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1.
Gene ; 736: 144448, 2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32032743

RESUMO

Dispose of unnecessary cells in multicellular organism take place through apoptosis as a mode of programmed cell death (PCD). This process is triggered through two main pathway including extrinsic pathway or death receptor pathway and intrinsic or mitochondrial pathway. An alternative role for mitochondrial pathway of cell death is its involvement in cell differentiation. Biochemistry of cell differentiation indicates a common origin for differentiation and apoptosis. miRNAs are a group of small non coding mediator RNAs in regulation of many routes such as apoptosis and differentiation. By using bioinformatics tools hsa-miR-766-5p was predicted to target the BAX, BAK and BOK genes involved in mitochondrial apoptosis pathway. RT-qPCR and dual luciferase assay showed targeting of BAX, BAK and BOK 3'UTRs via hsa-miR-766, detected in SW480 and HEK293T cell lines. Caspases 3/7 and 9 activity assay revealed the involvement of hsa-miR-766-5p in mitochondrial apoptosis pathway regulation detected following overexpression and downregulation of this miRNA, detected in SW480 cells treated with 1 µM doxorubicin. Flow cytometry and MTT assay indicated cell death reduction and viability elevation effect of hsa-miR-766 in SW480 cells after its overexpression. Endogenous expression of hsa-miR-766 during the course of human embryonic stem cells (hESCs) differentiation into cardiomyocytes revealed an inverse expression status of this miRNA with BOK. However, the expression of this miRNA was inversely related to BAX and BAK for some time points of differentiation. Overall this results show the involvement of hsa-miR-766 in regulation of mitochondrial apoptosis pathway.


Assuntos
Apoptose/genética , Morte Celular/genética , Diferenciação Celular/genética , MicroRNAs/genética , Mitocôndrias/genética , Miócitos Cardíacos/fisiologia , Linhagem Celular , Biologia Computacional/métodos , Regulação para Baixo/genética , Células HEK293 , Células-Tronco Embrionárias Humanas/fisiologia , Humanos
2.
PLoS Genet ; 15(11): e1008454, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31697683

RESUMO

α-catenin is a key protein of adherens junctions (AJs) with mechanosensory properties. It also acts as a tumor suppressor that limits tissue growth. Here we analyzed the function of Drosophila α-Catenin (α-Cat) in growth regulation of the wing epithelium. We found that different α-Cat levels led to a differential activation of Hippo/Yorkie or JNK signaling causing tissue overgrowth or degeneration, respectively. α-Cat can modulate Yorkie-dependent tissue growth through recruitment of Ajuba, a negative regulator of Hippo signaling to AJs but also through a mechanism independent of Ajuba recruitment to AJs. Both mechanosensory regions of α-Cat, the M region and the actin-binding domain (ABD), contribute to growth regulation. Whereas M is dispensable for α-Cat function in the wing, individual M domains (M1, M2, M3) have opposing effects on growth regulation. In particular, M1 limits Ajuba recruitment. Loss of M1 causes Ajuba hyper-recruitment to AJs, promoting tissue-tension independent overgrowth. Although M1 binds Vinculin, Vinculin is not responsible for this effect. Moreover, disruption of mechanosensing of the α-Cat ABD affects tissue growth, with enhanced actin interactions stabilizing junctions and leading to tissue overgrowth. Together, our findings indicate that α-Cat acts through multiple mechanisms to control tissue growth, including regulation of AJ stability, mechanosensitive Ajuba recruitment, and dynamic direct F-actin interactions.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas com Domínio LIM/genética , Asas de Animais/crescimento & desenvolvimento , alfa Catenina/genética , Citoesqueleto de Actina/genética , Actinas/genética , Junções Aderentes/genética , Animais , Morte Celular/genética , Citoesqueleto/genética , Drosophila melanogaster/crescimento & desenvolvimento , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Sistema de Sinalização das MAP Quinases/genética , Mecanotransdução Celular/genética , Proteínas Nucleares/genética , Domínios Proteicos/genética , Proteínas Serina-Treonina Quinases/genética , Transativadores/genética , Vinculina/genética , Asas de Animais/metabolismo
3.
PLoS Biol ; 17(10): e3000492, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31626642

RESUMO

Naturally occurring cell death is a fundamental developmental mechanism for regulating cell numbers and sculpting developing organs. This is particularly true in the nervous system, where large numbers of neurons and oligodendrocytes are eliminated via apoptosis during normal development. Given the profound impact of death upon these two major cell populations, it is surprising that developmental death of another major cell type-the astrocyte-has rarely been studied. It is presently unclear whether astrocytes are subject to significant developmental death, and if so, how it occurs. Here, we address these questions using mouse retinal astrocytes as our model system. We show that the total number of retinal astrocytes declines by over 3-fold during a death period spanning postnatal days 5-14. Surprisingly, these astrocytes do not die by apoptosis, the canonical mechanism underlying the vast majority of developmental cell death. Instead, we find that microglia engulf astrocytes during the death period to promote their developmental removal. Genetic ablation of microglia inhibits astrocyte death, leading to a larger astrocyte population size at the end of the death period. However, astrocyte death is not completely blocked in the absence of microglia, apparently due to the ability of astrocytes to engulf each other. Nevertheless, mice lacking microglia showed significant anatomical changes to the retinal astrocyte network, with functional consequences for the astrocyte-associated vasculature leading to retinal hemorrhage. These results establish a novel modality for naturally occurring cell death and demonstrate its importance for the formation and integrity of the retinal gliovascular network.


Assuntos
Astrócitos/citologia , Morte Celular/genética , Microglia/citologia , Retina/citologia , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/fisiopatologia , Comunicação Celular , Contagem de Células , Toxina Diftérica/toxicidade , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Hemorragia Retiniana/genética , Hemorragia Retiniana/metabolismo , Hemorragia Retiniana/fisiopatologia , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
4.
Nat Commun ; 10(1): 4157, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519887

RESUMO

Receptor-interacting protein kinase 1 (RIPK1) is a critical regulator of cell death through its kinase activity. However, how its kinase activity is regulated remains poorly understood. Here, we generate Ripk1K376R/K376R knock-in mice in which the Lys(K)63-linked ubiquitination of RIPK1 is impaired. The knock-in mice display an early embryonic lethality due to massive cell death that is resulted from reduced TAK1-mediated suppression on RIPK1 kinase activity and forming more TNFR1 complex II in Ripk1K376R/K376R cells in response to TNFα. Although TNFR1 deficiency delays the lethality, concomitant deletion of RIPK3 and Caspase8 fully prevents embryonic lethality of Ripk1K376R/K376R mice. Notably, Ripk1K376R/- mice are viable but develop severe systemic inflammation that is mainly driven by RIPK3-dependent signaling pathway, indicating that K63-linked ubiquitination on Lys376 residue of RIPK1 also contributes to inflammation process. Together, our study reveals the mechanism by which K63-linked ubiquitination on K376 regulates RIPK1 kinase activity to control cell death programs.


Assuntos
Morte Celular/fisiologia , Desenvolvimento Embrionário/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Ubiquitinação/fisiologia , Animais , Morte Celular/genética , Desenvolvimento Embrionário/genética , Citometria de Fluxo , Células HEK293 , Humanos , Imunoprecipitação , Inflamação/genética , Inflamação/metabolismo , Camundongos , Biologia Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Ubiquitinação/genética
5.
Int J Mol Sci ; 20(18)2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31491992

RESUMO

Hepatocyte death is critical for the pathogenesis of liver disease progression, which is closely associated with endoplasmic reticulum (ER) stress responses. However, the molecular basis for ER stress-mediated hepatocyte injury remains largely unknown. This study investigated the effect of ER stress on dual-specificity phosphatase 5 (DUSP5) expression and its role in hepatocyte death. Analysis of Gene Expression Omnibus (GEO) database showed that hepatic DUSP5 levels increased in the patients with liver fibrosis, which was verified in mouse models of liver diseases with ER stress. DUSP5 expression was elevated in both fibrotic and acutely injured liver of mice treated with liver toxicants. Treatment of ER stress inducers enhanced DUSP5 expression in hepatocytes, which was validated in vivo condition. The induction of DUSP5 by ER stress was blocked by either treatment with a chemical inhibitor of the protein kinase RNA-like endoplasmic reticulum kinase (PERK) pathway, or knockdown of C/EBP homologous protein (CHOP), whereas it was not affected by the silencing of IRE1 or ATF6. In addition, DUSP5 overexpression decreased extracellular-signal-regulated kinase (ERK) phosphorylation, but increased cleaved caspase-3 levels. Moreover, the reduction of cell viability under ER stress condition was attenuated by DUSP5 knockdown. In conclusion, DUSP5 expression is elevated in hepatocytes by ER stress through the PERK-CHOP pathway, contributing to hepatocyte death possibly through ERK inhibition.


Assuntos
Fosfatases de Especificidade Dupla/genética , Estresse do Retículo Endoplasmático , Hepatócitos/metabolismo , Transdução de Sinais , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/metabolismo , Animais , Apoptose/genética , Morte Celular/genética , Expressão Gênica , Hepatócitos/patologia , Humanos , Hepatopatias/etiologia , Hepatopatias/metabolismo , Camundongos
6.
Nucleic Acids Res ; 47(19): 10010-10026, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31552418

RESUMO

Transcriptional perturbation signatures are valuable data sources for functional genomics. Linking perturbation signatures to screenings opens the possibility to model cellular phenotypes from expression data and to identify efficacious drugs. We linked perturbation transcriptomics data from the LINCS-L1000 project with cell viability information upon genetic (Achilles project) and chemical (CTRP screen) perturbations yielding more than 90 000 signature-viability pairs. An integrated analysis showed that the cell viability signature is a major factor underlying perturbation signatures. The signature is linked to transcription factors regulating cell death, proliferation and division time. We used the cell viability-signature relationship to predict viability from transcriptomics signatures, and identified and validated compounds that induce cell death in tumor cell lines. We showed that cellular toxicity can lead to unexpected similarity of signatures, confounding mechanism of action discovery. Consensus compound signatures predicted cell-specific drug sensitivity, even if the signature is not measured in the same cell line, and outperformed conventional drug-specific features. Our results can help in understanding mechanisms behind cell death and removing confounding factors of transcriptomic perturbation screens. To interactively browse our results and predict cell viability in new gene expression samples, we developed CEVIChE (CEll VIability Calculator from gene Expression; https://saezlab.shinyapps.io/ceviche/).


Assuntos
Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/genética , Software , Transcriptoma/genética , Morte Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Descoberta de Drogas , Humanos
7.
Cell Physiol Biochem ; 53(3): 496-507, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31486324

RESUMO

BACKGROUND/AIMS: Like nucleated cells, erythrocytes (red blood cells, RBCs) are capable of executing programmed cell death pathways. RBCs undergo necroptosis in response to CD59-specific pore-forming toxins (PFTs). The relationship between blood bank storage and RBC necroptosis was explored in this study. METHODS: Human RBCs were stored in standard blood bank additive solutions (AS-1, AS-3, or AS-5) for 1 week and hemolysis was evaluated in the context of necroptosis inhibitors and reactive oxygen species (ROS) scavengers. Activation of key factors including RIP1, RIP3, and MLKL was determined using immunoprecipitations and western blot. RBC vesiculation and formation of echinocytes was determined using phase-contrast microscopy. The effect of necroptosis and storage on RBC clearance was determined using a murine transfusion model. RESULTS: Necroptosis is associated with increased RBC clearance post-transfusion. Moreover, storage in AS-1, AS-3, or AS-5 sensitizes RBCs for necroptosis. Importantly, storage-sensitized RBCs undergo necroptosis in response to multiple PFTs, regardless of specificity for CD59. Storage-sensitized RBCs undergo necroptosis via NADPH oxidase-generated ROS. RBC storage led to RIP1 phosphorylation and necrosome formation in an NADPH oxidase-dependent manner suggesting the basis for this sensitization. In addition, storage led to increased RBC clearance post-transfusion. Clearance of these RBCs was due to Syk-dependent echinocyte formation. CONCLUSION: Storage-induced sensitization to RBC necroptosis and clearance is important as it may be relevant to hemolytic transfusion reactions.


Assuntos
Antígenos CD59/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Necrose/metabolismo , Adjuvantes Imunológicos , Animais , Apoptose/fisiologia , Bancos de Sangue , Western Blotting , Morte Celular/genética , Morte Celular/fisiologia , Células Cultivadas , Hemólise/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação/genética , Fosforilação/fisiologia , Espécies Reativas de Oxigênio/metabolismo
8.
Int J Radiat Oncol Biol Phys ; 105(5): 1119-1125, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31425731

RESUMO

PURPOSE: This study seeks to identify biological factors that may yield a therapeutic advantage of proton therapy versus photon therapy. Specifically, we address the role of nonhomologous end-joining (NHEJ) and homologous recombination (HR) in the survival of cells in response to clinical photon and proton beams. METHODS AND MATERIALS: We irradiated HT1080, M059K (DNA-PKcs+/+), and HCC1937 human cancer cell lines and their isogenic counterparts HT1080-shDNA-PKcs, HT1080-shRAD51IND, M059J (DNA-PKcs-/-), and HCC1937-BRCA1 (BRCA1 complemented) to assess cell clonogenic survival and γ-H2AX radiation-induced foci. Cells were irradiated with either clinically relevant photons or 1 of 3 proton linear energy transfer (LET) values. RESULTS: Our results indicate that NHEJ deficiency is more important in dictating cell survival than proton LET. Cells with disrupted HR through BRCA1 mutation showed increased radiosensitivity only for high-LET protons whereas RAD51 depletion showed increased radiosensitivity for both photons and protons. DNA double strand breaks, assessed by γ-H2AX radiation-induced foci, showed greater numbers after 24 hours in cells exposed to higher LET protons. We also observed that NHEJ-deficient cells were unable to repair the vast majority of double strand breaks after 24 hours. CONCLUSIONS: BRCA1 mutation significantly sensitizes cells to protons, but not photons. Loss of NHEJ renders cells hypersensitive to radiation, whereas the relative importance of HR increases with LET across several cell lines. This may be attributable to the more clustered damage induced by higher LET protons, which are harder to repair through NHEJ. This highlights the importance of tumor biology in dictating treatment modality and suggests BRCA1 as a potential biomarker for proton therapy response. Our data also support the use of pharmacologic inhibitors of DNA repair to enhance the sensitivity to different radiation types, although this raises issues for normal tissue toxicity.


Assuntos
Morte Celular/genética , Reparo do DNA por Junção de Extremidades/fisiologia , Genes BRCA1 , Recombinação Homóloga/fisiologia , Transferência Linear de Energia , Fótons , Prótons , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Sobrevivência Celular/efeitos da radiação , Quebras de DNA de Cadeia Dupla , Inativação Gênica , Histonas/análise , Humanos , Mutação , Rad51 Recombinase/genética , Tolerância a Radiação/genética , Tolerância a Radiação/efeitos da radiação , Fatores de Tempo
9.
Elife ; 82019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31318332

RESUMO

Acid-sensing ion channels have important functions in physiology and pathology, but the molecular composition of acid-activated chloride channels had remained unclear. We now used a genome-wide siRNA screen to molecularly identify the widely expressed acid-sensitive outwardly-rectifying anion channel PAORAC/ASOR. ASOR is formed by TMEM206 proteins which display two transmembrane domains (TMs) and are expressed at the plasma membrane. Ion permeation-changing mutations along the length of TM2 and at the end of TM1 suggest that these segments line ASOR's pore. While not belonging to a gene family, TMEM206 has orthologs in probably all vertebrates. Currents from evolutionarily distant orthologs share activation by protons, a feature essential for ASOR's role in acid-induced cell death. TMEM206 defines a novel class of ion channels. Its identification will help to understand its physiological roles and the diverse ways by which anion-selective pores can be formed.


Assuntos
Membrana Celular/genética , Canais de Cloreto/genética , Cloretos/metabolismo , Ácidos/metabolismo , Animais , Ânions/metabolismo , Morte Celular/genética , Membrana Celular/metabolismo , Canais de Cloreto/química , Canais de Cloreto/metabolismo , Genoma Humano/genética , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Prótons
10.
Int J Mol Sci ; 20(14)2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340436

RESUMO

Status epilepticus may decrease mitochondrial biogenesis, resulting in neuronal cell death occurring in the hippocampus. Sirtuin 1 (SIRT1) functionally interacts with peroxisome proliferator-activated receptors and γ coactivator 1α (PGC-1α), which play a crucial role in the regulation of mitochondrial biogenesis. In Sprague-Dawley rats, kainic acid was microinjected unilaterally into the hippocampal CA3 subfield to induce bilateral seizure activity. SIRT1, PGC-1α, and other key proteins involving mitochondrial biogenesis and the amount of mitochondrial DNA were investigated. SIRT1 antisense oligodeoxynucleotide was used to evaluate the relationship between SIRT1 and mitochondrial biogenesis, as well as the mitochondrial function, oxidative stress, and neuronal cell survival. Increased SIRT1, PGC-1α, and mitochondrial biogenesis machinery were found in the hippocampus following experimental status epilepticus. Downregulation of SIRT1 decreased PGC-1α expression and mitochondrial biogenesis machinery, increased Complex I dysfunction, augmented the level of oxidized proteins, raised activated caspase-3 expression, and promoted neuronal cell damage in the hippocampus. The results suggest that the SIRT1 signaling pathway may play a pivotal role in mitochondrial biogenesis, and could be considered an endogenous neuroprotective mechanism counteracting seizure-induced neuronal cell damage following status epilepticus.


Assuntos
Região CA3 Hipocampal/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Sirtuína 1/genética , Estado Epiléptico/genética , Animais , Região CA3 Hipocampal/metabolismo , Região CA3 Hipocampal/patologia , Caspase 3/genética , Caspase 3/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Complexo I de Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Injeções Intraventriculares , Ácido Caínico/administração & dosagem , Masculino , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neurônios/metabolismo , Neurônios/patologia , Biogênese de Organelas , Estresse Oxidativo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Sirtuína 1/antagonistas & inibidores , Sirtuína 1/metabolismo , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/metabolismo , Estado Epiléptico/patologia , Técnicas Estereotáxicas
11.
Int J Mol Sci ; 20(14)2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340550

RESUMO

Cysteine cathepsins are lysosomal enzymes belonging to the papain family. Their expression is misregulated in a wide variety of tumors, and ample data prove their involvement in cancer progression, angiogenesis, metastasis, and in the occurrence of drug resistance. However, while their overexpression is usually associated with highly aggressive tumor phenotypes, their mechanistic role in cancer progression is still to be determined to develop new therapeutic strategies. In this review, we highlight the literature related to the role of the cysteine cathepsins in cancer biology, with particular emphasis on their input into tumor biology.


Assuntos
Catepsinas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Lisossomos/enzimologia , Neoplasias/genética , Neovascularização Patológica/genética , Animais , Antineoplásicos/uso terapêutico , Catepsinas/química , Catepsinas/classificação , Catepsinas/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular Tumoral , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Humanos , Metástase Linfática , Lisossomos/efeitos dos fármacos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/enzimologia , Neovascularização Patológica/patologia , Conformação Proteica , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais
12.
Curr Med Sci ; 39(4): 513-522, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31346984

RESUMO

Necroptosis is a non-apoptotic programmed cell death pathway, which causes necrosis-like morphologic changes and triggers inflammation in the surrounding tissues. Accumulating evidence has demonstrated that necroptosis is involved in a number of pathological processes that lead to cardiovascular diseases. However, the exact molecular pathways linking them remain unknown. Herein, this review summarizes the necroptosis-related pathways involved in the development of various cardiovascular diseases, including atherosclerosis, cardiac ischemia-reperfusion injury, cardiac hypertrophy, dilated cardiomyopathy and myocardial infarction, and may shed light on the diagnosis and treatment of these diseases.


Assuntos
Apoptose/genética , Doenças Cardiovasculares/genética , Morte Celular/genética , /genética , Aterosclerose/genética , Aterosclerose/fisiopatologia , Cardiomegalia/genética , Cardiomegalia/fisiopatologia , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/fisiopatologia , Doenças Cardiovasculares/fisiopatologia , Humanos , Infarto do Miocárdio/genética , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/fisiopatologia , Transdução de Sinais/genética
13.
Invest Ophthalmol Vis Sci ; 60(8): 3034-3045, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31311035

RESUMO

Purpose: Visual (retinoid) cycle anomalies induce aberrant build-up of all-trans retinal (atRAL) in the retinal pigment epithelium (RPE), which is a cause of RPE atrophy in Stargardt disease type 1 and age-related macular degeneration. NLR family pyrin domain containing 3 (NLRP3) inflammasome activation is implicated in the etiology of age-related macular degeneration. Here, we elucidated the relationship between NLRP3 inflammasome activation and atRAL-induced death of RPE cells. Methods: Cellular toxicities were assessed by MTS or MTT assays. Expression levels of mRNAs and proteins were determined by quantitative reverse transcription-polymerase chain reaction, Western blotting, or enzyme-linked immunosorbent assay. Fluorescence microscopy was used to examine intracellular signals. Ultrastructural features of organelles were examined by transmission electron microscope. Results: Abnormal accumulation of atRAL was associated with a significant increase in the proportion of human ARPE-19 cells exhibiting features of apoptosis and Caspase-3/gasdermin E (GSDME)-mediated pyroptosis. These cells also exhibited elevated expression of NLRP3, ASC, cleaved Caspase-1/poly ADP-ribose polymerase (PARP)/Caspase-3/GSDME, interleukin-1ß (IL-1ß), and IL-18, as well as NLRP3 inflammasome-related genes (IL1B and IL18). After exposure of human ARPE-19 cells to excess atRAL, reactive oxygen species (ROS) (including mitochondrial ROS) and cathepsins released from lysosomes transmitted signals leading to NLRP3 inflammasome activation. Suppressing the production of ROS, NLRP3 inflammasome, Caspase-1, cathepsin B, or cathepsin D protected ARPE-19 cells against atRAL-associated cytotoxicity. Damage to mitochondria, lysosomes, and endoplasmic reticulum in atRAL-exposed ARPE-19 cells was partially alleviated by treatment with MCC950, a selective NLRP3 inflammasome inhibitor. Conclusions: Aberrant build-up of atRAL promotes the death of RPE cells via NLRP3 inflammasome activation.


Assuntos
Morte Celular/genética , Regulação da Expressão Gênica , Inflamassomos/metabolismo , Degeneração Macular/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Epitélio Pigmentado da Retina/ultraestrutura , Western Blotting , Células Cultivadas , Humanos , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Microscopia Eletrônica de Transmissão , Proteína 3 que Contém Domínio de Pirina da Família NLR/biossíntese , RNA/genética , Epitélio Pigmentado da Retina/metabolismo , Transdução de Sinais
14.
Immunity ; 50(6): 1352-1364, 2019 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-31216460

RESUMO

Caspases are an evolutionary conserved family of cysteine proteases that are centrally involved in cell death and inflammation responses. A wealth of foundational insight into the molecular mechanisms that control caspase activation has emerged in recent years. Important advancements include the identification of additional inflammasome platforms and pathways that regulate activation of inflammatory caspases; the discovery of gasdermin D as the effector of pyroptosis and interleukin (IL)-1 and IL-18 secretion; and the existence of substantial crosstalk between inflammatory and apoptotic initiator caspases. A better understanding of the mechanisms regulating caspase activation has supported initial efforts to modulate dysfunctional cell death and inflammation pathways in a suite of communicable, inflammatory, malignant, metabolic, and neurodegenerative diseases. Here, we review current understanding of caspase biology with a prime focus on the inflammatory caspases and outline important topics for future experimentation.


Assuntos
Caspases/metabolismo , Suscetibilidade a Doenças , Inflamação/etiologia , Inflamação/metabolismo , Animais , Apoptose , Biomarcadores , Caspases/química , Caspases/genética , Morte Celular/genética , Citocinas/metabolismo , Humanos , Inflamassomos/metabolismo , Inflamação/tratamento farmacológico , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Terapia de Alvo Molecular , Piroptose , Transdução de Sinais/efeitos dos fármacos
15.
Mol Carcinog ; 58(9): 1691-1700, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31237025

RESUMO

Non-melanoma skin cancer frequently results from chronic exposure to ultraviolet (UV) irradiation. UV-induced DNA damage activates cell cycle arrest checkpoints through degradation of the cyclin-dependent kinase activators, the cell division cycle 25 (CDC25) phosphatases. We previously reported increased CDC25A in nonmelanoma skin cancer, but CDC25B and CDC25C had not been previously examined. Consequently, we hypothesized that increased expression of CDC25B and CDC25C increases tumor cell proliferation and skin tumor growth. We found that CDC25B and CDC25C were increased in mouse and human skin cancers. CDC25B was primarily cytoplasmic in skin and skin tumors and was significantly increased in the squamous cell carcinoma (SCC), while CDC25C was mostly nuclear in the skin, with an increased cytoplasmic signal in the premalignant and malignant tumors. Surprisingly, forced expression of CDC25B or CDC25C in cultured SCC cells did not affect proliferation, but instead suppressed apoptosis, while CDC25C silencing increased apoptosis without impacting proliferation. Targeting CDC25C to the nucleus via mutation of its nuclear export sequence, however, increased proliferation in SCC cells. Overexpression of CDC25C in the nuclear compartment did not hinder the ability of CDC25C to suppress apoptosis, neither did mutation of sites necessary for its interaction with 14-3-3 proteins. Analysis of apoptotic signaling pathways revealed that CDC25C increased activating phosphorylation of Akt on Ser473 , increased inhibitory phosphorylation of proapoptotic BAD on Ser136 , and increased the survival protein Survivin. Silencing of CDC25C significantly reduced Survivin levels. Taken together, these data suggest that increased expression of CDC25B or CDC25C are mechanisms by which skin cancers evade apoptotic cell death.


Assuntos
Morte Celular/genética , Neoplasias Cutâneas/genética , Fosfatases cdc25/genética , Animais , Apoptose/genética , Carcinoma de Células Escamosas/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Proliferação de Células/genética , Citoplasma/genética , Dano ao DNA/genética , Humanos , Camundongos , Camundongos Transgênicos/genética , Fosforilação/genética , Transdução de Sinais/genética
16.
Curr Protoc Mol Biol ; 127(1): e90, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31237424

RESUMO

Cell death involves the release of short DNA fragments into blood, termed circulating cell-free DNA (cfDNA). Sequencing of cfDNA in the plasma has recently emerged as a liquid biopsy for detecting fetal chromosomal aberrations, tumor DNA, and graft rejection. However, in cases where cfDNA is derived from tissues with a normal genome, its primary sequence is not informative regarding the tissue of origin. We developed a method of determining the tissue origins of cfDNA, allowing inference of tissue-specific cell death, based on tissue-specific methylation patterns. We have previously described a version of the method that uses next generation sequencing (NGS) to determine methylation patterns in specific marker loci. Here we describe a rapid and simple procedure for cfDNA methylation analysis using droplet digital PCR (ddPCR) on bisulfite treated cfDNA to accurately count the number of molecules carrying a specific methylation signature. Specificity and sensitivity of the assay increases by simultaneously interrogating four to six cytosines in the same molecule using two fluorescent probes. cfDNA methylation analysis using ddPCR can find multiple applications in the non-invasive study of human tissue dynamics in health and disease. © 2019 by John Wiley & Sons, Inc.


Assuntos
Morte Celular/genética , Ácidos Nucleicos Livres/sangue , Metilação de DNA/genética , DNA/sangue , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reação em Cadeia da Polimerase/métodos , Humanos , Análise de Sequência de DNA/métodos
17.
Int J Mol Sci ; 20(13)2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31248062

RESUMO

Regulated neuronal cell death plays an essential role in biological processes in normal physiology, including the development of the nervous system. However, the deregulation of neuronal apoptosis by various factors leads to neurodegenerative diseases such as ischemic stroke and Alzheimer's disease (AD). Death-associated protein kinase 1 (DAPK1) is a calcium/calmodulin (Ca2+/CaM)-dependent serine/threonine (Ser/Thr) protein kinase that activates death signaling and regulates apoptotic neuronal cell death. Although DAPK1 is tightly regulated under physiological conditions, DAPK1 deregulation in the brain contributes to the development of neurological disorders. In this review, we describe the molecular mechanisms of DAPK1 regulation in neurons under various stresses. We also discuss the role of DAPK1 signaling in the phosphorylation-dependent and phosphorylation-independent regulation of its downstream targets in neuronal cell death. Moreover, we focus on the major impact of DAPK1 deregulation on the progression of neurodegenerative diseases and the development of drugs targeting DAPK1 for the treatment of diseases. Therefore, this review summarizes the DAPK1 phosphorylation signaling pathways in various neurodegenerative diseases.


Assuntos
Proteínas Quinases Associadas com Morte Celular/metabolismo , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/metabolismo , Neurônios/metabolismo , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Animais , Apoptose/genética , Biomarcadores , Morte Celular/genética , Proteínas Quinases Associadas com Morte Celular/química , Proteínas Quinases Associadas com Morte Celular/genética , Humanos , Família Multigênica , Doenças Neurodegenerativas/patologia , Neurônios/patologia , Fosforilação , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/metabolismo , Relação Estrutura-Atividade
18.
Genes (Basel) ; 10(6)2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31207907

RESUMO

Photoreceptor physiology and pathophysiology is intricately linked to guanosine-3',5'-cyclic monophosphate (cGMP)-signaling. Here, we discuss the importance of cGMP-signaling for the pathogenesis of hereditary retinal degeneration. Excessive accumulation of cGMP in photoreceptors is a common denominator in cell death caused by a variety of different gene mutations. The cGMP-dependent cell death pathway may be targeted for the treatment of inherited photoreceptor degeneration, using specifically designed and formulated inhibitory cGMP analogues. Moreover, cGMP-signaling and its down-stream targets may be exploited for the development of novel biomarkers that could facilitate monitoring of disease progression and reveal the response to treatment in future clinical trials. We then briefly present the importance of appropriate formulations for delivery to the retina, both for drug and biomarker applications. Finally, the review touches on important aspects of future clinical translation, highlighting the need for interdisciplinary cooperation of researchers from a diverse range of fields.


Assuntos
GMP Cíclico/metabolismo , Células Fotorreceptoras/metabolismo , Retina/metabolismo , Degeneração Retiniana/genética , Morte Celular/genética , GMP Cíclico/genética , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Humanos , Células Fotorreceptoras/patologia , Retina/patologia , Degeneração Retiniana/metabolismo , Degeneração Retiniana/fisiopatologia , Transdução de Sinais/genética
19.
J Exp Clin Cancer Res ; 38(1): 252, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31196136

RESUMO

BACKGROUND: Optimal therapeutic strategies for hepatocellular carcinoma (HCC) patients are still challenging due to the high recurrence rate after surgical resection and chemotherapy resistance. Growing evidence shows that genetic and epigenetic alterations are involved in HCC progression and resistance to therapy, however the molecular mechanisms underlying resistance to therapy have not been fully understood. METHODS: Expression of SIRT7 in 17 paired paraffin-embedded HCC tissues and adjacent nontumoral liver tissues was examined by immunohistochemistry and Western blot. The mRNA expression of SIRT7 in 20 paired frozen HCC tissues and adjacent nontumoral liver tissues was analyzed by quantitative RT-PCR. The biologic consequences of overexpression and knockdown of SIRT7 in HCC therapy sensitivity were studied in vitro and in vivo. Interaction between SIRT7 and p53 were studied in HCC cell lines. RESULTS: SIRT7 expression was frequently upregulated in clinical HCC samples, and its expression was highly associated with TACE-resistance and poor survival (P = 0.008.) Depletion of SIRT7 from multiple liver cancer cell lines significantly increased doxorubicin toxicity while overexpression of SIRT7 largely abolished doxorubicin induced apoptosis. At the molecular level, we observed that SIRT7 interacts with and induces deacetylation of p53 at lysines 320 and 373. Deacetylated p53 showed significantly less affinity for the NOXA promoter and its transcription. In mouse xenografts, SIRT7 suppression increased doxorubicin induced p53 activation, inhibited tumor growth and induced apoptosis. CONCLUSION: The newly identified SIRT7-p53-NOXA axis partially illustrates the molecular mechanism of HCC resistance to therapy and represents a novel potential therapeutic target for HCC treatment.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Transdução de Sinais , Sirtuínas/genética , Proteína Supressora de Tumor p53/genética , Idoso , Animais , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/terapia , Morte Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Modelos Animais de Doenças , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/terapia , Masculino , Camundongos , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Ligação Proteica , Sirtuínas/metabolismo , Proteína Supressora de Tumor p53/metabolismo
20.
Phytopathology ; 109(10): 1751-1759, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31199201

RESUMO

The wheat Sr2 locus confers partial resistance to four biotrophic pathogens: wheat stem rust (Puccinia graminis f. sp. tritici), leaf rust (P. triticina), stripe rust (P. striiformis f. sp. tritici), and powdery mildew (Blumeria graminis f. sp. tritici). In addition, Sr2 is linked with a brown coloration of ears and stems, termed pseudo-black chaff (PBC). PBC, initially believed to be elicited by stem rust infection, was subsequently recognized to occur in the absence of pathogen infection. The current study demonstrates that the resistance response to stem rust is associated with the death of photosynthetic cells around rust infection sites in the inoculated leaf sheath. Similarly, Sr2-dependent resistance to powdery mildew was associated with the death of leaf mesophyll cells around mildew infection sites. We demonstrate that PBC occurring in the absence of pathogen inoculation also corresponds with death and the collapse of photosynthetic cells in the affected parts of stems and ears. In addition, Sr2-dependent necrosis was inducible in leaves by application of petroleum jelly or by heat treatments. Thus, Sr2 was found to be associated with cell death, which could be triggered by either biotic or abiotic stresses. Our results suggest a role for the Sr2 locus in controlling cell death in response to stress.


Assuntos
Basidiomycota , Resistência à Doença , Genes de Plantas , Triticum , Morte Celular/genética , Resistência à Doença/genética , Genes de Plantas/genética , Fenótipo , Doenças das Plantas/microbiologia , Estresse Fisiológico , Triticum/genética , Triticum/microbiologia
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