Systematic analysis of a mitochondrial disease-causing ND6 mutation in mitochondrial deficiency.

BACKGROUND: The m.14487T>C mutation is recognized as a diagnostic mutation of mitochondrial disease during the past 16 years, emerging evidence suggests that mutant loads of m.14487T>C and disease phenotype are not closely correlated. METHODS: Immortalized lymphocytes were generated by coculturing the Epstein-Barr virus and lymphocytes from m.14487T>C carrier Chinese patient with Leigh syndrome. Fifteen cytoplasmic hybrid (cybrid) cell lines were generated by fusing mtDNA lacking 143B cells with platelets donated by patients. Mitochondrial function was systematically analyzed at transcriptomic, metabolomic, and biochemical levels. RESULTS: Unlike previous reports, we found that the assembly of mitochondrial respiratory chain complexes, mitochondrial respiration, and mitochondrial OXPHOS function was barely affected in cybrid cells carrying homoplastic m.14487T>C mutation. Mitochondrial dysfunction associated transcriptomic and metabolomic reprogramming were not detected in cybrid carrying homoplastic m.14487T>C. However, we found that mitochondrial function was impaired in patient-derived immortalized lymphocytes. CONCLUSION: Our data revealed that m.14487T>C mutation is insufficient to cause mitochondrial deficiency; additional modifier genes may be involved in m.14487T>C-associated mitochondrial disease. Our results further demonstrated that a caution should be taken by solely use of m.14487T>C mutation for molecular diagnosis of mitochondrial disease.

Expression of soluble native protein in Escherichia coli using a cold-shock SUMO tag-fused expression vector.

At present, approximately 30% of eukaryotic proteins can be expressed in a soluble form in Escherichia coli. In this study, a pCold-SUMOa plasmid was constructed in order to express heterologous proteins fused with SUMO by a cold-shock expression vector. The human cysteine desulfurase NFS1 and a chimeric cysteine desulfurase namely, EH-IscS were successfully expressed in E. coli. The proteins were particularly difficult to be produced functionally, due to their readily sequestered nature. The recombinant cysteine desulfurases that were generated by pCold-SUMOa exhibited higher activity, solubility and stability compared with the well-known plasmid pCold I. In contrast to the pCold TF plasmid, the SUMO tag conferred no biological activity with regard to the conformation of the cysteine desulfurases. Furthermore, the SUMO protease 1 can efficiently recognize the tertiary structure of SUMO and cleave it. The data indicate that the pCold-SUMOa vector is a promising tool for native eukaryotic protein production.