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1.
Nat Rev Mol Cell Biol ; 25(3): 223-245, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38001393

RESUMEN

Every cell must satisfy basic requirements for nutrient sensing, utilization and recycling through macromolecular breakdown to coordinate programmes for growth, repair and stress adaptation. The lysosome orchestrates these key functions through the synchronised interplay between hydrolytic enzymes, nutrient transporters and signalling factors, which together enable metabolic coordination with other organelles and regulation of specific gene expression programmes. In this Review, we discuss recent findings on lysosome-dependent signalling pathways, focusing on how the lysosome senses nutrient availability through its physical and functional association with mechanistic target of rapamycin complex 1 (mTORC1) and how, in response, the microphthalmia/transcription factor E (MiT/TFE) transcription factors exert feedback regulation on lysosome biogenesis. We also highlight the emerging interactions of lysosomes with other organelles, which contribute to cellular homeostasis. Lastly, we discuss how lysosome dysfunction contributes to diverse disease pathologies and how inherited mutations that compromise lysosomal hydrolysis, transport or signalling components lead to multi-organ disorders with severe metabolic and neurological impact. A deeper comprehension of lysosomal composition and function, at both the cellular and organismal level, may uncover fundamental insights into human physiology and disease.


Asunto(s)
Lisosomas , Transducción de Señal , Humanos , Transducción de Señal/fisiología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Lisosomas/metabolismo , Homeostasis/fisiología , Autofagia/fisiología
2.
EMBO Rep ; 25(8): 3651-3677, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-39039299

RESUMEN

Endoplasmic reticulum (ER) remodeling is vital for cellular organization. ER-phagy, a selective autophagy targeting ER, plays an important role in maintaining ER morphology and function. The FAM134 protein family, including FAM134A, FAM134B, and FAM134C, mediates ER-phagy. While FAM134B mutations are linked to hereditary sensory and autonomic neuropathy in humans, the physiological role of the other FAM134 proteins remains unknown. To address this, we investigate the roles of FAM134 proteins using single and combined knockouts (KOs) in mice. Single KOs in young mice show no major phenotypes; however, combined Fam134b and Fam134c deletion (Fam134b/cdKO), but not the combination including Fam134a deletion, leads to rapid neuromuscular and somatosensory degeneration, resulting in premature death. Fam134b/cdKO mice show rapid loss of motor and sensory axons in the peripheral nervous system. Long axons from Fam134b/cdKO mice exhibit expanded tubular ER with a transverse ladder-like appearance, whereas no obvious abnormalities are present in cortical ER. Our study unveils the critical roles of FAM134C and FAM134B in the formation of tubular ER network in axons of both motor and sensory neurons.


Asunto(s)
Axones , Retículo Endoplásmico , Proteínas de la Membrana , Animales , Humanos , Ratones , Axones/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Noqueados
3.
EMBO J ; 40(4): e105120, 2021 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-33368531

RESUMEN

Autophagy is a lysosome-dependent degradation pathway essential to maintain cellular homeostasis. Therefore, either defective or excessive autophagy may be detrimental for cells and tissues. The past decade was characterized by significant advances in molecular dissection of stimulatory autophagy inputs; however, our understanding of the mechanisms that restrain autophagy is far from complete. Here, we describe a negative feedback mechanism that limits autophagosome biogenesis based on the selective autophagy-mediated degradation of ATG13, a component of the ULK1 autophagy initiation complex. We demonstrate that the centrosomal protein OFD1 acts as bona fide autophagy receptor for ATG13 via direct interaction with the Atg8/LC3/GABARAP family of proteins. We also show that patients with Oral-Facial-Digital type I syndrome, caused by mutations in the OFD1 gene, display excessive autophagy and that genetic inhibition of autophagy in a mouse model of the disease, significantly ameliorates polycystic kidney, a clinical manifestation of the disorder. Collectively, our data report the discovery of an autophagy self-regulated mechanism and implicate dysregulated autophagy in the pathogenesis of renal cystic disease in mammals.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagosomas/fisiología , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Autofagia , Proteínas Asociadas a Microtúbulos/metabolismo , Enfermedades Renales Poliquísticas/patología , Proteínas/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Humanos , Lisosomas/metabolismo , Lisosomas/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Asociadas a Microtúbulos/genética , Enfermedades Renales Poliquísticas/etiología , Enfermedades Renales Poliquísticas/metabolismo , Proteínas/genética
4.
Nat Rev Mol Cell Biol ; 14(5): 283-96, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23609508

RESUMEN

For a long time, lysosomes were considered merely to be cellular 'incinerators' involved in the degradation and recycling of cellular waste. However, now there is compelling evidence indicating that lysosomes have a much broader function and that they are involved in fundamental processes such as secretion, plasma membrane repair, signalling and energy metabolism. Furthermore, the essential role of lysosomes in autophagic pathways puts these organelles at the crossroads of several cellular processes, with significant implications for health and disease. The identification of a master regulator, transcription factor EB (TFEB), that regulates lysosomal biogenesis and autophagy has revealed how the lysosome adapts to environmental cues, such as starvation, and targeting TFEB may provide a novel therapeutic strategy for modulating lysosomal function in human disease.


Asunto(s)
Lisosomas/fisiología , Animales , Metabolismo Energético , Humanos , Lisosomas/metabolismo , Transducción de Señal
5.
Traffic ; 23(5): 238-269, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35343629

RESUMEN

Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground-breaking discoveries on an organelle that continues to surprise and excite cell biologists.


Asunto(s)
Lisosomas , Redes y Vías Metabólicas , Lisosomas/metabolismo , Transducción de Señal
6.
EMBO J ; 39(5): e104546, 2020 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-32073155

RESUMEN

To maintain cellular homeostasis, the endoplasmic reticulum (ER) necessitates a continuous removal of ER fragments via a selective, receptor-mediated, form of autophagy known as ER-phagy. In this issue of The EMBO Journal, Jiang et al (2020) shed light on how the best characterized autophagy receptor FAM134B mediates ER membrane fragmentation, the earliest event during ER-phagy. They propose a dynamic model for FAM134B protein oligomerization and ER membrane scission, which are driven by CAMK2B-mediated phosphorylation of the receptor and are altered in sensory neuropathy.


Asunto(s)
Autofagia , Proteínas de la Membrana , Proteínas Portadoras , Retículo Endoplásmico , Homeostasis
7.
EMBO J ; 39(17): e105696, 2020 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-32716134

RESUMEN

Lysosomal degradation of the endoplasmic reticulum (ER) via autophagy (ER-phagy) is emerging as a critical regulator of cell homeostasis and function. The recent identification of ER-phagy receptors has shed light on the molecular mechanisms underlining this process. However, the signaling pathways regulating ER-phagy in response to cellular needs are still largely unknown. We found that the nutrient responsive transcription factors TFEB and TFE3-master regulators of lysosomal biogenesis and autophagy-control ER-phagy by inducing the expression of the ER-phagy receptor FAM134B. The TFEB/TFE3-FAM134B axis promotes ER-phagy activation upon prolonged starvation. In addition, this pathway is activated in chondrocytes by FGF signaling, a critical regulator of skeletal growth. FGF signaling induces JNK-dependent proteasomal degradation of the insulin receptor substrate 1 (IRS1), which in turn inhibits the PI3K-PKB/Akt-mTORC1 pathway and promotes TFEB/TFE3 nuclear translocation and enhances FAM134B transcription. Notably, FAM134B is required for protein secretion in chondrocytes, and cartilage growth and bone mineralization in medaka fish. This study identifies a new signaling pathway that allows ER-phagy to respond to both metabolic and developmental cues.


Asunto(s)
Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Núcleo Celular/metabolismo , Retículo Endoplásmico/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal , Transporte Activo de Núcleo Celular , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Núcleo Celular/genética , Retículo Endoplásmico/genética , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Proteínas de la Membrana/genética , Ratones , Oryzias
8.
Annu Rev Neurosci ; 39: 277-95, 2016 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-27090953

RESUMEN

Recent studies of autophagic and lysosomal pathways have significantly changed our understanding of lysosomes; once thought to be simple degradative and recycling centers, lysosomes are now known to be organelles capable of influencing signal transduction, via the mammalian target of rapamycin complex 1 (mTORC1), and regulating gene expression, via transcription factor EB (TFEB) and other transcription factors. These pathways are particularly relevant to maintaining brain homeostasis, as dysfunction of the endolysosomal and autophagic pathways has been associated with common neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's, and lysosomal storage disorders, a group of inherited disorders characterized by the intralysosomal buildup of partially degraded metabolites. This review focuses on the cellular biology of lysosomes and discusses the possible mechanisms by which disruption of their function contributes to neurodegeneration. We also review and discuss how targeting TFEB and lysosomes may offer innovative therapeutic approaches for treating a wide range of neurological conditions.


Asunto(s)
Autofagia/fisiología , Encefalopatías/fisiopatología , Encéfalo/fisiopatología , Lisosomas/metabolismo , Animales , Encéfalo/metabolismo , Encefalopatías/metabolismo , Expresión Génica/fisiología , Homeostasis/fisiología , Humanos
9.
EMBO J ; 38(3)2019 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-30591554

RESUMEN

Transcription factor TFEB is thought to control cellular functions-including in the vascular bed-primarily via regulation of lysosomal biogenesis and autophagic flux. Here, we report that TFEB also orchestrates a non-canonical program that controls the cell cycle/VEGFR2 pathway in the developing vasculature. In endothelial cells, TFEB depletion halts proliferation at the G1-S transition by inhibiting the CDK4/Rb pathway. TFEB-deficient cells attempt to compensate for this limitation by increasing VEGFR2 levels at the plasma membrane via microRNA-mediated mechanisms and controlled membrane trafficking. TFEB stimulates expression of the miR-15a/16-1 cluster, which limits VEGFR2 transcript stability and negatively modulates expression of MYO1C, a regulator of VEGFR2 trafficking to the cell surface. Altered levels of miR-15a/16-1 and MYO1C in TFEB-depleted cells cause increased expression of plasma membrane VEGFR2, but in a manner associated with low signaling strength. An endothelium-specific Tfeb-knockout mouse model displays defects in fetal and newborn mouse vasculature caused by reduced endothelial proliferation and by anomalous function of the VEGFR2 pathway. These previously unrecognized functions of TFEB expand its role beyond regulation of the autophagic pathway in the vascular system.


Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/fisiología , Proliferación Celular , Embrión de Mamíferos/citología , Endotelio Vascular/citología , Regulación del Desarrollo de la Expresión Génica , Neovascularización Fisiológica , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos/fisiología , Endotelio Vascular/fisiología , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética
10.
EMBO J ; 38(2)2019 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-30559329

RESUMEN

Autophagy is a cytosolic quality control process that recognizes substrates through receptor-mediated mechanisms. Procollagens, the most abundant gene products in Metazoa, are synthesized in the endoplasmic reticulum (ER), and a fraction that fails to attain the native structure is cleared by autophagy. However, how autophagy selectively recognizes misfolded procollagens in the ER lumen is still unknown. We performed siRNA interference, CRISPR-Cas9 or knockout-mediated gene deletion of candidate autophagy and ER proteins in collagen producing cells. We found that the ER-resident lectin chaperone Calnexin (CANX) and the ER-phagy receptor FAM134B are required for autophagy-mediated quality control of endogenous procollagens. Mechanistically, CANX acts as co-receptor that recognizes ER luminal misfolded procollagens and interacts with the ER-phagy receptor FAM134B. In turn, FAM134B binds the autophagosome membrane-associated protein LC3 and delivers a portion of ER containing both CANX and procollagen to the lysosome for degradation. Thus, a crosstalk between the ER quality control machinery and the autophagy pathway selectively disposes of proteasome-resistant misfolded clients from the ER.


Asunto(s)
Calnexina/metabolismo , Retículo Endoplásmico/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Procolágeno/metabolismo , Animales , Autofagia , Calnexina/genética , Línea Celular , Técnicas de Silenciamiento del Gen , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Oryzias , Pliegue de Proteína
11.
EMBO Rep ; 22(9): e52289, 2021 09 06.
Artículo en Inglés | MEDLINE | ID: mdl-34338405

RESUMEN

Degradation of the endoplasmic reticulum (ER) via selective autophagy (ER-phagy) is vital for cellular homeostasis. We identify FAM134A/RETREG2 and FAM134C/RETREG3 as ER-phagy receptors, which predominantly exist in an inactive state under basal conditions. Upon autophagy induction and ER stress signal, they can induce significant ER fragmentation and subsequent lysosomal degradation. FAM134A, FAM134B/RETREG1, and FAM134C are essential for maintaining ER morphology in a LC3-interacting region (LIR)-dependent manner. Overexpression of any FAM134 paralogue has the capacity to significantly augment the general ER-phagy flux upon starvation or ER-stress. Global proteomic analysis of FAM134 overexpressing and knockout cell lines reveals several protein clusters that are distinctly regulated by each of the FAM134 paralogues as well as a cluster of commonly regulated ER-resident proteins. Utilizing pro-Collagen I, as a shared ER-phagy substrate, we observe that FAM134A acts in a LIR-independent manner and compensates for the loss of FAM134B and FAM134C, respectively. FAM134C instead is unable to compensate for the loss of its paralogues. Taken together, our data show that FAM134 paralogues contribute to common and unique ER-phagy pathways.


Asunto(s)
Proteínas de la Membrana , Proteómica , Autofagia/genética , Colágeno , Retículo Endoplásmico/genética , Proteínas de la Membrana/genética , Control de Calidad
12.
Annu Rev Cell Dev Biol ; 25: 629-48, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19575648

RESUMEN

In the past few years, our molecular understanding of bone formation has continued to increase. This review aims to present a comprehensive view of the current state of knowledge in the field. Thus, it will cover our current knowledge of chondrogenesis and osteoblastogenesis. It will also cover the most salient aspects of osteoblast function.


Asunto(s)
Condrogénesis , Regulación del Desarrollo de la Expresión Génica , Osteogénesis , Animales , Humanos , Osteoblastos/metabolismo , Factores de Transcripción/metabolismo
13.
EMBO J ; 36(17): 2544-2552, 2017 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-28754656

RESUMEN

TFEB is a master regulator for transcription of genes involved in autophagy and lysosome biogenesis. Activity of TFEB is inhibited upon its serine phosphorylation by mTOR The overall mechanisms by which TFEB activity in the cell is regulated are not well elucidated. Specifically, the mechanisms of TFEB turnover and how they might influence its activity remain unknown. Here, we show that STUB1, a chaperone-dependent E3 ubiquitin ligase, modulates TFEB activity by preferentially targeting inactive phosphorylated TFEB for degradation by the ubiquitin-proteasome pathway. Phosphorylated TFEB accumulated in STUB1-deficient cells and in tissues of STUB1-deficient mice resulting in reduced TFEB activity. Conversely, cellular overexpression of STUB1 resulted in reduced phosphorylated TFEB and increased TFEB activity. STUB1 preferentially interacted with and ubiqutinated phosphorylated TFEB, targeting it to proteasomal degradation. Consistent with reduced TFEB activity, accumulation of phosphorylated TFEB in STUB1-deficient cells resulted in reduced autophagy and reduced mitochondrial biogenesis. These studies reveal that the ubiquitin-proteasome pathway participates in regulating autophagy and lysosomal functions by regulating the activity of TFEB.


Asunto(s)
Autofagia/fisiología , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Lisosomas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Fibroblastos/metabolismo , Células HEK293 , Células HeLa , Humanos , Ratones Noqueados , Fosforilación , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética
14.
Nature ; 528(7581): 272-5, 2015 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-26595272

RESUMEN

Skeletal growth relies on both biosynthetic and catabolic processes. While the role of the former is clearly established, how the latter contributes to growth-promoting pathways is less understood. Macroautophagy, hereafter referred to as autophagy, is a catabolic process that plays a fundamental part in tissue homeostasis. We investigated the role of autophagy during bone growth, which is mediated by chondrocyte rate of proliferation, hypertrophic differentiation and extracellular matrix (ECM) deposition in growth plates. Here we show that autophagy is induced in growth-plate chondrocytes during post-natal development and regulates the secretion of type II collagen (Col2), the major component of cartilage ECM. Mice lacking the autophagy related gene 7 (Atg7) in chondrocytes experience endoplasmic reticulum storage of type II procollagen (PC2) and defective formation of the Col2 fibrillary network in the ECM. Surprisingly, post-natal induction of chondrocyte autophagy is mediated by the growth factor FGF18 through FGFR4 and JNK-dependent activation of the autophagy initiation complex VPS34-beclin-1. Autophagy is completely suppressed in growth plates from Fgf18(-/-) embryos, while Fgf18(+/-) heterozygous and Fgfr4(-/-) mice fail to induce autophagy during post-natal development and show decreased Col2 levels in the growth plate. Strikingly, the Fgf18(+/-) and Fgfr4(-/-) phenotypes can be rescued in vivo by pharmacological activation of autophagy, pointing to autophagy as a novel effector of FGF signalling in bone. These data demonstrate that autophagy is a developmentally regulated process necessary for bone growth, and identify FGF signalling as a crucial regulator of autophagy in chondrocytes.


Asunto(s)
Autofagia/fisiología , Desarrollo Óseo/fisiología , Factores de Crecimiento de Fibroblastos/genética , Transducción de Señal , Animales , Autofagia/genética , Proteína 7 Relacionada con la Autofagia , Desarrollo Óseo/genética , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Condrocitos/citología , Condrocitos/metabolismo , Colágeno Tipo II/metabolismo , Embrión de Mamíferos , Matriz Extracelular/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Placa de Crecimiento/citología , Placa de Crecimiento/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo
15.
Genes Dev ; 27(8): 955-69, 2013 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-23599343

RESUMEN

Bone resorption by osteoclasts requires a large number of lysosomes that release proteases in the resorption lacuna. Whether lysosomal biogenesis is a consequence of the action of transcriptional regulators of osteoclast differentiation or is under the control of a different and specific transcriptional pathway remains unknown. We show here, through cell-based assays and cell-specific gene deletion experiments in mice, that the osteoclast differentiation factor RANKL promotes lysosomal biogenesis once osteoclasts are differentiated through the selective activation of TFEB, a member of the MITF/TFE family of transcription factors. This occurs following PKCß phosphorylation of TFEB on three serine residues located in its last 15 amino acids. This post-translational modification stabilizes and increases the activity of this transcription factor. Supporting these biochemical observations, mice lacking in osteoclasts--either TFEB or PKCß--show decreased lysosomal gene expression and increased bone mass. Altogether, these results uncover a RANKL-dependent signaling pathway taking place in differentiated osteoclasts and culminating in the activation of TFEB to enhance lysosomal biogenesis-a necessary step for proper bone resorption.


Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Regulación de la Expresión Génica , Lisosomas/metabolismo , Osteoclastos/fisiología , Proteína Quinasa C/metabolismo , Ligando RANK/metabolismo , Transducción de Señal , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Diferenciación Celular , Línea Celular , Femenino , Eliminación de Gen , Ratones , Ratones Endogámicos C57BL , Osteoclastos/citología , Osteoclastos/metabolismo , Fosforilación , Proteína Quinasa C/genética , Proteína Quinasa C beta , Procesamiento Proteico-Postraduccional , Ligando RANK/genética
16.
Mol Ther ; 24(12): 2054-2063, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27658524

RESUMEN

Enzyme replacement therapy (ERT) is the standard of care for several lysosomal storage diseases (LSDs). ERT, however, requires multiple and costly administrations and has limited efficacy. We recently showed that a single high dose administration of adeno-associated viral vector serotype 8 (AAV2/8) is at least as effective as weekly ERT in a mouse model of mucopolysaccharidosis type VI (MPS VI). However, systemic administration of high doses of AAV might result in both cell-mediated immune responses and insertional mutagenesis. Here we evaluated whether the combination of low doses of AAV2/8 with a less frequent (monthly) than canonical (weekly) ERT schedule may be as effective as the single treatments at high doses or frequent regimen. A greater reduction of both urinary glycosaminoglycans, considered a sensitive biomarker of therapeutic efficacy, and storage in the myocardium and heart valves was observed in mice receiving the combined than the single therapies. Importantly, these levels of correction were similar to those we obtained in a previous study following either high doses of AAV2/8 or weekly ERT. Our data show that low-dose gene therapy can be used as a means to rarify ERT administration, thus reducing both the risks and costs associated with either therapies.


Asunto(s)
Terapia Combinada/métodos , Terapia de Reemplazo Enzimático/métodos , Terapia Genética/métodos , Enfermedades por Almacenamiento Lisosomal/terapia , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Vectores Genéticos/administración & dosificación , Glicosaminoglicanos/orina , Humanos , Enfermedades por Almacenamiento Lisosomal/orina , Ratones , Resultado del Tratamiento
17.
EMBO J ; 31(5): 1095-108, 2012 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-22343943

RESUMEN

The lysosome plays a key role in cellular homeostasis by controlling both cellular clearance and energy production to respond to environmental cues. However, the mechanisms mediating lysosomal adaptation are largely unknown. Here, we show that the Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis, colocalizes with master growth regulator mTOR complex 1 (mTORC1) on the lysosomal membrane. When nutrients are present, phosphorylation of TFEB by mTORC1 inhibits TFEB activity. Conversely, pharmacological inhibition of mTORC1, as well as starvation and lysosomal disruption, activates TFEB by promoting its nuclear translocation. In addition, the transcriptional response of lysosomal and autophagic genes to either lysosomal dysfunction or pharmacological inhibition of mTORC1 is suppressed in TFEB-/- cells. Interestingly, the Rag GTPase complex, which senses lysosomal amino acids and activates mTORC1, is both necessary and sufficient to regulate starvation- and stress-induced nuclear translocation of TFEB. These data indicate that the lysosome senses its content and regulates its own biogenesis by a lysosome-to-nucleus signalling mechanism that involves TFEB and mTOR.


Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Núcleo Celular/fisiología , Lisosomas/fisiología , Proteínas/metabolismo , Transducción de Señal , Animales , Línea Celular , Humanos , Inmunoprecipitación , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Microscopía Fluorescente , Modelos Biológicos , Complejos Multiproteicos , Unión Proteica , Mapeo de Interacción de Proteínas , Serina-Treonina Quinasas TOR
19.
Arterioscler Thromb Vasc Biol ; 34(9): 1942-1952, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25060788

RESUMEN

OBJECTIVE: Recent reports of a proatherogenic phenotype in mice with macrophage-specific autophagy deficiency have renewed interest in the role of the autophagy-lysosomal system in atherosclerosis. Lysosomes have the unique ability to process both exogenous material, including lipids and autophagy-derived cargo such as dysfunctional proteins/organelles. We aimed to understand the effects of an atherogenic lipid environment on macrophage lysosomes and to evaluate novel ways to modulate this system. APPROACH AND RESULTS: Using a variety of complementary techniques, we show that oxidized low-density lipoproteins and cholesterol crystals, commonly encountered lipid species in atherosclerosis, lead to profound lysosomal dysfunction in cultured macrophages. Disruptions in lysosomal pH, proteolytic capacity, membrane integrity, and morphology are readily seen. Using flow cytometry, we find that macrophages isolated from atherosclerotic plaques also display features of lysosome dysfunction. We then investigated whether enhancing lysosomal function can be beneficial. Transcription factor EB (TFEB) is the only known transcription factor that is a master regulator of lysosomal biogenesis although its role in macrophages has not been studied. Lysosomal stress induced by chloroquine or atherogenic lipids leads to TFEB nuclear translocation and activation of lysosomal and autophagy genes. TFEB overexpression in macrophages further augments this prodegradative response and rescues several deleterious effects seen with atherogenic lipid loading as evidenced by blunted lysosomal dysfunction, reduced secretion of the proinflammatory cytokine interleukin-1ß, enhanced cholesterol efflux, and decreased polyubiquitinated protein aggregation. CONCLUSIONS: Taken together, these data demonstrate that lysosomal function is markedly impaired in atherosclerosis and suggest that induction of a lysosomal biogenesis program in macrophages has antiatherogenic effects.


Asunto(s)
Aterosclerosis/metabolismo , Lisosomas/fisiología , Macrófagos Peritoneales/fisiología , Animales , Apolipoproteínas E/deficiencia , Aterosclerosis/patología , Autofagia , Proteína 5 Relacionada con la Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Permeabilidad de la Membrana Celular , Cloroquina/farmacología , Colesterol/metabolismo , Concentración de Iones de Hidrógeno , Cuerpos de Inclusión/metabolismo , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Lípidos , Lipoproteínas LDL/metabolismo , Lisosomas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología , Poliubiquitina/metabolismo , Proteolisis , Esterol Esterasa/metabolismo , Transcripción Genética
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