Mutation in subdomain G' of mitochondrial elongation factor G1 is associated with combined OXPHOS deficiency in fibroblasts but not in muscle - PubMed (original) (raw)

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Mutation in subdomain G' of mitochondrial elongation factor G1 is associated with combined OXPHOS deficiency in fibroblasts but not in muscle

Paulien Smits et al. Eur J Hum Genet. 2011 Mar.

Abstract

The mitochondrial translation system is responsible for the synthesis of 13 proteins required for oxidative phosphorylation (OXPHOS), the major energy-generating process of our cells. Mitochondrial translation is controlled by various nuclear encoded proteins. In 27 patients with combined OXPHOS deficiencies, in whom complex II (the only complex that is entirely encoded by the nuclear DNA) showed normal activities, and mutations in the mitochondrial genome as well as polymerase gamma were excluded, we screened all mitochondrial translation factors for mutations. Here, we report a mutation in mitochondrial elongation factor G1 (GFM1) in a patient affected by severe, rapidly progressive mitochondrial encephalopathy. This mutation is predicted to result in an Arg250Trp substitution in subdomain G' of the elongation factor G1 protein and is presumed to hamper ribosome-dependent GTP hydrolysis. Strikingly, the decrease in enzyme activities of complex I, III and IV detected in patient fibroblasts was not found in muscle tissue. The OXPHOS system defects and the impairment in mitochondrial translation in fibroblasts were rescued by overexpressing wild-type GFM1, establishing the GFM1 defect as the cause of the fatal mitochondrial disease. Furthermore, this study evinces the importance of a thorough diagnostic biochemical analysis of both muscle tissue and fibroblasts in patients suspected to suffer from a mitochondrial disorder, as enzyme deficiencies can be selectively expressed.

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Figures

Figure 1

Mutational analysis of GFM1. (a) DNA sequence chromatogram from the patient (homozygous c.748C>T) and both parents (heterozygous). (b) EFG1 protein structure with the functional domains showing the predicted Arg250Trp substitution in the G' subdomain of domain I. (c) Protein sequence alignment from humans to bacteria. The alignment shows that mutated Arg250Trp is highly conserved among different species. (d) Immunoblot analysis in patient (P) and control (C) fibroblasts reveals the absence of the EFG1 protein. The 70 kDa subunit of complex II (CII) was used as the loading control.

Figure 2

Computational modeling based on the crystal structure of the T. thermophilus EFG protein. The left panel shows the structure of the entire human EFG1 protein and the right panel is a close-up of the outlined region containing the mutation. The wild-type Arg 250 residue is depicted in green and the mutant Trp is depicted in red (bound GDP is depicted in yellow). The right panel shows that the Trp residue fits in the structure; however, the hydrogen bond that Arg forms with Glu 153 is lost.

Figure 3

: Rescue of the mitochondrial translation defect and combined OXPHOS deficiency in patient fibroblasts by expression of GFM1. Control (C) and patient (P) fibroblasts were transduced with a retroviral vector expressing the wild-type GFM1 cDNA and analyzed by a mitochondrial translation assay (a) and BN-PAGE (b). The mitochondrial translation products are indicated on the left: seven subunits of complex I (ND), one subunit of complex III (cyt b), three subunits of complex IV (COX) and two subunits of complex V (ATP). Co I – Co V, complex I to V.

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Mutation in subdomain G' of mitochondrial elongation factor G1 is associated with combined OXPHOS deficiency in fibroblasts but not in muscle - PubMed (original) (raw)