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1.
Mol Biol Cell ; 35(1): ar5, 2024 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-37910189

RESUMO

The interaction between aggregated low-density lipoprotein (agLDL) and macrophages in arteries plays a major role in atherosclerosis. Macrophages digest agLDL and generate free cholesterol in an extracellular, acidic, hydrolytic compartment known as the lysosomal synapse. Macrophages form a tight seal around agLDL through actin polymerization and deliver lysosomal contents into this space in a process termed digestive exophagy. Our laboratory has identified TLR4 activation of MyD88/Syk as critical for digestive exophagy. Here we use pharmacological agents and siRNA knockdown to characterize signaling pathways downstream of Syk that are involved in digestive exophagy. Syk activates Bruton's tyrosine kinase (BTK) and phospholipase Cγ2 (PLCγ2). We show that PLCγ2 and to a lesser extent BTK regulate digestive exophagy. PLCγ2 cleaves PI(4,5)P2 into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). Soluble IP3 activates release of Ca2+ from the endoplasmic reticulum (ER). We demonstrate that Ca2+ release from the ER is upregulated by agLDL and plays a key role in digestive exophagy. Both DAG and Ca2+ activate protein kinase Cα (PKCα). We find that PKCα is an important regulator of digestive exophagy. These results expand our understanding of the mechanisms of digestive exophagy, which could be useful in developing therapeutic interventions to slow development of atherosclerosis.


Assuntos
Aterosclerose , Proteína Quinase C-alfa , Humanos , Proteína Quinase C-alfa/metabolismo , Fosfolipase C gama/metabolismo , Macrófagos/metabolismo , Transdução de Sinais , Lipoproteínas LDL/metabolismo , Tirosina Quinase da Agamaglobulinemia/metabolismo , Aterosclerose/metabolismo , Digestão
2.
Front Cardiovasc Med ; 10: 1148304, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36926046

RESUMO

Cardiovascular diseases remain the leading cause of death throughout the world. Accumulation of lipoprotein-associated lipids and their interaction with macrophages are early steps in the development of atherosclerotic lesions. For decades, it has been known that aggregates of lipoproteins in the subendothelial space are found in early plaques, and these aggregates are tightly associated with extracellular matrix fibers. Additionally, most of the cholesterol in these subendothelial aggregates is unesterified, in contrast to the core of low-density lipoproteins (LDL), in which cholesteryl esters predominate. This suggests that the hydrolysis of cholesteryl esters occurs extracellularly. At the cellular level, macrophages in early plaques engage with the LDL and ingest large amounts of cholesterol, which is esterified and stored in lipid droplets. When excessive lipid droplets have accumulated, endoplasmic reticulum stress responses are activated, leading to cell death. The cholesterol-laden dead cells must be cleared by other macrophages. For many years, it was unclear how unesterified (free) cholesterol could be formed extracellularly in early lesions. Papers in the past decade have shown that macrophages form tightly sealed extracellular attachments to aggregates of LDL. These sealed regions become acidified, and lysosomal contents are secreted into these compartments. Lysosomal acid lipase hydrolyzes the cholesteryl esters, and the free cholesterol is transported into the macrophages. High concentrations of cholesterol can also lead to formation of crystals of cholesterol hydrate, and these crystals have been observed in atherosclerotic blood vessels. Characterization of this process may lead to novel therapies for the prevention and treatment of atherosclerosis.

4.
Bio Protoc ; 11(11): e4039, 2021 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-34250205

RESUMO

Maturation of secretory granules is a crucial process that ensures the bioactivity of cargo proteins undergoing regulated secretion. In Drosophila melanogaster, the larval salivary glands produce secretory granules that are up to four-fold larger in cross-sectional area after maturation. Therefore, we developed a live imaging microscopy approach to quantitate the size of secretory granules with a view to identifying genes involved in their maturation. Here, we describe the procedures of larval salivary gland dissection and sample preparation for live imaging with a fluorescence confocal microscope. Furthermore, we describe the workflow for measuring the size of secretory granules by cross-sectional surface area and statistical analysis. Our live imaging microscopy method provides a reliable read-out for the status of secretory granule maturation in Drosophila larval salivary glands.

6.
J Cell Biol ; 219(3)2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32045479

RESUMO

Regulated secretion is a fundamental cellular process in which biologically active molecules stored in long-lasting secretory granules (SGs) are secreted in response to external stimuli. Many studies have described mechanisms responsible for biogenesis and secretion of SGs, but how SGs mature remains poorly understood. In a genetic screen, we discovered a large number of endolysosomal trafficking genes required for proper SG maturation, indicating that maturation of SGs might occur in a manner similar to lysosome-related organelles (LROs). CD63, a tetraspanin known to decorate LROs, also decorates SG membranes and facilitates SG maturation. Moreover, CD63-mediated SG maturation requires type II phosphatidylinositol 4 kinase (PI4KII)-dependent early endosomal sorting and accumulation of phosphatidylinositol 4-phosphate (PI4P) on SG membranes. In addition, the PI4P effector Past1 is needed for formation of stable PI4KII-containing endosomal tubules associated with this process. Our results reveal that maturation of post-Golgi-derived SGs requires trafficking via the endosomal system, similar to mechanisms employed by LROs.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endossomos/metabolismo , Glândulas Salivares/metabolismo , Vesículas Secretórias/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Endossomos/genética , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transporte Proteico , Glândulas Salivares/embriologia , Vesículas Secretórias/genética , Tetraspanina 30/genética , Tetraspanina 30/metabolismo , Fatores de Tempo
7.
Arterioscler Thromb Vasc Biol ; 33(11): 2481-90, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23990208

RESUMO

OBJECTIVE: The hypothesis that cholesterol that enters the cell within low-density lipoprotein (LDL) particles rapidly equilibrates with the regulatory pool of intracellular cholesterol and maintains cholesterol homeostasis by reducing cholesterol and LDL receptor synthesis was validated in the fibroblast but not in the hepatocyte. Accordingly, the present studies were designed to compare the effects of cholesterol that enters the hepatocyte within an LDL particle with those of cholesterol that enters via other lipoprotein particles. APPROACH AND RESULTS: We measured cholesterol synthesis and esterification in hamster hepatocytes treated with LDL and other lipoprotein particles, including chylomicron remnants and VLDL. Endogenous cholesterol synthesis was not significantly reduced by uptake of LDL, but cholesterol esterification (280%) and acyl CoA:cholesterol acyltransferase 2 expression (870%) were increased. In contrast, cholesterol synthesis was significantly reduced (70% decrease) with other lipoprotein particles. Furthermore, more cholesterol that entered the hepatocyte within LDL particles was secreted within VLDL particles (480%) compared with cholesterol from other sources. CONCLUSIONS: Much of the cholesterol that enters the hepatocyte within LDL particles is shunted through the cell and resecreted within VLDL particles without reaching equilibrium with the regulatory pool.


Assuntos
LDL-Colesterol/metabolismo , VLDL-Colesterol/metabolismo , Hepatócitos/metabolismo , Homeostase/fisiologia , Metabolismo dos Lipídeos/fisiologia , Animais , Ésteres do Colesterol/biossíntese , Ésteres do Colesterol/metabolismo , HDL-Colesterol/biossíntese , HDL-Colesterol/metabolismo , LDL-Colesterol/biossíntese , VLDL-Colesterol/biossíntese , Quilomícrons/metabolismo , Cricetinae , Fibroblastos/metabolismo , Homeostase/genética , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Metabolismo dos Lipídeos/genética , Receptores de LDL/genética , Receptores de LDL/metabolismo
8.
Development ; 139(16): 3040-50, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22791894

RESUMO

Type II phosphatidylinositol 4-kinase (PI4KII) produces the lipid phosphatidylinositol 4-phosphate (PI4P), a key regulator of membrane trafficking. Here, we generated genetic models of the sole Drosophila melanogaster PI4KII gene. A specific requirement for PI4KII emerged in larval salivary glands. In PI4KII mutants, mucin-containing glue granules failed to reach normal size, with glue protein aberrantly accumulating in enlarged Rab7-positive late endosomes. Presence of PI4KII at the Golgi and on dynamic tubular endosomes indicated two distinct foci for its function. First, consistent with the established role of PI4P in the Golgi, PI4KII is required for sorting of glue granule cargo and the granule-associated SNARE Snap24. Second, PI4KII also has an unforeseen function in late endosomes, where it is required for normal retromer dynamics and for formation of tubular endosomes that are likely to be involved in retrieving Snap24 and Lysosomal enzyme receptor protein (Lerp) from late endosomes to the trans-Golgi network. Our genetic analysis of PI4KII in flies thus reveals a novel role for PI4KII in regulating the fidelity of granule protein trafficking in secretory tissues.


Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Vesículas Secretórias/metabolismo , 1-Fosfatidilinositol 4-Quinase/genética , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Endossomos/metabolismo , Proteínas do Grude Salivar de Drosophila/metabolismo , Complexo de Golgi/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Biológicos , Transporte Proteico , Glândulas Salivares/crescimento & desenvolvimento , Glândulas Salivares/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
9.
Biochem Cell Biol ; 90(5): 636-45, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22607224

RESUMO

Mechanisms to increase plasma high-density lipoprotein (HDL) or to promote egress of cholesterol from cholesterol-loaded cells (e.g., foam cells from atherosclerotic lesions) remain an important target to regress heart disease. Reconstituted HDL (rHDL) serves as a valuable vehicle to promote cellular cholesterol efflux in vitro and in vivo. rHDL were prepared with wild type apolipoprotein (apo) A-I and the rare variant, apoA-I Milano (M), and each apolipoprotein was reconstituted with phosphatidylcholine (PC) or sphingomyelin (SM). The four distinct rHDL generated were incubated with CHO cells, J774 macrophages, and BHK cells in cellular cholesterol efflux assays. In each cell type, apoA-I(M) SM-rHDL promoted the greatest cholesterol efflux. In BHK cells, the cholesterol efflux capacities of all four distinct rHDL were greatly enhanced by increased expression of ABCG1. Efflux to PC-containing rHDL was stimulated by transfection of a nonfunctional ABCA1 mutant (W590S), suggesting that binding to ABCA1 represents a competing interaction. This interpretation was confirmed by binding experiments. The data show that cholesterol efflux activity is dependent upon the apoA-I protein employed, as well as the phospholipid constituent of the rHDL. Future studies designed to optimize the efflux capacity of therapeutic rHDL may improve the value of this emerging intervention strategy.


Assuntos
Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Células CHO , Células Cultivadas , Cricetinae , Transfecção
10.
J Biol Chem ; 287(24): 20636-51, 2012 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-22451657

RESUMO

Transforming growth factor ß (TGF-ß) is a key regulatory molecule with pleiotropic effects on cell growth, migration, and invasion. As a result, impairment of proper TGF-ß signaling is central to tumorigenesis and metastasis. The TGF-ß receptor V (TGFBRV or LRP1) has been shown to be responsible for TGF-ß-mediated cell growth inhibition in Chinese hamster ovary (CHO) cells. The LRP1 adapter protein GULP mediates internalization of the various LRP1-specific ligands, and we hypothesize that GULP acts as a novel regulator of TGF-ß signaling in ovarian cells. CHO cells that overexpress exogenous GULP (FL) demonstrate enhancement in growth inhibition, migration, and invasion from TGF-ß treatment, whereas cells that lack GULP (AS) show impairment of growth inhibition and decreased migration and invasion. The enhanced TGF-ß response in FL cells was confirmed by a prolonged TGF-ß-induced SMAD3 phosphorylation, whereas a shortening of the phosphorylation event is observed in AS cells. Mechanistically, the presence of GULP retains the TGF-ß in a signaling-competent early endosome for enhanced signaling. To address this mechanism in a physiological setting, TGF-ß insensitive ovarian adenocarcinoma cells (HEY) have a very low GULP expression level, similar to the observation made in a wide selection of human ovarian adenocarcinomas. Transfection of GULP into the HEY cells restored the TGF-ß responsiveness, as measured by SMAD3 phosphorylation and impairment of cell growth. Because GULP expression positively regulates TGF-ß signaling leading to growth inhibition, this may represent an attractive target to achieve TGF-ß responsiveness in ovarian cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/biossíntese , Adenocarcinoma/metabolismo , Movimento Celular , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Células CHO , Cricetinae , Cricetulus , Endossomos/genética , Endossomos/metabolismo , Endossomos/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/patologia , Proteína Smad3/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/genética
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