Overexpression of Twinkle-helicase protects cardiomyocytes from genotoxic stress caused by reactive oxygen species

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.Mammalian mitochondrial DNA (mtDNA) is a 16.5-kb circular double-stranded molecule that exists in thousands of copies per cell. It is essential for ATP production in mitochondria because it encodes 13 subunits of the protein complexes required for oxidative phosphorylation, as well as tRNAs and rRNAs necessary for mitochondrial protein biosynthesis. Mitochondria are especially abundant in the heart, the most energy demanding tissue in the mammalian body. Efficient mitochondrial activity is essential for normal heart function and embryonic development (1).The majority of O₂ in mitochondria is consumed by complex IV of the electron transport chain (ETC) during controlled reduction of O₂ to water. However, because of electron leaks at complexes I and III, some oxygen molecules are reduced to superoxide anions (O₂⁻), which, in turn, are converted to H₂O₂ by mitochondrial superoxide dismutase (SOD2) and further into water in a reaction facilitated by catalase (2, 3). Both O₂⁻ and H₂O₂ can react to form highly destructive OH· radicals, and all three are therefore commonly referred to as reactive oxygen species (ROS). Mitochondrial ROS directly damage mtDNA, oxidize disulfides in proteins, and cause peroxidation of membrane fatty acids (4, 5). Oxidative damage has been suggested to be a major source of somatic mtDNA mutations because it cross-links DNA and causes nucleotide modifications as well as single- and double-strand DNA breaks (6). The importance of ROS damage specifically to the heart is particularly evident in Sod2 knockout mice. Complete lack of SOD2 in homozygous knockout mice results in early postnatal lethality, whereas reduction of SOD2 activity in heterozygous (Sod2^+/−) mice causes dilated cardiomyopathy during aging (7, 8). Other tissues in Sod2^+/− mice are not markedly affected under physiological conditions with the exception of an increased rate of tumor formation in aged mice (9).Oxidative mtDNA damage increases during postnatal heart development in rats when mitochondrial biogenesis is up-regulated and a metabolic switch from carbohydrate metabolism to β-oxidation of fatty acids occurs (10). In rodents, the developmental increase in ROS exposure temporarily elicits mtDNA repair responses but does not lead to major changes in mtDNA topology or replication during aging (10). In contrast, human heart mtDNA topology changes considerably during postnatal development (10). Although adult human heart mtDNA is organized in complex networks and shows high levels of junctional molecules, the mtDNA organization in the hearts of newborn babies is simple, resembling the situation in rodents (11–13).Although four-way junctions and complex mtDNA molecules are not present at detectable levels in normal mouse heart, they can be induced by transgenic overexpression of the TWINKLE helicase (12). Besides being necessary for the maintenance of four-way junctions in human heart (10), TWINKLE also possesses strand-annealing activity in vitro, making it an attractive candidate conferring mitochondrial recombination activity (14).We have hypothesized previously that enhanced recombination protects human heart mtDNA from chronic ROS exposure during long lifetime (11, 15). This view is also supported by the acquisition of complex mtDNA organization in postnatal human hearts concomitant with the increase in oxidative metabolism (12, 13) and ROS-dependent activation of recombination-dependent replication (RDR) in yeast (16). To test whether the mtDNA organization seen in human hearts protects against ROS, we took advantage of TWINKLE overexpressing (Tw⁺) mice, which recapitulate the structural phenotype of human heart mtDNA and crossed them with heterozygous Sod2^+/− mice. We found that TWINKLE overexpression essentially eliminated the elevated mtDNA mutation load in Sod2^+/− mouse hearts, changed the type of mtDNA rearrangements, and rescued cardiomyopathy in Sod2^+/− mice, most likely by preventing apoptosis of cardiomyocytes via p21-dependent signaling. Our results indicate that TWINKLE maintains mtDNA integrity, hence promoting cardiomyocyte survival.     

Palmitoyl acyltransferase Aph2 in cardiac function and the development of cardiomyopathy

Protein palmitoylation regulates many aspects of cell function and is carried out by acyl transferases that contain zf-DHHC motifs. The in vivo physiological function of protein palmitoylation is largely unknown. Here we generated mice deficient in the acyl transferase Aph2 (Ablphilin 2 or zf-DHHC16) and demonstrated an essential role for Aph2 in embryonic/postnatal survival, eye development, and heart development. Aph2^−/− embryos and pups showed cardiomyopathy and cardiac defects including bradycardia. We identified phospholamban, a protein often associated with human cardiomyopathy, as an interacting partner and a substrate of Aph2. Aph2-mediated palmitoylation of phospholamban on cysteine 36 differentially alters its interaction with PKA and protein phosphatase 1 α, augmenting serine 16 phosphorylation, and regulates phospholamban pentamer formation. Aph2 deficiency results in phospholamban hypophosphorylation, a hyperinhibitory form. Ablation of phospholamban in Aph2^−/− mice histologically and functionally alleviated the heart defects. These findings establish Aph2 as a critical in vivo regulator of cardiac function and reveal roles for protein palmitoylation in the development of other organs including eyes.Protein S-acylation on cysteine residues by palmitate regulates substrate protein localization, trafficking, and protein–protein interactions (1–3) and could potentially play important roles in vivo (4–9). Palmitoylation is the only reversible lipid modification that can be removed by protein palmitoyl thioesterases. Recently, several palmitoyl acyltransferases (PATs) containing a unique zinc finger domain called zf-DHHC have been identified (10). Comparative genome searches have uncovered 23 PAT-like proteins in mammals (11). However, the physiological function of these PATs remains poorly understood (4).Heart disease is a leading cause of morbidity and mortality worldwide (12). Recent studies have established a pivotal role for defects of calcium cycling in the onset of heart disease (12–16), with a particularly critical role for the regulation of the sarcoplasmic reticulum (SR) calcium ATPase (SERCA). SERCA2a activity is controlled by phospholamban (PLN), an abundant SR protein. Upon β-adrenergic signaling, PKA can phosphorylate serine 16 of PLN, leading to PLN pentamer formation and loss of ability to inhibit SERCA2a (13, 14, 17). Ser16 phosphorylation is removed by phosphatases such as protein phosphatase 1 α (PP1α) (18, 19).It has been reported that some cardiac proteins are palmitoylated, including Na pump regulatory subunit phospholemman and β1-adrenergic receptor (20–22). However, there is a lack of genetic evidence that protein palmitoylation plays a role in heart development and function. Here we have taken a reverse genetic approach to study the physiological function of Aph2 (Ablphilin 2), first identified as an interacting protein of nonreceptor tyrosine kinase c-Abl (23), and provide direct evidence that Aph2 is a PAT, with PLN as one substrate, and that Aph2 is essential for embryonic/postnatal survival, eye development, proper embryonic heart development, and in vivo cardiac function. Some of the heart phenotypes can be rescued by PLN deficiency. Thus, Aph2-deficient mice represent an animal model for cardiomyopathy, whose pathogenesis involves defective PLN palmitoylation.     

Target specificity among canonical nuclear poly(A) polymerases in plants modulates organ growth and pathogen response

Polyadenylation of pre-mRNAs is critical for efficient nuclear export, stability, and translation of the mature mRNAs, and thus for gene expression. The bulk of pre-mRNAs are processed by canonical nuclear poly(A) polymerase (PAPS). Both vertebrate and higher-plant genomes encode more than one isoform of this enzyme, and these are coexpressed in different tissues. However, in neither case is it known whether the isoforms fulfill different functions or polyadenylate distinct subsets of pre-mRNAs. Here we show that the three canonical nuclear PAPS isoforms in Arabidopsis are functionally specialized owing to their evolutionarily divergent C-terminal domains. A strong loss-of-function mutation in PAPS1 causes a male gametophytic defect, whereas a weak allele leads to reduced leaf growth that results in part from a constitutive pathogen response. By contrast, plants lacking both PAPS2 and PAPS4 function are viable with wild-type leaf growth. Polyadenylation of SMALL AUXIN UP RNA (SAUR) mRNAs depends specifically on PAPS1 function. The resulting reduction in SAUR activity in paps1 mutants contributes to their reduced leaf growth, providing a causal link between polyadenylation of specific pre-mRNAs by a particular PAPS isoform and plant growth. This suggests the existence of an additional layer of regulation in plant and possibly vertebrate gene expression, whereby the relative activities of canonical nuclear PAPS isoforms control de novo synthesized poly(A) tail length and hence expression of specific subsets of mRNAs.The poly(A) tail at the 3′ end is an essential feature of virtually all eukaryotic mRNAs that influences stability, nuclear export, and translational efficiency of the mRNAs (1, 2). It is synthesized after RNA polymerase II has transcribed past the cleavage and polyadenylation site and associated signal sequences (3, 4). These sequences are bound by several protein complexes, including Cleavage-stimulation Factor (CstF) and Cleavage and Polyadenylation Specificity Factor (CPSF) in animals and their counterparts in yeast and presumably in plants (2, 5). The complexes cleave the nascent pre-mRNA at the prospective polyadenylation site and recruit poly(A) polymerase (PAPS) to add the poly(A) tail.The poly(A) tail is synthesized by PAPSs, with the bulk of cellular pre-mRNAs being polyadenylated by canonical nuclear PAPSs (cPAPSs) (5, 6) that share substantial sequence identity with human poly(A) polymerase-α (PAPOLA), bovine poly(A) polymerase, or the yeast enzyme Pap1p (7–9). Although the Saccharomyces cerevisiae and Drosophila melanogaster genomes only encode one cPAPS, which is essential for growth (7, 10), three such cPAPSs are found in humans: PAPOLA (PAPα), PAPOLB (PAPβ), and PAPOLG (PAPγ) (11). Of these, PAPOLA is thought to be the main PAPS in somatic cells. PAPOLA and PAPOLG proteins contain a C-terminal regulatory region next to the highly conserved catalytic N-terminal domain and are found either in both nucleus and cytoplasm (PAPOLA) or only in the nucleus (PAPOLG) of cells throughout the human body (9, 11–14). By contrast, PAPOLB lacks the C-terminal region, is exclusively cytoplasmic, and is only found in testis cells, where it is required to extend the poly(A) tail of cytoplasmic mRNAs encoding sperm-related proteins (15); as a consequence, male mice mutant for PAPOLB are sterile.The Arabidopsis thaliana genome encodes four cPAPS proteins, termed PAPS1 to PAPS4 (16, 17). PAPS3 resembles PAPOLB in lacking an extended C-terminal region, being localized in the cytoplasm and expressed mainly in the male gametophytes (the pollen). By contrast, PAPS1, PAPS2, and PAPS4 all contain an extended C-terminal region, localize exclusively to the nucleus, and are expressed throughout the plant (2, 16–18). All four proteins have nonspecific polyadenylation activity in vitro, suggesting that they represent functional cPAPSs (16, 19). On the basis of the failure to identify homozygous transfer DNA (T-DNA) insertion mutants for any of the three genes, it was concluded that all of them are essential for plant growth and development (17).Gene expression can be regulated via a number of mechanisms impinging on the mRNA 3′ end. The choice between alternative 3′ end cleavage sites is widely used to regulate gene expression in both animal and plant development, for example via the exclusion or inclusion of microRNA target sites in the resulting 3′ UTRs (20–24). Additionally, modulating the length of the poly(A) tails on mRNAs in the cytoplasm by the opposing actions of cytoplasmic PAPS (e.g., PAPOLB) and deadenylases can be used to control the expression of the encoded proteins (1, 15). However, it is currently unclear whether polyadenylation by nuclear cPAPS can also contribute to the control of specific gene expression. In principle, this could occur in either of two ways. First, pre-mRNAs could be differentially sensitive to variations in the total cPAPS activity provided by one or more functionally interchangeable cPAPS isoforms; such a mechanism may underlie specific developmental phenotypes in weak mutants of D. melanogaster cPAPS (25). Second, some mRNAs may be exclusively or preferentially polyadenylated by one cPAPS in organisms with more than one isoform. Given such target specificity, modulating the balance of activities between the isoforms could then be used to alter the length of the de novo synthesized poly(A) tails, and hence ultimately gene expression, of subsets of mRNAs. Target specificity has at present only been observed for noncanonical PAPS (6, 26), such as Star-PAP, which is required for the cellular response to oxidative stress.Here we provide evidence for functional specialization and target specificity among A. thaliana nuclear cPAPS isoforms. Mutations affecting different isoforms cause very different phenotypes that depend on the divergent C-terminal domains of the proteins. In particular, reduction of PAPS1 activity disrupts polyadenylation of SMALL AUXIN UP RNA (SAUR) mRNAs and causes leaf growth defects due to reduced SAUR function and a constitutive pathogen response. We propose that this specificity of PAPS isoforms provides an additional level of regulating plant gene expression.