Protein quality control systems maintain a functional proteome through detection and removal of abnormal proteins. While typically only misfolded or damaged molecules are thought of as abnormal, recent work has revealed that also mislocalized proteins are subject to quality control. Mislocalized proteins are defined as proteins that fail to reach their native compartment or fail to assemble into their native complex, and thus cannot function normally. Protein mislocalization is a constitutive problem caused by inefficiencies of cellular processes and increases with aging. Proteins can also mislocalize due to mutations, as seen in various metabolic, cardiovascular and neurodegenerative diseases, and some types of cancer. Despite the ubiquity of protein mislocalization, the systems performing quality control of mislocalized proteins are unknown for most of the proteome because quality control substrates are usually rare, thus difficult to identify, and there is considerable redundancy built into quality control systems. Here, I propose to systematically dissect quality control mechanisms of mislocalized proteins through a combination of molecular biology, genetics, biochemistry and computational biology in yeast and human cells. We will establish a platform for conditional protein mislocalization and apply it (i) to identify quality control substrates proteome-wide, (ii) to dissect redundancies in quality control systems, (iii) to identify the machinery responsible for quality control of mislocalized proteins and (iv) to map the features involved in substrate recognition by the quality control machinery. Finally, we will exploit our findings to selectively target aneuploid cancer cells, which exhibit a high burden of mislocalized proteins. This work will provide a comprehensive picture of quality control systems for mislocalized proteins and shed light on their roles under both normal and perturbed conditions.

The mammalian SWI/SNF chromatin remodellers, the BAF complexes, are critical regulators of gene expression by modulating the accessibility of regulatory regions, especially of cell identity genes. Their importance for cellular maintenance and differentiation is emphasised by the fact that they are frequently associated with disease. Mutations in genes encoding BAF subunits are found in over 20% of cancers and are causative for neurodevelopmental disorders (NDD). The prevalence for specific NDD with unique clinical features depends on the mutant subunit. The molecular changes leading to the disease phenotypes are largely unresolved. The functions of BAF complexes and specific subunits during human brain development are also still unclear. SWItchFate thus aims to systematically identify the role of individual BAF subunits and their mutations in brain development and abnormalities. To this end, isogenic wild type, mutant and engineered human induced pluripotent stem cell (hiPSC)-derived cerebral organoids will be used in combination with various bulk and single-cell epigenomics and proteomics tools. SWItchFate will investigate gene regulatory mechanisms altered during brain development with CRISPR/Cas-based loss-of-function screens for all BAF subunits. Using protein degradation tools targeting specific BAF subunits, SWItchFate will pinpoint vulnerable processes and adaptation mechanisms. In addition, cell type- and BAF subtype-specific composition, interaction partners and target sites along brain development will be mapped to decipher BAF-dependent gene regulatory networks. Finally, the molecular changes in BAF mutation-induced NDD that cause the phenotypic changes in patients will be examined and conserved mechanisms across different genotypes will be deciphered using patient-derived hiPSC. Thus, SWItchFate will decode the regulatory functions of BAF complexes in the context of cell fate decisions in development and disease, paving the way for new therapeutics.