Biophysical characterization Of Alpers encephalopathy associated mutants of human mitochondrial phenylalanyl-tRNA synthetase.

Mutations in nucleus-encoded mitochondrial aminoacyl-tRNA synthetases (mitaaRSs) lead to defects in mitochondrial translation affecting the expression and function of 13 subunits of the respiratory chain complex leading to diverse pathological conditions. Mutations in the FARS2 gene encoding human mitochondrial phenylalanyl-tRNA synthetase (HsmitPheRS) have been found to be associated with two different clinical representations, infantile Alpers encephalopathy and spastic paraplegia. Here we have studied three pathogenic mutants (Tyr144Cys, Ile329Thr, and Asp391Val) associated with Alpers encephalopathy to understand how these variants affect the biophysical properties of the enzyme. These mutants have already been reported to have reduced aminoacylation activity. Our study established that the mutants are significantly more thermolabile compared to the wild-type enzyme with reduced solubility in vitro. The presence of aggregation-prone insoluble HsmitPheRS variants could have a detrimental impact on organellar translation, and potentially impact normal mitochondrial function. © 2019 IUBMB Life, 71(8): 1141-1149, 2019 © 2019 IUBMB Life, 71(8):1141-1149, 2019.

Reversible inactivation of yeast mitochondrial phenylalanyl-tRNA synthetase under oxidative stress.

BACKGROUND: Under oxidative stress cytoplasmic aminoacyl-tRNA synthetase (aaRSs) substrate specificity can be compromised, leading to tRNA mischarging and mistranslation of the proteome. Whether similar processes occur in mitochondria, which are major cellular sources of reactive oxygen species (ROS), is unknown. However, relaxed substrate specificity in yeast mitochondrial phenylalanyl-tRNA synthetase (ScmitPheRS) has been reported to increase tRNA mischarging and blocks mitochondrial biogenesis. METHODS: Non-reducing denaturing PAGE, cysteine reactivity studies, MALDI-TOF mass spectrometry, enzyme assay, western blot, growth assay, circular dichroism, dynamic light scattering and fluorescence spectroscopy were used to study the effect of oxidative stress on ScmitPheRS activity. RESULTS: ScmitPheRS is reversibly inactivated under oxidative stress. The targets for oxidative inactivation are two conserved cysteine residues resulting in reversible intra-molecular disulfide bridge formation. Replacement of either conserved cysteine residue increased viability during growth under oxidative stress. CONCLUSION: Formation of intra-molecular disulfide bridge under oxidative stress hinders the tRNAPhe binding of the enzyme, thus inactivating ScmitPheRS reversibly. GENERAL SIGNIFICANCE: The ScmitPheRS activity is compromised under oxidative stress due to formation of intra-molecular disulfide bridge. The sensitivity of ScmitPheRS to oxidation may provide a protective mechanism against error-prone translation under oxidative stress.

p-Benzoquinone-induced aggregation and perturbation of structure and chaperone function of α-crystallin is a causative factor of cigarette smoke-related cataractogenesis.

Cigarette smoking is a significant risk factor for cataract. However, the mechanism by which cigarette smoke (CS) causes cataract remains poorly understood. We had earlier shown that in CS-exposed guinea pig, p-benzoquinone (p-BQ) derived from CS in the lungs is carried by the circulatory system to distant organs and induces various smoke-related pathogeneses. Here, we observed that CS exposure caused accumulation of the p-BQ-protein adduct in the eye lens of guinea pigs. We also observed accumulation of the p-BQ-protein adduct in resected lens from human smokers with cataract. No such accumulation was observed in the lens of never smokers. p-BQ is a strong arylating agent that forms Michael adducts with serum albumin and haemoglobin resulting in alterations of structure and function. A major protein in the mammalian eye lens is αA-crystallin, which is a potent molecular chaperone. αA-crystallin plays a key role in maintaining the integrity and transparency of the lens. SDS-PAGE indicated that p-BQ induced aggregation of αA-crystallin. Various biophysical techniques including UV-vis spectroscopy, fluorescence spectroscopy, FT-IR, bis-ANS titration suggested a perturbation of structure and chaperone function of αA-crystallin upon p-BQ modification. Our results indicate that p-BQ is a causative agent involved in the modification of αA-crystallin and pathogenesis of CS-induced cataract. Our findings would educate public about the impacts of smoking on eye health and help to discourage them from smoking. The study might also help scientists to develop new drugs for the intervention of CS-induced cataract at an early stage.