RESUMO
Steroidogenic tissues contain cytosolic lipid droplets that are important for steroidogenesis. Perilipin 2 (PLIN2), a structural coat protein located on the surface of lipid droplets in mammalian cells, plays a crucial role in regulating lipid droplet formation and contributing to various cellular processes such as lipid storage and energy homeostasis. Herein, we examine the role that PLIN2 plays in regulating progesterone synthesis in the bovine corpus luteum. Utilizing gene array databases and Western blotting, we have delineated the expression pattern of PLIN2 throughout the follicular to luteal transition. Our findings reveal the presence of PLIN2 in both ovarian follicular and steroidogenic luteal cells, demonstrating an increase in its levels as follicular cells transition into the luteal phase. Moreover, the depletion of PLIN2 via siRNA enhanced progesterone production in small luteal cells, whereas adenovirus-mediated overexpression of both PLIN2 and Perilipin 3 (PLIN3) induced an increase in cytosolic lipid droplet accumulation and decreased hormone-induced progesterone synthesis in these cells. Lastly, in vivo administration of the luteolytic hormone prostaglandin F2α resulted in an upregulation of PLIN2 mRNA and protein expression, accompanied by a decline in serum progesterone. Our findings highlight the pivotal role of PLIN2 in regulating progesterone synthesis in the bovine corpus luteum, as supported by its dynamic expression pattern during the follicular to luteal transition and its responsiveness to luteotropic and luteolytic hormones. We suggest PLIN2 as a potential therapeutic target for modulating luteal function.
Assuntos
Células Lúteas , Perilipina-2 , Progesterona , Animais , Feminino , Bovinos , Progesterona/metabolismo , Perilipina-2/metabolismo , Perilipina-2/genética , Células Lúteas/metabolismo , Gotículas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Perilipina-3/metabolismo , Corpo Lúteo/metabolismo , Células CultivadasRESUMO
Lipid droplets (LDs) are fat-storing organelles enclosed by a phospholipid monolayer, which harbors membrane-associated proteins that regulate distinct LD functions. LD proteins are degraded by the ubiquitin-proteasome system (UPS) and/or by lysosomes. Because chronic ethanol (EtOH) consumption diminishes the hepatic functions of the UPS and lysosomes, we hypothesized that continuous EtOH consumption slows the breakdown of lipogenic LD proteins targeted for degradation, thereby causing LD accumulation. Here, we report that LDs from livers of EtOH-fed rats exhibited higher levels of polyubiquitylated-proteins, linked at either lysine 48 (directed to proteasome) or lysine 63 (directed to lysosomes) than LDs from pair-fed control rats. MS proteomics of LD proteins, immunoprecipitated with UB remnant motif antibody (K-ε-GG), identified 75 potential UB proteins, of which 20 were altered by chronic EtOH administration. Among these, hydroxysteroid 17ß-dehydrogenase 11 (HSD17ß11) was prominent. Immunoblot analyses of LD fractions revealed that EtOH administration enriched HSD17ß11 localization to LDs. When we overexpressed HSD17ß11 in EtOH-metabolizing VA-13 cells, the steroid dehydrogenase 11 became principally localized to LDs, resulting in elevated cellular triglycerides (TGs). Ethanol exposure augmented cellular TG, while HSD17ß11 siRNA decreased both control and EtOH-induced TG accumulation. Remarkably, HSD17ß11 overexpression lowered the LD localization of adipose triglyceride lipase. EtOH exposure further reduced this localization. Reactivation of proteasome activity in VA-13 cells blocked the EtOH-induced rises in both HSD17ß11 and TGs. Our findings indicate that EtOH exposure blocks HSD17ß11 degradation by inhibiting the UPS, thereby stabilizing HSD17ß11 on LD membranes, to prevent lipolysis by adipose triglyceride lipase and promote cellular LD accumulation.
Assuntos
17-Hidroxiesteroide Desidrogenases , Etanol , Fígado Gorduroso , Animais , Ratos , Etanol/farmacologia , Etanol/metabolismo , Fígado Gorduroso/metabolismo , Lipase/genética , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Lisina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , 17-Hidroxiesteroide Desidrogenases/metabolismoRESUMO
Lipophagy is a central cellular process for providing the cell with a readily utilized, high energy source of neutral lipids. Since its discovery over a decade ago, we are just starting to understand the molecular components that drive lipophagy, how it is activated in response to nutrient availability, and its potential as a therapeutic target in disease. In this Cell Science at a Glance article and the accompanying poster, we first provide a brief overview of the different structural and enzymatic proteins that comprise the lipid droplet (LD) proteome and reside within the limiting phospholipid monolayer of this complex organelle. We then highlight key players in the catabolic breakdown of LDs during the functionally linked lipolysis and lipophagy processes. Finally, we discuss what is currently known about macro- and micro-lipophagy based on findings in yeast, mammalian and other model systems, and how impairment of these important functions can lead to disease states.
Assuntos
Gotículas Lipídicas , Lipólise , Animais , Autofagia/fisiologia , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/fisiologia , Mamíferos/metabolismo , Fosfolipídeos/metabolismo , Proteínas/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
BACKGROUND & AIMS: Although T-cell intrinsic expression of G9a has been associated with murine intestinal inflammation, mechanistic insight into the role of this methyltransferase in human T-cell differentiation is ill defined, and manipulation of G9a function for therapeutic use against inflammatory disorders is unexplored. METHODS: Human naive T cells were isolated from peripheral blood and differentiated in vitro in the presence of a G9a inhibitor (UNC0642) before being characterized via the transcriptome (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin by sequencing), protein expression (cytometry by time of flight, flow cytometry), metabolism (mitochondrial stress test, ultrahigh performance liquid chromatography-tandem mas spectroscopy) and function (T-cell suppression assay). The in vivo role of G9a was assessed using 3 murine models. RESULTS: We discovered that pharmacologic inhibition of G9a enzymatic function in human CD4 T cells led to spontaneous generation of FOXP3+ T cells (G9a-inibitors-T regulatory cells [Tregs]) in vitro that faithfully reproduce human Tregs, functionally and phenotypically. Mechanistically, G9a inhibition altered the transcriptional regulation of genes involved in lipid biosynthesis in T cells, resulting in increased intracellular cholesterol. Metabolomic profiling of G9a-inibitors-Tregs confirmed elevated lipid pathways that support Treg development through oxidative phosphorylation and enhanced lipid membrane composition. Pharmacologic G9a inhibition promoted Treg expansion in vivo upon antigen (gliadin) stimulation and ameliorated acute trinitrobenzene sulfonic acid-induced colitis secondary to tissue-specific Treg development. Finally, Tregs lacking G9a expression (G9a-knockout Tregs) remain functional chronically and can rescue T-cell transfer-induced colitis. CONCLUSION: G9a inhibition promotes cholesterol metabolism in T cells, favoring a metabolic profile that facilitates Treg development in vitro and in vivo. Our data support the potential use of G9a inhibitors in the treatment of immune-mediated conditions including inflammatory bowel disease.
Assuntos
Linfócitos T CD4-Positivos , Colite , Camundongos , Humanos , Animais , Metabolismo dos Lipídeos , Linfócitos T Reguladores/metabolismo , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/genética , Cromatina , Inflamação , Colesterol , Lipídeos , Fatores de Transcrição Forkhead/metabolismoRESUMO
Hepatocytes metabolize energy-rich cytoplasmic lipid droplets (LDs) in the lysosome-directed process of autophagy. An organelle-selective form of this process (macrolipophagy) results in the engulfment of LDs within double-membrane delimited structures (autophagosomes) before lysosomal fusion. Whether this is an exclusive autophagic mechanism used by hepatocytes to catabolize LDs is unclear. It is also unknown whether lysosomes alone might be sufficient to mediate LD turnover in the absence of an autophagosomal intermediate. We performed live-cell microscopy of hepatocytes to monitor the dynamic interactions between lysosomes and LDs in real-time. We additionally used a fluorescent variant of the LD-specific protein (PLIN2) that exhibits altered fluorescence in response to LD interactions with the lysosome. We find that mammalian lysosomes and LDs undergo interactions during which proteins and lipids can be transferred from LDs directly into lysosomes. Electron microscopy (EM) of primary hepatocytes or hepatocyte-derived cell lines supports the existence of these interactions. It reveals a dramatic process whereby the lipid contents of the LD can be "extruded" directly into the lysosomal lumen under nutrient-limited conditions. Significantly, these interactions are not affected by perturbations to crucial components of the canonical macroautophagy machinery and can occur in the absence of double-membrane lipoautophagosomes. These findings implicate the existence of an autophagic mechanism used by mammalian cells for the direct transfer of LD components into the lysosome for breakdown. This process further emphasizes the critical role of lysosomes in hepatic LD catabolism and provides insights into the mechanisms underlying lipid homeostasis in the liver.
Assuntos
Autofagia/fisiologia , Hepatócitos/metabolismo , Gotículas Lipídicas/metabolismo , Lisossomos/metabolismo , Animais , Autofagossomos/metabolismo , Linhagem Celular , Metabolismo dos Lipídeos , Camundongos , Microscopia Confocal , Transporte Proteico , Ratos Sprague-DawleyRESUMO
Epithelial cells, such as liver-resident hepatocytes, rely heavily on the Rab family of small GTPases to perform membrane trafficking events that dictate cell physiology and metabolism. Not surprisingly, disruption of several Rab proteins can manifest in metabolic diseases or cancer. Rab32 is expressed in many secretory epithelial cells but its role in cellular metabolism is virtually unknown. In this study, we find that Rab32 associates with lysosomes and regulates proliferation and cell size of Hep3B hepatoma and HeLa cells. Specifically, we identify that Rab32 supports the mechanistic target of rapamycin complex 1 (mTORC1) signaling under basal and amino acid-stimulated conditions. Consistent with inhibited mTORC1, an increase in nuclear TFEB localization and lysosome biogenesis is also observed in Rab32-depleted cells. Finally, we find that Rab32 interacts with mTOR kinase, and that loss of Rab32 reduces the association of mTOR and mTORC1 pathway proteins with lysosomes, suggesting that Rab32 regulates lysosomal mTOR trafficking. In summary, these findings suggest that Rab32 functions as a novel regulator of cellular metabolism through supporting mTORC1 signaling.This article has an associated First Person interview with the first author of the paper.
Assuntos
Proteínas Monoméricas de Ligação ao GTP , Células HeLa , Humanos , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transdução de Sinais , Proteínas rab de Ligação ao GTPRESUMO
In liver steatosis (i.e. fatty liver), hepatocytes accumulate many large neutral lipid storage organelles known as lipid droplets (LDs). LDs are important in the maintenance of energy homeostasis, but the signaling mechanisms that stimulate LD metabolism in hepatocytes are poorly defined. In adipocytes, catecholamines target the ß-adrenergic (ß-AR)/cAMP pathway to activate cytosolic lipases and induce their recruitment to the LD surface. Therefore, the goal of this study was to determine whether hepatocytes, like adipocytes, also undergo cAMP-mediated lipolysis in response to ß-AR stimulation. Using primary rat hepatocytes and human hepatoma cells, we found that treatment with the ß-AR agent isoproterenol caused substantial LD loss via activation of cytosolic lipases adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL). ß-Adrenergic stimulation rapidly activated PKA, which led to the phosphorylation of ATGL and HSL and their recruitment to the LD surface. To test whether this ß-AR-dependent lipolysis pathway was altered in a model of alcoholic fatty liver, primary hepatocytes from rats fed a 6-week EtOH-containing Lieber-DeCarli diet were treated with cAMP agonists. Compared with controls, EtOH-exposed hepatocytes showed a drastic inhibition in ß-AR/cAMP-induced LD breakdown and the phosphorylation of PKA substrates, including HSL. This observation was supported in VA-13 cells, an EtOH-metabolizing human hepatoma cell line, which displayed marked defects in both PKA activation and isoproterenol-induced ATGL translocation to the LD periphery. In summary, these findings suggest that ß-AR stimulation mobilizes cytosolic lipases for LD breakdown in hepatocytes, and perturbation of this pathway could be a major consequence of chronic EtOH insult leading to fatty liver.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , AMP Cíclico/agonistas , Fígado Gorduroso Alcoólico/metabolismo , Hepatócitos/efeitos dos fármacos , Lipólise/efeitos dos fármacos , Receptores Adrenérgicos beta/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Fígado Gorduroso Alcoólico/patologia , Feminino , Hepatócitos/citologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Lipase/química , Lipase/metabolismo , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/patologia , Masculino , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Receptores Adrenérgicos beta/química , Esterol Esterase/química , Esterol Esterase/metabolismoRESUMO
Clathrin-mediated endocytosis of transferrin (Tf) and its cognate receptor (TfR1) is a central pathway supporting the uptake of trophic iron. It has generally been assumed that this is a constitutive process. However, we have reported that the non-receptor tyrosine kinase, Src, is activated by Tf to facilitate the internalization of the Tf-TfR1 ligand-receptor complex. As an extension of these findings, we have tested whether subsequent trafficking steps might be regulated by additional kinase-dependent cascades, and we observed a significant endocytic block by inhibiting c-Abl kinase by a variety of methods. Importantly, Tf internalization was reduced significantly in all of these cell models and could be restored by re-expression of WT c-Abl. Surprisingly, this attenuated Tf-TfR1 endocytosis was due to a substantial drop in both the surface and total cellular receptor levels. Additional studies with the LDL receptor showed a similar effect. Surprisingly, immunofluorescence microscopy of imatinib-treated cells revealed a marked colocalization of internalized TfR1 with late endosomes/lysosomes, whereas attenuating the lysosome function with several inhibitors reduced this receptor loss. Importantly, inhibition of c-Abl resulted in a striking redistribution of the chaperone Hsc70 from a diffuse cytosolic localization to an association with the TfR1 at the late endosome-lysosome. Pharmacological inhibition of Hsc70 ATPase activity in cultured cells by the drug VER155008 prevents this chaperone-receptor interaction, resulting in an accumulation of the TfR1 in the early endosome. Thus, inhibition of c-Abl minimizes receptor recycling pathways and results in chaperone-dependent trafficking of the TfR1 to the lysosome for degradation. These findings implicate a novel role for c-Abl and Hsc70 as an unexpected regulator of Hsc70-mediated transport of trophic receptor cargo between the early and late endosomal compartments.
Assuntos
Antígenos CD/metabolismo , Endocitose/fisiologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Receptores da Transferrina/metabolismo , Animais , Antígenos CD/genética , Endossomos/genética , Endossomos/metabolismo , Proteínas de Choque Térmico HSC70/genética , Proteínas de Choque Térmico HSC70/metabolismo , Células HeLa , Humanos , Lisossomos/genética , Lisossomos/metabolismo , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas c-abl/genética , Ratos , Receptores da Transferrina/genéticaRESUMO
BACKGROUND: Previous reports suggest that lipid droplets (LDs) in the hepatocyte can be catabolized by a direct engulfment from nearby endolysosomes (microlipophagy). Further, it is likely that this process is compromised by chronic ethanol (EtOH) exposure leading to hepatic steatosis. This study investigates the hepatocellular machinery supporting microlipophagy and EtOH-induced alterations in this process with a focus on the small, endosome-associated, GTPase Rab5. METHODS AND RESULTS: Here we report that this small Ras-related GTPase is a resident component of LDs, and its activity is important for hepatocellular LD-lysosome proximity and physical interactions. We find that Rab5 siRNA knockdown causes an accumulation of LDs in hepatocytes by inhibiting lysosome dependent LD catabolism. Importantly, Rab5 appears to support this process by mediating the recruitment of early endosomal and or multivesicular body compartments to the LD surface before lysosome fusion. Interestingly, while wild-type or a constituently active GTPase form (Q79L) of Rab5 supports LD-lysosome transport, this process is markedly reduced in cells expressing a GTPase dead (S34N) Rab5 protein or in hepatocytes exposed to chronic EtOH. CONCLUSIONS: These findings support the novel premise of an early endosomal/multivesicular body intermediate compartment on the LD surface that provides a "docking" site for lysosomal trafficking, not unlike the process that occurs during the hepatocellular degradation of endocytosed ligands that is also known to be compromised by EtOH exposure.
Assuntos
Etanol , Hepatócitos , Lisossomos , Proteínas rab5 de Ligação ao GTP , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Lisossomos/metabolismo , Lisossomos/efeitos dos fármacos , Etanol/farmacologia , Hepatócitos/metabolismo , Hepatócitos/efeitos dos fármacos , Humanos , Gotículas Lipídicas/metabolismo , Autofagia/efeitos dos fármacos , Animais , Endossomos/metabolismoRESUMO
Progesterone is an essential steroid hormone that is required to initiate and maintain pregnancy in mammals and serves as a metabolic intermediate in the synthesis of endogenously produced steroids, including sex hormones and corticosteroids. Steroidogenic luteal cells of the corpus luteum have the tremendous capacity to synthesize progesterone. These specialized cells are highly enriched with lipid droplets that store lipid substrate, which can be used for the synthesis of steroids. We recently reported that hormone-stimulated progesterone synthesis by luteal cells requires protein kinase A-dependent mobilization of cholesterol substrate from lipid droplets to mitochondria. We hypothesize that luteal lipid droplets are enriched with steroidogenic enzymes and facilitate the synthesis of steroids in the corpus luteum. In the present study, we analyzed the lipid droplet proteome, conducted the first proteomic analysis of lipid droplets under acute cyclic adenosine monophosphate (cAMP)-stimulated conditions, and determined how specific lipid droplet proteins affect steroidogenesis. Steroidogenic enzymes, cytochrome P450 family 11 subfamily A member 1 and 3 beta-hydroxysteroid dehydrogenase (HSD3B), were highly abundant on lipid droplets of the bovine corpus luteum. High-resolution confocal microscopy confirmed the presence of active HSD3B on the surface of luteal lipid droplets. We report that luteal lipid droplets have the capacity to synthesize progesterone from pregnenolone. Lastly, we analyzed the lipid droplet proteome following acute stimulation with cAMP analog, 8-Br-cAMP, and report increased association of HSD3B with luteal lipid droplets following stimulation. These findings provide novel insights into the role of luteal lipid droplets in steroid synthesis.
Assuntos
Gotículas Lipídicas , Progesterona , Gravidez , Feminino , Bovinos , Animais , Progesterona/metabolismo , Gotículas Lipídicas/metabolismo , Proteoma/metabolismo , Proteômica , Corpo Lúteo/metabolismo , Esteroides , Hormônios/metabolismo , Mamíferos/metabolismoRESUMO
Lipid droplets (LDs) are dynamic fat-storage organelles that interact readily with numerous cellular structures and organelles. A prominent LD contact site is with degradative vesicles such as autophagosomes, lysosomes, autolysosomes, and late endosomes. These contacts support lipid catabolism through the selective autophagy of LDs (i.e., lipophagy) or the recruitment of cytosolic lipases to the LD surface (i.e., lipolysis). However, LD-autophagosome contacts serve additional functions beyond lipid catabolism, including the supply of lipids for autophagosome biogenesis. In this review, we discuss the molecular mediators of LD contacts with autophagosomes and other degradative organelles as well as the diverse cellular functions of these contact sites in health and disease.
RESUMO
The liver performs numerous vital functions, including the detoxification of blood before access to the brain while simultaneously secreting and internalizing scores of proteins and lipids to maintain appropriate blood chemistry. Furthermore, the liver also synthesizes and secretes bile to enable the digestion of food. These diverse attributes are all performed by hepatocytes, the parenchymal cells of the liver. As predicted, these cells possess a remarkably well-developed and complex membrane trafficking machinery that is dedicated to moving specific cargos to their correct cellular locations. Importantly, while most epithelial cells secrete nascent proteins directionally toward a single lumen, the hepatocyte secretes both proteins and bile concomitantly at its basolateral and apical domains, respectively. In this Beyond the Cell review, we will detail these central features of the hepatocyte and highlight how membrane transport processes play a key role in healthy liver function and how they are affected by disease.
Assuntos
Membrana Celular/genética , Hepatócitos/metabolismo , Proteínas de Membrana Transportadoras/genética , Transporte Proteico/genética , Animais , Membrana Celular/metabolismo , Movimento Celular/genética , Humanos , Fígado/metabolismo , Proteínas de Membrana Transportadoras/química , Tecido Parenquimatoso/metabolismoRESUMO
Lipid droplet (LD) catabolism in hepatocytes is mediated by a combination of lipolysis and a selective autophagic mechanism called lipophagy, but the relative contributions of these seemingly distinct pathways remain unclear. We find that inhibition of lipolysis, lipophagy, or both resulted in similar overall LD content but dramatic differences in LD morphology. Inhibition of the lipolysis enzyme adipose triglyceride lipase (ATGL) resulted in large cytoplasmic LDs, whereas lysosomal inhibition caused the accumulation of numerous small LDs within the cytoplasm and degradative acidic vesicles. Combined inhibition of ATGL and LAL resulted in large LDs, suggesting that lipolysis targets these LDs upstream of lipophagy. Consistent with this, ATGL was enriched in larger-sized LDs, whereas lipophagic vesicles were restricted to small LDs as revealed by immunofluorescence, electron microscopy, and Western blot of size-separated LDs. These findings provide new evidence indicating a synergistic relationship whereby lipolysis targets larger-sized LDs to produce both size-reduced and nascently synthesized small LDs that are amenable for lipophagic internalization.
Assuntos
Hepatócitos/citologia , Hepatócitos/metabolismo , Gotículas Lipídicas/metabolismo , Lipólise , Animais , Feminino , Ratos , Ratos Sprague-Dawley , Triglicerídeos/análise , Triglicerídeos/metabolismoRESUMO
Alcohol consumption is a well-established risk factor for the onset and progression of fatty liver disease. An estimated 90% of heavy drinkers are thought to develop significant liver steatosis. For these reasons, an increased understanding of the molecular basis for alcohol-induced hepatic steatosis is important. It has become clear that autophagy, a catabolic process of intracellular degradation and recycling, plays a key role in hepatic lipid metabolism. We have shown that Rab7, a small guanosine triphosphatase known to regulate membrane trafficking, acts as a key orchestrator of hepatocellular lipophagy, a selective form of autophagy in which lipid droplets (LDs) are specifically targeted for turnover by the autophagic machinery. Nutrient starvation results in Rab7 activation on the surface of the LD and lysosomal compartments, resulting in the mobilization of triglycerides stored within the LDs for energy production. Here, we examine whether the steatotic effects of alcohol exposure are a result of perturbations to the Rab7-mediated lipophagic pathway. Rats chronically fed an ethanol-containing diet accumulated significantly higher levels of fat in their hepatocytes. Interestingly, hepatocytes isolated from these ethanol-fed rats contained juxtanuclear lysosomes that exhibited impaired motility. These changes are similar to those we observed in Rab7-depleted hepatocytes. Consistent with these defects in the lysosomal compartment, we observed a marked 80% reduction in Rab7 activity in cultured hepatocytes as well as a complete block in starvation-induced Rab7 activation in primary hepatocytes isolated from chronic ethanol-fed animals. Conclusion: A mechanism is supported whereby ethanol exposure inhibits Rab7 activity, resulting in the impaired transport, targeting, and fusion of the autophagic machinery with LDs, leading to an accumulation of hepatocellular lipids and hepatic steatosis. (Hepatology Communications 2017;1:140-152).
RESUMO
Scaffold proteins play a critical role in cellular homeostasis by anchoring signaling enzymes in close proximity to downstream effectors. In addition to anchoring static enzyme complexes, some scaffold proteins also form dynamic signalosomes that can traffic to different subcellular compartments upon stimulation. Gravin (AKAP12), a multivalent scaffold, anchors PKA and other enzymes to the plasma membrane under basal conditions, but upon [Ca(2+)]i elevation, is rapidly redistributed to the cytosol. Because gravin redistribution also impacts PKA localization, we postulate that gravin acts as a calcium "switch" that modulates PKA-substrate interactions at the plasma membrane, thus facilitating a novel crosstalk mechanism between Ca(2+) and PKA-dependent pathways. To assess this, we measured the impact of gravin-V5/His expression on compartmentalized PKA activity using the FRET biosensor AKAR3 in cultured cells. Upon treatment with forskolin or isoproterenol, cells expressing gravin-V5/His showed elevated levels of plasma membrane PKA activity, but cytosolic PKA activity levels were reduced compared with control cells lacking gravin. This effect required both gravin interaction with PKA and localization at the plasma membrane. Pretreatment with calcium-elevating agents thapsigargin or ATP caused gravin redistribution away from the plasma membrane and prevented gravin from elevating PKA activity levels at the membrane. Importantly, this mode of Ca(2+)/PKA crosstalk was not observed in cells expressing a gravin mutant that resisted calcium-mediated redistribution from the cell periphery. These results reveal that gravin impacts subcellular PKA activity levels through the spatial targeting of PKA, and that calcium elevation modulates downstream ß-adrenergic/PKA signaling through gravin redistribution, thus supporting the hypothesis that gravin mediates crosstalk between Ca(2+) and PKA-dependent signaling pathways. Based on these results, AKAP localization dynamics may represent an important paradigm for the regulation of cellular signaling networks.
Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Transferência Ressonante de Energia de Fluorescência , Proteínas de Ancoragem à Quinase A/genética , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/genética , HumanosRESUMO
The autophagic digestion of lipid droplets (LDs) through lipophagy is an essential process by which most cells catabolize lipids as an energy source. However, the cellular machinery used for the envelopment of LDs during autophagy is poorly understood. We report a novel function for a small Rab guanosine triphosphatase (GTPase) in the recruitment of adaptors required for the engulfment of LDs by the growing autophagosome. In hepatocytes stimulated to undergo autophagy, Rab10 activity is amplified significantly, concomitant with its increased recruitment to nascent autophagic membranes at the LD surface. Disruption of Rab10 function by small interfering RNA knockdown or expression of a GTPase-defective variant leads to LD accumulation. Finally, Rab10 activation during autophagy is essential for LC3 recruitment to the autophagosome and stimulates its increased association with the adaptor protein EHBP1 (EH domain binding protein 1) and the membrane-deforming adenosine triphosphatase EHD2 (EH domain containing 2) that, together, are essential in driving the activated "engulfment" of LDs during lipophagy in hepatocytes.
RESUMO
A-Kinase Anchoring Proteins (AKAPs) direct the flow of cellular information by positioning multiprotein signaling complexes into proximity with effector proteins. However, certain AKAPs are not stationary but can undergo spatiotemporal redistribution in response to stimuli. Gravin, a 300kD AKAP that intersects with a diverse signaling array, is localized to the plasma membrane but has been shown to translocate to the cytosol following the elevation of intracellular calcium ([Ca(2+)]i). Despite the potential for gravin redistribution to impact multiple signaling pathways, the dynamics of this event remain poorly understood. In this study, quantitative microscopy of cells expressing gravin-EGFP revealed that Ca(2+) elevation caused the complete translocation of gravin from the cell cortex to the cytosol in as little as 60s of treatment with ionomycin or thapsigargin. In addition, receptor mediated signaling was also shown to cause gravin redistribution following ATP treatment, and this event required both [Ca(2+)]i elevation and PKC activation. To understand the mechanism for Ca(2+) mediated gravin dynamics, deletion of calmodulin-binding domains revealed that a fourth putative calmodulin binding domain called CB4 (a.a. 670-694) is critical for targeting gravin to the cell cortex despite its location downstream of gravin's membrane-targeting domains, which include an N-terminal myristoylation site and three polybasic domains. Finally, confocal microscopy of cells co-transfected with gravin-EYFP and PKA RII-ECFP revealed that gravin redistribution mediated by ionomycin, thapsigargin, and ATP each triggered the gravin-dependent loss of PKA localized at the cell cortex. Our results support the hypothesis that gravin redistribution regulates cross-talk between PKA-dependent signaling and receptor-mediated events involving Ca(2+) and PKC.