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Mitochondrial Assembly Disorders

Final Activity Report Summary - MAD (Mitochondrial Assembly Disorders)

Mitochondria are the main source of energy in eukaryotic cells. ATP is generated by means of the oxidative phosphorylation (OXPHOS) system located in the mitochondrial inner membrane. The OXPHOS system is composed of four large multimeric respiratory chain complexes, which are responsible for electron transport and generation of the proton gradient in the mitochondrial intermembrane space, and of the ATP synthase that uses this proton gradient to produce ATP. The assembly of the OXPHOS system in the mitochondrial inner membrane is an intricate process in which many factors must interact. The respiratory complexes are composed of mitochondrially- and nuclearly-encoded structural subunits and some proteins acting as assembly factors/chaperones are needed for the correct gathering of the different subunits to give rise to the functional complex. Some human disorders are caused by dysfunction of the OXPHOS system, and many of them are associated with altered assembly of one or more components of the OXPHOS system. The study of assembly defects in patients and in animal models has been useful in unraveling and/or gaining a complete understanding of the processes by which these large multimeric complexes are formed.

Assembly defects of mitochondrial respiratory chain complex III (CIII) and complex IV (CIV or COX) were studied in human samples derived from patients carrying mutations in the BCS1L gene, encoding a protein necessary for CIII biogenesis, and in the COX6B1 gene, that encodes a small COX structural subunit. Furthermore, a COX15-deficient animal model to study the consequences of the lack of this CIV assembly factor was generated in the laboratory. Also, the association of CIV with other OXPHOS complexes into super-complexes in the SURF1 knock-out mouse (a mitochondrial CIV deficiency model) was analysed.%:%: Two patients showing mitochondrial CIII deficiency and isolated mitochondrial encephalopathy were found to carry pathological mutations in the BCS1L gene that encodes a protein known to be essential for the correct formation of CIII. Several mutations in this gene were found to be associated with different clinical presentations as the GRACILE syndrome, (growth retardation, aminoaciduria, cholestasis, iron overload, lactic acidosis, and early death), complex III deficiency associated with congenital metabolic acidosis, neonatal proximal tubulopathy, liver failure, and encephalopathy or the Björnstad syndrome (sensorineural hearing loss and pili torti). With the work developed in this project, the defect in CIII assembly was characterized at the molecular level. By studying patient-derived skeletal muscle biopsies and cultured skin fibroblasts and lymphoblasts, it was determined that the role of the BCS1L protein is to insert the catalytic subunit UQCRFS1 (Rieske Fe-S protein) into the 'pre-complex III' as one of the last steps of the CIII assembly process. The catalytically inactive 'pre-complexIII' was found to be accumulated in the patient samples and the lack of insertion of the Rieske protein made the complex become unstable, as sub-complexes containing CIII subunits were present in the patients and absent in the controls. These studies also revealed that the BCS1L protein was part of a large protein complex in which CIII subunits were absent.

Two siblings showing mitochondrial CIV deficiency and a severe encephalomyopathy were studied. Mutational screening in the mitochondrial DNA-encoded structural subunits and in the nucleus-encoded CIV assembly factors was negative. Mutations were searched for in nucleus-encoded structural subunits and a homozygous mutation was found in the COX6B1 gene. The pathogeneicity of the amino acid change and the CIV structural abnormality associated to it was thoroughly demonstrated. In many occasions, mutations in nucleus-encoded COX subunits were sought for but never found in CIV-defective patients, leading to the conjecture that they may be incompatible with extra-uterine survival. This work represents a new and relevant finding as this was the first time that a mutation in a nuclear encoded COX subunit (COX6B1) was associated to mitochondrial disease and it indicates that these proteins should be reconsidered as possible candidates in COX-deficiency associated disorders.