Structure–Function Defects of the TWINKLE Linker Region in Progressive External Ophthalmoplegia

Korhonen, Jenny; Pande, Vineet; Holmlund, Teresa; Farge, Géraldine; Pham, Xuan Hoi; Nilsson, Lennart; Falkenberg, Maria

doi:10.1016/j.jmb.2008.01.035

Cited by 58 publications

(100 citation statements)

References 44 publications

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“…The demonstrated DNA helicase activities of the W315L, K319E, I367T, S369P, R374Q, and L381P variants (Fig. 4C) disagree with published reports that these amino acid substitutions significantly impair or even inactivate helicase function (13,24,29). A, the ability of WT p72 to unwind a forked oligonucleotide substrate was determined as described under "Experimental Procedures," except that reactions contained 3 (lanes 1-4), 6 (lanes 5-8), 12 (lanes 9 -12), or 20 nM (lanes 13-16) WT p72 hexamers.…”

Section: Resultscontrasting

confidence: 93%

“…Wild type p72 exhibited a K d of 5.7 Ϯ 2.5 nM on our single-stranded DNA substrate and 4.9 Ϯ 1.9 nM on our double-stranded DNA substrate. Both of these values are in good agreement with the ϳ5 nM affinity of p72 hexamers for 30-nt single-stranded or double-stranded oligonucleotide substrates or for an oligo(dT) 30 homopolymer, as estimated by electrophoretic mobility shift assay (24,25). The replicative DNA helicase of bacteriophage T7, gp4, also exhibits a similar K d (DNA) of 6.4 nM for a singlestranded 15-mer oligonucleotide in a filter binding assay (26).…”

Section: Resultssupporting

confidence: 76%

“…In the present work, we also found the affinity of the S369P variant to be higher than that of the WT enzyme for both single-stranded DNA and double-stranded DNA (Table 2). Similarly, singlestranded DNA binding by the R374Q and W315L forms is reported to be both deficient (24,29) and proficient (13) by EMSA. Although the current work concurs with the near WT binding affinity previously observed for the A359T (13,24) and R334Q (29) variants, the weak DNA binding reported for the K319E (13), L381P and I367T (24), and P335L (29) variants is difficult to reconcile with the high affinity binding that we observed (Table 1).…”

Section: Discussionmentioning

confidence: 97%

“…Previous strategies for recombinant p72 have included incorporation of single or dual affinity tags to facilitate rapid purification or supplementing buffers with glycerol, NaCl or 0.1 M arginine to stabilize the protein (11, 13, 24, 31). Nevertheless, preparation of certain adPEO variants has proven difficult, such as the inability to make R354P p72 in soluble form with baculovirus-infected insect cells (24). To establish an E. coli expression system for recombinant p72, we surveyed a variety of conditions by trial and error to maximize expression, solubility, and activity of the protein.…”

Section: Discussionmentioning

confidence: 99%

“…For example, published reports of the DNA binding strength of certain mutant forms of p72 are inconsistent. As determined by electrophoretic mobility shift assay (EMSA), the S369P protein was reported to have low affinity for single-stranded DNA relative to the wild type enzyme (24). However, another group reported higher than wild type DNA binding affinity for this protein using comparable experimental conditions (13).…”

Section: Discussionmentioning

confidence: 99%

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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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Section: Resultscontrasting

confidence: 93%

Section: Resultssupporting

confidence: 76%

Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

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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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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In Vitro Assays of TWINKLE Function

Uhler

Alexandersson

Falkenberg

2023

Methods in Molecular Biology

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Structure–Function Defects of the TWINKLE Linker Region in Progressive External Ophthalmoplegia

Cited by 58 publications

References 44 publications

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

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

In Vitro Assays of TWINKLE Function

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