Development of a bifunctional photo-reactive amino acid to identify cell-type-specific proteomics and interactomics

Abstract

Protein, as perhaps the most important biomolecule involved in almost every biological process, performs its function by interacting with other biomolecules. In multicellular organisms, each specific cell or tissue must express the exact amounts of proteins at the right moment throughout their development stages, to ensure that they can deliver the proper functions. Therefore, studying the proteomes dynamics of specific cell types and analyzing their complicated interactomics are key to understanding both the unique characteristics of these cells and their regulatory process in multi-cellular organisms. Recently, with the development of specific and controllable biorthogonal reactions, such as “click” chemistry, residue-specific incorporation of non-canonical amino acid method has been developed rapidly for global labeling and analysis of newly synthesized proteins in complex and behaving organisms, such as Drosophila melanogaster or C. elegans. However, it remains an attractive challenge to analyze these protein-protein interactions (PPIs) in living cells because interactions between two proteins can occur rapidly and usually are weak, transient or dynamic. In addition, it is more challenging to investigate some interactions between proteins from different cells or even from different tissues because it is hard to distinguish these proteins from each other. In this thesis, a powerful chemical tool to address these questions has been developed and characterized. In chapter 2, I developed and characterized a new bifunctional photo-reactive amino acid reporter, called diazirine 2-aminononynoic acid (dzANA), as a powerful tool for efficient labeling proteomes with spatiotemporal selectivity in living Caenorhabditis elegans and establishing global protein-protein interaction maps. I showed that dzANA could be cell-type-specific incorporated in C. elegans proteins with the help of a mutant CeMetRS, which expressed restrictedly in cells we are interested in by using cell-selective promoters. The study in this chapter also demonstrated that dzANA coupled with stable-isotope labeling of amino acids in cell culture (SILAC) and state-of-the-art mass spectrometry, could allow the proteome-wide identification of proteins expressed in specific cells. To further apply this chemical tool to more complex organisms, in chapter 3, I focused on studying the host-pathogen protein-protein interactions and identify bacterial pathogen virulence factors and their target proteins during the infection of mammalian cells. I demonstrated that dzANA can be accepted by one single mutant methionyl-tRNA synthetase L13G in the bacterial pathogen Salmonella Typhimurium and examined the biological functions of some pathogen effectors during infection. The studies described in chapter 4 are the incorporation of photo-reactive amino acid dzANA into mammalian proteins with the help of engineered translation machinery and examining the ability of dzANA for studying dynamic or transient protein-protein interactions in a cell-type-specific manner. dzANA has shown enormous potential for analyzing protein-protein interaction at a high spatial and temporal level in this chapter.