Dissecting Molecular Regulation of Unconventional Protein Secretion

Many elegant studies have revealed conserved molecular principles underlying the classical ER-Golgi-Secretory pathway for protein secretion. 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, understanding the molecular regulation of unconventional cytoplasmic protein secretion (UCPS) is of considerable interest. Our current studies are focused on 1) identifying unconventionally secreted proteins using an unbiased high-throughput mass-spectrometry based approach and 2) identifying regulators of UCPS using whole-genome CRISPR screening. By comparing levels of a given protein in conditioned media and cell lysates, our analysis effectively discriminates between authentically secreted proteins and non-secreted proteins released into the extracellular media from dead or dying cells. Coupling this approach with treatment of cells with Brefeldin-A, an inhibitor of ER-Golgi traffic, we have identified Brefeldin-A resistant peptides derived from candidate unconventionally secreted proteins from Huh7 (hepatocyte) and THP1 (immune) cell lines and primary endothelial cells. As expected, most highly secreted proteins from these cell lines were observed to contain signal peptides, presumably mediating traffic through the conventional ER/Golgi secretory pathway, with a minor fraction of the secretome consisting of unconventionally secreted proteins. Our analysis has identified several novel candidate UCPS cargoes, as well as confirming previously reported UCPS cargoes including metallothionein and leukocyte elastase inhibitor. A preliminary whole genome CRISPR screen for modulators of secretion for the known UCPS cargo, interleukin-1β (IL-1β), identified PIK3C2A (a lipid kinase known to be localized to the endo-lysosomal system), molecular motor proteins, and other protein kinases as candidate regulators of UCPS. Our preliminary studies also suggest significant heterogeneity in the regulation of UCPS across cell types, with PIK3C2A selectively influencing IL-1β secretion in non-pyroptotic cells.