Investigating protein phase separation and aggregation in age dependent human disease

PROJECT SUMMARY Our overarching goal is to determine whether a newly discovered form of macromolecular organization – protein phase-separation – contributes to the age dependency of human diseases. Recently, it has been appreciated that many proteins related to age-dependent disease have the capacity to self-associate, condensing to form separate ‘phase-separated’ compartments called biomolecular condensates. In vitro, some of these protein condensates can mature over time into toxic aggregates. However, the generality of this property is unclear, as is the degree to which it may be altered for mutant proteins associated with different ages of disease onset and progression. Our proposal leverages comprehensive libraries of human disease alleles along with biochemistry, cell biology, and physiological modeling to investigate and define the contribution of protein phase separation, and aggregation, to the age dependency of human disease processes. Many human diseases involve defects in protein folding that can be amplified with age-dependent declines in protein quality control. Moreover, many disease alleles arise within intrinsically disordered regions of proteins, which often control phase separation behaviors. Yet whether disease alleles commonly create imbalances in protein phase separation and aggregation processes is unclear. Taking advantage of comprehensive libraries of mutant proteins associated with diverse human diseases, we recently investigated the phase-separation processes of more than two hundred disease-associated proteins. These data revealed a strong enrichment for phase separation properties among proteins with later ages of disease onset. In targeted follow-up experiments we also validated that disease variants in those proteins had strong effects on their phase separation and aggregation behaviors. To define the relationships between protein phase separation and the age dependency of human disease we will conduct the following experiments: 1. Define phase separation properties of proteins driving diverse human diseases; 2. Characterize aggregation and prion-like ‘seeding’ associated with human disease alleles; 3. Identify protein- autonomous and age-related systemic drivers of altered phase separation in disease. We will also investigate whether protein phase separation provides a mechanism to understand and potentially block the impact of dominant disease alleles in the context of aging. Together, our proposal will define biophysical and systemic features driving phase separation and aggregation, a critical aspect of many age-related disease pathologies.