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1.
Mol Cell ; 77(6): 1251-1264.e9, 2020 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-32023484

RESUMO

Lipid droplets (LDs) store lipids for energy and are central to cellular lipid homeostasis. The mechanisms coordinating lipid storage in LDs with cellular metabolism are unclear but relevant to obesity-related diseases. Here we utilized genome-wide screening to identify genes that modulate lipid storage in macrophages, a cell type involved in metabolic diseases. Among ∼550 identified screen hits is MLX, a basic helix-loop-helix leucine-zipper transcription factor that regulates metabolic processes. We show that MLX and glucose-sensing family members MLXIP/MondoA and MLXIPL/ChREBP bind LDs via C-terminal amphipathic helices. When LDs accumulate in cells, these transcription factors bind to LDs, reducing their availability for transcriptional activity and attenuating the response to glucose. Conversely, the absence of LDs results in hyperactivation of MLX target genes. Our findings uncover a paradigm for a lipid storage response in which binding of MLX transcription factors to LD surfaces adjusts the expression of metabolic genes to lipid storage levels.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Gotículas Lipídicas/metabolismo , Proteoma/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Células Cultivadas , Testes Genéticos , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Ligação Proteica , Proteoma/análise , RNA Interferente Pequeno , Transcrição Gênica
2.
Proc Natl Acad Sci U S A ; 117(50): 31914-31922, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33257571

RESUMO

Inhibiting membrane association of RAS has long been considered a rational approach to anticancer therapy, which led to the development of farnesyltransferase inhibitors (FTIs). However, FTIs proved ineffective against KRAS-driven tumors. To reveal alternative therapeutic strategies, we carried out a genome-wide CRISPR-Cas9 screen designed to identify genes required for KRAS4B membrane association. We identified five enzymes in the prenylation pathway and SAFB, a nuclear protein with both DNA and RNA binding domains. Silencing SAFB led to marked mislocalization of all RAS isoforms as well as RAP1A but not RAB7A, a pattern that phenocopied silencing FNTA, the prenyltransferase α subunit shared by farnesyltransferase and geranylgeranyltransferase type I. We found that SAFB promoted RAS membrane association by controlling FNTA expression. SAFB knockdown decreased GTP loading of RAS, abrogated alternative prenylation, and sensitized RAS-mutant cells to growth inhibition by FTI. Our work establishes the prenylation pathway as paramount in KRAS membrane association, reveals a regulator of prenyltransferase expression, and suggests that reduction in FNTA expression may enhance the efficacy of FTIs.


Assuntos
Membrana Celular/metabolismo , Dimetilaliltranstransferase/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Neoplasias/patologia , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Receptores de Estrogênio/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Sistemas CRISPR-Cas/genética , Biologia Computacional , Conjuntos de Dados como Assunto , Técnicas de Silenciamento de Genes , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/genética , Neoplasias/genética , Proteínas Associadas à Matriz Nuclear/genética , Prenilação de Proteína , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Receptores de Estrogênio/genética
3.
Proc Natl Acad Sci U S A ; 113(31): E4558-66, 2016 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-27432991

RESUMO

Bromodomain and extraterminal domain protein inhibitors (BETi) hold great promise as a novel class of cancer therapeutics. Because acquired resistance typically limits durable responses to targeted therapies, it is important to understand mechanisms by which tumor cells adapt to BETi. Here, through pooled shRNA screening of colorectal cancer cells, we identified tripartite motif-containing protein 33 (TRIM33) as a factor promoting sensitivity to BETi. We demonstrate that loss of TRIM33 reprograms cancer cells to a more resistant state through at least two mechanisms. TRIM33 silencing attenuates down-regulation of MYC in response to BETi. Moreover, loss of TRIM33 enhances TGF-ß receptor expression and signaling, and blocking TGF-ß receptor activity potentiates the antiproliferative effect of BETi. These results describe a mechanism for BETi resistance and suggest that combining inhibition of TGF-ß signaling with BET bromodomain inhibition may offer new therapeutic benefits.


Assuntos
Azepinas/farmacologia , Proteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Triazóis/farmacologia , Azepinas/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Resistência a Medicamentos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Células HEK293 , Humanos , Estrutura Molecular , Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Interferência de RNA , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genética , Triazóis/química
4.
Proc Natl Acad Sci U S A ; 110(18): 7452-7, 2013 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-23569269

RESUMO

Despite major advances in our understanding of many aspects of human papillomavirus (HPV) biology, HPV entry is poorly understood. To identify cellular genes required for HPV entry, we conducted a genome-wide screen for siRNAs that inhibited infection of HeLa cells by HPV16 pseudovirus. Many retrograde transport factors were required for efficient infection, including multiple subunits of the retromer, which initiates retrograde transport from the endosome to the trans-Golgi network (TGN). The retromer has not been previously implicated in virus entry. Furthermore, HPV16 capsid proteins arrive in the TGN/Golgi in a retromer-dependent fashion during entry, and incoming HPV proteins form a stable complex with retromer subunits. We propose that HPV16 directly engages the retromer at the early or late endosome and traffics to the TGN/Golgi via the retrograde pathway during cell entry. These results provide important insights into HPV entry, identify numerous potential antiviral targets, and suggest that the role of the retromer in infection by other viruses should be assessed.


Assuntos
Genoma Humano/genética , Papillomaviridae/fisiologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Internalização do Vírus , Complexo de Golgi/virologia , Células HeLa , Papillomavirus Humano 16/fisiologia , Humanos , Infecções por Papillomavirus/genética , Infecções por Papillomavirus/virologia , Ligação Proteica , Transporte Proteico , Reprodutibilidade dos Testes , Proteínas Virais/metabolismo
5.
Dev Cell ; 57(3): 387-397.e4, 2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-35134345

RESUMO

Lipid droplets (LDs) are organelles of cellular lipid storage with fundamental roles in energy metabolism and cell membrane homeostasis. There has been an explosion of research into the biology of LDs, in part due to their relevance in diseases of lipid storage, such as atherosclerosis, obesity, type 2 diabetes, and hepatic steatosis. Consequently, there is an increasing need for a resource that combines datasets from systematic analyses of LD biology. Here, we integrate high-confidence, systematically generated human, mouse, and fly data from studies on LDs in the framework of an online platform named the "Lipid Droplet Knowledge Portal" (https://lipiddroplet.org/). This scalable and interactive portal includes comprehensive datasets, across a variety of cell types, for LD biology, including transcriptional profiles of induced lipid storage, organellar proteomics, genome-wide screen phenotypes, and ties to human genetics. This resource is a powerful platform that can be utilized to identify determinants of lipid storage.


Assuntos
Bases de Dados como Assunto , Gotículas Lipídicas/metabolismo , Animais , Ésteres do Colesterol/metabolismo , Mineração de Dados , Genoma , Humanos , Inflamação/patologia , Metabolismo dos Lipídeos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Fenótipo , Fosforilação , Interferência de RNA
6.
Cell Rep ; 34(13): 108928, 2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33789117

RESUMO

Flux through the RAF-MEK-ERK protein kinase cascade is shaped by phosphatases acting on the core components of the pathway. Despite being an established drug target and a hub for crosstalk regulation, little is known about dephosphorylation of MEK, the central kinase within the cascade. Here, we identify PPP6C, a phosphatase frequently mutated or downregulated in melanoma, as a major MEK phosphatase in cells exhibiting oncogenic ERK pathway activation. Recruitment of MEK to PPP6C occurs through an interaction with its associated regulatory subunits. Loss of PPP6C causes hyperphosphorylation of MEK at activating and crosstalk phosphorylation sites, promoting signaling through the ERK pathway and decreasing sensitivity to MEK inhibitors. Recurrent melanoma-associated PPP6C mutations cause MEK hyperphosphorylation, suggesting that they promote disease at least in part by activating the core oncogenic pathway driving melanoma. Collectively, our studies identify a key negative regulator of ERK signaling that may influence susceptibility to targeted cancer therapies.


Assuntos
Carcinogênese/patologia , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Linhagem Celular Tumoral , Células HEK293 , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Fosfoproteínas Fosfatases/genética , Fosforilação , RNA Interferente Pequeno/metabolismo , Especificidade por Substrato
7.
J Biomed Sci ; 16: 21, 2009 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-19272191

RESUMO

BACKGROUND: The endocardial endothelium that lines the inner cavity of the heart is distinct from the microvascular endothelial cells and modulates cardiac muscle performance in a manner similar to the vascular endothelial modulation of vascular structure and vasomotor tone. Although the modulatory effects of endocardial endothelium (EE) on cardiomyocytes are firmly established, the regulatory effects of endocardial endothelium on the cardiac interstitium and its cellular components remain ill defined. METHODS AND RESULTS: We investigated whether the stimulatory effect of EE on cardiac fibroblasts would be altered when EECs are activated by the cytokine tumor necrosis factor-alpha (TNF-alpha) or the endotoxin bacterial lipopolysaccharide (LPS). Both TNF-alpha and LPS were found to independently attenuate the stimulatory effect of EE on cardiac fibroblasts. These agents lowered the synthesis or release of ET-1 and increased the secretion of TGF-beta and NO. CONCLUSION: The findings of this study using endocardial endothelial cells (EECs) and neonatal cardiac fibroblasts demonstrate that pro-inflammatory cytokines cause altered secretion of paracrine factors by EECs and inhibit proliferation and lower collagen synthesis in fibroblasts. These changes may influence fibroblast response and extra cellular matrix remodeling in pathological conditions of the heart.


Assuntos
Endocárdio/citologia , Células Endoteliais/metabolismo , Fibroblastos/metabolismo , Lipopolissacarídeos/metabolismo , Miocárdio/citologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Meios de Cultivo Condicionados/química , Endocárdio/metabolismo , Células Endoteliais/citologia , Fibroblastos/citologia , Miocárdio/metabolismo , Nitritos/metabolismo , Comunicação Parácrina , Ratos , Ratos Wistar , Suínos
8.
Cell Biochem Biophys ; 47(1): 65-72, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17406060

RESUMO

Given that vascular endothelial cells play an important role in the modulation of vascular structure and function, we hypothesized that endocardial endothelial cells (EECs) may have a modulator role in regulating the cardiac interstitial cells. Endocardial endothelial cells were isolated from freshly collected pig hearts and cardiac fibroblasts were isolated from 3- to 4-d-old Wistar rats. Fibroblasts were cultured in the presence or absence of conditioned medium from EECs. Proliferation of cardiac fibroblasts was measured by the incorporation of [3H]- Thymidine and collagen synthesis was assayed by the incorporation of [3H]-Proline. To determine the involvement of signaling mediators, in separate experiments, cardiac fibroblasts were incubated with BQ123 (selective ETA receptor antagonist), PD142893 (nonselective ETA/ETB receptor antagonist), Bis-indolylmaleimide (PKC inhibitor), PD 098059 (MEK inhibitor), or neutralizing anti-transforming growth factor (TGF)-beta-antibody. Endocardial endothelium-derived factors endothelin (ET)-1, TGF-beta, and Angiotensin (Ang)-II in the conditioned medium were assayed by enzyme-linked immunosorbent assay using commercially available kits. We report here evidence that suggest that endocardial endothelial cells stimulate both proliferation and collagen synthesis of cardiac fibroblasts. The response seems to be mediated by endothelin through its ETA receptor. Our results also indicate that protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) pathways are essential for the EEC-induced proliferation of cardiac fibroblasts.


Assuntos
Colágeno/metabolismo , Endocárdio/metabolismo , Células Endoteliais/citologia , Endotélio Vascular/metabolismo , Miocárdio/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Meios de Cultivo Condicionados/metabolismo , Células Endoteliais/metabolismo , Inibidores Enzimáticos/farmacologia , Fibroblastos/metabolismo , Proteína Quinase C/metabolismo , Ratos , Ratos Wistar , Fator de Crescimento Transformador beta/metabolismo
9.
Endothelium ; 14(1): 35-43, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17364895

RESUMO

Endocardial endothelial cells (EECs), which form the inner lining of the cavities of the heart, are a distinct cell population whose dysfunction can be critical in pathological conditions of heart. Insights into the role and organization of these cells in pathological states of the heart are limited mainly due to a dearth of experimental models. To date no endocardial endothelial cell line is available. The authors attempted to immortalize porcine ventricular EECs by transfecting the cells with human telomerase reverse transcriptase (hTERT). EECs immortalized by ectopic expression of hTERT exhibit phenotypic and functional characteristics similar to primary EECs. The EE cell line could be useful for the study of mechanisms involved in the interaction of EECs with the underlying myocardium and cardiac interstitium and as useful tools in understanding their role in diseased states of heart.


Assuntos
Endocárdio/citologia , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Endotélio Vascular/citologia , Ventrículos do Coração/citologia , Telomerase/metabolismo , Animais , Western Blotting , Divisão Celular/fisiologia , Linhagem Celular Transformada , Fluoresceína-5-Isotiocianato , Técnica Direta de Fluorescência para Anticorpo , Corantes Fluorescentes , Humanos , Imuno-Histoquímica , Cinética , Nitritos/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos , Telomerase/análise , Telomerase/genética , Transfecção
10.
Biol Trace Elem Res ; 114(1-3): 85-92, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17205990

RESUMO

Cerium has been implicated in the pathogenesis of cardiac disorders such as acute myocardial infarction and endomyocardial fibrosis (EMF). A geochemical hypothesis for the causation of EMF linked the cardiac lesions to magnesium deficiency consequent to malnutrition and increased cardiac levels of cerium derived from monazite soils in the coastal regions of the tropics. We tested the hypothesis that the stimulus for fibroblast proliferation and enhanced collagen synthesis in EMF is derived from cardiac endothelial cells activated or injured by cerium. We explored whether endocardial endothelial cells exposed to cerium secrete factors responsible for the increased proliferation and collagen synthesis in cardiac fibroblasts. Our results suggest that the growth response of cardiac fibroblasts to cerium is not mediated through growth factors secreted by endocardial endothelium and that the cardiac lesions in EMF result from direct stimulation of subendocardial fibroblasts by cerium.


Assuntos
Proliferação de Células , Cério/toxicidade , Endotélio Vascular/efeitos dos fármacos , Coração/efeitos dos fármacos , Miocárdio/citologia , Animais , Células Cultivadas , Colágeno/biossíntese , Meios de Cultivo Condicionados , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Miocárdio/metabolismo , Suínos
11.
Nat Commun ; 7: 13516, 2016 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-27869117

RESUMO

In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL.


Assuntos
Receptores de Activinas Tipo II/metabolismo , LDL-Colesterol/metabolismo , Células Endoteliais/metabolismo , Receptores de Ativinas Tipo I/genética , Receptores de Ativinas Tipo I/metabolismo , Receptores de Activinas Tipo II/genética , Animais , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Transporte Biológico , Células Cultivadas , LDL-Colesterol/genética , Clonagem Molecular , Técnicas de Silenciamento de Genes , Estudo de Associação Genômica Ampla , Humanos , Masculino , Camundongos , Interferência de RNA
12.
Mol Cell Biochem ; 253(1-2): 113-23, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14619961

RESUMO

Endothelium is now recognized as a massive, regionally specific, multifunctional organ. Given its strategic anatomic location between the circulating blood components and the vascular smooth muscle or the cardiac muscle, it is a biologically significant interface whose dysfunction can be a critical factor in various pathological conditions. Two types of endothelial cells are recognized in the heart, the endocardial endothelial (EE) cells and the microvascular endothelial cells (MVE). Both produce common autacoids and share similar roles in signal transduction induced by neurotransmitters, hormones or mechanical stimuli. They are however two distinct cell populations with dissimilar embryological origin, cytoskeletal organization, receptor mediated functions and electrophysiological properties. Both the MVE and EE are modulators of cardiac performance. Myocardial contraction may be modulated by cardioactive agents such as nitric oxide, prostanoids, endothelin, natriuretic peptides, angiotensin II, kinins, reactive oxygen species and adenyl purines released from the cardiac endothelium. Two mechanisms have been proposed for the signal transduction from EE to the underlying myocytes: stimulus-secretion-contraction coupling and blood-heart barrier. Nitric oxide, bradykinin and myofilament desensitizing agent are probably important in short-term regulation of myocardial functions. Endothelin and Angiotensin II are probably involved in long-term regulation. Besides its sensory function and paracrine modulation of myocardial performance, EE as a blood-heart barrier could be of significance for the ionic homeostasis of the cardiac interstitium. In cardiac diseases, the damage to EE or MVE leading to failure of the endothelial cells to perform its regulatory and modulator functions may have serious consequences. A better understanding of the endothelial signaling pathways in cardiac physiology and pathophysiology may lead to the development of novel therapeutic strategies.


Assuntos
Endotélio Vascular/metabolismo , Contração Miocárdica/fisiologia , Miocárdio/metabolismo , Animais , Comunicação Celular/fisiologia , Humanos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia
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