RESUMO
Small-molecule competitors of protein-protein interactions are urgently needed for functional analysis of large-scale genomics and proteomics data. Particularly abundant, yet so far undruggable, targets include domains specialized in recognizing proline-rich segments, including Src-homology 3 (SH3), WW, GYF, and Drosophila enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) homology 1 (EVH1) domains. Here, we present a modular strategy to obtain an extendable toolkit of chemical fragments (ProMs) designed to replace pairs of conserved prolines in recognition motifs. As proof-of-principle, we developed a small, selective, peptidomimetic inhibitor of Ena/VASP EVH1 domain interactions. Highly invasive MDA MB 231 breast-cancer cells treated with this ligand showed displacement of VASP from focal adhesions, as well as from the front of lamellipodia, and strongly reduced cell invasion. General applicability of our strategy is illustrated by the design of an ErbB4-derived ligand containing two ProM-1 fragments, targeting the yes-associated protein 1 (YAP1)-WW domain with a fivefold higher affinity.
Assuntos
Domínios Proteicos Ricos em Prolina , Mapeamento de Interação de Proteínas , Animais , Moléculas de Adesão Celular/química , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Cristalografia por Raios X , Drosophila melanogaster/metabolismo , Esterificação , Imunofluorescência , Humanos , Cinética , Ligantes , Proteínas dos Microfilamentos/química , Modelos Moleculares , Peso Molecular , Peptídeos/química , Fosfoproteínas/química , Ligação Proteica , Estrutura Terciária de Proteína , Pseudópodes , Fibras de Estresse/metabolismo , Zixina/químicaRESUMO
The cyclodepsipeptide cotransin was described to inhibit the biosynthesis of a small subset of proteins by a signal sequence-discriminatory mechanism at the Sec61 protein-conducting channel. However, it was not clear how selective cotransin is, i.e. how many proteins are sensitive. Moreover, a consensus motif in signal sequences mediating cotransin sensitivity has yet not been described. To address these questions, we performed a proteomic study using cotransin-treated human hepatocellular carcinoma cells and the stable isotope labelling by amino acids in cell culture technique in combination with quantitative mass spectrometry. We used a saturating concentration of cotransin (30 micromolar) to identify also less-sensitive proteins and to discriminate the latter from completely resistant proteins. We found that the biosynthesis of almost all secreted proteins was cotransin-sensitive under these conditions. In contrast, biosynthesis of the majority of the integral membrane proteins was cotransin-resistant. Cotransin sensitivity of signal sequences was neither related to their length nor to their hydrophobicity. Instead, in the case of signal anchor sequences, we identified for the first time a conformational consensus motif mediating cotransin sensitivity.
Assuntos
Peptídeos Cíclicos/análise , Proteômica , Sequência de Aminoácidos , Aquaporina 2/genética , Aquaporina 2/metabolismo , Isótopos de Carbono/química , Cromatografia Líquida de Alta Pressão , Células HEK293 , Células Hep G2 , Humanos , Marcação por Isótopo , Microscopia Confocal , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Isótopos de Nitrogênio/química , Peptídeos Cíclicos/química , Peptídeos Cíclicos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Alinhamento de Sequência , Espectrometria de Massas em TandemRESUMO
We have recently developed a soluble mimic of the corticotropin-releasing factor receptor type 1 (CRF1), a membrane-spanning G protein-coupled receptor, which allowed investigations on receptor-ligand interactions. The CRF1 mimic consists of the receptor N-terminus and three synthetic extracellular loops (ECL1-3), which constitute the extracellular receptor domains (ECDs) of CRF1, coupled to a linear peptide template. Here, we report the synthesis of a modified CRF1 mimic, which is more similar to the native receptor possessing a cyclic template that displays the ECDs in a more physiological conformation compared with the initial linear design. In order to facilitate detailed biophysical investigations on CRF1 mimics, we have further established a cost-efficient access to the CRF1 mimic, which is suitable for isotopic labeling for NMR spectroscopy. To this end, the loop-mimicking cyclic peptide of the ECL2 of CRF1 was produced recombinantly and cyclized by expressed protein ligation. Cyclic ECL2 was obtained in milligram scale, and CRF1 mimics synthesized from this material displayed the same binding properties as synthetic CRF1 constructs.
Assuntos
Mimetismo Molecular , Receptores de Hormônio Liberador da Corticotropina/biossíntese , Receptores de Hormônio Liberador da Corticotropina/química , Sequência de Aminoácidos , Biomimética , Ligantes , Dados de Sequência Molecular , Conformação Proteica , Engenharia de Proteínas , Estrutura Terciária de Proteína , Receptores de Hormônio Liberador da Corticotropina/genética , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
Molecular determinants regulating the activation of class B G-protein-coupled receptors (GPCRs) by native peptide agonists are largely unknown. We have investigated here the interaction between the corticotropin releasing factor receptor type 1 (CRF1R) and its native 40-mer peptide ligand Urocortin-I directly in mammalian cells. By incorporating unnatural amino acid photochemical and new click-chemical probes into the intact receptor expressed in the native membrane of live cells, 44 intermolecular spatial constraints have been derived for the ligand-receptor interaction. The data were analyzed in the context of the recently resolved crystal structure of CRF1R transmembrane domain and existing extracellular domain structures, yielding a complete conformational model for the peptide-receptor complex. Structural features of the receptor-ligand complex yield molecular insights on the mechanism of receptor activation and the basis for discrimination between agonist and antagonist function.
Assuntos
Modelos Moleculares , Receptores de Hormônio Liberador da Corticotropina/química , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Urocortinas/química , Urocortinas/metabolismo , Sequência de Aminoácidos , Animais , Química Click/métodos , Reagentes de Ligações Cruzadas/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Ratos , Receptores de Hormônio Liberador da Corticotropina/genética , Alinhamento de SequênciaRESUMO
The human endothelin receptors are members of the rhodopsin class A of G-protein coupled receptors and key modulators of blood pressure regulation. Their functional in vitro characterization has widely been limited by the availability of high quality samples. We have optimized cell-free expression protocols for the human endothelin A and endothelin B receptors by implementing co-translational association approaches of the synthesized proteins with supplied liposomes or nanodiscs. Efficiency of membrane association and ligand binding properties of the receptors have systematically been studied in correlation to different membrane environments and lipid types. Ligand binding was analyzed by a number of complementary assays including radioassays, surface plasmon resonance and fluorescence measurements. High affinity binding of the peptide ligand ET-1 to both endothelin receptors could be obtained with several conditions and the highest Bmax values were measured in association with nanodiscs. We could further obtain the characteristic differential binding pattern of the two endothelin receptors with a panel of selected agonists and antagonists. Two intrinsic properties of the functionally folded endothelin B receptor, the proteolytic processing based on conformational recognition as well as the formation of SDS-resistant complexes with the peptide ligand ET-1, were observed with samples obtained from several cell-free expression conditions. High affinity and specific binding of ligands could furthermore be obtained with non-purified receptor samples in crude cell-free reaction mixtures, thus providing new perspectives for fast in vitro screening applications.
Assuntos
Endotelina-1/química , Lipossomos/química , Receptor de Endotelina A/química , Receptor de Endotelina B/química , Sistema Livre de Células/metabolismo , Detergentes/química , Endotelina-1/metabolismo , Expressão Gênica , Humanos , Cinética , Nanoestruturas/química , Ligação Proteica , Dobramento de Proteína , Receptor de Endotelina A/biossíntese , Receptor de Endotelina A/genética , Receptor de Endotelina B/biossíntese , Receptor de Endotelina B/genéticaRESUMO
Multivalent peptide-oligosaccharide conjugates were prepared and used to investigate the multivalency effect concerning the activity of Bid-BH3 peptides in live cells. Dextran oligosaccharides were carboxyethylated selectively in the 2-position of the carbohydrate units and activated for the ligation of N-terminally cysteinylated peptides. Ligation through maleimide coupling was found to be superior to the native chemical ligation protocol. Monomeric Bid-BH3 peptides were virtually inactive, whereas pentameric peptide conjugates induced apoptosis up to 20-fold stronger at identical peptide concentrations. Comparison of lowly multivalent and highly multivalent peptide dextrans proved a multivalency effect in life cells which was specific for the BH3 peptide sequence.
Assuntos
Apoptose/efeitos dos fármacos , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/química , Dextranos/química , Fragmentos de Peptídeos/química , Proteínas Proto-Oncogênicas/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Dextranos/síntese química , Dextranos/farmacologia , Relação Dose-Resposta a Droga , Eletroporação , Células HeLa , Humanos , Células Jurkat , Maleimidas/química , Microscopia Confocal , Oligopeptídeos/química , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/administração & dosagem , Proteínas Proto-Oncogênicas/síntese química , Proteínas Proto-Oncogênicas/farmacologia , Espectrometria de FluorescênciaRESUMO
Aggregation of amyloid ß (Aß(1-42)), causing toxicity, is a critical step in Alzheimer's disease (AD). AD studies are difficult to compare because Aß(1-42) aggregation is poorly controllable under physiological conditions. To control aggregation and toxicity, we engineered light-switchable Aß(1-42) analogues that enable controllable conversion of nontoxic fibrils into toxic oligomers simply by illumination.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/toxicidade , Luz , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/toxicidade , Engenharia de Proteínas , Multimerização Proteica/efeitos da radiação , Sequência de Aminoácidos , Linhagem Celular Tumoral , Humanos , Dados de Sequência Molecular , Estrutura Secundária de Proteína/efeitos da radiaçãoRESUMO
Photoswitchable click amino acids (PSCaa) are amino acids bearing a side chain consisting of a photoswitchable unit elongated with a functional group that allows for a specific click reaction, such as an alkene that can react with the thiol group of a cysteine residue. An intramolecular click reaction results in the formation of a photoswitchable bridge, which can be used for controlling conformational domains in peptides and proteins. The ability to control conformations as well as the efficiency of the intramolecular bridging depends on the length of the PSCaa side chain and the distance to the cysteine residue to be clicked with. On comparing i,i+4 and i,i+7 spacings of PSCaa and cysteine in a model peptide without a preferred conformation, it was seen that the thiol-ene click reaction takes place efficiently in both cases. Upon induction of an α-helical structure by the addition of trifluoroethanol, the thiol click reaction occurs preferentially with the i,i+4 spacing. Even in the presence of glutathione as an additional thiol the click reaction of the PSCaa occurs intramolecularly with the cysteine rather than with the glutathione, indicating that the click reaction may be used even under reducing conditions occurring in living cells.
RESUMO
Sperm, navigating in a chemical gradient, are exposed to a periodic stream of chemoattractant molecules. The periodic stimulation entrains Ca(2+) oscillations that control looping steering responses. It is not known how sperm sample chemoattractant molecules during periodic stimulation and adjust their sensitivity. We report that sea urchin sperm sampled molecules for 0.2-0.6 s before a Ca(2+) response was produced. Additional molecules delivered during a Ca(2+) response reset the cell by causing a pronounced Ca(2+) drop that terminated the response; this reset was followed by a new Ca(2+) rise. After stimulation, sperm adapted their sensitivity following the Weber-Fechner law. Taking into account the single-molecule sensitivity, we estimate that sperm can register a minimal gradient of 0.8 fM/µm and be attracted from as far away as 4.7 mm. Many microorganisms sense stimulus gradients along periodic paths to translate a spatial distribution of the stimulus into a temporal pattern of the cell response. Orchestration of temporal sampling, resetting, and adaptation might control gradient sensing in such organisms as well.
Assuntos
Ouriços-do-Mar/fisiologia , Espermatozoides/fisiologia , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Fatores Quimiotáticos/metabolismo , Limiar Diferencial/fisiologia , Masculino , Ouriços-do-Mar/metabolismo , Transdução de Sinais , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/metabolismoRESUMO
Here we report a simple procedure for generating colorimetric histone deacetylase (HDAC) substrates by solid-phase peptide synthesis based on racemization-free couplings of amino acid chlorides. We demonstrate the applicability of these substrates in HDAC assays.
Assuntos
Colorimetria , Histona Desacetilases/metabolismo , Benzenossulfonatos/síntese química , Benzenossulfonatos/química , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , NAD/química , Técnicas de Síntese em Fase Sólida , Especificidade por SubstratoRESUMO
The paracellular flux of solutes through tissue barriers is limited by transmembrane tight junction proteins. Within the family of tight junction proteins, claudin-1 seems to be a key protein for tightness formation and integrity. In the peripheral nervous system, the nerve fibers are surrounded with a barrier formed by the perineurium which expresses claudin-1. To enhance the access of hydrophilic pharmaceutical agents via the paracellular route, a claudin-1 specific modulator was developed. For this purpose, we designed and investigated the claudin-1 derived peptide C1C2. It transiently increased the paracellular permeability for ions and high and low molecular weight compounds through a cellular barrier model. Structural studies revealed a ß-sheet potential for the functionality of the peptide. Perineurial injection of C1C2 in rats facilitated the effect of hydrophilic antinociceptive agents and raised mechanical nociceptive thresholds. The mechanism is related to the internalization of C1C2 and to a vesicle-like distribution within the cells. The peptide mainly colocalized with intracellular claudin-1. C1C2 decreased membrane-localized claudin-1 of cells in culture and in vivo in the perineurium of rats after perineurial injection. In conclusion, a novel tool was developed to improve the delivery of pharmaceutical agents through the perineurial barrier by transient modulation of claudin-1.
Assuntos
Analgesia/métodos , Peptídeos/farmacologia , Peptidomiméticos/química , Peptidomiméticos/metabolismo , Nervos Periféricos/metabolismo , Junções Íntimas/metabolismo , Animais , Western Blotting , Células CACO-2 , Linhagem Celular , Dicroísmo Circular , Claudina-1/química , Humanos , Imuno-Histoquímica , Masculino , Microscopia Confocal , Peptídeos/química , Nervos Periféricos/efeitos dos fármacos , Ratos , Ratos Wistar , Nervo Isquiático/efeitos dos fármacos , Nervo Isquiático/metabolismoRESUMO
Covalent immobilization of cationic antimicrobial peptides (CAPs) at sufficient density and distance from the solid matrix has been suggested as a successful strategy for the generation of biocidal surfaces. To test the hypothesis that the mode of peptide action is decisive for the selection of an appropriate tethering position on solid surfaces, melittin (MEL), a channel-forming peptide, buforin 2 (BUF2), a peptide able to translocate bacterial membranes without permeabilization and targeting nucleic acids, and tritrpticin (TP), described to be membrane-lytic and to have intracellular targets, were C- and N-terminally immobilized on TentaGel S NH(2) resin beads as model surface. The peptide termini were modified with aminooxyacetic acid (AOA) and coupled via oxime-forming ligation. The comparison of the activities of the three peptides and their AOA-modified analogues with a KLAL model peptide which permeabilizes membranes by a so-called "carpet-like" mode provided the following results: The peptides in solution state were active against Bacillus subtilis and Escherichia coli at micromolar concentrations. MEL and TP but not BUF2-derived peptides permeabilized the inner and outer membrane of E. coli and enhanced the permeability of lipid bilayers at concentrations around their antimicrobial values (MICs). Immobilization reduced peptide activity to millimolar MICs. The activity reduction for KLAL was independent of the tethering position and comparably low, as reflected by a low ratio of MIC(tethered)/MIC(free). In contrary, the pore-forming MEL was much less active when immobilized at the N-terminus compared with the C-terminally tethered peptide. C- and N-terminal TP tethering caused an identical but much pronounced activity decrease. The tethered BUF2 peptides were inactive at the tested concentrations suggesting that the peptides could not reach the intracellular targets. In conclusion, membrane active peptides seem to be most suitable for the generation of antimicrobial surfaces, but knowledge about their mode of membrane insertion and positioning is required to identify optimal tethering positions. The relationship between the mechanism of action and position of immobilization is highly relevant for the establishment of a general approach to obtain efficient biocidal solid matrices loaded with CAPs.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Peptídeos Catiônicos Antimicrobianos/síntese química , Peptídeos Catiônicos Antimicrobianos/química , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cinética , Bicamadas Lipídicas/metabolismo , Testes de Sensibilidade Microbiana , Propriedades de SuperfícieRESUMO
G-protein-coupled receptors sense extracellular chemical or physical stimuli and transmit these signals to distinct trimeric G-proteins. Activated Gα-proteins route signals to interconnected effector cascades, thus regulating thresholds, amplitudes and durations of signalling. Gαs- or Gαi-coupled receptor cascades are mechanistically conserved and mediate many sensory processes, including synaptic transmission, cell proliferation and chemotaxis. Here we show that a central, conserved component of Gαs-coupled receptor cascades, the regulatory subunit type-II (RII) of protein kinase A undergoes adenosine 3'-5'-cyclic monophosphate (cAMP)-dependent binding to Gαi. Stimulation of a mammalian Gαi-coupled receptor and concomitant cAMP-RII binding to Gαi, augments the sensitivity, amplitude and duration of Gαi:ßγ activity and downstream mitogen-activated protein kinase signalling, independent of protein kinase A kinase activity. The mechanism is conserved in budding yeast, causing nutrient-dependent modulation of a pheromone response. These findings suggest a direct mechanism by which coincident activation of Gαs-coupled receptors controls the precision of adaptive responses of activated Gαi-coupled receptor cascades.
Assuntos
Adaptação Fisiológica/genética , Proteína Quinase Tipo II Dependente de AMP Cíclico/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Receptores Acoplados a Proteínas G/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais/fisiologia , Clonagem Molecular , AMP Cíclico/metabolismo , Proteína Quinase Tipo II Dependente de AMP Cíclico/genética , Escherichia coli , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Mutação , Fosforilação , Plasmídeos , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores Acoplados a Proteínas G/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transformação BacterianaRESUMO
The ligation of two polypeptides in a chemoselective manner by the bacterial transpeptidase sortase A has become a versatile tool for protein engineering approaches. When sortase-mediated ligation is used for protein semisynthesis, up to four mutations resulting from the strict requirement of the LPxTG sorting motif are introduced into the target protein. Here we report the directed evolution of a mutant sortase A that possesses broad substrate selectivity. A phage-display screen of a mutant sortase library that was randomized in the substrate recognition loop was used to isolate this mutant. The altered substrate selectivity represents a gain-of-function that was exploited for the traceless semisynthesis of histone H3. Our report is a decisive step toward a platform of engineered sortases with distinct ligation properties that will conceivably allow for more versatile assemblies of modified proteins in biotechnological approaches.
Assuntos
Aminoaciltransferases/genética , Proteínas de Bactérias/genética , Cisteína Endopeptidases/genética , Evolução Molecular Direcionada/métodos , Staphylococcus aureus/enzimologia , Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Mutação , Engenharia de Proteínas , Especificidade por SubstratoRESUMO
Click the switch: By using a photoswitchable click amino acid (PSCaa) a light-induced intramolecular thiol-ene click reaction with a neighboring cysteine under very mild conditions results in an azobenzene bridge. By expanding the genetic code for PSCaa the specific incorporation of photoswitch units into proteins in living cells can result in an exciting approach for studying light-controllable activity, in vivo.
Assuntos
Aminoácidos/química , Luz , Compostos Azo/química , Química Click , Cisteína/química , Isomerismo , Conformação Molecular , Processos Fotoquímicos , Compostos de Sulfidrila/química , Urocortinas/químicaRESUMO
The specific inhibition of the biosynthesis of target proteins is a relatively novel strategy in pharmacology and is based mainly on antisense approaches (e.g. antisense oligonucleotides or RNA interference). Recently, a novel class of substances was described acting at a later step of protein biosynthesis. The cyclic heptadepsipeptides CAM741 and cotransin were shown to inhibit selectively the biosynthesis of a small subset of secretory proteins by preventing stable insertion of the nascent chains into the Sec61 translocon complex at the endoplasmic reticulum membrane (Besemer, J., Harant, H., Wang, S., Oberhauser, B., Marquardt, K., Foster, C. A., Schreiner, E. P., de Vries, J. E., Dascher-Nadel, C., and Lindley, I. J. (2005) Nature 436, 290-293; Garrison, J. L., Kunkel, E. J., Hegde, R. S., and Taunton, J. (2005) Nature 436, 285-289). These peptides act in a signal sequence-discriminatory manner, which explains their selectivity. Here, we have analyzed the cotransin sensitivity of various G protein-coupled receptors in transfected HEK 293 cells. We show that the biosynthesis of the human endothelin B receptor (ET(B)R) is highly sensitive to cotransin, in contrast to that of the other G protein-coupled receptors analyzed. Using a novel biosynthesis assay based on fusions with the photoconvertible Kaede protein, we show that the IC(50) value of cotransin action on ET(B)R biosynthesis is 5.4 µm and that ET(B)R signaling could be completely blocked by treating cells with 30 µm cotransin. Taken together, our data add an integral membrane protein, namely the ET(B)R, to the small group of cotransin-sensitive proteins.
Assuntos
Peptídeos Cíclicos/farmacologia , Biossíntese de Proteínas/efeitos dos fármacos , Receptor de Endotelina B/biossíntese , Células HEK293 , Humanos , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Biossíntese de Proteínas/genética , Receptor de Endotelina B/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
Evidence is presented that components of fetal calf serum (FCS) can significantly enhance the splicing correction activity of peptide nucleic acids (PNA) in HeLa pLuc 705 cells. The effect proved more pronounced for PNAs bearing fluorescence tags and relies on the ability of specific components of FCS to mediate a mainly nonendocytotic intracellular delivery of PNA. Attempts to isolate and characterize the active serum components using PNA-loaded beads and nano-LC-ESI mass spectrometry revealed the growth-factor related inter-alpha-trypsin inhibitor and the adhesion protein fibronectin to be substantially responsible for the delivery activity of FCS.