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1.
Nat Commun ; 11(1): 4416, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32887881

RESUMO

Despite the clear association between myocardial injury, heart failure and depressed myocardial energetics, little is known about upstream signals responsible for remodeling myocardial metabolism after pathological stress. Here, we report increased mitochondrial calmodulin kinase II (CaMKII) activation and left ventricular dilation in mice one week after myocardial infarction (MI) surgery. By contrast, mice with genetic mitochondrial CaMKII inhibition are protected from left ventricular dilation and dysfunction after MI. Mice with myocardial and mitochondrial CaMKII overexpression (mtCaMKII) have severe dilated cardiomyopathy and decreased ATP that causes elevated cytoplasmic resting (diastolic) Ca2+ concentration and reduced mechanical performance. We map a metabolic pathway that rescues disease phenotypes in mtCaMKII mice, providing insights into physiological and pathological metabolic consequences of CaMKII signaling in mitochondria. Our findings suggest myocardial dilation, a disease phenotype lacking specific therapies, can be prevented by targeted replacement of mitochondrial creatine kinase or mitochondrial-targeted CaMKII inhibition.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomiopatia Dilatada/metabolismo , Infarto do Miocárdio/fisiopatologia , Animais , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Insuficiência Cardíaca/metabolismo , Ventrículos do Coração/fisiopatologia , Camundongos , Camundongos Transgênicos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Infarto do Miocárdio/cirurgia , Transdução de Sinais
2.
Nat Commun ; 11(1): 3866, 2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32737287

RESUMO

Upon severe head injury (HI), blood vessels of the meninges and brain parenchyma are inevitably damaged. While limited vascular regeneration of the injured brain has been studied extensively, our understanding of meningeal vascular regeneration following head injury is quite limited. Here, we identify key pathways governing meningeal vascular regeneration following HI. Rapid and complete vascular regeneration in the meninges is predominantly driven by VEGFR2 signaling. Substantial increase of VEGFR2 is observed in both human patients and mouse models of HI, and endothelial cell-specific deletion of Vegfr2 in the latter inhibits meningeal vascular regeneration. We further identify the facilitating, stabilizing and arresting roles of Tie2, PDGFRß and Dll4 signaling, respectively, in meningeal vascular regeneration. Prolonged inhibition of this angiogenic process following HI compromises immunological and stromal integrity of the injured meninges. These findings establish a molecular framework for meningeal vascular regeneration after HI, and may guide development of wound healing therapeutics.


Assuntos
Traumatismos Craniocerebrais/genética , Células Endoteliais/metabolismo , Neovascularização Fisiológica/genética , Regeneração/genética , Transdução de Sinais/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Circulação Cerebrovascular , Traumatismos Craniocerebrais/metabolismo , Traumatismos Craniocerebrais/patologia , Modelos Animais de Doenças , Células Endoteliais/patologia , Regulação da Expressão Gênica/genética , Humanos , Macrófagos/metabolismo , Macrófagos/patologia , Meninges/lesões , Meninges/metabolismo , Camundongos , Camundongos Knockout , Receptor beta de Fator de Crescimento Derivado de Plaquetas/genética , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor TIE-2/genética , Receptor TIE-2/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Cicatrização/genética
3.
PLoS One ; 15(8): e0237847, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833985

RESUMO

PROM is one of the common complications of perinatal period, which seriously threatens the mother and newborn. The purpose of this study was to identify the role of NLRC4 inflammasomes in this process and their underlying mechanisms. We performed high-throughput RNA sequencing of fetal membrane tissue from 3 normal pregnant women and 3 term-premature rupture of fetal membrane (TPROM) patients who met the inclusion criteria, and found that NLRC4 was significantly up-regulated in TPROM patients. An observational study of TPROM patients (PROM group, n = 30) and normal pregnant women (control group, n = 30) was performed at the Xuzhou Maternal and Child Health Hospital affiliated to Xuzhou Medical University from May 2018 to May 2019. The expression of genes involved in inflammasome complex including NLRC1, NLRC3, AIM2, NLRC4, ASC, caspase-1, IL-6, IL-18 and IL-1ßwas determined via real-time PCR, immunohistochemistry and immunofluorescence. Measurement of NLRC4 level in serum was conducted by ELISA assay. The results showed that the NLRC4, ASC, caspase-1, IL-1ß and IL-18 levels in fetal membrane, placental tissues and maternal serum were markedly higher in the PROM group than that in the control group. In conclusion, NLRC4 is a markedly up-regulated gene in TPROM fetal membrane tissue, suggesting that NLRC4 is involved in the occurrence and development of TPROM; NLRC4 levels in maternal blood serum are closely related to TPROM and have the potential to assist doctors in predicting and diagnosing PROM.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Ruptura Prematura de Membranas Fetais/metabolismo , Inflamassomos/metabolismo , Adulto , Proteínas Adaptadoras de Sinalização CARD/sangue , Proteínas de Ligação ao Cálcio/sangue , Feminino , Ruptura Prematura de Membranas Fetais/sangue , Humanos , Placenta/metabolismo , Gravidez
4.
PLoS Biol ; 18(7): e3000782, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32692742

RESUMO

Tight regulation of gene transcription and mRNA splicing is essential for plant growth and development. Here we demonstrate that a plant-specific protein, EMBRYO DEFECTIVE 1579 (EMB1579), controls multiple growth and developmental processes in Arabidopsis. We demonstrate that EMB1579 forms liquid-like condensates both in vitro and in vivo, and the formation of normal-sized EMB1579 condensates is crucial for its cellular functions. We found that some chromosomal and RNA-related proteins interact with EMB1579 compartments, and loss of function of EMB1579 affects global gene transcription and mRNA splicing. Using floral transition as a physiological process, we demonstrate that EMB1579 is involved in FLOWERING LOCUS C (FLC)-mediated repression of flowering. Interestingly, we found that EMB1579 physically interacts with a homologue of Drosophila nucleosome remodeling factor 55-kDa (p55) called MULTIPLE SUPPRESSOR OF IRA 4 (MSI4), which has been implicated in repressing the expression of FLC by forming a complex with DNA Damage Binding Protein 1 (DDB1) and Cullin 4 (CUL4). This complex, named CUL4-DDB1MSI4, physically associates with a CURLY LEAF (CLF)-containing Polycomb Repressive Complex 2 (CLF-PRC2). We further demonstrate that EMB1579 interacts with CUL4 and DDB1, and EMB1579 condensates can recruit and condense MSI4 and DDB1. Furthermore, emb1579 phenocopies msi4 in terms of the level of H3K27 trimethylation on FLC. This allows us to propose that EMB1579 condensates recruit and condense CUL4-DDB1MSI4 complex, which facilitates the interaction of CUL4-DDB1MSI4 with CLF-PRC2 and promotes the role of CLF-PRC2 in establishing and/or maintaining the level of H3K27 trimethylation on FLC. Thus, we report a new mechanism for regulating plant gene transcription, mRNA splicing, and growth and development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Proteínas de Ligação ao Cálcio/metabolismo , Desenvolvimento Vegetal/genética , Processamento de RNA/genética , Transcrição Genética , Proteínas de Arabidopsis/genética , Proteínas de Ligação ao Cálcio/genética , Núcleo Celular/metabolismo , Flores/fisiologia , Histonas/metabolismo , Mutação com Perda de Função , Lisina/metabolismo , Metilação , Proteínas Nucleares/metabolismo , Fenótipo , Raízes de Plantas/citologia , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Repetitivas de Aminoácidos
5.
PLoS Comput Biol ; 16(7): e1008048, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32658888

RESUMO

Heart failure (HF) is associated with an increased propensity for atrial fibrillation (AF), causing higher mortality than AF or HF alone. It is hypothesized that HF-induced remodelling of atrial cellular and tissue properties promotes the genesis of atrial action potential (AP) alternans and conduction alternans that perpetuate AF. However, the mechanism underlying the increased susceptibility to atrial alternans in HF remains incompletely elucidated. In this study, we investigated the effects of how HF-induced atrial cellular electrophysiological (with prolonged AP duration) and tissue structural (reduced cell-to-cell coupling caused by atrial fibrosis) remodelling can have an effect on the generation of atrial AP alternans and their conduction at the cellular and one-dimensional (1D) tissue levels. Simulation results showed that HF-induced atrial electrical remodelling prolonged AP duration, which was accompanied by an increased sarcoplasmic reticulum (SR) Ca2+ content and Ca2+ transient amplitude. Further analysis demonstrated that HF-induced atrial electrical remodelling increased susceptibility to atrial alternans mainly due to the increased sarcoplasmic reticulum Ca2+-ATPase (SERCA) Ca2+ reuptake, modulated by increased phospholamban (PLB) phosphorylation, and the decreased transient outward K+ current (Ito). The underlying mechanism has been suggested that the increased SR Ca2+ content and prolonged AP did not fully recover to their previous levels at the end of diastole, resulting in a smaller SR Ca2+ release and AP in the next beat. These produced Ca2+ transient alternans and AP alternans, and further caused AP alternans and Ca2+ transient alternans through Ca2+→AP coupling and AP→Ca2+ coupling, respectively. Simulation of a 1D tissue model showed that the combined action of HF-induced ion channel remodelling and a decrease in cell-to-cell coupling due to fibrosis increased the heart tissue's susceptibility to the formation of spatially discordant alternans, resulting in an increased functional AP propagation dispersion, which is pro-arrhythmic. These findings provide insights into how HF promotes atrial arrhythmia in association with atrial alternans.


Assuntos
Remodelamento Atrial , Insuficiência Cardíaca/fisiopatologia , Potenciais de Ação , Algoritmos , Animais , Fibrilação Atrial/fisiopatologia , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Simulação por Computador , Cães , Condutividade Elétrica , Átrios do Coração/fisiopatologia , Ventrículos do Coração/fisiopatologia , Humanos , Camundongos , Modelos Cardiovasculares , Contração Miocárdica , Miócitos Cardíacos/patologia , Fosforilação , Retículo Sarcoplasmático/metabolismo
6.
PLoS One ; 15(7): e0230400, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32639965

RESUMO

Alterations in the cortico-cerebellar-thalamic-cortical circuit might underlie the diversity of symptoms in schizophrenia. However, molecular changes in cerebellar neuronal circuits, part of this network, have not yet been fully determined. Using LC-MS/MS, we screened altered candidates in pooled grey matter of cerebellum from schizophrenia subjects who committed suicide (n = 4) and healthy individuals (n = 4). Further validation by immunoblotting of three selected candidates was performed in two cohorts comprising schizophrenia (n = 20), non-schizophrenia suicide (n = 6) and healthy controls (n = 21). We found 99 significantly altered proteins, 31 of them previously reported in other brain areas by proteomic studies. Transport function was the most enriched category, while cell communication was the most prevalent function. For validation, we selected the vacuolar proton pump subunit 1 (VPP1), from transport, and two EF-hand calcium-binding proteins, calmodulin and parvalbumin, from cell communication. All candidates showed significant changes in schizophrenia (n = 7) compared to controls (n = 7). VPP1 was altered in the non-schizophrenia suicide group and increased levels of parvalbumin were linked to antipsychotics. Further validation in an independent cohort of non-suicidal chronic schizophrenia subjects (n = 13) and non-psychiatric controls (n = 14) showed that parvalbumin was increased, while calmodulin was decreased in schizophrenia. Our findings provide evidence of calcium-binding protein dysregulation in the cerebellum in schizophrenia, suggesting an impact on normal calcium-dependent synaptic functioning of cerebellar circuits. Our study also links VPP1 to suicide behaviours, suggesting a possible impairment in vesicle neurotransmitter refilling and release in these phenotypes.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cerebelo/metabolismo , Esquizofrenia/patologia , Adulto , Calmodulina/metabolismo , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Regulação para Baixo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Parvalbuminas/metabolismo , Proteoma/análise , Esquizofrenia/metabolismo , Tentativa de Suicídio , Espectrometria de Massas em Tandem , Regulação para Cima
7.
Tumour Biol ; 42(7): 1010428320937863, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32686600

RESUMO

Maintaining intracellular pH is crucial for preserving healthy cellular behavior and, when dysregulated, results in increased proliferation, migration, and invasion. The Na+/H+ exchanger isoform 1 is a highly regulated transmembrane antiporter that maintains pH homeostasis by exporting protons in response to intra- and extracellular signals. Activation of Na+/H+ exchanger isoform 1 is exquisitely regulated by the extracellular environment and protein cofactors, including calcineurin B homologous proteins 1 and 2. While Na+/H+ exchanger isoform 1 and calcineurin B homologous protein 1 are ubiquitously expressed, calcineurin B homologous protein 2 shows tissue-specific expression and upregulation in a variety of cancer cells. In addition, calcineurin B homologous protein 2 expression is modulated by tumorigenic extracellular conditions like low nutrients. To understand the role of calcineurin B homologous protein 2 in tumorigenesis and survival in lung cancer, we surveyed existing databases and formed a comprehensive report of Na+/H+ exchanger isoform 1, calcineurin B homologous protein 1, and calcineurin B homologous protein 2 expression in diseased and non-diseased tissues. We show that calcineurin B homologous protein 2 is upregulated during oncogenesis in many adeno and squamous carcinomas. To understand the functional role of calcineurin B homologous protein 2 upregulation, we evaluated the effect of Na+/H+ exchanger isoform 1 and calcineurin B homologous protein 2 depletion on cellular function during cancer progression in situ. Here, we show that calcineurin B homologous protein 2 functions through Na+/H+ exchanger isoform 1 to effect cell proliferation, cell migration, steady-state pHi, and anchorage-independent tumor growth. Finally, we present evidence that calcineurin B homologous protein 2 depletion in vivo has potential to reduce tumor burden in a xenograft model. Together, these data support the tumor-promoting potential of aberrant calcineurin B homologous protein 2 expression and position calcineurin B homologous protein 2 as a potential therapeutic target for the treatment of non-small cell lung cancer.


Assuntos
Calcineurina/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Trocador 1 de Sódio-Hidrogênio/metabolismo , Animais , Calcineurina/genética , Proteínas de Ligação ao Cálcio/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Nus , Transplante de Neoplasias , Isoformas de Proteínas/metabolismo , Transplante Heterólogo
8.
Arterioscler Thromb Vasc Biol ; 40(9): 2212-2226, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32640908

RESUMO

OBJECTIVE: The ductus arteriosus (DA) is a fetal artery connecting the aorta and pulmonary arteries. Progressive matrix remodeling, that is, intimal thickening (IT), occurs in the subendothelial region of DA to bring anatomic DA closure. IT is comprised of multiple ECMs (extracellular matrices) and migrated smooth muscle cells (SMCs). Because glycoprotein fibulin-1 binds to multiple ECMs and regulates morphogenesis during development, we investigated the role of fibulin-1 in DA closure. Approach and Results: Fibulin-1-deficient (Fbln1-/-) mice exhibited patent DA with hypoplastic IT. An unbiased transcriptome analysis revealed that EP4 (prostaglandin E receptor 4) stimulation markedly increased fibulin-1 in DA-SMCs via phospholipase C-NFκB (nuclear factor κB) signaling pathways. Fluorescence-activated cell sorting (FACS) analysis demonstrated that fibulin-1 binding protein versican was derived from DA-endothelial cells (ECs). We examined the effect of fibulin-1 on directional migration toward ECs in association with versican by using cocultured DA-SMCs and ECs. EP4 stimulation promoted directional DA-SMC migration toward ECs, which was attenuated by either silencing fibulin-1 or versican. Immunofluorescence demonstrated that fibulin-1 and versican V0/V1 were coexpressed at the IT of wild-type DA, whereas 30% of versican-deleted mice lacking a hyaluronan binding site displayed patent DA. Fibulin-1 expression was attenuated in the EP4-deficient mouse (Ptger4-/-) DA, which exhibits patent DA with hypoplastic IT, and fibulin-1 protein administration restored IT formation. In human DA, fibulin-1 and versican were abundantly expressed in SMCs and ECs, respectively. CONCLUSIONS: Fibulin-1 contributes to DA closure by forming an environment favoring directional SMC migration toward the subendothelial region, at least, in part, in combination with EC-derived versican and its binding partner hyaluronan.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Permeabilidade do Canal Arterial/metabolismo , Canal Arterial/metabolismo , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Canal Arterial/anormalidades , Permeabilidade do Canal Arterial/genética , Permeabilidade do Canal Arterial/patologia , Células Endoteliais/patologia , Matriz Extracelular/genética , Matriz Extracelular/patologia , Humanos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/patologia , NF-kappa B/metabolismo , Técnicas de Cultura de Órgãos , Proteína Quinase C/metabolismo , Ratos Wistar , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo
10.
Anticancer Res ; 40(6): 3119-3128, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32487606

RESUMO

BACKGROUND/AIM: Myoferlin (MYOF) has emerged as an oncogenic protein in various human cancer types. This study was conducted to investigate comprehensively the expression and functional properties of MYOF in clear-cell renal-cell carcinoma (ccRCC) with respect to its value as diagnostic biomarker and therapeutic target. MATERIALS AND METHODS: mRNA and protein expression of MYOF were assessed by quantitative polymerase chain reaction and immunohistochemistry. siRNA-mediated knockdown of MYOF was performed in the RCC cell line ACHN followed by proliferation, migration and invasion assays. RESULTS: MYOF mRNA and protein expression were significantly up-regulated in ccRCC. Higher mRNA levels were measured in advanced tumors. MYOF protein expression was increased in tumors with higher histological grades, and those with positive lymph node and surgical margin status. MYOF knockdown led to reduction of migration and invasion in ACHN cells, whereas expression of angiogenesis-associated genes tyrosine-protein kinase receptor-2 (TIE2), angiopoietin 2 (ANG2) and caveolin-1 (CAV1) was up-regulated following knockdown. CONCLUSION: MYOF may serve as a diagnostic biomarker of tumor progression and a potential therapeutic target in ccRCC.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Carcinoma de Células Renais/genética , Neoplasias Renais/genética , Proteínas de Membrana/metabolismo , Proteínas Musculares/metabolismo , Carcinoma de Células Renais/patologia , Movimento Celular , Feminino , Humanos , Neoplasias Renais/patologia , Masculino , Invasividade Neoplásica
11.
Nat Struct Mol Biol ; 27(5): 500-510, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32367068

RESUMO

Replication-dependent histones (RDH) are required for packaging of newly synthetized DNA into nucleosomes during the S phase when their expression is highly upregulated. However, the mechanisms of this upregulation in metazoan cells remain poorly understood. Using iCLIP and ChIP-seq, we found that human cyclin-dependent kinase 11 (CDK11) associates with RNA and chromatin of RDH genes primarily in the S phase. Moreover, its amino-terminal region binds FLASH, an RDH-specific 3'-end processing factor, which keeps the kinase on the chromatin. CDK11 phosphorylates serine 2 (Ser2) of the carboxy-terminal domain of RNA polymerase II (RNAPII), which is initiated when RNAPII reaches the middle of RDH genes and is required for further RNAPII elongation and 3'-end processing. CDK11 depletion leads to decreased number of cells in S phase, likely owing to the function of CDK11 in RDH gene expression. Thus, the reliance of RDH expression on CDK11 could explain why CDK11 is essential for the growth of many cancers.


Assuntos
Quinases Ciclina-Dependentes/metabolismo , Histonas/genética , Transcrição Genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Sítios de Ligação , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Cromatina/genética , Cromatina/metabolismo , Quinases Ciclina-Dependentes/genética , Replicação do DNA , Regulação da Expressão Gênica , Histonas/metabolismo , Humanos , Fosforilação , RNA/genética , RNA/metabolismo , Fase S , Serina/metabolismo
12.
PLoS One ; 15(5): e0233422, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32437418

RESUMO

SPARCL1 is a matricellular protein with anti-adhesive, anti-proliferative and anti-tumorigenic functions and is frequently downregulated in tumors such as colorectal carcinoma or non-small cell lung cancer. Studies have identified SPARCL1 as an angiocrine tumor suppressor secreted by tumor vessel endothelial cells, thereby exerting inhibitory activity on angiogenesis and tumor growth, in colorectal carcinoma. It is unknown whether SPARCL1 may exert these homeostatic functions in all organs and in other species. Therefore, SPARCL1 expression was comparatively analysed between humans and mice in a systematic manner. Murine Sparcl1 (mSparcl1) is most strongly expressed in the lung; expressed at an intermediate level in most organs, including the large intestine; and absent in the liver. In human tissues, SPARCL1 (hSPARCL1) was detected in all organs, with the strongest expression in the stomach, large intestine and lung, mostly consistent with the murine expression pattern. A striking difference between human and murine tissues was the absence of mSparcl1 expression in murine livers, while human livers showed moderate expression. Furthermore, mSparcl1 was predominantly associated with mural cells, whereas hSPARCL1 was detected in both mural and endothelial cells. Human SPARCL1 expression was downregulated in different carcinomas, including lung and colon cancers. In conclusion, this study revealed species-, organ- and cell-type-dependent expression of SPARCL1, suggesting that its function may not be similar between humans and mice.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Mucosa Intestinal/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas da Matriz Extracelular/genética , Mucosa Gástrica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Especificidade da Espécie
13.
PLoS Pathog ; 16(5): e1008214, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32379809

RESUMO

Calcium signaling plays a key role in many essential processes in almost all eukaryotic systems. It is believed that it may also be an important signaling system of the protist parasite Entamoeba histolytica. Motility, adhesion, cytolysis, and phagocytosis/trogocytosis are important steps in invasion and pathogenesis of E. histolytica, and Ca2+ signaling is thought to be associated with these processes leading to tissue invasion. There are a large number of Ca2+-binding proteins (CaBPs) in E. histolytica, and a number of these proteins appear to be associated with different steps in pathogenesis. The genome encodes 27 EF-hand-containing CaBPs in addition to a number of other Ca2+-binding domain/motif-containing proteins, which suggest intricate calcium signaling network in this parasite. Unlike other eukaryotes, a typical calmodulin-like protein has not been seen in E. histolytica. Though none of the CaBPs display sequence similarity with a typical calmodulin, extensive structural similarity has been seen in spite of lack of significant functional overlap with that of typical calmodulins. One of the unique features observed in E. histolytica is the identification of CaBPs (EhCaBP1, EhCaBP3) that have the ability to directly bind actin and modulate actin dynamics. Direct interaction of CaBPs with actin has not been seen in any other system. Pseudopod formation and phagocytosis are some of the processes that require actin dynamics, and some of the amoebic CaBPs (EhC2Pk, EhCaBP1, EhCaBP3, EhCaBP5) participate in this process. None of these E. histolytica CaBPs have any homolog in organisms other than different species of Entamoeba, suggesting a novel Ca2+ signaling pathway that has evolved in this genus.


Assuntos
Cálcio/metabolismo , Entamoeba histolytica/metabolismo , Entamebíase/metabolismo , Actinas/metabolismo , Cálcio/fisiologia , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Calmodulina/metabolismo , Entamoeba histolytica/genética , Entamoeba histolytica/patogenicidade , Fagocitose , Proteínas de Protozoários/metabolismo
14.
PLoS One ; 15(5): e0233152, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32453755

RESUMO

Obesity is associated with significantly higher mortality rates, and excess adipose tissue is involved in respective pathologies. Here we established a human adipose tissue slice cultures (HATSC) model ex vivo. HATSC match the in vivo cell composition of human adipose tissue with, among others, mature adipocytes, mesenchymal stem cells as well as stroma tissue and immune cells. This is a new method, optimized for live imaging, to study adipose tissue and cell-based mechanisms of obesity in particular. HATSC survival was tested by means of conventional and immunofluorescence histological techniques, functional analyses and live imaging. Surgery-derived tissue was cut with a tissue chopper in 500 µm sections and transferred onto membranes building an air-liquid interface. HATSC were cultured in six-well plates filled with Dulbecco's Modified Eagle's Medium (DMEM), insulin, transferrin, and selenium, both with and without serum. After 0, 1, 7 and 14 days in vitro, slices were fixated and analyzed by morphology and Perilipin A for tissue viability. Immunofluorescent staining against IBA1, CD68 and Ki67 was performed to determine macrophage survival and proliferation. These experiments showed preservation of adipose tissue as well as survival and proliferation of monocytes and stroma tissue for at least 14 days in vitro even in the absence of serum. The physiological capabilities of adipocytes were functionally tested by insulin stimulation and measurement of Phospho-Akt on day 7 and 14 in vitro. Viability was further confirmed by live imaging using Calcein-AM (viable cells) and propidium iodide (apoptosis/necrosis). In conclusion, HATSC have been successfully established by preserving the monovacuolar form of adipocytes and surrounding macrophages and connective tissue. This model allows further analysis of mature human adipose tissue biology ex vivo.


Assuntos
Adipócitos , Tecido Adiposo , Modelos Biológicos , Obesidade , Técnicas de Cultura de Tecidos , Adipócitos/metabolismo , Adipócitos/patologia , Tecido Adiposo/metabolismo , Tecido Adiposo/patologia , Adolescente , Adulto , Idoso , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Sobrevivência Celular , Feminino , Humanos , Antígeno Ki-67/metabolismo , Masculino , Proteínas dos Microfilamentos/metabolismo , Pessoa de Meia-Idade , Obesidade/metabolismo , Obesidade/patologia
15.
Physiol Genomics ; 52(5): 217-221, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: covidwho-47305
16.
Nat Commun ; 11(1): 1797, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286273

RESUMO

Mutations that inactivate negative translation regulators cause autism spectrum disorders (ASD), which predominantly affect males and exhibit social interaction and communication deficits and repetitive behaviors. However, the cells that cause ASD through elevated protein synthesis resulting from these mutations remain unknown. Here we employ conditional overexpression of translation initiation factor eIF4E to increase protein synthesis in specific brain cells. We show that exaggerated translation in microglia, but not neurons or astrocytes, leads to autism-like behaviors in male mice. Although microglial eIF4E overexpression elevates translation in both sexes, it only increases microglial density and size in males, accompanied by microglial shift from homeostatic to a functional state with enhanced phagocytic capacity but reduced motility and synapse engulfment. Consequently, cortical neurons in the mice have higher synapse density, neuroligins, and excitation-to-inhibition ratio compared to control mice. We propose that functional perturbation of male microglia is an important cause for sex-biased ASD.


Assuntos
Transtorno Autístico/metabolismo , Comportamento Animal , Microglia/metabolismo , Biossíntese de Proteínas , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Movimento Celular , Feminino , Perfilação da Expressão Gênica , Genótipo , Homeostase , Masculino , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Neurônios/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fagocitose , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/ultraestrutura , Comportamento Social , Sinapses/metabolismo
17.
PLoS One ; 15(4): e0231597, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32287325

RESUMO

Resident microglia of the central nervous system are being increasingly recognized as key players in diseases such as neuropathic pain. Biochemical and behavioral studies in neuropathic pain rodent models have documented compelling evidence of the critical role of ATP mediated-P2X4R-brain-derived neurotrophic factor (BDNF) signaling pathway in the initiation and maintenance of pain hypersensitivity, a feature driving neuropathic pain-related behavior. The goal of this study was to develop and characterize an in vitro cell line model of activated microglia that can be subsequently utilized for screening neuropathic pain therapeutics. In the present study, we characterized the SIM-A9 microglia cell line for key molecules in the P2X4R-BDNF signaling axis using a combination of biochemical techniques and developed an ATP-activated SIM-A9 microglia model. We present three novel findings: first, SIM-A9 cells expressed P2X4R and BDNF proteins, second, ATP, but not LPS, was cytocompatible with SIM-A9 cells and third, exposure of cells to optimized ATP concentrations for defined periods increased intracellular expression of Iba1 and BDNF proteins. Increased Iba1 levels confirmed microglia activation and increased BDNF expression confirmed ATP-mediated stimulation of the P2X4R signaling pathway. We propose that this ATP-activated SIM-A9 cell line model system can be utilized for screening both small- as well as macro-molecular neuropathic pain therapeutics targeting BDNF and/or P2X4R knockdown.


Assuntos
Microglia/metabolismo , Neuralgia/metabolismo , Transdução de Sinais , Trifosfato de Adenosina/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Técnicas de Cultura de Células/métodos , Lipopolissacarídeos/farmacologia , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Microglia/citologia , Microglia/efeitos dos fármacos , Neuralgia/patologia , Receptores Purinérgicos P2X4/genética , Receptores Purinérgicos P2X4/metabolismo
18.
Nat Commun ; 11(1): 1941, 2020 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-32321914

RESUMO

Cytokinesis requires the constriction of ESCRT-III filaments on the side of the midbody, where abscission occurs. After ESCRT recruitment at the midbody, it is not known how the ESCRT-III machinery localizes to the abscission site. To reveal actors involved in abscission, we obtained the proteome of intact, post-abscission midbodies (Flemmingsome) and identified 489 proteins enriched in this organelle. Among these proteins, we further characterized a plasma membrane-to-ESCRT module composed of the transmembrane proteoglycan syndecan-4, ALIX and syntenin, a protein that bridges ESCRT-III/ALIX to syndecans. The three proteins are highly recruited first at the midbody then at the abscission site, and their depletion delays abscission. Mechanistically, direct interactions between ALIX, syntenin and syndecan-4 are essential for proper enrichment of the ESCRT-III machinery at the abscission site, but not at the midbody. We propose that the ESCRT-III machinery must be physically coupled to a membrane protein at the cytokinetic abscission site for efficient scission, uncovering common requirements in cytokinesis, exosome formation and HIV budding.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Citocinese , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Organelas/metabolismo , Sindecana-4/metabolismo , Sinteninas/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ciclo Celular/genética , Membrana Celular/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Endossomos/genética , Endossomos/metabolismo , Células HeLa , Humanos , Organelas/genética , Ligação Proteica , Sindecana-4/genética , Sinteninas/genética
19.
Invest Ophthalmol Vis Sci ; 61(3): 54, 2020 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-32232349

RESUMO

Purpose: To investigate the effects and mechanisms of the peroxisome proliferator-activated receptor alpha (PPAR-α) agonist fenofibrate on the formation of ocular surface squamous metaplasia induced by topical benzalkonium chloride (BAC) in a mouse model. Methods: Ocular surface squamous metaplasia was induced in 16 days by topical BAC application in mice. During the period of induction, mice were divided into four groups: no additional treatment (BAC+UT), topical vehicle (BAC+Vehicle), topical fenofibrate (BAC+Feno), or topical fenofibrate plus intraperitoneal injection of MK886 (BAC+Feno+MK886). The parameters of tear film were evaluated on day 16, and eye specimens were collected. Histologic investigation; PAS assays; immunostaining for cytokeratin 10 (K10), Ki67, and F4/80; and PCR assays for TNF-α and IL-6 were performed. Cell Counting Kit 8 (CCK-8) assays were performed to evaluate the inhibitory effects of fenofibrate on RAW264.7 cells. Results: Fenofibrate suppressed the formation of BAC-induced instable tear film. In the BAC+Feno group, the expression of K10 and Ki67 was lower than in the other three groups. The number of goblet cells was reduced in eyes of the BAC+UT and BAC+Vehicle groups but was maintained in eyes of the BAC+Feno group. The number of F4/80-positive cells and the levels of TNF-α and IL-6 mRNA were significantly reduced in the cornea of the BAC+Feno group. These effects of fenofibrate could be attenuated by MK886. The cell viability of RAW264.7 cells could be significantly inhibited by fenofibrate in a dose-dependent pattern. Conclusions: Topical application of fenofibrate suppressed the formation of ocular surface squamous metaplasia, which might be mediated through the PPAR-α signaling pathway.


Assuntos
Epitélio Anterior/efeitos dos fármacos , Fenofibrato/farmacologia , Hipolipemiantes/farmacologia , PPAR alfa/agonistas , Animais , Anti-Infecciosos Locais/toxicidade , Compostos de Benzalcônio/toxicidade , Proteínas de Ligação ao Cálcio/metabolismo , Contagem de Células , Linhagem Celular , Sobrevivência Celular , Modelos Animais de Doenças , Epitélio Anterior/metabolismo , Epitélio Anterior/patologia , Humanos , Imuno-Histoquímica , Interleucina-6/genética , Queratina-10/metabolismo , Antígeno Ki-67/metabolismo , Masculino , Metaplasia/induzido quimicamente , Metaplasia/tratamento farmacológico , Metaplasia/metabolismo , Metaplasia/patologia , Camundongos , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase em Tempo Real , Receptores Acoplados a Proteínas-G/metabolismo , Fator de Necrose Tumoral alfa/genética
20.
Physiol Genomics ; 52(5): 217-221, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32275178
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