Differential Phenotypes of Active Site and Human Autosomal Dominant Progressive External Ophthalmoplegia Mutations in Drosophila Mitochondrial DNA Helicase Expressed in Schneider Cells

Matsushima, Yohkazu; Kaguni, Laurie S.

doi:10.1074/jbc.m610550200

Cited by 40 publications

(86 citation statements)

References 37 publications

(45 reference statements)

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“…2A). Even in the uninduced condition, the cells carrying pMt/invLon/ Hy suppressed expression of Lon by >10-fold, most likely due to leaky expression (32)(33)(34)(35). In contrast, expression of β-tubulin was unchanged.…”

Section: Resultsmentioning

confidence: 97%

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Mitochondrial Lon protease regulates mitochondrial DNA copy number and transcription by selective degradation of mitochondrial transcription factor A (TFAM)

Matsushima

Goto

Kaguni

2010

Proc. Natl. Acad. Sci. U.S.A.

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247

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Lon is the major protease in the mitochondrial matrix in eukaryotes, and is well conserved among species. Although a role for Lon in mitochondrial biogenesis has been proposed, the mechanistic basis is unclear. Here, we demonstrate a role for Lon in mtDNA metabolism. An RNA interference (RNAi) construct was designed that reduces Lon to less than 10% of its normal level in Drosophila Schneider cells. RNAi knockdown of Lon results in increased abundance of mitochondrial transcription factor A (TFAM) and mtDNA copy number. In a corollary manner, overexpression of Lon reduces TFAM levels and mtDNA copy number. Notably, induction of mtDNA depletion in Lon knockdown cells does not result in degradation of TFAM, thereby causing a dramatic increase in the TFAM∶mtDNA ratio. The increased TFAM∶mtDNA ratio in turn causes inhibition of mitochondrial transcription. We conclude that Lon regulates mitochondrial transcription by stabilizing the mitochondrial TFAM∶ mtDNA ratio via selective degradation of TFAM.AAA+ protease | mtDNA maintenance | quality control

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Section: Resultsmentioning

confidence: 97%

“…Southern blotting was performed as described previously (32), and the data were analyzed using a PhosphoImager (Molecular Dynamics). Blots were probed with radiolabeled DNAs for the mitochondrial gene Cytb and the nuclear histone gene cluster.…”

Section: Methodsmentioning

confidence: 99%

Mitochondrial Lon protease regulates mitochondrial DNA copy number and transcription by selective degradation of mitochondrial transcription factor A (TFAM)

Matsushima

Goto

Kaguni

2010

Proc. Natl. Acad. Sci. U.S.A.

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247

214

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“…In fact, evaluation of mtDNA copy number in cultured Schneider cells following overexpression of Drosophila mtDNA helicase genes bearing missense mutations suggested that heterohexamers with only one or two mutant subunits retained some helicase activity in vivo (12,42). However, targeted mutations that substantially inactivate catalytic function may prove enlightening in subunit mixing experiments.…”

Section: Discussionmentioning

confidence: 99%

“…The C10orf2 gene product dynamically colocalizes with mtDNA in nucleoprotein structures known as mitochondrial nucleoids (5,9), and knocking down expression of C10orf2 by RNAi results in the rapid decrease in mtDNA copy number in cultured human osteosarcoma (143B) cells (10). Overexpression of catalytic mutants and dominant disease variants of the mtDNA helicase in cultured human or Schneider cells results in stalled mtDNA replication or depletion of mtDNA (11)(12)(13), which emulates the disease state. However, only two of five adPEO mutants exhibited a dominant negative phenotype with mtDNA depletion in Schneider cells (12).…”

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confidence: 99%

“…Overexpression of catalytic mutants and dominant disease variants of the mtDNA helicase in cultured human or Schneider cells results in stalled mtDNA replication or depletion of mtDNA (11)(12)(13), which emulates the disease state. However, only two of five adPEO mutants exhibited a dominant negative phenotype with mtDNA depletion in Schneider cells (12). Overexpression of two adPEO variants in a mouse transgenic model recapitulated many of the symptoms observed in human PEO, including accumulation of multiple mtDNA deletions, progressive respiratory dysfunction, and cytochrome c oxidase deficiency (14,15).…”

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Disease Variants of the Human Mitochondrial DNA Helicase Encoded by C10orf2 Differentially Alter Protein Stability, Nucleotide Hydrolysis, and Helicase Activity

Longley

Humble

Sharief

et al. 2010

Journal of Biological Chemistry

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Missense mutations in the human C10orf2 gene, encoding the mitochondrial DNA (mtDNA) helicase, co-segregate with mitochondrial diseases such as adult-onset progressive external ophthalmoplegia, hepatocerebral syndrome with mtDNA depletion syndrome, and infantile-onset spinocerebellar ataxia. To understand the biochemical consequences of C10orf2 mutations, we overproduced wild type and 20 mutant forms of human mtDNA helicase in Escherichia coli and developed novel schemes to purify the recombinant enzymes to near homogeneity. A combination of molecular crowding, non-ionic detergents, Mg 2؉ ions, and elevated ionic strength was required to combat insolubility and intrinsic instability of certain mutant variants. A systematic biochemical assessment of the enzymes included analysis of DNA binding affinity, DNA helicase activity, the kinetics of nucleotide hydrolysis, and estimates of thermal stability. In contrast to other studies, we found that all 20 mutant variants retain helicase function under optimized in vitro conditions despite partial reductions in DNA binding affinity, nucleotide hydrolysis, or thermal stability for some mutants. Such partial defects are consistent with the delayed presentation of mitochondrial diseases associated with mutation of C10orf2.Chronic disruption of mitochondrial function can result in a broad array of neuromuscular degenerative disorders known as mitochondrial diseases, including progressive external ophthalmoplegia (PEO), 2 Alpers syndrome, parkinsonism, and several complex ataxia neuropathy syndromes (1-3). A heritable form of PEO was first mapped to a chromosomal interval near position 10q24 in a Finnish pedigree (4). One molecular hallmark of PEO was the age-dependent accumulation of mtDNA deletions in somatic tissues of affected individuals, which suggested that this locus was needed for replication or maintenance of mtDNA (4). In 2001, autosomal dominant PEO with mtDNA deletions was shown to co-segregate with 11 different missense mutations in the C10orf2 gene (5), which encoded a protein containing predicted amino acid sequences homologous to portions of the bacteriophage T7 gene product 4 and other superfamily 4 DNA helicases (5, 6). Since that time, numerous reports have identified 23 additional missense mutations in C10orf2 associated with heritable mitochondrial diseases such as adPEO, hepatocerebral mtDNA depletion syndrome (MDS), and infantile-onset spinocerebellar ataxia (IOSCA) (7, 8) (see Fig. 1).Several lines of evidence more directly link C10orf2 function to maintenance of mtDNA. The C10orf2 gene product dynamically colocalizes with mtDNA in nucleoprotein structures known as mitochondrial nucleoids (5, 9), and knocking down expression of C10orf2 by RNAi results in the rapid decrease in mtDNA copy number in cultured human osteosarcoma (143B) cells (10). Overexpression of catalytic mutants and dominant disease variants of the mtDNA helicase in cultured human or Schneider cells results in stalled mtDNA replication or depletion of mtDNA (11-13), which emulates the...

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Clinical phenotype of autosomal dominant progressive external ophthalmoplegia in a family with a novel mutation in the C10orf2 gene

Hong

Yao

et al. 2009

Muscle and Nerve

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Autosomal dominant progressive external ophthalmoplegia (adPEO) is a mitochondrial disorder caused by mutations in nuclear genes. Here we report the clinical and genetic features of adPEO in a Chinese family. All patients had gradual onset of ptosis, with or without ophthalmoplegia, around age 30. Thirteen patients had limb weakness around age 40. Eight patients developed dysphagia around age 50. Four patients died of cardiac abnormalities around age 60. Muscle biopsy of the proband indicated mitochondrial myopathy characterized by ragged-red fibers, cytochrome c oxidase-negative fibers, and multiple deletions of mitochondrial DNA. A heterozygous missense mutation of c.1342A>G in the C10orf2 gene resulting in the p.448N>D mutation in the protein was found in the proband and four other affected family members. In summary, we identified an adPEO family with a novel C10orf2 gene mutation that manifested an age-dependent phenotype. It suggests that greater attention must be paid to cardiac abnormalities in the late stages of this disease.

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Differential Phenotypes of Active Site and Human Autosomal Dominant Progressive External Ophthalmoplegia Mutations in Drosophila Mitochondrial DNA Helicase Expressed in Schneider Cells

Cited by 40 publications

References 37 publications

Mitochondrial Lon protease regulates mitochondrial DNA copy number and transcription by selective degradation of mitochondrial transcription factor A (TFAM)

Mitochondrial Lon protease regulates mitochondrial DNA copy number and transcription by selective degradation of mitochondrial transcription factor A (TFAM)

Disease Variants of the Human Mitochondrial DNA Helicase Encoded by C10orf2 Differentially Alter Protein Stability, Nucleotide Hydrolysis, and Helicase Activity

Clinical phenotype of autosomal dominant progressive external ophthalmoplegia in a family with a novel mutation in the C10orf2 gene

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