Giulia D'amati - Convegno Mitocon 2014

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

• Premises

• Experimental evidence

• Future directions

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.

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Note di presentazione

Consequently, the biochemical deficiency is due to mitochondrial protein synthesis defects, which manifest as aberrant cellular respiration and ATP synthesis. Genetically correcting an mtDNA defect is not currently a therapeutic option. Moreover human mtDNA encodes 22 different tRNAs, therefore, it would be ideal if a single approach could be developed that would be equally effective with any pathogenic mt-tRNA mutation

COMPLEX IV COMPLEX III COMPLEX II COMPLEX I

ATP SYNTHASE

s

2 proteins

1 protein 3 proteins

7 proteins

G.P. 16 years, hypertrophic cardiomyopathy and hearing impairment.

Homoplasmic m.4277T>C mutation in mt-tRNAIle

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Figure 1. Echocardiogram and histochemical and molecular analyses of skeletal muscle Two-dimensional echocardiogram from the index case showing symmetrical left ventricular hypertrophy (LVH). LV posterior wall (asterisk) is 20 mm thick. Sequential COX/SDH histochemistry on skeletal muscle biopsy from the proband (top) and his mother (bottom). Muscle from the proband reveals numerous COX-deficient (blue) muscle fibers. Note one COX-deficient fiber with subsarcolemmal accumulation of mitochondria (20X magnification). Mitochondrial DNA copy number in muscle homogenate from the proband (P, black), his mother (M, white) and age-matched controls (C, n=11, grey). The amount of mtDNA was evaluated by quantitative real time PCR and expressed as mtDNA/nuclear DNA ratio. Relative expression of PPARGC1A gene in muscle homogenate from the proband, his mother and age-matched controls (n=9). In all samples, the relative expression of PPARGC1A was evaluated with respect to one control (reference sample) by quantitative real-time PCR, using the comparative threshold cycle (ΔCt) method. Values are normalized to VDAC1 gene. C = controls; PPARGC1A= peroxisome proliferator-activated receptor gamma, coactivator 1 alpha; VDAC1= mitochondrial voltage-dependent anion channel 1.

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

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Sequence electropherogram of mt-tRNAIle showing the m.4277T>C mutation in the proband’s skeletal muscle (mutation is indicated by an asterisk). WT= wild-type. Secondary structure of mt-tRNAIle with the position of the m.4277T>C mutation highlighted in the D-loop. Quantification by last hot cycle PCR/RFLP of m.4277 T>C mutation levels in skeletal muscle from the proband (P) and his unaffected mother (M). Wild-type 161-bp PCR products, amplified with mismatched forward and reverse primers, contain a single Tsp45I restriction site, which cleaves the amplicons into two fragments of 125 and 36-bp. The m.4277T>C mutation introduces a second Tsp45I recognition site that cuts the 125-bp fragment into two smaller fragments of 103 and 22-bp. Labeled products were separated through a 12% non-denaturing polyacrylamide gel. Fragment sizes (bp) are shown on the left. Determination of steady-state mt-tRNAIle levels in skeletal muscle from the patient (P), his mother (M) and three age-matched controls by high-resolution Northern blot analysis. For each sample, the mt-tRNAIle signal has been normalized to that of mt-tRNALeu(UUR) and expressed as a percentage of one of the controls.

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.

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Expression level of cognate aaRS modulates the effect of the mitochondrial tRNA mutation Previous work by our group has shown that the over expression of cognate aaRSs can rescue the defects of yeast mt-tRNA mutants (35,36). Different groups confirmed these findings on human cell lines (23,28,30). Based on these results, we speculated that the expression level of mitochondrial isoleucyl-tRNA synthetase could modulate the penetrance of m.4277T>C mt-tRNAIle mutation in this small family. Concordant with this hypothesis, we observed a significantly higher expression of the cognate IleRS in skeletal muscle and fibroblasts from the unaffected mother as compared to the proband. This is the first observation that constitutively high levels of aaRSs in human tissues are able to suppress the phenotypic expression of a homoplasmic mt-tRNA point mutation. Since isoleucylation does not seems to be affected by the m.4277T>C transition, the suppressive effect of IleRSs may be mediated by a stabilizing, chaperone-like activity on the structurally altered tRNA (29). In agreement with this hypothesis, we found a direct relationship between steady-state levels of mutated mt-tRNAIle and expression levels of its cognate synthetase, both in skeletal muscle, fibroblasts and transmitochondrial cybrids.

Essential enzymes performing the attachment of amino acids to their cognate tRNA molecules, the first step of

protein synthesis.

MITOCHONDRIAL AMINO ACYL-tRNA SYNTHETASES

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.

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Expression levels of mitochondrial isoleucyl-tRNA synthetase gene (IARS2) in skeletal muscle from the m.4277T>C proband (P, black circle) and his unaffected mother (M, white circle), from two m.4300A>G patients (V14, IV9, triangle), and from controls (grey square). In all samples, the relative expression of each target gene was evaluated with respect to one control (reference sample) by quantitative real-time PCR, using the comparative threshold cycle (ΔCt) method. All values were normalized to VDAC1 gene. Each point represents a single observation. Red arrows indicate M values that are significantly higher than both P (p<0.001) and control (p<0.01) values. Similar results were obtained using the housekeeping gene HPRT1 (data not shown). Experiments were performed at least in duplicate. Immunofluorescent staining with an antibody against isoleucyl-tRNA synthetase (IleRS) on skeletal muscle from the m.4277T>C proband (P), his mother (M) and a control subject (C). Nuclei are stained with DAPI (blue). The cells were examined and imaged with an Olympus IX50 fluorescence microscope (original magnification 40X). Prior to these experiments, we confirmed localization of the anti-IleRS antibody to mitochondria (see Supplemental Figure S5). Western blot analysis of IleRS protein performed on fibroblast extracts from the m.4277T>C proband (P), his mother (M) and a control subject (C).

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mock pIARS2

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pIARS2

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mock pIARS2

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Over-expression of exogenous IARS2 ameliorates viability of mutant transmitochondrial cybrids in galactose medium

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A large number of nuclear factors required for the correct biogenesis and function of mt tRNAs have been identified and characterized, including aminoacyl-tRNA synthetases (aaRSs) (Suzuki et al., 2011). Aminoacyl-tRNA synthetases are very complex, ubiquitously expressed, essential enzymes performing the first step of protein synthesis, namely the attachment of amino acids to their cognate tRNA molecules. The accuracy of the aminoacylation reaction, on which the fidelity of protein synthesis is based, relies on the aaRS ability to specifically recognize and link tRNA molecules with the cognate aminoacids. Based on the presence of common structural/functional features, aaRSs have been assigned to two classes (I and II) and further divided into subclasses (Francklyn et al, 1997). Mutations in mt aaRSs are emerging as an important cause of disease (Suzuki et al, 2011). Pathogenic mutations described so far affect the catalytic or editing domain of mt aaRSs (Gotz et al., 2011, Scheper et al., 2007, Riley et al, 2010) or the mt targeting sequence (Messmer et al., 2011), or result in immature, truncated proteins (Edvardson et al., 2007). A severe reduction in the steady-state levels of the

Phenotype correction of MTTI cybrids by overexpression of non-cognate human mt-ValRS and mt-LeuRS

catalytic domain anticodon-

binding domain

editing domain C-term

T.Thermophilus LeuRS

PDBid: 2bte

unknown domain

LeuRS Cterm Domain Improves Cell Viability and mt Bioenergetics of Cybrids Carrying Mutations in MTTI Gene

LeuRS Cterm Domain Improves Cell Viability and mt Bioenergetics of Cybrids Carrying m.3243A>G Mutation in

MTTL1 Gene

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Anti-mitochondria Anti-Cterm Merge

MpC

termpM

TS-Cterm

*

*

Anti-mitochondria Anti-Cterm Merge

MpC

termpM

TS-Cterm

*

LeuRS Cterm Domain Targets to Mitochondria

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

FUTURE DIRECTIONS • To identify the minimal C-terminal fragment that still

retains both mitochondrial localization and rescue ability.

β30-31

β32-33

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

FUTURE DIRECTIONS

• To optimize the delivery method.

• To analyze in detail the mechanisms of rescue.

CELLULAR AND ANIMAL MODELS ARE NEEDED!

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

Giulia D'amati - Convegno Mitocon 2014

Health & Medicine

Transcript of Giulia D'amati - Convegno Mitocon 2014