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NMR ANALYSIS OF INTRACELLULAR AMYLOID-BETA PEPTIDE

Agatisa-Boyle, Colin Gerard
http://rave.ohiolink.edu/etdc/view?acc_num=case1499265031796677

Abstract Details

2017, Doctor of Philosophy, Case Western Reserve University, Chemistry.
The primary goal of the present research is to study the earliest events of aggregation and eventual amyloid formation the Aß peptide inside of living human cells using primarily NMR. The secondary goal is to examine the binding interaction between the Aß peptide and melatonin in order to achieve a greater understanding of how melatonin behaves as an Aß aggregation inhibitor. Prior to using in-cell NMR to characterize the Aß inside of living cells, the conditions used to introduce the Aß peptide into living cells using streptolysin-O (SLO) were further optimized on previous work of Dr. Fang Han. Previously, we were able to introduce the Aß into cells and reseal the cellular membrane in about 45% of SLO treated cells, as assessed by flow cytometry (FCM). Further efforts to optimize the SLO technique, improved this previous result and introduced Aß into 57.6% of viable resealed cells. In order to confirm that the Aß peptide was entering the cytoplasm and not sticking to the outer plasma membrane, images were acquired using confocal microscopy. By counter staining the plasma membranes with CellMask™ Deep Red and acquiring a z-stack series of images, that the Aß was distributed throughout the interior of the cells. After optimizing the SLO technique, uniformly 15N labeled Aß 1 – 40 was introduced into living cells, and 2D NMR heteronuclear single quantum coherence (HSQC) spectrum was acquired. The results showed twelve identifiable amino acid signals that had similar chemical shifts to the control Aß spectrum. This result indicated the Aß peptide was not adopting a new and folded tertiary structure inside the living cells. The effect of macromolecular crowding on Aß was examined using polyethylene glycol (PEG) in an effort to aid in exploring the basis for the intracellular loss of signals. These studies involved acquiring HSQC of Aß in solutions with various concentrations of PEG. Analysis of these spectra showed that a crowded molecular environment could not account for the loss of signals observed for intracellular Aß. Following these macromolecular crowding studies, the binding interaction between Aß and cellular organelles was examined. Nuclei and mitochondria were individually isolated using differential centrifugation, and allowed to incubate in solution with Aß, and finally 2D HSQC NMR spectra were acquired. The resulting spectra showed that three Aß signals disappeared when the peptide was in the presence of isolated nuclei, and twelve signals disappeared when the peptide was in the presence of isolated mitochondria. Immediately following the completion of the HSQC experiments, the samples were centrifuged and an HSQC of the supernatants were acquired. In both cases, no signals were observed indicating that the previous results were consistent with the Aß bound to the cellular organelles and not from free Aß in solution. Further examination of the cellular organelle spectra, it was observed that these organelles had a significant impact on the Leu-17 to Ala-21 region of the Aß peptide. Previous studies have shown that this region is crucial to the aggregation of Aß. Following this result, we had a F19A mutant Aß 1 – 40 peptide, 15N labeled from Leu-17 to Ala-21, produced in order to determine if a mutation in this crucial region will prevent Aß from binding to cellular organelles. This study is currently ongoing, but our initial results suggest that this mutation may in fact prevent Aß from binding to the organelles. The second project examined the interaction between melatonin (a hormone produced in the brain) and the Aß peptide. Previous studies established that melatonin inhibits the aggregation of Aß. Saturation transfer difference (STD) NMR was performed and showed that the melatonin-6H proton was in close proximity to the His-4H protons of Aß. Following this result, we hypothesized that melatonin is able to prevent Aß aggregation by inhibiting the formation of Asp-His salt bridges. This hypothesis was tested by performing 1D 13C NMR on a mixture of melatonin and 13C labeled at the side chain carbonyl of Asp-23 Aß peptide. The result showed that in the presence of melatonin, the Asp-23 side chain carbonyl peak shifted when compared to the control, indicating that interruption of Asp-His salt bridges may be the mechanism by which melatonin inhibits Aß aggregation. The third project involved studying an experimental drug IXR4204 and its ability to inhibit Aß aggregation. At the start of this project, this drug was undergoing a clinical trial for treatment of prostate cancer. For the present study HSQC NMR and circular dichroism (CD) were utilized. Overall the results showed that the drug interacts with Aß monomers, but unfortunately accelerated Aß aggregation. The fourth project examined the abilities of different spices found in traditional Indian cuisine to inhibit Aß aggregation. A variety of different techniques including 1H NMR, CD, and atomic force microscopy (AFM) were utilized and demonstrated that curcumin, anethole, tartaric acid and acetic acid all demonstrated some ability to inhibit Aß aggregation.
Michael Zagorski, Ph.D. (Advisor)
Anthony Pearson, Ph.D. (Committee Chair)
Tolbert Blanton, Ph.D. (Committee Member)
Carey Paul, Ph.D. (Committee Member)
Mieyal John, Ph.D. (Committee Member)
252 p.
Biochemistry; Chemistry
Amyloid Beta; In-cell NMR; Alzheimers disease

Recommended Citations

Citations

  • Agatisa-Boyle, C. G. (2017). NMR ANALYSIS OF INTRACELLULAR AMYLOID-BETA PEPTIDE [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1499265031796677

    APA Style (7th edition)

  • Agatisa-Boyle, Colin. NMR ANALYSIS OF INTRACELLULAR AMYLOID-BETA PEPTIDE. 2017. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1499265031796677.

    MLA Style (8th edition)

  • Agatisa-Boyle, Colin. "NMR ANALYSIS OF INTRACELLULAR AMYLOID-BETA PEPTIDE." Doctoral dissertation, Case Western Reserve University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=case1499265031796677

    Chicago Manual of Style (17th edition)

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