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Item A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH(Elsevier, 2019-07-19) Ueda, Mitsuharu; Okada, Masamitsu; Mizuguchi, Mineyuki; Kluve-Beckerman, Barbara; Kanenawa, Kyosuke; Isoguchi, Aito; Misumi, Yohei; Tasaki, Masayoshi; Ueda, Akihiko; Kanai, Akinori; Sasaki, Ryoko; Masuda, Teruaki; Inoue, Yasuteru; Nomura, Toshiya; Shinriki, Satoru; Shuto, Tsuyoshi; Kai, Hirofumi; Yamashita, Taro; Matsui, Hirotaka; Benson, Merrill D.; Ando, Yukio; Pathology and Laboratory Medicine, School of MedicineTransthyretin (TTR) is a major amyloidogenic protein associated with hereditary (ATTRm) and nonhereditary (ATTRwt) intractable systemic transthyretin amyloidosis. The pathological mechanisms of ATTR-associated amyloid fibril formation are incompletely understood, and there is a need for identifying compounds that target ATTR. C-terminal TTR fragments are often present in amyloid-laden tissues of most patients with ATTR amyloidosis, and on the basis of in vitro studies, these fragments have been proposed to play important roles in amyloid formation. Here, we found that experimentally-formed aggregates of full-length TTR are cleaved into C-terminal fragments, which were also identified in patients' amyloid-laden tissues and in SH-SY5Y neuronal and U87MG glial cells. We observed that a 5-kDa C-terminal fragment of TTR, TTR81–127, is highly amyloidogenic in vitro, even at neutral pH. This fragment formed amyloid deposits and induced apoptosis and inflammatory gene expression also in cultured cells. Using the highly amyloidogenic TTR81–127 fragment, we developed a cell-based high-throughput screening method to discover compounds that disrupt TTR amyloid fibrils. Screening a library of 1280 off-patent drugs, we identified two candidate repositioning drugs, pyrvinium pamoate and apomorphine hydrochloride. Both drugs disrupted patient-derived TTR amyloid fibrils ex vivo, and pyrvinium pamoate also stabilized the tetrameric structure of TTR ex vivo in patient plasma. We conclude that our TTR81–127–based screening method is very useful for discovering therapeutic drugs that directly disrupt amyloid fibrils. We propose that repositioning pyrvinium pamoate and apomorphine hydrochloride as TTR amyloid-disrupting agents may enable evaluation of their clinical utility for managing ATTR amyloidosis.Item Electrostatic Modeling of Protein Aggregation(2004-12) Vanam, Ram; Dubin, Paul L.Electrostatic modeling was done with Delphi of insight II to explain and predict protein aggregation, measured here for β-lactoglobulin and insulin using turbidimetry and stopped flow spectrophotometry. The initial rate of aggregation of β-Lactoglobulin was studied between pH 3.8 and 5.2 in 4.5mM NaCl; and for ionic strengths from 4.5 to 500mM NaCl at pH 5.0. The initial slope of the turbidity vs. time curve was used to define the initial rate of aggregation. The highest initial rate was observed near pH < pI i.e., 4.6 (< 5.2). The decrease in aggregation rate when the pH was increased from 4.8 to 5.0 was large compared to its decrease when the pH was reduced from 4.4 to 4.2; i.e., the dependence of initial rate on pH was highly asymmetric. The initial rate of aggregation at pH 5.0 increased linearly with the reciprocal of ionic strength in the range I = 0.5 to 0.0045M. Protein electrostatic potential distributions are used to understand the pH and ionic strength dependence of the initial rate of aggregation. Similar studies were done with insulin. In contrast to BLG, the highest initial aggregation rate for insulin was observed at pH = pI. Electrostatic computer modeling shows that these differences arise from the distinctly different surface charge distributions of insulin and BLG.Item Structure-based inhibitors halt prion-like seeding by Alzheimer’s disease–and tauopathy–derived brain tissue samples(The American Society for Biochemistry and Molecular Biology, 2019-11) Seidler, Paul Matthew; Boyer, David R.; Murray, Kevin A.; Yang, Tianxiao P.; Bentzel, Megan; Sawaya, Michael R.; Rosenberg, Gregory; Cascio, Duilio; Williams, Christopher Kazu; Newell, Kathy L.; Ghetti, Bernardino; DeTure, Michael A.; Dickson, Dennis W.; Vinters, Harry V.; Eisenberg, David S.; Pathology and Laboratory Medicine, School of MedicineIn Alzheimer's disease (AD) and tauopathies, tau aggregation accompanies progressive neurodegeneration. Aggregated tau appears to spread between adjacent neurons and adjacent brain regions by prion-like seeding. Hence, inhibitors of this seeding offer a possible route to managing tauopathies. Here, we report the 1.0 Å resolution micro-electron diffraction structure of an aggregation-prone segment of tau with the sequence SVQIVY, present in the cores of patient-derived fibrils from AD and tauopathies. This structure illuminates how distinct interfaces of the parent segment, containing the sequence VQIVYK, foster the formation of distinct structures. Peptide-based fibril-capping inhibitors designed to target the two VQIVYK interfaces blocked proteopathic seeding by patient-derived fibrils. These VQIVYK inhibitors add to a panel of tau-capping inhibitors that targets specific polymorphs of recombinant and patient-derived tau fibrils. Inhibition of seeding initiated by brain tissue extracts differed among donors with different tauopathies, suggesting that particular fibril polymorphs of tau are associated with certain tauopathies. Donors with progressive supranuclear palsy exhibited more variation in inhibitor sensitivity, suggesting that fibrils from these donors were more polymorphic and potentially vary within individual donor brains. Our results suggest that a subset of inhibitors from our panel could be specific for particular disease-associated polymorphs, whereas inhibitors that blocked seeding by extracts from all of the tauopathies tested could be used to broadly inhibit seeding by multiple disease-specific tau polymorphs. Moreover, we show that tau-capping inhibitors can be transiently expressed in HEK293 tau biosensor cells, indicating that nucleic acid–based vectors can be used for inhibitor delivery.