Martin L. Duennwald

Aminoacyl-tRNA synthetases are essential enzymes responsible for charging amino acids onto cognate tRNAs during protein synthesis. In histidyl-tRNA synthetase (HARS), autosomal dominant mutations V133F, V155G, Y330C and S356N in the HARS catalytic domain cause Charcot–Marie–Tooth disease type 2 W (CMT2W), while tRNA-binding domain mutation Y454S causes recessive Usher syndrome type IIIB. In a yeast model, all human HARS variants complemented a genomic deletion of the yeast ortholog HTS1 at high expression levels. CMT2W associated mutations, but not Y454S, resulted in reduced growth. We show mistranslation of histidine to glutamine and threonine in V155G and S356N but not Y330C mutants in yeast. Mistranslating V155G and S356N mutants lead to accumulation of insoluble proteins, which was rescued by histidine. Mutants V133F and Y330C showed the most significant growth defect and decreased HARS abundance in cells. Here, histidine supplementation led to insoluble protein aggregation and further reduced viability, indicating histidine toxicity associated with these mutants. V133F proteins displayed reduced thermal stability in vitro, which was rescued by tRNA. Our data will inform future treatment options for HARS patients, where histidine supplementation may either have a toxic or compensating effect depending on the nature of the causative HARS variant.

Nrf2 is the master transcriptional regulator of cellular responses against oxidative stress. It is chiefly regulated by Keap1, a substrate adaptor protein that mediates Nrf2 degradation. Nrf2 activity is also influenced by many other protein interactions that provide Keap1-independent regulation. To study Nrf2 regulation, we established and characterized yeast models expressing human Nrf2 (also known as NFE2L2), Keap1 and other proteins that interact with and regulate Nrf2. Yeast models have been well established as powerful tools to study protein function and genetic and physical protein-protein interactions. In this work, we recapitulate previously described Nrf2 interactions in yeast and discover that Nrf2 interacts with the molecular chaperone Hsp90. Our work establishes yeast as a useful tool to study Nrf2 interactions and provides new insight into the crosstalk between the antioxidant response and the heat shock response.