Cellular handling of protein aggregation gatekeepers

Amyloid-like protein aggregation is a hallmark of more than 30 human pathologies, but also occurs as a functional protein assembly mechanism in all kingdoms of life. The process is mediated by short aggregation-prone regions (APRs) of the polypeptide chain that form intermolecular b-sheet structures. Aggregation gatekeepers (GKs) are the evolutionary countermeasure that reduces unwanted aggregation of APRs: these residues that flank almost all APRs in the human proteome reduce the aggregation rate of the APR. Our lab discovered GKs as a novel functional and evolutionarily enriched class. We have demonstrated that substrate recognition of several chaperones is tuned towards GK chemistry and that GKs affect protein synthesis, aggregation and degradation. With the current project we want to assess the role of the cellular machinery on protein homeostasis regulation by the GKs. We will develop a high-content imaging assay in mammalian cells that allows to quantify expression level, aggregation status and inclusion body formation of aggregation-prone model constructs, containing different GKs. The assay will be adapted to high-throughput screening format and we will use it to perform a functional genomics screen to obtain an unbiased identification of cellular factors that recognize APRs and GKs. This project will reveal new elements of protein homeostasis regulation and unravel the role of the aggregation gatekeepers in this process.