Aminoacyl-transfer ribonucleic acid synthetases (ARSes) are enzymes involved in translation of mRNAs into proteins. Lysyl-transfer RNA synthetase (LysRS) encoded by KARS loads lysine to its cognate tRNA. In contrast to other ARSes, which are encoded by two genes, two LysRS isoforms generated by alternative splicing of KARS pre-mRNA localizes either in cytosol (cytKARS, NM_005548.2) or in mitochondria (mtKARS, NM_001130089.1). To date, different KARS pathogenic variants have been associated to wide spectrum of clinical manifestations including sensorineural hearing loss, visual loss, neuropathy, seizures, leukodystrophy with spinal and brainstem calcification. In the context of an international collaborative project, ten further cases with a severe neurodegenerative and multi-system disease due to KARS bi-allelic variants were collected. The mapping of twelve amino acids affected in KARS patients on the crystal structure of KARS-p38 complex (PDB: 4dpg) showed that the majority of them localize in the catalytic domain or in the anticodon binding domain, probably affecting the aminoacylation reaction, the binding affinity for lysine or compromising the protein structure and its interaction with other proteins. Moreover, since the exact contribution of mitochondrial and/or cytosolic LysRS deficiency to KARS-related phenotype is difficult to untangle, we investigated the impact of each mutation on the activity of both compartments, modelling each mutation in the yeast Saccharomyces cerevisiae. While in human KARS encodes for both cytoplasmic and mitochondrial isoforms of lysyl-tRNA synthetase, in yeast two different genes, KRS1 and MSK1, encode the cytoplasmic and mitochondrial lysyl-tRNA synthetase, respectively. Taking advantage of this, we studied the effects of mutated alleles mtKARS and cytKARS separately through heterologous complementation in strains deleted in MSK1 and KRS1, respectively. The results obtained showed that most mutant strains displayed a growth defect, though at very different extent, and none affected only a single isoform. However, for several mutations, a variable degree of impairment was observed when the same variant was expressed by the cytKARS or the mtKARS, suggesting that defects of both cytKARS or the mtKARS contribute to the phenotype but also that the impairment of either cytKARS or the mtKARS activity was predominant. Moreover the detrimental effects of two variants in cytKARS, were partially improved by lysine supplementation in the medium, suggesting that mutations of these two amino acids, which are in the catalytic domain, may decrease the binding affinity for lysine, and thus supporting the therapeutic potential of lysine supplementation in patients.

Modeling in yeast of KARS pathogenic variants associated with a progressive and multi-systemic disease: impact on cytosolic and mitochondrial isoforms / CECCATELLI BERTI, Camilla; Cappuccio, Gerarda; Baruffini, Enrico; Figuccia, Sonia; Brunetti-Pierri, Nicola; Goffrini, Paola. - (2020). ((Intervento presentato al convegno Mitochondrial Medicine 2020 tenutosi a VIRTUAL event nel 30 November–02 December 2020.

Modeling in yeast of KARS pathogenic variants associated with a progressive and multi-systemic disease: impact on cytosolic and mitochondrial isoforms

Camilla Ceccatelli Berti;Enrico Baruffini;Sonia Figuccia;Paola Goffrini
2020

Abstract

Aminoacyl-transfer ribonucleic acid synthetases (ARSes) are enzymes involved in translation of mRNAs into proteins. Lysyl-transfer RNA synthetase (LysRS) encoded by KARS loads lysine to its cognate tRNA. In contrast to other ARSes, which are encoded by two genes, two LysRS isoforms generated by alternative splicing of KARS pre-mRNA localizes either in cytosol (cytKARS, NM_005548.2) or in mitochondria (mtKARS, NM_001130089.1). To date, different KARS pathogenic variants have been associated to wide spectrum of clinical manifestations including sensorineural hearing loss, visual loss, neuropathy, seizures, leukodystrophy with spinal and brainstem calcification. In the context of an international collaborative project, ten further cases with a severe neurodegenerative and multi-system disease due to KARS bi-allelic variants were collected. The mapping of twelve amino acids affected in KARS patients on the crystal structure of KARS-p38 complex (PDB: 4dpg) showed that the majority of them localize in the catalytic domain or in the anticodon binding domain, probably affecting the aminoacylation reaction, the binding affinity for lysine or compromising the protein structure and its interaction with other proteins. Moreover, since the exact contribution of mitochondrial and/or cytosolic LysRS deficiency to KARS-related phenotype is difficult to untangle, we investigated the impact of each mutation on the activity of both compartments, modelling each mutation in the yeast Saccharomyces cerevisiae. While in human KARS encodes for both cytoplasmic and mitochondrial isoforms of lysyl-tRNA synthetase, in yeast two different genes, KRS1 and MSK1, encode the cytoplasmic and mitochondrial lysyl-tRNA synthetase, respectively. Taking advantage of this, we studied the effects of mutated alleles mtKARS and cytKARS separately through heterologous complementation in strains deleted in MSK1 and KRS1, respectively. The results obtained showed that most mutant strains displayed a growth defect, though at very different extent, and none affected only a single isoform. However, for several mutations, a variable degree of impairment was observed when the same variant was expressed by the cytKARS or the mtKARS, suggesting that defects of both cytKARS or the mtKARS contribute to the phenotype but also that the impairment of either cytKARS or the mtKARS activity was predominant. Moreover the detrimental effects of two variants in cytKARS, were partially improved by lysine supplementation in the medium, suggesting that mutations of these two amino acids, which are in the catalytic domain, may decrease the binding affinity for lysine, and thus supporting the therapeutic potential of lysine supplementation in patients.
Modeling in yeast of KARS pathogenic variants associated with a progressive and multi-systemic disease: impact on cytosolic and mitochondrial isoforms / CECCATELLI BERTI, Camilla; Cappuccio, Gerarda; Baruffini, Enrico; Figuccia, Sonia; Brunetti-Pierri, Nicola; Goffrini, Paola. - (2020). ((Intervento presentato al convegno Mitochondrial Medicine 2020 tenutosi a VIRTUAL event nel 30 November–02 December 2020.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11381/2907367
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