Exogenous peptides are able to penetrate human cell and mitochondrial membranes, stabilize mitochondrial tRNA structures, and rescue severe mitochondrial defects

Perli, Elena; Pisano, Annalinda; Pignataro, Maria Gemma; Campese, Antonio Francesco; Pelullo, Maria; Genovese, Ilaria; Turris, Valeria de; Ghelli, Anna; Cerbelli, Bruna; Giordano, Carla; Colotti, Gianni; Morea, Veronica; d’Amati, Giulia

doi:10.1096/fj.201903270r

Cited by 6 publications

(9 citation statements)

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“…Here, as a starting point of our investigation, we showed that overexpressed Cterm was capable to interact with the mutated cognate mt-tRNALeu (UUR) in cultured cybrids; this finding is in line with the obtained in vitro results and proposed chaperonic effect of Cterm domain [ 14 , 15 , 16 , 17 ]. Then, we assessed whether Cterm was able to relieve mitochondrial molecular defects in a MELAS cell line characterised by depressed mitochondrial translation.…”

Section: Discussionsupporting

confidence: 81%

“…We also evaluated the effect of Cterm overexpression on a range of mitochondrial processes such as mitochondrial translation, steady-state level and aminoacylation efficiency of mt-tRNALeu (UUR) , bioenergetics, and mitophagy. In agreement with previous findings [ 14 , 15 , 16 , 17 ], a complete recovery of cell viability was observed. Unexpectedly, despite an improvement of protein synthesis, the other analysed mitochondrial processes were not affected by Cterm expression.…”

Section: Introductionsupporting

confidence: 93%

“…Numerous studies performed in yeast [ 34 ] and human cell lines [ 13 , 14 , 15 , 16 ] highlighted the ability of the LARS2 carboxy-terminal domain (Cterm) to interact with several mt-tRNAs, including the cognate mt-tRNALeu (UUR) , and to relieve cell growth and respiration defects due to mt-tRNA mutations. Furthermore, a similar rescuing effect by short Cterm-derived peptides was also observed in human MELAS cybrids [ 17 ].…”

Section: Discussionmentioning

confidence: 64%

“…Therefore, we decided to assess the impact of Cterm peptide on mitochondrial translation efficiency. Since the cybrid lines used for RIP experiments and in previous work [ 14 , 15 , 16 , 17 ] do not show defects in mitochondrial protein synthesis, from here on we employed a MELAS cybrid cell line (kindly provided by Prof. A. Filipovska, University of Western Australia) that displays a depressed mitochondrial translation phenotype (see below). In these cells, mtDNA heteroplasmy analysis indicated a 3243A>G mutation load of ~94% ( Figure S3 ).…”

Section: Resultsmentioning

confidence: 99%

“…The correction activity was observed following both overexpression and exogenous administration to cells carrying the m.3243A>G MELAS mutation in the mt-tRNALeu (UUR) , as well as the m.8344A>G MERRF mutation in mt-tRNALys. To explain the rescuing effect of the Cterm and its peptides, a chaperonic activity towards mutated tRNAs were proposed on the basis of in vitro evidences, which could result in the stabilization of a wild-type-like conformation of the tRNA [ 14 , 15 , 16 , 17 ].…”

Section: Introductionmentioning

confidence: 99%

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Exploring the Ability of LARS2 Carboxy-Terminal Domain in Rescuing the MELAS Phenotype

Capriglia

Rizzo

Petrosillo

et al. 2021

Life

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The m.3243A>G mutation within the mitochondrial mt-tRNALeu(UUR) gene is the most prevalent variant linked to mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) syndrome. This pathogenic mutation causes severe impairment of mitochondrial protein synthesis due to alterations of the mutated tRNA, such as reduced aminoacylation and a lack of post-transcriptional modification. In transmitochondrial cybrids, overexpression of human mitochondrial leucyl-tRNA synthetase (LARS2) has proven effective in rescuing the phenotype associated with m.3243A>G substitution. The rescuing activity resides in the carboxy-terminal domain (Cterm) of the enzyme; however, the precise molecular mechanisms underlying this process have not been fully elucidated. To deepen our knowledge on the rescuing mechanisms, we demonstrated the interactions of the Cterm with mutated mt-tRNALeu(UUR) and its precursor in MELAS cybrids. Further, the effect of Cterm expression on mitochondrial functions was evaluated. We found that Cterm ameliorates de novo mitochondrial protein synthesis, whilst it has no effect on mt-tRNALeu(UUR) steady-state levels and aminoacylation. Despite the complete recovery of cell viability and the increase in mitochondrial translation, Cterm-overexpressing cybrids were not able to recover bioenergetic competence. These data suggest that, in our MELAS cell model, the beneficial effect of Cterm may be mediated by factors that are independent of the mitochondrial bioenergetics.

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Section: Discussionsupporting

confidence: 81%

Section: Introductionsupporting

confidence: 93%

Section: Discussionmentioning

confidence: 64%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Exploring the Ability of LARS2 Carboxy-Terminal Domain in Rescuing the MELAS Phenotype

Capriglia

Rizzo

Petrosillo

et al. 2021

Life

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Restoration of mitochondrial function through activation of hypomodified tRNAs with pathogenic mutations associated with mitochondrial diseases

Tomoda

Nagao

Shirai

et al. 2023

Nucleic Acids Research

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Mutations in mitochondrial (mt-)tRNAs frequently cause mitochondrial dysfunction. Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), and myoclonus epilepsy associated with ragged red fibers (MERRF) are major clinical subgroups of mitochondrial diseases caused by pathogenic point mutations in tRNA genes encoded in mtDNA. We previously reported a severe reduction in the frequency of 5-taurinomethyluridine (τm5U) and its 2-thiouridine derivative (τm5s2U) in the anticodons of mutant mt-tRNAs isolated from the cells of patients with MELAS and MERRF, respectively. The hypomodified tRNAs fail to decode cognate codons efficiently, resulting in defective translation of respiratory chain proteins in mitochondria. To restore the mitochondrial activity of MELAS patient cells, we overexpressed MTO1, a τm5U-modifying enzyme, in patient-derived myoblasts. We used a newly developed primer extension method and showed that MTO1 overexpression almost completely restored the τm5U modification of the MELAS mutant mt-tRNALeu(UUR). An increase in mitochondrial protein synthesis and oxygen consumption rate suggested that the mitochondrial function of MELAS patient cells can be activated by restoring the τm5U of the mutant tRNA. In addition, we confirmed that MTO1 expression restored the τm5s2U of the mutant mt-tRNALys in MERRF patient cells. These findings pave the way for epitranscriptomic therapies for mitochondrial diseases.

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Rapamycin rescues mitochondrial dysfunction in cells carrying the m.8344A > G mutation in the mitochondrial tRNALys

Capristo

Dotto

Tropeano

et al. 2022

Mol Med

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Background Myoclonus, Epilepsy and Ragged-Red-Fibers (MERRF) is a mitochondrial encephalomyopathy due to heteroplasmic mutations in mitochondrial DNA (mtDNA) most frequently affecting the tRNALys gene at position m.8344A > G. Defective tRNALys severely impairs mitochondrial protein synthesis and respiratory chain when a high percentage of mutant heteroplasmy crosses the threshold for full-blown clinical phenotype. Therapy is currently limited to symptomatic management of myoclonic epilepsy, and supportive measures to counteract muscle weakness with co-factors/supplements. Methods We tested two therapeutic strategies to rescue mitochondrial function in cybrids and fibroblasts carrying different loads of the m.8344A > G mutation. The first strategy was aimed at inducing mitochondrial biogenesis directly, over-expressing the master regulator PGC-1α, or indirectly, through the treatment with nicotinic acid, a NAD+ precursor. The second was aimed at stimulating the removal of damaged mitochondria through prolonged rapamycin treatment. Results The first approach slightly increased mitochondrial protein expression and respiration in the wild type and intermediate-mutation load cells, but was ineffective in high-mutation load cell lines. This suggests that induction of mitochondrial biogenesis may not be sufficient to rescue mitochondrial dysfunction in MERRF cells with high-mutation load. The second approach, when administered chronically (4 weeks), induced a slight increase of mitochondrial respiration in fibroblasts with high-mutation load, and a significant improvement in fibroblasts with intermediate-mutation load, rescuing completely the bioenergetics defect. This effect was mediated by increased mitochondrial biogenesis, possibly related to the rapamycin-induced inhibition of the Mechanistic Target of Rapamycin Complex 1 (mTORC1) and the consequent activation of the Transcription Factor EB (TFEB). Conclusions Overall, our results point to rapamycin-based therapy as a promising therapeutic option for MERRF.

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Exogenous peptides are able to penetrate human cell and mitochondrial membranes, stabilize mitochondrial tRNA structures, and rescue severe mitochondrial defects

Cited by 6 publications

References 30 publications

Exploring the Ability of LARS2 Carboxy-Terminal Domain in Rescuing the MELAS Phenotype

Exploring the Ability of LARS2 Carboxy-Terminal Domain in Rescuing the MELAS Phenotype

Restoration of mitochondrial function through activation of hypomodified tRNAs with pathogenic mutations associated with mitochondrial diseases

Rapamycin rescues mitochondrial dysfunction in cells carrying the m.8344A > G mutation in the mitochondrial tRNALys

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