Macquarie University
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Proximity-dependent biotinylation and identification of protein- protein interactions

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posted on 2022-08-16, 04:44 authored by Emmanuel Prikas

A crucial step towards understanding complex biological processes in living systems is to map the underlying protein-protein interactions (PPIs). Established PPI research methods include co-immunoprecipitation (co-IP) and imaging; yet co-IP is not amenable to identifying transient interactions and imaging methods often require prior knowledge of interactors. Proximity-dependent biotin ligation and identification (BioID) of putative interactors offers unique advantages and can be used in parallel with other PPI research methods. BioID allows identification of both transient and stable interactions within cells of multiple biological systems. (I) To establish this method with a previously characterised interactome, we fused a modified biotin ligase enzyme (BioID2) to p38α and expressed the fusion protein in cultured cells. Biotinylated peptides were isolated and identified by mass spectrometry. We created a p38α interactome following network and gene ontology (GO) analyses; establishing the BioID technique in vitro. We evaluated the GO results and focussed on the interaction of p38α and the UV-mediated DNA damage repair protein XPA. (II) To address interactions of a key protein in neurodegenerative diseases, we fused BioID2 to tau, a protein shown to aggregate and cause cell death in Alzheimer’s disease, and expressed it in primary neurons and mouse brain. The resulting biotin-tagged tau interactome and data analysis corroborated known tau associations with the neuronal cytoskeleton, and additionally revealed potential interactions of tau with synaptic vesicle proteins. Understanding the complexity of intracellular PPIs requires placing them in the context of their interaction networks. Labelling interactions as they occur in vivo is a key step towards understanding how the proteome supports cellular function. This is possible with BioID and hence an applicable method for using PPIs to study physiological and pathological processes in cellular and animal model systems.


Table of Contents

Introduction -- Aims -- Methods -- Chapter 1. Proximity-labelling identifies p38 MAP kinase signalling factors and maps Zinc-finger protein gene regulators as indirect p38α targets -- Chapter 2. Proximity-labelled tau interactions in vivo reveal extensive associations with pre-synaptic proteins -- Supplementary information -- Conclusions -- References -- Appendices


Presented for the degree of: Master of Research

Awarding Institution

Macquarie University

Degree Type

Thesis MRes


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

Department, Centre or School

Department of Biomedical Sciences

Year of Award


Principal Supervisor

Arne Ittner


Copyright: Emmanuel Prikas Copyright disclaimer:




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