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1.
Redox Biol ; 64: 102759, 2023 08.
Article in English | MEDLINE | ID: mdl-37302345

ABSTRACT

Regulation of mitochondrial redox balance is emerging as a key event for cell signaling in both physiological and pathological conditions. However, the link between the mitochondrial redox state and the modulation of these conditions remains poorly defined. Here, we discovered that activation of the evolutionary conserved mitochondrial calcium uniporter (MCU) modulates mitochondrial redox state. By using mitochondria-targeted redox and calcium sensors and genetic MCU-ablated models, we provide evidence of the causality between MCU activation and net reduction of mitochondrial (but not cytosolic) redox state. Redox modulation of redox-sensitive groups via MCU stimulation is required for maintaining respiratory capacity in primary human myotubes and C. elegans, and boosts mobility in worms. The same benefits are obtained bypassing MCU via direct pharmacological reduction of mitochondrial proteins. Collectively, our results demonstrate that MCU regulates mitochondria redox balance and that this process is required to promote the MCU-dependent effects on mitochondrial respiration and mobility.


Subject(s)
Caenorhabditis elegans , Mitochondria , Animals , Humans , Caenorhabditis elegans/metabolism , Calcium/metabolism , Mitochondria/metabolism , Oxidation-Reduction , Respiration
2.
Nat Chem Biol ; 16(12): 1385-1393, 2020 12.
Article in English | MEDLINE | ID: mdl-32778841

ABSTRACT

Mitochondrial membrane potential (ΔΨm) is a universal selective indicator of mitochondrial function and is known to play a central role in many human pathologies, such as diabetes mellitus, cancer and Alzheimer's and Parkinson's diseases. Here, we report the design, synthesis and several applications of mitochondria-activatable luciferin (MAL), a bioluminescent probe sensitive to ΔΨm, and partially to plasma membrane potential (ΔΨp), for non-invasive, longitudinal monitoring of ΔΨm in vitro and in vivo. We applied this new technology to evaluate the aging-related change of ΔΨm in mice and showed that nicotinamide riboside (NR) reverts aging-related mitochondrial depolarization, revealing another important aspect of the mechanism of action of this potent biomolecule. In addition, we demonstrated application of the MAL probe for studies of brown adipose tissue (BAT) activation and non-invasive in vivo assessment of ΔΨm in animal cancer models, opening exciting opportunities for understanding the underlying mechanisms and for discovery of effective treatments for many human pathologies.


Subject(s)
Aging/genetics , Diagnostic Imaging/methods , Firefly Luciferin/chemistry , Fluorescent Dyes/chemistry , Mammary Neoplasms, Experimental/diagnostic imaging , Membrane Potential, Mitochondrial/genetics , Adipocytes/cytology , Adipocytes/drug effects , Adipocytes/metabolism , Adipose Tissue, Brown/cytology , Adipose Tissue, Brown/diagnostic imaging , Adipose Tissue, Brown/drug effects , Adipose Tissue, Brown/metabolism , Aging/drug effects , Aging/metabolism , Animals , Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacology , Dioxoles/pharmacology , Female , Firefly Luciferin/metabolism , Fluorescent Dyes/metabolism , Luciferases/genetics , Luciferases/metabolism , Luminescent Measurements , Mammary Neoplasms, Experimental/metabolism , Membrane Potential, Mitochondrial/drug effects , Membrane Potentials/drug effects , Mice , Mice, Nude , Mitochondria/drug effects , Mitochondria/metabolism , Niacinamide/analogs & derivatives , Niacinamide/pharmacology , Nigericin/pharmacology , Pyridinium Compounds
3.
Nutrients ; 12(2)2020 Feb 19.
Article in English | MEDLINE | ID: mdl-32093050

ABSTRACT

Pancreatic ß-cells secrete insulin to lower blood glucose, following a meal. Maintenance of ß-cell function is essential to preventing type 2 diabetes. In pancreatic ß-cells, mitochondrial matrix calcium is an activating signal for insulin secretion. Recently, the molecular identity of the mitochondrial calcium uniporter (MCU), the transporter that mediates mitochondrial calcium uptake, was revealed. Its role in pancreatic ß-cell signal transduction modulation was clarified, opening new perspectives for intervention. Here, we investigated the effects of a mitochondrial Ca2+-targeted nutritional intervention strategy on metabolism/secretion coupling, in a model of pancreatic insulin-secreting cells (INS-1E). Acute treatment of INS-1E cells with the natural plant flavonoid and MCU activator kaempferol, at a low micromolar range, increased mitochondrial calcium rise during glucose stimulation, without affecting the expression level of the MCU and with no cytotoxicity. Enhanced mitochondrial calcium rises potentiated glucose-induced insulin secretion. Conversely, the MCU inhibitor mitoxantrone inhibited mitochondrial Ca2+ uptake and prevented both glucose-induced insulin secretion and kaempferol-potentiated effects. The kaempferol-dependent potentiation of insulin secretion was finally validated in a model of a standardized pancreatic human islet. We conclude that the plant product kaempferol activates metabolism/secretion coupling in insulin-secreting cells by modulating mitochondrial calcium uptake.


Subject(s)
Calcium Channels/metabolism , Calcium/metabolism , Insulin Secretion/drug effects , Insulin-Secreting Cells/drug effects , Kaempferols/pharmacology , Animals , Cell Culture Techniques , Humans , Mitochondria/metabolism
4.
Br J Pharmacol ; 176(17): 3250-3263, 2019 09.
Article in English | MEDLINE | ID: mdl-31166006

ABSTRACT

BACKGROUND AND PURPOSE: Quinic acid (QA) is an abundant natural compound from plant sources which may improve metabolic health. However, little attention has been paid to its effects on pancreatic beta-cell functions, which contribute to the control of metabolic health by lowering blood glucose. Strategies targeting beta-cell signal transduction are a new approach for diabetes treatment. This study investigated the efficacy of QA to stimulate beta-cell function by targeting the basic molecular machinery of metabolism-secretion coupling. EXPERIMENTAL APPROACH: We measured bioenergetic parameters and insulin exocytosis in a model of insulin-secreting beta-cells (INS-1E), together with Ca2+ homeostasis, using genetically encoded sensors, targeted to different subcellular compartments. Islets from mice chronically infused with QA were also assessed. KEY RESULTS: QA triggered transient cytosolic Ca2+ increases in insulin-secreting cells by mobilizing Ca2+ from intracellular stores, such as endoplasmic reticulum. Following glucose stimulation, QA increased glucose-induced mitochondrial Ca2+ transients. We also observed a QA-induced rise of the NAD(P)H/NAD(P)+ ratio, augmented ATP synthase-dependent respiration, and enhanced glucose-stimulated insulin secretion. QA promoted beta-cell function in vivo as islets from mice infused with QA displayed improved glucose-induced insulin secretion. A diet containing QA improved glucose tolerance in mice. CONCLUSIONS AND IMPLICATIONS: QA modulated intracellular Ca2+ homeostasis, enhancing glucose-stimulated insulin secretion in both INS-1E cells and mouse islets. By increasing mitochondrial Ca2+ , QA activated the coordinated stimulation of oxidative metabolism, mitochondrial ATP synthase-dependent respiration, and therefore insulin secretion. Bioactive agents raising mitochondrial Ca2+ in pancreatic beta-cells could be used to treat diabetes.


Subject(s)
Biological Products/pharmacology , Calcium/metabolism , Insulin Secretion/drug effects , Insulin-Secreting Cells/drug effects , Mitochondria/drug effects , Quinic Acid/pharmacology , Actinidia/chemistry , Animals , Biological Products/chemistry , Biological Products/isolation & purification , Cells, Cultured , Coffee/chemistry , Dose-Response Relationship, Drug , Hippophae/chemistry , Insulin-Secreting Cells/metabolism , Male , Mice , Mice, Inbred C57BL , Mitochondria/metabolism , Prunus/chemistry , Quinic Acid/chemistry , Quinic Acid/isolation & purification , Rats , Structure-Activity Relationship , Vaccinium macrocarpon/chemistry , Vaccinium myrtillus/chemistry
5.
FASEB J ; 33(4): 4660-4674, 2019 04.
Article in English | MEDLINE | ID: mdl-30589571

ABSTRACT

In pancreatic ß-cells, mitochondria generate signals that promote insulin granule exocytosis. Here we study how lysine acetylation of mitochondrial proteins mechanistically affects metabolism-secretion coupling in insulin-secreting cells. Using mass spectrometry-based proteomics, we identified lysine acetylation sites in rat insulinoma cell line clone 1E cells. In cells lacking the mitochondrial lysine deacetylase sirtuin-3 (SIRT3), several matrix proteins are hyperacetylated. Disruption of the SIRT3 gene has a deleterious effect on mitochondrial energy metabolism and Ca2+ signaling. Under resting conditions, SIRT3 deficient cells are overactivated, which elevates the respiratory rate and enhances calcium signaling and basal insulin secretion. In response to glucose, the SIRT3 knockout cells are unable to mount a sustained cytosolic ATP response. Calcium signaling is strongly reduced and the respiratory response as well as insulin secretion are blunted. We propose mitochondrial protein lysine acetylation as a control mechanism in ß-cell energy metabolism and Ca2+ signaling.-De Marchi, U., Galindo, A. N., Thevenet, J., Hermant, A., Bermont, F., Lassueur, S., Domingo, J. S., Kussmann, M., Dayon, L., Wiederkehr, A. Mitochondrial lysine deacetylation promotes energy metabolism and calcium signaling in insulin-secreting cells.


Subject(s)
Calcium Signaling/physiology , Insulin-Secreting Cells/metabolism , Lysine/metabolism , Mitochondria/metabolism , Acetylation , Adenosine Triphosphate/metabolism , Calcium Signaling/drug effects , Cell Line , Energy Metabolism/physiology , Glucose/pharmacology , Humans , Insulin/metabolism , Insulin-Secreting Cells/drug effects , Oxygen Consumption/drug effects , Sirtuin 3/metabolism , Tandem Mass Spectrometry
6.
Oncotarget ; 8(31): 50359-50375, 2017 Aug 01.
Article in English | MEDLINE | ID: mdl-28881568

ABSTRACT

Estrogen-related receptor alpha (ERR1) is an orphan nuclear receptor that can bind transcriptional co-activators constitutively. ERR1 expression correlates with poor patient outcomes in breast cancer, heightening interest in this nuclear receptor as a therapeutic target. Because ERR1 has no known regulatory ligand, a major challenge in targeting its activity is to find cellular or synthetic modulators of its function. We identified an interaction between ERR1 and KIF17, a kinesin-2 family microtubule motor, in a yeast-2-hybrid screen. We confirmed the interaction using in vitro biochemical assays and determined that binding is mediated by the ERR1 ligand-binding/AF2 domain and the KIF17 C-terminal tail. Expression of KIF17 tail domain in either ER-negative or ER-positive breast cancer epithelial cells attenuated nuclear accumulation of newly synthesized ERR1 and inhibited ERR1 transcriptional activity. Conversely, ERR1 transcriptional activity was elevated significantly in KIF17 knock-out cells. Sequence analysis of the KIF17 tail domain revealed it contains a nuclear receptor box with a conserved LXXLL motif found in transcriptional co-activators. Expression of a 12 amino-acid peptide containing this motif was sufficient to inhibit ERR1 transcriptional activity and cell invasion, while deletion of this region from the KIF17 tail resulted in increased ERR1 activity. Together, these data suggest KIF17 modifies ERR1 function by two possible, non-exclusive mechanisms: (i) by regulating nuclear-cytoplasmic distribution or (ii) by competing with transcriptional co-activators for binding to ERR1. Thus targeting the ERR1-KIF17 interaction has potential as a novel strategy for treating breast cancer.

7.
J Cell Sci ; 130(11): 1929-1939, 2017 Jun 01.
Article in English | MEDLINE | ID: mdl-28404787

ABSTRACT

Pancreatic ß-cells sense glucose, promoting insulin secretion. Glucose sensing requires the sequential stimulation of glycolysis, mitochondrial metabolism and Ca2+ entry. To elucidate how mitochondrial activation in ß-cells contributes to insulin secretion, we compared the effects of glucose and the mitochondrial substrate methylsuccinate in the INS-1E insulin-secreting cell line at the respective concentrations at which they maximally activate mitochondrial respiration. Both substrates induced insulin secretion with distinct respiratory profiles, mitochondrial hyperpolarization, NADH production and ATP-to-ADP ratios. In contrast to glucose, methylsuccinate failed to induce large [Ca2+] rises and exocytosis proceeded largely independently of mitochondrial ATP synthesis. Both glucose- and methylsuccinate-induced secretion was blocked by diazoxide, indicating that Ca2+ is required for exocytosis. Dynamic assessment of the redox state of mitochondrial thiols revealed a less marked reduction in response to methylsuccinate than with glucose. Our results demonstrate that insulin exocytosis can be promoted by two distinct mechanisms one of which is dependent on mitochondrial ATP synthesis and large Ca2+ transients, and one of which is independent of mitochondrial ATP synthesis and relies on small Ca2+ signals. We propose that the combined effects of Ca2+ and redox reactions can trigger insulin secretion by these two mechanisms.


Subject(s)
Calcium/metabolism , Glucose/pharmacology , Insulin-Secreting Cells/metabolism , Insulin/metabolism , Mitochondria/metabolism , Succinates/pharmacology , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/biosynthesis , Animals , Cell Line, Tumor , Diazoxide/pharmacology , Exocytosis/drug effects , Glucose/metabolism , Glycolysis/drug effects , Glycolysis/physiology , Insulin Secretion , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/drug effects , Membrane Potential, Mitochondrial/drug effects , Mitochondria/drug effects , Molecular Imaging , Oxygen Consumption/drug effects , Rats , Single-Cell Analysis , Succinates/metabolism
8.
Adv Healthc Mater ; 6(4)2017 Feb.
Article in English | MEDLINE | ID: mdl-27995762

ABSTRACT

The development of cell-based biosensors that give insight into cell and tissue function in vivo is an attractive technology for biomedical research. Here, the development of a cell line expressing a fluorescent calcium sensor for the study of beta-cell function in vivo is reported. The bioresponsive cell model is based on INS-1E pancreatic beta-cells, stably expressing the genetically encoded cameleon-based fluorescent sensor YC3.6cyto . Following single-cell selection and expansion, functional testing and in vitro encapsulation experiments are used to identify a suitable clone of INS-1E cells expressing the calcium sensor. This clone is transplanted subcutaneous in mouse using a cell macroencapsulation system based on flat sheet porous membranes. Cells in the implanted capsules are able to respond to glucose in vivo by secreting insulin and thereby contributing to the regulation of glycaemia in the mice. Furthermore, fluorescence imaging of explanted devices shows that encapsulated cells maintain high level expression of YC3.6cyto in vivo. In conclusion, these data show that encapsulated INS-1E cells stably expressing a genetically encoded calcium sensor can be successfully implanted in vivo, and therefore serve as biosensing element or in vivo model to longitudinally monitor the function of pancreatic beta-cells.


Subject(s)
Calcium-Binding Proteins/biosynthesis , Calcium/metabolism , Cells, Immobilized , Insulin-Secreting Cells , Insulin/metabolism , Luminescent Proteins/biosynthesis , Animals , Calcium-Binding Proteins/genetics , Cell Line, Tumor , Cells, Immobilized/metabolism , Cells, Immobilized/transplantation , Heterografts , Insulin Secretion , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/transplantation , Luminescent Proteins/genetics , Mice , Mice, SCID , Rats
9.
PLoS One ; 11(11): e0165420, 2016.
Article in English | MEDLINE | ID: mdl-27812135

ABSTRACT

NOD2 contributes to the innate immune response and to the homeostasis of the intestinal mucosa. In response to its bacterial ligand, NOD2 interacts with RICK and activates the NF-κB and MAPK pathways, inducing gene transcription and synthesis of proteins required to initiate a balanced immune response. Mutations in NOD2 have been associated with an increased risk of Crohn's Disease (CD), a disabling inflammatory bowel disease (IBD). Because NOD2 signaling plays a key role in CD, it is important to further characterize the network of protein interacting with NOD2. Using yeast two hybrid (Y2H) screens, we identified new NOD2 interacting proteins (NIP). The primary interaction was confirmed by coimmunoprecipitation and/or bioluminescence resonance energy transfer (BRET) experiments for 11 of these proteins (ANKHD1, CHMP5, SDCCAG3, TRIM41, LDOC1, PPP1R12C, DOCK7, VIM, KRT15, PPP2R3B, and C10Orf67). These proteins are involved in diverse functions, including endosomal sorting complexes required for transport (ESCRT), cytoskeletal architecture and signaling regulation. Additionally, we show that the interaction of 8 NIPs is compromised with the 3 main CD associated NOD2 mutants (R702W, G908R and 1007fs). Furthermore, to determine whether these NOD2 protein partners could be encoded by IBD susceptibility genes, a transmission disequilibrium test (TDT) was performed on 101 single nucleotide polymorphisms (SNPs) and the main corresponding haplotypes in genes coding for 15 NIPs using a set of 343 IBD families with 556 patients. Overall this work did not increase the number of IBD susceptibility genes but extends the NOD2 protein interaction network and suggests that NOD2 interactome and signaling depend upon the NOD2 mutation profile in CD.


Subject(s)
Crohn Disease/genetics , Crohn Disease/metabolism , Nod2 Signaling Adaptor Protein/metabolism , Protein Interaction Mapping , Cell Line , Humans , Macrophages/metabolism , Mutation , NF-kappa B/genetics , NF-kappa B/metabolism , Nod2 Signaling Adaptor Protein/genetics , Polymorphism, Single Nucleotide
10.
J Biol Chem ; 289(13): 9182-94, 2014 Mar 28.
Article in English | MEDLINE | ID: mdl-24554722

ABSTRACT

Mitochondrial energy metabolism is essential for glucose-induced calcium signaling and, therefore, insulin granule exocytosis in pancreatic beta cells. Calcium signals are sensed by mitochondria acting in concert with mitochondrial substrates for the full activation of the organelle. Here we have studied glucose-induced calcium signaling and energy metabolism in INS-1E insulinoma cells and human islet beta cells. In insulin secreting cells a surprisingly large fraction of total respiration under resting conditions is ATP synthase-independent. We observe that ATP synthase-dependent respiration is markedly increased after glucose stimulation. Glucose also causes a very rapid elevation of oxidative metabolism as was followed by NAD(P)H autofluorescence. However, neither the rate of the glucose-induced increase nor the new steady-state NAD(P)H levels are significantly affected by calcium. Our findings challenge the current view, which has focused mainly on calcium-sensitive dehydrogenases as the target for the activation of mitochondrial energy metabolism. We propose a model of tight calcium-dependent regulation of oxidative metabolism and ATP synthase-dependent respiration in beta cell mitochondria. Coordinated activation of matrix dehydrogenases and respiratory chain activity by calcium allows the respiratory rate to change severalfold with only small or no alterations of the NAD(P)H/NAD(P)(+) ratio.


Subject(s)
ATP Synthetase Complexes/metabolism , Calcium/metabolism , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/biosynthesis , Adenosine Triphosphate/metabolism , Animals , Calcium Signaling/drug effects , Cell Respiration/drug effects , Cytosol/drug effects , Cytosol/metabolism , Glucose/pharmacology , Humans , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/drug effects , NADP/metabolism , Oxidation-Reduction/drug effects , Rats
11.
J Immunol ; 192(5): 2177-85, 2014 Mar 01.
Article in English | MEDLINE | ID: mdl-24501198

ABSTRACT

TNF-stimulated gene/protein-6 (TSG-6) is expressed by many different cell types in response to proinflammatory cytokines and plays an important role in the protection of tissues from the damaging consequences of acute inflammation. Recently, TSG-6 was identified as being largely responsible for the beneficial effects of multipotent mesenchymal stem cells, for example in the treatment of animal models of myocardial infarction and corneal injury/allogenic transplant. The protective effect of TSG-6 is due in part to its inhibition of neutrophil migration, but the mechanisms underlying this activity remain unknown. In this study, we have shown that TSG-6 inhibits chemokine-stimulated transendothelial migration of neutrophils via a direct interaction (KD, ∼ 25 nM) between TSG-6 and the glycosaminoglycan binding site of CXCL8, which antagonizes the association of CXCL8 with heparin. Furthermore, we found that TSG-6 impairs the binding of CXCL8 to cell surface glycosaminoglycans and the transport of CXCL8 across an endothelial cell monolayer. In vivo this could limit the formation of haptotactic gradients on endothelial heparan sulfate proteoglycans and, hence, integrin-mediated tight adhesion and migration. We further observed that TSG-6 suppresses CXCL8-mediated chemotaxis of neutrophils; this lower potency effect might be important at sites where there is high local expression of TSG-6. Thus, we have identified TSG-6 as a CXCL8-binding protein, making it, to our knowledge, the first soluble mammalian chemokine-binding protein to be described to date. We have also revealed a potential mechanism whereby TSG-6 mediates its anti-inflammatory and protective effects. This could inform the development of new treatments for inflammation in the context of disease or following transplantation.


Subject(s)
Cell Adhesion Molecules/immunology , Cell Movement/physiology , Interleukin-8/immunology , Neutrophils/immunology , Allografts , Binding Sites , Biological Transport, Active/physiology , Cell Adhesion/physiology , HL-60 Cells , Heparin , Human Umbilical Vein Endothelial Cells , Humans , Inflammation , Neutrophils/cytology , Stem Cell Transplantation
12.
Methods Mol Biol ; 1013: 67-92, 2013.
Article in English | MEDLINE | ID: mdl-23625494

ABSTRACT

This chapter describes assays that focus on the characterization of compounds identified in high--throughput screening campaigns, and the subsequent medicinal chemistry programs. They cover methods to determine potency in buffer, the effect of whole blood on the compounds' activity and finally the pharmacokinetic (PK)/pharmacodynamic (PD) -relationship of the compounds in a rodent species.


Subject(s)
Drug Discovery/methods , High-Throughput Screening Assays , Receptors, Chemokine/antagonists & inhibitors , Animals , Automation, Laboratory , Cell Culture Techniques , Cell Migration Assays , Cells, Cultured , Chemokines/metabolism , Chemotaxis/drug effects , Dielectric Spectroscopy , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Humans , Ligands , Pharmacokinetics , Protein Binding , Receptors, Chemokine/metabolism , Signal Transduction/drug effects
13.
Protein Expr Purif ; 75(2): 192-203, 2011 Feb.
Article in English | MEDLINE | ID: mdl-20851186

ABSTRACT

Production of correctly folded and biologically active proteins in Escherichiacoli can be a challenging process. Frequently, proteins are recovered as insoluble inclusion bodies and need to be denatured and refolded into the correct structure. To address this, a refolding screening process based on a 96-well assay format supported by design of experiments (DOE) was developed for identification of optimal refolding conditions. After a first generic screen of 96 different refolding conditions the parameters that produced the best yield were further explored in a focused DOE-based screen. The refolding efficiency and the quality of the refolded protein were analyzed by RP-HPLC and SDS-PAGE. The results were analyzed by the DOE software to identify the optimal concentrations of the critical additives. The optimal refolding conditions suggested by DOE were verified in medium-scale refolding tests, which confirmed the reliability of the predictions. Finally, the refolded protein was purified and its biological activity was tested in vitro. The screen was applied for the refolding of Interleukin 17F (IL-17F), stromal-cell-derived factor-1 (SDF-1α/CXCL12), B cell-attracting chemokine 1 (BCA-1/CXCL13), granulocyte macrophage colony stimulating factor (GM-CSF) and the complement factor C5a. This procedure identified refolding conditions for all the tested proteins. For the proteins where refolding conditions were already available, the optimized conditions identified in the screening process increased the yields between 50% and 100%. Thus, the method described herein is a useful tool to determine the feasibility of refolding and to identify high-yield scalable refolding conditions optimized for each individual protein.


Subject(s)
Anaphylatoxins/chemistry , Anaphylatoxins/metabolism , Chemokine CXCL12/chemistry , Chemokine CXCL12/metabolism , Chemokine CXCL13/chemistry , Chemokine CXCL13/metabolism , Granulocyte Colony-Stimulating Factor/chemistry , Granulocyte Colony-Stimulating Factor/metabolism , High-Throughput Screening Assays , Inclusion Bodies/chemistry , Interleukin-17/chemistry , Interleukin-17/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Research Design , Anaphylatoxins/genetics , Anaphylatoxins/isolation & purification , Biological Assay , Chemokine CXCL12/genetics , Chemokine CXCL12/isolation & purification , Chemokine CXCL13/genetics , Chemokine CXCL13/isolation & purification , Cloning, Molecular , Escherichia coli , Granulocyte Colony-Stimulating Factor/genetics , Granulocyte Colony-Stimulating Factor/isolation & purification , Humans , Inclusion Bodies/metabolism , Interleukin-17/genetics , Interleukin-17/isolation & purification , Protein Renaturation , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Reducing Agents/chemistry , Reducing Agents/metabolism
14.
J Biol Chem ; 285(43): 33230-33241, 2010 Oct 22.
Article in English | MEDLINE | ID: mdl-20699222

ABSTRACT

Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several "hot spots," such as Val(42), Met(44), Ala(71), Lys(93), Gly(94), and Tyr(96), forming a continuous protein-protein surface of about 830 Å(2), bearing very conserved amino acids among coronaviruses. Because nsp16 carries RNA cap 2'-O-methyltransferase (2'O-MTase) activity only in the presence of its interacting partner nsp10 (Bouvet, M., Debarnot, C., Imbert, I., Selisko, B., Snijder, E. J., Canard, B., and Decroly, E. (2010) PLoS Pathog. 6, e1000863), functional consequences of mutations on this surface were evaluated biochemically. Most changes that disrupted the nsp10-nsp16 interaction without structural perturbations were shown to abrogate stimulation of nsp16 RNA cap 2'O-MTase activity. More strikingly, the Y96A mutation abrogates stimulation of nsp16 2'O-MTase activity, whereas Y96F overstimulates it. Thus, the nsp10-nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses.


Subject(s)
Methyltransferases/metabolism , Severe acute respiratory syndrome-related coronavirus/metabolism , Viral Nonstructural Proteins/metabolism , Cell Line , Enzyme Activation , Humans , Methyltransferases/genetics , Mutagenesis , Mutation, Missense , Severe acute respiratory syndrome-related coronavirus/genetics , Viral Nonstructural Proteins/genetics
15.
FEBS Lett ; 583(14): 2326-32, 2009 Jul 21.
Article in English | MEDLINE | ID: mdl-19555689

ABSTRACT

To further characterize the molecular events supporting the tumor suppressor activity of Scrib in mammals, we aim to identify new binding partners. We isolated MCC, a recently identified binding partner for beta-catenin, as a new interacting protein for Scrib. MCC interacts with both Scrib and the NHERF1/NHERF2/Ezrin complex in a PDZ-dependent manner. In T47D cells, MCC and Scrib proteins colocalize at the cell membrane and reduced expression of MCC results in impaired cell migration. By contrast to Scrib, MCC inhibits cell directed migration independently of Rac1, Cdc42 and PAK activation. Altogether, these results identify MCC as a potential scaffold protein regulating cell movement and able to bind Scrib, beta-catenin and NHERF1/2.


Subject(s)
Cell Movement/physiology , Epithelial Cells/physiology , Membrane Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Animals , Cell Line , Enzyme Activation , Epithelial Cells/cytology , Humans , Membrane Proteins/genetics , Phosphoproteins/genetics , Phosphoproteins/metabolism , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sodium-Hydrogen Exchangers/genetics , Sodium-Hydrogen Exchangers/metabolism , Tumor Suppressor Proteins/genetics , beta Catenin/metabolism , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolism
16.
Biochem Biophys Res Commun ; 378(3): 360-5, 2009 Jan 16.
Article in English | MEDLINE | ID: mdl-19013433

ABSTRACT

In this work, we describe how the Erbin PDZ domain interacts with Smad3, a transductor of the Transforming Growth Factor-beta (TGFbeta) pathway, via its MH2 domain. This interaction was described as important for TGFbeta signaling as it could potentially repress the transcriptional activity of the growth factor. In order to clarify our preliminary experimental observations pointing this interaction, we built a 3D model of the Erbin PDZ/Smad3 MH2 complex and checked its stability using molecular dynamics simulations. This model pointed out charged residues in Smad3 and Erbin which could be important for the interaction. By introducing point mutations of these residues within the proposed binding domains, we experimentally confirmed that arginine 279, glutamic acid 246 in Smad3 and glutamic acid 1321 in Erbin are important for the binding. These data suggest a possible novel interface of binding in the Erbin PDZ domain and reveal an unconventional mode of interaction for a PDZ domain and its ligand.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , PDZ Domains , Smad3 Protein/metabolism , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Sequence , Arginine/genetics , Arginine/metabolism , Cell Line , Glutamic Acid/genetics , Glutamic Acid/metabolism , Humans , Protein Binding , Protein Interaction Mapping , Smad3 Protein/genetics , Transforming Growth Factor beta/metabolism
17.
Proteomics ; 8(15): 3071-81, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18654987

ABSTRACT

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease that shows minimal response to chemotherapy. Genetic changes involved in the progression of PDAC concern genes that encode proteins related to signal transduction networks. This fact reveals the importance in identifying the role and the relations between multiple signaling cascades in PDAC. One of the major factors that modulate signaling events is multidomain scaffold proteins that function by binding several proteins simultaneously, inducing their proximity and influencing the outcome of signaling. A particular group among them, containing multiple Src homology 3 (SH3) domains that can bind proteins containing proline-rich motifs, was associated to different aspects of cancer cell homeostasis. In this work, using a microarray-based analysis, we have shown that 13 multiple SH3 domain containing scaffold proteins are expressed in PDAC cells. Using a yeast two-hybrid approach, we have identified proteins that interact with these adaptor proteins. Among them we have found several molecules that modulate cell proliferation and survival (CIZ1, BIRC6, RBBP6), signaling (LTBP4, Notch2, TOM1L1, STK24) and membrane dynamics (PLSCR1, DDEF2, VCP). Our results indicate that interactions mediated by multi-SH3 domain-containing proteins could lead to the formation of dynamic protein complexes that function in pancreatic cancer cell signaling. The identification of such protein complexes is of paramount importance in deciphering pancreatic cancer biology and designing novel therapeutic approaches.


Subject(s)
Carrier Proteins/genetics , Proteins/genetics , Proteomics/methods , src Homology Domains/genetics , Blotting, Western , Carrier Proteins/isolation & purification , Carrier Proteins/metabolism , Cell Line , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Humans , Immunoprecipitation , Oligonucleotide Array Sequence Analysis , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Protein Array Analysis/methods , Protein Binding , Proteins/metabolism , Two-Hybrid System Techniques
18.
Cancer Res ; 67(3): 1145-54, 2007 Feb 01.
Article in English | MEDLINE | ID: mdl-17283149

ABSTRACT

Mantle cell lymphoma (MCL) is one of the most frequent of the newly recognized non-Hodgkin's lymphomas. The major problem of MCL therapy is the occurrence of relapse and subsequent resistance to chemotherapy and immunotherapy in virtually all cases. Here, we show that one injection of anti-human transferrin receptor (TfR) monoclonal antibody A24 totally prevented xenografted MCL tumor establishment in nude mice. It also delayed and inhibited tumor progression of established tumors, prolonging mice survival. In vitro, A24 induced up to 85% reduction of MCL cell proliferation (IC(50) = 3.75 nmol/L) independently of antibody aggregation, complement-dependent or antibody-dependent cell-mediated cytotoxicity. A24 induced MCL cell apoptosis through caspase-3 and caspase-9 activation, either alone or synergistically with chemotherapeutic agents. A24 induced TfR endocytosis via the clathrin adaptor protein-2 complex pathway followed by transport to lysosomal compartments. Therefore, A24-based therapies alone or in association with classic chemotherapies could provide a new alternative strategy against MCL, particularly in relapsing cases.


Subject(s)
Antibodies, Monoclonal/pharmacology , Immunization, Passive/methods , Lymphoma, Mantle-Cell/prevention & control , Lysosomes/metabolism , Receptors, Transferrin/metabolism , Animals , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Growth Processes/drug effects , Cell Line, Tumor , Endocytosis/drug effects , Female , Humans , Lymph Nodes/metabolism , Lymph Nodes/pathology , Lymphoma, Mantle-Cell/metabolism , Lymphoma, Mantle-Cell/pathology , Mice , Mice, Nude , Receptors, Transferrin/immunology , Xenograft Model Antitumor Assays
19.
Proc Natl Acad Sci U S A ; 103(36): 13497-502, 2006 Sep 05.
Article in English | MEDLINE | ID: mdl-16938850

ABSTRACT

Understanding the mechanisms of Salmonella virulence is an important challenge. The capacity of this intracellular bacterial pathogen to cause diseases depends on the expression of virulence factors including the second type III secretion system (TTSS-2), which is used to translocate into the eukaryotic cytosol a set of effector proteins that divert the biology of the host cell and shape the bacterial replicative niche. Yet little is known about the eukaryotic functions affected by individual Salmonella effectors. Here we report that the TTSS-2 effector PipB2 interacts with the kinesin light chain, a subunit of the kinesin-1 motor complex that drives anterograde transport along microtubules. Translocation of PipB2 is both necessary and sufficient for the recruitment of kinesin-1 to the membrane of the Salmonella-containing vacuole. In vivo, PipB2 contributes to the attenuation of Salmonella mutant strains in mice. Taken together, our data indicate that the TTSS-2-mediated fine-tuning of kinesin-1 activity associated with the bacterial vacuole is crucial for the virulence of Salmonella.


Subject(s)
Bacterial Proteins/metabolism , Kinesins/metabolism , Salmonella/metabolism , Salmonella/pathogenicity , Animals , Bacterial Proteins/genetics , Bone Marrow Cells/cytology , Cell Differentiation , Cell Line , Cells, Cultured , Female , Femur/cytology , Gene Deletion , HeLa Cells , Humans , Macrophages/metabolism , Macrophages/microbiology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Salmonella/classification , Salmonella/genetics , Salmonella/growth & development , Salmonella Infections, Animal/microbiology , Vacuoles/microbiology , Virulence
20.
Blood ; 108(3): 1065-72, 2006 Aug 01.
Article in English | MEDLINE | ID: mdl-16597595

ABSTRACT

Two classes of oncogenic mutations of the c-kit tyrosine kinase have been described: the juxtamembrane domain V560G mutation, which is preferentially found in gastrointestinal stromal tumors (GISTs), and the kinase domain D816V mutation, which is highly representative of systemic mastocytosis (SM). Here we show that both mutations constitutively activate the mammalian target of rapamycin (mTOR) signaling pathway. Surprisingly, the mTOR inhibitor rapamycin induces only apoptosis in HMC-1 cells bearing the D816V but not the V560G mutation. In support of this unexpected selectivity, rapamycin inhibits the phosphorylation of 4E-BP1, a downstream substrate of the mTOR pathway, but only in D816V HMC-1 cells. Importantly, D816V mast cells isolated from SM patients or from transgenic mice are sensitive to rapamycin whereas normal human or mouse mast cells are not. Thus, rapamycin inhibition appears specific to the D816V mutation. At present there is no effective cure for SM patients with the D816V mutation. The data presented here provide a rationale to test whether rapamycin could be a possible treatment for SM and other hematologic malignancies with the D816V mutation.


Subject(s)
Mastocytosis, Systemic/drug therapy , Mastocytosis, Systemic/genetics , Mutation, Missense , Pharmacogenetics , Proto-Oncogene Proteins c-kit/genetics , Sirolimus/pharmacology , Animals , Apoptosis/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Humans , Mast Cells/drug effects , Mast Cells/pathology , Mastocytosis, Systemic/pathology , Mice , Mice, Transgenic , Protein Kinases/metabolism , Signal Transduction , TOR Serine-Threonine Kinases , Tumor Cells, Cultured
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