Short peptides from leucyl-tRNA synthetase rescue disease-causing mitochondrial tRNA point mutations

Human Molecular Genetics, 2016, Vol. 25, No. 5 903–915 doi: 10.1093/hmg/ddv619 Advance Access Publication Date: 31 December 2015 Original Article ORIGINAL ARTICLE Short peptides from leucyl-tRNA synthetase rescue disease-causing mitochondrial tRNA point mutations 1 Department of Radiology, Oncology and Pathology, 2Department of Biochemical Sciences “A. Rossi Fanelli”, Department of Biology and Biotechnologies ‘Charles Darwin’ and 4Department of Molecular Medicine, Sapienza University of Rome, Rome 00161, Italy, 5Pasteur Institute-Cenci Bolognetti Foundation, Rome 00161, Italy, 6 Wellcome Trust Center for Mitochondrial Research, Institute for Ageing and Health, Newcastle University, Newcastle upon Tyne NE1 7RU, UK and 7National Research Council of Italy, Institute of Molecular Biology and Pathology, Rome 00185, Italy 3 *To whom correspondence should be addressed at: Department of Radiology, Oncology and Pathology, Sapienza University of Rome, Policlinico Umberto I, Viale Regina Elena 324, 00161 Rome, Italy. Tel: +39 0649973332; Fax: +39 064461484; Email: (G.d.A.); National Research Council of Italy, Institute of Molecular Biology and Pathology, P.le A. Moro 5, 00185 Rome, Italy. Tel: +39 0649910910; Fax: +39 064440062; Email: gianni.colotti@uniroma1. it (G.C.) Abstract Mutations in mitochondrial (mt) genes coding for mt-tRNAs are responsible for a range of syndromes, for which no effective treatment is available. We recently showed that the carboxy-terminal domain (Cterm) of human mt-leucyl tRNA synthetase rescues the pathologic phenotype associated either with the m.3243A>G mutation in mt-tRNALeu(UUR) or with mutations in the mt-tRNAIle, both of which are aminoacylated by Class I mt-aminoacyl-tRNA synthetases (mt-aaRSs). Here we show, by using the human transmitochondrial cybrid model, that the Cterm is also able to improve the phenotype caused by the m.8344A>G mutation in mt-tRNALys, aminoacylated by a Class II aaRS. Importantly, we demonstrate that the same rescuing ability is retained by two Cterm-derived short peptides, β30_31 and β32_33, which are effective towards both the m.8344A>G and the m.3243A>G mutations. Furthermore, we provide in vitro evidence that these peptides bind with high affinity wild-type and mutant human mt-tRNALeu(UUR) and mt-tRNALys, and stabilize mutant mt-tRNALeu(UUR). In conclusion, we demonstrate that small Cterm-derived peptides can be effective tools to rescue cellular defects caused by mutations in a wide range of mt-tRNAs. Introduction Mitochondrial (mt) diseases are multi-system disorders due to mutations in nuclear or mtDNA genes. Among the latter, >50% are located in transfer RNA (tRNA) genes [(1); URL: http://www. mitomap.org] and are responsible for a wide range of clinical phenotypes, such as the severe Mitochondrial Encephalopathy with Received: November 17, 2015. Revised and Accepted: December 18, 2015 © The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact 903 Elena Perli1, Annarita Fiorillo2, Carla Giordano1, Annalinda Pisano1, Arianna Montanari3,5, Paola Grazioli4, Antonio F. Campese4, Patrizio Di Micco2, Helen A. Tuppen6, Ilaria Genovese2, Elena Poser2, Carmela Preziuso1, Robert W. Taylor6, Veronica Morea7, Gianni Colotti7,* and Giulia d’Amati1,5, * 904 | Human Molecular Genetics, 2016, Vol. 25, No. 5 Results Pathological phenotype of m.8344A>G MTTK mutant cybrids First, we investigated the Cterm ability to rescue the pathological phenotype of cybrid cells bearing a mutation in mt-tRNALys, which is aminoacylated by class II LysRS. To this end, we manipulated m.8344A>G osteosarcoma-derived cybrids (kind gift from Valeria Tiranti) using ethidium bromide (EtBr) to obtain different cell lines bearing either high or low levels of mutated mtDNA (see the Materials and Methods section). The assessment of m.8344A>G mutation load was performed by quantitative pyrosequencing. The selected clones showed 34% (L-8344, low-mutant percentage) and 86% (H-8344, high-mutant percentage) m.8344A>G mutation loads, respectively. To investigate the phenotype of mutant cell lines, cybrids were grown in glucosefree medium supplemented with galactose (galactose medium), a condition forcing cells to rely on the mt respiratory chain for ATP synthesis. In this condition, cell growth of H-8344 cybrids was severely impaired whereas L-8344 cybrids were indistinguishable from WT cells (Supplementary Material, Fig. S1A), providing us with an isogenic control for further experiments. Decreased viability of H-8344 cybrids was associated with an increase in apoptosis and impaired respiratory chain activity, as compared with isogenic L-8344 cybrids (Supplementary Material, Fig. S1A–C). To evaluate the effect of the m.8344A>G mutation on mt-tRNALys steady-state levels we performed high-resolution northern blot analysis. We showed that high-mutation levels are associated with a 50% decrease in mt-tRNALys steady-state levels relative to the amount of 5SrRNA (Supplementary Material, Fig. S1D). The carboxy-terminal domain of human mt-LeuRS is able to rescue the phenotype of m.8344A>G MTTK mutant cybrids We transiently expressed the Cterm in H-8344 and L-8344 cybrids. This resulted in increased cell viability of H-8344 cybrids after 24 h incubation in galactose medium (up to 1.5-fold as compared to mock transformants) and decreased apoptosis after 6 h (up to 1.6-fold when compared with mock transformants) (Fig. 1A and B). Moreover, Cterm transformants showed a significant increase in oxygen consumption when compared with mock transformants (up to 1.4-fold increase) (Fig. 1C). Conversely, Cterm overexpression did not affect viability of isogenic L-8344 cybrids (Fig. 1A and C). For each experiment, we measured transfection efficiency by quantitative real-time polymerase chain reaction (PCR) (Fig. 1D). Cterm-derived peptides are endowed with rescuing ability towards both m.3243A>G MTTL1 and m.8344A>G MTTK mutant cybrid phenotypes The structural basis of the interaction between the Cterm of human mt-LeuRS and the cognate tRNA can be inferred from the analysis of the experimentally determined 3D structures of LeuRS-tRNALeu complexes from the bacteria Thermus thermophilus (14) and Escherichia coli (15) available from the Protein Data Bank [PDB; (18)]. As shown in Figure 2, two pairs of β-strands in the carboxy-terminal domain of bacterial LeuRS (whose human homologues were named β30_31 and β32_33 (16) comprise most of the residues that are involved in contacts with the elbow region of tRNALeu. Thus, we tested the hypothesis that the short peptides from the human enzyme were responsible for the Cterm ability Lactic Acido (...truncated)