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Mesoscopic Properties and Molecular Mechanisms of IAPP Amyloid Inhibition and Remodeling with Small Molecules
340a
Tuesday, February 14, 2017
1665-Plat Mesoscopic Properties and Molecular Mechanisms of IAPP Amyloid Inhibition and Remodeling with Small Molecules Aleksandr Kakinen1, Bo Wang2, Xinwei Ge2, Raffaele Mezzenga3, Thomas P. Davis1,4, Feng Ding2, Pu Chun Ke1. 1 Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia, 2Department of Physics and Astronomy, Clemson University, Clemson, SC, USA, 3Department of Health Science & Technology, ETH Zurich, Zurich, Switzerland, 4Department of Chemistry, Warwick University, Coventry, United Kingdom. The amyloid aggregation of proteins is associated with a number of neurodegenerative diseases such like Alzheimer’s, Huntington’s and Parkinson’s diseases, and also type-2 diabetes (T2D). Despite the physical and structural properties of the aggregating proteins, the corresponding amyloid aggregates feature some common characteristics including the formation of cross-b architecture and cytotoxicity to human cells, suggesting a common amyloid mechanism. Experimental studies have shown that several naturally-occurring small molecules, such as epigallocatechin gallate (EGCG), have an inhibitory effect on the aggregation of a wide range of those amyloid proteins, including islet amyloid polypeptide (IAPP, a.k.a. amylin) in T2D, which is one of the most aggregation-prone peptides. Furthermore, EGCG was also reported having a novel function as amyloid fibrils remodeling, indicating a promising therapeutic approach against amyloid diseases, and importance of understanding the molecular mechanism of such anti-aggregation effect. In this study, we applied experiments and desecrated molecular dynamic (DMD) simulations to investigate the effects of EGCG on IAPP fibrillization. Our high- resolution transmission electron microscopy (TEM) images showed that EGCG inhibited IAPP aggregation by rendering shorter and softer structures, while remodeled mature IAPP fibrils by compromising fibril contour length and triggering splitting without altering the twisted fibril morphology. DMD simulations further revealed that, upon binding with EGCG, IAPP monomers and oligomers tended to form disordered off-pathway clusters instead of cross-b structure due to competing interactions with amyloidogenic sequences. EGCG could also target fibril ends through capping and stacking the exposed sites and enable fibril remodeling. Our combined computational and experimental study offers a detailed mechanistic insight into the inhibitory effect of EGCG on IAPP fibrillization as well as fibril remodeling, suggesting a promising approach for the mitigation of IAPP aggregation and future T2D therapy. 1666-Plat Conformational Plasticity of the MAGE-A3 Protein as a Therapeutic Strategy in Multiple Myeloma Roman Osman1, Hearn J. Cho2, Opher Gileadi3. 1 Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 2Medicine, Hematology/Oncology, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 3Structural Genomics Consortium, University of Oxford, Oxford, United Kingdom. Type I MAGE proteins interact with the RING domain protein Kap1 through their conserved MAGE Homology Domains (MHD) to form E3 ubiquitin (Ub) ligases, which ubiquitinylate p53 targeting it for proteasomal degradation. RNAi experiments demonstrated that MAGE-A3 inhibits p53dependent and independent mechanisms of apoptosis and confers resistance to chemotherapy-induced apoptosis in human myeloma cell lines. Since MAGE expression correlates with progression of multiple myeloma (MM), preventing the interaction with Kap1 is a promising therapeutic intervention against MM. The MHD of MAGE proteins are made of two winged helix domains (WH) linked by a flexible b-hairpin. Structures of MAGE proteins suggest that the WH domains need to undergo a conformational change from a closed to an open form to interact with Kap1 and activate its Ub-ligase activity. Virtual screening on the closed form of MAGE-A3 identified two compounds that recapitulate the RNAi experiments, suggesting that they may inhibit the interaction of MAGE-A3 with Kap1 leading to the apoptosis of MM cells. Further experiments are being conducted to elucidate the nature of the observed effect. MD simulations of the complexes of MAGE-A3 with the small molecules show binding modes in the groove between the two WH domains. These may be responsible for inhibiting the conformational transition. Further simulations to estimate the effect of the small molecules on the conformational change as well as refinement of the initial leads to improve the affinity and selectivity of the compounds are under way. Supported by NIH R21 CA191898.
Workshop: Beyond Calcium: Imaging Voltage and Other Ions 1667-Wkshp Fluorescent Visualization of Cellular Efflux William R. Kobertz. Biochemistry and Molecular Pharmacology, UMASS Medical School, Worcester, MA, USA. The fluorescent visualization of calcium ions entering the cytoplasm has reimaged our basic understanding of the inner workings of calcium signaling in cells, tissues and living organisms. In contrast, there is a dearth of tools to fluorescently visualize ions exiting cells. Part of the challenge stems from the fact that cellular egress is contrary to the pervasive intracellular-centric experimental paradigm. Recently, we have been using glycan engineering to install chemical handles into the cell’s glycocalyx that directly abuts the plasma membrane in all cells. Subsequent chemical modfication of these unnatural sugars ideally positions molecular probes within nanometers of the extracellular vestibules of ion channels and transporters. My laboratory’s efforts to fluorescently visualize ions exiting cells using this technology will be presented. 1668-Wkshp Critical Evaluation of FRET-Based Biosensor Performance: Implications for Measuring Ion Concentrations Amy E. Palmer. Chemistry and Biochemistry, University of Colorado, Boulder, CO, USA. Fluorescent sensors are widely used to visualize, quantify, and reveal the dynamics of small molecules, secondary metabolites, metals, and ions. One of the great promises of such sensors is the ability to quantify cellular signals in precise locations with high temporal resolution, enabling the creation of realtime dynamic maps of cellular signals. Yet this is coupled with the challenge of how to ensure that sensors are measuring what we think they are measuring, developing robust approaches for quantification, and assessing whether sensors are perturbing the underlying biology. This talk will highlight our efforts to develop genetically encoded FRET-based sensors for quantitative mapping of zinc ions in cells. I will discuss approaches for defining whether sensors perturb cellular ions, the importance of carefully defining dynamic range, strategies to compare sensor functionality in vitro, in cells, and in organelles, and the specific challenges associated with quantifying ions in cellular organelles. 1669-Wkshp Light Up the Brain with Genetically Encoded Sensors of Neural Activity Lin Tian. University of California, Davis, Davis, CA, USA. A central challenge in neuroscience is to understand how neural circuits extract information from the environment and generate appropriate behaviors. To address this challenge, one would like to measure the complex spatiotemporal neural activity on many different scales, from single synapses to microcircuits to entire brains, in behaving animals. Recent breakthroughs in modern microscope and protein based fluorescence sensors including calcium, voltage and neurotransmitters, permit optically large-scale recording of neural activity with needed molecular and cell type specificity and spatiotemporal resolutions in behaving animals. To further expand the kinds of neural activity that can be measured with genetically encoded indicators, we applied the established sensor design and optimization platform to the development of a set of specific, targetable and sensitive sensors for direct measurement neuromodulators of the biogenic amine family and activation of corresponding G-protein coupled receptors. We have characterized the expression, signal-to-noise ratio, dynamic range and kinetics of these sensors in dissociated neuronal culture, acute brain slice and in vivo in zebrafish and mouse. A broad application of these imaging tools will enable neuroscientists to obtain a dynamic and comprehensive view of synaptic transmission in action to decipher the codes for transferring information across neural circuitry and systems. 1670-Wkshp Functional Cortical Connectomics through Co-Expression of Genetically Expressed Voltage and Calcium Indicators Sam Barnes, Chenchen Song, Thomas Kno¨pfel. Imperial College London, United Kingdom, London, United Kingdom. The flow of information through networks of cortical neurons is thought to provide the processing power that underpins cognition. Traditional structural methods to map connectivity are blind to the functional dynamics of cortical circuits. Emerging approaches use genetically encoded activity indicators such as GCaMP. However they are limited to reporting spiking activity and
Tuesday, February 14, 2017
1665-Plat Mesoscopic Properties and Molecular Mechanisms of IAPP Amyloid Inhibition and Remodeling with Small Molecules Aleksandr Kakinen1, Bo Wang2, Xinwei Ge2, Raffaele Mezzenga3, Thomas P. Davis1,4, Feng Ding2, Pu Chun Ke1. 1 Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia, 2Department of Physics and Astronomy, Clemson University, Clemson, SC, USA, 3Department of Health Science & Technology, ETH Zurich, Zurich, Switzerland, 4Department of Chemistry, Warwick University, Coventry, United Kingdom. The amyloid aggregation of proteins is associated with a number of neurodegenerative diseases such like Alzheimer’s, Huntington’s and Parkinson’s diseases, and also type-2 diabetes (T2D). Despite the physical and structural properties of the aggregating proteins, the corresponding amyloid aggregates feature some common characteristics including the formation of cross-b architecture and cytotoxicity to human cells, suggesting a common amyloid mechanism. Experimental studies have shown that several naturally-occurring small molecules, such as epigallocatechin gallate (EGCG), have an inhibitory effect on the aggregation of a wide range of those amyloid proteins, including islet amyloid polypeptide (IAPP, a.k.a. amylin) in T2D, which is one of the most aggregation-prone peptides. Furthermore, EGCG was also reported having a novel function as amyloid fibrils remodeling, indicating a promising therapeutic approach against amyloid diseases, and importance of understanding the molecular mechanism of such anti-aggregation effect. In this study, we applied experiments and desecrated molecular dynamic (DMD) simulations to investigate the effects of EGCG on IAPP fibrillization. Our high- resolution transmission electron microscopy (TEM) images showed that EGCG inhibited IAPP aggregation by rendering shorter and softer structures, while remodeled mature IAPP fibrils by compromising fibril contour length and triggering splitting without altering the twisted fibril morphology. DMD simulations further revealed that, upon binding with EGCG, IAPP monomers and oligomers tended to form disordered off-pathway clusters instead of cross-b structure due to competing interactions with amyloidogenic sequences. EGCG could also target fibril ends through capping and stacking the exposed sites and enable fibril remodeling. Our combined computational and experimental study offers a detailed mechanistic insight into the inhibitory effect of EGCG on IAPP fibrillization as well as fibril remodeling, suggesting a promising approach for the mitigation of IAPP aggregation and future T2D therapy. 1666-Plat Conformational Plasticity of the MAGE-A3 Protein as a Therapeutic Strategy in Multiple Myeloma Roman Osman1, Hearn J. Cho2, Opher Gileadi3. 1 Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 2Medicine, Hematology/Oncology, Icahn School of Medicine at Mount Sinai, New York, NY, USA, 3Structural Genomics Consortium, University of Oxford, Oxford, United Kingdom. Type I MAGE proteins interact with the RING domain protein Kap1 through their conserved MAGE Homology Domains (MHD) to form E3 ubiquitin (Ub) ligases, which ubiquitinylate p53 targeting it for proteasomal degradation. RNAi experiments demonstrated that MAGE-A3 inhibits p53dependent and independent mechanisms of apoptosis and confers resistance to chemotherapy-induced apoptosis in human myeloma cell lines. Since MAGE expression correlates with progression of multiple myeloma (MM), preventing the interaction with Kap1 is a promising therapeutic intervention against MM. The MHD of MAGE proteins are made of two winged helix domains (WH) linked by a flexible b-hairpin. Structures of MAGE proteins suggest that the WH domains need to undergo a conformational change from a closed to an open form to interact with Kap1 and activate its Ub-ligase activity. Virtual screening on the closed form of MAGE-A3 identified two compounds that recapitulate the RNAi experiments, suggesting that they may inhibit the interaction of MAGE-A3 with Kap1 leading to the apoptosis of MM cells. Further experiments are being conducted to elucidate the nature of the observed effect. MD simulations of the complexes of MAGE-A3 with the small molecules show binding modes in the groove between the two WH domains. These may be responsible for inhibiting the conformational transition. Further simulations to estimate the effect of the small molecules on the conformational change as well as refinement of the initial leads to improve the affinity and selectivity of the compounds are under way. Supported by NIH R21 CA191898.
Workshop: Beyond Calcium: Imaging Voltage and Other Ions 1667-Wkshp Fluorescent Visualization of Cellular Efflux William R. Kobertz. Biochemistry and Molecular Pharmacology, UMASS Medical School, Worcester, MA, USA. The fluorescent visualization of calcium ions entering the cytoplasm has reimaged our basic understanding of the inner workings of calcium signaling in cells, tissues and living organisms. In contrast, there is a dearth of tools to fluorescently visualize ions exiting cells. Part of the challenge stems from the fact that cellular egress is contrary to the pervasive intracellular-centric experimental paradigm. Recently, we have been using glycan engineering to install chemical handles into the cell’s glycocalyx that directly abuts the plasma membrane in all cells. Subsequent chemical modfication of these unnatural sugars ideally positions molecular probes within nanometers of the extracellular vestibules of ion channels and transporters. My laboratory’s efforts to fluorescently visualize ions exiting cells using this technology will be presented. 1668-Wkshp Critical Evaluation of FRET-Based Biosensor Performance: Implications for Measuring Ion Concentrations Amy E. Palmer. Chemistry and Biochemistry, University of Colorado, Boulder, CO, USA. Fluorescent sensors are widely used to visualize, quantify, and reveal the dynamics of small molecules, secondary metabolites, metals, and ions. One of the great promises of such sensors is the ability to quantify cellular signals in precise locations with high temporal resolution, enabling the creation of realtime dynamic maps of cellular signals. Yet this is coupled with the challenge of how to ensure that sensors are measuring what we think they are measuring, developing robust approaches for quantification, and assessing whether sensors are perturbing the underlying biology. This talk will highlight our efforts to develop genetically encoded FRET-based sensors for quantitative mapping of zinc ions in cells. I will discuss approaches for defining whether sensors perturb cellular ions, the importance of carefully defining dynamic range, strategies to compare sensor functionality in vitro, in cells, and in organelles, and the specific challenges associated with quantifying ions in cellular organelles. 1669-Wkshp Light Up the Brain with Genetically Encoded Sensors of Neural Activity Lin Tian. University of California, Davis, Davis, CA, USA. A central challenge in neuroscience is to understand how neural circuits extract information from the environment and generate appropriate behaviors. To address this challenge, one would like to measure the complex spatiotemporal neural activity on many different scales, from single synapses to microcircuits to entire brains, in behaving animals. Recent breakthroughs in modern microscope and protein based fluorescence sensors including calcium, voltage and neurotransmitters, permit optically large-scale recording of neural activity with needed molecular and cell type specificity and spatiotemporal resolutions in behaving animals. To further expand the kinds of neural activity that can be measured with genetically encoded indicators, we applied the established sensor design and optimization platform to the development of a set of specific, targetable and sensitive sensors for direct measurement neuromodulators of the biogenic amine family and activation of corresponding G-protein coupled receptors. We have characterized the expression, signal-to-noise ratio, dynamic range and kinetics of these sensors in dissociated neuronal culture, acute brain slice and in vivo in zebrafish and mouse. A broad application of these imaging tools will enable neuroscientists to obtain a dynamic and comprehensive view of synaptic transmission in action to decipher the codes for transferring information across neural circuitry and systems. 1670-Wkshp Functional Cortical Connectomics through Co-Expression of Genetically Expressed Voltage and Calcium Indicators Sam Barnes, Chenchen Song, Thomas Kno¨pfel. Imperial College London, United Kingdom, London, United Kingdom. The flow of information through networks of cortical neurons is thought to provide the processing power that underpins cognition. Traditional structural methods to map connectivity are blind to the functional dynamics of cortical circuits. Emerging approaches use genetically encoded activity indicators such as GCaMP. However they are limited to reporting spiking activity and