Effetto di peptidi derivati dalla Effetto di peptidi derivati dalla mt-Leucyl-tRNA sintetasi su cellule umane con mt-Leucyl-tRNA sintetasi su cellule umane con

mutazioni di mt-tRNA: aggiornamentimutazioni di mt-tRNA: aggiornamenti

Giulia d’AmatiGiulia d’Amati

Dip. Scienze Radiologiche, Oncologiche e Dip. Scienze Radiologiche, Oncologiche e Anatomo Patologiche Anatomo Patologiche

Mitochondrial (mt) diseases are multisystem Mitochondrial (mt) diseases are multisystem disorders due to mutations in nuclear or mtDNA disorders due to mutations in nuclear or mtDNA genes. genes. Among the latter, more than 50% are located in Among the latter, more than 50% are located in transfer RNA (tRNA) genes and are responsible transfer RNA (tRNA) genes and are responsible for a wide range of syndromes, for which no for a wide range of syndromes, for which no effective treatment is available at present. effective treatment is available at present.

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

protein synthesisprotein synthesis..

AMINO ACYL-tRNA SYNTHETASES AMINO ACYL-tRNA SYNTHETASES (aaRSs)(aaRSs)

Two major classes of aaRSsBased on distinct structural motifs

• characteristic Rossman fold catalytic domain

• two highly conserved sequence motifs

• aminoacylates at the 2'-OH of a the tRNA terminal adenosine

• mostly monomeric

Class I

These proteins differ widely in size and oligomeric state, and have limited sequence homology

LeuRSPDBID: 2bte

• anti-parallel beta-sheet fold flanked by alpha-helices.

• three highly conserved sequence motifs.

• aminoacylate at the 3'-OH of tRNA terminal adenosine (exception: PheRS)

• usually dimeric or tetrameric (two or four subunits, respectively).

Class II

Anticodon binding domain, which is separate from the

catalytic unit, appears to be a later addition to synthetases

Interestingly

AspRSPDBID: 1asy

P M C

IleRS

porin

βactin

P M CEndogenous levels of mt isoleucyl-tRNA synthetase (IARS2)

are significantly higher in skeletal muscle and fibroblasts of the unaffected mother as compared to the proband.

Defects due to point mutations in several mt-tRNAs (mt-Defects due to point mutations in several mt-tRNAs (mt-tRNALeu(UUR), mt-tRNAIle, mt-tRNAVal) are rescued by the tRNALeu(UUR), mt-tRNAIle, mt-tRNAVal) are rescued by the cognate human mt-aaRS in both the yeast model and human cognate human mt-aaRS in both the yeast model and human

cells.cells.

catalytic domaincatalytic domain

anticodon-binding domain

anticodon-binding domain

editing domainediting domainC-termC-term

T.ThermophilusLeuRS

PDBid: 2bte

unknown domainunknown domain

The mt-tRNAThe mt-tRNALeuLeu interacts interacts extensively with the carboxy-extensively with the carboxy-terminal domain of LeuRS terminal domain of LeuRS (LeuRS-Cterm), and in (LeuRS-Cterm), and in particular with residues particular with residues within within β-β-strands strands β30, β31, β30, β31, β32 β32 and and β33.β33.

HYPOTHESIS: The severe HYPOTHESIS: The severe phenotype caused by the phenotype caused by the m.3243A>G point mutation in mt-m.3243A>G point mutation in mt-tRNAtRNALeu(UUR)Leu(UUR) can be rescued by can be rescued by overexpression of LeuRS-Cterm in overexpression of LeuRS-Cterm in human cells.human cells.

LeuRS-Cterm rescues the pathological phenotype of cybrids carrying the severe m.3243A>G point mutation in mt-tRNALeu(UUR)

M

M

pLARS2 pCterm

pCterm

M

M

pLARS2 pCterm

pCterm

Perli e. et al, EMBO Mol Med 2014

Viability Apoptosis Oxygen Consumption

LeuRS-Cterm localizes to mitochondria even in the absence of a canonical N-terminal mt targeting sequence

*

Anti-mitochondria Anti-Cterm Merge

Mp

Cterm

pM

TS

-Cterm

*

*

Anti-mitochondria Anti-Cterm Merge

Mp

Cterm

pM

TS

-Cterm

*

Perli e. et al, EMBO Mol Med 2014

tRN

ALe

u(U

UR

)

ste

ady-s

tate

level %

* *

Perli et al, unpublished results

LeuRS-Cterm directly and specifically interacts with human mt-tRNALeu(UUR) in vitro with high affinity and stability.

Perli e. et al, EMBO Mol Med 2014

Surface Plasmon Resonance

HYPOTHESIS (2): Defects due to point HYPOTHESIS (2): Defects due to point mutations in mutations in non-cognate non-cognate mt-tRNAs mt-tRNAs can be rescued by overexpression of can be rescued by overexpression of LeuRS-Cterm in human cells.LeuRS-Cterm in human cells.

LeuRS-Cterm LeuRS-Cterm Rescues the pathological phenotype of cybrids Rescues the pathological phenotype of cybrids carrying the m.4277T>C and m.4300A>G mutations in mt-carrying the m.4277T>C and m.4300A>G mutations in mt-tRNAtRNAIleIle..

Perli e. et al, EMBO Mol Med 2014

Viability Apoptosis

Oxygen Consumption

LeuRS-Cterm LeuRS-Cterm interacts with human mt-tRNAinteracts with human mt-tRNAIleIle in vitro in vitro

Surface Plasmon Resonance

Perli e. et al, EMBO Mol Med 2014

In the yeast model the isolated human mt LeuRS-In the yeast model the isolated human mt LeuRS-Cterm rescues defects due to point mutations in Cterm rescues defects due to point mutations in several mt-tRNAs aminoacylated by aaRSs several mt-tRNAs aminoacylated by aaRSs belonging to either class I or class II.belonging to either class I or class II.

Di Micco et al. 2014

HYPOTHESIS (3): Overexpression of LeuRS-Cterm can also rescue defects due to mutations in mt-tRNAs aminoacylated by class II aaRSs.

Ia Ib Ic

Class I ValRS MetRS GluRS TyrRS

IleRS CysRS GlnRS TrpRS

LeuRS ArgRS LysRS-I

Class II HisRS ProRS Asp PheRS

SerRS GlyRS Asn PylRS

ThrRS AlaRS LysRS-II SepRS

IIa IIb IIc

LeuRS-Cterm rescues the pathological phenotype of cybrids LeuRS-Cterm rescues the pathological phenotype of cybrids carrying the severe MERRF m.8344A>G point mutation in mt-carrying the severe MERRF m.8344A>G point mutation in mt-tRNAtRNALysLys..

Viability Apoptosis

Oxygen Consumption Gene expression levels

(34 %) (86 %)

Perli et al, manuscript in preparation

HYPOTHESIS (4)HYPOTHESIS (4):: The pathologic phenotype of both The pathologic phenotype of both the m.3243A>G in mt-tRNAthe m.3243A>G in mt-tRNALeu(UUR) Leu(UUR) andand m.8344A>G m.8344A>G point mutation in mt-tRNApoint mutation in mt-tRNALys Lys is rescued by is rescued by overexpression of short peptides, derived from overexpression of short peptides, derived from LeuRS-Cterm. LeuRS-Cterm.

β30-31 (15 aa)β30-31 (15 aa)

β32-33 (16 aa)β32-33 (16 aa)

Peptides Peptides β30-31 β30-31 and and β32-33 β32-33 ameliorate viability and ameliorate viability and energetic proficiency of human cells carrying either energetic proficiency of human cells carrying either m.3243A>G mutation in mt-tRNAm.3243A>G mutation in mt-tRNALeu(UUR) Leu(UUR) or m. 8344A>G or m. 8344A>G mutation in mt-tRNAmutation in mt-tRNALysLys

Viability

Apoptosis

Perli et al, manuscript in preparation

Peptides Peptides β30-31β30-31 and and β32-33β32-33 ameliorate viability and ameliorate viability and energetic proficiency of human cells carrying either energetic proficiency of human cells carrying either m.3243A>G mutation in mt-tRNAm.3243A>G mutation in mt-tRNALeu(UUR) Leu(UUR) or m. 8344A>G or m. 8344A>G mutation in mt-tRNAmutation in mt-tRNALysLys

Oxygen Consumption

Perli et al, manuscript in preparation

Immunofluorescence stain (anti-Flag antibody)

MTS -GGSGG-rescuing_peptide-GGSGG-2x(FLAG)

Immunofluorescence stain (anti human LeuRS Cterm domain)

Peptides Peptides β30-31 β30-31 and and β32-33 β32-33 interact in vitro with: interact in vitro with: •human mt-tRNAhuman mt-tRNALeu(UUR) Leu(UUR) with high affinity;with high affinity;•m.3243A>G mutant, with lower affinity; m.3243A>G mutant, with lower affinity; • human mt-tRNAhuman mt-tRNALysLys

Peptides Peptides β30-31β30-31 and and β32-33 β32-33 restore the stability and restore the stability and structure of mt-tRNAstructure of mt-tRNALeu(UUR)Leu(UUR),, which are strongly impaired which are strongly impaired by the m.3243A>G mutationby the m.3243A>G mutation

Short peptides derived from LeuRS-Cterm represent attractive new candidates for future therapeutic applications against syndromes associated with mt-tRNA mutation.

Transfer of these results to the clinical setting requires the following studies to be performed.a. Implementation of a system to deliver the β30-31 and β32-33 peptides to the mt matrix where they can perform their therapeutic action by directly interacting with mutated mt-tRNA molecules. The following strategies are currently under investigation:i. conjugation of β30-31 or β32-33 peptide to protein-based nanoparticles endowed with specific mt targeting moieties;ii. delivery of DNA sequences coding for the peptides via appropriate vectors (gene therapy).b. Investigation of the toxicity and pharmacokinetic properties of the developed systems in vivo.c. Assessment of the rescuing activity of the developed systems in animal models, when available.

Carla Giordano Elena Perli Annalinda Pisano Arianna Montanari Carmela PreziusoVeronica Morea Gianni Colotti Annarita Fiorillo Patrizio Di Micco Pierpaolo Ceci Annarita Fiorillo Antonio Campese Laura Frontali Silvia Francisci

Massimo ZevianiAurelio Reyes

Robert W TaylorHelen Tuppen