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1.
Cancer Lett ; 360(1): 48-59, 2015 Apr 28.
Article in English | MEDLINE | ID: mdl-25684663

ABSTRACT

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) emerges as one of the most-promising experimental cancer therapeutic drugs and is currently being tested in clinical trials. However, both intrinsic and acquired resistance of human cancer cells to TRAIL-induced apoptosis poses a huge problem in establishing clinically efficient TRAIL therapies. To assess the regulation of TRAIL-resistance in human pancreatic cancer cells, we studied the TRAIL resistant pancreatic cell line PANC-1. We show that treatment with PH11, a novel Focal Adhesion Kinase (FAK) inhibitor in association with TRAIL rapidly induces apoptosis in TRAIL-resistant PANC-1 cells, but not in normal human fibroblast cells. To explain sensitization, we showed that PH11 restores TRAIL apoptotic pathway in PANC-1 cells through down-regulation of c-FLIP via inhibition of FAK and the phosphatidylinositol-3 kinase (PI3K)/AKT pathways. These findings suggest that this combined treatment may offer an attractive therapeutic strategy for safely and efficiently treating pancreatic cancer.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Drug Resistance, Neoplasm/drug effects , Focal Adhesion Kinase 1/antagonists & inhibitors , Imidazoles/pharmacology , Pancreatic Neoplasms/enzymology , Pancreatic Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Triazines/pharmacology , CASP8 and FADD-Like Apoptosis Regulating Protein/genetics , CASP8 and FADD-Like Apoptosis Regulating Protein/metabolism , Caspases/metabolism , Cell Line, Tumor , Dose-Response Relationship, Drug , Enzyme Activation , Focal Adhesion Kinase 1/genetics , Focal Adhesion Kinase 1/metabolism , Humans , Molecular Targeted Therapy , Pancreatic Neoplasms/genetics , Phosphatidylinositol 3-Kinase/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA Interference , Signal Transduction/drug effects , Time Factors , Transfection
2.
Anal Biochem ; 423(1): 54-60, 2012 Apr 01.
Article in English | MEDLINE | ID: mdl-22266292

ABSTRACT

Citrulline, a key amino acid of the urea cycle, has been shown to play a regulatory role in protein and energy metabolism in mammals. We questioned whether N-carbamoyl-putrescine (NCP), the decarboxylated derivative of citrulline, could play a role in the biological properties of this amino acid. To evidence the presence of NCP in mammalian tissues, we developed a sensitive reverse-phase high-performance liquid chromatography (HPLC) with fluorimetric detection method with precolumn dansyl derivatization and solid-phase extraction for the determination of NCP together with polyamines in biological samples. Dansyl NCP was identified with a 5.85-min retention time. Linearity was obtained in a concentration range of 0.125 to 12.5 µM. Intraday and day-to-day relative coefficients of variation ranged from 8.9% to 12.3% and from 14% to 14.3%, respectively. Recovery rates in serum ranged from 75% to 83%. Thereafter, we used this method to search for the presence of NCP in serum, muscle, liver, jejunum, and ileum in rats after both short-term intraperitoneal injection and long-term oral citrulline supplementation. We failed to detect NCP in these animals. These data suggest that NCP is not a significant citrulline metabolite in rats.


Subject(s)
Chromatography, High Pressure Liquid , Chromatography, Reverse-Phase , Citrulline/metabolism , Putrescine/analogs & derivatives , Animals , Citrulline/chemistry , Dansyl Compounds/chemistry , Putrescine/analysis , Putrescine/isolation & purification , Rats , Rats, Sprague-Dawley , Solid Phase Extraction
3.
Med Image Comput Comput Assist Interv ; 11(Pt 2): 1066-74, 2008.
Article in English | MEDLINE | ID: mdl-18982710

ABSTRACT

In most approaches, tissue and subcortical structure segmentations of MR brain scans are handled globally over the entire brain volume through two relatively independent sequential steps. We propose a fully Bayesian joint model that integrates local tissue and structure segmentations and local intensity distributions. It is based on the specification of three conditional Markov Random Field (MRF) models. The first two encode cooperations between tissue and structure segmentations and integrate a priori anatomical knowledge. The third model specifies a Markovian spatial prior over the model parameters that enables local estimations while ensuring their consistency, handling this way nonuniformity of intensity without any bias field modelization. The complete joint model provides a sound theoretical framework for carrying out tissue and structure segmentation by distributing a set of local and cooperative MRF models. The evaluation, using a previously affine-registred atlas of 17 structures and performed on both phantoms and real 3T brain scans, shows good results.


Subject(s)
Algorithms , Brain/anatomy & histology , Image Interpretation, Computer-Assisted/methods , Pattern Recognition, Automated/methods , Subtraction Technique , Bayes Theorem , Humans , Image Enhancement/methods , Models, Neurological , Models, Statistical , Reproducibility of Results , Sensitivity and Specificity
4.
Med Image Comput Comput Assist Interv ; 10(Pt 1): 219-27, 2007.
Article in English | MEDLINE | ID: mdl-18051062

ABSTRACT

We propose to carry out cooperatively both tissue and structure segmentations by distributing a set of local and cooperative models in a unified MRF framework. Tissue segmentation is performed by partitionning the volume into subvolumes where local MRFs are estimated in cooperation with their neighbors to ensure consistency. Local estimation fits precisely to the local intensity distribution and thus handles nonuniformity of intensity without any bias field modelization. Structure segmentation is performed via local MRFs that integrate localization constraints provided by a priori general fuzzy description of brain anatomy. Structure segmentation is not reduced to a postprocessing step but cooperates with tissue segmentation to gradually and conjointly improve models accuracy. The evaluation was performed using phantoms and real 3T brain scans. It shows good results and in particular robustness to nonuniformity and noise with a low computational cost.


Subject(s)
Algorithms , Artificial Intelligence , Brain/anatomy & histology , Image Enhancement/methods , Image Interpretation, Computer-Assisted/methods , Imaging, Three-Dimensional/methods , Magnetic Resonance Imaging/methods , Pattern Recognition, Automated/methods , Humans , Reproducibility of Results , Sensitivity and Specificity
5.
Article in English | MEDLINE | ID: mdl-18002276

ABSTRACT

The problem addressed in this paper is the automatic segmentation of stroke lesions on MR multi-sequences. Lesions enhance differently depending on the MR modality and there is an obvious gain in trying to account for various sources of information in a single procedure. To this aim, we propose a multimodal Markov random field model which includes all MR modalities simultaneously. The results of the multimodal method proposed are compared with those obtained with a mono-dimensional segmentation applied on each MRI sequence separately. We constructed an Atlas of blood supply territories to help clinicians in the determination of stroke subtypes and potential functional deficit.


Subject(s)
Brain Ischemia/pathology , Brain/pathology , Image Interpretation, Computer-Assisted/methods , Magnetic Resonance Imaging/methods , Pattern Recognition, Automated/methods , Stroke/pathology , Subtraction Technique , Algorithms , Artificial Intelligence , Brain Ischemia/etiology , Humans , Image Enhancement/methods , Reproducibility of Results , Sensitivity and Specificity , Stroke/complications
6.
Anal Biochem ; 351(1): 93-9, 2006 Apr 01.
Article in English | MEDLINE | ID: mdl-16480678

ABSTRACT

Growth factor receptor-bound protein 2 (Grb2) is an adapter protein involved in the Ras-dependent signaling pathway that plays an important role in human cancers initiated by oncogenic receptors. Grb2 is constituted by one Src homology 2 domain surrounded by two SH3 domains, and the inhibition of the interactions produced by these domains could provide an antitumor approach. In evaluating chemical libraries, to search for potential Grb2 inhibitors, it was necessary to elaborate a rapid test for their screening. We have developed, first, a batch method based on the use of an affinity column bearing a Grb2-SH3 peptide ligand to isolate highly purified Grb2. We subsequently describe a very rapid 96-well screening of inhibitors based on a simple competition between purified Grb2 and a peroxidase-coupled proline-rich peptide.


Subject(s)
Chromatography, Affinity/methods , GRB2 Adaptor Protein/isolation & purification , Horseradish Peroxidase/chemistry , Amino Acid Sequence , Electrophoresis, Polyacrylamide Gel , GRB2 Adaptor Protein/chemistry , Spectrometry, Fluorescence , src Homology Domains
7.
Biochemistry ; 43(23): 7336-44, 2004 Jun 15.
Article in English | MEDLINE | ID: mdl-15182177

ABSTRACT

This paper describes the design of the highest affinity ligands for Grb2 SH3 domains reported so far. These compounds were designed by combining N-alkyl amino acid incorporation in a proline-rich sequence with subsequent dimerization of the peptoid sequence based on structural data and molecular modeling. Optimization of the linker size is discussed, and the N-alkyl amino acid incorporation into both monomeric halves is reported. Because the affinity for Grb2 of the optimized compounds was too high to be measured using the fluorescent modifications that they induce on the Grb2 emission spectrum, a competition assay was developed. In this test, Grb2 is pulled down from a cellular extract by the initial VPPPVPPRRR peptide bound to Sepharose beads. In the presence of competitors, the test quantifies the amount of Grb2 displaced from the beads. It has enabled us to determine a K(i) value in the 10(-10) M range for the highest affinity Grb2 peptoid analogue dimer.


Subject(s)
Adaptor Proteins, Signal Transducing , Peptoids/analogs & derivatives , Peptoids/metabolism , Proteins/chemistry , Proteins/metabolism , src Homology Domains , Alkylation , Amino Acid Sequence , Animals , Cell Line , Cricetinae , Dimerization , GRB2 Adaptor Protein , Humans , Molecular Sequence Data , Peptoids/chemical synthesis , Peptoids/chemistry , Proline/chemistry , Protein Structure, Tertiary , Proteins/antagonists & inhibitors , Spectrometry, Fluorescence , src Homology Domains/drug effects
8.
Artif Intell Med ; 27(3): 229-32, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12667737
9.
Biochem J ; 360(Pt 2): 499-506, 2001 Dec 01.
Article in English | MEDLINE | ID: mdl-11716779

ABSTRACT

In the vitamin K-dependent protein family, only protein S (PS) contains a thrombin-sensitive region (TSR), located between the domain containing the gamma-carboxyglutamic acid and the first epidermal growth factor-like domain. To better define the role of TSR in the PS molecule, we expressed a recombinant human PS (rHPS) and its analogue lacking TSR (rTSR-less), and prepared factor Xa- and thrombin-cleaved rHPS. A peptide reproducing TSR (TSR-peptide) was also synthesized in an attempt to obtain direct evidence of the domain involvement in PS anticoagulant activity. In a coagulation assay, both rTSR-less and factor Xa-cleaved PS were devoid of activated protein C cofactor activity. The TSR-peptide did not inhibit rHPS activity, showing that TSR must be embedded in the native protein to promote interaction with activated protein C. The binding of rHPS to activated platelets and to phospholipid vesicles was not modified after factor Xa- or thrombin-mediated TSR cleavage, whereas the binding of rTSR-less was markedly reduced. This suggested a role for TSR in conferring to PS a strong affinity for phospholipid membranes. TSR-peptide did not directly bind to activated platelets or compete with rHPS for phospholipid binding. The results of the present study show that TSR may not interact directly with membranes, but probably constrains the gamma-carboxyglutamic acid-rich domain in a conformation allowing optimal interaction with phospholipids.


Subject(s)
1-Carboxyglutamic Acid/metabolism , Protein S/chemistry , Protein S/metabolism , Thrombin/physiology , Antibodies, Monoclonal/metabolism , Anticoagulants/chemistry , Anticoagulants/metabolism , Blood Platelets/metabolism , Cell Line , Cell Membrane/enzymology , Cell Membrane/genetics , Cell Membrane/metabolism , Enzyme Activation , Epitope Mapping , Factor Xa/metabolism , Humans , Hydrolysis , Liposomes/metabolism , Mutagenesis, Site-Directed , Phospholipids/metabolism , Platelet Activation , Protein Binding/genetics , Protein C/metabolism , Protein Conformation , Protein S/genetics , Protein S/physiology , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiology , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Sequence Deletion
10.
Crit Rev Oncol Hematol ; 40(2): 175-86, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11682324

ABSTRACT

The Src homology domains SH2 and SH3 are small modular protein motifs about 100 and 60 amino acids long, respectively. SH2 domains interact with phosphotyrosine residues, whereas SH3 domains recognize proline-rich motifs of their interacting partners. SH2 and SH3 domains are frequently found in signaling proteins such as small adaptors and in enzymes such as kinases, lipases and phosphatases, in which they differ from the catalytic motif and constitute recognition modules. SH2 and SH3 domains are also found in oncoproteins and in proteins overexpressed in deregulated signaling pathways in tumor cells. The highly folded structures of these domains have been characterized alone and complexed with the essential fragments of their targets. Therefore, based on molecular data, inhibitors of interactions with SH2 and SH3 domains are considered to be potential antitumor agents. Current results are very promising, as inhibitors with very efficient anti-proliferative activity in tumor cells have been reported. This paper describes SH2 and/or SH3 domain-containing proteins that may constitute targets for anticancer therapeutics. It also deals with the essential structural data concerning SH2 and SH3 domains, and the rational design of inhibitors. Some of the more recent pharmacological results obtained with these compounds are also discussed.


Subject(s)
Antineoplastic Agents/pharmacology , Growth Inhibitors/pharmacology , Neoplasm Proteins/antagonists & inhibitors , src Homology Domains/drug effects , Amino Acid Sequence , Animals , Antineoplastic Agents/chemistry , Cell Division/drug effects , Drug Design , Growth Inhibitors/chemistry , Hydrolysis , Ligands , Models, Biological , Models, Molecular , Molecular Sequence Data , Neoplasm Proteins/chemistry , Neoplasm Proteins/physiology , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/enzymology , Neoplastic Stem Cells/pathology , Phosphopeptides/chemistry , Phosphopeptides/pharmacology , Phosphoric Monoester Hydrolases/metabolism , Prodrugs/chemistry , Prodrugs/pharmacokinetics , Protein Conformation , Signal Transduction/drug effects , src-Family Kinases/antagonists & inhibitors , src-Family Kinases/chemistry , src-Family Kinases/physiology
11.
Cell Signal ; 13(1): 51-5, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11257447

ABSTRACT

The adaptor protein Grb2 plays a central role in cell proliferation and/or cell cycle progression. In this study, we investigate the role of Grb2 in signalling pathways involved in meiotic reinitiation. For that purpose, Xenopus Grb2 cRNA and its mutated forms or human Grb2 protein was microinjected into immature Xenopus oocytes. Reinitiation of meiosis was seen in unstimulated oocytes. Induction of the meiosis was time dependent and Ras dependent, and the presence in Grb2 of SH2 and SH3 domains was required. Several tyrosine phosphorylated proteins were solely detected in oocytes responsive to Grb2 injection. Our results are in favour of an unusual recruitment and initiation of the Grb2 transduction cascade independent of a receptor tyrosine kinase (RTK) stimulation.


Subject(s)
Adaptor Proteins, Signal Transducing , Oocytes/cytology , Proteins/agonists , Proteins/physiology , Animals , Female , GRB2 Adaptor Protein , Humans , Meiosis , Protein-Tyrosine Kinases/physiology , Signal Transduction/physiology , Xenopus laevis
12.
Psychopharmacology (Berl) ; 153(2): 170-9, 2001 Jan 01.
Article in English | MEDLINE | ID: mdl-11205416

ABSTRACT

RATIONALE: A previous study in the rat has shown that systemic injection of two CCK-B agonists, BC264 and BC197, induced opposing effects on the retrieval phase of a spatial recognition memory task. OBJECTIVE: The present study was designed to investigate the mechanisms underlying these effects at the level of the dopaminergic system. METHODS: Rats were injected IPly with BC264 (0.3 microg/kg) or BC197 (30 microg/kg) and with D1 or D2 agonists and antagonists. The cognitive performances of rat were analysed on the retrieval phase of a spatial recognition memory task. The extracellular levels of dopamine were quantified in the anterior nucleus accumbens after injection of BC197 (3, 30 and 300 microg/kg IP), using the microdialysis technique on freely moving rats. Local injection of the D2 antagonist, sulpiride (2.5 ng/microl) was performed in the anterior nucleus accumbens and the cognitive performances analysed following systemic injection of BC264 (0.3 microg/kg). RESULTS: The improvement and the impairment of performance induced respectively by BC264 and BC197 were suppressed by peripheral administration of sulpiride, showing that these opposing effects were both mediated by the stimulation of D2-like receptors. However, different dopaminergic pathways seem to be involved in the effects of the two CCK-B agonists. Indeed, systemic administration of BC197 did not induce the increase of extracellular dopamine levels observed with BC264. Furthermore, local injection of sulpiride, in the anterior nucleus accumbens, completely suppressed the cognitive enhancing effect of BC264. CONCLUSION: These findings suggest that the D2-mediated deficit in the performance induced by BC197 involves brain structures other than the anterior nucleus accumbens. They also demonstrate a critical role of dopaminergic transmission within the anterior nucleus accumbens in the improving effect induced by BC264 in a spatial memory task.


Subject(s)
Cholecystokinin/analogs & derivatives , Memory/drug effects , Nucleus Accumbens/physiology , Receptors, Cholecystokinin/agonists , Receptors, Dopamine D2/drug effects , Space Perception/drug effects , 3,4-Dihydroxyphenylacetic Acid/metabolism , Animals , Biotransformation , Cholecystokinin/pharmacology , Dopamine/metabolism , Drug Implants , Homovanillic Acid/metabolism , Male , Microinjections , Peptide Fragments/pharmacology , Rats , Rats, Wistar , Receptor, Cholecystokinin B , Stereotaxic Techniques
13.
J Med Chem ; 43(20): 3614-23, 2000 Oct 05.
Article in English | MEDLINE | ID: mdl-11020275

ABSTRACT

Recent advances in the field of cholecystokinin have indicated the possible occurrence of multiple affinity states of the CCK(2) receptor. Besides, numerous pharmacological experiments performed "in vitro" and "in vivo" support the eventuality of different pharmacological profiles associated to CCK(2) ligands. Indeed, some agonists are essentially anxiogenic and uneffective in memory tests, whereas others are not anxiogenic and appear as able to reinforce memory. The reference compound for the latter profile is the CCK-8 analogue BC 264 (Boc-Tyr(SO(3)H)-gNle-mGly-Trp-(NMe)Nle-Asp-Phe-NH(2)). However, although tetrapeptide ligands based on CCK-4 (Trp-Met-Asp-Phe-NH(2)) are known to possess sufficient structural features for CCK(2) recognition, none shares the properties of BC 264. Hence we have developed new short peptidic or pseudo-peptidic derivatives containing the C-terminal tetrapeptide of BC 264. Our results indicate that some compounds characterized by the presence of two carbonyl groups at the N-terminus, as in 2b (HO(2)C-CH(2)-CONH-Trp-(NMe)Nle-Asp-Phe-NH(2)), are likely to show a BC 264-like profile, bind to the CCK(2) receptor in a specific way, and display remarkable affinities (2b: 0.28 nM on guinea-pig cortex membrane preparations). This original binding mode is discussed and further enlightened by NMR and molecular modeling studies.


Subject(s)
Cholecystokinin/chemistry , Glycine/chemistry , Oligopeptides/chemical synthesis , Peptide Fragments/chemistry , Receptors, Cholecystokinin/agonists , Animals , Aspartic Acid/analogs & derivatives , Aspartic Acid/chemical synthesis , Aspartic Acid/chemistry , Aspartic Acid/metabolism , CHO Cells , Cerebral Cortex/metabolism , Cricetinae , Guinea Pigs , In Vitro Techniques , Inositol Phosphates/biosynthesis , Membranes , Models, Molecular , Molecular Conformation , Oligopeptides/chemistry , Oligopeptides/metabolism , Pancreas/metabolism , Rats , Receptors, Cholecystokinin/metabolism , Structure-Activity Relationship
14.
Biochem Pharmacol ; 60(8): 1165-9, 2000 Oct 15.
Article in English | MEDLINE | ID: mdl-11007954

ABSTRACT

Anarchic cell proliferation, observed in some leukemia and in breast and ovarian cancers, has been related to dysfunctioning of cytoplasmic or receptor tyrosine kinase activities coupled to p21 Ras. The growth factor receptor-bound protein 2 (Grb2) adaptor when complexed with Sos (Son of sevenless), the exchange factor of Ras, conveys the signal induced by tyrosine kinase-activated receptor to Ras by recruiting Sos to the membrane, allowing activation of Ras. This review shows how it is possible to stop the Ras-deregulated signaling pathway to obtain potential antitumor agents. Grb2 protein is comprised of one SH2 surrounded by two SH3 domains and interacts by means of its Src homology (SH2) domain with phosphotyrosine residues of target proteins such as the epidermal growth factor (EGF) receptor or the Shc adaptor. By means of its SH3 domains, Grb2 recognizes proline-rich sequences of Sos, leading to Ras activation. Inhibitors of SH2 and SH3 domains were designed with the aim of interrupting Grb2 recognition. On the one hand, using structural data and molecular modeling, peptide dimers or "peptidimers", made up of two proline-rich sequences from Sos linked by an optimized spacer, were developed. On the other, using the structure of the Grb2 SH2 domain complexed with a phosphotyrosine (pTyr)-containing peptide and molecular modeling studies, a series of N-protected tripeptides containing two phosphotyrosine or mimetic residues, with one pTyr sterically constrained, were devised. These compounds show very high affinities for Grb2 in vitro. They have been targeted into cells showing selective antiproliferative activity on tumor cells. These results suggest that inhibiting SH2 or SH3 domains of signaling proteins might provide antitumor agents.


Subject(s)
Adaptor Proteins, Signal Transducing , Antineoplastic Agents/pharmacology , Signal Transduction/drug effects , ras Proteins/antagonists & inhibitors , Animals , GRB2 Adaptor Protein , Humans , Models, Molecular , Protein Conformation , Proteins/antagonists & inhibitors , Proteins/chemistry , Proteins/metabolism , src Homology Domains/drug effects
15.
Biochemistry ; 39(26): 7722-35, 2000 Jul 04.
Article in English | MEDLINE | ID: mdl-10869177

ABSTRACT

SH3 (src homology domain 3) domains are small protein modules that interact with proline-rich peptides. The structure of the N-terminal SH3 domain from growth factor receptor-binding protein 2 (Grb2), an adapter protein in the intracellular signaling pathway to Ras, was investigated by circular dichroic (CD) spectroscopy. The compact native beta-barrel conformation, previously elucidated by NMR spectroscopy, was largely predominant at pH = 4.8, in the absence of salt. From the structural changes induced by varying pH, ionic strength, temperature, or hydrophobicity of the environment, evidence for the existence of distinct nonnative conformations was obtained in the far- and near-UV domains. Along the free energy scale, these appear to distribute into two conformational ensembles, depending on the extent of structural and thermodynamic differences compared to the native conformation. The first ensemble consists of non-native conformations with a nativelike secondary structure, and the second is composed of partially unfolded conformations having short alpha-helical fragments or turnlike motifs in their nonnative secondary structure. Most of the observed nonnative conformations exist in mild or nondenaturing conditions. They probably have distinct compactness of their inner structure, depending on the strength of nonlocal interactions, but only the native all-beta conformation possesses a condensed protein exterior, appropriate for the binding to the VPPPVPPRRR decapeptide from Sos. Upon binding, the native conformation undergoes a local tertiary structure change in a hydrophobic pocket at the binding site. This is accompanied by the PP-II helix folding of the proline-rich peptide. Interestingly, in the near-UV domain, a significant change in the spectral contribution of an aromatic exciton was observed, thus allowing quantitative tracking of the binding process.


Subject(s)
Adaptor Proteins, Signal Transducing , Guanine Nucleotide Exchange Factors/chemistry , Peptides/chemistry , Proteins/chemistry , src Homology Domains/physiology , Circular Dichroism , GRB2 Adaptor Protein , Guanine Nucleotide Exchange Factors/metabolism , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy , Mutation , Osmolar Concentration , Peptides/metabolism , Proline-Rich Protein Domains , Protein Conformation , Protein Denaturation , Protein Folding , Proteins/genetics , Proteins/metabolism , Temperature , Ultraviolet Rays
16.
J Biol Chem ; 275(29): 21946-52, 2000 Jul 21.
Article in English | MEDLINE | ID: mdl-10781580

ABSTRACT

Proteins of the Wiskott-Aldrich Syndrome protein (WASp) family connect signaling pathways to the actin polymerization-driven cell motility. The ubiquitous homolog of WASp, N-WASp, is a multidomain protein that interacts with the Arp2/3 complex and G-actin via its C-terminal WA domain to stimulate actin polymerization. The activity of N-WASp is enhanced by the binding of effectors like Cdc42-guanosine 5'-3-O-(thio)triphosphate, phosphatidylinositol bisphosphate, or the Shigella IcsA protein. Here we show that the SH3-SH2-SH3 adaptor Grb2 is another activator of N-WASp that stimulates actin polymerization by increasing the amount of N-WASp. Arp2/3 complex. The concentration dependence of N-WASp activity, sedimentation velocity and cross-linking experiments together suggest that N-WASp is subject to self-association, and Grb2 enhances N-WASp activity by binding preferentially to its active monomeric form. Use of peptide inhibitors, mutated Grb2, and isolated SH3 domains demonstrate that the effect of Grb2 is mediated by the interaction of its C-terminal SH3 domain with the proline-rich region of N-WASp. Cdc42 and Grb2 bind simultaneously to N-WASp and enhance actin polymerization synergistically. Grb2 shortens the delay preceding the onset of Escherichia coli (IcsA) actin-based reconstituted movement. These results suggest that Grb2 may activate Arp2/3 complex-mediated actin polymerization downstream from the receptor tyrosine kinase signaling pathway.


Subject(s)
Actins/metabolism , Adaptor Proteins, Signal Transducing , Cytoskeletal Proteins , Proteins/metabolism , Signal Transduction , Actin-Related Protein 2 , Actin-Related Protein 3 , Animals , ErbB Receptors/metabolism , GRB2 Adaptor Protein , Humans , Rabbits , Recombinant Proteins/metabolism , Wiskott-Aldrich Syndrome , Wiskott-Aldrich Syndrome Protein
17.
Bioorg Med Chem Lett ; 10(7): 669-72, 2000 Apr 03.
Article in English | MEDLINE | ID: mdl-10762050

ABSTRACT

Phosphopeptide prodrugs bearing two S-acyl-2-thioethyl (SATE) biolabile phosphate protections were developed. They are capable to inhibit the Shc/Grb2 interaction and MAP kinases (ERK1 and ERK2) phosphorylation in cellular assay. The S-acetyl-2-thioethyl (MeSATE) analogue showed an IC50 of 1 microM in the inhibition of the colony formation of tumor cell line NIH3T3/HER2.


Subject(s)
Adaptor Proteins, Signal Transducing , Antineoplastic Agents/pharmacology , Phosphopeptides/pharmacology , Prodrugs/pharmacology , 3T3 Cells , Animals , Antineoplastic Agents/chemistry , GRB2 Adaptor Protein , Intramolecular Transferases/metabolism , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Phosphopeptides/chemistry , Phosphorylation/drug effects , Prodrugs/chemistry , Proteins/metabolism , Stem Cells/drug effects , ras Proteins/antagonists & inhibitors , ras Proteins/pharmacology
18.
Artif Intell Med ; 20(1): 77-93, 2000 Aug.
Article in English | MEDLINE | ID: mdl-11185422

ABSTRACT

Automatic segmentation of MRI brain scans is a complex task for two main reasons: the large variability of the human brain anatomy, which limits the use of general knowledge and, inherent to MRI acquisition, the artifacts present in the images that are difficult to process. To tackle these difficulties, we propose to mix, in a cooperative framework, several types of information and knowledge provided and used by complementary individual systems: presently, a multi-agent system, a deformable model and an edge detector. The outcome is a cooperative segmentation performed by a set of region and edge agents constrained automatically and dynamically by both, the specific gray levels in the considered image, statistical models of the brain structures and general knowledge about MRI brain scans. Interactions between the individual systems follow three modes of cooperation: integrative, augmentative and confrontational cooperation, combined during the three steps of the segmentation process namely, the specialization of the seeded-region-growing agents, the fusion of heterogeneous information and the retroaction over slices. The described cooperative framework allows the dynamic adaptation of the segmentation process to the own characteristics of each MRI brain scan. Its evaluation using realistic brain phantoms is reported.


Subject(s)
Brain/anatomy & histology , Brain/physiology , Computer Simulation , Magnetic Resonance Imaging , Artificial Intelligence , Automation , Humans , Magnetic Resonance Imaging/methods , Phantoms, Imaging
19.
J Med Chem ; 42(18): 3737-41, 1999 Sep 09.
Article in English | MEDLINE | ID: mdl-10479306

ABSTRACT

A series of small peptides with the sequence mAZ-pTyr-Xaa-Asn-NH(2), where Xaa denotes alpha-methylphosphotyrosine or its carboxylic mimetics, were synthesized as inhibitors of the Grb2 SH2 domain. Peptide 3 with (alpha-Me)pTyr as Xaa has the highest affinity for Grb2 (K(d) = 3 +/- 1 nM) and exhibits to date the best inhibitory activity (IC(50) = 11 +/- 1 nM) to displace PSpYVNVQN-Grb2 interaction in an ELISA test. The lower affinities of peptides with (alpha-Me)Tyr, (alpha-Me)Phe(4-CO(2)H), or (alpha-Me)Phe(4-CH(2)CO(2)H) as Xaa demonstrate the importance of a double charged phosphate group at the pY+1 position. Molecular modeling showed additional hydrogen bond interactions provided by the (alpha-Me)pTyr residue with the Grb2 SH2 domain. These results thus show that the effect of hydrophobic pY+1 residues, initially put forth to increased the binding affinities, can be further enhanced by a (-Me)pTyr residue which has both hydrophobic and hydrophilic properties.


Subject(s)
Adaptor Proteins, Signal Transducing , Peptides/chemistry , Phosphotyrosine/chemistry , Proteins/antagonists & inhibitors , src Homology Domains , Binding Sites , GRB2 Adaptor Protein , Models, Molecular , Peptides/pharmacology , Protein Binding
20.
J Mol Biol ; 290(3): 717-30, 1999 Jul 16.
Article in English | MEDLINE | ID: mdl-10395825

ABSTRACT

Quantitative analysis of Grb2/dynamin interaction through plasmon resonance analysis (BIAcore) using Grb2 mutants showed that the high affinity measured between Grb2 and dynamin is essentially mediated by the N-SH3 domain of Grb2. In order to study the interactions between Grb2 and either dynamin or Sos in more detail, Grb2 N-SH3 domains containing different mutations have been analysed. Two mutations were located on the hydrophobic platform binding proline-rich peptides (Y7V and P49L) and one (E40T) located in a region that we had previously shown to be essential for Grb2/dynamin interactions. Through NMR analysis, we have clearly demonstrated that the structure of the P49L mutant is not folded, while the other E40T and Y7V mutants adopt folded structures that are quite similar to that described for the reference domain. Nevertheless, these point mutations were shown to alter the overall stability of these domains by inducing an equilibrium between a folded and an unfolded form. The complex formed between the peptide VPPPVPPRRR, derived from Sos, and the E40T mutant was shown to have the same 3D structure as that described for the wild-type SH3 domain. However, the VPPPVPPRRR peptide adopts a slightly different orientation when it is complexed with the Y7V mutant. Finally, the affinity of the proline-rich peptide GPPPQVPSRPNR, derived from dynamin, for the Grb2 N-SH3 domain was too low to be analyzed by NMR. Thus, the interaction between either Sos or dynamin and the SH3 mutants were tested on a cellular homogenate by means of a far-Western blot analysis. In these conditions, the P49L mutant was shown to be devoid of affinity for Sos as well as for dynamin. The Y7V SH3 mutant displayed a decrease of affinity for both Sos and dynamin, while the E40T mutant exhibited a decrease of affinity only for dynamin. These results support the existence of two binding sites between dynamin and the Grb2 N-SH3 domain.


Subject(s)
Adaptor Proteins, Signal Transducing , GTP Phosphohydrolases/chemistry , Membrane Proteins/chemistry , Proteins/genetics , src Homology Domains , Amino Acid Sequence , Animals , Cell Line , Cricetinae , Dynamins , GRB2 Adaptor Protein , GTP Phosphohydrolases/metabolism , Magnetic Resonance Spectroscopy , Membrane Proteins/metabolism , Models, Molecular , Protein Binding , Proteins/chemistry , Proteins/metabolism , Son of Sevenless Proteins , Spectrometry, Fluorescence , Surface Plasmon Resonance
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