Understanding the roles of protein phosphorylation
One of the areas in which mass spectrometry has made the greatest impact is in the identification of post-translationally modified proteins and the localization of these modifications to specific amino acid residues. Proteins can be modified by the addition of phosphoryl, acetyl, methyl, and many other chemical groups as well as by isopeptide linkages to other proteins such as ubiquitin and SUMO. These modifications can have profound impacts on cellular proteins and cellular physiology; they are used to activate and deactivate enzymes, control sub-cellular localization, mark proteins for degradation, direct protein trafficking, and modulate protein-protein interactions. Through these and other functions, they regulate nearly every aspect of cellular growth and proliferation, as well as cellular responses to changing environmental conditions. In recent years the application of mass spectrometric methods has generated an explosion of data identifying many thousands of protein phosphorylation, acetylation, and ubiquitylation sites. However, the details and physiological relevance of the vast majority of these sites is completely unknown. Uncovering these relationships is one of the main goals of our research. We use multiplexed quantitative phosphoproteomics in vivo and in vitro to identify protein kinase substrates and downstream signaling events in different cellular contexts. Parallel analyses of protein interactions and localization using proteomic and classical biochemical and cell biological methods allow us to identify sites that affect cellular physiology.
Immunopeptidomics - Radiotherapy induced changes in phosphoantigenic MHC-I peptides
The adaptive immune response is an essential safeguard against cancer. A critical requirement for T cell recognition is the expression of novel peptides that arise from mutational changes to endogenous protein sequences. These altered sequence peptides, AKA "neoantigens", are presented by major histocompatibility complex class-I (MHC-I) molecules and recognized by CD8+ T cells as non-self, leading to clearance of tumorigenic cells. More recently, it has been shown that in addition to changes in amino acid sequence, posttranslationally modified peptides, notably phosphorylated sequences, can elicit similar T cell responses. Irradiated tumor cells undergo significant alteration of their intracellular signaling pathways, including DNA damage repair pathways and stress pathways, which operate principally through protein phosphorylation. Thus, we hypothesize that generation of novel phosphoantigens may contribute to the ability of radiation to enhance tumor immunogenicity. Radiation is expected to increase the number and diversity of phosphoantigens a hypothesis supported by our preliminary data.
Identifying the roles of altered macroautophagy in neurodegenerative disease
Many lines of evidence indicate that disruption of the lysosome-mediated degradation pathway, autophagy, is a major component of neurodegenerative disease etiology, but it is unclear how it contributes to disease. In Alzheimer's Disease (AD), autophagic dysregulation has been noted in patient samples and large scale sequencing efforts have identified multiple genetic variants in components of the autophagic machinery in familial disease. In experimental model systems, genetic modulation of autophagy exacerbates the toxicity of phospho-tau and amyloidosis. Similar evidence suggests roles for autophagy in the pathogenesis of a broad array of neurodegenerative diseases such as Frontotemporal Dementia, Amyotrophic Lateral Sclerosis, the synucleinopathies, and Huntington’s disease. How dysregulation of a fundamental, widely active homeostatic pathway leads to diseases with discrete neuropathological and neurological changes remains unclear.
In collaboration with Ai Yamamoto at Columbia University, we are profiling the protein contents of highly purified autophagosomes from mouse brains. By identifying differences in autophagic cargoes in mouse models of genetic disease, we hope to understand how changes in autophagy lead to the neuronal dysfunction and death that give rise to disease.