Mitochondrial Encephalopathy with Lactic Acidosis and Stroke like Episodes (MELAS) is a mitochondrial disease caused by point mutations in the tRNALeu(UUR) with a prevalence in position 3243 (A>G). These mutations cause severe impairment of mitochondrial protein synthesis due to alterations of the mutated tRNA such as reduced aminoacylation and lack of post-transcriptional modification. It has been shown that overexpression of leucyl-tRNA-synthetase (mt-LeuRS) is able to rescue defects of pathogenic mutation of tRNALeu(UUR) in MELAS cybrids [1]. The rescuing activity of human mt-LeuRS resides in a small (<70 amino acids) part of the carboxy-terminal domain (Cterm) of the enzyme and in short (15-16 aa) C-term-derived peptides. However, the molecular mechanisms underlying the rescuing process are still unknown. On these bases, we started a project aimed at elucidating the ability of mt-LeuRS C-term and its derived peptides to correct the pathological phenotype in MELAS. To address this issue we intend: i) to investigate in vivo the interactions of rescuing molecules with mutated mt-tRNA; ii) to study the effect of overexpression of rescuing molecules on the rate of synthesis and stability of de novo synthesized mt-polypeptides; iii) to investigate the effect of rescuing molecules on the steady-state level, the aminoacylation level and the post-transcriptional modifications of the mutated tRNA. To date we have characterized the profile of the de novo synthesized mt-polypeptides from MELAS cybrids, finding an overall decreased rate of synthesis and the appearance of aberrant mitochondrial translation product. We have also observed a decreased steady-state level of NDUFB8 (Complex I) and COXI (Complex IV). Finally, we have demonstrated in MELAS cybrids constitutively expressing C-term, the existence of a preferential interaction between Cterm polypeptide and tRNALeu(UUR) by means of RNA immunoprecipitation experiments.

Elucidating the molecular mechanisms underlying the biological activity of rescuing peptides in MELAS mitochondrial disease

Francesco Capriglia
;
Francesco Bruni;Marina Roberti;Paola Loguercio Polosa;Palmiro Cantatore
2019-01-01

Abstract

Mitochondrial Encephalopathy with Lactic Acidosis and Stroke like Episodes (MELAS) is a mitochondrial disease caused by point mutations in the tRNALeu(UUR) with a prevalence in position 3243 (A>G). These mutations cause severe impairment of mitochondrial protein synthesis due to alterations of the mutated tRNA such as reduced aminoacylation and lack of post-transcriptional modification. It has been shown that overexpression of leucyl-tRNA-synthetase (mt-LeuRS) is able to rescue defects of pathogenic mutation of tRNALeu(UUR) in MELAS cybrids [1]. The rescuing activity of human mt-LeuRS resides in a small (<70 amino acids) part of the carboxy-terminal domain (Cterm) of the enzyme and in short (15-16 aa) C-term-derived peptides. However, the molecular mechanisms underlying the rescuing process are still unknown. On these bases, we started a project aimed at elucidating the ability of mt-LeuRS C-term and its derived peptides to correct the pathological phenotype in MELAS. To address this issue we intend: i) to investigate in vivo the interactions of rescuing molecules with mutated mt-tRNA; ii) to study the effect of overexpression of rescuing molecules on the rate of synthesis and stability of de novo synthesized mt-polypeptides; iii) to investigate the effect of rescuing molecules on the steady-state level, the aminoacylation level and the post-transcriptional modifications of the mutated tRNA. To date we have characterized the profile of the de novo synthesized mt-polypeptides from MELAS cybrids, finding an overall decreased rate of synthesis and the appearance of aberrant mitochondrial translation product. We have also observed a decreased steady-state level of NDUFB8 (Complex I) and COXI (Complex IV). Finally, we have demonstrated in MELAS cybrids constitutively expressing C-term, the existence of a preferential interaction between Cterm polypeptide and tRNALeu(UUR) by means of RNA immunoprecipitation experiments.
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11586/243267
 Attenzione

Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo

Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact