RESUMEN
Fatty acids (FAs) are central cellular metabolites that contribute to lipid synthesis, and can be stored or harvested for metabolic energy. Dysregulation in FA processing and storage causes toxic FA accumulation or altered membrane compositions and contributes to metabolic and neurological disorders. Saturated lipids are particularly detrimental to cells, but how lipid saturation levels are maintained remains poorly understood. Here, we identify the cerebellar ataxia spinocerebellar ataxia, autosomal recessive 20 (SCAR20)-associated protein Snx14, an endoplasmic reticulum (ER)-lipid droplet (LD) tethering protein, as a factor required to maintain the lipid saturation balance of cell membranes. We show that following saturated FA (SFA) treatment, the ER integrity of SNX14KO cells is compromised, and both SNX14KO cells and SCAR20 disease patient-derived cells are hypersensitive to SFA-mediated lipotoxic cell death. Using APEX2-based proximity labeling, we reveal the protein composition of Snx14-associated ER-LD contacts and define a functional interaction between Snx14 and Δ-9 FA desaturase SCD1. Lipidomic profiling reveals that SNX14KO cells increase membrane lipid saturation following exposure to palmitate, phenocopying cells with perturbed SCD1 activity. In line with this, SNX14KO cells manifest delayed FA processing and lipotoxicity, which can be rescued by SCD1 overexpression. Altogether, these mechanistic insights reveal a role for Snx14 in FA and ER homeostasis, defects in which may underlie the neuropathology of SCAR20.
RESUMEN
Mutations in SNX14 cause the autosomal recessive cerebellar ataxia 20 (SCAR20). Mutations generally result in loss of protein although several coding region deletions have also been reported. Patient-derived fibroblasts show disrupted autophagy, but the precise function of SNX14 is unknown. The yeast homolog, Mdm1, functions in endoplasmic reticulum (ER)-lysosome/vacuole inter-organelle tethering, but functional conservation in mammals is still required. Here, we show that loss of SNX14 alters but does not block autophagic flux. In addition, we find that SNX14 is an ER-associated protein that functions in neutral lipid homeostasis and inter-organelle crosstalk. SNX14 requires its N-terminal transmembrane helices for ER localization, while the Phox homology (PX) domain is dispensable for subcellular localization. Both SNX14-mutant fibroblasts and SNX14KO HEK293 cells accumulate aberrant cytoplasmic vacuoles, suggesting defects in endolysosomal homeostasis. However, ER-late endosome/lysosome contact sites are maintained in SNX14KO cells, indicating that it is not a prerequisite for ER-endolysosomal tethering. Further investigation of SNX14- deficiency indicates general defects in neutral lipid metabolism. SNX14KO cells display distinct perinuclear accumulation of filipin in LAMP1-positive lysosomal structures indicating cholesterol accumulation. Consistent with this, SNX14KO cells display a slight but detectable decrease in cholesterol ester levels, which is exacerbated with U18666A. Finally, SNX14 associates with ER-derived lipid droplets (LD) following oleate treatment, indicating a role in ER-LD crosstalk. We therefore identify an important role for SNX14 in neutral lipid homeostasis between the ER, lysosomes and LDs that may provide an early intervention target to alleviate the clinical symptoms of SCAR20.
Asunto(s)
Retículo Endoplásmico/genética , Metabolismo de los Lípidos/genética , Nexinas de Clasificación/genética , Ataxias Espinocerebelosas/genética , Autofagia/genética , Retículo Endoplásmico/metabolismo , Endosomas , Técnicas de Inactivación de Genes , Células HEK293 , Homeostasis/efectos de los fármacos , Humanos , Proteínas de Filamentos Intermediarios/genética , Gotas Lipídicas/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/genética , Mutación , Ácido Oléico/farmacología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Nexinas de Clasificación/deficiencia , Nexinas de Clasificación/metabolismo , Ataxias Espinocerebelosas/metabolismo , Ataxias Espinocerebelosas/fisiopatologíaRESUMEN
Cholesterol depletion by methyl-ß-cyclodextrin (MßCD) remodels the plasma membrane's mechanics in cells and its interactions with the underlying cytoskeleton, whereas in red blood cells, it is also known to cause lysis. Currently it's unclear if MßCD alters membrane tension or only enhances membrane-cytoskeleton interactions-and how this relates to cell lysis. We map membrane height fluctuations in single cells and observe that MßCD reduces temporal fluctuations robustly but flattens spatial membrane undulations only slightly. Utilizing models explicitly incorporating membrane confinement besides other viscoelastic factors, we estimate membrane mechanical parameters from the fluctuations' frequency spectrum. This helps us conclude that MßCD enhances membrane tension and does so even on ATP-depleted cell membranes where this occurs despite reduction in confinement. Additionally, on cholesterol depletion, cell membranes display higher intracellular heterogeneity in the amplitude of spatial undulations and membrane tension. MßCD also has a strong impact on the cell membrane's tenacity to mechanical stress, making cells strongly prone to rupture on hypo-osmotic shock with larger rupture diameters-an effect not hindered by actomyosin perturbations. Our study thus demonstrates that cholesterol depletion increases membrane tension and its variability, making cells prone to rupture independent of the cytoskeletal state of the cell.
Asunto(s)
Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Colesterol/metabolismo , beta-Ciclodextrinas/farmacología , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , Eritrocitos/citología , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Presión Osmótica/efectos de los fármacos , Estrés MecánicoRESUMEN
Mutations in the SNX14 gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20) in both humans and dogs. Studies implicating the phenotypic consequences of SNX14 mutations to be consequences of subcellular disruption to autophagy and lipid metabolism have been limited to in vitro investigation of patient-derived dermal fibroblasts, laboratory engineered cell lines and developmental analysis of zebrafish morphants. SNX14 homologues Snz (Drosophila) and Mdm1 (yeast) have also been conducted, demonstrated an important biochemical role during lipid biogenesis. In this study we report the effect of loss of SNX14 in mice, which resulted in embryonic lethality around mid-gestation due to placental pathology that involves severe disruption to syncytiotrophoblast cell differentiation. In contrast to other vertebrates, zebrafish carrying a homozygous, maternal zygotic snx14 genetic loss-of-function mutation were both viable and anatomically normal. Whilst no obvious behavioural effects were observed, elevated levels of neutral lipids and phospholipids resemble previously reported effects on lipid homeostasis in other species. The biochemical role of SNX14 therefore appears largely conserved through evolution while the consequences of loss of function varies between species. Mouse and zebrafish models therefore provide valuable insights into the functional importance of SNX14 with distinct opportunities for investigating its cellular and metabolic function in vivo.
Asunto(s)
Viabilidad Fetal/genética , Metabolismo de los Lípidos/genética , Placenta/anomalías , Nexinas de Clasificación/genética , Ataxias Espinocerebelosas/genética , Animales , Animales Modificados Genéticamente , Diferenciación Celular/genética , Desarrollo Embrionario/genética , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Animales , Fenotipo , Fosfolípidos/sangre , Embarazo , Trofoblastos/citología , Pez CebraRESUMEN
PURPOSE: The natural history of caliceal diverticula in children is unknown. We review our series of children with caliceal diverticula to examine the presentation, natural history and management. MATERIALS AND METHODS: We retrospectively reviewed the records of 22 children presenting with caliceal diverticula between 1983 and 2006. All pertinent clinical data were recorded, including demographics, imaging studies, treatment and clinical outcome. RESULTS: A total of 22 children had 23 caliceal diverticula. There were 14 girls and 8 boys who presented at a mean age of 5.4 +/- 3.1 years (range 0.2 to 12). At clinical presentation 10 patients had febrile urinary tract infection, 2 had hematuria, 2 had abdominal pain, 1 had flank pain and 8 were asymptomatic. Mean diameter of all caliceal diverticula was 2.2 +/- 1.7 cm. All patients underwent voiding cystourethrogram. Two patients (9%) had concomitant ipsilateral caliceal diverticula and vesicoureteral reflux. A total of 10 patients with caliceal diverticula (43%) were treated at a mean of 3.0 +/- 2.3 years after initial presentation due to symptomatic enlargement in 5, symptomatic calculus in 3, complicated abscess in 1 and urosepsis in 1. Treatment modalities included percutaneous ablation, open marsupialization/ablation, partial nephrectomy and laparoscopic marsupialization/ablation. There were no recurrences during a mean followup of 3.1 +/- 2.9 years (range 0.1 to 10.1) in these 10 patients. In the 13 caliceal diverticula (57%) that were observed mean followup was 6 +/- 3.7 years (range 1.2 to 10.5). These caliceal diverticula were stable in size and remained asymptomatic. CONCLUSIONS: Caliceal diverticula in children are rare. Most caliceal diverticula remain stable and asymptomatic but approximately 20% may have symptomatic enlargement that may warrant surgical management. Given the morbidity associated with caliceal diverticula and concomitant vesicoureteral reflux, evaluation for ipsilateral reflux is mandatory. Overall approximately 43% of children with caliceal diverticula require surgical intervention, and various treatment options are available.
Asunto(s)
Divertículo/diagnóstico , Divertículo/cirugía , Cálices Renales , Enfermedades Renales/diagnóstico , Enfermedades Renales/cirugía , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Estudios RetrospectivosRESUMEN
Lipid droplets (LDs) are nutrient reservoirs used by cells to maintain homeostasis. Nascent droplets form on the endoplasmic reticulum (ER) and grow following an influx of exogenous fatty acids (FAs). The budding of LDs requires extensive ER-LD crosstalk, but how this is regulated remains poorly understood. Here, we show that sorting nexin protein Snx14, an ER-resident protein associated with the cerebellar ataxia SCAR20, localizes to ER-LD contacts following FA treatment, where it promotes LD maturation. Using proximity-based APEX technology and topological dissection, we show that Snx14 accumulates specifically at ER-LD contacts independently of Seipin, where it remains ER-anchored and binds LDs in trans. SNX14KO cells exhibit perturbed LD morphology, whereas Snx14 overexpression promotes LD biogenesis and extends ER-LD contacts. Multi-time point imaging reveals that Snx14 is recruited to ER microdomains containing the fatty acyl-CoA ligase ACSL3, where nascent LDs bud. We propose that Snx14 is a novel marker for ER-LD contacts and regulates FA-stimulated LD growth.
Asunto(s)
Retículo Endoplásmico/metabolismo , Ácidos Grasos/metabolismo , Gotas Lipídicas/metabolismo , Nexinas de Clasificación/metabolismo , Línea Celular Tumoral , Coenzima A Ligasas/genética , Coenzima A Ligasas/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/genética , Retículo Endoplásmico/ultraestructura , Ácidos Grasos/toxicidad , Subunidades gamma de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Humanos , Gotas Lipídicas/efectos de los fármacos , Gotas Lipídicas/ultraestructura , Ácido Oléico/metabolismo , Ácido Oléico/toxicidad , Nexinas de Clasificación/genética , Factores de TiempoRESUMEN
Lipid droplets (LDs) serve as cytoplasmic reservoirs for energy-rich fatty acids (FAs) stored in the form of triacylglycerides (TAGs). During nutrient stress, yeast LDs cluster adjacent to the vacuole/lysosome, but how this LD accumulation is coordinated remains poorly understood. The ER protein Mdm1 is a molecular tether that plays a role in clustering LDs during nutrient depletion, but its mechanism of function remains unknown. Here, we show that Mdm1 associates with LDs through its hydrophobic N-terminal region, which is sufficient to demarcate sites for LD budding. Mdm1 binds FAs via its Phox-associated domain and coenriches with fatty acyl-coenzyme A ligase Faa1 at LD bud sites. Consistent with this, loss of MDM1 perturbs free FA activation and Dga1-dependent synthesis of TAGs, elevating the cellular FA level, which perturbs ER morphology and sensitizes yeast to FA-induced lipotoxicity. We propose that Mdm1 coordinates FA activation adjacent to the vacuole to promote LD production in response to stress, thus maintaining ER homeostasis.
Asunto(s)
Retículo Endoplásmico/metabolismo , Ácidos Grasos/metabolismo , Proteínas de Filamentos Intermediarios/metabolismo , Gotas Lipídicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Triglicéridos/metabolismo , Coenzima A Ligasas/genética , Coenzima A Ligasas/metabolismo , Diacilglicerol O-Acetiltransferasa/genética , Diacilglicerol O-Acetiltransferasa/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/genética , Retículo Endoplásmico/ultraestructura , Ácidos Grasos/toxicidad , Homeostasis , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de Filamentos Intermediarios/genética , Gotas Lipídicas/ultraestructura , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Phox homology (PX) domains are membrane interacting domains that bind to phosphatidylinositol phospholipids or phosphoinositides, markers of organelle identity in the endocytic system. Although many PX domains bind the canonical endosome-enriched lipid PtdIns3P, others interact with alternative phosphoinositides, and a precise understanding of how these specificities arise has remained elusive. Here we systematically screen all human PX domains for their phospholipid preferences using liposome binding assays, biolayer interferometry and isothermal titration calorimetry. These analyses define four distinct classes of human PX domains that either bind specifically to PtdIns3P, non-specifically to various di- and tri-phosphorylated phosphoinositides, bind both PtdIns3P and other phosphoinositides, or associate with none of the lipids tested. A comprehensive evaluation of PX domain structures reveals two distinct binding sites that explain these specificities, providing a basis for defining and predicting the functional membrane interactions of the entire PX domain protein family.
Asunto(s)
Fosfatidilinositoles/química , Sitios de Unión , Calorimetría , Humanos , Interferometría , Modelos Moleculares , Fosfatidilinositoles/metabolismo , Dominios Proteicos , Análisis de Secuencia de Proteína , Nexinas de Clasificación/química , Nexinas de Clasificación/metabolismoRESUMEN
Adipocytes store nutrients as lipid droplets (LDs), but how they organize their LD stores to balance lipid uptake, storage, and mobilization remains poorly understood. Here, using Drosophila fat body (FB) adipocytes, we characterize spatially distinct LD populations that are maintained by different lipid pools. We identify peripheral LDs (pLDs) that make close contact with the plasma membrane (PM) and are maintained by lipophorin-dependent lipid trafficking. pLDs are distinct from larger cytoplasmic medial LDs (mLDs), which are maintained by FASN1-dependent de novo lipogenesis. We find that sorting nexin CG1514 or Snazarus (Snz) associates with pLDs and regulates LD homeostasis at ER-PM contact sites. Loss of SNZ perturbs pLD organization, whereas Snz over-expression drives LD expansion, triacylglyceride production, starvation resistance, and lifespan extension through a DESAT1-dependent pathway. We propose that Drosophila adipocytes maintain spatially distinct LD populations and identify Snz as a regulator of LD organization and inter-organelle crosstalk.
Asunto(s)
Adipocitos/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Gotas Lipídicas/metabolismo , Nexinas de Clasificación/metabolismo , Animales , Drosophila , Proteínas de Drosophila/genética , Retículo Endoplásmico/metabolismo , Ácido Graso Desaturasas/genética , Ácido Graso Desaturasas/metabolismo , Longevidad , Unión Proteica , Nexinas de Clasificación/genéticaRESUMEN
AIMS: To demonstrate the correlation of anemia and intact parathormone with left ventricular hypertrophy in a cohort of Chronic Kidney Disease (CKD) patients in a tertiary care centre. METHODS: A cross-sectional study was done over 2 years on 230 renal failure patients (160 males, 70 females), aged 15-75 years, who had elevated serum creatinine and reduced GFR. The patients were assessed based on clinical history and a number of laboratory parameters including serum creatinine, calcium, iPTH level, Hb, Hct, GFR and LVMI. Settings : Patients were seen as inpatients and outpatients in a tertiary care centre. RESULTS: In CKD stages I, II and III, 51% of the patients had anemia Hb<11gm/dl), 16%of the patients had elevated iPTH, 79% of male patients and 71% of female patients had LVH. In Stage IV CKD, 55% of the patients had anemia, 25% of the patients had elevated iPTH, 74% of male patients and 100% of female patients had LVH. In stage V CKD, 76% of the patients had anemia, 31% of the patients had elevated iPTH, 77% of male patients and 96% of female patients had LVH. In all five stages, 78% of male patients and 71% of female patients with elevated iPTH had LVH, 81% of male patients and 90% of female patients with anemia had LVH. Systemic hypertension was present in 69% of the patients. CONCLUSION: Anemia is widely prevalent in our cohort of CKD patients. Severity of anemia is correlated to LVH and secondary hyperparathyroidism in these patients.