🇬🇧 Proteostasis analyzed through deep mutational scanning

Abstract

More than half of disease-causing missense variants are thought to lead to protein degradation, but the molecular mechanism of how these variants are recognized by the cell remains enigmatic. To approach this issue we have applied deep mutational scanning experiments to test the degradation of thousands of missense protein variants in large multiplexed experiments in cultured human cells. We use a model protein where known missense variants result in an autosomal recessive neurodegenerative disorder. The resulting mutational map comprises >99% of all possible single-amino-acid substitution and nonsense variants.

With a few notable exceptions, the majority of the destabilizing mutations are located within the structured domains of the protein, while flexible and disordered linker regions are more tolerant to mutations. The cellular abundance data correlate with the protein’s structural stability, evolutionary conservation, and separates known disease-linked variants from harmless variants. Systematic mapping of degradation signals (degrons) shows that inherent primary degrons in the protein largely overlap with regions that are highly sensitive to mutations. The vast majority of low abundant variants are degraded through the ubiquitin-proteasome system and are stabilized at lowered temperatures. In conclusion, in addition to providing a diagnostic tool for rare genetic disorders, deep mutational scanning technologies have the potential to reveal both protein specific and general information on the specificity of the protein quality control network and the ubiquitin-proteasome system. Examples of this will be presented.