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

J Inherit Metab Dis. 2021 Mar;44(2):450-468. doi: 10.1002/jimd.12361. Epub 2021 Jan 25.

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 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.

Keywords: CHIP; ETF:QO; late-onset multiple acyl-CoA dehydrogenase deficiency; protein degradation; ubiquitin-proteasome pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Adult
  • Child
  • Electron-Transferring Flavoproteins / genetics
  • Electron-Transferring Flavoproteins / metabolism*
  • Female
  • HSP70 Heat-Shock Proteins / metabolism
  • Humans
  • Iron-Sulfur Proteins / genetics
  • Iron-Sulfur Proteins / metabolism*
  • Male
  • Mitochondria / metabolism
  • Multiple Acyl Coenzyme A Dehydrogenase Deficiency / genetics*
  • Mutation / genetics*
  • Oxidoreductases Acting on CH-NH Group Donors / genetics
  • Oxidoreductases Acting on CH-NH Group Donors / metabolism*
  • Riboflavin / metabolism
  • Ubiquinone / metabolism
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism*
  • Young Adult

Substances

  • Electron-Transferring Flavoproteins
  • HSP70 Heat-Shock Proteins
  • Iron-Sulfur Proteins
  • Ubiquinone
  • Oxidoreductases Acting on CH-NH Group Donors
  • electron-transferring-flavoprotein dehydrogenase
  • STUB1 protein, human
  • Ubiquitin-Protein Ligases
  • Riboflavin