RESUMO
Seminal plasma contains a heterogeneous population of extracellular vesicles (sEVs) that remains poorly characterized. This study aimed to characterize the lipidomic profile of two subsets of differently sized sEVs, small (S-) and large (L-), isolated from porcine seminal plasma by size-exclusion chromatography and characterized by an orthogonal approach. High-performance liquid chromatography-high-resolution mass spectrometry was used for lipidomic analysis. A total of 157 lipid species from 14 lipid classes of 4 major categories (sphingolipids, glycerophospholipids, glycerolipids, and sterols) were identified. Qualitative differences were limited to two cholesteryl ester species present only in S-sEVs. L-sEVs had higher levels of all quantified lipid classes due to their larger membrane surface area. The distribution pattern was different, especially for sphingomyelins (more in S-sEVs) and ceramides (more in L-sEVs). In conclusion, this study reveals differences in the lipidomic profile of two subsets of porcine sEVs, suggesting that they differ in biogenesis and functionality.
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Vesículas Extracelulares , Lipidômica , Lipídeos , Sêmen , Animais , Vesículas Extracelulares/metabolismo , Suínos , Sêmen/metabolismo , Sêmen/química , Masculino , Lipídeos/análise , Lipídeos/química , Lipidômica/métodos , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas , Cromatografia em GelRESUMO
A non-pathogenic Mycoplasma pneumoniae-based chassis is leading the development of live biotherapeutic products (LBPs) for respiratory diseases. However, reports connecting Guillain-Barré syndrome (GBS) cases to prior M. pneumoniae infections represent a concern for exploiting such a chassis. Galactolipids, especially galactocerebroside (GalCer), are considered the most likely M. pneumoniae antigens triggering autoimmune responses associated with GBS development. In this work, we generated different strains lacking genes involved in galactolipids biosynthesis. Glycolipid profiling of the strains demonstrated that some mutants show a complete lack of galactolipids. Cross-reactivity assays with sera from GBS patients with prior M. pneumoniae infection showed that certain engineered strains exhibit reduced antibody recognition. However, correlation analyses of these results with the glycolipid profile of the engineered strains suggest that other factors different from GalCer contribute to sera recognition, including total ceramide levels, dihexosylceramide (DHCer), and diglycosyldiacylglycerol (DGDAG). Finally, we discuss the best candidate strains as potential GBS-free Mycoplasma chassis.
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Glicolipídeos , Síndrome de Guillain-Barré , Mycoplasma pneumoniae , Síndrome de Guillain-Barré/microbiologia , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/imunologia , Humanos , Glicolipídeos/metabolismo , Galactosilceramidas , Reações Cruzadas , Pneumonia por Mycoplasma/microbiologia , Pneumonia por Mycoplasma/imunologia , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologiaRESUMO
Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by impaired motor coordination due to neurological defects and cerebellar dysfunction caused by the accumulation of cholesterol in endolysosomes. Besides the increase in lysosomal cholesterol, mitochondria are also enriched in cholesterol, which leads to decreased membrane fluidity, impaired mitochondrial function and loss of GSH, and has been shown to contribute to the progression of NPC disease. S-Adenosyl-l-methionine (SAM) regulates membrane physical properties through the generation of phosphatidylcholine (PC) from phosphatidylethanolamine (PE) methylation and functions as a GSH precursor by providing cysteine in the transsulfuration pathway. However, the role of SAM in NPC disease has not been investigated. Here we report that Npc1-/- mice exhibit decreased brain SAM levels but unchanged S-adenosyl-l-homocysteine content and lower expression of Mat2a. Brain mitochondria from Npc1-/- mice display decreased mitochondrial GSH levels and liquid chromatography-high resolution mass spectrometry analysis reveal a lower PC/PE ratio in mitochondria, contributing to increased mitochondrial membrane order. In vivo treatment of Npc1-/- mice with SAM restores SAM levels in mitochondria, resulting in increased PC/PE ratio, mitochondrial membrane fluidity and subsequent replenishment of mitochondrial GSH levels. In vivo SAM treatment improves the decline of locomotor activity, increases Purkinje cell survival in the cerebellum and extends the average and maximal life spam of Npc1-/- mice. These findings identify SAM as a potential therapeutic approach for the treatment of NPC disease.
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Encéfalo , Glutationa , Fluidez de Membrana , Membranas Mitocondriais , Doença de Niemann-Pick Tipo C , S-Adenosilmetionina , Animais , Camundongos , S-Adenosilmetionina/metabolismo , Membranas Mitocondriais/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/genética , Glutationa/metabolismo , Encéfalo/metabolismo , Mitocôndrias/metabolismo , Proteína C1 de Niemann-Pick , Modelos Animais de Doenças , Camundongos Knockout , Fosfatidilcolinas/metabolismoRESUMO
Lipid amidases of therapeutic relevance include acid ceramidase (AC), N-acylethanolamine-hydrolyzing acid amidase, and fatty acid amide hydrolase (FAAH). Although fluorogenic substrates have been developed for the three enzymes and high-throughput methods for screening have been reported, a platform for the specific detection of these enzyme activities in intact cells is lacking. In this article, we report on the coumarinic 1-deoxydihydroceramide RBM1-151, a 1-deoxy derivative and vinilog of RBM14-C12, as a novel substrate of amidases. This compound is hydrolyzed by AC (appKm = 7.0 µM; appVmax = 99.3 nM/min), N-acylethanolamine-hydrolyzing acid amidase (appKm = 0.73 µM; appVmax = 0.24 nM/min), and FAAH (appKm = 3.6 µM; appVmax = 7.6 nM/min) but not by other ceramidases. We provide proof of concept that the use of RBM1-151 in combination with reported irreversible inhibitors of AC and FAAH allows the determination in parallel of the three amidase activities in single experiments in intact cells.
Assuntos
Amidoidrolases , Corantes Fluorescentes , Etanolaminas/química , LipídeosRESUMO
Dihydroceramide desaturase 1 (Des1) catalyzes the formation of a CC double bond in dihydroceramide to furnish ceramide. Inhibition of Des1 is related to cell cycle arrest and programmed cell death. The lack of the Des1 crystalline structure, as well as that of a close homologue, hampers the detailed understanding of its inhibition mechanism and difficults the design of new inhibitors, thus making Des1 a strategic target. Based on previous structure-activity studies, different ceramides containing rigid scaffolds were designed. The synthesis and evaluation of these compounds as Des1 inhibitors allowed the identification of PR280 as a better Des 1 inhibitor in vitro (IC50 = 700 nM) than GT11 and XM462, the current reference inhibitors. This cyclopropenone ceramide was obtained in a 6-step synthesis with a 24 % overall yield. The highly confident 3D structure of Des1, recently predicted by AlphaFold2, served as the basis for conducting docking studies of known Des1 inhibitors and the ceramide derivatives synthesized by us in this study. For this purpose, a complete holoprotein structure was previously constructed. This study has allowed a better knowledge of key ligand-enzyme interactions for Des1 inhibitory activity. Furthermore, it sheds some light on the inhibition mechanism of GT11.
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Ceramidas , Oxirredutases , Ceramidas/farmacologia , Ceramidas/química , Oxirredutases/metabolismo , Ciclopropanos/farmacologiaRESUMO
INTRODUCTION: This study has investigated the temporal disruptive effects of tributyltin (TBT) on lipid homeostasis in Daphnia magna. To achieve this, the study used Liquid Chromatography-Mass Spectrometry (LC-MS) analysis to analyze biological samples of Daphnia magna treated with TBT over time. The resulting data sets were multivariate and three-way, and were modeled using bilinear and trilinear non-negative factor decomposition chemometric methods. These methods allowed for the identification of specific patterns in the data and provided insight into the effects of TBT on lipid homeostasis in Daphnia magna. OBJECTIVES: Investigation of how are the changes in the lipid concentrations of Daphnia magna pools when they were exposed with TBT and over time using non-targeted LC-MS and advanced chemometric analysis. METHODS: The simultaneous analysis of LC-MS data sets of Daphnia magna samples under different experimental conditions (TBT dose and time) were analyzed using the ROIMCR method, which allows the resolution of the elution and mass spectra profiles of a large number of endogenous lipids. Changes obtained in the peak areas of the elution profiles of these lipids caused by the dose of TBT treatment and the time after its exposure are analyzed by principal component analysis, multivariate curve resolution-alternative least square, two-way ANOVA and ANOVA-simultaneous component analysis. RESULTS: 87 lipids were identified. Some of these lipids are proposed as Daphnia magna lipidomic biomarkers of the effects produced by the two considered factors (time and dose) and by their interaction. A reproducible multiplicative effect between these two factors is confirmed and the optimal approach to model this dataset resulted to be the application of the trilinear factor decomposition model. CONCLUSION: The proposed non-targeted LC-MS lipidomics approach resulted to be a powerful tool to investigate the effects of the two factors on the Daphnia magna lipidome using chemometric methods based on bilinear and trilinear factor decomposition models, according to the type of interaction between the design factors.
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Daphnia , Lipidômica , Animais , Cromatografia Líquida , Espectrometria de Massas em Tandem , Metabolômica/métodos , Lipídeos/análiseRESUMO
The Rad50 interacting protein 1 (Rint1) is a key player in vesicular trafficking between the ER and Golgi apparatus. Biallelic variants in RINT1 cause infantile-onset episodic acute liver failure (ALF). Here, we describe 3 individuals from 2 unrelated families with novel biallelic RINT1 loss-of-function variants who presented with early onset spastic paraplegia, ataxia, optic nerve hypoplasia, and dysmorphic features, broadening the previously described phenotype. Our functional and lipidomic analyses provided evidence that pathogenic RINT1 variants induce defective lipid-droplet biogenesis and profound lipid abnormalities in fibroblasts and plasma that impact both neutral lipid and phospholipid metabolism, including decreased triglycerides and diglycerides, phosphatidylcholine/phosphatidylserine ratios, and inhibited Lands cycle. Further, RINT1 mutations induced intracellular ROS production and reduced ATP synthesis, affecting mitochondria with membrane depolarization, aberrant cristae ultrastructure, and increased fission. Altogether, our results highlighted the pivotal role of RINT1 in lipid metabolism and mitochondria function, with a profound effect in central nervous system development.
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Paraplegia Espástica Hereditária , Humanos , Paraplegia Espástica Hereditária/genética , Paraplegia Espástica Hereditária/metabolismo , Metabolismo dos Lipídeos , Mutação , Complexo de Golgi/metabolismo , Lipídeos , Fenótipo , Proteínas de Ciclo Celular/metabolismoRESUMO
West Nile virus (WNV) is a neurotropic flavivirus transmitted by the bites of infected mosquitoes. Severe forms of West Nile disease (WND) can curse with meningitis, encephalitis or acute flaccid paralysis. A better understanding of the physiopathology associated with disease progression is mandatory to find biomarkers and effective therapies. In this scenario, blood derivatives (plasma and serum) constitute the more commonly used biofluids due to its ease of collection and high value for diagnostic purposes. Therefore, the potential impact of this virus in the circulating lipidome was addressed combining the analysis of samples from experimentally infected mice and naturally WND patients. Our results unveil dynamic alterations in the lipidome that define specific metabolic fingerprints of different infection stages. Concomitant with neuroinvasion in mice, the lipid landscape was dominated by a metabolic reprograming that resulted in significant elevations of circulating sphingolipids (ceramides, dihydroceramides, and dihydrosphingomyelins), phosphatidylethanolamines and triacylglycerols. Remarkably, patients suffering from WND also displayed an elevation of ceramides, dihydroceramides, lactosylceramides, and monoacylglycerols in their sera. The dysregulation of sphingolipid metabolism by WNV may provide new therapeutic opportunities and supports the potential of certain lipids as novel peripheral biomarkers of WND progression.
Assuntos
Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Animais , Camundongos , Vírus do Nilo Ocidental/genética , Esfingolipídeos/metabolismo , Esfingolipídeos/uso terapêutico , Ceramidas/metabolismo , Ceramidas/uso terapêutico , Biomarcadores/metabolismoRESUMO
Acid sphingomyelinase deficiency (ASMD) is a lysosomal storage disorder caused by mutations in the gene-encoding acid sphingomyelinase (ASM). ASMD impacts peripheral organs in all patients, including the liver and spleen. The infantile and chronic neurovisceral forms of the disease also lead to neuroinflammation and neurodegeneration for which there is no effective treatment. Cellular accumulation of sphingomyelin (SM) is a pathological hallmark in all tissues. SM is the only sphingolipid comprised of a phosphocholine group linked to ceramide. Choline is an essential nutrient that must be obtained from the diet and its deficiency promotes fatty liver disease in a process dependent on ASM activity. We thus hypothesized that choline deprivation could reduce SM production and have beneficial effects in ASMD. Using acid sphingomyelinase knock-out (ASMko) mice, which mimic neurovisceral ASMD, we have assessed the safety of a choline-free diet and its effects on liver and brain pathological features such as altered sphingolipid and glycerophospholipid composition, inflammation and neurodegeneration. We found that the choline-free diet was safe in our experimental conditions and reduced activation of macrophages and microglia in the liver and brain, respectively. However, there was no significant impact on sphingolipid levels and neurodegeneration was not prevented, arguing against the potential of this nutritional strategy to assist in the management of neurovisceral ASMD patients.
Assuntos
Doença de Niemann-Pick Tipo A , Doenças de Niemann-Pick , Camundongos , Animais , Doença de Niemann-Pick Tipo A/genética , Esfingomielina Fosfodiesterase/genética , Colina , Esfingolipídeos , Esfingomielinas , Dieta , Camundongos Knockout , Modelos Animais de DoençasRESUMO
The SARS-CoV-2 pandemic made evident that there are only a few drugs against coronavirus. Here we aimed to identify a cost-effective antiviral with broad spectrum activity and high safety profile. Starting from a list of 116 drug candidates, we used molecular modelling tools to rank the 44 most promising inhibitors. Next, we tested their efficacy as antivirals against α and ß coronaviruses, such as the HCoV-229E and SARS-CoV-2 variants. Four drugs, OSW-1, U18666A, hydroxypropyl-ß-cyclodextrin (HßCD) and phytol, showed in vitro antiviral activity against HCoV-229E and SARS-CoV-2. The mechanism of action of these compounds was studied by transmission electron microscopy and by fusion assays measuring SARS-CoV-2 pseudoviral entry into target cells. Entry was inhibited by HßCD and U18666A, yet only HßCD inhibited SARS-CoV-2 replication in the pulmonary Calu-3 cells. Compared to the other cyclodextrins, ß-cyclodextrins were the most potent inhibitors, which interfered with viral fusion via cholesterol depletion. ß-cyclodextrins also prevented infection in a human nasal epithelium model ex vivo and had a prophylactic effect in the nasal epithelium of hamsters in vivo. All accumulated data point to ß-cyclodextrins as promising broad-spectrum antivirals against different SARS-CoV-2 variants and distant alphacoronaviruses. Given the wide use of ß-cyclodextrins for drug encapsulation and their high safety profile in humans, our results support their clinical testing as prophylactic antivirals.
Assuntos
COVID-19 , Fármacos Dermatológicos , beta-Ciclodextrinas , Humanos , SARS-CoV-2 , Antivirais/farmacologia , Antivirais/uso terapêutico , beta-Ciclodextrinas/farmacologia , beta-Ciclodextrinas/uso terapêuticoRESUMO
Obesity, which is a worldwide public health issue, is associated with chronic inflammation that contribute to long-term complications, including insulin resistance, type 2 diabetes and non-alcoholic fatty liver disease. We hypothesized that obesity may also influence the sensitivity to food contaminants, such as fumonisin B1 (FB1), a mycotoxin produced mainly by the Fusarium verticillioides. FB1, a common contaminant of corn, is the most abundant and best characterized member of the fumonisins family. We investigated whether diet-induced obesity could modulate the sensitivity to oral FB1 exposure, with emphasis on gut health and hepatotoxicity. Thus, metabolic effects of FB1 were assessed in obese and non-obese male C57BL/6J mice. Mice received a high-fat diet (HFD) or normal chow diet (CHOW) for 15 weeks. Then, during the last three weeks, mice were exposed to these diets in combination or not with FB1 (10 mg/kg body weight/day) through drinking water. As expected, HFD feeding induced significant body weight gain, increased fasting glycemia, and hepatic steatosis. Combined exposure to HFD and FB1 resulted in body weight loss and a decrease in fasting blood glucose level. This co-exposition also induces gut dysbiosis, an increase in plasma FB1 level, a decrease in liver weight and hepatic steatosis. Moreover, plasma transaminase levels were significantly increased and associated with liver inflammation in HFD/FB1-treated mice. Liver gene expression analysis revealed that the combined exposure to HFD and FB1 was associated with reduced expression of genes involved in lipogenesis and increased expression of immune response and cell cycle-associated genes. These results suggest that, in the context of obesity, FB1 exposure promotes gut dysbiosis and severe liver inflammation. To our knowledge, this study provides the first example of obesity-induced hepatitis in response to a food contaminant.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Diabetes Mellitus Tipo 2 , Fumonisinas , Camundongos , Masculino , Animais , Fumonisinas/toxicidade , Fumonisinas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Disbiose , Camundongos Endogâmicos C57BL , Fígado/metabolismo , Obesidade/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Inflamação/induzido quimicamenteRESUMO
Acid sphingomyelinase deficiency (ASMD) is a lysosomal storage disorder caused by mutations in the SMPD1 gene encoding for the acid sphingomyelinase (ASM). While intravenous infusion of recombinant ASM is an effective treatment for the peripheral disease, the neurological complications of ASMD remain unaddressed. It has been shown that aberrantly high level of total brain sphingomyelin (SM) is a key pathological event leading to neurodegeneration. Using mice lacking ASM (ASMko), which mimic the disease, we here demonstrate that among the SM species, SM16:0 shows the highest accumulation and toxicity in ASMko neurons. By targeting lysosomes, SM16:0 causes permeabilization and exocytosis of these organelles and induces oxidative stress and cell death. We also show that genetic silencing of Ceramide Synthase 5, which is involved in SM16:0 synthesis and overexpressed in the ASMko brain, prevents disease phenotypes in ASMko cultured neurons and mice. The levels of SM16:0 in plasma also show a strong correlation with those in brain that is higher than in liver, even at early stages of the disease. These results identify SM16:0 both as a novel therapeutic target and potential biomarker of brain pathology in ASMD.
Assuntos
Doença de Niemann-Pick Tipo A , Camundongos , Animais , Doença de Niemann-Pick Tipo A/genética , Doença de Niemann-Pick Tipo A/metabolismo , Doença de Niemann-Pick Tipo A/patologia , Esfingomielinas/metabolismo , Camundongos Knockout , Esfingomielina Fosfodiesterase/metabolismo , Encéfalo/metabolismo , Lisossomos/metabolismoRESUMO
The flavivirus life cycle is strictly dependent on cellular lipid metabolism. Polyphenols like gallic acid and its derivatives are promising lead compounds for new therapeutic agents as they can exert multiple pharmacological activities, including the alteration of lipid metabolism. The evaluation of our collection of polyphenols against West Nile virus (WNV), a representative medically relevant flavivirus, led to the identification of N,N'-(dodecane-1,12-diyl)bis(3,4,5-trihydroxybenzamide) and its 2,3,4-trihydroxybenzamide regioisomer as selective antivirals with low cytotoxicity and high antiviral activity (half-maximal effective concentrations [EC50s] of 2.2 and 0.24 µM, respectively, in Vero cells; EC50s of 2.2 and 1.9 µM, respectively, in SH-SY5Y cells). These polyphenols also inhibited the multiplication of other flaviviruses, namely, Usutu, dengue, and Zika viruses, exhibiting lower antiviral or negligible antiviral activity against other RNA viruses. The mechanism underlying their antiviral activity against WNV involved the alteration of sphingolipid metabolism. These compounds inhibited ceramide desaturase (Des1), promoting the accumulation of dihydrosphingomyelin (dhSM), a minor component of cellular sphingolipids with important roles in membrane properties. The addition of exogenous dhSM or Des1 blockage by using the reference inhibitor GT-11 {N-[(1R,2S)-2-hydroxy-1-hydroxymethyl-2-(2-tridecyl-1-cyclopropenyl)ethyl]octanamide} confirmed the involvement of this pathway in WNV infection. These results unveil the potential of novel antiviral strategies based on the modulation of the cellular levels of dhSM and Des1 activity for the control of flavivirus infection.
Assuntos
Flavivirus , Neuroblastoma , Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Infecção por Zika virus , Zika virus , Animais , Chlorocebus aethiops , Humanos , Febre do Nilo Ocidental/tratamento farmacológico , Antivirais/uso terapêutico , Células Vero , Neuroblastoma/tratamento farmacológico , Infecção por Zika virus/tratamento farmacológico , Replicação ViralRESUMO
Sphingosine 1-phosphate lyase (SGPL1) insufficiency (SPLIS) is a syndrome which presents with adrenal insufficiency, steroid-resistant nephrotic syndrome, hypothyroidism, neurological disease, and ichthyosis. Where a skin phenotype is reported, 94% had abnormalities such as ichthyosis, acanthosis, and hyperpigmentation. To elucidate the disease mechanism and the role SGPL1 plays in the skin barrier we established clustered regularly interspaced short palindromic repeats-Cas9 SGPL1 KO and a lentiviral-induced SGPL1 overexpression (OE) in telomerase reverse-transcriptase immortalised human keratinocytes (N/TERT-1) and thereafter organotypic skin equivalents. Loss of SGPL1 caused an accumulation of S1P, sphingosine, and ceramides, while its overexpression caused a reduction of these species. RNAseq analysis showed perturbations in sphingolipid pathway genes, particularly in SGPL1_KO, and our gene set enrichment analysis revealed polar opposite differential gene expression between SGPL1_KO and _OE in keratinocyte differentiation and Ca2+ signaling genesets. SGPL1_KO upregulated differentiation markers, while SGPL1_OE upregulated basal and proliferative markers. The advanced differentiation of SGPL1_KO was confirmed by 3D organotypic models that also presented with a thickened and retained stratum corneum and a breakdown of E-cadherin junctions. We conclude that SPLIS associated ichthyosis is a multifaceted disease caused possibly by sphingolipid imbalance and excessive S1P signaling, leading to increased differentiation and an imbalance of the lipid lamellae throughout the epidermis.
Assuntos
Ictiose , Esfingolipídeos , Humanos , Cálcio/metabolismo , Aldeído Liases/genética , Aldeído Liases/metabolismo , Lisofosfolipídeos/metabolismo , Esfingosina/genética , Esfingosina/metabolismo , Ictiose/genéticaRESUMO
OBJECTIVE: Adrenocortical carcinomas (ACCs) are invasive tumours arising in the adrenal cortex, and steroidogenic tumours are associated with worse prognostic outcomes. Loss-of-function mutations in sphingosine-1-phosphate lyase (SGPL1) cause primary adrenal insufficiency and as a key degradative enzyme in the sphingolipid pathway, SGPL1 also influences the balance of pro-proliferative and pro-apoptotic sphingolipids. We, therefore, hypothesized increased SGPL1 may be linked to increased disease severity in ACC. DESIGN: Analyse SGPL1 expression impact on patient survival and adrenal cancer cell phenotype. We analysed two ACC cohorts with survival and corresponding transcriptomic data, focusing on SGPL1 and sphingolipid pathway genes. In vitro, we generated SGPL1-knockout and overexpressing H295R adrenocortical cells to investigate the role of SGPL1 in cell signalling in ACCs. RESULTS: We found increased expression of several sphingolipid pathway receptors and enzymes, most notably SGPL1 correlated with reduced patient survival in both cohorts. Overexpression of SGPL1 in the H295R cell line increased proliferation and migration while reducing apoptosis, while SGPL1 knockout had the opposite effect. RNA-seq revealed a global increase in the expression of genes in the electron transport chain in overexpressing cells, correlating with increased aerobic respiration and glycolysis. Furthermore, the opposite phenotype was seen in cells lacking SGPL1. We subsequently found the increased proliferation is linked to metabolic substrate availability and increased capacity to use different fuel sources, but particularly glucose, in overexpressing cells. CONCLUSIONS: We, therefore, propose that SGPL1-overexpressing ACC tumours reduce patient survival by increasing fuel usage for anabolism and energy production to facilitate growth and invasion.
Assuntos
Neoplasias do Córtex Suprarrenal , Carcinoma Adrenocortical , Humanos , Carcinoma Adrenocortical/genética , Aldeído Liases/genética , Aldeído Liases/metabolismo , Esfingolipídeos , Neoplasias do Córtex Suprarrenal/genéticaRESUMO
Breast cancer (BC) is the most common malignancy in women worldwide. While the main systemic treatment option is anthracycline-containing chemotherapy, chemoresistance continues to be an obstacle to patient survival. Carnitine palmitoyltransferase 1C (CPT1C) has been described as a poor-prognosis marker for several tumour types, as it favours tumour growth and hinders cells from entering senescence. At the molecular level, CPT1C has been associated with lipid metabolism regulation and important lipidome changes. Since plasma membrane (PM) rigidity has been associated with reduced drug uptake, we explored whether CPT1C expression could be involved in PM remodelling and drug chemoresistance. Liquid chromatography-high resolution mass spectrometry (LC-HRMS) lipid analysis of PM-enriched fractions of MDA-MB-231 BC cells showed that CPT1C silencing increased PM phospholipid saturation, suggesting a rise in PM rigidity. Moreover, CPT1C silencing increased cell survival against doxorubicin (DOX) treatment in different BC cells due to reduced drug uptake. These findings, further complemented by ROC plotter analysis correlating lower CPT1C expression with a lower pathological complete response to anthracyclines in patients with more aggressive types of BC, suggest CPT1C as a novel predictive biomarker for BC chemotherapy.
Assuntos
Neoplasias da Mama , Carnitina O-Palmitoiltransferase , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Antraciclinas/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Membrana Celular/metabolismo , Regulação para BaixoRESUMO
Ceramide has a key role in the regulation of cellular senescence and apoptosis. As Ceramide levels are lowered by the action of acid ceramidase (AC), abnormally expressed in various cancers, the identification of AC inhibitors has attracted increasing interest. However, this finding has been mainly hampered by the lack of formats suitable for the screening of large libraries. We have overcome this drawback by adapting a fluorogenic assay to a 384-well plate format. The performance of this optimised platform has been proven by the screening a library of 4100 compounds. Our results show that the miniaturised platform is well suited for screening purposes and it led to the identification of several hits, that belong to different chemical classes and display potency ranges of 2-25 µM. The inhibitors also show selectivity over neutral ceramidase and retain activity in cells and can therefore serve as a basis for further chemical optimisation.
Assuntos
Ceramidase Ácida , Neoplasias , Humanos , Ceramidase Ácida/antagonistas & inibidores , Apoptose , Ceramidas/química , Bibliotecas de Moléculas PequenasRESUMO
Candida albicans is a commensal yeast that inhabits the gastrointestinal tract of humans; increased colonization of this yeast in this niche has implicated the master regulator of the white-opaque transition, Wor1, by mechanisms not completely understood. We have addressed the role that this transcription factor has on commensalism by the characterization of strains overexpressing this gene. We show that WOR1 overexpression causes an alteration of the total lipid content of the fungal cell and significantly alters the composition of structural and reserve molecular species lipids as determined by lipidomic analysis. These cells are hypersensitive to membrane-disturbing agents such as SDS, have increased tolerance to azoles, an augmented number of peroxisomes, and increased phospholipase activity. WOR1 overexpression also decreases mitochondrial activity and results in altered susceptibility to certain oxidants. All together, these changes reflect drastic alterations in the cellular physiology that facilitate adaptation to the gastrointestinal tract environment.
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Mutations in the GBA gene that encodes the lysosomal enzyme ß-glucocerebrosidase (GCase) are a major genetic risk factor for Parkinson's disease (PD). In this study, we generated a set of differentiated and stable human dopaminergic cell lines that express the two most prevalent GBA mutations as well as GBA knockout cell lines as a in vitro disease modeling system to study the relationship between mutant GBA and the abnormal accumulation of α-synuclein. We performed a deep analysis of the consequences triggered by the presence of mutant GBA protein and the loss of GCase activity in different cellular compartments, focusing primarily on the lysosomal compartment, and analyzed in detail the lysosomal activity, composition, and integrity. The loss of GCase activity generates extensive lysosomal dysfunction, promoting the loss of activity of other lysosomal enzymes, affecting lysosomal membrane stability, promoting intralysosomal pH changes, and favoring the intralysosomal accumulation of sphingolipids and cholesterol. These local events, occurring only at a subcellular level, lead to an impairment of autophagy pathways, particularly chaperone-mediated autophagy, the main α-synuclein degradative pathway. The findings of this study highlighted the role of lysosomal function and lipid metabolism in PD and allowed us to describe a molecular mechanism to understand how mutations in GBA can contribute to an abnormal accumulation of different α-synuclein neurotoxic species in PD pathology.
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Methuosis is a type of programmed cell death in which the cytoplasm is occupied by fluid-filled vacuoles that originate from macropinosomes (cytoplasmic vacuolation). A few molecules have been reported to behave as methuosis inducers in cancer cell lines. Jaspine B (JB) is a natural anhydrous sphingolipid (SL) derivative reported to induce cytoplasmic vacuolation and cytotoxicity in several cancer cell lines. Here, we have investigated the mechanism and signalling pathways involved in the cytotoxicity induced by the natural sphingolipid Jaspine B (JB) in lung adenocarcinoma A549 cells, which harbor the G12S K-Ras mutant. The effect of JB on inducing cytoplasmic vacuolation and modifying cell viability was determined in A549 cells, as well as in mouse embryonic fibroblasts (MEF) lacking either the autophagy-related gene ATG5 or BAX/BAK genes. Apoptosis was analyzed by flow cytometry after annexin V/propidium iodide staining, in the presence and absence of z-VAD. Autophagy was monitored by LC3-II/GFP-LC3-II analysis, and autophagic flux experiments using protease inhibitors. Phase contrast, confocal, and transmission electron microscopy were used to monitor cytoplasmic vacuolation and the uptake of Lucifer yellow to assess macropinocyosis. We present evidence that cytoplasmic vacuolation and methuosis are involved in Jaspine B cytotoxicity over A549 cells and that activation of 5' AMP-activated protein kinase (AMPK) could be involved in Jaspine-B-induced vacuolation, independently of the phosphatidylinositol 3-kinase/protein kinase B/mechanistic target of rapamycin complex 1 (PI3K/Akt/mTORC1) axis.