RESUMEN
The ability to sense and respond to osmotic fluctuations is critical for the maintenance of cellular integrity. We used gene co-essentiality analysis to identify an unappreciated relationship between TSC22D2, WNK1, and NRBP1 in regulating cell volume homeostasis. All of these genes have paralogs and are functionally buffered for osmo-sensing and cell volume control. Within seconds of hyperosmotic stress, TSC22D, WNK, and NRBP family members physically associate into biomolecular condensates, a process that is dependent on intrinsically disordered regions (IDRs). A close examination of these protein families across metazoans revealed that TSC22D genes evolved alongside a domain in NRBPs that specifically binds to TSC22D proteins, which we have termed NbrT (NRBP binding region with TSC22D), and this co-evolution is accompanied by rapid IDR length expansion in WNK-family kinases. Our study reveals that TSC22D, WNK, and NRBP genes evolved in metazoans to co-regulate rapid cell volume changes in response to osmolarity.
Asunto(s)
Tamaño de la Célula , Proteína Quinasa Deficiente en Lisina WNK 1 , Humanos , Animales , Proteína Quinasa Deficiente en Lisina WNK 1/metabolismo , Proteína Quinasa Deficiente en Lisina WNK 1/genética , Evolución Molecular , Células HEK293 , Unión Proteica , Familia de Multigenes , Presión OsmóticaRESUMEN
The Nedd4 family of E3 ubiquitin ligases, consisting of a C2-WW(n)-HECT domain architecture, includes the closely related Nedd4/Nedd4-1 and Nedd4L/Nedd4-2, which play critical roles in human physiology and pathophysiology.This review focuses on the regulation of enzymatic activity of these Nedd4 proteins, as well as on their roles in regulating stability and function of membrane and other signaling proteins, such as ion channels, ion transporters, and growth factor receptors. The diseases caused by impairment of such regulation are discussed, as well as opportunities and challenges for targeting these enzymes for therapy.
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Complejos de Clasificación Endosomal Requeridos para el Transporte , Ubiquitina , Humanos , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/genética , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Nedd4-2 is an E3 ubiquitin ligase in which missense mutation is related to familial epilepsy, indicating its critical role in regulating neuronal network activity. However, Nedd4-2 substrates involved in neuronal network function have yet to be identified. Using mouse lines lacking Nedd4-1 and Nedd4-2, we identified astrocytic channel proteins inwardly rectifying K+ channel 4.1 (Kir4.1) and Connexin43 as Nedd4-2 substrates. We found that the expression of Kir4.1 and Connexin43 is increased upon conditional deletion of Nedd4-2 in astrocytes, leading to an elevation of astrocytic membrane ion permeability and gap junction activity, with a consequent reduction of γ-oscillatory neuronal network activity. Interestingly, our biochemical data demonstrate that missense mutations found in familial epileptic patients produce gain-of-function of the Nedd4-2 gene product. Our data reveal a process of coordinated astrocytic ion channel proteostasis that controls astrocyte function and astrocyte-dependent neuronal network activity and elucidate a potential mechanism by which aberrant Nedd4-2 function leads to epilepsy.
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Astrocitos , Permeabilidad de la Membrana Celular , Conexina 43 , Ubiquitina-Proteína Ligasas Nedd4 , Canales de Potasio de Rectificación Interna , Animales , Humanos , Ratones , Conexina 43/genética , Mutación Missense , Proteostasis , Canales de Potasio de Rectificación Interna/genética , Ubiquitina-Proteína Ligasas Nedd4/genética , EpilepsiaRESUMEN
Nedd4 (Nedd4-1) is an E3 ubiquitin ligase involved in crucial biological processes such as growth factor receptor signaling. While canonical Nedd4-1 comprises a C2-WW(4)-HECT domain architecture, alternative splicing produces non-canonical isoforms that are poorly characterized. Here we characterized Nedd4-1(NE), a primate-specific isoform of Nedd4-1 that contains a large N-terminal Extension (NE) that replaces most of the C2 domain. We show that Nedd4-1(NE) mRNA is ubiquitously expressed in human tissues and cell lines. Moreover, we found that Nedd4-1(NE) is more active than the canonical Nedd4-1 isoform, likely due to the absence of a C2 domain-mediated autoinhibitory mechanism. Additionally, we identified two Thr/Ser phosphoresidues in the NE region that act as binding sites for 14-3-3 proteins, and show that phosphorylation on these sites reduces substrate binding. Finally, we show that the NE region can act as a binding site for the RPB2 subunit of RNA polymerase II, a unique substrate of Nedd4-1(NE) but not the canonical Nedd4-1. Taken together, our results demonstrate that alternative splicing of the ubiquitin ligase Nedd4-1 can produce isoforms that differ in their catalytic activity, binding partners and substrates, and mechanisms of regulation.
Asunto(s)
Proteínas 14-3-3 , Empalme Alternativo , Animales , Humanos , Proteínas 14-3-3/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Ubiquitina-Proteína Ligasas Nedd4/genética , Fosforilación , Primates , Unión Proteica , Isoformas de Proteínas/genética , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
p38-MAPK is a stress-response kinase activated by hyperosmolarity. Here we interrogated the pathways involved. We show that p38-MAPK signaling is activated by hyperosmotic stimulation in various solutions, cell types and colonic organoids. Hyperosmolarity sensing is detected at the level of the upstream activators of p38-MAPK: TRAF2/ASK1 (but not Rac1) and MKK3/6/4. While WNK kinases are known osmo-sensors, we found, unexpectedly, that short (2 h) inhibition of WNKs (with WNK463) led to elevated p38-MAPK activity under hyperosmolarity, which was mediated by WNK463-dependent stimulation of TAK1 or TRAF2/ASK1, the upstream activators of MKK3/6/4. However, this effect was temporary and was reversed by long-term (2 days) incubation with WNK463. Accordingly, 2 days (but not 2 h) inhibition of p38-MAPK or its upstream activators ASK1 or TAK1, or WNKs, diminished regulatory volume increase (RVI) following cell shrinkage under hyperosmolarity. We also show that RVI mediated by the ion transporter NKCC1 is dependent on p38-MAPK. Since WNKs are known activators of NKCC1, we propose a WNK- > NKCC1- > p38-MAPK pathway that controls RVI. This pathway is augmented by NHE1. Additionally, hyperosmolarity inhibited mTORC1 activation and cell proliferation. Thus, activation of p38-MAPK and WNKs is important for RVI and for cell proliferation.
Asunto(s)
Sistema de Señalización de MAP Quinasas , Proteínas Quinasas p38 Activadas por Mitógenos , Activación Enzimática , MAP Quinasa Quinasa 3/metabolismo , Factor 2 Asociado a Receptor de TNF/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Regulation of catalytic activity of E3 ubiquitin ligases is critical for their cellular functions. We identified an unexpected mode of regulation of E3 catalytic activity by ions and osmolarity; enzymatic activity of the HECT family E3 Nedd4-2/Nedd4L is enhanced by increased intracellular Na+ ([Na+]i) and by hyperosmolarity. This stimulated activity is mediated by activation of p38-MAPK and is inhibited by WNKs. Moreover, protease (Furin)-mediated activation of the epithelial Na+ channel ENaC (a bona fide Nedd4-2 substrate), which leads to increased [Na+]i and osmolarity, results in enhanced Nedd4-2 catalytic activity. This enhancement is inhibited by a Furin inhibitor, by a protease-resistant ENaC mutant, or by treatment with the ENaC inhibitor amiloride. Moreover, WNK inhibition, which stimulates catalytic activity of Nedd4-2, leads to reduced levels of cell-surface ENaC and reduced channel activity. ENaC activity does not affect Nedd4-2:ENaC binding. Therefore, these results demonstrate activation of a ubiquitin ligase by Na+ and osmotic changes. Importantly, they reveal a negative feedback loop in which active ENaC leads to stimulation of catalytic activity of its own suppressor, Nedd4-2, to protect cells from excessive Na+ loading and hyperosmotic stress and to protect the animal from hypertension.
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Ubiquitina-Proteína Ligasas Nedd4 , Sodio , Animales , Catálisis , Cationes/metabolismo , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Furina/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/genética , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Concentración Osmolar , Sodio/metabolismoRESUMEN
Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis (CF)-causing mutant proteins. However, to date, CF-iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease-relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins. Specifically, we show how this assay can be employed to study CFTR and ENaC channels and an electrogenic acid transporter in the same iPSC-derived intestinal tissue. This phenotypic platform promises to expand CF therapy discovery to include strategies that target multiple determinants of epithelial fluid transport.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Intestinos/metabolismo , Organoides/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Animales , Diferenciación Celular , Perros , Canales Epiteliales de Sodio/metabolismo , Edición Génica , Humanos , Células de Riñón Canino Madin DarbyRESUMEN
Split reporter protein-based genetic section systems are widely used to identify and characterize protein-protein interactions (PPI). The assembly of split markers that antagonize toxins, rather than required for synthesis of missing metabolites, facilitates the seeding of high density of cells and selective growth. Here we present a newly developed split chloramphenicol acetyltransferase (split-CAT) -based genetic selection system. The N terminus fragment of CAT is fused downstream of the protein of interest and the C terminus fragment is tethered upstream to its postulated partner. We demonstrate the system's advantages for the study of PPIs. Moreover, we show that co-expression of a functional ubiquitylation cascade where the target and ubiquitin are tethered to the split-CAT fragments results in ubiquitylation-dependent selective growth. Since proteins do not have to be purified from the bacteria and due to the high sensitivity of the split-CAT reporter, detection of challenging protein cascades and post-translation modifications is enabled. In addition, we demonstrate that the split-CAT system responds to small molecule inhibitors and molecular glues (GLUTACs). The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli significantly simplify the interpretation of the results. We harnessed the developed system to demonstrate that like NEDD4, UBE3B also undergoes self-ubiquitylation-dependent inactivation. We show that self-ubiquitylation of UBE3B on K665 induces oligomerization and inactivation in yeast and mammalian cells respectively. Finally, we showcase the advantages of split-CAT in the study of human diseases by demonstrating that mutations in UBE3B that cause Kaufman oculocerebrofacial syndrome exhibit clear E. coli growth phenotypes.
Asunto(s)
Bioensayo/métodos , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , Expresión Génica , Genes Reporteros , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Activación Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Procesamiento Proteico-Postraduccional , ProteolisisRESUMEN
The Epithelial Na+ Channel, ENaC, comprised of 3 subunits (αßγ, or sometimes δßγENaC), plays a critical role in regulating salt and fluid homeostasis in the body. It regulates fluid reabsorption into the blood stream from the kidney to control blood volume and pressure, fluid absorption in the lung to control alveolar fluid clearance at birth and maintenance of normal airway surface liquid throughout life, and fluid absorption in the distal colon and other epithelial tissues. Moreover, recent studies have also revealed a role for sodium movement via ENaC in nonepithelial cells/tissues, such as endothelial cells in blood vessels and neurons. Over the past 25 years, major advances have been made in our understanding of ENaC structure, function, regulation, and role in human disease. These include the recently solved three-dimensional structure of ENaC, ENaC function in various tissues, and mutations in ENaC that cause a hereditary form of hypertension (Liddle syndrome), salt-wasting hypotension (PHA1), or polymorphism in ENaC that contributes to other diseases (such as cystic fibrosis). Moreover, great strides have been made in deciphering the regulation of ENaC by hormones (e.g., the mineralocorticoid aldosterone, glucocorticoids, vasopressin), ions (e.g., Na+ ), proteins (e.g., the ubiquitin-protein ligase NEDD4-2, the kinases SGK1, AKT, AMPK, WNKs & mTORC2, and proteases), and posttranslational modifications [e.g., (de)ubiquitylation, glycosylation, phosphorylation, acetylation, palmitoylation]. Characterization of ENaC structure, function, regulation, and role in human disease, including using animal models, are described in this article, with a special emphasis on recent advances in the field. © 2021 American Physiological Society. Compr Physiol 11:1-29, 2021.
Asunto(s)
Canales Epiteliales de Sodio , Hipertensión , Animales , Células Endoteliales/metabolismo , Canales Epiteliales de Sodio/genética , Humanos , Iones , Sodio/metabolismoRESUMEN
The eukaryotic elongation factor-2 kinase, eEF2K, which restricts protein translation elongation, has been identified as a potential therapeutic target for diverse types of malignancies including triple negative breast cancer (TNBC). However, the contexts in which eEF2K inhibition is essential in TNBC and its consequences on the proteome are largely unknown. Here we show that genetic or pharmacological inhibition of eEF2K cooperated with glutamine (Gln) starvation, and synergized with glutaminase (GLS1) inhibitors to suppress growth of diverse TNBC cell lines. eEF2K inhibition also synergized with depletion of eukaryotic translation initiation factor 4E-binding protein 1 (eIF4EBP1; 4EBP1), a suppressor of eukaryotic protein translation initiation factor 4E (eIF4E), to induce c-MYC and Cyclin D1 expression, yet attenuate growth of TNBC cells. Proteomic analysis revealed that whereas eEF2K depletion alone uniquely induced Cyclin Dependent Kinase 1 (CDK1) and 6 (CDK6), combined depletion of eEF2K and 4EBP1 resulted in overlapping effects on the proteome, with the highest impact on the 'Collagen containing extracellular matrix' pathway (e.g. COL1A1), as well as the amino-acid transporter, SLC7A5/LAT1, suggesting a regulatory loop via mTORC1. In addition, combined depletion of eEF2K and 4EBP1 indirectly reduced the levels of IFN-dependent innate immune response-related factors. Thus, eEF2K inhibition triggers cell cycle arrest/death under unfavourable metabolic conditions such as Gln-starvation/GLS1 inhibition or 4EBP1 depletion, uncovering new therapeutic avenues for TNBC and underscoring a pressing need for clinically relevant eEF2K inhibitors.
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Proteínas Adaptadoras Transductoras de Señales/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteínas de Ciclo Celular/genética , Quinasa del Factor 2 de Elongación/antagonistas & inhibidores , Glutaminasa/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Bencenoacetamidas/administración & dosificación , Bencenoacetamidas/farmacología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Ciclina D1/metabolismo , Ciclopentanos/farmacología , Sinergismo Farmacológico , Quinasa del Factor 2 de Elongación/genética , Femenino , Silenciador del Gen , Humanos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas/análisis , Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Sulfuros/administración & dosificación , Sulfuros/farmacología , Tiadiazoles/administración & dosificación , Tiadiazoles/farmacología , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/metabolismo , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Cystic Fibrosis is a lethal monogenic autosomal recessive disease linked to mutations in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) protein. The most frequent mutation is the deletion of phenylalanine at position 508 of the protein. This F508del-CFTR mutation leads to misfolded protein that is detected by the quality control machinery within the endoplasmic reticulum and targeted for destruction by the proteasome. Modulating quality control proteins as molecular chaperones is a promising strategy for attenuating the degradation and stabilizing the mutant CFTR at the plasma membrane. Among the molecular chaperones, the small heat shock protein HspB1 and HspB4 were shown to promote degradation of F508del-CFTR. Here, we investigated the impact of HspB5 expression and phosphorylation on transport to the plasma membrane, function and stability of F508del-CFTR. We show that a phosphomimetic form of HspB5 increases the transport to the plasma membrane, function and stability of F508del-CFTR. These activities are further enhanced in presence of therapeutic drugs currently used for the treatment of cystic fibrosis (VX-770/Ivacaftor, VX-770+VX-809/Orkambi). Overall, this study highlights the beneficial effects of a phosphorylated form of HspB5 on F508del-CFTR rescue and its therapeutic potential in cystic fibrosis.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Chaperonas Moleculares/metabolismo , Fenilalanina/metabolismo , Fosforilación/fisiología , Aminofenoles/farmacología , Aminopiridinas/farmacología , Animales , Benzodioxoles/farmacología , Línea Celular , Membrana Celular/metabolismo , Cristalinas/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Combinación de Medicamentos , Células HEK293 , Proteínas de Choque Térmico/metabolismo , Humanos , Masculino , Ratones , Chaperonas Moleculares/genética , Mutación/genética , Fenilalanina/genética , Fosforilación/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Quinolonas/farmacologíaRESUMEN
Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease characterized by patchy scarring of the distal lung with limited therapeutic options and poor prognosis. Here, we show that conditional deletion of the ubiquitin ligase Nedd4-2 (Nedd4l) in lung epithelial cells in adult mice produces chronic lung disease sharing key features with IPF including progressive fibrosis and bronchiolization with increased expression of Muc5b in peripheral airways, honeycombing and characteristic alterations in the lung proteome. NEDD4-2 is implicated in the regulation of the epithelial Na+ channel critical for proper airway surface hydration and mucus clearance and the regulation of TGFß signaling, which promotes fibrotic remodeling. Our data support a role of mucociliary dysfunction and aberrant epithelial pro-fibrotic response in the multifactorial disease pathogenesis. Further, treatment with the anti-fibrotic drug pirfenidone reduced pulmonary fibrosis in this model. This model may therefore aid studies of the pathogenesis and therapy of IPF.
Asunto(s)
Células Epiteliales/patología , Fibrosis Pulmonar Idiopática/genética , Pulmón/patología , Ubiquitina-Proteína Ligasas Nedd4/genética , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Adulto , Anciano , Animales , Biopsia , Modelos Animales de Enfermedad , Canales Epiteliales de Sodio/metabolismo , Humanos , Fibrosis Pulmonar Idiopática/tratamiento farmacológico , Fibrosis Pulmonar Idiopática/patología , Pulmón/citología , Ratones , Ratones Noqueados , Persona de Mediana Edad , Mucina 5B/metabolismo , Proteómica , Piridonas/administración & dosificación , UbiquitinaciónRESUMEN
mTORC1 regulates cellular growth and is activated by growth factors and by essential amino acids such as Leu. Leu enters cells via the Leu transporter LAT1-4F2hc (LAT1). Here we show that the Na+/K+/2Cl- cotransporter NKCC1 (SLC12A2), a known regulator of cell volume, is present in complex with LAT1. We further show that NKCC1 depletion or deletion enhances LAT1 activity, as well as activation of Akt and Erk, leading to activation of mTORC1 in cells, colonic organoids, and mouse colon. Moreover, NKCC1 depletion reduces intracellular Na+ concentration and cell volume (size) and mass and stimulates cell proliferation. NKCC1, therefore, suppresses mTORC1 by inhibiting its key activating signaling pathways. Importantly, by linking ion transport and cell volume regulation to mTORC1 function, NKCC1 provides a long-sought link connecting cell volume (size) to cell mass regulation.
Asunto(s)
Tamaño de la Célula , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Aminoácidos Esenciales/metabolismo , Animales , Bumetanida/farmacología , Línea Celular , Proliferación Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Transporte Iónico , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptor de Insulina/metabolismo , Transducción de SeñalRESUMEN
Drosophila Nedd4 (dNedd4) is a HECT E3 ubiquitin ligase present in two major isoforms: short (dNedd4S) and long (dNedd4Lo), with the latter containing two unique regions (N terminus and Middle). Although dNedd4S promotes neuromuscular synaptogenesis (NMS), dNedd4Lo inhibits it and impairs larval locomotion. To explain how dNedd4Lo inhibits NMS, MS analysis was performed to find its binding partners and identified SH3PX1, which binds dNedd4Lo unique Middle region. SH3PX1 contains SH3, PX, and BAR domains and is present at neuromuscular junctions, where it regulates active zone ultrastructure and presynaptic neurotransmitter release. Here, we demonstrate direct binding of SH3PX1 to the dNedd4Lo Middle region (which contains a Pro-rich sequence) in vitro and in cells, via the SH3PX1-SH3 domain. In Drosophila S2 cells, dNedd4Lo overexpression reduces SH3PX1 levels at the cell periphery. In vivo overexpression of dNedd4Lo post-synaptically, but not pre-synaptically, reduces SH3PX1 levels at the subsynaptic reticulum and impairs neurotransmitter release. Unexpectedly, larvae that overexpress dNedd4Lo post-synaptically and are heterozygous for a null mutation in SH3PX1 display increased neurotransmission compared with dNedd4Lo or SH3PX1 mutant larvae alone, suggesting a compensatory effect from the remaining SH3PX1 allele. These results suggest a post-synaptic-specific regulation of SH3PX1 by dNedd4Lo.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulación de la Expresión Génica , Larva/metabolismo , Ubiquitina-Proteína Ligasas Nedd4/metabolismo , Unión Neuromuscular/metabolismo , Transmisión Sináptica/fisiología , Animales , Proteínas Portadoras/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Péptidos y Proteínas de Señalización Intracelular , Larva/genética , Ubiquitina-Proteína Ligasas Nedd4/genética , Unión Neuromuscular/genética , Unión Proteica , Sinapsis/fisiología , Dominios Homologos srcRESUMEN
mTORC1 plays a crucial role in protein synthesis and cell proliferation and growth. It is activated by growth factors and amino acids, including essential amino acids (EAAs), such as leucine; Leu enters cells via the Leu transporter LAT1-4F2hc (also known as SLC7A5-SLC3A2) and potentially via endocytosis. Here, we investigated the contribution of the different routes of Leu entry into cells to mTORC1 activation using pharmacological inhibitors and cells that lack LAT1 or dynamin-1, -2 and -3. Our results show that LAT1 is the major route of Leu entry into cells and mTORC1 activation (â¼70%), whereas dynamin-dependent endocytosis and macropinocytosis contribute minimally to both (5-15%). However, macropinocytosis contributes significantly (â¼40%) to activation of mTORC1 by other EAAs. Surprisingly, the dynamin inhibitors dynasore and Dyngo 4A, which minimally inhibited Leu uptake, abolished mTORC1 activation independently of dynamin. Instead, dynasore inhibited RagA binding to Raptor, reduced mTORC1 recruitment to the lysosome, and inhibited Akt activation and TSC2-S939 phosphorylation; this resulted in inhibition of Rheb and mTORC1 activity. Our results suggest that these commonly used inhibitors of dynamin and endocytosis are potent suppressors of mTORC1 activation via off-target effects and not via dynamin inhibition.This article has an associated First Person interview with the first author of the paper.
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Dinaminas/metabolismo , Endocitosis , Cadena Pesada de la Proteína-1 Reguladora de Fusión/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Leucina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Animales , Transporte Biológico , Línea Celular Tumoral , Dinaminas/antagonistas & inhibidores , Humanos , Hidrazonas/farmacología , Lisosomas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/efectos de los fármacos , Proteínas de Unión al GTP Monoméricas , Naftoles/farmacologíaRESUMEN
BACKGROUND: Epithelial sodium channel (ENaC, Scnn1) hyperactivity in the lung leads to airway surface dehydration and mucus accumulation in cystic fibrosis (CF) patients and in mice with CF-like lung disease. METHODS: We identified several potent ENaC specific antisense oligonucleotides (ASOs) and tested them by inhalation in mouse models of CF-like lung disease. RESULTS: The inhaled ASOs distributed into lung airway epithelial cells and decreased ENaC expression by inducing RNase H1-dependent degradation of the targeted Scnn1a mRNA. Aerosol delivered ENaC ASO down-regulated mucus marker expression and ameliorated goblet cell metaplasia, inflammation, and airway hyper-responsiveness. Lack of systemic activity of ASOs delivered via the aerosol route ensures the safety of this approach. CONCLUSIONS: Our results demonstrate that antisense inhibition of ENaC in airway epithelial cells could be an effective and safe approach for the prevention and reversal of lung symptoms in CF and potentially other inflammatory diseases of the lung.
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Fibrosis Quística , Canales Epiteliales de Sodio/metabolismo , Oligonucleótidos Antisentido/farmacología , Mucosa Respiratoria , Administración por Inhalación , Animales , Fibrosis Quística/metabolismo , Fibrosis Quística/fisiopatología , Modelos Animales de Enfermedad , Bloqueadores del Canal de Sodio Epitelial/farmacología , Ratones , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/fisiopatología , Fármacos del Sistema Respiratorio/farmacología , Resultado del TratamientoRESUMEN
Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac IKS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the α1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the IKS channel, thus confirming the functional importance of E3-ligase autoinhibition.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas de Microfilamentos/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Multimerización de Proteína , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Humanos , Proteínas de Microfilamentos/química , Ubiquitina-Proteína Ligasas Nedd4 , Canales de Potasio con Entrada de Voltaje/química , Complejo de la Endopetidasa Proteasomal/química , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/química , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
The ubiquitin ligase Nedd4-like (Nedd4L, or Nedd4-2) binds to and regulates stability of the epithelial Na+ channel (ENaC) in salt-absorbing epithelia in the kidney, lung, and other tissues. Its role in the distal colon, which also absorbs salt and fluid and expresses ENaC, is unknown. Using a conditional knock-out approach to knock out Nedd4L in mice intestinal epithelium (Nedd4Lf/f ;Vil-CreERT2 ) we show here that Nedd4L depletion leads to a higher steady-state short circuit current (Isc) in mouse distal colon tissue relative to controls. This higher Isc was partially reduced by the addition of apical amiloride and strongly reduced by basolateral bumetanide as well as by depletion of basolateral Cl-, suggesting that Na+/K+/2Cl- (NKCC1/SLC12A2) co-transporter and ENaC are targets of Nedd4L in the colon. In accordance, NKCC1 (and γENaC) protein abundance in the colon of the Nedd4L knock-out animals was increased, indicating that Nedd4L normally suppresses these proteins. However, we did not observe co-immunoprecipitation between Nedd4L and NKCC1, suggesting that Nedd4L indirectly suppresses NKCC1 expression. Low salt diet resulted in a strong increase in ß and γ (but not α) ENaC mRNA and protein expression and ENaC activity. Although salt restriction also increased NKCC1 protein and mRNA abundance, it did not lead to its elevated activity (Isc). These results identify NKCC1 as a novel target for Nedd4L-mediated down-regulation in vivo, which modulates ion and fluid transport in the distal colon together with ENaC.
Asunto(s)
Colon/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Canales Epiteliales de Sodio/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Cloruros/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Canales Epiteliales de Sodio/genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ubiquitina-Proteína Ligasas Nedd4 , Potasio/metabolismo , Sales (Química)/metabolismo , Transducción de Señal , Sodio/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/genética , Ubiquitina-Proteína Ligasas/genética , Regulación hacia ArribaRESUMEN
HECT-family E3 ligases ubiquitinate protein substrates to control virtually every eukaryotic process and are misregulated in numerous diseases. Nonetheless, understanding of HECT E3s is limited by a paucity of selective and potent modulators. To overcome this challenge, we systematically developed ubiquitin variants (UbVs) that inhibit or activate HECT E3s. Structural analysis of 6 HECT-UbV complexes revealed UbV inhibitors hijacking the E2-binding site and activators occupying a ubiquitin-binding exosite. Furthermore, UbVs unearthed distinct regulation mechanisms among NEDD4 subfamily HECTs and proved useful for modulating therapeutically relevant targets of HECT E3s in cells and intestinal organoids, and in a genetic screen that identified a role for NEDD4L in regulating cell migration. Our work demonstrates versatility of UbVs for modulating activity across an E3 family, defines mechanisms and provides a toolkit for probing functions of HECT E3s, and establishes a general strategy for systematic development of modulators targeting families of signaling proteins.
Asunto(s)
Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Animales , Dominio Catalítico , Línea Celular , Movimiento Celular , Perros , Células HCT116 , Humanos , Células de Riñón Canino Madin Darby , Modelos Moleculares , Organoides/citología , Organoides/metabolismo , Biblioteca de Péptidos , Ubiquitina/química , Ubiquitina/genéticaRESUMEN
Drosophila Nedd4 (dNedd4) is a HECT ubiquitin ligase with two main splice isoforms: dNedd4-short (dNedd4S) and -long (dNedd4Lo). DNedd4Lo has a unique N-terminus containing a Pro-rich region. We previously showed that whereas dNedd4S promotes neuromuscular synaptogenesis, dNedd4Lo inhibits it and impairs larval locomotion. To delineate the cause of the impaired locomotion, we searched for binding partners to the N-terminal unique region of dNedd4Lo in larval lysates using mass spectrometry and identified Amphiphysin (dAmph). dAmph is a postsynaptic protein containing SH3-BAR domains and regulates muscle transverse tubule (T-tubule) formation in flies. We validated the interaction by coimmunoprecipitation and showed direct binding between dAmph-SH3 domain and dNedd4Lo N-terminus. Accordingly, dNedd4Lo was colocalized with dAmph postsynaptically and at muscle T-tubules. Moreover, expression of dNedd4Lo in muscle during embryonic development led to disappearance of dAmph and impaired T-tubule formation, phenocopying amph-null mutants. This effect was not seen in muscles expressing dNedd4S or a catalytically-inactive dNedd4Lo(CâA). We propose that dNedd4Lo destabilizes dAmph in muscles, leading to impaired T-tubule formation and muscle function.