RESUMEN
The sorting nexins (SNXs) are a family of peripheral membrane proteins that direct protein trafficking decisions within the endocytic network. Emerging evidence in yeast and mammalian cells implicates a subgroup of SNXs in selective and non-selective forms of autophagy. Using siRNA and CRISPR-Cas9, we demonstrate that the SNX-BAR protein SNX4 is needed for efficient LC3 (also known as MAP1LC3) lipidation and autophagosome assembly in mammalian cells. SNX-BARs exist as homo- and hetero-dimers, and we show that SNX4 forms functional heterodimers with either SNX7 or SNX30 that associate with tubulovesicular endocytic membranes. Detailed image-based analysis during the early stages of autophagosome assembly reveals that SNX4-SNX7 is an autophagy-specific SNX-BAR heterodimer, required for efficient recruitment and/or retention of core autophagy regulators at the nascent isolation membrane. SNX4 partially colocalises with juxtanuclear ATG9A-positive membranes, with our data linking the autophagy defect upon SNX4 disruption to the mis-trafficking and/or retention of ATG9A in the Golgi region. Taken together, our findings show that the SNX4-SNX7 heterodimer coordinates ATG9A trafficking within the endocytic network to establish productive autophagosome assembly sites, thus extending knowledge of SNXs as positive regulators of autophagy.
Asunto(s)
Autofagosomas , Endosomas , Nexinas de Clasificación , Animales , Autofagosomas/metabolismo , Autofagia , Endosomas/metabolismo , Transporte de Proteínas , Nexinas de Clasificación/genética , Nexinas de Clasificación/metabolismoRESUMEN
AIMS/HYPOTHESIS: Podocytes are insulin-responsive cells of the glomerular filtration barrier and are key in preventing albuminuria, a hallmark feature of diabetic nephropathy. While there is evidence that a loss of insulin signalling to podocytes is detrimental, the molecular mechanisms underpinning the development of podocyte insulin resistance in diabetes remain unclear. Thus, we aimed to further investigate podocyte insulin responses early in the context of diabetic nephropathy. METHODS: Conditionally immortalised human and mouse podocyte cell lines and glomeruli isolated from db/db DBA/2J mice were studied. Podocyte insulin responses were investigated with western blotting, cellular glucose uptake assays and automated fluorescent imaging of the actin cytoskeleton. Quantitative (q)RT-PCR was employed to investigate changes in mRNA. Human cell lines stably overproducing the insulin receptor (IR) and nephrin were also generated, using lentiviral constructs. RESULTS: Podocytes exposed to a diabetic environment (high glucose, high insulin and the proinflammatory cytokines TNF-α and IL-6) become insulin resistant with respect to glucose uptake and activation of phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling. These podocytes lose expression of the IR as a direct consequence of prolonged exposure to high insulin concentrations, which causes an increase in IR protein degradation via a proteasome-dependent and bafilomycin-sensitive pathway. Reintroducing the IR into insulin-resistant human podocytes rescues upstream phosphorylation events, but not glucose uptake. Stable expression of nephrin is also required for the insulin-stimulated glucose uptake response in podocytes and for efficient insulin-stimulated remodelling of the actin cytoskeleton. CONCLUSIONS/INTERPRETATION: Together, these results suggest that IR degradation, caused by high levels of insulin, drives early podocyte insulin resistance, and that both the IR and nephrin are required for full insulin sensitivity of this cell. This could be highly relevant for the development of nephropathy in individuals with type 2 diabetes, who are commonly hyperinsulinaemic in the early phases of their disease.
Asunto(s)
Resistencia a la Insulina/fisiología , Insulina/farmacología , Podocitos/efectos de los fármacos , Podocitos/metabolismo , Receptor de Insulina/metabolismo , Animales , Western Blotting , Células Cultivadas , Nefropatías Diabéticas/metabolismo , Humanos , Inmunoprecipitación , Masculino , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacosRESUMEN
The erythrocyte is one of the best characterized human cells. However, studies of the process whereby human reticulocytes mature to erythrocytes have been hampered by the difficulty of obtaining sufficient numbers of cells for analysis. In the present study, we describe an in vitro culture system producing milliliter quantities of functional mature human adult reticulocytes from peripheral blood CD34(+) cells. We show that the final stage of reticulocyte maturation occurs by a previously undescribed mechanism in which large glycophorin A-containing vesicles forming at the cytosolic face of the plasma membrane are internalized and fuse with autophagosomes before expulsion of the autophagosomal contents by exocytosis. Early reticulocyte maturation is characterized by the selective elimination of unwanted plasma membrane proteins (CD71, CD98, and ß1 integrin) through the endosome-exosome pathway. In contrast, late maturation is characterized by the generation of large glycophorin A-decorated vesicles of autophagic origin.
Asunto(s)
Exocitosis/fisiología , Glicoforinas/metabolismo , Fusión de Membrana/fisiología , Fagosomas/fisiología , Reticulocitos/fisiología , Vesículas Transportadoras/fisiología , Adulto , Diferenciación Celular , Membrana Celular/metabolismo , Eritrocitos/fisiología , Eritrocitos/ultraestructura , Humanos , Microscopía Confocal , Oxígeno/metabolismo , Fagosomas/metabolismo , Reticulocitos/metabolismo , Reticulocitos/ultraestructura , Vesículas Transportadoras/metabolismoRESUMEN
Insulin signaling to the glomerular podocyte via the insulin receptor (IR) is critical for kidney function. In this study we show that near-complete knockout of the closely related insulin-like growth factor 1 receptor (IGF1R) in podocytes is detrimental, resulting in albuminuria in vivo and podocyte cell death in vitro. In contrast, partial podocyte IGF1R knockdown confers protection against doxorubicin-induced podocyte injury. Proteomic analysis of cultured podocytes revealed that while near-complete loss of podocyte IGF1R results in the downregulation of mitochondrial respiratory complex I and DNA damage repair proteins, partial IGF1R inhibition promotes respiratory complex expression. This suggests that altered mitochondrial function and resistance to podocyte stress depends on the level of IGF1R suppression, the latter determining whether receptor inhibition is protective or detrimental. Our work suggests that the partial suppression of podocyte IGF1R could have therapeutic benefits in treating albuminuric kidney disease.
RESUMEN
Disruption of the insulin-PI3K-Akt signalling pathway in kidney podocytes causes endoplasmic reticulum (ER) stress, leading to podocyte apoptosis and proteinuria in diabetic nephropathy. We hypothesised that by improving insulin sensitivity we could protect podocytes from ER stress. Here we use established activating transcription factor 6 (ATF6)- and ER stress element (ERSE)-luciferase assays alongside a novel high throughput imaging-based C/EBP homologous protein (CHOP) assay to examine three models of improved insulin sensitivity. We find that by improving insulin sensitivity at the level of the insulin receptor (IR), either by IR over-expression or by knocking down the negative regulator of IR activity, protein tyrosine-phosphatase 1B (PTP1B), podocytes are protected from ER stress caused by fatty acids or diabetic media containing high glucose, high insulin and inflammatory cytokines TNFα and IL-6. However, contrary to this, knockdown of the negative regulator of PI3K-Akt signalling, phosphatase and tensin homolog deleted from chromosome 10 (PTEN), sensitizes podocytes to ER stress and apoptosis, despite increasing Akt phosphorylation. This indicates that protection from ER stress is conferred through not just the PI3K-Akt pathway, and indeed we find that inhibiting the MEK/ERK signalling pathway rescues PTEN knockdown podocytes from ER stress.
Asunto(s)
Estrés del Retículo Endoplásmico , Fosfatidilinositol 3-Quinasas/metabolismo , Podocitos/fisiología , Receptor de Insulina/metabolismo , Transducción de Señal , Animales , Apoptosis , Células Cultivadas , Insulina/metabolismo , Ratones , Fosfohidrolasa PTEN/metabolismo , Fosforilación , Podocitos/citología , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismoRESUMEN
Wholesale depletion of membrane organelles and extrusion of the nucleus are hallmarks of mammalian erythropoiesis. Using quantitative EM and fluorescence imaging we have investigated how autophagy contributes to organelle removal in an ex vivo model of human erythroid differentiation. We found that autophagy is induced at the polychromatic erythroid stage, and that autophagosomes remain abundant until enucleation. This stimulation of autophagy was concomitant with the transcriptional upregulation of many autophagy genes: of note, expression of all ATG8 mammalian paralog family members was stimulated, and increased expression of a subset of ATG4 family members (ATG4A and ATG4D) was also observed. Stable expression of dominant-negative ATG4 cysteine mutants (ATG4B (C74A) ; ATG4D (C144A) ) did not markedly delay or accelerate differentiation of human erythroid cells; however, quantitative EM demonstrated that autophagosomes are assembled less efficiently in ATG4B (C74A) -expressing progenitor cells, and that cells expressing either mutant accumulate enlarged amphisomes that cannot be degraded. The appearance of these hybrid autophagosome/endosome structures correlated with the contraction of the lysosomal compartment, suggesting that the actions of ATG4 family members (particularly ATG4B) are required for the control of autophagosome fusion with late, degradative compartments in differentiating human erythroblasts.
Asunto(s)
Autofagia , Diferenciación Celular , Cisteína Endopeptidasas/metabolismo , Fagosomas/metabolismo , Homología de Secuencia de Aminoácido , Proteínas Relacionadas con la Autofagia , Compartimento Celular , Cisteína/genética , Eritroblastos/citología , Eritroblastos/metabolismo , Eritroblastos/ultraestructura , Células HEK293 , Células HeLa , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Proteínas Mutantes/metabolismo , Fagosomas/ultraestructura , Regulación hacia ArribaRESUMEN
The Atg4 cysteine proteases play crucial roles in the processing of Atg8 proteins during autophagy, but their regulation during cellular stress and differentiation remains poorly understood. We have found that two Atg4 family members--Atg4C and Atg4D--contain cryptic mitochondrial targeting sequences immediately downstream of their canonical (DEVD) caspase cleavage sites. Consequently, caspase-cleaved Atg4D (ΔN63 Atg4D) localizes to the mitochondrial matrix when expressed in mammalian cells, where it undergoes further processing to a ~42 kDa mitochondrial form. Interestingly, caspase cleavage is not needed for Atg4D mitochondrial import, because ~42 kDa mitochondrial Atg4D is observed in cells treated with caspase inhibitors and in cells expressing caspase-resistant Atg4D (DEVA(63)). Using HeLa cell lines stably expressing ΔN63 Atg4D, we showed that mitochondrial Atg4D sensitizes cells to cell death in the presence of the mitochondrial uncoupler, CCCP, and that mitochondrial cristae are less extensive in these cells. We further showed that the organization of mitochondrial cristae is altered during the mitochondrial clearance phase in differentiating primary human erythroblasts stably expressing ΔN63 Atg4D, and that these cells have elevated levels of mitochondrial reactive oxygen species (ROS) during late stages of erythropoiesis. Together these data suggest that the import of Atg4D during cellular stress and differentiation may play important roles in the regulation of mitochondrial physiology, ROS, mitophagy and cell viability.
Asunto(s)
Caspasas/metabolismo , Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/metabolismo , Mitocondrias/metabolismo , Estrés Oxidativo , Señales de Clasificación de Proteína , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Autofagia , Proteínas Relacionadas con la Autofagia , Supervivencia Celular , Eritroblastos/metabolismo , Eritroblastos/ultraestructura , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Mitocondrias/ultraestructura , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Transporte de ProteínasRESUMEN
The maturation of reticulocytes into functional erythrocytes is a complex process requiring extensive cytoplasmic and plasma membrane remodeling, cytoskeletal rearrangements and changes to cellular architecture. Autophagy is implicated in the sequential removal of erythroid organelles during erythropoiesis, although how this is regulated during late stages of erythroid differentiation, and the potential contribution of autophagy during reticulocyte maturation, remain unclear. Using an optimized ex vivo differentiation system for human erythropoiesis, we have observed that maturing reticulocytes are characterized by the presence of one or few large vacuolar compartments. These label strongly for glycophorin A (GYPA/GPA) which is internalized from the plasma membrane; however, they also contain organellar remnants (ER, Golgi, mitochondria) and stain strongly for LC3, suggesting that they are endocytic/autophagic hybrid structures. Interestingly, we observed the release of these vacuoles by exocytosis in maturing reticulocytes, and speculate that autophagy is needed to concentrate the final remnants of the reticulocyte endomembrane system in autophagosome/endosome hybrid compartments that are primed to undergo exocytosis.
Asunto(s)
Exocitosis/fisiología , Glicoforinas/metabolismo , Fusión de Membrana/fisiología , Fagosomas/fisiología , Reticulocitos/fisiología , Vesículas Transportadoras/fisiología , HumanosRESUMEN
The Atg4 family of endopeptidases regulates autophagosome biogenesis by priming newly synthesized Atg8 to enable covalent attachment of phosphatidylethanolamine, and by delipidating Atg8 at the lysosomal fusion step. Control of Atg4 activity is therefore crucial, although little is known about how these molecules are regulated in living cells. We have found that one human Atg4 family member (Atg4D) is cleaved at DEVD(63)K by caspase-3 during apoptosis. Importantly, our studies suggest that native Atg4D is enzymatically inactive, but gains GABARAP-L1 priming/delipidation activity following caspase cleavage. Caspase-cleaved Atg4D is also highly cytotoxic; however, toxicity is not due to enhanced autophagy, but is mediated by a putative C-terminal BH3 domain, and is associated with transient recruitment of Atg4D to mitochondria.
Asunto(s)
Apoptosis/fisiología , Autofagia/fisiología , Cisteína Endopeptidasas/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Relacionadas con la Autofagia , Beclina-1 , Humanos , Proteínas de la Membrana/metabolismoRESUMEN
Autophagy is an important catabolic process with roles in cell survival and cell death. It sequesters cytosol and organelles within double-membrane autophagosomes that deliver their contents to lysosomes for degradation. Autophagosome biogenesis is coordinated by the autophagy-related protein 4 (Atg4) family of C54 endopeptidases (Atg4A-Atg4D). These enzymes prime and then later delipidate the autophagosome marker, Atg8. Here, we show that one family member, Atg4D, is cleaved by caspase-3 in vitro and in apoptotic cells. Atg4D is a poor priming and delipidation enzyme in vitro, but truncated DeltaN63 Atg4D displays increased activity against the Atg8 paralogue, gamma-aminobutyric acid receptor-associated protein-like 1 (GABARAP-L1). In living cells, DeltaN63 Atg4D stimulates the delipidation of GABARAP-L1, whereas siRNA silencing of the gene expressing Atg4D abrogates GABARAP-L1 autophagosome formation and sensitises cells to starvation and staurosporine-induced cell death. Interestingly, Atg4D overexpression induces apoptosis, which is preceded by the caspase-independent recruitment of Atg4D to mitochondria and is facilitated by a putative C-terminal Bcl-2 homology 3 (BH3) domain. Atg4D also acquires affinity for damaged mitochondria in cells treated with hydrogen peroxide. These data suggest that Atg4D is an autophagy regulator that links mitochondrial dysfunction with apoptosis.