Holmstroem 2014 Abstract MiP2014
|Homozygous missense mutation in the human NARS2 gene results in reduced homodimerization of mitochondrial AsnRS enzyme in a patient with Alpers syndrome.|
Mitochondrial function in energy production and biosynthesis is dependent on coordination of nuclear and mitochondrial gene expression. Defects in either genome can result in a wide spectrum of disease phenotypes. Alpers syndrome is a progressive, neurodegenerative disorder that is characterized by psychomotor regression, seizures and diffuse degeneration of cerebral grey matter . Although the genetic etiology is unknown in most patients, several mutations targeting components of mtDNA maintenance and expression have been described . Using whole-exome sequencing, a patient with Alpers syndrome was found to have a homozygous missense mutation in the asparaginyl-tRNA synthetase gene, encoding mitochondrial asparagine-tRNA ligase (AsnRS) . AsnRS is a class II aminoacyl tRNA-synthetase whose function is poorly characterized. By similarity to other class II enzymes, it is predicted to function as a homodimer. The crystal structure of the P. horikoshii  AsnRS homolog and in silico modeling of AsnRS suggest that the affected Pro214 residue is located at the base of a loop that protrudes out from the main body of the folded protein, suggesting it may participate in monomer-monomer interaction. In this study, we aimed to characterize the AsnRS p.P214L mutation in fibroblasts derived from the patient.
Mitochondrial polarography, performed previously on isolated mitochondria from skeletal muscle, revealed decreased oxygen consumption in the presence of Complex I and IV substrates as well as decreased respiratory enzyme activities. Protein abundance, measured by Western blot on fibroblast lysates, of a marker of Complex II was unaltered, whereas markers of Complex IV (COX IV – nuclear gene, and MT-CO1 – mitochondrial) were both increased in the patient compared to the control. Abundance of AsnRS protein was reduced by 63% in the patient, suggesting altered regulation of protein stability. Finally, preliminary results from size exclusion gel filtration under non-denaturing conditions indicated a shift in motility of AsnRS in the patient, suggestive of reduced dimerization.
In combination with previous clinical data, our results show that the AsnRS p.P214L mutation found in the patient is correlated with altered mitochondrial protein expression and regulation. Awaiting confirmation of our results, we hope to demonstrate that AsnRS indeed exists as a homodimer in the mitochondria. Future experiments should address the mechanistic link between disturbed dimerization and tRNA aminoacylation.
Labels: MiParea: mtDNA;mt-genetics, mt-Medicine, Patients Pathology: Neurodegenerative
Organism: Human Tissue;cell: Fibroblast Preparation: Isolated mitochondria Enzyme: Complex II;succinate dehydrogenase, Complex IV;cytochrome c oxidase
Event: A1, Oral MiP2014: RCE
1-Dep Clinical Chem Transfusion Medicine, Sahlgrenska Univ Hospital; 2-Dep Medical Biochem Cell Biol; Gothenburg Univ, Sweden. - [email protected]
References and acknowledgements
Supported by LUA/ALF grant from Sahlgrenska University Hospital.
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