RESUMEN
The lysosome is an acid organelle that contains a variety of hydrolytic enzymes and plays a significant role in intracellular degradation to maintain cellular homeostasis. Genetic variants in lysosome-related genes can lead to severe congenital diseases, such as lysosomal storage diseases. In the present study, we investigated the impact of depleting lysosomal acid lipase A (LIPA), a lysosomal esterase that metabolizes esterified cholesterol or triglyceride, on lysosomal function. Under nutrient-rich conditions, LIPA gene KO (LIPAKO) cells exhibited impaired autophagy, whereas, under starved conditions, they showed normal autophagy. The cause underlying the differential autophagic activity was increased sensitivity of LIPAKO cells to ammonia, which was produced from l-glutamine in the medium. Further investigation revealed that ammonia did not affect upstream signals involved in autophagy induction, autophagosome-lysosome fusion, and hydrolytic enzyme activities in LIPAKO cells. On the other hand, LIPAKO cells showed defective lysosomal acidity upon ammonia loading. Microscopic analyses revealed that lysosomes of LIPAKO cells enlarged, whereas the amount of lysosomal proton pump V-ATPase did not proportionally increase. Since the enlargement of lysosomes in LIPAKO cells was not normalized under starved conditions, this is the primary change that occurred in the LIPAKO cells, and autophagy was affected by impaired lysosomal function under the specific conditions. These findings expand our comprehension of the pathogenesis of Wolman's disease, which is caused by a defect in the LIPA gene, and suggest that conditions, such as hyperlipidemia, may easily disrupt lysosomal functions.
Asunto(s)
Autofagia , Lipasa , Lisosomas , Humanos , Amoníaco/metabolismo , Autofagia/fisiología , Lipasa/genética , Lipasa/metabolismo , Lisosomas/química , Lisosomas/enzimología , Enfermedad de Wolman/enzimología , Enfermedad de Wolman/genética , Células HeLa , Concentración de Iones de Hidrógeno , Técnicas de Inactivación de GenesRESUMEN
Autophagy dysfunction is associated with human diseases and conditions including neurodegenerative diseases, metabolic issues, and chronic infections. Additionally, the decline in autophagic activity contributes to tissue and organ dysfunction and aging-related diseases. Several factors, such as down-regulation of autophagy components and activators, oxidative damage, microinflammation, and impaired autophagy flux, are linked to autophagy decline. An autophagy flux impairment (AFI) has been implicated in neurological disorders and in certain other pathological conditions. Here, to enhance our understanding of AFI, we conducted a comprehensive literature review of findings derived from two well-studied cellular stress models: glucose deprivation and replicative senescence. Glucose deprivation is a condition in which cells heavily rely on oxidative phosphorylation for ATP generation. Autophagy is activated, but its flux is hindered at the autolysis step, primarily due to an impairment of lysosomal acidity. Cells undergoing replicative senescence also experience AFI, which is also known to be caused by lysosomal acidity failure. Both glucose deprivation and replicative senescence elevate levels of reactive oxygen species (ROS), affecting lysosomal acidification. Mitochondrial alterations play a crucial role in elevating ROS generation and reducing lysosomal acidity, highlighting their association with autophagy dysfunction and disease conditions. This paper delves into the underlying molecular and cellular pathways of AFI in glucose-deprived cells, providing insights into potential strategies for managing AFI that is driven by lysosomal acidity failure. Furthermore, the investigation on the roles of mitochondrial dysfunction sheds light on the potential effectiveness of modulating mitochondrial function to overcome AFI, offering new possibilities for therapeutic interventions.
Asunto(s)
Glucosa , Mitofagia , Humanos , Especies Reactivas de Oxígeno/metabolismo , Glucosa/metabolismo , Autofagia/fisiología , Lisosomas/metabolismo , Concentración de Iones de HidrógenoRESUMEN
Macroautophagic/autophagic turnover of endoplasmic reticulum (reticulophagy) is critical for cell health. Herein we reported a sensitive fluorescence-on imaging of reticulophagy using a small molecule probe (ER-proRed) comprised of green-emissive fluorinated rhodol for ER targeting and nonfluorescent rhodamine-lactam prone to lysosome-triggered red fluorescence. Partitioned in ER to exhibit green fluorescence, ER-proRed gives intense red fluorescence upon co-delivery with ER into acidic lysosomes. Serving as the signal of reticulophagy, the turning on of red fluorescence enables discernment of reticulophagy induced by starvation, varied levels of reticulophagic receptors, and chemical agents such as etoposide and sodium butyrate. These results show ER probes optically activatable in lysosomes, such as ER-proRed, offer a sensitive and simplified tool for studying reticulophagy in biology and diseases.Abbreviations: Baf-A1, bafilomycin A1; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CQ, chloroquine diphosphate; ER, endoplasmic reticulum; FHR, fluorinated hydrophobic rhodol; GFP, green fluorescent protein; Reticulophagy, selective autophagy of ER; RFP, red fluorescent protein; ROX, X-rhodamine; UPR, unfolded protein response.
Asunto(s)
Autofagia , Respuesta de Proteína Desplegada , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Proteínas Portadoras/metabolismoRESUMEN
Cadmium (Cd) exposure induced lipid metabolic disorder with changes in lipid composition, as well as triglyceride (TG) levels. Liver is the main organ maintaining body TG level and previous studies suggested that Cd exposure might increase TG synthesis but reduce TG uptake in liver. However, the effects of Cd exposure on TG secretion from liver and underlying mechanism are still unclear. In the present study, the data revealed that Cd exposure increased TG levels in the HepG2 cells and the cultured medium by increasing the expression of microsomal triglyceride transfer protein (MTTP), which was abrogated by siRNA knockdown of MTTP. MTTP was synergistically accumulated after Cd exposure or treated with proteasome inhibitor MG132 and lysosome inhibitor chloroquine (CQ), which suggested the Cd increased MTTP protein stability by inhibiting both the proteasome and the lysosomal protein degradation pathways. In addition, our results demonstrated that Cd exposure inhibited the lysosomal acidic degradation pathway through disrupting endoplastic reticulum (ER) Ca2+ homeostasis. Cd-induced MTTP protein and TG levels were significantly reduced by pretreatments of BAPTA/AM chelation of intracellular Ca2+, 2-APB inhibition of ER Ca2+ release channel inositol 1,4,5-trisphosphate receptor (IP3R) and CDN1163 activation of ER Ca2+ reuptake pump sarcoplasmic reticulum Ca2+-ATPase (SERCA). These results suggest that Cd-induced ER Ca2+ release impaired the lysosomal acidity, which associated with MTTP protein accumulation and contributed to increased TG levels.
Asunto(s)
Cadmio/farmacología , Proteínas Portadoras/metabolismo , Retículo Endoplásmico/metabolismo , Homeostasis/efectos de los fármacos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Triglicéridos/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Células Hep G2 , Humanos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismoRESUMEN
The lysosome is an acidic multi-functional organelle with roles in macromolecular digestion, nutrient sensing, and signaling. However, why cells require acidic lysosomes to proliferate and which nutrients become limiting under lysosomal dysfunction are unclear. To address this, we performed CRISPR-Cas9-based genetic screens and identified cholesterol biosynthesis and iron uptake as essential metabolic pathways when lysosomal pH is altered. While cholesterol synthesis is only necessary, iron is both necessary and sufficient for cell proliferation under lysosomal dysfunction. Remarkably, iron supplementation restores cell proliferation under both pharmacologic and genetic-mediated lysosomal dysfunction. The rescue was independent of metabolic or signaling changes classically associated with increased lysosomal pH, uncoupling lysosomal function from cell proliferation. Finally, our experiments revealed that lysosomal dysfunction dramatically alters mitochondrial metabolism and hypoxia inducible factor (HIF) signaling due to iron depletion. Altogether, these findings identify iron homeostasis as the key function of lysosomal acidity for cell proliferation.
Asunto(s)
Proliferación Celular/fisiología , Hierro/metabolismo , Lisosomas/metabolismo , Colesterol/biosíntesis , Colesterol/metabolismo , Células HEK293 , Células HeLa , Homeostasis , Humanos , Concentración de Iones de Hidrógeno , Células Jurkat , Lisosomas/fisiología , Mitocondrias/metabolismo , Transducción de Señal/genéticaRESUMEN
The loss of vacuolar/lysosomal acidity is an early event during aging that has been linked to mitochondrial dysfunction. However, it is unclear how loss of vacuolar acidity results in age-related dysfunction. Through unbiased genetic screens, we determined that increased iron uptake can suppress the mitochondrial respiratory deficiency phenotype of yeast vma mutants, which have lost vacuolar acidity due to genetic disruption of the vacuolar ATPase proton pump. Yeast vma mutants exhibited nuclear localization of Aft1, which turns on the iron regulon in response to iron-sulfur cluster (ISC) deficiency. This led us to find that loss of vacuolar acidity with age in wild-type yeast causes ISC defects and a DNA damage response. Using microfluidics to investigate aging at the single-cell level, we observe grossly divergent trajectories of iron homeostasis within an isogenic and environmentally homogeneous population. One subpopulation of cells fails to mount the expected compensatory iron regulon gene expression program, and suffers progressively severe ISC deficiency with little to no activation of the iron regulon. In contrast, other cells show robust iron regulon activity with limited ISC deficiency, which allows extended passage and survival through a period of genomic instability during aging. These divergent trajectories suggest that iron regulation and ISC homeostasis represent a possible target for aging interventions.
Asunto(s)
Homeostasis , Hierro , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Hierro/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , AzufreRESUMEN
Human exposure to cadmium (Cd) could lead to alterations in lipid metabolism. However, the underlying mechanism is still unclear. In the present study, the data revealed that Cd exposure induced cholesterol redistribution both in vivo from mouse liver tissue into the serum, and in vitro from the HepG2 cells to the cultured medium, which were associated with modulating the expressions of cholesterol efflux proteins, including upregulating cholesterol exporter ATP-binding cassette transporter A1 (ABCA1) and downregulating oxysterol-binding protein (OSBP). Further investigation in HepG2 cells revealed that Cd upregulated ABCA1 expression with increased stability by inhibiting lysosomal pathway, and downregulated OSBP expression by increasing ubiquitination. Cd-induced cholesterol redistribution was completely inhibited by knockdown of ABCA1 expression using siRNA, and was significantly reduced by overexpression of OSBP. Taken together, these results suggested that Cd induced cholesterol redistribution by upregulating ABCA1 and downregulating OSBP.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Cadmio/toxicidad , Colesterol/metabolismo , Receptores de Esteroides/metabolismo , Regulación hacia Abajo , Células Hep G2 , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
In principle, not only efficient but rapid transfection is required since it can maximize the bioavailability of vector-carried gene prior to the cellular excretion. However, the "rapid" goal has been paid few attentions so far in the research field of vector-aided transfection. As a pioneering attempt, the present study designed a lysosome-targeting acidity-responsive nanoassembly as gene vectors, which proved the amazing potency to mediate the "Superfast" transnuclear gene transport and gene transfection with high efficiency in vitro and in vivo. The nanoassembly was constructed on the pH-reversible covalent boronic acid-diol coupling between 1,3-diol-rich oligoethylenimine (OEI-EHDO) and phenylboronic acid modified cholesterol (Chol-PBA). The rapid and efficient nuclei-tropic delivery and transfection was demonstrated to highly rely on the lysosome-acidity induced assembly destruction followed by the easy liberation of gene payloads inside cells. The nanoassembly-mediated transfection at 8 h can afford the outcome even comparable to that achieved at 48 h by the golden standard of PEI25k, and the transfection efficiency can still remain at a high level during 48 h. In contrast, time-dependent efficiency enhancement was identified for the transfections using PEI25k and OEI-EHDO as delivery vectors. Moreover, owing to the hydroxyl-rich surface, this delivery nanosystem presented strong tolerance to the serum-induced transfection inhibition that frequently occurred for the polycationic gene vectors such as PEI25k. The in vitro and in vivo results manifested the low toxicity of this bio-decomposable nanoassembly.
Asunto(s)
Núcleo Celular/metabolismo , Transfección/métodos , Animales , Aziridinas/química , Ácidos Borónicos/química , Muerte Celular/efectos de los fármacos , Línea Celular , Núcleo Celular/efectos de los fármacos , Colesterol/química , ADN/metabolismo , Dispersión Dinámica de Luz , Electroforesis en Gel de Agar , Femenino , Glucosa/farmacología , Humanos , Concentración de Iones de Hidrógeno , Ratones Endogámicos BALB C , Nanopartículas/química , Nanopartículas/ultraestructura , Suero/metabolismo , SolucionesRESUMEN
The present study reported a lysosome-acidity-targeting bio-responsive nanovehicle self-assembled from dextran (Dex) and phenylboronic acid modified cholesterol (Chol-PBA), aiming at the nucleus-tropic drug delivery. The prominent advantage of this assembled nanoconstruction arose from its susceptibility to acidity-labile dissociation concurrently accompanied with the fast liberation of encapsulated drugs, leading to efficient nuclear drug translocation and consequently favorable drug efficacy. By elaborately exploiting NH4Cl pretreatment to interfere with the cellular endosomal acidification progression, this study clearly evidenced at a cellular level the strong lysosomal-acidity dependency of nuclear drug uptake efficiency, which was shown to be the main factor influencing the drug efficacy. The boronate-linked nanoassembly displayed nearly no cytotoxicity and can remain structural stability under the simulated physiological conditions including 10% serum and the normal blood sugar concentration. The cellular exposure to cholesterol was found to bate the cellular uptake of nanoassembly in a dose-dependent manner, suggesting a cholesterol-associated mechanism of the intracellular internalization. The in vivo antitumor assessment in xenograft mouse models revealed the significant superiority of DOX-loaded Dex/Chol-PBA nanoassembly over the controls including free DOX and the DOX-loaded non-sensitive Dex-Chol, as reflected by the more effective tumor-growth inhibition and the better systematic safety. In terms of the convenient preparation, sensitive response to lysosomal acidity and efficient nuclear drug translocation, Dex/Chol-PBA nanoassembly derived from natural materials shows promising potentials as the nanovehicle for nucleus-tropic drug delivery especially for antitumor agents. More attractively, this study offers a deeper insight into the mechanism concerning the contribution of acidity-responsive delivery to the enhanced chemotherapy performance.
Asunto(s)
Boro/química , Núcleo Celular/metabolismo , Colesterol/química , Dextranos/química , Portadores de Fármacos/química , Nanopartículas/química , Transporte Activo de Núcleo Celular , Animales , Antineoplásicos/administración & dosificación , Ácidos Borónicos/química , Carbamatos/química , Núcleo Celular/efectos de los fármacos , Citoplasma/metabolismo , Endosomas/metabolismo , Femenino , Células HeLa , Humanos , Lisosomas/química , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Micelas , Microscopía Electrónica de Transmisión , Nanomedicina , Trasplante de Neoplasias , Tamaño de la PartículaRESUMEN
Intracellular acidic vesicles, constituted mostly by lysosomes, mediated a variety of biological events ranging from endocytosis, apoptosis, to cancer metastasis, etc. A chimeric molecular pH-meter (Lyso-DR), comprised of a dansyl fluorophore and proton activatable rhodamine-lactam, was prepared for ratiometric reporting of intralysosomal acidity. Exclusively confined in lysosomes, Lyso-DR exhibited pH dependent dual fluorescence emission bands which enable resolution of individual lysosomes in terms of acidity and quantitation of the overall intracellular lysosomal acidity, e.g. the lysosomal pH of HeLa cells is around pH 5.0 whereas that of L929 cells is around pH 6.2. Lyso-DR effectively differentiated the lysosomal pH changes of cells undergoing apoptosis vs necrosis, suggesting its utility in investigations on lysosome involved biomedical processes.