ABSTRACT
Caveolin-1 (Cav-1) is a scaffolding protein and a major component of caveolae/lipid rafts. Previous reports have shown that endothelial dysfunction in Cav-1-deficient (Cav-1-/-) mice is mediated by elevated oxidative stress through endothelial nitric oxide synthase (eNOS) uncoupling and increased NADPH oxidase. Oxidant stress is the net balance of oxidant generation and scavenging, and the role of Cav-1 as a regulator of antioxidant enzymes in vascular tissue is poorly understood. Extracellular SOD (SOD3) is a copper (Cu)-containing enzyme that is secreted from vascular smooth muscle cells/fibroblasts and subsequently binds to the endothelial cells surface, where it scavenges extracellular [Formula: see text] and preserves endothelial function. SOD3 activity is dependent on Cu, supplied by the Cu transporter ATP7A, but whether Cav-1 regulates the ATP7A-SOD3 axis and its role in oxidative stress-mediated vascular dysfunction has not been studied. Here we show that the activity of SOD3, but not SOD1, was significantly decreased in Cav-1-/- vessels, which was rescued by re-expression of Cav-1 or Cu supplementation. Loss of Cav-1 reduced ATP7A protein, but not mRNA, and this was mediated by ubiquitination of ATP7A and proteasomal degradation. ATP7A bound to Cav-1 and was colocalized with SOD3 in caveolae/lipid rafts or perinucleus in vascular tissues or cells. Impaired endothelium-dependent vasorelaxation in Cav-1-/- mice was rescued by gene transfer of SOD3 or by ATP7A-overexpressing transgenic mice. These data reveal an unexpected role of Cav-1 in stabilizing ATP7A protein expression by preventing its ubiquitination and proteasomal degradation, thereby increasing SOD3 activity, which in turn protects against vascular oxidative stress-mediated endothelial dysfunction.
Subject(s)
Caveolin 1/genetics , Copper-Transporting ATPases/genetics , Endothelial Cells/metabolism , Superoxide Dismutase-1/genetics , Superoxide Dismutase/genetics , Animals , Aorta/cytology , Aorta/metabolism , Caveolin 1/deficiency , Copper/pharmacology , Copper Transport Proteins/genetics , Copper Transport Proteins/metabolism , Copper-Transporting ATPases/metabolism , Endothelial Cells/cytology , Endothelial Cells/drug effects , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/metabolism , Gene Expression Regulation , Male , Mesenteric Arteries/cytology , Mesenteric Arteries/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Molecular Chaperones/genetics , Molecular Chaperones/metabolism , Oxidative Stress , Primary Cell Culture , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Signal Transduction , Superoxide Dismutase/metabolism , Superoxide Dismutase-1/metabolism , Ubiquitination/drug effects , Vasodilation/drug effectsABSTRACT
RAS GTPases are important mediators of oncogenesis in humans. However, pharmacological inhibition of RAS has proved challenging. Here we describe a functionally critical region, located outside the effector lobe of RAS, that can be targeted for inhibition. We developed NS1, a synthetic binding protein (monobody) that bound with high affinity to both GTP- and GDP-bound states of H-RAS and K-RAS but not N-RAS. NS1 potently inhibited growth factor signaling and oncogenic H-RAS- and K-RAS-mediated signaling and transformation but did not block oncogenic N-RAS, BRAF or MEK1. NS1 bound the α4-ß6-α5 region of RAS, which disrupted RAS dimerization and nanoclustering and led to blocking of CRAF-BRAF heterodimerization and activation. These results establish the importance of the α4-ß6-α5 interface in RAS-mediated signaling and define a previously unrecognized site in RAS for inhibiting RAS function.
Subject(s)
Allosteric Site/drug effects , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/pharmacology , ras Proteins/antagonists & inhibitors , ras Proteins/chemistry , Animals , Antibodies, Monoclonal/chemistry , COS Cells , Cells, Cultured , Chlorocebus aethiops , HEK293 Cells , Humans , Mice , NIH 3T3 Cells , ras Proteins/metabolismABSTRACT
OBJECTIVE: Copper transporter ATP7A (copper-transporting/ATPase) is required for full activation of SOD3 (extracellular superoxide dismutase), which is secreted from vascular smooth muscle cells (VSMCs) and anchors to endothelial cell surface to preserve endothelial function by scavenging extracellular superoxide. We reported that ATP7A protein expression and SOD3 activity are decreased in insulin-deficient type 1 diabetes mellitus vessels, thereby, inducing superoxide-mediated endothelial dysfunction, which are rescued by insulin treatment. However, it is unknown regarding the mechanism by which insulin increases ATP7A expression in VSMCs and whether ATP7A downregulation is observed in T2DM (type2 diabetes mellitus) mice and human in which insulin-Akt (protein kinase B) pathway is selectively impaired. APPROACH AND RESULTS: Here we show that ATP7A protein is markedly downregulated in vessels isolated from T2DM patients, as well as those from high-fat diet-induced or db/db T2DM mice. Akt2 (protein kinase B beta) activated by insulin promotes ATP7A stabilization via preventing ubiquitination/degradation as well as translocation to plasma membrane in VSMCs, which contributes to activation of SOD3 that protects against T2DM-induced endothelial dysfunction. Downregulation of ATP7A in T2DM vessels is restored by constitutive active Akt or PTP1B-/- (protein-tyrosine phosphatase 1B-deficient) T2DM mice, which enhance insulin-Akt signaling. Immunoprecipitation, in vitro kinase assay, and mass spectrometry analysis reveal that insulin stimulates Akt2 binding to ATP7A to induce phosphorylation at Ser1424/1463/1466. Furthermore, SOD3 activity is reduced in Akt2-/- vessels or VSMCs, which is rescued by ATP7A overexpression. CONCLUSION: Akt2 plays a critical role in ATP7A protein stabilization and translocation to plasma membrane in VSMCs, which contributes to full activation of vascular SOD3 that protects against endothelial dysfunction in T2DM.
Subject(s)
Copper-Transporting ATPases/metabolism , Diabetes Mellitus, Experimental/enzymology , Diabetes Mellitus, Type 2/enzymology , Diabetic Angiopathies/enzymology , Endothelium, Vascular/enzymology , Muscle, Smooth, Vascular/enzymology , Proto-Oncogene Proteins c-akt/metabolism , Superoxide Dismutase/metabolism , Animals , Aorta, Thoracic/enzymology , Aorta, Thoracic/physiopathology , Cells, Cultured , Copper-Transporting ATPases/genetics , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/physiopathology , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/physiopathology , Diabetic Angiopathies/genetics , Diabetic Angiopathies/physiopathology , Diabetic Angiopathies/prevention & control , Endothelium, Vascular/drug effects , Endothelium, Vascular/physiopathology , Enzyme Stability , Female , Humans , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Male , Mesenteric Arteries/enzymology , Mesenteric Arteries/physiopathology , Mice, Inbred C57BL , Mice, Knockout , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/physiopathology , Phosphorylation , Protein Transport , Proto-Oncogene Proteins c-akt/deficiency , Proto-Oncogene Proteins c-akt/genetics , Rats, Sprague-Dawley , Signal Transduction , Superoxide Dismutase/deficiency , Superoxide Dismutase/genetics , VasodilationABSTRACT
Mitochondria are highly dynamic multifaceted organelles with various functions including cellular energy metabolism, reactive oxygen species (ROS) generation, calcium homeostasis, and apoptosis. Because of these diverse functions, mitochondria are key regulators of cell survival and death, and their dysfunction is implicated in numerous diseases, particularly neurodegenerative disorders such as Alzheimer's Disease, Parkinson's Disease, and Huntington's Disease. One of the most common neurodegenerative disorders is sensorineural hearing loss (SNHL). SNHL primarily originates from the degenerative changes in the cochlea, which is the auditory portion of the inner ear. Many cochlear cells contain an abundance of mitochondria and are metabolically highly active, rendering them susceptible to mitochondrial dysfunction. Indeed, the causal role of mitochondrial dysfunction in SNHL progression is well established, and therefore, targeted for treatment. In this review, we aim to compile the emerging findings in the literature indicating the role of mitochondrial dysfunction in the progression of sensorineural hearing loss and highlight potential therapeutics targeting mitochondrial dysfunction for hearing loss treatment.
Subject(s)
Hearing Loss, Sensorineural , Neurodegenerative Diseases , Parkinson Disease , Humans , Mitochondria/metabolism , Hearing Loss, Sensorineural/metabolism , Neurodegenerative Diseases/metabolism , Reactive Oxygen Species/metabolismABSTRACT
VEGFR2 (KDR/Flk1) signaling in endothelial cells (ECs) plays a central role in angiogenesis. The P-type ATPase transporter ATP7A regulates copper homeostasis, and its role in VEGFR2 signaling and angiogenesis is entirely unknown. Here, we describe the unexpected crosstalk between the Copper transporter ATP7A, autophagy, and VEGFR2 degradation. The functional significance of this Copper transporter was demonstrated by the finding that inducible EC-specific ATP7A deficient mice or ATP7A-dysfunctional ATP7Amut mice showed impaired post-ischemic neovascularization. In ECs, loss of ATP7A inhibited VEGF-induced VEGFR2 signaling and angiogenic responses, in part by promoting ligand-induced VEGFR2 protein degradation. Mechanistically, VEGF stimulated ATP7A translocation from the trans-Golgi network to the plasma membrane where it bound to VEGFR2, which prevented autophagy-mediated lysosomal VEGFR2 degradation by inhibiting autophagic cargo/adapter p62/SQSTM1 binding to ubiquitinated VEGFR2. Enhanced autophagy flux due to ATP7A dysfunction in vivo was confirmed by autophagy reporter CAG-ATP7Amut -RFP-EGFP-LC3 transgenic mice. In summary, our study uncovers a novel function of ATP7A to limit autophagy-mediated degradation of VEGFR2, thereby promoting VEGFR2 signaling and angiogenesis, which restores perfusion recovery and neovascularization. Thus, endothelial ATP7A is identified as a potential therapeutic target for treatment of ischemic cardiovascular diseases.
Subject(s)
Autophagy/genetics , Blood Vessels/metabolism , Copper-Transporting ATPases/genetics , P-type ATPases/genetics , Vascular Endothelial Growth Factor Receptor-2/genetics , Animals , Blood Vessels/drug effects , Blood Vessels/physiology , COS Cells , Cells, Cultured , Chlorocebus aethiops , Copper-Transporting ATPases/metabolism , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Endothelial Cells/physiology , Humans , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Microtubule-Associated Proteins/metabolism , P-type ATPases/metabolism , RNA Interference , Signal Transduction/genetics , Vascular Endothelial Growth Factor A/pharmacology , Vascular Endothelial Growth Factor Receptor-2/metabolismABSTRACT
Metabolic dysfunction is a primary feature of Werner syndrome (WS), a human premature aging disease caused by mutations in the gene encoding the Werner (WRN) DNA helicase. WS patients exhibit severe metabolic phenotypes, but the underlying mechanisms are not understood, and whether the metabolic deficit can be targeted for therapeutic intervention has not been determined. Here we report impaired mitophagy and depletion of NAD+, a fundamental ubiquitous molecule, in WS patient samples and WS invertebrate models. WRN regulates transcription of a key NAD+ biosynthetic enzyme nicotinamide nucleotide adenylyltransferase 1 (NMNAT1). NAD+ repletion restores NAD+ metabolic profiles and improves mitochondrial quality through DCT-1 and ULK-1-dependent mitophagy. At the organismal level, NAD+ repletion remarkably extends lifespan and delays accelerated aging, including stem cell dysfunction, in Caenorhabditis elegans and Drosophila melanogaster models of WS. Our findings suggest that accelerated aging in WS is mediated by impaired mitochondrial function and mitophagy, and that bolstering cellular NAD+ levels counteracts WS phenotypes.
Subject(s)
Aging, Premature/metabolism , Mitophagy , NAD/metabolism , Werner Syndrome Helicase/metabolism , Werner Syndrome/metabolism , Aging, Premature/genetics , Animals , Autophagy-Related Protein-1 Homolog/genetics , Autophagy-Related Protein-1 Homolog/metabolism , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Disease Models, Animal , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Mutation , Nicotinamide-Nucleotide Adenylyltransferase/genetics , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , Werner Syndrome/genetics , Werner Syndrome Helicase/geneticsABSTRACT
Phosphatidylinositol 3-kinases (PI3Ks) play important roles in human tumorigenesis. Activation of the PI3K target AKT is frequent in neuroblastoma (NB) and correlates with poor prognosis. PI3K pan-inhibitors reduce NB tumor formation but present severe toxicity, which limits their therapeutic potential. Therefore, defining the importance of specific PI3K isoforms may aid in developing more effective therapeutic strategies. We previously demonstrated that PI3K Class IIß (PI3KC2ß) and its regulator intersectin 1 (ITSN1) are highly expressed in primary NB tumors and cell lines. Silencing ITSN1 dramatically reduced the tumorigenic potential of NB cells. Interestingly, overexpression of PI3KC2ß rescued the anchorage-independent growth of ITSN1-silenced cells suggesting that PI3KC2ß mediates ITSN1's function in NB cells. To address the importance of PI3KC2ß in NBs, we generated PI3KC2ß-silenced lines and examined their biologic activity. Herein, we demonstrate that PI3KC2ß-silencing inhibits early stages of NB tumorigenic growth. We also show that loss of endogenous PI3KC2ß or ITSN1 reduces AKT activation but does not impact ERK-MAPK activation. These data reveal a novel role for PI3KC2ß in human NB tumorigenesis.
Subject(s)
Carcinogenesis/metabolism , Neuroblastoma/enzymology , Phosphatidylinositol 3-Kinases/physiology , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Cell Line, Tumor , Cell Proliferation , Class II Phosphatidylinositol 3-Kinases , Gene Knockdown Techniques , Humans , Isoenzymes/physiology , MAP Kinase Signaling System , Mice, Nude , Neoplasm Transplantation , Neuroblastoma/pathology , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
Ubiquitylation of receptor tyrosine kinases (RTKs) regulates their trafficking and lysosomal degradation. The multidomain scaffolding protein intersectin 1 (ITSN1) is an important regulator of this process. ITSN1 stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN1 enhances Cbl activity is unclear. Here, we demonstrate that ITSN1 interacts with and recruits the Shp2 tyrosine phosphatase to Spry2 to enhance its dephosphorylation, thereby disrupting the inhibitory effect of Spry2 on Cbl and enhancing EGFR ubiquitylation. In contrast, expression of a catalytically inactive Shp2 mutant reversed the effect of ITSN1 on Spry2 dephosphorylation and decreased Cbl-mediated EGFR ubiquitylation. In addition, disruption of ITSN1 binding to Spry2 through point mutation of the Pro-rich ITSN1 binding site in Spry2 resulted in decreased Shp2-Spry2 interaction and enhanced Spry2 tyrosine phosphorylation. This study demonstrates that ITSN1 enhances Cbl activity, in part, by modulating the interaction of Cbl with Spry2 through recruitment of Shp2 phosphatase to the Cbl-Spry2 complex. These findings reveal a new level of complexity in the regulation of RTKs by Cbl through ITSN1 binding with Shp2 and Spry2.
Subject(s)
Adaptor Proteins, Vesicular Transport/physiology , ErbB Receptors/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Proto-Oncogene Proteins c-cbl/metabolism , Ubiquitination , Animals , COS Cells , Chlorocebus aethiops , Epidermal Growth Factor/physiology , Humans , Mice , Mice, Knockout , Phosphorylation , Protein Binding , Protein Isoforms , Protein Transport , Receptor Protein-Tyrosine Kinases/metabolismABSTRACT
Ubiquitylation of receptor tyrosine kinases plays a critical role in regulating the trafficking and lysosomal degradation of these important signaling molecules. We identified the multidomain scaffolding protein intersectin 1 (ITSN1) as an important regulator of this process (N. P. Martin et al., Mol. Pharmacol. 70:1463-1653, 2006) ITSN1 stimulates ubiquitylation of the epidermal growth factor receptor (EGFR) through enhancing the activity of the Cbl E3 ubiquitin ligase. However, the precise mechanism through which ITSN1 enhances Cbl activity was unclear. In this study, we found that ITSN1 enhances Cbl activity through disrupting the interaction of Cbl with the Sprouty2 (Spry2) inhibitory protein. We demonstrate that ITSN1 binds Pro-rich regions in both Cbl and Spry2 and that interaction of ITSN1 with Spry2 disrupts Spry2-Cbl interaction, resulting in enhanced ubiquitylation of the EGFR. Disruption of ITSN1 binding to Spry2 through point mutation of the Pro-rich ITSN1 binding site in Spry2 results in enhanced Cbl-Spry2 interaction and inhibition of receptor ubiquitylation. This study demonstrates that ITSN1 enhances Cbl activity by modulating the interaction of Cbl with Spry2. In addition, our results reveal a new level of complexity in the regulation of Cbl through the interaction with ITSN1 and Spry2.
Subject(s)
Adaptor Proteins, Vesicular Transport/metabolism , ErbB Receptors/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Proto-Oncogene Proteins c-cbl/metabolism , Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport/chemistry , Adaptor Proteins, Vesicular Transport/genetics , Amino Acid Substitution , Animals , Base Sequence , COS Cells , Chlorocebus aethiops , DNA Primers/genetics , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Mutagenesis, Site-Directed , Protein Interaction Domains and Motifs , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins c-cbl/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Two-Hybrid System Techniques , UbiquitinationABSTRACT
Members of the intersectin (ITSN) family of scaffold proteins consist of multiple modular domains, each with distinct ligand preferences. Although ITSNs were initially implicated in the regulation of endocytosis, subsequent studies have revealed a more complex role for these scaffold proteins in regulation of additional biochemical pathways. In this study, we performed a high throughput yeast two-hybrid screen to identify additional pathways regulated by these scaffolds. Although several known ITSN binding partners were identified, we isolated more than 100 new targets for the two mammalian ITSN proteins, ITSN1 and ITSN2. We present the characterization of several of these new targets which implicate ITSNs in the regulation of the Rab and Arf GTPase pathways as well as regulation of the disrupted in schizophrenia 1 (DISC1) interactome. In addition, we demonstrate that ITSN proteins form homomeric and heteromeric complexes with each other revealing an added level of complexity in the function of these evolutionarily conserved scaffolds.