LAUSR

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 mutant ETF:QO proteins were unstable and easily degraded in patient fibroblasts and in vitro expression systems by ubiquitin‐proteasome pathway, and identified the specific ubiquitin E3 ligase as CHIP, which forms complex to control mutant ETF:QO degradation through poly‐ubiquitination. CHIP‐dependent degradation of mutant ETF:QO proteins was confirmed by MS and site‐directed mutagenesis of ubiquitination sites. Hsp70 is directly involved in this process as molecular chaperone of CHIP. CHIP plays an important role in ubiquitin‐proteasome pathway dependent degradation of mutant ETF:QO by working as a chaperone‐assisted E3 ligase, which reveals CHIP's potential role in pathological mechanisms of late‐onset MADD.