Defining tissue specific protein-protein interactomes in vivo and in disease

Werner H, Chojnowski A, Sobota R, Burke B and Stewart CL

Institute of Medical Biology, 8A Biomedical Grove, Immunos, Singapore 138648.

The genomic era identified and defined the composition of the genome. The post-genomic era seeks to understand how the organism’s genome functions in both building and maintaining the organism through the actions of the proteins encoded by the genome. To accomplish these ends, proteins interact extensively with each other resulting in the establishment of complex protein-protein interaction (PPI) networks or interactomes. We are using the BioID technique to describe and decipher PPI to define the LaminA interactome and to understand how different mutations in the LMNA gene result in a range of tissue specific diseases called the laminopathies. BioID involves the fusion of a promiscuous variant of the biotinylation enzyme Bira Ligase (BirA) to the protein of interest. The resulting fusion protein is expressed in vivo in any cell. Proteins within the 10-20nm of the fusion protein are potentially biotinylated, isolated by streptavidin pull down, and identified by MassSpec, establishing them as potential interactors with the protein of interest. We inserted the BirA gene in frame into the N-terminus of the murine Lmna gene in ES cells. From these we derived a mouse line expressing a hybrid LaminA/C-BirA fusion protein that correctly localizes to the nuclear lamina in different tissues. Endogenous levels of biotin within the various murine tissues were sufficient to result in the biotinylation and identification of proteins that are known interactors of the A-type lamins. With these mice, we are defining the tissue specific interactome of the A-type lamins and find that the interactome varies between different tissues. We are also determining how the interactome is altered by lamins carrying specific mutations that result in disease.