Characterizing Notch1 Singaling Regulation And Amyloid Beta Cleavage

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Gamma secretase cleaves numerous substrates that regulate a variety of cellular processes. The involvement of Gamma secretase in so many different signaling systems makes it a challenging enzyme to target without toxic side-effects. The two predominantly studied substrates are Notch and Amyloid precursor protein (APP). Dysregulated cleavage of the first can lead to cancer, while cleavage of the second results Amyloid Beta (A?) production which contributes to Alzheimer’s Disease (AD). Therefore, the development of substrate specific gamma secretase modulators is of primary importance. To find a modulator of Notch signaling we screened a 20-amino acid displaying bacteriophage library for a peptide sequence that recognizes Notch1. Through this approach we identified a sequence that specifically binds to Notch1 and none of the other isoforms of Notch. The phage displaying this sequence co-localized and co-immunoprecipitated with Notch1 and was able to reduce Notch1 signaling in a Notch reporter system. Therefore, we have identified a novel sequence that can specifically modulate Notch1 signaling without interfering with cleavage of similar proteins. The AD field has been primarily restricted to studying two A? species: A?-40 and A?-42, that are produced after gamma secretase cleavage of two sites on APP. While other cleavage sites have been reported, the ability to study and characterize them, especially in relation to Alzheimer’s Disease, has been hampered due to the lack of tools specific to these alternate sites. To address this issue, we generated, and characterized, monoclonal antibodies to these cleavage sites. We confirmed that these antibodies are selective for the alternative species, including A?-43 and A?-45, and utilized Alpha-LISA technology to quantify binding strength. We were also able to utilize some of these antibodies in immunohistochemistry and aggregation binding studies to further investigate difference in behavior of these amyloid species. The development of these antibodies will enable us to focus on alternative cleavage sites of A? and study their contribution to AD.

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Alzheimer's Disease; Amyloid beta 45; Amyloid beta 46; Amyloid beta 48; Bacteriophage; Notch1


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Doctor of Philosophy

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Government Document




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Attribution-NonCommercial-NoDerivatives 4.0 International


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