This work describes the analysis of a novel, isolated, autosomal dominant form of Fanconi´s syndrome, a disorder of the renal proximal tubule associated with decreased reapsorption of solutes from the primary urine. This yet unknown Fanconi´s syndrome is evoked by a mutation in the third codon of the peroxisomal protein enoyl-CoA hydratase / L-3-hydroxyacyl-CoA dehydrogenase (EHHADH), also called "Fanconi-associated protein", which results in the substitution of a glutamic acid residue with

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... ne (p.E3K). By complementing proteomic and metabolomic analyses of wildtype- and mutant-EHHADH-expressing proximal tubular cell lines (LLC-PK1) with different biochemical and cell biological investigations, the underlying pathomechanism is elucidated. The E3K-mutation leads to the erroneous localization of peroxisomal EHHADH into mitochondria causing a mitochondriopathy. Upon mistargeting of EHHADHMUT into mitochondria, it replaces an alpha subunit of the mitochondrial trifunctional protein (MTP). The MTP normally builds a heterooctamer consisting of four alpha and four beta subunits and is involved in mitochondrial fatty acid β-oxidation. The incorporation into MTP impairs both mitochondrial β-oxidation and respiratory supercomplex assembly, leading to a decreased oxidative phosphorylation capacity. Impairment of the former is shown by the characteristic accumulation of hydroxyacyl-, enoyl- and acylcarnitines in the cell culture supernatant, thus resembling the situation in patients with MTP and/or LCHAD deficiency. The impaired mitochondrial β-oxidation consequently decreases cellular long-chain fatty acid uptake and the acetyl-CoA production in EHHADHMUT cell line. In addition, EHHADHMUT is also incorporated into respiratory supercomplexes, thereby disturbing their assembly, as shown by blue native PAGE. As a result of impaired mitochondrial β-oxidation and diminished supercomplex assembly the EHHADHMUT cell line shows a decreased oxidative phosphorylation capacity and reduced ATP generation. This mitochondriopathy resu [...]