RESUMEN
Endocytosis is a fundamentally important process through which material is internalized into cells from the extracellular environment. In the renal proximal tubule, endocytosis of the abundant scavenger receptor megalin and its co-receptor cubilin play a vital role in retrieving low molecular weight proteins from the renal filtrate. Although we know much about megalin and its ligands, the machinery and mechanisms by which the receptor is trafficked through the endosomal system remain poorly defined. In this study, we show that inositol phosphatase interacting protein of 27 kDa (Ipip27A), an interacting partner of the Lowe syndrome protein oculocerebrorenal syndrome of Lowe (OCRL), is required for endocytic traffic of megalin within the proximal renal tubule of zebrafish larvae. Knockout of Ipip27A phenocopies the endocytic phenotype seen upon loss of OCRL, with a deficit in uptake of both fluid-phase and protein cargo, which is accompanied by a reduction in megalin abundance and altered endosome morphology. Rescue and co-depletion experiments indicate that Ipip27A functions together with OCRL to support proximal tubule endocytosis. The results therefore identify Ipip27A as a new player in endocytic traffic in the proximal tubule in vivo and support the view that defective endocytosis underlies the renal tubulopathy in Lowe syndrome and Dent-2 disease.
Asunto(s)
Síndrome Oculocerebrorrenal , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Endocitosis/genética , Endosomas/genética , Endosomas/metabolismo , Femenino , Humanos , Fosfatos de Inositol/metabolismo , Túbulos Renales Proximales/metabolismo , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/genética , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Masculino , Síndrome Oculocerebrorrenal/genética , Síndrome Oculocerebrorrenal/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Proteínas/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Apolipoprotein L1 (ApoL1) is a circulating innate immunity protein protecting against trypanosome infection. However, two ApoL1 coding variants are associated with a highly increased risk of chronic kidney disease. Here we present X-ray and NMR structures of the N-terminal domain (NTD) of ApoL1 and of its closest relative ApoL2. In both proteins, four of the five NTD helices form a four-helix core structure which is different from the classical four-helix bundle and from the pore-forming domain of colicin A. The reactivity with a conformation-specific antibody and structural models predict that this four-helix motif is also present in the NTDs of ApoL3 and ApoL4, suggesting related functions within the small ApoL family. The long helix 5 of ApoL1 is conformationally flexible and contains the BH3-like region. This BH3-like α-helix resembles true BH3 domains only in sequence and structure but not in function, since it does not bind to the pro-survival members of the Bcl-2 family, suggesting a Bcl-2-independent role in cytotoxicity. These findings should expedite a more comprehensive structural and functional understanding of the ApoL immune protein family.
Asunto(s)
Apolipoproteína L1/química , Apolipoproteínas L/química , Dominios Proteicos , Apolipoproteína L1/genética , Apolipoproteína L1/metabolismo , Apolipoproteínas L/genética , Apolipoproteínas L/metabolismo , HumanosRESUMEN
BACKGROUND: APOL1 is found in human kidney podocytes and endothelia. Variants G1 and G2 of the APOL1 gene account for the high frequency of nondiabetic CKD among African Americans. Proposed mechanisms of kidney podocyte cytotoxicity resulting from APOL1 variant overexpression implicate different subcellular compartments. It is unclear where endogenous podocyte APOL1 resides, because previous immunolocalization studies utilized overexpressed protein or commercially available antibodies that crossreact with APOL2. This study describes and distinguishes the locations of both APOLs. METHODS: Immunohistochemistry, confocal and immunoelectron microscopy, and podocyte fractionation localized endogenous and transfected APOL1 using a large panel of novel APOL1-specific mouse and rabbit monoclonal antibodies. RESULTS: Both endogenous podocyte and transfected APOL1 isoforms vA and vB1 (and a little of isoform vC) localize to the luminal face of the endoplasmic reticulum (ER) and to the cell surface, but not to mitochondria, endosomes, or lipid droplets. In contrast, APOL2, isoform vB3, and most vC of APOL1 localize to the cytoplasmic face of the ER and are consequently absent from the cell surface. APOL1 knockout podocytes do not stain for APOL1, attesting to the APOL1-specificity of the antibodies. Stable re-transfection of knockout podocytes with inducible APOL1-G0, -G1, and -G2 showed no differences in localization among variants. CONCLUSIONS: APOL1 is found in the ER and plasma membrane, consistent with either the ER stress or surface cation channel models of APOL1-mediated cytotoxicity. The surface localization of APOL1 variants potentially opens new therapeutic targeting avenues.
Asunto(s)
Apolipoproteína L1/análisis , Membrana Celular/química , Retículo Endoplásmico/química , Podocitos/química , Animales , Anticuerpos/inmunología , Apolipoproteína L1/inmunología , Apolipoproteínas L/análisis , Células COS , Células Cultivadas , Chlorocebus aethiops , Reacciones Cruzadas , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Podocitos/ultraestructuraRESUMEN
Nurr1, a nuclear receptor essential for the development, maintenance, and survival of midbrain dopaminergic neurons, is a potential therapeutic target for Parkinson's disease, a neurological disorder characterized by the degeneration of these same neurons. Efforts to identify Nurr1 agonists have been hampered by the recognition that it lacks several classic regulatory elements of nuclear receptor function, including the canonical ligand-binding pocket. Here we report that the dopamine metabolite 5,6-dihydroxyindole (DHI) binds directly to and modulates the activity of Nurr1. Using biophysical assays and X-ray crystallography, we show that DHI binds to the ligand-binding domain within a non-canonical pocket, forming a covalent adduct with Cys566. In cultured cells and zebrafish, DHI stimulates Nurr1 activity, including the transcription of target genes underlying dopamine homeostasis. These findings suggest avenues for developing synthetic Nurr1 ligands to ameliorate the symptoms and progression of Parkinson's disease.
Asunto(s)
Dopamina/metabolismo , Indoles/metabolismo , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Animales , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Dopamina/química , Humanos , Indoles/química , Indoles/farmacología , Larva/metabolismo , Simulación de Dinámica Molecular , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/química , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Estrés Oxidativo/efectos de los fármacos , Dominios Proteicos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Termodinámica , Transcripción Genética/efectos de los fármacos , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismoRESUMEN
The endocannabinoid system (eCBs), named after the plant Cannabis sativa, comprises cannabinoid receptors, endogenous ligands known as "endocannabinoids", and enzymes involved in the biosynthesis and degradation of these ligands, as well as putative transporters for these ligands. ECBs proteins and small molecules have been detected in early embryonic stages of many vertebrate models. As a result, cannabinoid receptors and endogenous as well as exogenous cannabinoids influence development and behavior in many vertebrate species. Understanding the precise mechanisms of action for the eCBs will provide an invaluable guide towards elucidation of vertebrate development and will also help delineate how developmental exposure to marijuana might impact health and cognitive/executive functioning in adulthood. Here we review the developmental roles of the eCBs in vertebrates, focusing our attention on the zebrafish model. Since little is known regarding the eCBs in zebrafish, we provide new data on the expression profiles of eCBs genes during development and in adult tissue types of this model organism. We also highlight exciting areas for future investigations, including the synaptic regulation of eCBs, its role in reward and addiction, and in nervous system development and plasticity.
Asunto(s)
Endocannabinoides/metabolismo , Receptores de Cannabinoides/metabolismo , Pez Cebra/embriología , Animales , Dronabinol/metabolismo , Pez Cebra/metabolismoRESUMEN
Phosphoinositides (PtdIns) control fundamental cell processes, and inherited defects of PtdIns kinases or phosphatases cause severe human diseases, including Lowe syndrome due to mutations in OCRL, which encodes a PtdIns(4,5)P2 5-phosphatase. Here we unveil a lysosomal response to the arrival of autophagosomal cargo in which OCRL plays a key part. We identify mitochondrial DNA and TLR9 as the cargo and the receptor that triggers and mediates, respectively, this response. This lysosome-cargo response is required to sustain the autophagic flux and involves a local increase in PtdIns(4,5)P2 that is confined in space and time by OCRL. Depleting or inhibiting OCRL leads to an accumulation of lysosomal PtdIns(4,5)P2, an inhibitor of the calcium channel mucolipin-1 that controls autophagosome-lysosome fusion. Hence, autophagosomes accumulate in OCRL-depleted cells and in the kidneys of Lowe syndrome patients. Importantly, boosting the activity of mucolipin-1 with selective agonists restores the autophagic flux in cells from Lowe syndrome patients.
Asunto(s)
Autofagosomas/fisiología , Autofagia/fisiología , Lisosomas/metabolismo , Fosfatidilinositoles/genética , Monoéster Fosfórico Hidrolasas/genética , Receptor Toll-Like 9/genética , Animales , Autofagia/genética , Línea Celular , Humanos , Mutación/genética , Síndrome Oculocerebrorrenal/genética , Síndrome Oculocerebrorrenal/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Pez CebraRESUMEN
The ability to grow embryonic organs ex vivo provides an opportunity to follow their differentiation in a controlled environment, with resulting insights into normal development. Additionally, similar strategies can be used to assess effects on organogenesis of physical and chemical manipulations. This study aimed to create an organ culture model with which to test physical manipulations to enhance healing of gut segments, thus generating a single functional organ. Embryonic mouse jejunum was isolated and cut into 2-3 mm tubes, which were placed in pairs, separated by a small gap, on semi-permeable supports. Each pair was linked by a nylon suture threaded through their lumens. After 3 days in organ culture fed by defined serum-free media, the rudiments differentiated to form tubes of smooth muscle surrounding a core of rudimentary villi. Of 34 such pairs, 74% had touching and well aligned proximate ends. Of these joined structures, 80% (59% of the total pairs) had a continuous lumen, as assessed by observing the trajectories of fluorescent dextrans injected into their distal ends. Fused organ pairs formed a single functional unit, as assessed by spontaneous contraction waves propagated along their lengths. In these healed intestines, peripherin(+) neurons formed a nexus in the zone of fusion, linking the rudiment pairs. In future, this system could be used to test whether growth factors enhance fusion. Such results should in turn inform the design of novel treatments for short bowel syndrome, a potentially fatal condition with a currently limited and imperfect range of therapies.
Asunto(s)
Intestino Delgado/embriología , Intestino Delgado/patología , Cicatrización de Heridas , Animales , Técnica del Anticuerpo Fluorescente , Técnicas In Vitro , Yeyuno/patología , Ratones , Técnicas de Cultivo de ÓrganosRESUMEN
Lowe syndrome and Dent-2 disease are caused by mutation of the inositol 5-phosphatase OCRL1. Despite our increased understanding of the cellular functions of OCRL1, the underlying basis for the renal tubulopathy seen in both human disorders, of which a hallmark is low molecular weight proteinuria, is currently unknown. Here, we show that deficiency in OCRL1 causes a defect in endocytosis in the zebrafish pronephric tubule, a model for the mammalian renal tubule. This coincides with a reduction in levels of the scavenger receptor megalin and its accumulation in endocytic compartments, consistent with reduced recycling within the endocytic pathway. We also observe reduced numbers of early endocytic compartments and enlarged vacuolar endosomes in the sub-apical region of pronephric cells. Cell polarity within the pronephric tubule is unaffected in mutant embryos. The OCRL1-deficient embryos exhibit a mild ciliogenesis defect, but this cannot account for the observed impairment of endocytosis. Catalytic activity of OCRL1 is required for renal tubular endocytosis and the endocytic defect can be rescued by suppression of PIP5K. These results indicate for the first time that OCRL1 is required for endocytic trafficking in vivo, and strongly support the hypothesis that endocytic defects are responsible for the renal tubulopathy in Lowe syndrome and Dent-2 disease. Moreover, our results reveal PIP5K as a potential therapeutic target for Lowe syndrome and Dent-2 disease.