Mutational scanning pinpoints distinct binding sites of key ATGL regulators in lipolysis.

ATGL is a key enzyme in intracellular lipolysis and plays an important role in metabolic and cardiovascular diseases. ATGL is tightly regulated by a known set of protein-protein interaction partners with activating or inhibiting functions in the control of lipolysis. Here, we use deep mutational protein interaction perturbation scanning and generate comprehensive profiles of single amino acid variants that affect the interactions of ATGL with its regulatory partners: CGI-58, G0S2, PLIN1, PLIN5 and CIDEC. Twenty-three ATGL amino acid variants yield a specific interaction perturbation pattern when validated in co-immunoprecipitation experiments in mammalian cells. We identify and characterize eleven highly selective ATGL switch mutations which affect the interaction of one of the five partners without affecting the others. Switch mutations thus provide distinct interaction determinants for ATGL's key regulatory proteins at an amino acid resolution. When we test triglyceride hydrolase activity in vitro and lipolysis in cells, the activity patterns of the ATGL switch variants trace to their protein interaction profile. In the context of structural data, the integration of variant binding and activity profiles provides insights into the regulation of lipolysis and the impact of mutations in human disease.

Exploring absent protein function in yeast: assaying post translational modification and human genetic variation.

Yeast is a valuable eukaryotic model organism that has evolved many processes conserved up to humans, yet many protein functions, including certain DNA and protein modifications, are absent. It is this absence of protein function that is fundamental to approaches using yeast as an in vivo test system to investigate human proteins. Functionality of the heterologous expressed proteins is connected to a quantitative, selectable phenotype, enabling the systematic analyses of mechanisms and specificity of DNA modification, post-translational protein modifications as well as the impact of annotated cancer mutations and coding variation on protein activity and interaction. Through continuous improvements of yeast screening systems, this is increasingly carried out on a global scale using deep mutational scanning approaches. Here we discuss the applicability of yeast systems to investigate absent human protein function with a specific focus on the impact of protein variation on protein-protein interaction modulation.