PROJECT SUMMARY. Secreted proteins and polypeptides mediate fundamental axes of cell and tissue
crossltalk. In recent years, there has been a renewed interest in profiling the entire collection of secreted proteins
and proteins (e.g., the secretome) from cells and organs in vivo. To address this challenge, we have recently
described a high-sensitivity proximity labeling methodology using adeno-associated viruses (AAVs) to delivery
the proximity labeling enzyme TurboID into distinct subcellular compartments for cell type-specific secretome
profiling in vivo. Our methodology provides a direct biochemical readout of secretome composition and dynamics
in a cell type-specific manner, directly in a living animal. Here, we will generate and validate a suite of conditional
mouse models based on this secretome profiling methodology. This application and our proposed suite of
conditional secretome labeling mice directly responds to the Funding Opportunity Announcement PAR-19-369
which encourages the development of improved animal models and novel technologies to better understand
health and disease. These conditional mice are important to develop because many cell types still cannot be
transduced efficiently or quantitatively with AAVs, or are rapidly turned over such that transient AAV infection
does not result in stable labeling of those cellular populations. In Specific Aim 1, we will generate three
conditional secretome labeling mouse lines with TurboID localized to the secretory pathway, cytosol, or
membrane and use shotgun proteomics to benchmark their performance relative to our first-generation viral
reagents. In Specific Aim 2, we will use these conditional mice to address a fundamental and well-defined
question that had previously remained inaccessible experimentally: to what extent are the levels of secreted
proteins determined transcriptionally in vitro? Successful completion of this proposal will provide investigators
with a conditional animal model to answer previously inaccessible and fundamental questions regarding the
identity of secreted protein and polypeptide factors mediating cell and tissue crosstalk in their experimental
system of interest.