Functional Dissection of Metabolic-Sensing Proline Hydroxylation Pathways

University Of Minnesota, Minneapolis MN

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Abstract

Project Summary Proline hydroxylation (Hyp) is a fundamental posttranslational modification and regulatory mechanism that are highly responsive to the changes in cellular metabolic conditions. During tumor progression, the rapid proliferation of cancer cells creates a hypoxic microenvironment that inhibits the hydroxyproline-mediated degradation of HIFa proteins and activates hypoxia-response cellular pathways to promote cancer cell survival in hypoxia. In addition to oxygen, the modification enzyme prolyl hydroxylases are also sensitive to the concentration of iron and key mitochondria metabolites including succinate, fumarate, and alpha-ketoglutarate, making the pathway a critical metabolic sensor in cells. Extensive studies have demonstrated that proline hydroxylation regulates protein structural stability, protein-protein interactions, or proteasomal degradation of substrate proteins. Despite its important roles in cell physiology and success in the targeted analysis of individual substrates, system-wide characterization and functional quantification of the pathway have been hindered by the lack of effective tools and strategies for site-specific identification of proline hydroxylation targets. Our overall hypothesis and long-term goal is that systematic characterization of “proline hydroxylome” through the development of functional proteomics approaches will lead to the mechanistic understanding of novel Hyp-mediated metabolic regulations in development and diseases. Moving towards this goal, in the past years, we have established HypDB for functional annotation analysis of the Hyp proteome with the development of a streamlined workflow for systematic analysis of the Hyp substrates in cells and tissues. We have gained extensive experience in biochemical characterization of Hyp targets, the interactome of specific prolyl hydroxylase as well as its crosstalk with other PTM regulatory pathways. To continue our effort, we will expand the HypDB to quantify Hyp dynamics in mouse tissues and develop functional proteomics strategies to identify key Hyp sites in protein structural stability and prolyl hydroxylase targets. We will apply recently developed chemical and biochemical strategies to investigate the crosstalk between proline hydroxylation and other metabolic-sensing modifications in regulating substrate protein degradation and activity. Furthermore, we will study the physiological significance of a new Hyp-mediated epigenetic modification pathway in regulating gene expression and chromatin activity. Overall, we anticipate that the development and application of functional proteomics technology for system-wide analysis of proline hydroxylation proteome will reveal novel metabolic-sensing pathways and potentially lead to paradigm-shifting concepts in the fields of cancer, metabolic diseases, and development.