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