Article Text

PDF
B14 Mapping cell type-specific protein interactomes of huntingtin by live biotin ligase tagging: a proof-of-concept study in human neural progenitor cells
  1. Kagistia Hana Utami1,
  2. Bernice Sim Meng Qi1,
  3. Radoslaw M Sobota2,
  4. Mahmoud A Pouladi1,3
  1. 1Translational Laboratory in Genetic Medicine (TLGM), Agency for Science, Technology and Research (A*STAR), 8A Biomedical Grove, Immunos, Singapore, Singapore
  2. 2Systems Structural Biology Group, Institute of Molecular and Cell Biology (IMCB) Agency for Science, Technology and Research (A*STAR), Proteos, Singapore
  3. 3Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

Abstract

Background Misfolding and altered conformation of huntingtin (HTT) as a result of an expanded polyglutamine tract in its N-terminus is thought to be the primary cause of Huntington’s disease (HD). The misfolding of HTT in its mutant form leads to gain- and loss-of-function effects as a result of altered protein interactions. Protein-protein interaction studies using Y2H and AP-MS on whole tissues have revealed that HTT interacts with hundreds of proteins. As vulnerability to mutant HTT varies across tissues, defining HTT interactomes in a tissue and cell type-specific manner may provide insight into the basis of differential pathology.

Aim In this study, we used proximity-dependent biotin identification (BioID) to identify proximity interactions of HTT in the living cells.

Method This approach entails fusion of a promiscuous biotin ligase (BirA) to a protein of interest to enable biotinylation of proteins that are located within its immediate proximity, upon treatment with biotin. Using CRISPR-Cas9 and TALEN-mediated homologous recombination, we engineered human embryonic stem cell lines in which the endogenous HTT gene is tagged at the 3’ or 5’ end with BirA (BirA-HTT and HTT-BirA hESCs). This ensures that the full length HTT-BirA fusion protein is expressed at physiological levels and appropriate stoichiometry of HTT-interactors is maintained.

Results We have differentiated the engineered hESCs into neural progenitor cells and have verified protein and mRNA expression levels of tagged HTT and BirA in these cells. Following 24 hours of biotin labelling in living cells, biotinylated proteins were isolated by streptavidin-affinity capture and identified by mass spectrometry. By differentiating these cells into neural progenitor cells and neurons, we aim to generate cell-type specific interactome maps of HTT in a human context. This study will provide comprehensive cell type-specific resources to elucidate HTT-interacting proteins in human cells and may provide insight into the differential cellular vulnerability to mutant HTT effects in HD.

  • Interactome
  • Biotin Ligase
  • Human Neural Progenitor Cells
  • Genome Editing
  • Mass Spectrometry

Statistics from Altmetric.com

Request permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.