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As sessile organisms, plants constantly face a variety of abiotic stresses, such as drought, salinity, and metal/metalloid toxicity, all of which possess significant threats to plant growth and yield potential. Improving plant resilience to such abiotic stresses bears paramount importance in practicing sustainable agriculture worldwide. Acetic acid/acetate has been recognized as an important metabolite with multifaceted roles in regulating plant adaptation to diverse abiotic stresses. Recent studies have elucidated that acetic acid can potentiate plants' inherent mechanisms to withstand the adverse effects of abiotic stresses through the regulation of lipid metabolism, hormone signaling, epigenetic changes, and physiological defense mechanisms. Numerous studies also underpin the potential use of acetic acid in boosting crop production under unfavorable environmental conditions. This review provides a comprehensive update on the understanding of how acetic acid regulates plant photosynthesis, acts as an antitranspirant, detoxifies reactive oxygen species to alleviate oxidative stress, interacts with phytohormones to regulate physiological processes, and improves soil fertility and microbial diversity, with a specific focus on drought, salinity, and metal toxicity. We also highlight the eco-friendly and economic potential of acetic acid that may attract farmers from developing countries to harness the benefits of acetic acid application for boosting abiotic stress resistance in crops. Given that acetic acid is a widely accessible, inexpensive, and eco-friendly compound, the revelation of acetic acid-mediated regulatory pathways and its crosstalk with other signaling molecules will have significant importance in developing a sustainable strategy for mitigating abiotic stresses in crops.
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Lipid processing by the retinal pigment epithelium (RPE) is necessary to maintain retinal health and function. Dysregulation of retinal lipid homeostasis due to normal aging or age-related disease triggers lipid accumulation within the RPE, on Bruch's membrane (BrM), and in the subretinal space. In its role as a hub for lipid trafficking into and out of the neural retina, the RPE packages a significant amount of lipid into lipid droplets for storage and into apolipoprotein B (APOB)-containing lipoproteins (Blps) for export. Microsomal triglyceride transfer protein (MTP), encoded by the MTTP gene, is essential for Blp assembly. Herein we test the hypothesis that MTP expression in the RPE is essential to maintain lipid balance and retinal function using the newly generated RPEΔMttp mouse model. Using non-invasive ocular imaging, electroretinography, and histochemical and biochemical analyses we show that genetic depletion of Mttp from the RPE results in intracellular lipid accumulation, increased photoreceptor-associated cholesterol deposits, and photoreceptor cell death, and loss of rod but not cone function. RPE-specific reduction in Mttp had no significant effect on plasma lipids and lipoproteins. While APOB was decreased in the RPE, most ocular retinoids remained unchanged, with the exception of the storage form of retinoid, retinyl ester. Thus suggesting that RPE MTP is critical for Blp synthesis and assembly but is not directly involved in plasma lipoprotein metabolism. These studies demonstrate that RPE-specific MTP expression is necessary to establish and maintain retinal lipid homeostasis and visual function.
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Proteínas de Transporte , Retina , Epitélio Pigmentado da Retina , Animais , Camundongos , Retinoides , Apolipoproteínas B/genética , HomeostaseRESUMO
Recently, we reported that oral-epithelial cells (OE) are unique in their response to Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) in that cell cycle arrest (G2/M) occurs without leading to apoptosis. We now demonstrate that Cdt-induced cell cycle arrest in OE has a duration of at least 7 days with no change in viability. Moreover, toxin-treated OE develops a new phenotype consistent with cellular senescence; this includes increased senescence-associated ß-galactosidase (SA-ß-gal) activity and accumulation of the lipopigment, lipofuscin. Moreover, the cells exhibit a secretory profile associated with cellular senescence known as the senescence-associated secretory phenotype (SASP), which includes IL-6, IL-8 and RANKL. Another unique feature of Cdt-induced OE senescence is disruption of barrier function, as shown by loss of transepithelial electrical resistance and confocal microscopic assessment of primary gingival keratinocyte structure. Finally, we demonstrate that Cdt-induced senescence is dependent upon the host cell protein cellugyrin, a homologue of the synaptic vesicle protein synaptogyrin. Collectively, these observations point to a novel pathogenic outcome in oral epithelium that we propose contributes to both A. actinomycetemcomitans infection and periodontal disease progression.
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Lipid processing by the retinal pigment epithelium (RPE) is necessary to maintain retinal health and function. Dysregulation of retinal lipid homeostasis due to normal aging or to age-related disease triggers lipid accumulation within the RPE, on Bruch's membrane (BrM), and in the subretinal space. In its role as a hub for lipid trafficking into and out of the neural retina, the RPE packages a significant amount of lipid into lipid droplets for storage and into apolipoprotein B (apoB)-containing lipoproteins (Blps) for export. Microsomal triglyceride transfer protein (MTP), encoded by the MTTP gene, is essential for Blp assembly. Herein we test the hypothesis that MTP expression in the RPE is essential to maintain lipid balance and retinal function using the newly generated RPEΔMttp mouse model. Using non-invasive ocular imaging, electroretinography, and histochemical and biochemical analyses we show that genetic deletion of Mttp from the RPE results in intracellular lipid accumulation, increased photoreceptor -associated cholesterol deposits and photoreceptor cell death, and loss of rod but not cone function. RPE-specific ablation of Mttp had no significant effect on plasma lipids and lipoproteins. While, apoB was decreased in the RPE, ocular retinoid concentrations remained unchanged. Thus suggesting that RPE MTP is critical for Blp synthesis and assembly but not directly involved in ocular retinoid and plasma lipoprotein metabolism. These studies demonstrate that RPE-specific MTP expression is necessary to establish and maintain retinal lipid homeostasis and visual function.
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Oxidative stress has been implicated in the pathogenesis of age-related macular degeneration, the leading cause of blindness in older adults, with retinal pigment epithelium (RPE) cells playing a key role. To better understand the cytotoxic mechanisms underlying oxidative stress, we used cell culture and mouse models of iron overload, as iron can catalyze reactive oxygen species formation in the RPE. Iron-loading of cultured induced pluripotent stem cell-derived RPE cells increased lysosomal abundance, impaired proteolysis and reduced the activity of a subset of lysosomal enzymes, including lysosomal acid lipase (LIPA) and acid sphingomyelinase (SMPD1). In a liver-specific Hepc (Hamp) knockout murine model of systemic iron overload, RPE cells accumulated lipid peroxidation adducts and lysosomes, developed progressive hypertrophy and underwent cell death. Proteomic and lipidomic analyses revealed accumulation of lysosomal proteins, ceramide biosynthetic enzymes and ceramides. The proteolytic enzyme cathepsin D (CTSD) had impaired maturation. A large proportion of lysosomes were galectin-3 (Lgals3) positive, suggesting cytotoxic lysosomal membrane permeabilization. Collectively, these results demonstrate that iron overload induces lysosomal accumulation and impairs lysosomal function, likely due to iron-induced lipid peroxides that can inhibit lysosomal enzymes.
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Sobrecarga de Ferro , Proteômica , Camundongos , Animais , Estresse Oxidativo , Lisossomos/metabolismo , Ferro/metabolismo , Sobrecarga de Ferro/metabolismo , Sobrecarga de Ferro/patologia , Células Epiteliais/metabolismo , Pigmentos da Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismoRESUMO
MicroRNAs (miRNAs) are 21-24 nt small RNAs (sRNAs) that negatively regulate protein-coding genes and/or trigger phased small-interfering RNA (phasiRNA) production. Two thousand nine hundred miRNA families, of which â¼40 are deeply conserved, have been identified in â¼80 different plant species genomes. miRNA functions in response to abiotic stresses is less understood than their roles in development. Only seven peanut MIRNA families are documented in miRBase, yet a reference genome assembly is now published and over 480 plant-like MIRNA loci were predicted in the diploid peanut progenitor Arachis duranensis genome. We explored by computational analysis of a leaf sRNA library and publicly available sRNA, degradome, and transcriptome datasets the miRNA and phasiRNA space associated with drought and heat stresses in peanut. We characterized 33 novel candidate and 33 ancient conserved families of MIRNAs and present degradome evidence for their cleavage activities on mRNA targets, including several noncanonical targets and novel phasiRNA-producing noncoding and mRNA loci with validated novel targets such as miR1509 targeting serine/threonine-protein phosphatase7 and miRc20 and ahy-miR3514 targeting penta-tricopeptide repeats (PPRs), in contradistinction to other claims of miR1509/173/7122 superfamily miRNAs indirectly targeting PPRs via TAS-like noncoding RNA loci. We characterized the inverse correlations of significantly differentially expressed drought- and heat-regulated miRNAs, assayed by sRNA blots or transcriptome datasets, with target mRNA expressions in the same datasets. Meta-analysis of an expression atlas and over representation of miRNA target genes in co-expression networks suggest that miRNAs have functions in unique aspects of peanut gynophore development. Genome-wide MIRNA annotation of the published allopolyploid peanut genome can facilitate molecular breeding of value-added traits.
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MicroRNAs , MicroRNAs/genética , Arachis/genética , Secas , Resposta ao Choque Térmico , RNA Mensageiro/metabolismoRESUMO
LC3b (Map1lc3b) plays an essential role in canonical autophagy and is one of several components of the autophagy machinery that mediates non-canonical autophagic functions. Phagosomes are often associated with lipidated LC3b to promote phagosome maturation in a process called LC3-associated phagocytosis (LAP). Specialized phagocytes, such as mammary epithelial cells, retinal pigment epithelial (RPE) cells, and sertoli cells, utilize LAP for optimal degradation of phagocytosed material, including debris. In the visual system, LAP is critical to maintain retinal function, lipid homeostasis, and neuroprotection. In a mouse model of retinal lipid steatosis-mice lacking LC3b (LC3b-/-), we observed increased lipid deposition, metabolic dysregulation, and enhanced inflammation. Herein, we present a non-biased approach to determine if loss of LAP mediated processes modulate the expression of various genes related to metabolic homeostasis, lipid handling, and inflammation. A comparison of the RPE transcriptome of WT and LC3b-/- mice revealed 1533 DEGs, with ~73% upregulated and 27% downregulated. Enriched gene ontology (GO) terms included inflammatory response (upregulated DEGs), fatty acid metabolism, and vascular transport (downregulated DEGs). Gene set enrichment analysis (GSEA) identified 34 pathways; 28 were upregulated (dominated by inflammation/related pathways) and 6 were downregulated (dominated by metabolic pathways). Analysis of additional gene families identified significant differences for genes in the solute carrier family, RPE signature genes, and genes with a potential role in age-related macular degeneration. These data indicate that loss of LC3b induces robust changes in the RPE transcriptome contributing to lipid dysregulation and metabolic imbalance, RPE atrophy, inflammation, and disease pathophysiology.
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Proteínas Associadas aos Microtúbulos , Transcriptoma , Animais , Masculino , Camundongos , Autofagia/genética , Inflamação/genética , Inflamação/metabolismo , Lipídeos , Proteínas Associadas aos Microtúbulos/metabolismo , Fagocitose/genética , Epitélio Pigmentado da Retina/metabolismoRESUMO
LC3b ( Map1lc3b ) plays an essential role in canonical autophagy and is one of several components of the autophagy machinery that mediates non-canonical autophagic functions. Phagosomes are often associated with lipidated LC3b, to pro-mote phagosome maturation in a process called LC3-associated phagocytosis (LAP). Specialized phagocytes such as mammary epithelial cells, retinal pigment epithelial (RPE) cells, and sertoli cells utilize LAP for optimal degradation of phagocytosed material, including debris. In the visual system, LAP is critical to maintain retinal function, lipid homeostasis and neuroprotection. In a mouse model of retinal lipid steatosis - mice lacking LC3b ( LC3b -/- ), we observed increased lipid deposition, metabolic dysregulation and enhanced inflammation. Herein we present a non-biased approach to determine if loss of LAP mediated processes modulate the expression of various genes related to metabolic homeostasis, lipid handling, and inflammation. A comparison of the RPE transcriptome of WT and LC3b -/- mice revealed 1533 DEGs, with ~73% upregulated and 27% down-regulated. Enriched gene ontology (GO) terms included inflammatory response (upregulated DEGs), fatty acid metabolism and vascular transport (downregulated DEGs). Gene set enrichment analysis (GSEA) identified 34 pathways; 28 were upregulated (dominated by inflammation/related pathways) and 6 were downregulated (dominated by metabolic pathways). Analysis of additional gene families identified significant differences for genes in the solute carrier family, RPE signature genes, and genes with potential role in age-related macular degeneration. These data indicate that loss of LC3b induces robust changes in the RPE transcriptome contributing to lipid dysregulation and metabolic imbalance, RPE atrophy, inflammation, and disease pathophysiology.
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Visual function depends on the intimate structural, functional and metabolic interactions between the retinal pigment epithelium (RPE) and the neural retina. The daily phagocytosis of the photoreceptor outer segment tips by the overlaying RPE provides essential nutrients for the RPE itself and photoreceptors through intricate metabolic synergy. Age-related retinal changes are often characterized by metabolic dysregulation contributing to increased lipid accumulation and peroxidation as well as the release of proinflammatory cytokines. LGM2605 is a synthetic lignan secoisolariciresinol diglucoside (SDG) with free radical scavenging, antioxidant and anti-inflammatory properties demonstrated in diverse in vitro and in vivo inflammatory disease models. In these studies, we tested the hypothesis that LGM2605 may be an attractive small-scale therapeutic that protects RPE against inflammation and restores its metabolic capacity under lipid overload. Using an in vitro model in which loss of the autophagy protein, LC3B, results in defective phagosome degradation and metabolic dysregulation, we show that lipid overload results in increased gasdermin cleavage, IL-1 ß release, lipid accumulation and decreased oxidative capacity. The addition of LGM2605 resulted in enhanced mitochondrial capacity, decreased lipid accumulation and amelioration of IL-1 ß release in a model of defective lipid homeostasis. Collectively, these studies suggest that lipid overload decreases mitochondrial function and increases the inflammatory response, with LGM2605 acting as a protective agent.
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Lignanas/metabolismo , Metabolismo dos Lipídeos , Estresse Oxidativo/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Pigmentos da Retina/metabolismo , Antioxidantes/metabolismo , Autofagia , Butileno Glicóis/farmacologia , Linhagem Celular , Citocinas , Expressão Gênica , Glucosídeos/farmacologia , Humanos , Inflamação/metabolismo , Lignanas/química , Lipídeos , Mitocôndrias/metabolismo , Oxirredução , Fagocitose , Fagossomos/metabolismo , Pigmentos da Retina/genéticaRESUMO
The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is a heterotrimeric AB2 toxin capable of inducing cell cycle arrest and apoptosis in lymphocytes and other cell types. Recently, we have demonstrated that human macrophages are resistant to Cdt-induced apoptosis but are susceptible to toxin-induced pro-inflammatory cytokine response involving activation of the NLRP3 inflammasome. Exposure to Cdt results in binding to the cell surface followed by internalization and translocation of the active subunit, CdtB, to intracellular compartments. Internalization involves hijacking of retrograde pathways; treatment of cells with Retro-2 leads to a decrease in CdtB-Golgi association. These events are dependent upon toxin binding to cholesterol in the context of lipid rich membrane microdomains often referred to as lipid rafts. We now demonstrate that within 1 h of exposure of macrophages to Cdt, CdtB is internalized and found primarily within lipid rafts; concurrently, cellugyrin (synaptogyrin-2) also translocates into lipid rafts. Further analysis by immunoprecipitation indicates that CdtB associates with complexes containing both cellugyrin and Derlin-2. Moreover, a human macrophage cell line deficient in cellugyrin expression (THP-1Cg-) challenged with Cdt failed to internalize CdtB and was resistant to the Cdt-induced pro-inflammatory response. We propose that lipid rafts along with cellugyrin play a critical role in the internalization and translocation of CdtB to critical intracellular target sites in human macrophages. These studies provide the first evidence that cellugyrin is expressed in human macrophages and plays a critical role in Cdt toxicity of these cells.
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Toxinas Bacterianas/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Subunidades Proteicas/imunologia , Sinaptogirinas/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Citocinas/metabolismo , Humanos , Imunoprecipitação , Espaço Intracelular/metabolismo , Subunidades Proteicas/metabolismo , Transporte Proteico , Células THP-1RESUMO
Leukotoxin (LtxA), from oral pathogen Aggregatibacter actinomycetemcomitans, is a secreted membrane-damaging protein. LtxA is internalized by ß2 integrin LFA-1 (CD11a/CD18)-expressing leukocytes and ultimately causes cell death; however, toxin localization in the host cell is poorly understood and these studies fill this void. We investigated LtxA trafficking using multi-fluor confocal imaging, flow cytometry and Rab5a knockdown in human T lymphocyte Jurkat cells. Planar lipid bilayers were used to characterize LtxA pore-forming activity at different pHs. Our results demonstrate that the LtxA/LFA-1 complex gains access to the cytosol of Jurkat cells without evidence of plasma membrane damage, utilizing dynamin-dependent and presumably clathrin-independent mechanisms. Upon internalization, LtxA follows the LFA-1 endocytic trafficking pathways, as identified by co-localization experiments with endosomal and lysosomal markers (Rab5, Rab11A, Rab7, and Lamp1) and CD11a. Knockdown of Rab5a resulted in the loss of susceptibility of Jurkat cells to LtxA cytotoxicity, suggesting that late events of LtxA endocytic trafficking are required for toxicity. Toxin trafficking via the degradative endocytic pathway may culminate in the delivery of the protein to lysosomes or its accumulation in Rab11A-dependent recycling endosomes. The ability of LtxA to form pores at acidic pH may result in permeabilization of the endosomal and lysosomal membranes.
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The retinal pigment epithelium (RPE) supports the outer retina through essential roles in the retinoid cycle, nutrient supply, ion exchange, and waste removal. Each day the RPE removes the oldest ~10% of photoreceptor outer segment (OS) disk membranes through phagocytic uptake, which peaks following light onset. Impaired degradation of phagocytosed OS material by the RPE can lead to toxic accumulation of lipids, oxidative tissue damage, inflammation, and cell death. OSs are rich in very long chain fatty acids, which are preferentially catabolized in peroxisomes. Despite the importance of lipid degradation in RPE function, the regulation of peroxisome number and activity relative to diurnal OS ingestion is relatively unexplored. Using immunohistochemistry, immunoblot analysis, and catalase activity assays, we investigated peroxisome abundance and activity at 6 AM, 7 AM (light onset), 8 AM, and 3 PM, in wild-type (WT) mice and mice lacking microtubule-associated protein 1 light chain 3B (Lc3b), which have impaired phagosome degradation. We found that catalase activity, but not the amount of catalase protein, is 50% higher in the morning compared with 3 PM, in RPE of WT, but not Lc3b-/-, mice. Surprisingly, we found that peroxisome abundance was stable during the day in RPE of WT mice; however, numbers were elevated overall in Lc3b-/- mice, implicating LC3B in autophagic organelle turnover in RPE. Our data suggest that RPE peroxisome function is regulated in coordination with phagocytosis, possibly through direct enzyme regulation, and may serve to prepare RPE peroxisomes for daily surges in ingested lipid-rich OS.
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Autofagia/efeitos da radiação , Ritmo Circadiano/genética , Proteínas Associadas aos Microtúbulos/genética , Peroxissomos/efeitos da radiação , Fagocitose/efeitos da radiação , Epitélio Pigmentado da Retina/efeitos da radiação , Animais , Autofagia/genética , Catalase/genética , Catalase/metabolismo , Ácidos Graxos/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Luz , Transdução de Sinal Luminoso , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Oxirredução , Peroxissomos/metabolismo , Fagocitose/genética , Epitélio Pigmentado da Retina/metabolismoRESUMO
OBJECTIVES: Ameloblastoma is an aggressive odontogenic jaw neoplasm. Its unlimited growth confers high potential for malignant transformation and recurrence. It is unclear why ameloblastoma is highly recurrent despite surgical resection with a wide margin of normal tissue. While canonical autophagy can be used to degrade and eliminate damaged cellular components, it is also a protective mechanism that provides energy and vital metabolites for cell survival. We used ameloblastoma-derived cells to test the hypothesis that autophagic processes play a role in survival and reactivation of ameloblastoma. METHODS: Primary epithelial (EP-AMCs) and mesenchymal (MS-AMCs) ameloblastoma-derived cells were established from tissue samples of solid multicystic ameloblastoma. Clonogenic capacity and basal autophagic capacity were assessed in ameloblastoma-derived cells relative to human odontoma-derived cells (HODCs) and maxilla-mesenchymal stem cells (MX-MSCs). Ability of ameloblastoma-derived cells to survive and form new ameloblastoma was assessed in mouse tumor xenografts. RESULTS: EP-AMCs were highly clonogenic (p < 0.0001) and demonstrated enhanced basal levels of autophagic proteins microtubule-associated protein 1-light chain 3 (LC3) (p < 0.01), p62 (Sequestosome 1, SQSTM1) (p < 0.01), and the LC3-adapter, melanoregulin (MREG) (p < 0.05) relative to controls. EP-AMCs xenografts regenerated solid ameloblastoma-like tumor with histological features of columnar ameloblast-like cells, loose stellate reticulum-like cells and regions of cystic degeneration characteristic of follicular variant of solid multicystic ameloblastoma. The xenografts also displayed stromal epithelial invaginations strongly reactive to LC3 and p62 suggestive of epithelial-mesenchymal transition and neoplastic odontogenic epithelium. CONCLUSIONS: EP-AMCs exhibit altered autophagic processes that can support survival and recurrence of post-surgical ameloblastoma cells.
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Ameloblastoma , Autofagia/fisiologia , Sobrevivência Celular , Tumores Odontogênicos , Proteínas Adaptadoras de Transporte Vesicular , Ameloblastoma/patologia , Ameloblastos/metabolismo , Ameloblastos/patologia , Animais , Proteínas de Transporte/metabolismo , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal , Epitélio/metabolismo , Feminino , Xenoenxertos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Células-Tronco Mesenquimais , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Recidiva Local de Neoplasia , Proteína Sequestossoma-1/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Repeats-in-toxin leukotoxin (LtxA) produced by the oral bacterium Aggregatibacter actinomycetemcomitans kills human leukocytes in a lymphocyte function-associated antigen 1 (LFA-1, integrin αL /ß2 )-dependent manner, although the mechanism for this interaction has not been identified. The LtxA internalisation by LFA-1-expressing cells was explored with florescence resonance energy transfer (FRET) microscopy using a cell line that expresses LFA-1 with a cyan fluorescent protein-tagged cytosolic αL domain and a yellow fluorescent protein-tagged ß2 domain. Phorbol 12-myristate 13-acetate activation of LFA-1 caused transient cytosolic domain separation. However, addition of LtxA resulted in an increase in FRET, indicating that LtxA brings the cytosolic domains closer together, compared with the inactive state. Unlike activation, this effect was not transient, lasting more than 30 min. Equilibrium constants of LtxA binding to the cytoplasmic domains of both αL and ß2 were determined using surface plasmon resonance. LtxA has a strong affinity for the cytosolic domains of both the αL and ß2 subunits (Kd = 15 and 4.2 nM, respectively) and a significantly lower affinity for the cytoplasmic domains of other integrin αM , αX , and ß3 subunits (Kd = 400, 180, and 230 nM, respectively), used as controls. Peptide fragments of αL and ß2 show that LtxA binds membrane-proximal domain of αL and intermediate domain of ß2 .
Assuntos
Aggregatibacter actinomycetemcomitans/imunologia , Exotoxinas/metabolismo , Interações Hospedeiro-Patógeno , Imunossupressores/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Células Jurkat , Microscopia de Fluorescência , Ligação ProteicaRESUMO
Like other neurons, retinal cells utilize autophagic pathways to maintain cell homeostasis. The mammalian retina relies on heterophagy and selective autophagy to efficiently degrade and metabolize ingested lipids with disruption in autophagy associated degradation contributing to age related retinal disorders. The retinal pigment epithelium (RPE) supports photoreceptor cell renewal by daily phagocytosis of shed photoreceptor outer segments (OS). The daily ingestion of these lipid-rich OS imposes a constant degradative burden on these terminally differentiated cells. These cells rely on Microtubule-Associated Protein 1 Light Chain 3 (LC3) family of proteins for phagocytic clearance of the ingested OS. The LC3 family comprises of three highly homologous members, MAP1LC3A (LC3A), MAP1LC3B (LC3B), and MAP1LC3C (LC3C). The purpose of this study was to determine whether the LC3B isoform plays a specific role in maintaining RPE lipid homeostasis. We examined the RPE and retina of the LC3B -/- mouse as a function of age using in vivo ocular imaging and electroretinography coupled with ex vivo, lipidomic analyses of lipid mediators, assessment of bisretinoids as well as imaging of lipid aggregates. Deletion of LC3B resulted in defects within the RPE including increased phagosome accumulation, decreased fatty acid oxidation and a subsequent increase in RPE and sub-RPE lipid deposits. Age-dependent RPE changes included elevated levels of oxidized cholesterol, deposition of 4-HNE lipid peroxidation products, bisretinoid lipofuscin accumulation, and subretinal migration of microglia, collectively likely contributing to loss of retinal function. These observations are consistent with a critical role for LC3B-dependent processes in the maintenance of normal lipid homeostasis in the aging RPE, and suggest that LC3 isoform specific disruption in autophagic processes contribute to AMD-like pathogenesis.
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Canine bestrophinopathy (cBest) is an important translational model for BEST1-associated maculopathies in man that recapitulates the broad spectrum of clinical and molecular disease aspects observed in patients. Both human and canine bestrophinopathies are characterized by focal to multifocal separations of the retina from the RPE. The lesions can be macular or extramacular, and the specific pathomechanism leading to formation of these lesions remains unclear. We used the naturally occurring canine BEST1 model to examine factors that underlie formation of vitelliform lesions and addressed the susceptibility of the macula to its primary detachment in BEST1-linked maculopathies.
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Bestrofinas/deficiência , Doenças do Cão/patologia , Modelos Animais , Epitélio Pigmentado da Retina/patologia , Distrofia Macular Viteliforme/veterinária , Animais , Bestrofinas/genética , Bestrofinas/fisiologia , Proteínas do Citoesqueleto/metabolismo , Doenças do Cão/genética , Doenças do Cão/metabolismo , Cães , Matriz Extracelular/patologia , Proteínas do Olho/metabolismo , Genes Recessivos , Humanos , Microvilosidades/patologia , Transportadores de Ácidos Monocarboxílicos/metabolismo , Células Fotorreceptoras Retinianas Cones/patologia , Descolamento Retiniano/etiologia , Epitélio Pigmentado da Retina/metabolismo , Especificidade da Espécie , Simportadores/metabolismo , Distrofia Macular Viteliforme/genética , Distrofia Macular Viteliforme/metabolismo , Distrofia Macular Viteliforme/patologiaRESUMO
Microtubule-associated protein 1 light chain 3 (MAP1LC3), a human homologue of yeast Atg8, is an essential component of autophagy. LC3 plays a critical role in hybrid degradation pathways in which some but not all components of autophagy are coupled with phagocytosis in a process known as LC3-associated phagocytosis (LAP). LC3 exists as three highly homologous isoforms in human (LC3A, LC3B, and LC3C) with two of these (LC3A and LC3B) in mouse. LC3B predominated in both fetal and adult human retinal pigment epithelium (RPE) relative to LC3A and LC3C, while in mouse RPE and neural retina, LC3A and LC3B were expressed at approximately equivalent levels. In situ hybridization studies localized LC3A and LC3B transcripts in the retina and RPE. LC3B protein was detected in C57Bl6/J RPE and retinal lysates and was absent in the LC3BKO mouse.
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Proteínas do Olho/análise , Proteínas Associadas aos Microtúbulos/análise , Retina/química , Processamento Alternativo , Animais , Autofagia , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Immunoblotting , Hibridização In Situ , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Isoformas de Proteínas/análise , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Retina/ultraestrutura , Epitélio Pigmentado da Retina/química , Epitélio Pigmentado da Retina/ultraestruturaRESUMO
MAIN CONCLUSION: Based on yeast one-hybrid assays, we show that the presence of C-terminal AHA motifs is not a prerequisite for transactivation potential in rice heat shock factors. Transcriptional activation or transactivation (TA) of heat stress responsive genes takes place by binding of heat shock factors (Hsfs) to heat shock elements. Analysis of TA potential of thirteen rice (Oryza sativa L.) Hsfs (OsHsfs) carried out in this study by yeast one-hybrid assay showed that OsHsfsA3 possesses strong TA potential while OsHsfs A1a, A2a, A2b, A4a, A4d, A5, A7b, B1, B2a, B2b, B2c and B4d lack TA potential. From a near complete picture of TA potential of the OsHsf family (comprising of 25 members) emerging from this study and an earlier report from our group (Mittal et al. in FEBS J 278(17):3076-3085, 2011), it is concluded that (1) overall, six OsHsfs, namely A3, A6a, A6b, A8, C1a and C1b possess TA potential; (2) four class A OsHsfs, namely A3, A6a, A6b and A8 have TA potential out of which A6a and A6b contain AHA motifs while A3 and A8 lack AHA motifs; (3) nine class A OsHsfs, namely A1a, A2a, A2b, A2e, A4a, A4d, A5, A7a and A7b containing AHA motif(s) lack TA function in the yeast assay system; (4) all class B OsHsfs lack AHA motifs and TA potential (B4a not analyzed) and (5) though all class C OsHsf members lack AHA motifs, two members C1a and C1b possess TA function, while one member C2a lacks TA potential (C2b not analyzed). Thus, the presence or absence of AHA motif is possibly not the only factor determining TA potential of OsHsfs. Our findings will help to identify the transcriptional activators of rice heat shock response.
Assuntos
Regulação da Expressão Gênica de Plantas , Fatores de Transcrição de Choque Térmico/metabolismo , Oryza/genética , Ativação Transcricional , Genes Reporter , Fatores de Transcrição de Choque Térmico/genética , Resposta ao Choque Térmico , Oryza/fisiologia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
The Aggregatibacter actinomycetemcomitans cytolethal distending toxin (Cdt) is a heterotrimeric AB2 toxin capable of inducing lymphocytes, and other cell types, to undergo cell cycle arrest and apoptosis. Exposure to Cdt results in binding to the cell surface followed by internalization and translocation of the active subunit, CdtB, to intracellular compartments. These events are dependent upon toxin binding to cholesterol in the context of lipid rich membrane microdomains often referred to as lipid rafts. We now demonstrate that, in addition to binding to the plasma membrane of lymphocytes, another early and critical event initiated by Cdt is the translocation of the host cell protein, cellugyrin (synaptogyrin-2) to the same cholesterol-rich microdomains. Furthermore, we demonstrate that cellugyrin is an intracellular binding partner for CdtB as demonstrated by immunoprecipitation. Using CRISPR/cas9 gene editing we established a Jurkat cell line deficient in cellugyrin expression (JurkatCg-); these cells were capable of binding Cdt, but unable to internalize CdtB. Furthermore, JurkatCg- cells were not susceptible to Cdt-induced toxicity; these cells failed to exhibit blockade of the PI-3K signaling pathway, cell cycle arrest or cell death. We propose that cellugyrin plays a critical role in the internalization and translocation of CdtB to critical intracellular target sites. These studies provide critical new insight into the mechanism by which Cdt, and in particular, CdtB is able to induce toxicity.
Assuntos
Aggregatibacter actinomycetemcomitans/metabolismo , Toxinas Bacterianas/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Vesículas Sinápticas/metabolismo , Sinaptogirinas/metabolismo , Aggregatibacter actinomycetemcomitans/patogenicidade , Apoptose/efeitos dos fármacos , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Proteínas de Transporte , Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Colesterol/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Citoplasma/metabolismo , Edição de Genes , Células HeLa , Humanos , Células Jurkat , Linfócitos/metabolismo , Linfócitos/microbiologia , Microdomínios da Membrana/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Porphyromonas gingivalis is a keystone pathogen that contributes to periodontal pathogenesis by disrupting host-microbe homeostasis and promoting dysbiosis. The virulence of P. gingivalis likely reflects an alteration in the lipid A composition of its lipopolysaccharide (LPS) from the penta-acylated (PgLPS1690) to the tetra-acylated (PgLPS1435/1449) form. Mast cells play an important role in periodontitis, but the mechanisms of their activation and regulation remain unknown. The expression of epithelium- and neutrophil-derived host defense peptides (HDPs) (LL-37 and human ß-defensin-3), which activate mast cells via Mas-related G protein-coupled receptor X2 (MRGPRX2), is increased in periodontitis. We found that MRGPRX2-expressing mast cells are present in normal gingiva and that their numbers are elevated in patients with chronic periodontitis. Furthermore, HDPs stimulated degranulation in a human mast cell line (LAD2) and in RBL-2H3 cells stably expressing MRGPRX2 (RBL-MRGPRX2). PgLPS1690 caused substantial inhibition of HDP-induced mast cell degranulation, but PgLPS1435/1449 had no effect. A fluorescently labeled HDP (FAM-LL-37) bound to RBL-MRGPRX2 cells, and PgLPS1690 inhibited this binding, but PgLPS1435/1449 had no effect. These findings suggest that low-level inflammation induced by HDP/MRGPRX2-mediated mast cell degranulation contributes to gingival homeostasis but that sustained inflammation due to elevated levels of both HDPs and MRGPRX2-expressing mast cells promotes periodontal disease. Furthermore, differential regulation of HDP-induced mast cell degranulation by PgLPS1690 and PgLPS1435/1449 may contribute to the modulation of disease progression.