RESUMO
Microsatellite repeat expansions within genes contribute to a number of neurological diseases1,2. The accumulation of toxic proteins and RNA molecules with repetitive sequences, and/or sequestration of RNA-binding proteins by RNA molecules containing expanded repeats are thought to be important contributors to disease aetiology3-9. Here we reveal that the adenosine in CAG repeat RNA can be methylated to N1-methyladenosine (m1A) by TRMT61A, and that m1A can be demethylated by ALKBH3. We also observed that the m1A/adenosine ratio in CAG repeat RNA increases with repeat length, which is attributed to diminished expression of ALKBH3 elicited by the repeat RNA. Additionally, TDP-43 binds directly and strongly with m1A in RNA, which stimulates the cytoplasmic mis-localization and formation of gel-like aggregates of TDP-43, resembling the observations made for the protein in neurological diseases. Moreover, m1A in CAG repeat RNA contributes to CAG repeat expansion-induced neurodegeneration in Caenorhabditis elegans and Drosophila. In sum, our study offers a new paradigm of the mechanism through which nucleotide repeat expansion contributes to neurological diseases and reveals a novel pathological function of m1A in RNA. These findings may provide an important mechanistic basis for therapeutic intervention in neurodegenerative diseases emanating from CAG repeat expansion.
Assuntos
Adenosina , Caenorhabditis elegans , Proteínas de Ligação a DNA , Drosophila melanogaster , Doenças Neurodegenerativas , RNA , Expansão das Repetições de Trinucleotídeos , Animais , Humanos , Adenosina/análogos & derivados , Adenosina/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , RNA/química , RNA/genética , RNA/metabolismo , Expansão das Repetições de Trinucleotídeos/genética , Citoplasma/metabolismo , Modelos Animais de DoençasRESUMO
Chromothripsis is a catastrophic mutational process that promotes tumorigenesis and causes congenital disease1-4. Chromothripsis originates from aberrations of nuclei called micronuclei or chromosome bridges5-8. These structures are associated with fragile nuclear envelopes that spontaneously rupture9,10, leading to DNA damage when chromatin is exposed to the interphase cytoplasm. Here we identify a mechanism explaining a major fraction of this DNA damage. Micronuclei accumulate large amounts of RNA-DNA hybrids, which are edited by adenine deaminases acting on RNA (ADAR enzymes) to generate deoxyinosine. Deoxyinosine is then converted into abasic sites by a DNA base excision repair (BER) glycosylase, N-methyl-purine DNA glycosylase11,12 (MPG). These abasic sites are cleaved by the BER endonuclease, apurinic/apyrimidinic endonuclease12 (APE1), creating single-stranded DNA nicks that can be converted to DNA double strand breaks by DNA replication or when closely spaced nicks occur on opposite strands13,14. This model predicts that MPG should be able to remove the deoxyinosine base from the DNA strand of RNA-DNA hybrids, which we demonstrate using purified proteins and oligonucleotide substrates. These findings identify a mechanism for fragmentation of micronuclear chromosomes, an important step in generating chromothripsis. Rather than breaking any normal chromosome, we propose that the eukaryotic cytoplasm only damages chromosomes with pre-existing defects such as the DNA base abnormality described here.
Assuntos
Cromossomos , Cromotripsia , Citoplasma , Dano ao DNA , Reparo do DNA , Cromatina/metabolismo , Cromatina/patologia , Cromossomos/metabolismo , Citoplasma/metabolismo , DNA/metabolismo , DNA Glicosilases/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , RNA/metabolismoRESUMO
Ageing of the immune system, or immunosenescence, contributes to the morbidity and mortality of the elderly1,2. To define the contribution of immune system ageing to organism ageing, here we selectively deleted Ercc1, which encodes a crucial DNA repair protein3,4, in mouse haematopoietic cells to increase the burden of endogenous DNA damage and thereby senescence5-7 in the immune system only. We show that Vav-iCre+/-;Ercc1-/fl mice were healthy into adulthood, then displayed premature onset of immunosenescence characterized by attrition and senescence of specific immune cell populations and impaired immune function, similar to changes that occur during ageing in wild-type mice8-10. Notably, non-lymphoid organs also showed increased senescence and damage, which suggests that senescent, aged immune cells can promote systemic ageing. The transplantation of splenocytes from Vav-iCre+/-;Ercc1-/fl or aged wild-type mice into young mice induced senescence in trans, whereas the transplantation of young immune cells attenuated senescence. The treatment of Vav-iCre+/-;Ercc1-/fl mice with rapamycin reduced markers of senescence in immune cells and improved immune function11,12. These data demonstrate that an aged, senescent immune system has a causal role in driving systemic ageing and therefore represents a key therapeutic target to extend healthy ageing.
Assuntos
Envelhecimento/imunologia , Envelhecimento/fisiologia , Sistema Imunitário/imunologia , Sistema Imunitário/fisiologia , Imunossenescência/imunologia , Imunossenescência/fisiologia , Especificidade de Órgãos/imunologia , Especificidade de Órgãos/fisiologia , Envelhecimento/efeitos dos fármacos , Envelhecimento/patologia , Animais , Dano ao DNA/imunologia , Dano ao DNA/fisiologia , Reparo do DNA/imunologia , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Feminino , Envelhecimento Saudável/imunologia , Envelhecimento Saudável/fisiologia , Homeostase/imunologia , Homeostase/fisiologia , Sistema Imunitário/efeitos dos fármacos , Imunossenescência/efeitos dos fármacos , Masculino , Camundongos , Especificidade de Órgãos/efeitos dos fármacos , Rejuvenescimento , Sirolimo/farmacologia , Baço/citologia , Baço/transplanteRESUMO
Matrix effect (ME) is commonly caused by coelution of compounds with target analytes, resulting in either suppression or enhancement of analyte ionization. Thus, to achieve the desired accuracy, precision, and sensitivity, ME needs to be evaluated and controlled during bioanalytical method development. As the application of supercritical fluid chromatography-mass spectrometry (SFC-MS) for analysis of biological samples has increased, ME using SFC-MS has also been investigated with a focus on the difference in ME in SFC-MS compared to other chromatographic techniques used for achiral separation in biological samples. Here, we provide a summary of the status of ME evaluation and mitigation in SFC-MS methods. This review presents an overview of the phenomenon of ME and methods for evaluating ME in bioanalysis. Next, the factors that can impact ME in SFC-MS-based bioanalytical methods are discussed in detail with an emphasis on SFC. A literature review of the evaluation of ME in targeted bioanalytical methods using SFC-MS is included at the end. Robust instrumentation, effective sample preparation, and superb separation selectivity are the foundations of reliable analytical methods as well as the ability to mitigate detrimental ME in SFC-MS methods.
Assuntos
Cromatografia com Fluido Supercrítico , Cromatografia com Fluido Supercrítico/métodos , Espectrometria de Massa com Cromatografia LíquidaRESUMO
During bioanalytical assay development and validation, maintaining the stability of the parent drug and metabolites of interest is critical. While stability of the parent drug has been thoroughly investigated, the stability of unanalyzed metabolites is often overlooked. When an unstable metabolite is known or suspected to interfere with measurement of the parent drug or other metabolites of interest through back-conversion or other routes, additional tests with these unstable metabolites should be conducted. Here, the development and validation of two assays for quantification of rosuvastatin, one in human plasma and one in human urine, was reported. To this end, additional sets of quality control samples were added during assay validation to ensure the reliability of the assays. Acid treatment of samples is shown to be necessary for rosuvastatin quantification. In this regard, stability issues caused by the metabolite, rosuvastatin lactone, may have been overlooked if assay development and validation had only considered the parent drug, rosuvastatin. These assays represent a case study for how to develop and validate assays with unstable metabolites. Taken together, unstable metabolites should be included in all applicable stability tests.
Assuntos
Espectrometria de Massa com Cromatografia Líquida , Espectrometria de Massas em Tandem , Humanos , Rosuvastatina Cálcica , Cromatografia Líquida , Reprodutibilidade dos TestesRESUMO
A wide spectrum of DNA lesions can be generated from byproducts of endogenous metabolism and/or from environmental exposure. A DNA adductomic approach for the robust quantification of DNA adducts in cellular and tissue DNA may facilitate the use of DNA adducts for biomonitoring studies and enable comprehensive assessment about DNA repair. Normalized retention time (iRT) has been widely used in scheduled selected-reaction monitoring (SRM) methods for highly sensitive and high-throughput analyses of protein samples in complicated matrices. By using a similar method, we established the iRT scores for 36 modified nucleosides from the retention times of the four canonical 2'-deoxynucleosides on a nanoflow liquid chromatography-nanospray ionization-tandem mass spectrometry (nLC-NSI-MS/MS) system. The iRT scores facilitated reliable prediction of retention time and were employed for establishing a scheduled SRM method for quantitative assessment of a subset of the DNA adductome. The quantification results of the scheduled SRM method were more accurate and precise than those from an unscheduled method.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Adutos de DNA/análise , Espectrometria de Massas em Tandem , Desoxiadenosinas/química , Desoxicitidina/química , Desoxiguanosina/química , Interações Hidrofóbicas e Hidrofílicas , Nanotecnologia , Estereoisomerismo , Timidina/químicaRESUMO
A variety of endogenous and exogenous agents can induce DNA damage and lead to genomic instability. Reactive oxygen species (ROS), an important class of DNA damaging agents, are constantly generated in cells as a consequence of endogenous metabolism, infection/inflammation, and/or exposure to environmental toxicants. A wide array of DNA lesions can be induced by ROS directly, including single-nucleobase lesions, tandem lesions, and hypochlorous acid (HOCl)/hypobromous acid (HOBr)-derived DNA adducts. ROS can also lead to lipid peroxidation, whose byproducts can also react with DNA to produce exocyclic DNA lesions. A combination of bioanalytical chemistry, synthetic organic chemistry, and molecular biology approaches have provided significant insights into the occurrence, repair, and biological consequences of oxidatively induced DNA lesions. The involvement of these lesions in the etiology of human diseases and aging was also investigated in the past several decades, suggesting that the oxidatively induced DNA adducts, especially bulky DNA lesions, may serve as biomarkers for exploring the role of oxidative stress in human diseases. The continuing development and improvement of LC-MS/MS coupled with the stable isotope-dilution method for DNA adduct quantification will further promote research about the clinical implications and diagnostic applications of oxidatively induced DNA adducts.
Assuntos
Dano ao DNA , Estresse Oxidativo , Humanos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Excessive intake of NaCl has been associated with the increased risk of several diseases, particularly hypertension. Strategies to reduce sodium intake include substitution of NaCl with other salts, such as KCl. In this study, the effects of NaCl reduction and its substitution with KCl on cell membranes of a cheese starter bacterium (Lactococcus lactis ssp. lactis), probiotic bacteria (Bifidobacterium longum, Lactobacillus acidophilus, and Lactobacillus casei), and a pathogenic bacterium (Escherichia coli) were investigated using Fourier-transform infrared (FTIR) spectroscopy. A critical NaCl concentration that inhibited the viability of E. coli without affecting the viability of probiotic bacteria significantly was determined. To find the critical NaCl concentration, de Man, Rogosa, and Sharpe (MRS) broth was supplemented with a range of NaCl concentrations [0 (control), 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0%], and the effect on cell viability and FTIR spectra was monitored for all bacteria. A NaCl concentration of 2.5% was found to be the critical level of NaCl to inhibit E. coli without significantly affecting the viability of most of the probiotic bacteria and the cheese starter bacterium. The FTIR spectral analysis also highlighted the changes that occurred mainly in the amide regions upon increasing the NaCl concentration from 2.5 to 3.0% in most of the bacteria. Escherichia coli and B. longum were more sensitive to substitution of NaCl with KCl, compared with Lb. acidophilus, Lb. casei, and Lc. lactis ssp. lactis. To evaluate the effect of substitution of NaCl with KCl, substitution was carried out at the critical total salt concentration (2.5%, wt/vol) at varying concentrations (0, 25, 50, 75, and 100% KCl). The findings suggest that 50% substitution of NaCl with KCl, at 2.5% total salt, could inhibit E. coli without affecting the probiotic bacteria.
Assuntos
Queijo/microbiologia , Escherichia coli , Bactérias Gram-Positivas , Viabilidade Microbiana/efeitos dos fármacos , Cloreto de Potássio/farmacologia , Cloreto de Sódio/farmacologia , Ração Animal/análise , Relação Dose-Resposta a Droga , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/fisiologia , Probióticos/química , Espectroscopia de Infravermelho com Transformada de FourierAssuntos
Adutos de DNA/análise , Dano ao DNA , DNA/química , DNA/efeitos dos fármacos , Alquilantes/efeitos adversos , Alquilantes/química , Aminas/efeitos adversos , Aminas/química , Animais , Técnicas de Química Analítica/métodos , Adutos de DNA/química , Compostos Heterocíclicos/efeitos adversos , Compostos Heterocíclicos/química , Humanos , Espécies Reativas de Oxigênio/efeitos adversos , Espécies Reativas de Oxigênio/químicaRESUMO
Cardiomyopathy is a progressive disease of the myocardium leading to impaired contractility. Genotoxic cancer therapies are known to be potent drivers of cardiomyopathy, whereas causes of spontaneous disease remain unclear. To test the hypothesis that endogenous genotoxic stress contributes to cardiomyopathy, we deleted the DNA repair gene Ercc1 specifically in striated muscle using a floxed allele of Ercc1 and mice expressing Cre under control of the muscle-specific creatinine kinase (Ckmm) promoter or depleted systemically (Ercc1-/D mice). Ckmm-Cre+/- ;Ercc1-/fl mice expired suddenly of heart disease by 7 months of age. As young adults, the hearts of Ckmm-Cre+/- ;Ercc1-/fl mice were structurally and functionally normal, but by 6-months-of-age, there was significant ventricular dilation, wall thinning, interstitial fibrosis, and systolic dysfunction indicative of dilated cardiomyopathy. Cardiac tissue from the tissue-specific or systemic model showed increased apoptosis and cardiac myocytes from Ckmm-Cre+/- ;Ercc1-/fl mice were hypersensitive to genotoxins, resulting in apoptosis. p53 levels and target gene expression, including several antioxidants, were increased in cardiac tissue from Ckmm-Cre+/- ;Ercc1-/fl and Ercc1-/D mice. Despite this, cardiac tissue from older mutant mice showed evidence of increased oxidative stress. Genetic or pharmacologic inhibition of p53 attenuated apoptosis and improved disease markers. Similarly, overexpression of mitochondrial-targeted catalase improved disease markers. Together, these data support the conclusion that DNA damage produced endogenously can drive cardiac disease and does so mechanistically via chronic activation of p53 and increased oxidative stress, driving cardiac myocyte apoptosis, dilated cardiomyopathy, and sudden death.
Assuntos
Cardiomiopatia Dilatada , Miócitos Cardíacos , Camundongos , Animais , Miócitos Cardíacos/metabolismo , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Miocárdio/metabolismo , Reparo do DNARESUMO
Investigations into post-transcriptional modifications of RNA and their regulatory proteins have revealed pivotal roles of these modifications in cellular functions. A robust method for the quantitative analysis of modified nucleosides in RNA may facilitate the assessment about their functions in RNA biology and disease etiology. Here, we developed a sensitive nano-liquid chromatography-multistage mass spectrometry (nLC-MS3) method for profiling simultaneously 27 modified ribonucleosides. We employed normalized retention time (iRT) and scheduled selected-reaction monitoring (SRM) to achieve high-throughput analysis, where we assigned iRT values for modified ribonucleosides based on their relative elution times with respect to the four canonical ribonucleosides. The iRT scores allowed for reliable predictions of retention times for modified ribonucleosides with the use of two types of stationary phase materials and various mobile phase gradients. The method enabled the identification of 20 modified ribonucleosides with the use of the enzymatic digestion mixture of 2.5 ng total RNA and facilitated robust quantification of modified cytidine derivatives in total RNA. Together, we established a scheduled SRM-based method for high-throughput analysis of modified ribonucleosides with the use of a few nanograms of RNA.
Assuntos
Cromatografia Líquida/métodos , Epigênese Genética , Espectrometria de Massas/métodos , Transcriptoma/genética , Grafite/química , Células HEK293 , Humanos , Ribonucleosídeos/análise , Fatores de TempoRESUMO
Mass spectrometry and tandem MS (MS/MS) have been widely employed for the identification and quantification of damaged nucleosides in DNA, including those induced by alkylating agents. Upon collisional activation, protonated ions of alkylated nucleosides frequently undergo facile neutral loss of a 2-deoxyribose in MS/MS, and further cleavage of the resulting protonated nucleobases in MS3 can sometimes be employed for differentiating regioisomeric alkylated DNA lesions. Herein, we investigated systematically the collision-induced dissociation (CID) of the protonated ions of O4-alkylthymidine (O4-alkyldT), O2-alkyldT, O6-alkyl-2'-deoxyguanosine (O6-alkyldG), and N2-alkyldG through MS3 analysis. The MS3 of O2- and O4-MedT exhibit different fragmentation patterns from each other and from other O2- and O4-alkyldT adducts carrying larger alkyl groups. Meanwhile, elimination of alkene via a six-membered ring transition state is the dominant fragmentation pathway for O2-alkyldT, O4-alkyldT, and O6-alkyldG adducts carrying larger alkyl groups, whereas O6-MedG mainly undergoes elimination of ammonia. The breakdown of N2-alkyldG is substantially influenced by the structure of the alkyl group, where the relative ease in eliminating ammonia and alkene is modulated by the chain length and branching of the alkyl groups. We also rationalize our observations with density functional theory (DFT) calculations.
Assuntos
Desoxiguanosina/química , Espectrometria de Massas em Tandem/métodos , Timidina/química , Alquilantes/química , Alquilação , Adutos de DNA/química , Teoria da Densidade Funcional , Oligodesoxirribonucleotídeos/química , Prótons , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
DNA damage is presumed to be one type of stochastic macromolecular damage that contributes to aging, yet little is known about the precise mechanism by which DNA damage drives aging. Here, we attempt to address this gap in knowledge using DNA repair-deficient C. elegans and mice. ERCC1-XPF is a nuclear endonuclease required for genomic stability and loss of ERCC1 in humans and mice accelerates the incidence of age-related pathologies. Like mice, ercc-1 worms are UV sensitive, shorter lived, display premature functional decline and they accumulate spontaneous oxidative DNA lesions (cyclopurines) more rapidly than wild-type worms. We found that ercc-1 worms displayed early activation of DAF-16 relative to wild-type worms, which conferred resistance to multiple stressors and was important for maximal longevity of the mutant worms. However, DAF-16 activity was not maintained over the lifespan of ercc-1 animals and this decline in DAF-16 activation corresponded with a loss of stress resistance, a rise in oxidant levels and increased morbidity, all of which were cep-1/ p53 dependent. A similar early activation of FOXO3A (the mammalian homolog of DAF-16), with increased resistance to oxidative stress, followed by a decline in FOXO3A activity and an increase in oxidant abundance was observed in Ercc1-/- primary mouse embryonic fibroblasts. Likewise, in vivo, ERCC1-deficient mice had transient activation of FOXO3A in early adulthood as did middle-aged wild-type mice, followed by a late life decline. The healthspan and mean lifespan of ERCC1 deficient mice was rescued by inactivation of p53. These data indicate that activation of DAF-16/FOXO3A is a highly conserved response to genotoxic stress that is important for suppressing consequent oxidative stress. Correspondingly, dysregulation of DAF-16/FOXO3A appears to underpin shortened healthspan and lifespan, rather than the increased DNA damage burden itself.