2 domain would be an excellent platform for PTEN membrane interaction, suggesting that PI is a better second binding partner than PS. We are using stopped-flow fluorescence spectrophotometry as an ensemble technique that provides us with information about the association and dissociation kinetics of PTEN when interacting with lipid model membranes of different composition. Using this technique we have found that PTEN binds to pure PS and PI vesicles with a similar binding constant. We see synergistic binding to 30%PS/5%PI(4,5)P2 as well as 30%PI 5%PI(4,5)P2. Additionally, we are utilizing single molecule TIRF microscopy to track PTEN on model membranes of different composition. From these data, we determine the PTEN diffusion coefficient, hopping frequency and dwell times. In the aggregate, this allows us to describe in detail PTEN binding mechanisms with differently composed lipid model membranes. 1928-Pos Board B248 Structure, Dynamics, and Interactions of GPI-Anchored Human Glypican-1 having N-Glycans and Heparan Sulfates in Membranes Hongjing Ma, Jumin Lee, Wonpil Im. Lehigh University, Bethlehem, PA, USA. The glypican family of cell-surface proteoglycans, anchored to the outer leaflet of eukaryotic cell membrane through a glycosylphosphatidylinosital (GPI) anchor, is involved in important cellular signaling pathways. Glypican-1 (Gpc1),
the predominant heparan sulfate (HS) proteoglycan in the developing and adult human brain, has two N-linked glycans and three chains of HS. Loss-offunction mutations show that both glypican core protein and their HS chains are important in shaping animal development. Here, to explore structure, dynamics, and interactions of Gpc1 in a membrane environment, we have performed molecular dynamics simulation studies of Gpc1 modeled with both N-glycans and HS chains and anchored to a bilayer via a GPI linker. Our analyses reveal that HS chains exhibit a pronounced flexibility, allowing great freedom for HS to reach out and accommodate binding to receptors and other signaling molecules. In addition, within the given simulation time, Gpc1 core ˚ above the membrane surface, showing a tenprotein is steadily located ~50 A dency of providing enough distance for HS assembly enzymes or a membrane receptor to interact with the membrane-proximal region of Gpc1. 1929-Pos Board B249 Assembly of Matrix Protein 1 of Influenza a Virus and its Role in Budding Process Liudmila A. Shilova1, Anna S. Lyushnyak1, Denis G. Knyazev2, Natalia V. Fedorova3, Liudmila A. Baratova3, Oleg V. Batishchev1. 1 A.N. Frumkin Institute of Physical Chemistry and Electrochemistry of RAS (IPCE RAS), Moscow, Russian Federation, 2Johannes Kepler University Linz, Institute of Biophysics, Linz, Austria, 3A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russian Federation. Influenza A virus is a serious pathogen belonging to the Ortomixoviridae family. It is an enveloped virus with outer shell represented by a lipid bilayer with incorporated glycoproteins and proton channel, while the inner one is a scaffold formed by matrix protein 1, M1. This protein, being the most abundant one among viral proteins, is known to possess a multifunctionality: along with being a mechanical skeleton of a virion, it acts as a factor, which triggers crucial viral processes, such as the release of viral RNA during infection and budding of newly assembled virions on a final step of viral maturation. We focused on this last stage of influenza virus life cycle: assembly and budding of new virions at the surface of infected cell. Our goal was to clarify the role of the M1 protein in formation of virus protein scaffold and budding of progeny virions. We performed experiments on giant unilamellar vesicles (GUVs) using confocal fluorescent microscopy. We succeed in detecting the budding process of virus-like particles by M1 protein in vitro under special conditions. We examined the possibility of matrix protein alone to cause budding on GUVs of different lipid composition, and the role of lipid nanodomains - rafts - in this process. This work was supported in part by RSF grant #15-14-00060, RFBR grant #15-54-74002 and grant of the President of the Russian Federation (MK6058.2016.4). 1930-Pos Board B250 Tubulin on Biomimetic Mitochondrial Membranes: Structural Features and Lipid Discrimination David P. Hoogerheide1, Sergei Yu Noskov2, Daniel Jacobs3, Hirsh Nanda1, Tatiana K. Rostovtseva3, Sergey M. Bezrukov3. 1 Center for Neutron Research, National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA, 2Biochemistry Department, University of Calgary, Calgary, AB, Canada, 3Section on Molecular Transport, Eunice Kennedy Shriver National Institute of Child Health and Human, National Institutes of Health, Bethesda, MD, USA. Dimeric tubulin, an abundant cytosolic water-soluble protein, has emerged as an important regulatory factor of the permeability of the voltage-dependent anion channel (VDAC) in the mitochondrial outer membrane, with implications for mitochondrial energetics as well as the Warburg effect observed in cancer cells. A wealth of recent in vitro and in vivo evidence points to a mechanism of this regulation, in which a charged C-terminal tail of tubulin inserts into the water-filled VDAC pore, thus blocking metabolite fluxes through this passive transport channel. The membrane-binding properties of tubulin have proven difficult to ascertain, as early work generated contradictory results regarding the tubulin-membrane association, including whether it was integral or peripheral in nature. Here we report on a comprehensive biophysical study of tubulin binding to lipid membranes with compositions that mimic the mitochondrial outer membrane. A combination of surface plasmon resonance, bilayer overtone (second harmonics) analysis, and single-channel recordings show that tubulin distinguishes between lamellar and non-lamellar lipid components of the membrane. To obtain the structural features of the tubulin heterodimer on the membrane surface, we have employed neutron reflectometry (NR) of tubulin on a tethered bilayer lipid membrane platform. The NR results definitively show that tubulin binds peripherally, and in combination with molecular dynamics (MD) simulations suggest the binding domain to be a highly conserved amphipathic a-helix on a-tubulin. Thus tubulin joins a short but
Tuesday, February 14, 2017
into the nanoparticles the interfacial adsorption capability of the complex increased slightly depending on the palmitoylation state of the protein. We have also analyzed the interaction of the complexes with surfactant monolayers and bilayers. Fluorescently labeled nanocomplexes seem to associate to both lipid structures with no apparent lipid transfer. These nanostructures could then constitute an efficient vehicle to direct molecules such as proteins or drugs to the respiratory air-liquid interface. 1926-Pos Board B246 Lipid Bilayer Association of the Alternatively Translated Region of PTEN-Long Anne-Marie Bryant, Arne Gericke. Worcester Polytechnic Institute, Worcester, MA, USA. PTEN-Long is a 576-amino acid translational variant of phosphatase and tension homolog on chromosome ten (PTEN), a tumor suppressor gene and antagonist to phosphoinositide-3-kinase (PI3K) signaling. PTEN-Long (PTEN-L) exhibits at the N-terminal end an alternative translation region (ATR) with additional 173 N-terminal amino acids to the normal PTEN open reading frame. PTEN-L is secreted from cells, can exist outside the cell and shows activity towards PI(3,4,5)P3 after cell entry. Therefore, PTEN-L may have therapeutic uses by restoring a functional tumor suppressor protein to tumor cells. The mechanism by which PTEN-L enters cells is currently unknown. The ATR region of PTEN-L is evolutionary conserved and contains a polyarginine stretch with homology to cell permeable peptides, suggesting that PTEN-L may be able to traverse the plasma membrane passively. It has been suggested that the N-terminal ATR part of PTEN-L is intrinsically disordered, which we confirmed using CD and FTIR spectroscopy. Our FTIR studies did not reveal any observable structural change of PTEN-L ATR upon lipid binding to PC, PS, and PI(4,5)P2. We further found that the integrity of model lipid bilayers is compromised upon interaction of PTEN-L’s ATR. We are currently further delineating the lipid binding preferences of the ATR part of PTEN-L, as well as the full-length protein using surface plasmon resonance (SPR) and FRET techniques. 1927-Pos Board B247 PTEN Binding Mechanism with Compositionally Diverse Lipid Model Membranes Brittany M. Neumann1, Katrice E. McLoughlin1, Vanessa Pinderi1, Rakesh Harishchandra1, Alonzo Ross2, Arne Gericke1. 1 Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, MA, USA, 2Chemistry and Biochemistry, University of Massachusetts Medical School, Worcester, MA, USA. PTEN is one of the most frequent mutated tumor suppressor proteins in human cancer. This project investigates how PTEN interacts with lipid model membranes of different composition and morphology. We hypothesize that PTEN binds synergistically to the plasma membrane (PM) by interacting with phosphatidylinositol-(4,5)-bisphosphate(PI(4,5)P2) through its PBM domain as well as non-specifically, electrostatically through its C2 domain with anionic lipids. While the currently accepted model is that PTEN’s C2 domain interacts with phosphatidylserine (PS), we hypothesize that the C2 domain can interact with any anionic lipid, as long as this lipid has a sufficiently high local concentration. PI(4,5)P2 is present in the PM at about 1%, while PS is found at about 30%. We observed in model membrane studies that PI(4,5)P2 and PS have only a limited tendency to co-localize in a domain. In contrast, phosphatidylinositol (PI) (about 8% of PM lipids), aids in the formation of PI(4,5)P2 enriched domains through headgroup hydrogen bond formation. A PI/PI(4,5)P2 domain would be an excellent platform for PTEN membrane interaction, suggesting that PI is a better second binding partner than PS. We are using stopped-flow fluorescence spectrophotometry as an ensemble technique that provides us with information about the association and dissociation kinetics of PTEN when interacting with lipid model membranes of different composition. Using this technique we have found that PTEN binds to pure PS and PI vesicles with a similar binding constant. We see synergistic binding to 30%PS/5%PI(4,5)P2 as well as 30%PI 5%PI(4,5)P2. Additionally, we are utilizing single molecule TIRF microscopy to track PTEN on model membranes of different composition. From these data, we determine the PTEN diffusion coefficient, hopping frequency and dwell times. In the aggregate, this allows us to describe in detail PTEN binding mechanisms with differently composed lipid model membranes. 1928-Pos Board B248 Structure, Dynamics, and Interactions of GPI-Anchored Human Glypican-1 having N-Glycans and Heparan Sulfates in Membranes Hongjing Ma, Jumin Lee, Wonpil Im. Lehigh University, Bethlehem, PA, USA. The glypican family of cell-surface proteoglycans, anchored to the outer leaflet of eukaryotic cell membrane through a glycosylphosphatidylinosital (GPI) anchor, is involved in important cellular signaling pathways. Glypican-1 (Gpc1),
the predominant heparan sulfate (HS) proteoglycan in the developing and adult human brain, has two N-linked glycans and three chains of HS. Loss-offunction mutations show that both glypican core protein and their HS chains are important in shaping animal development. Here, to explore structure, dynamics, and interactions of Gpc1 in a membrane environment, we have performed molecular dynamics simulation studies of Gpc1 modeled with both N-glycans and HS chains and anchored to a bilayer via a GPI linker. Our analyses reveal that HS chains exhibit a pronounced flexibility, allowing great freedom for HS to reach out and accommodate binding to receptors and other signaling molecules. In addition, within the given simulation time, Gpc1 core ˚ above the membrane surface, showing a tenprotein is steadily located ~50 A dency of providing enough distance for HS assembly enzymes or a membrane receptor to interact with the membrane-proximal region of Gpc1. 1929-Pos Board B249 Assembly of Matrix Protein 1 of Influenza a Virus and its Role in Budding Process Liudmila A. Shilova1, Anna S. Lyushnyak1, Denis G. Knyazev2, Natalia V. Fedorova3, Liudmila A. Baratova3, Oleg V. Batishchev1. 1 A.N. Frumkin Institute of Physical Chemistry and Electrochemistry of RAS (IPCE RAS), Moscow, Russian Federation, 2Johannes Kepler University Linz, Institute of Biophysics, Linz, Austria, 3A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russian Federation. Influenza A virus is a serious pathogen belonging to the Ortomixoviridae family. It is an enveloped virus with outer shell represented by a lipid bilayer with incorporated glycoproteins and proton channel, while the inner one is a scaffold formed by matrix protein 1, M1. This protein, being the most abundant one among viral proteins, is known to possess a multifunctionality: along with being a mechanical skeleton of a virion, it acts as a factor, which triggers crucial viral processes, such as the release of viral RNA during infection and budding of newly assembled virions on a final step of viral maturation. We focused on this last stage of influenza virus life cycle: assembly and budding of new virions at the surface of infected cell. Our goal was to clarify the role of the M1 protein in formation of virus protein scaffold and budding of progeny virions. We performed experiments on giant unilamellar vesicles (GUVs) using confocal fluorescent microscopy. We succeed in detecting the budding process of virus-like particles by M1 protein in vitro under special conditions. We examined the possibility of matrix protein alone to cause budding on GUVs of different lipid composition, and the role of lipid nanodomains - rafts - in this process. This work was supported in part by RSF grant #15-14-00060, RFBR grant #15-54-74002 and grant of the President of the Russian Federation (MK6058.2016.4). 1930-Pos Board B250 Tubulin on Biomimetic Mitochondrial Membranes: Structural Features and Lipid Discrimination David P. Hoogerheide1, Sergei Yu Noskov2, Daniel Jacobs3, Hirsh Nanda1, Tatiana K. Rostovtseva3, Sergey M. Bezrukov3. 1 Center for Neutron Research, National Institute of Standards and Technology (NIST), Gaithersburg, MD, USA, 2Biochemistry Department, University of Calgary, Calgary, AB, Canada, 3Section on Molecular Transport, Eunice Kennedy Shriver National Institute of Child Health and Human, National Institutes of Health, Bethesda, MD, USA. Dimeric tubulin, an abundant cytosolic water-soluble protein, has emerged as an important regulatory factor of the permeability of the voltage-dependent anion channel (VDAC) in the mitochondrial outer membrane, with implications for mitochondrial energetics as well as the Warburg effect observed in cancer cells. A wealth of recent in vitro and in vivo evidence points to a mechanism of this regulation, in which a charged C-terminal tail of tubulin inserts into the water-filled VDAC pore, thus blocking metabolite fluxes through this passive transport channel. The membrane-binding properties of tubulin have proven difficult to ascertain, as early work generated contradictory results regarding the tubulin-membrane association, including whether it was integral or peripheral in nature. Here we report on a comprehensive biophysical study of tubulin binding to lipid membranes with compositions that mimic the mitochondrial outer membrane. A combination of surface plasmon resonance, bilayer overtone (second harmonics) analysis, and single-channel recordings show that tubulin distinguishes between lamellar and non-lamellar lipid components of the membrane. To obtain the structural features of the tubulin heterodimer on the membrane surface, we have employed neutron reflectometry (NR) of tubulin on a tethered bilayer lipid membrane platform. The NR results definitively show that tubulin binds peripherally, and in combination with molecular dynamics (MD) simulations suggest the binding domain to be a highly conserved amphipathic a-helix on a-tubulin. Thus tubulin joins a short but