Initially in this lab, a protein complex map was developed from a Drosophila cell line. As this protein interaction characterization is not context-dependent, it is pivotal to extend the analysis beyond this static draft to the context of an organism. Therefore, in parallel to the cell culture efforts, for the same set of bait proteins, in collaboration with NCBS, Bangalore, India, more than one thousand stable transgenic Drosophila lines have been generated using the site-specific Attp integration. Expression of the bait protein is driven by an UAS promoter that enables tissue specific expression, under various genetic and physiological conditions (using specific Gal4 drivers). Drosophila brain is being used as a model tissue to develop a tissue-specific protein complex map. Protein complexes from brains are obtained by TAP affinity chromatography and the prey proteins are identified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Analysis of protein complexes found in the brain would provide insights on the major players and their ability to tune the cascade of others. Genetic validation would aid dissecting certain pathways in diseased state.
Proteome Research Projects
From the earliest embryo to the adult, the spatiotemporal expression of genes is essential for normal development and physiology. At the very basis of this is the regulation of transcription via transcription factors (TFs), proteins that physically bind DNA to activate or suppress gene expression. As the target of signaling pathways and the first step in synthesis of proteins and regulatory RNAs, transcription factors represent a crucial point of regulation relating to the vast majority of cellular processes. The majority of TFs function through interactions with other proteins. Consequently, the characterization of these protein-protein interactions is essential for understanding how TFs function to regulate gene expression and in turn, biology of the cell. Towards this end, we are currently defining the protein “interactome” of Drosophila transcription factors based on the isolation and mass spectrometric analysis of affinity tagged proteins.
Mutations in several signaling pathway components have been causally implicated in leukemia, lymphoma, myelomas and many solid tumors. Incomplete knowledge of the protein complexes that mediate the cellular functions of individual signaling components and a superficial understanding of signaling pathway cross-talk are obstacles in predicting specific output states of signaling. Knowledge of the protein complex composition would provide insights into the molecular circuitry essential for a mechanistic understanding of the biology and pathobiology of a given protein. We are investigating the protein interactions occurring in canonical signaling pathways, i.e. Dpp, EGFR, FGFR, Hh, Insulin, JAK/STAT, JNK, Notch and Wg using affinity purifications of protein complexes followed by tandem mass spectrometry (AP-MS) to identify constituent proteins. Using a collection of 200 tagged signaling proteins, already in hand, we hope to identify potential interacting proteins both in cell culture and transgenic flies. Bioinformatics analysis will integrate this data with existing information from other genome-scale analyses. The combined results will provide unprecedented insight into the protein interaction network of the major signaling pathways. Given the importance of signaling pathways in oncogenesis, this work has the potential of identifying novel therapeutic targets by virtue of their functional association with known tumor suppressors or oncogenes
The sequencing of the Drosophila melanogaster genome has made it possible to predict the numbers and types of proteins made by the fly, but not how these proteins interact to form functional units in living cells. To address this question, we have used Tandem Affinity Purification (“TAP”) to isolate protein complexes at the scale of the proteome. Our goal is to express each of ~5,000 Drosophila proteins fused to a “FLAG-HA” moiety so that it can be expressed and recovered from whole cells, or whole flies, with its binding partners and interactions intact. The resulting set of tens of thousands of protein-protein interactions will allow us to construct a map of a significant proportion of the Drosophila “Interactome.” These data are deposited in FlyBase for unrestricted public access, following validation. As an additional resource for the community, we are generating stable transgenic lines for the same set of tagged proteins where expression is driven by a UAS promoter to allow in vivo expression of the protein in a spatio-temporally regulated manner. Analysis of the resulting data set will allow us to better understand the fundamental ways in which individual proteins form complexes in this model organism and in many other biological systems in which corresponding interactions occur.