Overexpression of Twinkle-helicase protects cardiomyocytes from genotoxic stress caused by reactive oxygen species.
ABSTRACT: Mitochondrial DNA (mtDNA) in adult human heart is characterized by complex molecular forms held together by junctional molecules of unknown biological significance. These junctions are not present in mouse hearts and emerge in humans during postnatal development, concomitant with increased demand for oxidative metabolism. To analyze the role of mtDNA organization during oxidative stress in cardiomyocytes, we used a mouse model, which recapitulates the complex mtDNA organization of human hearts by overexpression of the mitochondrial helicase, TWINKLE. Overexpression of TWINKLE rescued the oxidative damage induced replication stalling of mtDNA, reduced mtDNA point mutation load, and modified mtDNA rearrangements in heterozygous mitochondrial superoxide dismutase knockout hearts, as well as ameliorated cardiomyopathy in mice superoxide dismutase knockout in a p21-dependent manner. We conclude that mtDNA integrity influences cell survival and reason that tissue specific modes of mtDNA maintenance represent an adaptation to oxidative stress.
Project description:Mammalian mitochondria contain a circular genome (mtDNA) which encodes subunits of the oxidative phosphorylation machinery. The replication and maintenance of mtDNA is carried out by a set of nuclear-encoded factors-of which, helicases form an important group. The TWINKLE helicase is the main helicase in mitochondria and is the only helicase required for mtDNA replication. Mutations in TWINKLE cause a number of human disorders associated with mitochondrial dysfunction, neurodegeneration and premature ageing. In addition, a number of other helicases with a putative role in mitochondria have been identified. In this review, we discuss our current knowledge of TWINKLE structure and function and its role in diseases of mtDNA maintenance. We also briefly discuss other potential mitochondrial helicases and postulate on their role(s) in mitochondria.
Project description:Newly synthesized mitochondrial RNA is concentrated in structures juxtaposed to nucleoids, called RNA granules, that have been implicated in mitochondrial RNA processing and ribosome biogenesis. Here we show that two classical mtDNA replication factors, the mtDNA helicase Twinkle and single-stranded DNA-binding protein mtSSB, contribute to RNA metabolism in mitochondria and to RNA granule biology. Twinkle colocalizes with both mitochondrial RNA granules and nucleoids, and it can serve as bait to greatly enrich established RNA granule proteins, such as G-rich sequence factor 1, GRSF1. Likewise, mtSSB also is not restricted to the nucleoids, and repression of either mtSSB or Twinkle alters mtRNA metabolism. Short-term Twinkle depletion greatly diminishes RNA granules but does not inhibit RNA synthesis or processing. Either mtSSB or GRSF1 depletion results in RNA processing defects, accumulation of mtRNA breakdown products as well as increased levels of dsRNA and RNA:DNA hybrids. In particular, the processing and degradation defects become more pronounced with both proteins depleted. These findings suggest that Twinkle is essential for RNA organization in granules, and that mtSSB is involved in the recently proposed GRSF1-mtRNA degradosome pathway, a route suggested to be particularly aimed at degradation of G-quadruplex prone long non-coding mtRNAs.
Project description:<h4>Background</h4>Mitochondrial DNA (mtDNA) copy number decreases in animal and human heart failure (HF), yet its role in cardiomyocytes remains to be elucidated. Thus, we investigated the cardioprotective function of increased mtDNA copy number resulting from the overexpression of human transcription factor A of mitochondria (TFAM) or Twinkle helicase in volume overload (VO)-induced HF.<h4>Methods and results</h4>Two strains of transgenic (TG) mice, one overexpressing TFAM and the other overexpressing Twinkle helicase, exhibit an approximately 2-fold equivalent increase in mtDNA copy number in heart. These TG mice display similar attenuations in eccentric hypertrophy and improved cardiac function compared to wild-type (WT) mice without any deterioration of mitochondrial enzymatic activities in response to VO, which was accompanied by a reduction in matrix-metalloproteinase (MMP) activity and reactive oxygen species after 8 weeks of VO. Moreover, acute VO-induced MMP-2 and MMP-9 upregulation was also suppressed at 24 h in both TG mice. In isolated rat cardiomyocytes, mitochondrial reactive oxygen species (mitoROS) upregulated MMP-2 and MMP-9 expression, and human TFAM (hTFAM) overexpression suppressed mitoROS and their upregulation. Additionally, mitoROS were equally suppressed in H9c2 rat cardiomyoblasts that overexpress hTFAM or rat Twinkle, both of which exhibit increased mtDNA copy number. Furthermore, mitoROS and mitochondrial protein oxidation from both TG mice were suppressed compared to WT mice.<h4>Conclusions</h4>The overexpression of TFAM or Twinkle results in increased mtDNA copy number and facilitates cardioprotection associated with limited mitochondrial oxidative stress. Our findings suggest that increasing mtDNA copy number could be a useful therapeutic strategy to target mitoROS in HF.
Project description:Mutations in the c10orf2 gene encoding the human mitochondrial DNA replicative helicase Twinkle are linked to several rare genetic diseases characterized by mitochondrial defects. In this study, we have examined the catalytic activity of Twinkle helicase on model replication fork and DNA repair structures. Although Twinkle behaves as a traditional 5' to 3' helicase on conventional forked duplex substrates, the enzyme efficiently dissociates D-loop DNA substrates irrespective of whether it possesses a 5' or 3' single-stranded tailed invading strand. In contrast, we report for the first time that Twinkle branch-migrates an open-ended mobile three-stranded DNA structure with a strong 5' to 3' directionality preference. To determine how well Twinkle handles potential roadblocks to mtDNA replication, we tested the ability of the helicase to unwind substrates with site-specific oxidative DNA lesions or bound by the mitochondrial transcription factor A. Twinkle helicase is inhibited by DNA damage in a unique manner that is dependent on the type of oxidative lesion and the strand in which it resides. Novel single molecule FRET binding and unwinding assays show an interaction of the excluded strand with Twinkle as well as events corresponding to stepwise unwinding and annealing. TFAM inhibits Twinkle unwinding, suggesting other replisome proteins may be required for efficient removal. These studies shed new insight on the catalytic functions of Twinkle on the key DNA structures it would encounter during replication or possibly repair of the mitochondrial genome and how well it tolerates potential roadblocks to DNA unwinding.
Project description:Mutations in the mitochondrial helicase Twinkle underlie autosomal dominant progressive external ophthalmoplegia (PEO), as well as recessively inherited infantile-onset spinocerebellar ataxia and rare forms of mitochondrial DNA (mtDNA) depletion syndrome. Familial PEO is typically associated with the occurrence of multiple mtDNA deletions, but the mechanism by which Twinkle dysfunction induces deletion formation has been under debate. Here we looked at the effects of Twinkle adPEO mutations in human cell culture and studied the mtDNA replication in the Deletor mouse model, which expresses a dominant PEO mutation in Twinkle and accumulates multiple mtDNA deletions during life. We show that expression of dominant Twinkle mutations results in the accumulation of mtDNA replication intermediates in cell culture. This indicated severe replication pausing or stalling and caused mtDNA depletion. A strongly enhanced accumulation of replication intermediates was evident also in six-week-old Deletor mice compared with wild-type littermates, even though mtDNA deletions accumulate in a late-onset fashion in this model. In addition, our results in cell culture pointed to a problem of transcription that preceded the mtDNA depletion phenotype and might be of relevance in adPEO pathophysiology. Finally, in vitro assays showed functional defects in the various Twinkle mutants and broadly agreed with the cell culture phenotypes such as the level of mtDNA depletion and the level of accumulation of replication intermediates. On the basis of our results we suggest that mtDNA replication pausing or stalling is the common consequence of Twinkle PEO mutations that predisposes to multiple deletion formation.
Project description:Myocardial mitochondrial DNA (mtDNA) copy number decreases in heart failure. In post-myocardial infarction mice, increasing mtDNA copy number by overexpressing mitochondrial transcription factor attenuates mtDNA deficiency and ameliorates pathological remodeling thereby markedly improving survival. However, the functional significance of increased mtDNA copy number in hypertensive heart disease remains unknown. We addressed this question using transgenic mice that overexpress Twinkle helicase (Twinkle; Tg), the mtDNA helicase, and examined whether Twinkle overexpression protects the heart from left ventricular (LV) remodeling and failure after pressure overload created by transverse aortic constriction (TAC). Twinkle overexpression increased mtDNA copy number by 2.2 ± 0.1-fold. Heart weight, LV diastolic volume and wall thickness were comparable between Tg and wild type littermates (WT) at 28 days after TAC operation. LV end-diastolic pressure increased in WT after TAC (8.6 ± 2.8 mmHg), and this increase was attenuated in Tg (4.6 ± 2.6 mmHg). Impaired LV fractional shortening after TAC operation was also suppressed in Tg, as measured by echocardiography (WT: 16.2 ± 7.2% vs Tg: 20.7 ± 6.2%). These LV functional improvements were accompanied by a decrease in interstitial fibrosis (WT: 10.6 ± 1.1% vs Tg: 3.0 ± 0.6%). In in vitro studies, overexpressing Twinkle using an adenovirus vector in cultured cardiac fibroblasts significantly suppressed mRNA of collagen 1a, collagen 3a and connective tissue growth factor, and angiotensin II-induced transforming growth factor ?1 expression. The findings suggest that Twinkle overexpression prevents LV function deterioration. In conclusion, Twinkle overexpression increases mtDNA copy number and ameliorates the progression of LV fibrosis and heart failure in a mouse pressure overload model. Increasing mtDNA copy number by Twinkle overexpression could be a novel therapeutic strategy for hypertensive heart disease.
Project description:Mitochondrial DNA replication is performed by a simple machinery, containing the TWINKLE DNA helicase, a single-stranded DNA-binding protein, and the mitochondrial DNA polymerase ?. In addition, mitochondrial RNA polymerase is required for primer formation at the origins of DNA replication. TWINKLE adopts a hexameric ring-shaped structure that must load on the closed circular mtDNA genome. In other systems, a specialized helicase loader often facilitates helicase loading. We here demonstrate that TWINKLE can function without a specialized loader. We also show that the mitochondrial replication machinery can assemble on a closed circular DNA template and efficiently elongate a DNA primer in a manner that closely resembles initiation of mtDNA synthesis in vivo.
Project description:The mitochondrial replicative helicase Twinkle is involved in strand separation at the replication fork of mitochondrial DNA (mtDNA). Twinkle malfunction is associated with rare diseases that include late onset mitochondrial myopathies, neuromuscular disorders and fatal infantile mtDNA depletion syndrome. We examined its 3D structure by electron microscopy (EM) and small angle X-ray scattering (SAXS) and built the corresponding atomic models, which gave insight into the first molecular architecture of a full-length SF4 helicase that includes an N-terminal zinc-binding domain (ZBD), an intermediate RNA polymerase domain (RPD) and a RecA-like hexamerization C-terminal domain (CTD). The EM model of Twinkle reveals a hexameric two-layered ring comprising the ZBDs and RPDs in one layer and the CTDs in another. In the hexamer, contacts in trans with adjacent subunits occur between ZBDs and RPDs, and between RPDs and CTDs. The ZBDs show important structural heterogeneity. In solution, the scattering data are compatible with a mixture of extended hexa- and heptameric models in variable conformations. Overall, our structural data show a complex network of dynamic interactions that reconciles with the structural flexibility required for helicase activity.
Project description:Defects of mitochondrial DNA (mtDNA) maintenance have recently been associated with inherited neurodegenerative and muscle diseases and the aging process. Twinkle is a nuclear-encoded mtDNA helicase, dominant mutations of which cause adult-onset progressive external ophthalmoplegia (PEO) with multiple mtDNA deletions. We have generated transgenic mice expressing mouse Twinkle with PEO patient mutations. Multiple mtDNA deletions accumulate in the tissues of these mice, resulting in progressive respiratory dysfunction and chronic late-onset mitochondrial disease starting at 1 year of age. The muscles of the mice faithfully replicate all of the key histological, genetic, and biochemical features of PEO patients. Furthermore, the mice have progressive deficiency of cytochrome c oxidase in distinct neuronal populations. These "deletor" mice do not, however, show premature aging, indicating that subtle accumulation of mtDNA deletions and progressive respiratory chain dysfunction are not sufficient to accelerate aging. This model is a valuable tool for therapy development and testing for adult-onset mitochondrial disorders.
Project description:TWINKLE is the helicase involved in replication and maintenance of mitochondrial DNA (mtDNA) in mammalian cells. Structurally, TWINKLE is closely related to the bacteriophage T7 gp4 protein and comprises a helicase and primase domain joined by a flexible linker region. Mutations in and around this linker region are responsible for autosomal dominant progressive external ophthalmoplegia (adPEO), a neuromuscular disorder associated with deletions in mtDNA. The underlying molecular basis of adPEO-causing mutations remains unclear, but defects in TWINKLE oligomerization are thought to play a major role. In this study, we have characterized these disease variants by single-particle electron microscopy and can link the diminished activities of the TWINKLE variants to altered oligomeric properties. Our results suggest that the mutations can be divided into those that (i) destroy the flexibility of the linker region, (ii) inhibit ring closure and (iii) change the number of subunits within a helicase ring. Furthermore, we demonstrate that wild-type TWINKLE undergoes large-scale conformational changes upon nucleoside triphosphate binding and that this ability is lost in the disease-causing variants. This represents a substantial advancement in the understanding of the molecular basis of adPEO and related pathologies and may aid in the development of future therapeutic strategies.