An Investigation of the domain-domain interactions of A Disintegrin Metalloproteinase domain-containing protein 10 (ADAM10) Open Access

Abstract

Alzheimer’s disease (AD) is the most common form of dementia and is characterized as a chronic neurodegenerative illness leading to memory loss and loss of cognitive function. A Disintegrin and Metalloproteinase domain-containing protein 10 (ADAM10) is a key protein involved in many biological processes including neuropathology, inflammatory response, and tumor progression. In neurons, ADAM10 acts as an α-secretase to mediate proteolytic processing of the amyloid precursor protein (APP). It plays a critical role in reducing the generation of amyloid-β (Aβ) peptides which helps reduce the pathogenesis of AD. ADAM10 is a membrane protein, and its extracellular component consists of four functional domains, including a pro domain (P), metalloprotease domain (M), a disintegrin domain (D) and a cysteine rich domain (C). The crystal structure of a truncated ADAM10 containing M, D, and C domains revealed the molecular details of how D and C domain regulates M domain activity. Studies have shown that ADAM10 is active only when the P domain is cleaved, however, how the P domain interacts with other domains remains unknown. To further explore the regulatory roles of the four functional domains on ADAM10 activity, we designed protein domain truncations of ADAM10 fused with various molecular tags and performed protein-protein interaction assays. The techniques used in this study include Gateway cloning, mammalian cell culture and overexpression of recombinant protein domains, GST pulldown, TR-FRET, SDS-PAGE, and Western blotting. The results showed that the P domain can interact with M, D and C domains but with a preference to M domain. This study helps to provide a better understanding on the regulation of ADAM10 activity by its functional domains. Due to the neuroprotective role of ADAM10, restoring its enzymatic activity could be beneficial. Information gained from this investigation could assist in discovery of a novel therapeutic strategy for treating AD.

Table of Contents

Introduction………………………………..………………………………………………………1

Materials and Methods…………………………………………………………………………….7

Results……………………………………………………………………………………………11

Discussion………………………………………………………………………………………..20

Future Directions and Conclusions………………………………………………………………25

Supplemental Information……………………………………………………………………….26

References………………………………………………………………………………………..33

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Molecular Chemistry, Biochemistry
Keyword	ADAM10 Alzheimer's disease
Committee Chair / Thesis Advisor	Fu, Haian, Emory University
Committee Members	Lynn, David, Emory University Raj, Monika, Emory University Harmon, Brenda, Emory University

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