Publications by authors named "Sanjula P Wickramasinghe"

6 Publications

  • Page 1 of 1

Measuring Interactions Between Tau and Aggregation Inducers with Single-Molecule Förster Resonance Energy Transfer.

Methods Mol Biol 2020 ;2141:755-775

Graduate Group in Biochemistry and Molecular Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Tau is an intrinsically disordered protein implicated in the pathogenesis of Alzheimer's disease and other neurodegenerative disorders. Here we describe the application of single-molecule Förster resonance energy transfer (smFRET) for the characterization of the interactions between tau and polyphosphate, an intracellular polymer that accelerates tau aggregation. We describe the design of tau constructs, purification and fluorescent labeling of tau, and details of acquisition and analysis of smFRET data. The protocols provided here outline an approach that may be applied to the study of other intrinsically disordered proteins and their binding partners.
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http://dx.doi.org/10.1007/978-1-0716-0524-0_39DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8386101PMC
March 2021

Targeting the ensemble of heterogeneous tau oligomers in cells: A novel small molecule screening platform for tauopathies.

Alzheimers Dement 2019 11 22;15(11):1489-1502. Epub 2019 Oct 22.

Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN, USA. Electronic address:

Objective: Understanding the heterogeneous pathology in Alzheimer's disease and related tauopathies is one of the most urgent and fundamental challenges facing the discovery of novel disease-modifying therapies. Through monitoring ensembles of toxic and nontoxic tau oligomers spontaneously formed in cells, our biosensor technology can identify tool compounds that modulate tau oligomer structure and toxicity, providing much needed insight into the nature and properties of toxic tau oligomers.

Background: Tauopathies are a group of neurodegenerative disorders characterized by pathologic aggregation of the microtubule binding protein tau. Recent studies suggest that tau oligomers are the primary toxic species in tauopathies.

New/updated Hypothesis: We hypothesize that tau biosensors capable of monitoring tau oligomer conformation are able to identify tool compounds that modulate the structure and conformation of these tau assemblies, providing key insight into the unique structural fingerprints of toxic tau oligomers. These fingerprints will provide gravely needed biomarker profiles to improve staging of early tauopathy pathology and generate lead compounds for potential new therapeutics. Our time-resolved fluorescence resonance energy transfer biosensors provide us an exquisitely sensitive technique to monitor minute structural changes in monomer and oligomer conformation. In this proof-of-concept study, we identified a novel tool compound, MK-886, which directly binds tau, perturbs the conformation of toxic tau oligomers, and rescues tau-induced cytotoxicity. Furthermore, we show that MK-886 alters the conformation of tau monomer at the proline-rich and microtubule binding regions, stabilizing an on-pathway oligomer.

Major Challenges For The Hypothesis: Our approach monitors changes in the ensemble of assemblies that are spontaneously formed in cells but does not specifically isolate or enrich unique toxic tau species. However, time-resolved fluorescence resonance energy transfer does not provide high-resolution, atomic scale information, requiring additional experimental techniques to resolve the structural features stabilized by different tool compounds.

Linkage To Other Major Theories: Our biosensor technology is broadly applicable to other areas of tauopathy therapeutic development. These biosensors can be readily modified for different isoforms of tau, specific post-translational modifications, and familial Alzheimer's disease-associated mutations. We are eager to explore tau interactions with chaperone proteins, monitor cross-reactivity with other intrinsically disordered proteins, and target seeded oligomer pathology.
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http://dx.doi.org/10.1016/j.jalz.2019.06.4954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038631PMC
November 2019

Polyphosphate Initiates Tau Aggregation through Intra- and Intermolecular Scaffolding.

Biophys J 2019 08 24;117(4):717-728. Epub 2019 Jul 24.

Biochemistry and Molecular Biophysics Graduate Group, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania. Electronic address:

The aggregation and deposition of tau is a hallmark of a class of neurodegenerative diseases called tauopathies. Despite intensive study, cellular and molecular factors that trigger tau aggregation are not well understood. Here, we provide evidence for two mechanisms relevant to the initiation of tau aggregation in the presence of cytoplasmic polyphosphates (polyP): changes in the conformational ensemble of monomer tau and noncovalent cross-linking of multiple tau monomers. We identified conformational changes throughout full-length tau, most notably diminishment of long-range interactions between the termini coupled with compaction of the microtubule binding and proline- rich regions. We found that while the proline-rich and microtubule binding regions both contain polyP binding sites, the proline-rich region is a requisite for compaction of the microtubule binding region upon binding. Additionally, both the magnitude of the conformational change and the aggregation of tau are dependent on the chain length of the polyP polymer. Longer polyP chains are more effective at intermolecular, noncovalent cross-linking of tau. These observations provide an understanding of the initial steps of tau aggregation through interaction with a physiologically relevant aggregation inducer.
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http://dx.doi.org/10.1016/j.bpj.2019.07.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6712485PMC
August 2019

Membrane Bending Moduli of Coexisting Liquid Phases Containing Transmembrane Peptide.

Biophys J 2018 05;114(9):2152-2164

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York. Electronic address:

A number of highly curved membranes in vivo, such as epithelial cell microvilli, have the relatively high sphingolipid content associated with "raft-like" composition. Given the much lower bending energy measured for bilayers with "nonraft" low sphingomyelin and low cholesterol content, observing high curvature for presumably more rigid compositions seems counterintuitive. To understand this behavior, we measured membrane rigidity by fluctuation analysis of giant unilamellar vesicles. We found that including a transmembrane helical GWALP peptide increases the membrane bending modulus of the liquid-disordered (Ld) phase. We observed this increase at both low-cholesterol fraction and higher, more physiological cholesterol fraction. We find that simplified, commonly used Ld and liquid-ordered (Lo) phases are not representative of those that coexist. When Ld and Lo phases coexist, GWALP peptide favors the Ld phase with a partition coefficient of 3-10 depending on mixture composition. In model membranes at high cholesterol fractions, Ld phases with GWALP have greater bending moduli than the Lo phase that would coexist.
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http://dx.doi.org/10.1016/j.bpj.2018.03.026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5961461PMC
May 2018

Line Tension Controls Liquid-Disordered + Liquid-Ordered Domain Size Transition in Lipid Bilayers.

Biophys J 2017 Apr;112(7):1431-1443

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York. Electronic address:

To better understand animal cell plasma membranes, we studied simplified models, namely four-component lipid bilayer mixtures. Here we describe the domain size transition in the region of coexisting liquid-disordered (Ld) + liquid-ordered (Lo) phases. This transition occurs abruptly in composition space with domains increasing in size by two orders of magnitude, from tens of nanometers to microns. We measured the line tension between coexisting Ld and Lo domains close to the domain size transition for a variety of lipid mixtures, finding that in every case the transition occurs at a line tension of ∼0.3 pN. A computational model incorporating line tension and dipole repulsion indicated that even small changes in line tension can result in domains growing in size by several orders of magnitude, consistent with experimental observations. We find that other properties of the coexisting Ld and Lo phases do not change significantly in the vicinity of the abrupt domain size transition.
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http://dx.doi.org/10.1016/j.bpj.2017.02.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5390056PMC
April 2017
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