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Defective Axonal Transport and Alzheimer's Disease Correlations: A Molecular Motor Point of View
492a
Wednesday, February 15, 2017
2419-Pos Board B26 Unfolding Efficacy of the Immunoglobulin Domain I27 Controlled by Force Directionality in Protein Remodeling by CLP ATPase Chaperones Abdolreza Javidialesaadi, George Stan. Department of Chemistry, University of Cincinnati, Cincinnati, OH, USA. Cellular protein quality control comprises a network of chaperones that maintain the proteome viability by performing key cellular tasks such as degrading or remodeling misfolded proteins. Bacterial Caseinolytic proteases (Clp) which are responsible for protein degradation include powerful ring-shaped AAAþ (ATPases Associated with diverse cellular Activities) motors with a central narrow pore that unfold and translocate tagged abnormal proteins. Clp ATPase machines thread substrate proteins (SPs) through their central channel by using repetitive ATP-driven subunit motions coupled with axial mechanical forces exerted onto the SP. Here we perform multiscale molecular simulations of ClpYDI and Titin I27 to mimic and contrast laser optical tweezer (LOT) experiments, in which the SP N-terminus is restrained, with in vivo ClpY-mediated unfolding and translocation, in which the SP is not restrained at the N-terminus. This allows us to shed light on the effects of restraining forces and SP mechanical direction probed on Clp-mediated unfolding mechanism. The external LOT restraint limits ClpY-mediated pulling along the N-C direction of the SP, which yields unfolding of I27 via a shearing mechanism. By contrast, in vivo-like ClpY-action results in pulling along softer mechanical directions and I27 is unfolded via an unzipping mechanism. We find that factors that affect these distinct mechanisms are SP-ClpY surface interactions, the size of the SP relative to the ClpY pore size, the SP mechanical resistance, and the presence of other substrate domains. 2420-Pos Board B27 Metastabilities in the Human Prion Protein N-Terminal Beta-Sheet are Dictated by the 129 Polymorphism S. Alexis Paz1, Eric Vanden-Eijnden2, Cameron Abrams3. 1 Mathematics and Physics, National University, Cordoba, Cordoba, Argentina, 2Courant Institute of Mathematical Sciences, New York University, New York, NY, USA, 3Chemical and Biological Engineering, Drexel University, Philadelphia, PA, USA. We study the thermodynamic stability of the native state of the human prion protein using a new free-energy method, replica-exchange on-the-fly paramaterization. This method is designed to overcome hidden-variable sampling limitations to yield nearly error-free free-energy profiles along a conformational coordinate. We confirm that all four (M129V,D178N) polymorphs have a ground-state conformation with three intact beta-sheet hydrogen bonds. Additionally, they are observed to have distinct metastabilities determined by the side-chain at position 129. We rationalize these findings with reference to the prion ‘‘strain’’ hypothesis, which links the variety of transmissible spongiform encephalopathy phenotypes to conformationally distinct infectious prion forms and classifies distinct phenotypes of sporadic Creutzfeldt-Jakob disease based solely on the 129 polymorphism. Because such metastable structures are not easily observed in structural experiments, our approach could potentially provide new insights into the conformational origins of prion diseases and other pathologies arising from protein misfolding and aggregation. 2421-Pos Board B28 Defective Axonal Transport and Alzheimer’s Disease Correlations: A Molecular Motor Point of View Marcelo Nakaema. School of Sciences and Technology (ECT - UFRN), University of Rio Grande do Norte at Natal, Natal, Brazil. Alzheimer’s disease (AD) is the principal factor for dementia during old age; it is characterized by a progressive impairment of cognitive skills. Several genetic and environmental factors contribute for the AD onset. Recently, several evidences were unraveled that correlated defective axonal transport (AT) caused by abnormal axonal swellings to the pathogenesis of AD. AT is an active process that utilizes the cellular cytoskeleton as a road network for the cytoskeletal motor proteins (dyneins and kinesins superfamilies). There are two types of AT: the anterograde and the retrograde axonal transport. Dynein molecular motors support the retrograde transport to cell bodies by walking to the minus end of microtubules while the kinesin motors drive the anterograde axonal transport towards the synapses by walking the microtubules to the plus end. The three major components of the neuronal cytoskeleton are microtubules, actin and intermediate filaments. One major feature about AT that has been challenging scientists is its bidirectional transport character and how it is efficiently organized. It is critical for axonal function the vesicular transport for long distances and this transport occurs mainly through the microtubule (MT) tracks. In this work, we model this bidirectional transport using a variant of TASEP (Totally Asymmetric Simple Exclusion Process) which consists of two parallel one
dimensional lattices with periodic boundary conditions (two lanes model). One lane represents the transport through the filament and the other lane mimics a diffusive environment. The filament lane also takes into account the filament dynamics. The results are discussed in terms of the efficiency of the bidirectional transport. 2422-Pos Board B29 Characterization of Human IgG1 Fc Region Stability and Aggregation Propensity Evan A. Wells, Anne S. Robinson. Chemical and Biomolecular Engineering, Tulane University, New Orleans, LA, USA. Monoclonal therapeutic antibodies (MAb) are highly valued for their efficacy in treating ailments ranging from arthritis to cancer. Unrivaled specificity and infrequent adverse effects benefit patients in ways traditional small molecule therapeutics cannot. However, formation of aggregates during expression, purification, or storage as a drug substance can lead to product loss and increased immunogenicity. Glycans present on the antibodies also contribute to both aggregation propensity and antibody effector functions. Understanding the importance of glycans in aggregation and protein function is important in limiting costs and maximizing therapeutic efficacy. Antibodies are comprised of two heavy chains and two light chains, and regions are alternatively classified into Fab (fragment antigen binding) and Fc (fragment crystallizable) regions. Only heavy chain subunits exist in the Fc region and remain constant between MAbs of the same isotype (commonly human IgG1). Uniformity of Fc regions between antibodies allows for creation of fusion proteins containing an Fc region that facilitates affinity purification using a standardized protocol. The Fc region is N-glycosylated at Asn-297, and glycoform identities drive effector functions via interactions with specific Fcg receptors (FcgRs). We have utilized mammalian cells to express the human IgG1 Fc region and will present initial studies on efforts to purify and characterize the aggregation propensity and stability of this antibody fragment under controlled stress conditions (e.g. thermal, mechanical, etc.). Data collected from these conditions has potential applications in building and modifying protein stability models and could improve the rational design of future recombinant therapeutic antibodies. 2423-Pos Board B30 Utilising Fluorescence Microscopy to Visualise the Dynamics and Interactions of Molecular Chaperones and a-Synuclein Quill Bowden1,2, Alex MacMillan3, Tobias Rosenkranz2, Till Bo¨cking2. 1 Single-molecule Biophysics, University of Wollongong, Wollongong, Australia, 2Single Molecule Science, University of New South Wales, Sydney, Australia, 3Biomedical Imaging Facility, University of Sydney, Sydney, Australia. In Parkinson’s Disease (PD), the protein a-synuclein and its early stage oligomers have been implicated as the primary cause of neuronal toxicity. Molecular chaperones play an important role in maintaining protein homeostasis in such diseases. The dynamic nature of the chaperone cycle and its interaction with the a-synuclein substrate makes it difficult to characterise by traditional biochemical techniques. Using a range of fluorescence microscopy approaches we show that a-synuclein exists in a concentration dependant dynamic equilibrium between monomeric and oligomeric states, with rapid exchange of subunits between species. Dilution leads to rapid monomerisation and loss of structure followed by a slow recovery of higher order species. Chaperones from the Hsp40/Hsc70 machinery interact only with the dissociated monomeric a-synuclein and delay the reassociation of subunits to higher order species implicating the multimer form as the potential unknown native state of a-synuclein. Using single-molecule techniques we studied the membrane binding properties of the larger a-synuclein oligomers associated with toxicity. This method allowed us to visualise in real-time the perforation of lipid vesicles in the presence of a-synuclein oligomers and characterise the ability of chaperones to prevent this process. A greater understanding of the mechanism of chaperone mediated modulation of disease proteins and their preferential interactions will provide insights into the early stages of the progression of Parkinson’s disease.
Protein-Small Molecule Interactions II 2424-Pos Board B31 Biomolecular Interaction Determination and Quantification by Microscale Thermophoresis Wyatt Strutz, Govind Shah. NanoTemper Technologies, South San Francisco, CA, USA. MicroScale Thermophoresis (MST), an immobilization-free technology, is used to rapidly quantify biomolecular interactions (pM-mM). MST, the
Wednesday, February 15, 2017
2419-Pos Board B26 Unfolding Efficacy of the Immunoglobulin Domain I27 Controlled by Force Directionality in Protein Remodeling by CLP ATPase Chaperones Abdolreza Javidialesaadi, George Stan. Department of Chemistry, University of Cincinnati, Cincinnati, OH, USA. Cellular protein quality control comprises a network of chaperones that maintain the proteome viability by performing key cellular tasks such as degrading or remodeling misfolded proteins. Bacterial Caseinolytic proteases (Clp) which are responsible for protein degradation include powerful ring-shaped AAAþ (ATPases Associated with diverse cellular Activities) motors with a central narrow pore that unfold and translocate tagged abnormal proteins. Clp ATPase machines thread substrate proteins (SPs) through their central channel by using repetitive ATP-driven subunit motions coupled with axial mechanical forces exerted onto the SP. Here we perform multiscale molecular simulations of ClpYDI and Titin I27 to mimic and contrast laser optical tweezer (LOT) experiments, in which the SP N-terminus is restrained, with in vivo ClpY-mediated unfolding and translocation, in which the SP is not restrained at the N-terminus. This allows us to shed light on the effects of restraining forces and SP mechanical direction probed on Clp-mediated unfolding mechanism. The external LOT restraint limits ClpY-mediated pulling along the N-C direction of the SP, which yields unfolding of I27 via a shearing mechanism. By contrast, in vivo-like ClpY-action results in pulling along softer mechanical directions and I27 is unfolded via an unzipping mechanism. We find that factors that affect these distinct mechanisms are SP-ClpY surface interactions, the size of the SP relative to the ClpY pore size, the SP mechanical resistance, and the presence of other substrate domains. 2420-Pos Board B27 Metastabilities in the Human Prion Protein N-Terminal Beta-Sheet are Dictated by the 129 Polymorphism S. Alexis Paz1, Eric Vanden-Eijnden2, Cameron Abrams3. 1 Mathematics and Physics, National University, Cordoba, Cordoba, Argentina, 2Courant Institute of Mathematical Sciences, New York University, New York, NY, USA, 3Chemical and Biological Engineering, Drexel University, Philadelphia, PA, USA. We study the thermodynamic stability of the native state of the human prion protein using a new free-energy method, replica-exchange on-the-fly paramaterization. This method is designed to overcome hidden-variable sampling limitations to yield nearly error-free free-energy profiles along a conformational coordinate. We confirm that all four (M129V,D178N) polymorphs have a ground-state conformation with three intact beta-sheet hydrogen bonds. Additionally, they are observed to have distinct metastabilities determined by the side-chain at position 129. We rationalize these findings with reference to the prion ‘‘strain’’ hypothesis, which links the variety of transmissible spongiform encephalopathy phenotypes to conformationally distinct infectious prion forms and classifies distinct phenotypes of sporadic Creutzfeldt-Jakob disease based solely on the 129 polymorphism. Because such metastable structures are not easily observed in structural experiments, our approach could potentially provide new insights into the conformational origins of prion diseases and other pathologies arising from protein misfolding and aggregation. 2421-Pos Board B28 Defective Axonal Transport and Alzheimer’s Disease Correlations: A Molecular Motor Point of View Marcelo Nakaema. School of Sciences and Technology (ECT - UFRN), University of Rio Grande do Norte at Natal, Natal, Brazil. Alzheimer’s disease (AD) is the principal factor for dementia during old age; it is characterized by a progressive impairment of cognitive skills. Several genetic and environmental factors contribute for the AD onset. Recently, several evidences were unraveled that correlated defective axonal transport (AT) caused by abnormal axonal swellings to the pathogenesis of AD. AT is an active process that utilizes the cellular cytoskeleton as a road network for the cytoskeletal motor proteins (dyneins and kinesins superfamilies). There are two types of AT: the anterograde and the retrograde axonal transport. Dynein molecular motors support the retrograde transport to cell bodies by walking to the minus end of microtubules while the kinesin motors drive the anterograde axonal transport towards the synapses by walking the microtubules to the plus end. The three major components of the neuronal cytoskeleton are microtubules, actin and intermediate filaments. One major feature about AT that has been challenging scientists is its bidirectional transport character and how it is efficiently organized. It is critical for axonal function the vesicular transport for long distances and this transport occurs mainly through the microtubule (MT) tracks. In this work, we model this bidirectional transport using a variant of TASEP (Totally Asymmetric Simple Exclusion Process) which consists of two parallel one
dimensional lattices with periodic boundary conditions (two lanes model). One lane represents the transport through the filament and the other lane mimics a diffusive environment. The filament lane also takes into account the filament dynamics. The results are discussed in terms of the efficiency of the bidirectional transport. 2422-Pos Board B29 Characterization of Human IgG1 Fc Region Stability and Aggregation Propensity Evan A. Wells, Anne S. Robinson. Chemical and Biomolecular Engineering, Tulane University, New Orleans, LA, USA. Monoclonal therapeutic antibodies (MAb) are highly valued for their efficacy in treating ailments ranging from arthritis to cancer. Unrivaled specificity and infrequent adverse effects benefit patients in ways traditional small molecule therapeutics cannot. However, formation of aggregates during expression, purification, or storage as a drug substance can lead to product loss and increased immunogenicity. Glycans present on the antibodies also contribute to both aggregation propensity and antibody effector functions. Understanding the importance of glycans in aggregation and protein function is important in limiting costs and maximizing therapeutic efficacy. Antibodies are comprised of two heavy chains and two light chains, and regions are alternatively classified into Fab (fragment antigen binding) and Fc (fragment crystallizable) regions. Only heavy chain subunits exist in the Fc region and remain constant between MAbs of the same isotype (commonly human IgG1). Uniformity of Fc regions between antibodies allows for creation of fusion proteins containing an Fc region that facilitates affinity purification using a standardized protocol. The Fc region is N-glycosylated at Asn-297, and glycoform identities drive effector functions via interactions with specific Fcg receptors (FcgRs). We have utilized mammalian cells to express the human IgG1 Fc region and will present initial studies on efforts to purify and characterize the aggregation propensity and stability of this antibody fragment under controlled stress conditions (e.g. thermal, mechanical, etc.). Data collected from these conditions has potential applications in building and modifying protein stability models and could improve the rational design of future recombinant therapeutic antibodies. 2423-Pos Board B30 Utilising Fluorescence Microscopy to Visualise the Dynamics and Interactions of Molecular Chaperones and a-Synuclein Quill Bowden1,2, Alex MacMillan3, Tobias Rosenkranz2, Till Bo¨cking2. 1 Single-molecule Biophysics, University of Wollongong, Wollongong, Australia, 2Single Molecule Science, University of New South Wales, Sydney, Australia, 3Biomedical Imaging Facility, University of Sydney, Sydney, Australia. In Parkinson’s Disease (PD), the protein a-synuclein and its early stage oligomers have been implicated as the primary cause of neuronal toxicity. Molecular chaperones play an important role in maintaining protein homeostasis in such diseases. The dynamic nature of the chaperone cycle and its interaction with the a-synuclein substrate makes it difficult to characterise by traditional biochemical techniques. Using a range of fluorescence microscopy approaches we show that a-synuclein exists in a concentration dependant dynamic equilibrium between monomeric and oligomeric states, with rapid exchange of subunits between species. Dilution leads to rapid monomerisation and loss of structure followed by a slow recovery of higher order species. Chaperones from the Hsp40/Hsc70 machinery interact only with the dissociated monomeric a-synuclein and delay the reassociation of subunits to higher order species implicating the multimer form as the potential unknown native state of a-synuclein. Using single-molecule techniques we studied the membrane binding properties of the larger a-synuclein oligomers associated with toxicity. This method allowed us to visualise in real-time the perforation of lipid vesicles in the presence of a-synuclein oligomers and characterise the ability of chaperones to prevent this process. A greater understanding of the mechanism of chaperone mediated modulation of disease proteins and their preferential interactions will provide insights into the early stages of the progression of Parkinson’s disease.
Protein-Small Molecule Interactions II 2424-Pos Board B31 Biomolecular Interaction Determination and Quantification by Microscale Thermophoresis Wyatt Strutz, Govind Shah. NanoTemper Technologies, South San Francisco, CA, USA. MicroScale Thermophoresis (MST), an immobilization-free technology, is used to rapidly quantify biomolecular interactions (pM-mM). MST, the