Investigating the role of protein stability on eEF1A2 function

Abstract

Spontaneous missense mutations, whereby a single nucleotide results in amino acid substitution, affect the normal functioning of the eukaryotic translation elongation factor eEF1A2. These mutations have varying disease severity causing neurodevelopmental disorders in children, including autism, intellectual disability, and epilepsy. Mutations D252H and E122K, located in binding hotspots of eEF1A2, have differing eEF1A2 expression levels across different tissues in mouse models. This thesis aims to compare the effect of D252H and E122K mutations on eEF1A2 protein stability and how differences in disease severity and tissue-specific expression arise by investigating eEF1A2 expression and changes in the interactome resulting from mutations. Molecular biology techniques, such as western blotting, conducted on mouse tissue and mutant transfected HEK293T cells were conducted to determine the expression levels of D252H.eEF1A2 and E122K.eEF1A2 mutants compared to wildtype. Moreover, co-immunoprecipitation and mass spectrometry was conducted to identify and compare the molecular binding partners of eEF1A2 mutants and revealed that proteins involved in translation were downregulated in D252H and E122K eEF1A2 mutants.

Mass spectrometry was also performed in mouse brain tissue to conduct a differential expression analysis of the brain proteome, which found translation and ribosomal proteins’ expression was significantly affected in E122K mutants.

In conclusion, D252H and E122K mutations result in differing expression patterns of eEF1A2 across mouse tissues and an altered eEF1A2 interactome.

The E122K.eEF1A2 mouse brain proteomic analysis suggested reduced translation efficiency is one of the major biological processes affected. Therefore, mutations could be targeted by identifying and grouping altered proteins to their common functions and molecular pathways.