Functional Analysis of Novel Variants Located in the Tetramerization Loop of ACAT1.

Patients with mitochondrial acetoacetyl-CoA thiolase (T2) deficiency, a rare inborn error of ketone body metabolism, sometimes experience severe ketoacidosis crises. The etiology of T2 deficiency is related to pathogenic variants of ACAT1. Typically, T2 homodimers form homotetramers through dimeric polymerization. Although the "tetramerization loop" comprising amino acid residues 160-177 is expected to be crucial for the formation and stabilization of tetramers, no functional analysis of this loop has been reported. We identified a novel homozygous ACAT1 c.508T>C (p.Y170H) variant in siblings with recurrent ketoacidosis, and further investigated the previously reported but not functionally analyzed heterozygous variant c.481T>C (p.Y161H), both of which are located in the T2 tetramerization loop. We evaluated T2 enzymatic activities and protein expression levels in patient-derived dermal fibroblasts expressing Y170H and in dermal fibroblasts with transient expression of variants Y161H and Y170H cultured at 37°C and 40°C. A silkworm expression system was used to confirm T2 enzymatic activity and evaluate the protein-stoichiometry of the two variants, which showed decreased enzymatic activity and protein stability. Native-polyacrylamide gel electrophoresis immunoblotting revealed structural differences between the wild-type and variant T2 proteins. Tetramerization loop variants Y161H and Y170H were found to be temperature sensitive, with compromised protein stability and obstruction of homotetramer formation. This study provides new insights into the functional importance of the T2 tetramerization loop in patients with T2 deficiency.