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This journal is published by theUniversity Library System of the University of Pittsburgh as part  

of its D-Scribe Digital Publishing Program and is cosponsored by the University of Pittsburgh Press. 

 

 

 

 

 

 

 

 

 

Proximity Utilizing Biotinylation of 

Nuclear Proteins in vivo 

 

Arman Kulyyassov1, Gulsamal 

Zhubanova1, Erlan Ramanculov1, 

Vasily Ogryzko2 
 
1National Center for Biotechnology, 
Astana, Kazakhstan; 2Institut Gustave 
Roussy, Villejuif, France 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Vol. 3, Suppl. (2014)   |   ISSN 2166-7403 (online)  

DOI 10.5195/cajgh.2014.165 |   http://cajgh.pitt.edu 

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KULYYASSOV 

 

 

This work is licensed under a Creative Commons Attribution 4.0 United States License. 

 

This journal is published by theUniversity Library System of the University of Pittsburgh as part  

of its D-Scribe Digital Publishing Program and is cosponsored by the University of Pittsburgh Press. 

 

Central Asian Journal of Global Health 

Volume 3, Suppl. (2014) |  ISSN 2166-7403 (online) | DOI 10.5195/cajgh.2014.165|http://cajgh.pitt.edu 

 

 

Abstract 

Introduction. The human genome consists of roughly 30,000 genes coding for over 500,000 different proteins, of which more than 

10,000 proteins can be produced by the cell at any given time (the cellular “proteome”). It has been estimated that over 80% of 

proteins do not operate alone, but in complexes. These protein-protein interactions (PPI) are regulated by several mechanisms. For 

example, post-translational modifications (methylation, acetylation, phosphorylation, or ubiquitination) or metal-binding can lead 

to conformational changes that alter the affinity and kinetic parameters of the interaction. Many PPIs are part of larger cellular 

networks of interactions or interactomes. Indeed, these interactions are at the core of the entire interactomics system of any living 

cell, and so, aberrant PPIs are the basis of multiple diseases, such as neurodegenerative diseases and cancer. The objective of this 

study was to develop a method of monitoring protein-protein interactions and proximity dependence in vivo. 

Methods. The biotin ligase BirA was fused to the protein of interest, and the Biotin Acceptor Peptide (BAP) was fused to an 

interacting partner to make the detection of its biotinylation possible by western blot or mass spectrometry.  

Results. Using several experimental systems (BirA.A + BAP.B), we showed that the biotinylation is interaction/proximity 

dependent. Here, A and B are the next nuclear proteins used in the experiments – 3 paralogues of heterochromatin protein HP1 

(CBX5), HP1 (CBX1), HP1 (CBX3), wild type and transcription mutant factor Kap1, translesion DNA polymerase PolH and 

E3, ubiquitin ligase RAD18, Proliferative Cell Nuclear Antigen (PCNA), ubiquitin Ub, SUMO-2/3, different types and isoforms 

of histones H2A, H2Az, H3.1, H3.3, CenpA, H2A.BBD, and macroH2A. The variant of this approach is termed PUB-NChIP 

(Proximity Utilizing Biotinylation with Native Chromatin Immuno-precipitation) and is designed to purify and study the protein 

composition of chromatin in proximity to the nuclear protein of interest. Using the RAD18 protein as a model, we demonstrated 

that the RAD18-proximal chromatin is enriched in some H4 acetylated species. Moreover, the RAD18-proximal chromatin 

containing a replacement histone H2Az has a different pattern of H4 acetylation. 

Conclusion. Progress in the last decade in cancer drug therapy has led us to the conclusion that the nucleus of eukaryotic cells is 

an active site for many cellular processes important to the development of cancer. These processes include changes in genetic and 

epigenetic landscape (e. g. methylation of DNA, modification of histones) and the expression levels of transcription factors, which 

regulates gene products (e.g. hypoxia-inducible factor 1α (HIF-1α) in chronic anemia, etc.) where protein-protein interactions play 

important role. Understanding the nature of protein-protein interactions may improve design strategies for small-molecule PPI 

modulators. PPI assay technologies that closely reflect physiological conditions hold the key to developing specific anti-cancer 

drugs.  

Keywords: protein-protein interaction, proximity, biotinylation 

 

 

 

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http://www.pitt.edu/
http://www.library.pitt.edu/articles/digpubtype/index.html
http://www.upress.pitt.edu/upressIndex.aspx
http://en.wikipedia.org/wiki/Interactomics
http://en.wikipedia.org/wiki/Alzheimer%E2%80%99s_disease
http://en.wikipedia.org/wiki/Cancer

