RESUMO
Lysophosphatidic acid (LPA) species accumulate in the ascites of ovarian high-grade serous cancer (HGSC) and are associated with short relapse-free survival. LPA is known to support metastatic spread of cancer cells by activating a multitude of signaling pathways via G-protein-coupled receptors of the LPAR family. Systematic unbiased analyses of the LPA-regulated signal transduction network in ovarian cancer cells have, however, not been reported to date. Methods: LPA-induced signaling pathways were identified by phosphoproteomics of both patient-derived and OVCAR8 cells, RNA sequencing, measurements of intracellular Ca2+ and cAMP as well as cell imaging. The function of LPARs and downstream signaling components in migration and entosis were analyzed by selective pharmacological inhibitors and RNA interference. Results: Phosphoproteomic analyses identified > 1100 LPA-regulated sites in > 800 proteins and revealed interconnected LPAR1, ROCK/RAC, PKC/D and ERK pathways to play a prominent role within a comprehensive signaling network. These pathways regulate essential processes, including transcriptional responses, actomyosin dynamics, cell migration and entosis. A critical component of this signaling network is MYPT1, a stimulatory subunit of protein phosphatase 1 (PP1), which in turn is a negative regulator of myosin light chain 2 (MLC2). LPA induces phosphorylation of MYPT1 through ROCK (T853) and PKC/ERK (S507), which is majorly driven by LPAR1. Inhibition of MYPT1, PKC or ERK impedes both LPA-induced cell migration and entosis, while interference with ROCK activity and MLC2 phosphorylation selectively blocks entosis, suggesting that MYPT1 figures in both ROCK/MLC2-dependent and -independent pathways. We finally show a novel pathway governed by LPAR2 and the RAC-GEF DOCK7 to be indispensable for the induction of entosis. Conclusion: We have identified a comprehensive LPA-induced signal transduction network controlling LPA-triggered cytoskeletal changes, cell migration and entosis in HGSC cells. Due to its pivotal role in this network, MYPT1 may represent a promising target for interfering with specific functions of PP1 essential for HGSC progression.
Assuntos
Actomiosina , Neoplasias Ovarianas , Humanos , Feminino , Actomiosina/metabolismo , Entose , Recidiva Local de Neoplasia , Transdução de Sinais , Neoplasias Ovarianas/metabolismo , Movimento Celular/fisiologiaRESUMO
Most mitochondrial proteins are encoded by nuclear genes, synthetized in the cytosol and targeted into the organelle. To characterize the spatial organization of mitochondrial gene products in zebrafish (Danio rerio), we sequenced RNA from different cellular fractions. Our results confirmed the presence of nuclear-encoded mRNAs in the mitochondrial fraction, which in unperturbed conditions, are mainly transcripts encoding large proteins with specific properties, like transmembrane domains. To further explore the principles of mitochondrial protein compartmentalization in zebrafish, we quantified the transcriptomic changes for each subcellular fraction triggered by the chchd4a -/- mutation, causing the disorders in the mitochondrial protein import. Our results indicate that the proteostatic stress further restricts the population of transcripts on the mitochondrial surface, allowing only the largest and the most evolutionary conserved proteins to be synthetized there. We also show that many nuclear-encoded mitochondrial transcripts translated by the cytosolic ribosomes stay resistant to the global translation shutdown. Thus, vertebrates, in contrast to yeast, are not likely to use localized translation to facilitate synthesis of mitochondrial proteins under proteostatic stress conditions.
Assuntos
Genes Mitocondriais , Peixe-Zebra , Animais , Peixe-Zebra/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Nucleares/genéticaRESUMO
Silencing of the Apoptosis associated Tyrosine Kinase gene (AATK) has been described in cancer. In our study, we specifically investigated the epigenetic inactivation of AATK in pancreatic adenocarcinoma, lower grade glioma, lung, breast, head, and neck cancer. The resulting loss of AATK correlates with impaired patient survival. Inhibition of DNA methyltransferases (DNMTs) reactivated AATK in glioblastoma and pancreatic cancer. In contrast, epigenetic targeting via the CRISPR/dCas9 system with either EZH2 or DNMT3A inhibited the expression of AATK. Via large-scale kinomic profiling and kinase assays, we demonstrate that AATK acts a Ser/Thr kinase that phosphorylates TP53 at Ser366. Furthermore, whole transcriptome analyses and mass spectrometry associate AATK expression with the GO term 'regulation of cell proliferation'. The kinase activity of AATK in comparison to the kinase-dead mutant mediates a decreased expression of the key cell cycle regulators Cyclin D1 and WEE1. Moreover, growth suppression through AATK relies on its kinase activity. In conclusion, the Ser/Thr kinase AATK represses growth and phosphorylates TP53. Furthermore, expression of AATK was correlated with a better patient survival for different cancer entities. This data suggests that AATK acts as an epigenetically inactivated tumor suppressor gene.
Assuntos
Adenocarcinoma , Proteínas Reguladoras de Apoptose , Neoplasias Pancreáticas , Proteínas Tirosina Quinases , Humanos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Ciclina D1/genética , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Neoplasias PancreáticasRESUMO
BACKGROUND: Transcoelomic spread is the major route of metastasis of ovarian high-grade serous carcinoma (HGSC) with the omentum as the major metastatic site. Its unique tumour microenvironment with its large populations of adipocytes, mesothelial cells and immune cells establishes an intercellular signaling network that is instrumental for metastatic growth yet poorly understood. METHODS: Based on transcriptomic analysis of tumour cells, tumour-associated immune and stroma cells we defined intercellular signaling pathways for 284 cytokines and growth factors and their cognate receptors after bioinformatic adjustment for contaminating cell types. The significance of individual components of this network was validated by analysing clinical correlations and potentially pro-metastatic functions, including tumour cell migration, pro-inflammatory signal transduction and TAM expansion. RESULTS: The data show an unexpected prominent role of host cells, and in particular of omental adipocytes, mesothelial cells and fibroblasts (CAF), in sustaining this signaling network. These cells, rather than tumour cells, are the major source of most cytokines and growth factors in the omental microenvironment (n = 176 vs. n = 13). Many of these factors target tumour cells, are linked to metastasis and are associated with a short survival. Likewise, tumour stroma cells play a major role in extracellular-matrix-triggered signaling. We have verified the functional significance of our observations for three exemplary instances. We show that the omental microenvironment (i) stimulates tumour cell migration and adhesion via WNT4 which is highly expressed by CAF; (ii) induces pro-tumourigenic TAM proliferation in conjunction with high CSF1 expression by omental stroma cells and (iii) triggers pro-inflammatory signaling, at least in part via a HSP70-NF-κB pathway. CONCLUSIONS: The intercellular signaling network of omental metastases is majorly dependent on factors secreted by immune and stroma cells to provide an environment that supports ovarian HGSC progression. Clinically relevant pathways within this network represent novel options for therapeutic intervention.
Assuntos
Redes Reguladoras de Genes/fisiologia , Metástase Neoplásica/fisiopatologia , Neoplasias Ovarianas/fisiopatologia , Movimento Celular/genética , Movimento Celular/fisiologia , Feminino , Redes Reguladoras de Genes/genética , Humanos , Metástase Neoplásica/imunologia , Neoplasias Ovarianas/imunologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Cardiac trabeculae are muscular ridge-like structures within the ventricular wall that are crucial for cardiac function. In zebrafish, these structures first form primarily through the delamination of compact wall cardiomyocytes (CMs). Although defects in proteasomal degradation have been associated with decreased cardiac function, whether they also affect cardiac development has not been extensively analyzed. Here we report a role during cardiac wall morphogenesis in zebrafish for the E3 ubiquitin-protein ligase Rbx1, which has been shown to regulate the degradation of key signaling molecules. Although development is largely unperturbed in zebrafish rbx1 mutant larvae, they exhibit CM multi-layering. This phenotype is not affected by blocking ErbB signaling, but fails to manifest itself in the absence of blood flow/cardiac contractility. Surprisingly, rbx1 mutants display ErbB independent Notch reporter expression in the myocardium. We generated tissue-specific rbx1 overexpression lines and found that endothelial, but not myocardial, specific rbx1 expression normalizes the cardiac wall morphogenesis phenotype. In addition, we found that pharmacological activation of Hedgehog signaling ameliorates the multi-layered myocardial wall phenotype in rbx1 mutants. Collectively, our data indicate that endocardial activity of Rbx1 is essential for cardiac wall morphogenesis.
Assuntos
Miocárdio/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proliferação de Células/genética , Endocárdio/metabolismo , Endotélio/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Genes erbB/genética , Coração/fisiologia , Ventrículos do Coração/metabolismo , Proteínas Hedgehog/metabolismo , Morfogênese/genética , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Organogênese/genética , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais/genética , Ubiquitina-Proteína Ligases/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Arachidonic acid (AA) is a polyunsaturated fatty acid present at high concentrations in the ovarian cancer (OC) microenvironment and associated with a poor clinical outcome. In the present study, we have unraveled a potential link between AA and macrophage functions. Methods: AA-triggered signal transduction was studied in primary monocyte-derived macrophages (MDMs) by phosphoproteomics, transcriptional profiling, measurement of intracellular Ca2+ accumulation and reactive oxygen species production in conjunction with bioinformatic analyses. Functional effects were investigated by actin filament staining, quantification of macropinocytosis and analysis of extracellular vesicle release. Results: We identified the ASK1 - p38δ/α (MAPK13/14) axis as a central constituent of signal transduction pathways triggered by non-metabolized AA. This pathway was induced by the Ca2+-triggered activation of calmodulin kinase II, and to a minor extent by ROS generation in a subset of donors. Activated p38 in turn was linked to a transcriptional stress response associated with a poor relapse-free survival. Consistent with the phosphorylation of the p38 substrate HSP27 and the (de)phosphorylation of multiple regulators of Rho family GTPases, AA impaired actin filament organization and inhibited actin-driven macropinocytosis. AA also affected the phosphorylation of proteins regulating vesicle biogenesis, and consistently, AA enhanced the release of tetraspanin-containing exosome-like vesicles. Finally, we identified phospholipase A2 group 2A (PLA2G2A) as the clinically most relevant enzyme producing extracellular AA, providing further potentially theranostic options. Conclusion: Our results suggest that AA contributes to an unfavorable clinical outcome of OC by impacting the phenotype of tumor-associated macrophages. Besides critical AA-regulated signal transduction proteins identified in the present study, PLA2G2A might represent a potential prognostic tool and therapeutic target to interfere with OC progression.
Assuntos
Ácido Araquidônico/farmacologia , Macrófagos/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Cálcio/metabolismo , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Feminino , Fosfolipases A2 do Grupo II/metabolismo , Humanos , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/metabolismo , Neoplasias Ovarianas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transcrição Gênica/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacosRESUMO
Ergosterol is a prominent component of the yeast plasma membrane and essential for yeast cell viability. It is synthesized in the endoplasmic reticulum and transported to the plasma membrane by nonvesicular mechanisms requiring carrier proteins. Oxysterol-binding protein homologues and yeast StARkin proteins have been proposed to function as sterol carriers. Although many of these proteins are capable of transporting sterols between synthetic lipid vesicles in vitro, they are not essential for ergosterol transport in cells, indicating that they may be functionally redundant with each other or with additional-as yet unidentified-sterol carriers. To address this point, we hypothesized that sterol transport proteins are also sterol-binding proteins (SBPs), and used an in vitro chemoproteomic strategy to identify all cytosolic SBPs. We generated a cytosol fraction enriched in SBPs and captured the proteins with a photoreactive clickable cholesterol analogue. Quantitative proteomics of the captured proteins identified 342 putative SBPs. Analysis of these identified proteins based on their annotated function, reported drug phenotypes, interactions with proteins regulating lipid metabolism, gene ontology, and presence of mammalian orthologues revealed a subset of 62 characterized and nine uncharacterized candidates. Five of the uncharacterized proteins play a role in maintaining plasma membrane integrity as their absence affects the ability of cells to grow in the presence of nystatin or myriocin. We anticipate that the dataset reported here will be a comprehensive resource for functional analysis of sterol-binding/transport proteins and provide insights into novel aspects of non-vesicular sterol trafficking.
Assuntos
Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte/genética , Citosol/metabolismo , Ergosterol/metabolismo , Transporte Proteico , Proteômica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
BACKGROUND: Cancer still is one of the leading causes of death and its death toll is predicted to rise further. We identified earlier the potential tumour suppressor zygote arrest 1 (ZAR1) to play a role in lung carcinogenesis through its epigenetic inactivation. RESULTS: We are the first to report that ZAR1 is epigenetically inactivated not only in lung cancer but also across cancer types, and ZAR1 methylation occurs across its complete CpG island. ZAR1 hypermethylation significantly correlates with its expression reduction in cancers. We are also the first to report that ZAR1 methylation and expression reduction are of clinical importance as a prognostic marker for lung cancer and kidney cancer. We further established that the carboxy (C)-terminally present zinc-finger of ZAR1 is relevant for its tumour suppression function and its protein partner binding associated with the mRNA/ribosomal network. Global gene expression profiling supported ZAR1's role in cell cycle arrest and p53 signalling pathway, and we could show that ZAR1 growth suppression was in part p53 dependent. Using the CRISPR-dCas9 tools, we were able to prove that epigenetic editing and reactivation of ZAR1 is possible in cancer cell lines. CONCLUSION: ZAR1 is a novel cancer biomarker for lung and kidney, which is epigenetically silenced in various cancers by DNA hypermethylation. ZAR1 exerts its tumour suppressive function in part through p53 and through its zinc-finger domain. Epigenetic therapy can reactivate the ZAR1 tumour suppressor in cancer.
Assuntos
Biomarcadores Tumorais/genética , Metilação de DNA , Proteínas do Ovo/genética , Proteínas do Ovo/metabolismo , Neoplasias Renais/diagnóstico , Neoplasias Pulmonares/diagnóstico , Células A549 , Sítios de Ligação , Ciclo Celular , Linhagem Celular Tumoral , Ilhas de CpG , Regulação para Baixo , Detecção Precoce de Câncer , Proteínas do Ovo/química , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HeLa , Humanos , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Prognóstico , Transdução de Sinais , Análise de Sobrevida , Proteína Supressora de Tumor p53/metabolismo , Dedos de ZincoRESUMO
Atrial fibrillation (AF) is the most common type of cardiac arrhythmia. The major AF susceptibility locus 4q25 establishes long-range interactions with the promoter of PITX2, a transcription factor gene with critical functions during cardiac development. While many AF-linked loci have been identified in genome-wide association studies, mechanistic understanding into how genetic variants, including those at the 4q25 locus, increase vulnerability to AF is mostly lacking. Here, we show that loss of pitx2c in zebrafish leads to adult cardiac phenotypes with substantial similarities to pathologies observed in AF patients, including arrhythmia, atrial conduction defects, sarcomere disassembly, and altered cardiac metabolism. These phenotypes are also observed in a subset of pitx2c+/- fish, mimicking the situation in humans. Most notably, the onset of these phenotypes occurs at an early developmental stage. Detailed analyses of pitx2c loss- and gain-of-function embryonic hearts reveal changes in sarcomeric and metabolic gene expression and function that precede the onset of cardiac arrhythmia first observed at larval stages. We further find that antioxidant treatment of pitx2c-/- larvae significantly reduces the incidence and severity of cardiac arrhythmia, suggesting that metabolic dysfunction is an important driver of conduction defects. We propose that these early sarcomere and metabolic defects alter cardiac function and contribute to the electrical instability and structural remodeling observed in adult fish. Overall, these data provide insight into the mechanisms underlying the development and pathophysiology of some cardiac arrhythmias and importantly, increase our understanding of how developmental perturbations can predispose to functional defects in the adult heart.
Assuntos
Arritmias Cardíacas/metabolismo , Proteínas de Homeodomínio/genética , Sarcômeros/metabolismo , Fatores de Transcrição/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Acetilcisteína/farmacologia , Animais , Animais Geneticamente Modificados , Antioxidantes/farmacologia , Arritmias Cardíacas/tratamento farmacológico , Arritmias Cardíacas/etiologia , Doença do Sistema de Condução Cardíaco/etiologia , Doença do Sistema de Condução Cardíaco/genética , Cardiomiopatias/genética , Cardiomiopatias/fisiopatologia , Modelos Animais de Doenças , Eletrocardiografia , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Larva/efeitos dos fármacos , Mitocôndrias Cardíacas/genética , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Sarcômeros/genética , Sarcômeros/patologia , Estresse Fisiológico/genética , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Hypertension is a primary risk factor for cardiovascular diseases including myocardial infarction and stroke. Major determinants of blood pressure are vasodilatory factors such as nitric oxide (NO) released from the endothelium under the influence of fluid shear stress exerted by the flowing blood. Several endothelial signaling processes mediating fluid shear stress-induced formation and release of vasodilatory factors have been described. It is, however, still poorly understood how fluid shear stress induces these endothelial responses. Here we show that the endothelial mechanosensitive cation channel PIEZO1 mediated fluid shear stress-induced release of adrenomedullin, which in turn activated its Gs-coupled receptor. The subsequent increase in cAMP levels promoted the phosphorylation of endothelial NO synthase (eNOS) at serine 633 through protein kinase A (PKA), leading to the activation of the enzyme. This Gs/PKA-mediated pathway synergized with the AKT-mediated pathways leading to eNOS phosphorylation at serine 1177. Mice with endothelium-specific deficiency of adrenomedullin, the adrenomedullin receptor, or Gαs showed reduced flow-induced eNOS activation and vasodilation and developed hypertension. Our data identify fluid shear stress-induced PIEZO1 activation as a central regulator of endothelial adrenomedullin release and establish the adrenomedullin receptor and subsequent Gs-mediated formation of cAMP as a critical endothelial mechanosignaling pathway regulating basal endothelial NO formation, vascular tone, and blood pressure.
Assuntos
Adrenomedulina/metabolismo , Pressão Sanguínea , Endotélio Vascular , Sistemas do Segundo Mensageiro , Estresse Mecânico , Animais , AMP Cíclico/metabolismo , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Hipertensão/metabolismo , Hipertensão/patologia , Hipertensão/fisiopatologia , Canais Iônicos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismoRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1007743.].
RESUMO
Pathways modulating glucose homeostasis independently of insulin would open new avenues to combat insulin resistance and diabetes. Here, we report the establishment, characterization, and use of a vertebrate 'insulin-free' model to identify insulin-independent modulators of glucose metabolism. insulin knockout zebrafish recapitulate core characteristics of diabetes and survive only up to larval stages. Utilizing a highly efficient endoderm transplant technique, we generated viable chimeric adults that provide the large numbers of insulin mutant larvae required for our screening platform. Using glucose as a disease-relevant readout, we screened 2233 molecules and identified three that consistently reduced glucose levels in insulin mutants. Most significantly, we uncovered an insulin-independent beneficial role for androgen receptor antagonism in hyperglycemia, mostly by reducing fasting glucose levels. Our study proposes therapeutic roles for androgen signaling in diabetes and, more broadly, offers a novel in vivo model for rapid screening and decoupling of insulin-dependent and -independent mechanisms.
Assuntos
Glucose/metabolismo , Hiperglicemia/genética , Insulina/genética , Receptores Androgênicos/genética , Antagonistas de Receptores de Andrógenos/química , Antagonistas de Receptores de Andrógenos/metabolismo , Animais , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Homeostase , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Resistência à Insulina/genética , Receptores Androgênicos/química , Transdução de Sinais/genética , Peixe-Zebra/genéticaRESUMO
Development and function of tissues and organs are powered by the activity of mitochondria. In humans, inherited genetic mutations that lead to progressive mitochondrial pathology often manifest during infancy and can lead to death, reflecting the indispensable nature of mitochondrial biogenesis and function. Here, we describe a zebrafish mutant for the gene mia40a (chchd4a), the life-essential homologue of the evolutionarily conserved Mia40 oxidoreductase which drives the biogenesis of cysteine-rich mitochondrial proteins. We report that mia40a mutant animals undergo progressive cellular respiration defects and develop enlarged mitochondria in skeletal muscles before their ultimate death at the larval stage. We generated a deep transcriptomic and proteomic resource that allowed us to identify abnormalities in the development and physiology of endodermal organs, in particular the liver and pancreas. We identify the acinar cells of the exocrine pancreas to be severely affected by mutations in the MIA pathway. Our data contribute to a better understanding of the molecular, cellular and organismal effects of mitochondrial deficiency, important for the accurate diagnosis and future treatment strategies of mitochondrial diseases.
RESUMO
Impaired alveolar formation and maintenance are features of many pulmonary diseases that are associated with significant morbidity and mortality. In a forward genetic screen for modulators of mouse lung development, we identified the non-muscle myosin II heavy chain gene, Myh10. Myh10 mutant pups exhibit cyanosis and respiratory distress, and die shortly after birth from differentiation defects in alveolar epithelium and mesenchyme. From omics analyses and follow up studies, we find decreased Thrombospondin expression accompanied with increased matrix metalloproteinase activity in both mutant lungs and cultured mutant fibroblasts, as well as disrupted extracellular matrix (ECM) remodeling. Loss of Myh10 specifically in mesenchymal cells results in ECM deposition defects and alveolar simplification. Notably, MYH10 expression is downregulated in the lung of emphysema patients. Altogether, our findings reveal critical roles for Myh10 in alveologenesis at least in part via the regulation of ECM remodeling, which may contribute to the pathogenesis of emphysema.
Assuntos
Matriz Extracelular/metabolismo , Pneumopatias/metabolismo , Cadeias Pesadas de Miosina/deficiência , Miosina não Muscular Tipo IIB/deficiência , Sequência de Aminoácidos , Animais , Regulação para Baixo/genética , Enfisema/patologia , Etilnitrosoureia , Feminino , Pneumopatias/patologia , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Mesoderma/metabolismo , Camundongos Endogâmicos C57BL , Mutagênese/genética , Mutação de Sentido Incorreto/genética , Cadeias Pesadas de Miosina/química , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosina não Muscular Tipo IIB/química , Miosina não Muscular Tipo IIB/genética , Miosina não Muscular Tipo IIB/metabolismo , Organogênese , Fenótipo , Alvéolos Pulmonares/embriologia , Alvéolos Pulmonares/metabolismo , Regulação para Cima/genéticaRESUMO
Disulfide bond formation is crucial for the biogenesis and structure of many proteins that are localized in the intermembrane space of mitochondria. The importance of disulfide bond formation within mitochondrial proteins was extended beyond soluble intermembrane space proteins. Tim22, a membrane protein and core component of the mitochondrial translocase TIM22, forms an intramolecular disulfide bond in yeast. Tim22 belongs to the Tim17/Tim22/Tim23 family of protein translocases. Here, we present evidence of the high evolutionary conservation of disulfide bond formation in Tim17 and Tim22 among fungi and metazoa. Topological models are proposed that include the location of disulfide bonds relative to the predicted transmembrane regions. Yeast and human Tim22 variants that are not oxidized do not properly integrate into the membrane complex. Moreover, the lack of Tim17 oxidation disrupts the TIM23 translocase complex. This underlines the importance of disulfide bond formation for mature translocase assembly through membrane stabilization of weak transmembrane domains.
Assuntos
Evolução Biológica , Dissulfetos/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Mitocondriais/metabolismo , Humanos , Oxirredução , Saccharomyces cerevisiae/metabolismoRESUMO
Autosomal recessively inherited glucocerebrosidase 1 (GBA1) mutations cause the lysosomal storage disorder Gaucher's disease (GD). Heterozygous GBA1 mutations (GBA1(+/-)) are the most common risk factor for Parkinson's disease (PD). Previous studies typically focused on the interaction between the reduction of glucocerebrosidase (enzymatic) activity in GBA1(+/-) carriers and alpha-synuclein-mediated neurotoxicity. However, it is unclear whether other mechanisms also contribute to the increased risk of PD in GBA1(+/-) carriers. The zebrafish genome does not contain alpha-synuclein (SNCA), thus providing a unique opportunity to study pathogenic mechanisms unrelated to alpha-synuclein toxicity. Here we describe a mutant zebrafish line created by TALEN genome editing carrying a 23 bp deletion in gba1 (gba1(c.1276_1298del)), the zebrafish orthologue of human GBA1. Marked sphingolipid accumulation was already detected at 5 days post-fertilization with accompanying microglial activation and early, sustained up-regulation of miR-155, a master regulator of inflammation. gba1(c.1276_1298del) mutant zebrafish developed a rapidly worsening phenotype from 8 weeks onwards with striking reduction in motor activity by 12 weeks. Histopathologically, we observed marked Gaucher cell invasion of the brain and other organs. Dopaminergic neuronal cell count was normal through development but reduced by >30% at 12 weeks in the presence of ubiquitin-positive, intra-neuronal inclusions. This gba1(c.1276_1298del) zebrafish line is the first viable vertebrate model sharing key pathological features of GD in both neuronal and non-neuronal tissue. Our study also provides evidence for early microglial activation prior to alpha-synuclein-independent neuronal cell death in GBA1 deficiency and suggests upregulation of miR-155 as a common denominator across different neurodegenerative disorders.
Assuntos
Modelos Animais de Doenças , Doença de Gaucher/genética , Glucosilceramidase/genética , Neurônios/patologia , Proteínas de Peixe-Zebra/genética , Peixe-Zebra , Animais , Morte Celular , Doença de Gaucher/patologia , MicroRNAs/genética , Microglia/metabolismo , Microglia/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Deleção de Sequência , Regulação para Cima , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Mitochondria are involved in many essential cellular activities. These broad functions explicate the need for the well-orchestrated biogenesis of mitochondrial proteins to avoid death and pathological consequences, both in unicellular and more complex organisms. Yeast as a model organism has been pivotal in identifying components and mechanisms that drive the transport and sorting of nuclear-encoded mitochondrial proteins. The machinery components that are involved in the import of mitochondrial proteins are generally evolutionarily conserved within the eukaryotic kingdom. However, topological and functional differences have been observed. We review the similarities and differences in mitochondrial translocases from yeast to human. Additionally, we provide a systematic overview of the contribution of mitochondrial import machineries to human pathologies, including cancer, mitochondrial diseases, and neurodegeneration.
Assuntos
Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Doenças Neurodegenerativas/metabolismo , Animais , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/patologia , Doenças Mitocondriais/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Doenças Neurodegenerativas/genética , Transporte Proteico , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Human cells lacking DNA polymerase η (polη) are sensitive to platinum-based cancer chemotherapeutic agents. Using DNA combing to directly investigate the role of polη in bypass of platinum-induced DNA lesions in vivo, we demonstrate that nascent DNA strands are up to 39% shorter in human cells lacking polη than in cells expressing polη. This provides the first direct evidence that polη modulates replication fork progression in vivo following cisplatin and carboplatin treatment. Severe replication inhibition in individual platinum-treated polη-deficient cells correlates with enhanced phosphorylation of the RPA2 subunit of replication protein A on serines 4 and 8, as determined using EdU labelling and immunofluorescence, consistent with formation of DNA strand breaks at arrested forks in the absence of polη. Polη-mediated bypass of platinum-induced DNA lesions may therefore represent one mechanism by which cancer cells can tolerate platinum-based chemotherapy.
Assuntos
Dano ao DNA/efeitos dos fármacos , Dano ao DNA/fisiologia , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , Platina/farmacologia , Carboplatina/farmacologia , Carboplatina/toxicidade , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Linhagem Celular , Cisplatino/farmacologia , Cisplatino/toxicidade , DNA Polimerase Dirigida por DNA/deficiência , DNA Polimerase Dirigida por DNA/genética , Sinergismo Farmacológico , Expressão Gênica , Humanos , Fosforilação , Platina/toxicidade , Proteína de Replicação A/metabolismoRESUMO
A series of new aromatic monoesters of alpha-aminoaralkylphosphonic acids were synthesized by selective hydrolysis of corresponding aromatic diesters of alpha-aminoaralkylphosphonic acids. New potential inhibitors of aminopeptidase N/CD13, an enzyme important in tumour angiogenesis, were developed. Some derivatives of the homophenylalanine and norleucine related monoaryl phosphonates displayed higher inhibition potency than corresponding alpha-aminoaralkylphosphonic acids toward aminopeptidase N/CD13. The effect of one of the new inhibitors on the growth of human PANC-1 and HT-1080 cell lines was examined, either alone or in combination with TNF-alpha.