Proteomic and Phosphoproteomic Remodeling Under Genomic Imbalance

Summary The imbalanced chromosome number, or aneuploidy, is associated with various human diseases, including cancers and trisomic syndromes. The genomic imbalance in these aneuploid cells leads to copy number alterations (CNAs) for a large number of genes, affecting both proteomic abundance and lifetime. In the past five years, we have developed a set of reproducible mass spectrometry (MS)-based proteomic methods for measuring the abundance and lifetime regulations of thousands of proteins and their post-translational modifications (PTMs), such as phosphorylation. We have exploited these toolkits in analyzing several meticulously selected isogenic aneuploidy models and have discovered characteristic proteostasis and cell signaling pathways that challenge current paradigms in the field. Our future research will expand in several directions. First, we will distinguish and determine the proteomic remodeling and tolerance mechanisms in chromosome Gain and Loss type aneuploidies. We observed a notable absence of protein degradation regulation in maintaining protein complex stoichiometry in a model of Chromosome 3p hemizygous deletion, in which we detected changes in protein thermal stability. We propose to extend the study of proteostasis in Loss type aneuploidies that are clinically important but have previously been underexplored and to further profile the protein interactome reformation and perturbation through cross-linking MS and other techniques. Second, we will apply phosphoproteomics to identify the direct dosage effects of kinases and phosphatases and their downstream signaling events. A specific focus will be the functional characterization of phosphosites on Death domain-associated protein 6 (DAXX), a histone H3.3 chaperone, in the context of human trisomies. The profiled phosphorylation regulations will be summarized to expose druggable vulnerabilities associated with trisomic syndromes and cancer aneuploidies. Our third line of research will adopt a cross-species strategy to study aneuploidy and gene CNAs. We will measure homologous aneuploid chromosome-induced effects in terms of protein and phosphosite abundance and turnover and apply an evolutionary biological perspective to understanding genome dosage imbalance. Finally, more broadly, we will develop bottom-up quantitative technologies based on limited proteolysis and matrix-assisted laser desorption ionization imaging mass spectrometry to assess how the native lipid environment impacts protein conformation and function in disease states. Collectively, our proposed technology developments and biological studies over the next five years are poised to significantly advance our understanding of the physiology and biology of genome imbalance stress and aneuploidy-associated abnormalities.