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Thesis Fang Han ETD.pdf (7.54 MB)

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AMYLOID A-BETA PEPTIDE: IN-CELL STUDIES AND MECHANISM OF POLYPHENOL-BASED INHIBITION TO AGGREGATION

Han, Fang
http://rave.ohiolink.edu/etdc/view?acc_num=case1404771350

Abstract Details

2014, Doctor of Philosophy, Case Western Reserve University, Chemistry.
The two major goals of my research work are to obtain in-cell nuclear magnetic resonance (NMR) spectra of the A-beta peptide, and to elucidate the molecular mechanism of the polyphenol-based inhibition to A-beta aggregation. To characterize the A-beta structure inside living cells, optimum conditions were established to get the A-beta peptide inside living cells using steptolysin-O (SLO) reversible membrane permeabilization and Ca2+ resealing of the target cells. This was confirmed by multi-complementary techniques, including confocal microscopy, flow cytometry, and NMR. The confocal images demonstrated that A-beta(1-40) was evenly distributed throughout the cell cytoplasm and the nucleus, indicating that the pore formation and resealing occurred properly. To assess the efficiency of the pore formation and resealing, flow cytometry (FCM) analysis was performed. The FCM showed that 100 ng/ml SLO yielded optimal resealing efficiency (86%). By comparing the cells without SLO treatment, FCM results also indicated that the pore formation and resealing occurred properly. Based on the 2D NMR hetero-single quantum coherence (HSQC) spectra of the cells and supernatant with Ala2 and Ala21 15N labeled A-beta(1-40), two peaks were observed from the cell samples, while no signals were observed from the supernatant, establishing that all of the NMR signals originated from the A-beta protein within the cells. Next, the HSQC spectrum of the uniformly 15N labeled A-beta(1-40) inside the cells showed only 5 peaks, and none had identical chemical shifts with control spectra. In an effort to elucidate the reasons for the peak disappearances and shifts, cells were lysed and further NMR studies were conducted with cell lysate. These studies involved HSQC of cell lysate and cell lysate treated with nuclease. Analysis of these spectra showed that the A-beta peptide did not bind to cell membranes during and after delivery into the cells and that the nuclease does not change the viscosity to alter the conformation and/or mobility of the peptide. To examine the influence of survival rate of cells on the structure of A-beta(1- 40) inside cells, NMR experiments were performed for different time spans. Comparison of the results suggested that no obvious changes occurred in the cellular environment during 8-20 hr after the cells were resealed. In regard to the second project with elucidating the molecular mechanism of polyphenol-based inhibition to the A-beta(1-42) aggregation, circular dichroism (CD), thioflavin-T (ThT), electron microscopy (EM), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and NMR were carried out. The CD and EM data established that the polyphenol, myricetin, inhibits A-beta(1-42) aggregation while the ThT and SDS-PAGE data demonstrated that myricetin also disaggregates preformed aggregates. HSQC showed that oxidation of the Met35 sulfur side chain to the sulfoxide occurred in A-beta(1-42) after incubating with myricetin, which was also confirmed by ESM-mass spectrometry (MS). However, when catalase (an H2O2 scavenger) was present in the mixture, the myricetin-induced inhibition of A-beta fibrillation was not altered, suggesting that oxidation of the Met35 side chain by H2O2 was not the only factor to alter A-beta fibrillation. CD experiments carried out under anaerobic conditions suggested that auto-oxidation of myricetin was a prerequisite for the myricetin-induced inhibition of the protein fibril. Moreover, the overlaid HSQC spectrum of selectively 13C-labeled A-beta(1-42) with and without myricetin is essentially the same, which indicates that Schiff base formation between the quinone section of myricetin, and the A-beta(1-42) did not occur. However, saturation transfer difference (STD) NMR studies demonstrated that myricetin weakly interacts with monomeric A-beta(1-40). In conclusion, the first part of this thesis work established optimal conditions for in-cell NMR studies of the A-beta peptides and constitutes the first step for additional work that may determine the structure inside living cells. The second part of this work established that myricetin oxidizes the Met35 side chain of the A-beta peptide which in part inhibits aggregation.
Michael Zagorski (Advisor)
156 p.
Chemistry
A-BETA PEPTIDE, IN-CELL NMR, INHIBITION, AGGREGATION

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Citations

  • Han, F. (2014). AMYLOID A-BETA PEPTIDE: IN-CELL STUDIES AND MECHANISM OF POLYPHENOL-BASED INHIBITION TO AGGREGATION [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1404771350

    APA Style (7th edition)

  • Han, Fang. AMYLOID A-BETA PEPTIDE: IN-CELL STUDIES AND MECHANISM OF POLYPHENOL-BASED INHIBITION TO AGGREGATION. 2014. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1404771350.

    MLA Style (8th edition)

  • Han, Fang. "AMYLOID A-BETA PEPTIDE: IN-CELL STUDIES AND MECHANISM OF POLYPHENOL-BASED INHIBITION TO AGGREGATION." Doctoral dissertation, Case Western Reserve University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=case1404771350

    Chicago Manual of Style (17th edition)

case1404771350
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This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.