Comprehensive analysis of proteins regulating protein synthesis, folding, and transport during cellular differentiation

Our principal objective is to identify and biochemically characterize proteins regulating changes in the secretory pathway using differentiating B-lymphocytes as a model system. The specific aims are: - further development of robust mass spectrometry-based methods to facilitate the analysis of protein expression and protein interaction maps - identification and functional characterization of novel ER folding factors - identification and functional characterization of proteins mediating transport of immunoglobulins to the cell surface B cell differentiation is an important process in the development of innate immunity during the first minutes-hours of exposure to infectious agents. This is essential for the subsequent adaptive immune response and survival of an individual. We will use the mouse I.29u+ B cell line as a model to investigate the host response against established microbial antigens such as LPS and CpG DNA. Engagement of B cells with bacterial pathogens elicits signal transduction pathways that are remarkably conserved between mammalian cells, fruit flies and plants and ultimately result in transcriptional activation of a large number of genes. One of the hallmarks of the response in mammalian cells is the enormous expansion of the endoplasmic reticulum (ER). This allows the production and secretion of immunoglobulin molecules that reach the cell surface via vesicular transport pathways through the Golgi complex and possibly other intracellular compartments. So far, little to nothing is known about the mechanism and the proteins involved in this response. Now that the human genome has been sequenced, it should theoretically be possible to assign functions to the proteins participating in the abovementioned signal transduction pathways. We aim to investigate the basic mechanisms that underlie the B cell secretory response: the biochemical reactions taking place inside cellular compartments and the protein machineries that establish and maintain the communication between them. We propose to analyze B cell activation and signal transduction processes at the protein level using proteomics. The basic proteomic techniques that we will use are routine in our labs, and are based on 2-D gel electrophoresis combined with state-of-the art mass spectrometric protein identification and characterization. Additionally, we will develop affinity-based techniques to select particular cell components and protein complexes and further develop tools to characterize relevant post-translational modifications. The integration of our joint expertise in protein mass spectrometry, chaperone assisted protein folding, membrane transport and signal transduction, and existing collaborations within our institutes will provide a unique opportunity for rapid and innovative progress on our objectives. We also expect that the novel techniques and assays developed in this application will be of immediate use to other proteome-driven projects in our and other institutes.