CHIP control degradation of mutant ETF:QO through ubiquitylation in late‐onset multiple acyl‐CoA dehydrogenase deficiency

Abstract

Late-onset multiple acyl-CoA dehydrogenase deficiency (MADD) is the most common form of lipid storage myopathy. The disease is mainly caused by mutations in electron-transfer flavoprotein dehydrogenase gene (ETFDH), which leads to decreased levels of ETF:QO in skeletal muscle. However, the specific underlying mechanisms triggering such degradation remain unknown. We constructed expression plasmids containing wild type ETF:QO and mutants ETF:QO-A84T, R175H, A215T, Y333C, and cultured patient-derived fibroblasts containing the following mutations in ETFDH: c.250G>A (p.A84T), c.998A>G (p.Y333C), c.770A>G (p.Y257C), c.1254_1257delAACT (p. L418TfsX10), c.524G>A (p.R175H), c.380T>A (p.L127P), and c.892C>T (p. P298S). We used in vitro expression systems and patient-derived fibroblasts to detect stability of ETF:QO mutants then evaluated their interaction with Hsp70 interacting protein CHIP with active/inactive ubiquitin E3 ligase carboxyl terminus using western blot and immunofluorescence staining. This interaction was confirmed in vitro and in vivo by co-immunoprecipitation and immunofluorescence staining. We confirmed the existence two ubiquitination sites in mutant ETF:QO using mass spectrometry (MS) analysis. We found that Xin-Yi Liu, Xue-Jiao Chen, Miao Zhao, and Zhi-qiang Wang have equally contributed to the work.

Clinical Presentations and Genetic Characteristics of Late-Onset MADD Due to ETFDH Mutations in Five Patients: A Case Series

“…ETF-QO protein, a component of the mitochondrial respiratory chain, is responsible for transferring electrons from ETF in the mitochondrial matrix to the ubiquinone pool located in the inner mitochondrial membrane ( 26 , 27 ), which is critical for fatty acid β-oxidation (FAO). However, RR-MADD–associated mutant ETF-QO, a protein with a gentle structural defect that partly but not wholly disrupts FAD-binding to flavoprotein ( 1 , 2 ), was unstable and easily degraded by the ubiquitin-proteasome pathway ( 28 ). Therefore, the impaired FAO resulted from ETFDH mutations impacts energy supply, resulting in lipid accumulation in muscle and induces an increase of its upstream substrates (short-, medium-, and long-chain acylcarnitine) and a decrease in free carnitine, which binds to acylcarnitine.…”

New insights into the nutritional genomics of adult-onset riboflavin-responsive diseases

“…In line with this proposal, increasing riboflavin intake would lead to increased intracellular FAD concentrations that would shift the equilibrium toward FAD binding to the apo-enzyme thereby stabilizing tertiary protein structure. In some cases, the ability of increased concentrations of riboflavin to restore flavoprotein stability has been directly observed in vitro in cells derived from individuals expressing ETFDH, FLAD1, ACAD9 and MTHFR variants that exhibit riboflavin-responsive phenotypes [ 54 , 60 , 90 , 91 ]. In addition, supplementation of FAD to MTHFR and EFT:QO in vitro has been shown to help rescue enzyme stability and activity following incubation at 46 °C and 40 °C respectively, particularly in variants known to be heat sensitive [ 54 , 72 ].…”