Examining Molecular Regulation of Unconventional Cytoplasmic Protein Secretion

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Abstract/Project Summary Secreted proteins serve a variety of critical biological roles and thus, the molecular regulation of protein secretion is intensively investigated. Many elegant studies have revealed conserved molecular principles underlying the classical secretory pathway. Proteins bearing signal peptides are co-translationally inserted into the endoplasmic reticulum (ER) where they are properly folded, transported to the Golgi apparatus, and packaged into secretory vesicles for export to various intracellular destinations or fusion with the plasma membrane to enable protein secretion into the extracellular space. However, over the past two decades, several cytoplasmic proteins involved in innate immunity, angiogenesis, and neuronal pathogenicity have been shown to be secreted via ER-Golgi independent, unconventional secretory routes. Thus, given the broad biological utility of unconventional cytoplasmic protein secretion (UCPS), understanding the molecular regulation of the process is of considerable interest. To this end, I developed a genome-scale CRISPR screen to identify novel regulators of UCPS. My preliminary work has identified a class II phosphatidylinositol-3-kinase (PI3K-C2α) as a regulator of the secretion of interleukin-1β, an unconventionally secreted cytokine that plays a major role in the inflammatory response. Furthermore, I have developed a mass-spectrometry based method that can identify unconventionally secreted proteins in a high-throughput, unbiased manner. My proposal will apply cutting-edge molecular genetic and proteomic tools to uncover the breadth of secreted proteins that use UCPS pathways in diverse cell types (Aim 1), and to characterize the role of PI3K-C2α in modulating UCPS (Aim 2). Furthermore, the CRISPR screening strategy that will be used in Aim 2 may identify novel regulators of UCPS. Together, these studies will address my central hypothesis that UCPS operates in diverse cell types, and is regulated by a conserved molecular machinery that supports the secretion of functionally diverse, signal-peptide lacking proteins. This research will be conducted under the guidance of my primary mentor and mentoring committee, who are experts in the fields of molecular genetics, protein trafficking, phosphoinositide biology, and proteomics. Their technical, academic, and professional guidance will allow me to successfully complete the proposed experiments, and help me transition to independence. As an independent investigator, the long-term goal of my research program will be to decipher the mechanisms by which the secretory pathway adapts to intrinsic and extrinsic stressors, and characterize the maladaptive responses that contribute to disease states.