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Establishing a proximity-ligation method to understand the interactome of protein aggregates in neurodegenerative diseases

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posted on 2022-09-09, 02:34 authored by Paulina Szwaja

Progressive supranuclear palsy (PSP) and amyotrophic lateral sclerosis (ALS) are progressive neurodegenerative diseases pathologically characterized by insoluble inclusions of phosphorylated-Tau (p-Tau) and TDP-43 in neurons respectively. Understanding the protein interactome of these inclusions may uncover important insight into the pathological mechanisms of these diseases. Traditional biochemical extraction methods have several limitations for isolation of detergent-insoluble proteins, with significant sample loss and non-specific binding. Thus to limit these problems, an unbiased proteomic approach was taken that enables labeling of insoluble proteins in situ prior to tissue homogenization. This enables targeting of all proteins within a small radius of the protein (inclusion) of interest. This approach, termed biotinylation by antibody recognition (BAR), is a recently described method that will be used in this project to label co-aggregating proteins within post-mortem PSP tissue and fibroblasts from ALS patients. BAR makes use of a primary antibody binding to the protein of interest (POI) to selectively biotinylate proteins in direct proximity to the POI. A secondary antibody conjugated to horseradish peroxidase (HRP) is attached to the primary antibody and the HRP, with the addition of hydrogen peroxide and biotin tyramide, facilitates biotinylation of proteins proximal to the POI. Then the proteins are biochemically isolated and identified using mass spectrometry. With this workflow and bioinformatic analysis, several proteins known to interact with p-Tau and TDP-43 directly and indirectly have been identified along with potentially novel interactors. The data here substantiates this unbiased approach of rapidly labeling and identifying components of aggregates within fixed tissue and cultured cells with a broad application to other diseases and interaction studies.


Table of Contents

Chapter 1 Introduction, overview and aims -- Chapter 2 Optimization of biotinylation-by-antibody recognition (BAR) technique for fixed tissue and cells -- Chapter 3 Biotinylation by antibody recognition to identify proteomic constituents of p-Tau inclusions in postmortem tissue: progressive supranuclear palsy -- Chapter 4 Biotinylation by antibody recognition to identify proteomic constituents of TDP-43 inclusions in patient-derived fibroblasts: amyotrophic lateral sclerosis -- Chapter 5 Discussion and future directions -- References


This thesis is presented to Macquarie University in fulfillment of the Master of Research in Biomedical Sciences

Awarding Institution

Macquarie University

Degree Type

Thesis MRes


Thesis (MRes), Macquarie University, Faculty of Medicine, Health and Human Sciences, 2020

Department, Centre or School

Department of Biomedical Sciences

Year of Award


Principal Supervisor

Roger Chung


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