Giulia D'amati - Convegno Mitocon 2014

1,529 views

Published on

Published in: Health & Medicine, Technology
0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
1,529
On SlideShare
0
From Embeds
0
Number of Embeds
913
Actions
Shares
0
Downloads
4
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Giulia D'amati - Convegno Mitocon 2014

  1. 1. IL POSSIBILE EFFETTO TERAPEUTICO DEL TERMINALE CARBOSSILICO ISOLATO DELLA mt-LEUCIL-tRNA SINTETASI NELLE MALATTIE DA MUTAZIONI DEI tRNA MITOCONDRIALI Giulia d’Amati Dip. Scienze Radiologiche, Oncologiche e Anatomo Patologiche
  2. 2. • Premises • Experimental evidence • Future directions
  3. 3. PREMISES Mitochondrial (mt) diseases are multisystem disorders due to mutations in nuclear or mtDNA genes. Among the latter, more than 50% are located in transfer RNA (tRNA) genes and are responsible for a wide range of syndromes, for which no effective treatment is available at present.
  4. 4. COMPLEX IV COMPLEX IIICOMPLEX IICOMPLEX I ATP SYNTHASE s 2 proteins 1 protein 3 proteins 7 proteins
  5. 5. G.P. 16 years, hypertrophic cardiomyopathy and hearing impairment. Homoplasmic m.4277T>C mutation in mt-tRNAIle
  6. 6. Leu(UUR) Ile P M controls 38 90 100 94 104 (% control) Leu/Ile Skeletal muscle from the patient’s clinically unaffected mother shows normal muscle respiratory chain activities and steady-state levels of mt-tRNAIle, whilst homoplasmic for the m.4277T>C. P M
  7. 7. Previous studies had shown that over-expression of mitochondrial aminoacyl-tRNA synthetases (aaRSs) can rescue the pathologic phenotype both in transmitochondrial cybrids and yeast models. We speculated that the penetrance of the m.4277T>C mt-tRNAIle mutation in this small kindred could be modulated by the expression levels of the mitochondrial isoleucyl-tRNA synthetase.
  8. 8. Essential enzymes performing the attachment of amino acids to their cognate tRNA molecules, the first step of protein synthesis. MITOCHONDRIAL AMINO ACYL-tRNA SYNTHETASES
  9. 9. P M C IleRS porin βactin P M C Endogenous levels of mt isoleucyl-tRNA synthetase (IARS2) are significantly higher in skeletal muscle and fibroblasts of the unaffected mother as compared to the proband.
  10. 10. fold-changeinIARS2expression mock pIARS2 mock pIARS2 b c a 160 140 120 100 80 60 40 20 0 %ofviablecellsingalactose mock pIARS2 %ofapoptoticcells mock pIARS2 120 100 80 60 40 20 0 Over-expression of exogenous IARS2 ameliorates viability of mutant transmitochondrial cybrids in galactose medium
  11. 11. Phenotype correction of MTTI cybrids by overexpression of non-cognate human mt-ValRS and mt-LeuRS
  12. 12. catalytic domain anticodon- binding domain editing domain C-term T.Thermophilus LeuRS PDBid: 2bte unknown domain
  13. 13. LeuRS Cterm Domain Improves Cell Viability and mt Bioenergetics of Cybrids Carrying Mutations in MTTI Gene
  14. 14. LeuRS Cterm Domain Improves Cell Viability and mt Bioenergetics of Cybrids Carrying m.3243A>G Mutation in MTTL1 Gene
  15. 15. * Anti-mitochondria Anti-Cterm Merge MpCtermpMTS-Cterm * * Anti-mitochondria Anti-Cterm Merge MpCtermpMTS-Cterm * LeuRS Cterm Domain Targets to Mitochondria
  16. 16. FUTURE DIRECTIONS • To verify whether the Cterm/Cterm peptides can modulate the detrimental effects of mutations in other tRNAs. Perli et al, EMM 2014 Hornig-Do TA, EMM 2014 Tyynismaa H, Schon EA, EMM 2014
  17. 17. FUTURE DIRECTIONS • To identify the minimal C-terminal fragment that still retains both mitochondrial localization and rescue ability. β30-31 β32-33
  18. 18. Surface Plasmon Resonance (SPR) shows the interaction of tRNAs with LARS2 C- term, C-term peptides and polyamines 1. SPR measures interactions between an immobilized ligand and an analyte in real time. 2. The wt mt-tRNALeu(UUR) interacts strongly with the C-term domain of LARS2. 3. The wt tRNA interacts strongly with the beta-strand peptides derived from the C-term. 4. The wt tRNA interacts with polyamines. 5. The 3243A>G mutant interacts with the C-term peptides with three time lower affinity than wt tRNA
  19. 19. FUTURE DIRECTIONS • To optimize the delivery method. • To analyze in detail the mechanisms of rescue. CELLULAR AND ANIMAL MODELS ARE NEEDED!
  20. 20. Elena Perli Annalinda Pisano Arianna Montanari Carmela Preziuso Veronica Morea Gianni Colotti Patrizio Di Micco Pierpaolo Ceci Annarita Fiorillo Antonio Campese Laura Frontali Silvia Francisci Massimo Zeviani Robert W Taylor

×