RESUMEN
Maintaining a highly acidic lysosomal pH is central to cellular physiology. Here, we use functional proteomics, single-particle cryo-EM, electrophysiology, and in vivo imaging to unravel a key biological function of human lysosome-associated membrane proteins (LAMP-1 and LAMP-2) in regulating lysosomal pH homeostasis. Despite being widely used as a lysosomal marker, the physiological functions of the LAMP proteins have long been overlooked. We show that LAMP-1 and LAMP-2 directly interact with and inhibit the activity of the lysosomal cation channel TMEM175, a key player in lysosomal pH homeostasis implicated in Parkinson's disease. This LAMP inhibition mitigates the proton conduction of TMEM175 and facilitates lysosomal acidification to a lower pH environment crucial for optimal hydrolase activity. Disrupting the LAMP-TMEM175 interaction alkalinizes the lysosomal pH and compromises the lysosomal hydrolytic function. In light of the ever-increasing importance of lysosomes to cellular physiology and diseases, our data have widespread implications for lysosomal biology.
Asunto(s)
Enfermedad de Parkinson , Humanos , Concentración de Iones de Hidrógeno , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Enfermedad de Parkinson/metabolismo , Canales de Potasio/metabolismoRESUMEN
The mechanisms whereby progesterone (P4), acting via the progesterone receptor (PR), inhibits proinflammatory/contractile gene expression during pregnancy are incompletely defined. Using immortalized human myometrial (hTERT-HM) cells stably expressing wild-type PR-A or PR-B (PRWT), we found that P4 significantly inhibited IL-1ß induction of the NF-κB target genes, COX-2 and IL-8 P4-PRWT transrepression occurred at the level of transcription initiation and was mediated by decreased recruitment of NF-κB p65 and RNA polymerase II to COX-2 and IL-8 promoters. However, in cells stably expressing a PR-A or PR-B DNA-binding domain mutant (PRmDBD), P4-mediated transrepression was significantly reduced, suggesting a critical role of the PR DBD. ChIP analysis of hTERT-HM cells stably expressing PRWT or PRmDBD revealed that P4 treatment caused equivalent recruitment of PRWT and PRmDBD to COX-2 and IL-8 promoters, suggesting that PR inhibitory effects were not mediated by its direct DNA binding. Using immunoprecipitation, followed by MS, we identified a transcriptional repressor, GATA zinc finger domain-containing 2B (GATAD2B), that interacted strongly with PRWT but poorly with PRmDBD P4 treatment of PRWT hTERT-HM cells caused enhanced recruitment of endogenous GATAD2B to COX-2 and IL-8 promoters. Further, siRNA knockdown of endogenous GATAD2B significantly reduced P4-PRWT transrepression of COX-2 and IL-8 Notably, GATAD2B expression was significantly decreased in pregnant mouse and human myometrium during labor. Our findings suggest that GATAD2B serves as an important mediator of P4-PR suppression of proinflammatory and contractile genes during pregnancy. Decreased GATAD2B expression near term may contribute to the decline in PR function, leading to labor.
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Regulación hacia Abajo , Factores de Transcripción GATA/metabolismo , Miometrio/metabolismo , Receptores de Progesterona/metabolismo , Proteínas Represoras/metabolismo , Contracción Uterina/genética , Animales , Células Cultivadas , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Femenino , Células HEK293 , Humanos , Interleucina-8/antagonistas & inhibidores , Interleucina-8/genética , Interleucina-8/metabolismo , Ratones , Miometrio/efectos de los fármacos , Progesterona/farmacología , Receptores de Progesterona/agonistasRESUMEN
The carbohydrate-response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays an essential role in converting excess carbohydrate to fat storage in the liver. In response to glucose levels, ChREBP is regulated by nuclear/cytosol trafficking via interaction with 14-3-3 proteins, CRM-1 (exportin-1 or XPO-1), or importins. Nuclear localization of ChREBP was rapidly inhibited when incubated in branched-chain α-ketoacids, saturated and unsaturated fatty acids, or 5-aminoimidazole-4-carboxamide ribonucleotide. Here, we discovered that protein-free extracts of high fat-fed livers contained, in addition to ketone bodies, a new metabolite, identified as AMP, which specifically activates the interaction between ChREBP and 14-3-3. The crystal structure showed that AMP binds directly to the N terminus of ChREBP-α2 helix. Our results suggest that AMP inhibits the nuclear localization of ChREBP through an allosteric activation of ChREBP/14-3-3 interactions and not by activation of AMPK. AMP and ketone bodies together can therefore inhibit lipogenesis by restricting localization of ChREBP to the cytoplasm during periods of ketosis.
Asunto(s)
Adenosina Monofosfato/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proteínas 14-3-3/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Regulación Alostérica , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/química , Núcleo Celular/metabolismo , Células Cultivadas , Cristalografía por Rayos X , Dieta Alta en Grasa , Sacarosa en la Dieta/administración & dosificación , Hepatocitos/metabolismo , Carioferinas/metabolismo , Cuerpos Cetónicos/metabolismo , Masculino , Modelos Biológicos , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteína Exportina 1RESUMEN
RATIONALE: Signal initiation by the high-density lipoprotein (HDL) receptor scavenger receptor class B, type I (SR-BI), which is important to actions of HDL on endothelium and other processes, requires cholesterol efflux and the C-terminal transmembrane domain. The C-terminal transmembrane domain uniquely interacts with plasma membrane (PM) cholesterol. OBJECTIVE: The molecular basis and functional significance of SR-BI interaction with PM cholesterol are unknown. We tested the hypotheses that the interaction is required for SR-BI signaling, and that it enables SR-BI to serve as a PM cholesterol sensor. METHODS AND RESULTS: In studies performed in COS-M6 cells, mutation of a highly conserved C-terminal transmembrane domain glutamine to alanine (SR-BI-Q445A) decreased PM cholesterol interaction with the receptor by 71% without altering HDL binding or cholesterol uptake or efflux, and it yielded a receptor incapable of HDL-induced signaling. Signaling prompted by cholesterol efflux to methyl-ß-cyclodextrin also was prevented, indicating that PM cholesterol interaction with the receptor enables it to serve as a PM cholesterol sensor. Using SR-BI-Q445A, we further demonstrated that PM cholesterol sensing by SR-BI does not influence SR-BI-mediated reverse cholesterol transport to the liver in mice. However, the PM cholesterol sensing does underlie apolipoprotein B intracellular trafficking in response to postprandial micelles or methyl-ß-cyclodextrin in cultured enterocytes, and it is required for HDL activation of endothelial NO synthase and migration in cultured endothelial cells and HDL-induced angiogenesis in vivo. CONCLUSIONS: Through interaction with PM cholesterol, SR-BI serves as a PM cholesterol sensor, and the resulting intracellular signaling governs processes in both enterocytes and endothelial cells.
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Membrana Celular/metabolismo , Colesterol/metabolismo , Células Endoteliales/metabolismo , Enterocitos/metabolismo , Receptores Depuradores de Clase B/metabolismo , Transducción de Señal , Alanina , Animales , Apolipoproteínas B/metabolismo , Células CACO-2 , Bovinos , Membrana Celular/efectos de los fármacos , HDL-Colesterol/metabolismo , Células Endoteliales/efectos de los fármacos , Enterocitos/efectos de los fármacos , Glutamina , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Hígado/metabolismo , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Mutación , Neovascularización Fisiológica , Óxido Nítrico Sintasa de Tipo III/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Receptores Depuradores de Clase B/química , Receptores Depuradores de Clase B/genética , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Transfección , beta-Ciclodextrinas/farmacologíaRESUMEN
Contact sites between the endoplasmic reticulum (ER) and the plasma membrane (PM) play a crucial role in governing calcium regulation and lipid homeostasis. Despite their significance, the factors regulating their spatial distribution on the PM remain elusive. Inspired by observations in cardiomyocytes, where ER-PM contact sites concentrate on tubular PM invaginations known as transverse tubules (T-tubules), we hypothesize that the PM curvature plays a role in ER-PM contact formation. Through precise control of PM invaginations, we show that PM curvatures locally induce the formation of ER-PM contacts in cardiomyocytes. Intriguingly, the junctophilin family of ER-PM tethering proteins, specifically expressed in excitable cells, is the key player in this process, while the ubiquitously expressed extended synaptotagmin 2 does not show a preference for PM curvature. At the mechanistic level, we find that the low complexity region (LCR) and the MORN motifs of junctophilins can independently bind to the PM, but both the LCR and MORN motifs are required for targeting PM curvatures. By examining the junctophilin interactome, we identify a family of curvature-sensing proteins, Eps15-homology domain containing proteins (EHDs), that interact with the MORN_LCR motifs and facilitate junctophilins' preferential tethering to curved PM. These findings highlight the pivotal role of PM curvature in the formation of ER-PM contacts in cardiomyocytes and unveil a novel mechanism for the spatial regulation of ER-PM contacts through PM curvature modulation.
RESUMEN
Homeostatic regulation of plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate (PIP2) in receptor-stimulated cells is mediated by the lipid transfer protein Nir2. Nir2 is dynamically recruited to endoplasmic reticulum-plasma membrane (ER-PM) junctions to facilitate replenishment of PM PIP2 hydrolyzed during receptor-mediated signaling. However, our knowledge regarding the activation and sustainment of Nir2-mediated replenishment of PM PIP2 is limited. Here, we describe the functions of Nir1 as a positive regulator of Nir2 and PIP2 homeostasis. In contrast to the family proteins Nir2 and Nir3, Nir1 constitutively localizes at ER-PM junctions. Nir1 potentiates Nir2 targeting to ER-PM junctions during receptor-mediated signaling and is required for efficient PM PIP2 replenishment. Live-cell imaging and biochemical analysis reveal that Nir1 interacts with Nir2 via a region between the FFAT motif and the DDHD domain. Combined, results from this study identify Nir1 as an ER-PM junction localized protein that promotes Nir2 recruitment for PIP2 homeostasis.
Asunto(s)
Retículo Endoplásmico , Proteínas de la Membrana , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Homeostasis , Proteínas de la Membrana/metabolismo , Fosfatidilinositoles/metabolismoRESUMEN
Sigma 1 receptor (S1R) is a 223-amino-acid-long transmembrane endoplasmic reticulum (ER) protein. S1R modulates activity of multiple effector proteins and is a well-established drug target. However, signaling functions of S1R in cells are poorly understood. Here, we test the hypothesis that biological activity of S1R in cells can be explained by its ability to interact with cholesterol and to form cholesterol-enriched microdomains in the ER membrane. By performing experiments in reduced reconstitution systems, we demonstrate direct effects of cholesterol on S1R clustering. We identify a novel cholesterol-binding motif in the transmembrane region of human S1R. Mutations of this motif impair association of recombinant S1R with cholesterol beads, affect S1R clustering in vitro and disrupt S1R subcellular localization. We demonstrate that S1R-induced membrane microdomains have increased local membrane thickness and that increased local cholesterol concentration and/or membrane thickness in these microdomains can modulate signaling of inositol-requiring enzyme 1α in the ER. Further, S1R agonists cause disruption of S1R clusters, suggesting that biological activity of S1R agonists is linked to remodeling of ER membrane microdomains. Our results provide novel insights into S1R-mediated signaling mechanisms in cells.
Asunto(s)
Colesterol/metabolismo , Retículo Endoplásmico/metabolismo , Receptores sigma/genética , Receptores sigma/metabolismo , Transducción de Señal , Células HEK293 , Humanos , Membranas Intracelulares/metabolismo , Microdominios de Membrana , Unión Proteica , Receptor Sigma-1RESUMEN
Pathogens find diverse niches for survival including inside a host cell where replication occurs in a relatively protective environment. Vibrio parahaemolyticus is a facultative intracellular pathogen that uses its type 3 secretion system 2 (T3SS2) to invade and replicate inside host cells. Analysis of the T3SS2 pathogenicity island encoding the T3SS2 appeared to lack a mechanism for egress of this bacterium from the invaded host cell. Using a combination of molecular tools, we found that VPA0226, a constitutively secreted lipase, is required for escape of V. parahaemolyticus from the host cells. This lipase must be delivered into the host cytoplasm where it preferentially uses fatty acids associated with innate immune response to esterify cholesterol, weakening the plasma membrane and allowing egress of the bacteria. This study reveals the resourcefulness of microbes and the interplay between virulence systems and host cell resources to evolve an ingenious scheme for survival and escape.
Asunto(s)
Proteínas Bacterianas/metabolismo , Colesterol/metabolismo , Ácidos Grasos/metabolismo , Lipasa/metabolismo , Vibrio parahaemolyticus/metabolismo , Esterificación , Islas Genómicas , Sistemas de Secreción Tipo III , Vibrio parahaemolyticus/enzimologíaRESUMEN
27-Hydroxycholesterol (27HC) is an abundant cholesterol metabolite and has detrimental effects on the cardiovascular system, whereas its impact on adiposity is not well known. In this study, we found that elevations in 27HC cause increased body weight gain in mice fed a high-fat/high-cholesterol diet in an estrogen receptor α-dependent manner. Regardless of diet type, body fat mass was increased by 27HC without changes in food intake or fat absorption. 27HC did not alter energy expenditure in mice fed a normal chow diet and increased visceral white adipose mass by inducing hyperplasia but not hypertrophy. Although 27HC did not augment adipocyte terminal differentiation, it increased the adipose cell population that differentiates to mature adipocytes. RNA sequencing analysis revealed that 27HC treatment of mice fed a normal chow diet induces inflammatory gene sets similar to those seen after high-fat/high-cholesterol diet feeding, whereas there was no overlap in inflammatory gene expression among any other 27HC administration/diet change combination. Histological analysis showed that 27HC treatment increased the number of total and M1-type macrophages in white adipose tissues. Thus, 27HC promotes adiposity by directly affecting white adipose tissues and by increasing adipose inflammatory responses. Lowering serum 27HC levels may lead to an approach targeting cholesterol to prevent diet-induced obesity.
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Adipogénesis/efectos de los fármacos , Adiposidad/efectos de los fármacos , Grasas de la Dieta/efectos adversos , Hidroxicolesteroles , Obesidad/inducido químicamente , Animales , Familia 7 del Citocromo P450/genética , Familia 7 del Citocromo P450/metabolismo , Dieta Alta en Grasa/efectos adversos , Grasas de la Dieta/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/inducido químicamente , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/efectos de los fármacos , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismoRESUMEN
Uterine mesenchymal tumors are genetically heterogenous; those with uniform cytomorphology, best exemplified by endometrial stromal tumors, often contain various fusion genes. Novel fusions involving ESR1 and GREB1, key factors in sex hormone pathways, have been implicated in rare uterine mesenchymal tumors. Particularly, the fusions between 5'-ESR1/GREB1 and 3'-NCOA2/NCOA3 were recently identified in 4 uterine tumors resembling ovarian sex-cord tumor (UTROSCT). By RNA sequencing, pathology review, and FISH screening, we identified 4 uterine sarcomas harboring rearranged GREB1, including GREB1-NCOA2 and the novel GREB1-NR4A3, GREB1-SS18, and GREB1-NCOA1, validated by RT-PCR and/or FISH. They occurred in the myometrium of postmenopausal women and were pathologically similar despite minor differences. Tumor cells were generally uniform and epithelioid, with vesicular nuclei and distinct to prominent nucleoli. Growth patterns included solid sheets, trabeculae/cords, nests, and fascicles. Only 1 tumor showed small foci of definitive sex-cord components featuring well-formed tubules, retiform structures, Leydig-like cells, and lipid-laden cells and exhibiting convincing immunoreactivity to sex-cord markers (calretinin, α-inhibin, and Melan-A). In contrast, all the 4 classic UTROSCT we collected occurred in premenopausal patients, consisted predominantly of unequivocal sex-cord elements, prominently expressed multiple sex-cord markers, and harbored ESR1-NCOA3 fusion. Combined with previously reported cases, GREB1-rearranged tumors involved significantly older women (P=0.001), tended to be larger and more mitotically active, showed more variable and often inconspicuous sex-cord differentiation, and appeared to behave more aggressively than ESR1-rearranged UTROSCT. Therefore, these 2 groups of tumors might deserve separate consideration, despite some overlapping features and the possibility of belonging to the same disease spectrum.
Asunto(s)
Biomarcadores de Tumor/genética , Diferenciación Celular , Fusión Génica , Reordenamiento Génico , Proteínas de Neoplasias/genética , Neoplasias Ováricas/genética , Sarcoma/genética , Tumores de los Cordones Sexuales y Estroma de las Gónadas/genética , Neoplasias Uterinas/genética , Anciano , Proteínas de Unión al ADN/genética , Femenino , Predisposición Genética a la Enfermedad , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Persona de Mediana Edad , Mitosis , Coactivador 1 de Receptor Nuclear/genética , Coactivador 2 del Receptor Nuclear/genética , Neoplasias Ováricas/patología , Fenotipo , Pronóstico , Proteínas Proto-Oncogénicas/genética , Receptores de Esteroides/genética , Receptores de Hormona Tiroidea/genética , Proteínas Represoras/genética , Sarcoma/patología , Análisis de Secuencia de ARN , Tumores de los Cordones Sexuales y Estroma de las Gónadas/patología , Taiwán , Carga Tumoral , Neoplasias Uterinas/patologíaRESUMEN
27-hydroxycholesterol (27HC) is an abundant cholesterol metabolite in human circulation and promotes breast cancer cell proliferation. Although lung is one of the organs that contain high levels of 27HC, the role of 27HC in lung is unknown. In this study, we found that 27HC promotes lung cancer cell proliferation in an estrogen receptor ß (ERß)-dependent manner. The expression of 27HC-generating enzyme CYP27A1 is higher in lung cancer cells than in normal lung cells. Treatment with 27HC increased cell proliferation in ERß-positive lung cancer cells, but not in ERα-positive or ER-negative cells. The effect on cell proliferation is specific to 27HC and another oxysterol, 25-hydroxycholesterol that has a similar oxysterol structure with 27HC. Moreover, among ligands for nuclear receptors tested, only estrogen had the proliferative effect, and the effect by 27HC and estrogen was inhibited by ERß-specific, but not ERα-specific, inhibitors. In addition, the effect by 27HC was not affected by membrane-bound estrogen receptor GPR30. Interestingly, despite the high expression of CYP27A1, endogenously produced 27HC was not the major contributor of the 27HC-induced cell proliferation. Using kinase inhibitors, we found that the effect by 27HC was mediated by the PI3K-Akt signaling pathway. These results suggest that 27HC promotes lung cancer cell proliferation via ERß and PI3K-Akt signaling. Thus, lowering 27HC levels may lead to a novel approach for the treatment of lung cancer.
RESUMEN
RAS association domain family 4 (RASSF4) is involved in tumorigenesis and regulation of the Hippo pathway. In this study, we identify new functional roles of RASSF4. First, we discovered that RASSF4 regulates store-operated Ca2+ entry (SOCE), a fundamental Ca2+ signaling mechanism, by affecting the translocation of the endoplasmic reticulum (ER) Ca2+ sensor stromal interaction molecule 1 (STIM1) to ER-plasma membrane (PM) junctions. It was further revealed that RASSF4 regulates the formation of ER-PM junctions and the ER-PM tethering function of extended synaptotagmins E-Syt2 and E-Syt3. Moreover, steady-state PM phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) levels, important for localization of STIM1 and E-Syts at ER-PM junctions, were reduced in RASSF4-knockdown cells. Furthermore, we demonstrated that RASSF4 interacts with and regulates the activity of adenosine diphosphate ribosylation factor 6 (ARF6), a small G protein and upstream regulator of type I phosphatidylinositol phosphate kinases (PIP5Ks) and PM PI(4,5)P2 levels. Overall, our study suggests that RASSF4 controls SOCE and ER-PM junctions through ARF6-dependent regulation of PM PI(4,5)P2 levels, pivotal for a variety of physiological processes.
Asunto(s)
Señalización del Calcio , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Factores de Ribosilacion-ADP/metabolismo , Femenino , Células HeLa , Humanos , Microscopía Fluorescente , Microscopía por Video , Proteínas de Neoplasias/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Transporte de Proteínas , Interferencia de ARN , Molécula de Interacción Estromal 1/genética , Sinaptotagmina II/genética , Sinaptotagmina II/metabolismo , Sinaptotagminas/genética , Sinaptotagminas/metabolismo , Factores de Tiempo , Imagen de Lapso de Tiempo , Transfección , Proteínas Supresoras de Tumor/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patologíaRESUMEN
Endoplasmic reticulum-plasma membrane (ER-PM) junctions mediate crucial activities ranging from Ca2+ signaling to lipid metabolism. Spatial organization of ER-PM junctions may modulate the extent and location of these cellular activities. However, the morphology and distribution of ER-PM junctions are not well characterized. Using photoactivated localization microscopy, we reveal that the contact area of single ER-PM junctions is mainly oblong with the dimensions of â¼120 nm × â¼80 nm in HeLa cells. Using total internal reflection fluorescence microscopy and structure illumination microscopy, we show that cortical actin contributes to spatial distribution and stability of ER-PM junctions. Further functional assays suggest that intact F-actin architecture is required for phosphatidylinositol 4,5-bisphosphate homeostasis mediated by Nir2 at ER-PM junctions. Together, our study provides quantitative information on spatial organization of ER-PM junctions that is in part regulated by F-actin. We envision that functions of ER-PM junctions can be differentially regulated through dynamic actin remodeling during cellular processes.
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Actinas/metabolismo , Retículo Endoplásmico/metabolismo , Análisis Espacial , Citoesqueleto de Actina/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/fisiología , Células HeLa , Homeostasis , Humanos , Proteínas de la Membrana/metabolismo , Fosfatidilinositoles/metabolismo , Transducción de SeñalRESUMEN
The majority of human breast cancer is estrogen receptor alpha (ER) positive. While anti-estrogens/aromatase inhibitors are initially effective, resistance to these drugs commonly develops. Therapy-resistant tumors often retain ER signaling, via interaction with critical oncogenic coregulator proteins. To address these mechanisms of resistance, we have developed a novel ER coregulator binding modulator, ERX-11. ERX-11 interacts directly with ER and blocks the interaction between a subset of coregulators with both native and mutant forms of ER. ERX-11 effectively blocks ER-mediated oncogenic signaling and has potent anti-proliferative activity against therapy-sensitive and therapy-resistant human breast cancer cells. ERX-11 is orally bioavailable, with no overt signs of toxicity and potent activity in both murine xenograft and patient-derived breast tumor explant models. This first-in-class agent, with its novel mechanism of action of disrupting critical protein-protein interactions, overcomes the limitations of current therapies and may be clinically translatable for patients with therapy-sensitive and therapy-resistant breast cancers.
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Neoplasias de la Mama/tratamiento farmacológico , Antagonistas del Receptor de Estrógeno/metabolismo , Receptores de Estrógenos/metabolismo , Administración Oral , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Antagonistas del Receptor de Estrógeno/administración & dosificación , Xenoinjertos , Humanos , Ratones , Trasplante de Neoplasias , Técnicas de Cultivo de Órganos , Unión Proteica , Transducción de Señal/efectos de los fármacosRESUMEN
Scavenger receptor class B, type I (SR-BI) and its adaptor protein PDZK1 mediate responses to HDL cholesterol in endothelium. Whether the receptor-adaptor protein tandem serves functions in other vascular cell types is unknown. The current work determined the roles of SR-BI and PDZK1 in vascular smooth muscle (VSM). To evaluate possible VSM functions of SR-BI and PDZK1 in vivo, neointima formation was assessed 21 days post-ligation in the carotid arteries of wild-type, SR-BI-/- or PDZK1-/- mice. Whereas neointima development was negligible in wild-type and SR-BI-/-, there was marked neointima formation in PDZK1-/- mice. PDZK1 expression was demonstrated in primary mouse VSM cells, and compared to wild-type cells, PDZK1-/- VSM displayed exaggerated proliferation and migration in response to platelet derived growth factor (PDGF). Tandem affinity purification-mass spectrometry revealed that PDZK1 interacts with breakpoint cluster region kinase (Bcr), which contains a C-terminal PDZ binding sequence and is known to enhance responses to PDGF in VSM. PDZK1 interaction with Bcr in VSM was demonstrated by pull-down and by coimmunoprecipitation, and the augmented proliferative response to PDGF in PDZK1-/- VSM was abrogated by Bcr depletion. Furthermore, compared with wild-type Bcr overexpression, the introduction of a Bcr mutant incapable of PDZK1 binding into VSM cells yielded an exaggerated proliferative response to PDGF. Thus, PDZK1 has novel SR-BI-independent function in VSM that affords protection from neointima formation, and this involves PDZK1 suppression of VSM cell proliferation via an inhibitory interaction with Bcr.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/fisiología , Músculo Liso Vascular/enzimología , Proteínas Proto-Oncogénicas c-bcr/antagonistas & inhibidores , Túnica Íntima/crecimiento & desarrollo , Animales , Movimiento Celular , Proliferación Celular , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/citología , Proteínas Proto-Oncogénicas c-bcr/metabolismoRESUMEN
Oxysterols are metabolites of cholesterol that are produced in liver and other peripheral tissues as a means to eliminate cholesterol to bile acid. Recent studies have revealed that the most abundant circulating oxysterol 27-hydroxycholesterol (27HC) is the first identified endogenous selective estrogen receptor modulator. 27HC levels correlate well with that of cholesterol, and also rise progressively with age. 27HC affects estrogen receptor function by the antagonism of estrogen action and also by the direct modulation of the receptor function, and similar to estrogen/estrogen receptors, 27HC has many actions in various tissues. This review article introduces the recent progress in the understanding of the role of 27HC in breast cancer and cardiovascular dysfunction.
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BACKGROUND: Antiphospholipid syndrome patients have antiphospholipid antibodies (aPLs) that promote thrombosis, and they have increased cardiovascular disease risk. Although the basis for the thrombosis has been well delineated, it is not known why antiphospholipid syndrome patients also have an increased prevalence of nonthrombotic vascular occlusion. The aims of this work were to determine if aPLs directly promote medial hypertrophy or neointima formation in mice and to identify the underlying mechanisms. METHODS AND RESULTS: Medial hypertrophy and neointima formation invoked by carotid artery endothelial denudation were evaluated in mice administered normal human IgG or aPLs. While aPLs had no effect on medial hypertrophy, they caused exaggerated neointima development. This was related to an aPL-induced impairment in reendothelialization post denudation, and scratch assays in cell culture revealed that there are direct effects of aPLs on endothelium that retard cell migration. Further experiments showed that aPL antagonism of endothelial migration and repair is mediated by antibody recognition of ß2-glycoprotein I, apolipoprotein E receptor 2, and a decline in bioavailable NO. Consistent with these mechanisms, the adverse impacts of aPLs on reendothelialization and neointima formation were fully prevented by the NO donor molsidomine. CONCLUSIONS: APLs blunt endothelial repair, and there is related aPL-induced exaggeration in neointima formation after endothelial injury in mice. The initiating process entails NO deficiency mediated by ß2-glycoprotein I recognition by aPLs and apolipoprotein E receptor 2. The modulation of endothelial apolipoprotein E receptor 2 function or NO bioavailability may represent new interventions to prevent the nonthrombotic vascular occlusion and resulting cardiovascular disorders that afflict antiphospholipid syndrome patients.
Asunto(s)
Anticuerpos Antifosfolípidos/metabolismo , Traumatismos de las Arterias Carótidas/patología , Movimiento Celular/fisiología , Endotelio Vascular/citología , Neointima/patología , Óxido Nítrico/metabolismo , Animales , Anticuerpos Antifosfolípidos/inmunología , Síndrome Antifosfolípido/inmunología , Síndrome Antifosfolípido/fisiopatología , Disponibilidad Biológica , Traumatismos de las Arterias Carótidas/fisiopatología , Movimiento Celular/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Hipertrofia/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Neointima/inmunología , Distribución Aleatoria , Sensibilidad y EspecificidadRESUMEN
A subpopulation of plasma membrane-associated estrogen receptor (ER)α interact directly with G proteins and mediate nonnuclear receptor signaling. This mechanism underlies numerous processes, including important cardiovascular protective actions of estradiol (E(2)), such as the activation of endothelial NO synthase (eNOS) and endothelial cell growth and migration. In the present work we sought a genetic approach to differentiate nonnuclear from nuclear ERα actions. We generated single alanine substitutions within the Gαi-binding domain of ERα (amino acids 251-260) and tested signaling to eNOS or ERK1,2 and activation of luciferase (Luc) reporters signifying transactivation via direct or indirect ERα-DNA binding in HeLa cells. The point mutants ERα-R256A, ERα-K257A, ERα-D258A, and ERα-R260A were all incapable of activating eNOS in response to E(2), and ERα-R256A and ERα-D258A also showed loss of ERK1,2 activation. In contrast, ERα-R256A, ERα-K257A, ERα-D258A, and ERα-R260A all displayed normal capacity to invoke E(2)-induced transactivation of estrogen response element (ERE)-Luc or Sp1-Luc. However, whereas activator protein 1-Luc activation by ERα-R256A and ERα-D258A was intact, ERα-K257A and ERα-R260A were incapable of activator protein 1-Luc activation. In in vitro pull-down assays with the two mutants that lack all nonnuclear functions tested and retain all nuclear functions tested, ERα-R256A and ERα-D258A, there was normal direct interaction between Gαi and ERα-R256A and an absence of interaction between Gαi and ERα-D258A. When expressed in endothelial cells, these two mutants prevented E(2)-induced migration and eNOS activation mediated by endogenous receptor, indicative of dominant-negative action. Thus, the point mutants ERα-R256A and ERα-D258A in the receptor GαI-binding domain provide genetic segregation of nonnuclear from nuclear ERα function.
Asunto(s)
Núcleo Celular/metabolismo , Receptor alfa de Estrógeno/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Mutación Puntual , Secuencia de Aminoácidos , Animales , Línea Celular , Chlorocebus aethiops , Estradiol/fisiología , Receptor alfa de Estrógeno/química , Receptor alfa de Estrógeno/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas , Mutagénesis Sitio-Dirigida , Óxido Nítrico Sintasa de Tipo III/metabolismo , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Activación TranscripcionalRESUMEN
Liver X receptors (LXR) are stimulated by cholesterol-derived oxysterols and serve as transcription factors to regulate gene expression in response to alterations in cholesterol. In the present study, we investigated the role of LXRs in vascular endothelial cells (ECs) and discovered that LXRß has nonnuclear function and stimulates EC migration by activating endothelial NOS (eNOS). This process is mediated by estrogen receptor-α (ERα). LXR activation promoted the direct binding of LXRß to the ligand-binding domain of ERα and initiated an extranuclear signaling cascade that requires ERα Ser118 phosphorylation by PI3K/AKT. Further studies revealed that LXRß and ERα are colocalized and functionally coupled in EC plasma membrane caveolae/lipid rafts. In isolated aortic rings, LXR activation of NOS caused relaxation, while in mice, LXR activation stimulated carotid artery reendothelialization via LXRß- and ERα-dependent processes. These studies demonstrate that LXRß has nonnuclear function in EC caveolae/lipid rafts that entails crosstalk with ERα, which promotes NO production and maintains endothelial monolayer integrity in vivo.
Asunto(s)
Células Endoteliales/enzimología , Receptor alfa de Estrógeno/metabolismo , Microdominios de Membrana/metabolismo , Receptores Nucleares Huérfanos/metabolismo , Animales , Aorta/citología , Caveolas/metabolismo , Línea Celular , Movimiento Celular , Núcleo Celular/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/citología , Activación Enzimática , Humanos , Técnicas In Vitro , Receptores X del Hígado , Masculino , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo III , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Receptor Cross-Talk , Transducción de Señal , VasodilataciónRESUMEN
Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of gene products involved in hepatic glycolysis and lipogenesis. Glucose affects the activity of ChREBP largely through post-translational mechanisms involving phosphorylation-dependent cellular localization. In this work we show that the N-terminal region of ChREBP (residues 1-251) regulates its subcellular localization via an interaction with 14-3-3. 14-3-3 binds an alpha-helix in this region (residues 125-135) to retain ChREBP in the cytosol, and binding of 14-3-3 is facilitated by phosphorylation of nearby Ser-140 and Ser-196. Phosphorylation of ChREBP at these sites was essential for its interaction with CRM1 for export to the cytosol, whereas nuclear import of ChREBP requires dephosphorylated ChREBP to interact with importin alpha. Notably, 14-3-3 appears to compete with importin alpha for ChREBP binding. 14-3-3beta bound to a synthetic peptide spanning residues 125-144 and bearing a phosphate at Ser-140 with a dissociation constant of 1.1 microm, as determined by isothermal calorimetry. The interaction caused a shift in the fluorescence maximum of the tryptophan residues of the peptide. The corresponding unphosphorylated peptide failed to bind 14-3-3beta. These results suggest that interactions with importin alpha and 14-3-3 regulate movement of ChREBP into and out of the nucleus, respectively, and that these interactions are regulated by the ChREBP phosphorylation status.