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Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large-scale deletions or point mutations.

ABSTRACT: We designed and engineered mitochondrially targeted obligate heterodimeric zinc finger nucleases (mtZFNs) for site-specific elimination of pathogenic human mitochondrial DNA (mtDNA). We used mtZFNs to target and cleave mtDNA harbouring the m.8993T>G point mutation associated with neuropathy, ataxia, retinitis pigmentosa (NARP) and the "common deletion" (CD), a 4977-bp repeat-flanked deletion associated with adult-onset chronic progressive external ophthalmoplegia and, less frequently, Kearns-Sayre and Pearson's marrow pancreas syndromes. Expression of mtZFNs led to a reduction in mutant mtDNA haplotype load, and subsequent repopulation of wild-type mtDNA restored mitochondrial respiratory function in a CD cybrid cell model. This study constitutes proof-of-principle that, through heteroplasmy manipulation, delivery of site-specific nuclease activity to mitochondria can alleviate a severe biochemical phenotype in primary mitochondrial disease arising from deleted mtDNA species.

SUBMITTER: Gammage PA

PROVIDER: S-EPMC3992073 | biostudies-literature | 2014 Apr

REPOSITORIES: biostudies-literature

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Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large-scale deletions or point mutations.

Gammage Payam A PA Rorbach Joanna J Vincent Anna I AI Rebar Edward J EJ Minczuk Michal M

EMBO molecular medicine 20140224 4

We designed and engineered mitochondrially targeted obligate heterodimeric zinc finger nucleases (mtZFNs) for site-specific elimination of pathogenic human mitochondrial DNA (mtDNA). We used mtZFNs to target and cleave mtDNA harbouring the m.8993T>G point mutation associated with neuropathy, ataxia, retinitis pigmentosa (NARP) and the "common deletion" (CD), a 4977-bp repeat-flanked deletion associated with adult-onset chronic progressive external ophthalmoplegia and, less frequently, Kearns-Say ...[more]

PMID: 24567072

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Project description:Targeting the tumor suppressor p53 to the mitochondria triggers a rapid apoptotic response as efficiently as transcription-dependent p53. (1, 2) p53 forms a complex with the antiapoptotic Bcl-XL, which leads to Bak and Bax oligomerization resulting in apoptosis via mitochondrial outer membrane permeabilization. (3, 4) Although p53 performs its main role in the mitochondrial outer membrane, it also interacts with different proteins in the mitochondrial inner membrane and matrix. (5, 6) To further investigate mitochondrial activity of p53, EGFP-p53 was fused to different mitochondrial targeting signals (MTSs) directing it to the mitochondrial outer membrane ("XL-MTS" from Bcl-XL; "TOM-MTS" from TOM20), the inner membrane ("CCO-MTS" from cytochrome c oxidase), or matrix ("OTC-MTS" from ornithine transcarbamylase). Fluorescence microscopy and a p53 reporter dual luciferase assay demonstrated that fusing MTSs to p53 increased mitochondrial localization and nuclear exclusion depending on which MTS was used. To examine if the MTSs initiate mitochondrial damage, we fused each individual MTS to EGFP (a nontoxic protein) as negative controls. We performed caspase-9, TUNEL, annexin-V, and 7-AAD apoptosis assays on T47D breast cancer cells transfected with mitochondrial constructs. Except for EGFP-XL, apoptotic potential was observed in all MTS-EGFP-p53 and MTS-EGFP constructs. In addition, EGFP-p53-XL showed the greatest significant increase in programmed cell death compared to its nontoxic MTS control (EGFP-XL). The apoptotic mechanism for each construct was further investigated using pifithrin-? (an inhibitor of p53 transcriptional activity), pifithrin-? (a small molecule that reduces binding of p53 to Bcl-2 and Bcl-XL), and overexpressing the antiapoptotic Bcl-XL. Unlike the MTSs from TOM, CCO, and OTC, which showed different apoptotic mechanisms, we conclude that p53 fused to the MTS from Bcl-XL performs its apoptotic potential exclusively through the p53/Bcl-XL specific pathway.

Dataset Information

Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large-scale deletions or point mutations.

Publications

Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large-scale deletions or point mutations.

Similar Datasets

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