RESUMO
Protein-protein interactions between SH2 domains and segments of proteins that include a post-translationally phosphorylated tyrosine residue (pY) underpin numerous signal transduction cascades that allow cells to respond to their environment. Dysregulation of the writing, erasing, and reading of these posttranslational modifications is a hallmark of human disease, notably cancer. Elucidating the precise role of the SH2 domain-containing adaptor proteins Crk and CrkL in tumor cell migration and invasion is challenging because there are no specific and potent antagonists available. Crk and CrkL SH2s interact with a region of the docking protein p130Cas containing 15 potential pY-containing tetrapeptide motifs. This chapter summarizes recent efforts toward peptide antagonists for this Crk/CrkL-p130Cas interaction. We describe our protocol for recombinant expression and purification of Crk and CrkL SH2s for functional assays and our procedure to determine the consensus binding motif from the p130Cas sequence. To develop a more potent antagonist, we employ methods often associated with structure-based drug design. Computational docking using Rosetta FlexPepDock, which accounts for peptides having a greater number of conformational degrees of freedom than small organic molecules that typically constitute libraries, provides quantitative docking metrics to prioritize candidate peptides for experimental testing. A battery of biophysical assays, including fluorescence polarization, differential scanning fluorimetry and saturation transfer difference nuclear magnetic resonance spectroscopy, were employed to assess the candidates. In parallel, GST pulldown competition assays characterized protein-protein binding in vitro. Taken together, our methodology yields peptide antagonists of the Crk/CrkL-p130Cas axis that will be used to validate targets, assess druggability, foster in vitro assay development, and potentially serve as lead compounds for therapeutic intervention.
Assuntos
Proteína Substrato Associada a Crk , Peptídeos , Fosfotirosina , Proteínas Proto-Oncogênicas c-crk , Domínios de Homologia de src , Proteína Substrato Associada a Crk/metabolismo , Proteína Substrato Associada a Crk/química , Proteínas Proto-Oncogênicas c-crk/metabolismo , Proteínas Proto-Oncogênicas c-crk/química , Humanos , Fosfotirosina/metabolismo , Fosfotirosina/química , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/metabolismo , Ligação Proteica , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Simulação de Acoplamento Molecular/métodos , Proteínas Nucleares/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/químicaRESUMO
AMPylation is a post-translational modification in which AMP is added to the amino acid side chains of proteins1,2. Here we show that, with ATP as the ligand and actin as the host activator, the effector protein LnaB of Legionella pneumophila exhibits AMPylase activity towards the phosphoryl group of phosphoribose on PRR42-Ub that is generated by the SidE family of effectors, and deubiquitinases DupA and DupB in an E1- and E2-independent ubiquitination process3-7. The product of LnaB is further hydrolysed by an ADP-ribosylhydrolase, MavL, to Ub, thereby preventing the accumulation of PRR42-Ub and ADPRR42-Ub and protecting canonical ubiquitination in host cells. LnaB represents a large family of AMPylases that adopt a common structural fold, distinct from those of the previously known AMPylases, and LnaB homologues are found in more than 20 species of bacterial pathogens. Moreover, LnaB also exhibits robust phosphoryl AMPylase activity towards phosphorylated residues and produces unique ADPylation modifications in proteins. During infection, LnaB AMPylates the conserved phosphorylated tyrosine residues in the activation loop of the Src family of kinases8,9, which dampens downstream phosphorylation signalling in the host. Structural studies reveal the actin-dependent activation and catalytic mechanisms of the LnaB family of AMPylases. This study identifies, to our knowledge, an unprecedented molecular regulation mechanism in bacterial pathogenesis and protein phosphorylation.
Assuntos
Monofosfato de Adenosina , Proteínas de Bactérias , Legionella pneumophila , Fosfotirosina , Transdução de Sinais , Humanos , Actinas/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , ADP-Ribosilação , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Hidrólise , Legionella pneumophila/enzimologia , Legionella pneumophila/metabolismo , Legionella pneumophila/patogenicidade , Ligantes , Modelos Moleculares , N-Glicosil Hidrolases/metabolismo , Fosforilação , Processamento de Proteína Pós-Traducional , Tirosina/metabolismo , Tirosina/química , Ubiquitina/metabolismo , Ubiquitinação , Enzimas Desubiquitinantes/metabolismo , Dobramento de Proteína , Fosfotirosina/química , Fosfotirosina/metabolismoRESUMO
Tyrosine phosphorylation (pTyr) regulates various signaling pathways under normal and cancerous states. Due to their low abundance and transient and dynamic natures, systematic profiling of pTyr sites is challenging. Antibody and engineered binding domain-based approaches have been well applied to pTyr peptide enrichment. However, traditional methods have the disadvantage of a long sample preparation process, which makes them unsuitable for processing limited amount of samples, especially in a high-throughput manner. In this study we developed a 96-well microplate-based approach to integrate all the sample preparation steps starting from cell culture to MS-compatible pTyr peptide enrichment in three consecutive 96-well microplates. By assembling an engineered SH2 domain onto a microplate, nonspecific adsorption of phosphopeptides is greatly reduced, which allows us to remove the Ti-IMAC purification and three C18 desalting steps (after digestion, pTyr enrichment, and Ti-IMAC purification) and, therefore, greatly simplifies the entire pTyr peptide enrichment workflow, especially when processing a large number of samples. Starting with 96-well microplate-cultured, pervanadate-stimulated cells, our approach could enrich 21% more pTyr sites than the traditional serial pTyr enrichment approach and showed good sensitivity and reproducibility in the range of 200 ng to 200 µg peptides. Importantly, we applied this approach to profile tyrosine kinase inhibitor-mediated EGFR signaling pathway and could well differentiate the distinct response of different pTyr sites. Collectively, the integrated 96-well microplate-based approach is valuable for profiling pTyr sites from limited biological samples and in a high-throughput manner.
Assuntos
Fosfopeptídeos , Tirosina , Receptores ErbB/metabolismo , Fosfopeptídeos/análise , Fosforilação , Fosfotirosina/química , Inibidores de Proteínas Quinases , Proteoma/análise , Reprodutibilidade dos Testes , Tirosina/químicaRESUMO
Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible fibrotic disease with high mortality. Currently, pirfenidone and nintedanib are the only approved drugs for IPF by the U.S. Food and Drug Administration (FDA), but their efficacy is limited. The activation of multiple phosphotyrosine (pY) mediated signaling pathways underlying the pathological mechanism of IPF has been explored. A Src homology-2 (SH2) superbinder, which contains mutations of three amino acids (AAs) of natural SH2 domain has been shown to be able to block phosphotyrosine (pY) pathway. Therefore, we aimed to introduce SH2 superbinder into the treatment of IPF. Methods: We analyzed the database of IPF patients and examined pY levels in lung tissues from IPF patients. In primary lung fibroblasts obtained from IPF patient as well as bleomycin (BLM) treated mice, the cell proliferation, migration and differentiation associated with pY were investigated and the anti-fibrotic effect of SH2 superbinder was also tested. In vivo, we further verified the safety and effectiveness of SH2 superbinder in multiple BLM mice models. We also compared the anti-fibrotic effect and side-effect of SH2 superbinder and nintedanib in vivo. Results: The data showed that the cytokines and growth factors pathways which directly correlated to pY levels were significantly enriched in IPF. High pY levels were found to induce abnormal proliferation, migration and differentiation of lung fibroblasts. SH2 superbinder blocked pY-mediated signaling pathways and suppress pulmonary fibrosis by targeting high pY levels in fibroblasts. SH2 superbinder had better therapeutic effect and less side-effect compare to nintedanib in vivo. Conclusions: SH2 superbinder had significant anti-fibrotic effects both in vitro and in vivo, which could be used as a promising therapy for IPF.
Assuntos
Fibrose Pulmonar Idiopática , Animais , Bleomicina/farmacologia , Proliferação de Células , Fibroblastos/metabolismo , Fibrose , Fibrose Pulmonar Idiopática/metabolismo , Camundongos , Fosfotirosina/química , Fosfotirosina/metabolismo , Fosfotirosina/farmacologiaRESUMO
Discovery of protein-binding fragments for precisely defined binding sites is an unmet challenge to date. Herein, formylglycine is investigated as a molecular probe for the sensitive detection of fragments binding to a spatially defined protein site . Formylglycine peptide 3 was derived from a phosphotyrosine-containing peptide substrate of protein tyrosine phosphatase PTP1B by replacing the phosphorylated amino acid with the reactive electrophile. Fragment ligation with formylglycine occurred inâ situ in aqueous physiological buffer. Structures and kinetics were validated by NMR spectroscopy. Screening and hit validation revealed fluorinated and non-fluorinated hit fragments being able to replace the native phosphotyrosine residue. The formylglycine probe identified low-affinity fragments with high spatial resolution as substantiated by molecular modelling. The best fragment hit, 4-amino-phenyl-acetic acid, was converted into a cellularly active, nanomolar inhibitor of the protein tyrosine phosphatase SHP2.
Assuntos
Aminoácidos , Peptídeos , Acetatos , Sítios de Ligação , Glicina/análogos & derivados , Sondas Moleculares , Peptídeos/química , Fosfotirosina/químicaRESUMO
Protein tyrosine phosphorylation (pTyr) plays a prominent role in signal transduction and regulation in all eukaryotic cells. In conventional immunoaffinity purification (IP) methods, phosphotyrosine peptides are isolated from the digest of cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. However, low sensitivity, poor reproducibility, and high cost are universal concerns for IP approaches. In this study, we presented an antibody-free approach to identify phosphotyrosine peptides by using protein tyrosine phosphatase (PTP). It was found that most of the PTPs including PTP1B, TCPTP, and SHP1 can efficiently and selectively dephosphorylate phosphotyrosine peptides. We then designed a workflow by combining two Ti4+-IMAC-based phosphopeptide enrichment steps with PTP-catalyzed dephosphorylation for tyrosine phosphoproteomics analysis. This workflow was first validated by selective detection of phosphotyrosine peptides from semicomplex samples and then applied to analyze the tyrosine phosphoproteome of Jurkat T cells. Around 1000 putative former phosphotyrosine peptides were identified from less than 500 µg of cell lysate. The tyrosine phosphosites on the majority of these peptides could be unambiguously determined for over 70% of them possessing only one tyrosine residue. It was also found that the tyrosine sites identified by this method were highly complementary to those identified by the SH2 superbinder-based method. Therefore, the combination of Ti4+-IMAC enrichment with PTP dephosphorylation provides an alternative and cost-effective approach for tyrosine phosphoproteomics analysis.
Assuntos
Proteômica , Tirosina , Humanos , Peptídeos/química , Fosforilação , Fosfotirosina/química , Proteínas Tirosina Fosfatases , Proteoma/análise , Proteômica/métodos , Reprodutibilidade dos Testes , Tirosina/químicaRESUMO
We developed peptide probes containing a non-hydrolyzable phosphotyrosine mimetic, 4-[difluoro(phosphono)methyl]-L-phenylalanine (F2 Pmp) for the enrichment of protein tyrosine phosphatases (PTPs). We found that different F2 Pmp probes can enrich different PTPs, depending on the probe sequence. Furthermore, proteins containing a Src homology 2 (SH2) domain were enriched together. Importantly, probes containing phosphotyrosine instead of F2 Pmp failed to enrich PTPs due to dephosphorylation during the pulldown step. This enrichment approach using peptides containing F2 Pmp could be a generic tool for tyrosine phosphatome analysis without the use of antibodies.
Assuntos
Proteínas Tirosina Fosfatases , Domínios de Homologia de src , Sequência de Aminoácidos , Peptídeos/química , Fosfotirosina/química , Proteínas Tirosina Fosfatases/metabolismo , Tirosina/químicaRESUMO
Tumorigenic risk of undifferentiated human induced pluripotent stem cells (iPSCs), being a major obstacle for clinical application of iPSCs, requires novel approaches for selectively eliminating undifferentiated iPSCs. Here, we show that an l-phosphopentapeptide, upon the dephosphorylation catalyzed by alkaline phosphatase (ALP) overexpressed by iPSCs, rapidly forms intranuclear peptide assemblies made of α-helices to selectively kill iPSCs. The phosphopentapeptide, consisting of four l-leucine residues and a C-terminal l-phosphotyrosine, self-assembles to form micelles/nanoparticles, which transform into peptide nanofibers/nanoribbons after enzymatic dephosphorylation removes the phosphate group from the l-phosphotyrosine. The concentration of ALP and incubation time dictates the morphology of the peptide assemblies. Circular dichroism and FTIR indicate that the l-pentapeptide in the assemblies contains a mixture of an α-helix and aggregated strands. Incubating the l-phosphopentapeptide with human iPSCs results in rapid killing of the iPSCs (=<2 h) due to the significant accumulation of the peptide assemblies in the nuclei of iPSCs. The phosphopentapeptide is innocuous to normal cells (e.g., HEK293 and hematopoietic progenitor cell (HPC)) because normal cells hardly overexpress ALP. Inhibiting ALP, mutating the l-phosphotyrosine from the C-terminal to the middle of the phosphopentapeptides, or replacing l-leucine to d-leucine in the phosphopentapeptide abolishes the intranuclear assemblies of the pentapeptides. Treating the l-phosphopentapeptide with cell lysate of normal cells (e.g., HS-5) confirms the proteolysis of the l-pentapeptide. This work, as the first case of intranuclear assemblies of peptides, not only illustrates the application of enzymatic noncovalent synthesis for selectively targeting nuclei of cells but also may lead to a new way to eliminate other pathological cells that express a high level of certain enzymes.
Assuntos
Fosfatase Alcalina/química , Nanofibras/química , Nanotubos de Carbono/química , Peptídeos/química , Terapia Baseada em Transplante de Células e Tecidos , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas , Organofosfatos/química , Fosfotirosina/química , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Spatiotemporal control of chemical assembly in living cells remains challenging. We have now developed an efficient and general platform to precisely control the formation of assemblies in living cells. We introduced an O-[bis(dimethylamino)phosphono]tyrosine protection strategy in the self-assembly motif as the Trojan horse, whereby the programmed precursors resist hydrolysis by phosphatases on and inside cells because the unmasking of the enzymatic cleavage site occurs selectively in the acidic environment of lysosomes. After demonstrating the multistage self-assembly processes inâ vitro by liquid chromatography/mass spectrometry (LC-MS), cryogenic electron microscopy (Cryo-EM), and circular dichroism (CD), we investigated the formation of site-specific self-assembly in living cells using confocal laser scanning microscopy (CLSM), LC-MS, and biological electron microscopy (Bio-EM). Controlling chemical assembly in living systems spatiotemporally may have applications in supramolecular chemistry, materials science, synthetic biology, and chemical biology.
Assuntos
Fosfatase Ácida/metabolismo , Fosfatase Alcalina/metabolismo , Fosfotirosina/metabolismo , Biocatálise , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Espectrometria de Massas , Microscopia Confocal , Microscopia Eletrônica , Estrutura Molecular , Fosfotirosina/químicaRESUMO
Access to phosphotyrosine (pTyr) mimetics requires multistep syntheses, and therefore late stage incorporation of these mimetics into peptides is not feasible. Here, we develop and employ metallaphotoredox catalysis using 4-halogenated phenylalanine to afford a variety of protected pTyr mimetics in one step. This methodology was shown to be tolerant of common protecting groups and applicable to the late stage pTyr mimetic modification of protected and unprotected peptides, and peptides of biological relevance.
Assuntos
Peptídeos/metabolismo , Fenilalanina/química , Fosfotirosina/metabolismo , Catálise , Estrutura Molecular , Peptídeos/química , Fosfotirosina/química , Transdução de Sinais/fisiologiaRESUMO
Protein engineering through directed evolution is an effective way to obtain proteins with novel functions with the potential applications as tools for diagnosis or therapeutics. Many natural proteins have undergone directed evolution in vitro in the test tubes in the laboratories worldwide, resulting in the numerous protein variants with novel or enhanced functions. we constructed here an SH2 variant library by randomizing 8 variable residues in its phosphotyrosine (pTyr) binding pocket. Selection of this library by a pTyr peptide led to the identification of SH2 variants with enhanced affinities measured by EC50. Fluorescent polarization was then applied to quantify the binding affinities of the newly identified SH2 variants. As a result, three SH2 variants, named V3, V13 and V24, have comparable binding affinities with the previously identified SH2 triple-mutant superbinder. Biolayer Interferometry assay was employed to disclose the kinetics of the binding of these SH2 superbinders to the phosphotyrosine peptide. The results indicated that all the SH2 superbinders have two-orders increase of the dissociation rate when binding the pTyr peptide while there was no significant change in their associate rates. Intriguingly, though binding the pTyr peptide with comparable affinity with other SH2 superbinders, the V3 does not bind to the sTyr peptide. However, variant V13 and V24 have cross-reactivity with both pTyr and sTyr peptides. The newly identified superbinders could be utilized as tools for the identification of pTyr-containing proteins from tissues under different physiological or pathophysiological conditions and may have the potential in the therapeutics.
Assuntos
Evolução Molecular Direcionada/métodos , Fosfotirosina , Proteínas Proto-Oncogênicas c-fyn , Proteínas Recombinantes , Domínios de Homologia de src/genética , Sítios de Ligação/genética , Técnicas de Visualização da Superfície Celular , Escherichia coli/genética , Humanos , Biblioteca de Peptídeos , Fosfotirosina/química , Fosfotirosina/metabolismo , Ligação Proteica/genética , Engenharia de Proteínas , Proteínas Proto-Oncogênicas c-fyn/química , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Engineering sequence-specific antibodies (Abs) against phosphotyrosine (pY) motifs embedded in folded polypeptides remains highly challenging because of the stringent requirement for simultaneous recognition of the pY motif and the surrounding folded protein epitope. Here, we present a method named phosphotyrosine Targeting by Recombinant Ab Pair, or pY-TRAP, for in vitro engineering of binders for native pY proteins. Specifically, we create the pY protein by unnatural amino acid misincorporation, mutagenize a universal pY-binding Ab to create a first binder B1 for the pY motif on the pY protein, and then select against the B1-pY protein complex for a second binder B2 that recognizes the composite epitope of B1 and the pY-containing protein complex. We applied pY-TRAP to create highly specific binders to folded Ub-pY59, a rarely studied Ub phosphoform exclusively observed in cancerous tissues, and ZAP70-pY248, a kinase phosphoform regulated in feedback signaling pathways in T cells. The pY-TRAPs do not have detectable binding to wild-type proteins or to other pY peptides or proteins tested. This pY-TRAP approach serves as a generalizable method for engineering sequence-specific Ab binders to native pY proteins.
Assuntos
Anticorpos/química , Fosfotirosina/química , Receptores de Trombina/química , Proteínas Recombinantes/química , Sequência de Aminoácidos , Sítios de Ligação , Biotinilação , Modelos Moleculares , Biblioteca de Peptídeos , Fosforilação , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Transdução de Sinais , Ubiquitina/químicaRESUMO
Protein tyrosine phosphorylation is key to activation of receptor tyrosine kinases (RTK) that drive development of some cancers. One challenge of RTK-targeted therapy is identification of those tumors that express non-mutated but activated RTKs. Phosphotyrosine (pTyr) RTK levels should be more predictive of the latter than expressed total protein. Western blotting (WB) with a pTyr antibody and enhanced chemiluminescence (ECL) detection is sufficiently sensitive to detect pTyr-RTKs in human tumor homogenates. Presentation of results by comparing WB images, however, is wanting. Here we describe the preparation of a new pTyr-protein standard, pTyr-ALK48-SB (pA), derived from a commercial anaplastic lymphoma kinase (ALK) recombinant fragment, and its use to quantify pTyr-epidermal growth factor receptor (pTyr-EGFR) in commercial A431 cell lysates. Linearity of one-dimensional (1D) WB plots of pA band density versus load as well as its lower level of detection (0.1 ng, 2 fmole) were determined for standardized conditions. Adding pA to two lots of A431 cell lysates with high and low pTyr-EGFR allowed normalization and quantification of the latter by expressing results as density ratios for both 1D and 2D WB. This approach is semi-quantitative because unknown RTKs may be outside the linear range of detection. Semiquantitative ratios are an improvement over comparisons of images without a reference standard and facilitate comparisons between samples.
Assuntos
Quinase do Linfoma Anaplásico/química , Western Blotting/métodos , Medições Luminescentes/normas , Fosfotirosina/química , Linhagem Celular Tumoral , Receptores ErbB/análise , Humanos , Luminescência , Proteínas Recombinantes/química , Padrões de ReferênciaRESUMO
The combination of immunotherapy and chemotherapy has shown great potential for treating solid tumors. Although various combination therapy regimens have been demonstrated to be effective, their implementation in practice always needs separate drugs or modalities, which in turn requires specific and complicated operation procedures. Here, we describe the use of enzyme-instructed self-assembly (EISA) of a phosphotyrosine-cholesterol (PTC) conjugate to combine immune microenvironment modulation and chemotherapy. The enzymatic dephosphorylation of PTC results in the assembly of its derivatives inside and outside the cells, which leads to repolarization of the macrophage phenotype and direct death of cancer cells. The generation of reactive oxygen species and actin disturbance induced by the assemblies has been verified relative to the macrophage repolarization. We also demonstrate its dual repolarization and inhibition effects in vivo using an ovarian cancer bearing immunocompetent murine model. The EISA of cholesterol conjugates is an effective yet simple approach for the combination of tumor microenvironment immunomodulation and chemotherapy.
Assuntos
Colesterol/administração & dosagem , Macrófagos/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Fosfotirosina/química , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Colesterol/química , Colesterol/farmacologia , Citocinas/genética , Feminino , Humanos , Macrófagos/metabolismo , Camundongos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/imunologia , Fosforilação , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Células THP-1 , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
C-type lectin-like receptor 2 (CLEC-2) plays a crucial role in different platelet-related physiological and pathological processes. It signals through a tyrosine kinase-mediated pathway that is highly dependent on the positive feedback exerted by the platelet-derived secondary mediators, adenosine diphosphate (ADP) and thromboxane A2 (TXA2). Here, we aimed to analyze the tyrosine phosphoproteome of platelets activated with the CLEC-2 agonist rhodocytin to identify relevant phosphorylated tyrosine residues (p-Tyr) and proteins involved in platelet activation downstream of this receptor. We identified 363 differentially p-Tyr residues, corresponding to the majority of proteins previously known to participate in CLEC-2 signaling and also novel ones, including adaptors (e.g., DAPP1, Dok1/3, CASS4, Nck1/2), kinases/phosphatases (e.g., FAK1, FES, FGR, JAK2, SHIP2), and membrane proteins (e.g., G6F, JAM-A, PECAM-1, TLT-1). To elucidate the contribution of ADP and TXA2 at different points of the CLEC-2 signaling cascade, we evaluated p-Tyr levels of residues identified in the analysis and known to be essential for the catalytic activity of kinases Syk(p-Tyr525+526) and Src(p-Tyr419), and for PLCγ2 activity (p-Tyr759). We demonstrated that Syk phosphorylation at Tyr525+526 also happens in the presence of ADP and TXA2 inhibitors, which is not the case for Src-pTyr419 and PLCγ2-pTyr759. Kinetics studies for the three phosphoproteins show some differences in the phosphorylation profile. Ca2+ mobilization assays confirmed the relevance of ADP and TXA2 for full CLEC-2-mediated platelet activation. The present study provides significant insights into the intracellular events that take place following CLEC-2 activation in platelets, contributing to elucidate in detail the CLEC-2 signalosome.
Assuntos
Plaquetas/metabolismo , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/metabolismo , Fosfoproteínas/química , Transdução de Sinais , Tirosina/química , Difosfato de Adenosina/química , Adulto , Cálcio/química , Cálcio/metabolismo , Feminino , Humanos , Cinética , Masculino , Pessoa de Meia-Idade , Fosforilação , Fosfotirosina/química , Ativação Plaquetária , Agregação Plaquetária , Proteoma , Tromboxano A2/química , Adulto JovemRESUMO
Covalent probes serve as valuable tools for global investigation of protein function and ligand binding capacity. Despite efforts to expand coverage of residues available for chemical proteomics (e.g., cysteine and lysine), a large fraction of the proteome remains inaccessible with current activity-based probes. Here, we introduce sulfur-triazole exchange (SuTEx) chemistry as a tunable platform for developing covalent probes with broad applications for chemical proteomics. We show modifications to the triazole leaving group can furnish sulfonyl probes with ~5-fold enhanced chemoselectivity for tyrosines over other nucleophilic amino acids to investigate more than 10,000 tyrosine sites in lysates and live cells. We discover that tyrosines with enhanced nucleophilicity are enriched in enzymatic, protein-protein interaction and nucleotide recognition domains. We apply SuTEx as a chemical phosphoproteomics strategy to monitor activation of phosphotyrosine sites. Collectively, we describe SuTEx as a biocompatible chemistry for chemical biology investigations of the human proteome.
Assuntos
Sondas Moleculares/química , Proteômica/métodos , Enxofre/química , Triazóis/química , Tirosina/análise , Tirosina/química , Células A549 , Sítios de Ligação , Flúor/química , Glutationa S-Transferase pi/genética , Glutationa S-Transferase pi/metabolismo , Células HEK293 , Humanos , Sondas Moleculares/síntese química , Fosforilação , Fosfotirosina/química , Fosfotirosina/metabolismo , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Ácidos Sulfínicos/química , Tirosina/metabolismoRESUMO
Fes and Fes-related (Fer) protein tyrosine kinases (PTKs) comprise a subfamily of nonreceptor tyrosine kinases characterized by a unique multidomain structure composed of an N-terminal Fer/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain, a central Src homology 2 (SH2) domain, and a C-terminal PTK domain. Fer is ubiquitously expressed, and upregulation of Fer has been implicated in various human cancers. The PTK activity of Fes has been shown to be positively regulated by the binding of phosphotyrosine-containing ligands to the SH2 domain. Here, the X-ray crystal structure of human Fer SH2 domain bound to a phosphopeptide that has D-E-pY-E-N-V-D sequence is reported at 1.37 å resolution. The asymmetric unit (ASU) contains six Fer-phosphopeptide complexes, and the structure reveals three distinct binding modes for the same phosphopeptide. At four out of the six binding sites in the ASU, the phosphopeptide binds to Fer SH2 domain in a type I ß-turn conformation, and this could be the optimal binding mode of this phosphopeptide. At the other two binding sites in the ASU, it appears that spatial proximity of neighboring SH2 domains in the crystal induces alternative modes of binding of this phosphopeptide.
Assuntos
Peptídeos/química , Fosfotirosina/química , Proteínas Tirosina Quinases/química , Cristalografia por Raios X , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Proteínas Tirosina Quinases/metabolismo , Domínios de Homologia de srcRESUMO
Low-abundance phosphotyrosine (pTyr)-mediated signaling protein complexes play critical roles in cancer signaling. The precise and comprehensive profiling of these pTyr-mediated protein complexes remains challenging because of their dynamic nature and weak binding affinity. Taking advantage of the SH2 domains modified with trifunctional chemical probes and genetic mutations (termed Photo-pTyr-scaffold), we developed a Photo-pTyr-scaffold-based forward-phase protein array that can be used to specifically capture complexes by developing an engineered SH2 domain, photoaffinity cross-linking, and antibody-based measuring weak pTyr-mediated protein complexes from complex biological samples in a 96-well microplate format. This platform demonstrated good precision for quantitation (R2 = 0.99) and high sensitivity by which only 5 µg of whole cell lysates is needed. We successfully applied the technology for profiling the dynamic EGF-stimulation-dependent EGFR signaling protein complexes across four different time courses (i.e., 0, 2, 5, 10, and 30 min) in a high-throughput manner. We further evaluated the modulation of EGFR-GRB2-SHC1 protein complexes by FDA-approved EGFR kinase inhibitor erlotinib, demonstrating the feasibility of this approach for high-throughput drug screening. The Photo-pTyr-scaffold-based forward-phase protein array could be generically applicable for exploring the dynamic pTyr signaling complexes in various biological systems and screening for related drugs in a high-throughput manner.
Assuntos
Fosfotirosina/metabolismo , Análise Serial de Proteínas/métodos , Raios Ultravioleta , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/química , Cloridrato de Erlotinib/metabolismo , Cloridrato de Erlotinib/farmacologia , Proteína Adaptadora GRB2/química , Proteína Adaptadora GRB2/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Fosfotirosina/química , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/química , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/metabolismo , Domínios de Homologia de srcRESUMO
The suppressor of cytokine signaling 2 (SOCS2) acts as substrate recognition subunit of a Cullin5 E3 ubiquitin ligase complex. SOCS2 binds to phosphotyrosine-modified epitopes as degrons for ubiquitination and proteasomal degradation, yet the molecular basis of substrate recognition has remained elusive. Here, we report co-crystal structures of SOCS2-ElonginB-ElonginC in complex with phosphorylated peptides from substrates growth hormone receptor (GHR-pY595) and erythropoietin receptor (EpoR-pY426) at 1.98 Å and 2.69 Å, respectively. Both peptides bind in an extended conformation recapitulating the canonical SH2 domain-pY pose, but capture different conformations of the EF loop via specific hydrophobic interactions. The flexible BG loop is fully defined in the electron density, and does not contact the substrate degron directly. Cancer-associated SNPs located around the pY pocket weaken substrate-binding affinity in biophysical assays. Our findings reveal insights into substrate recognition and specificity by SOCS2, and provide a blueprint for small molecule ligand design.
Assuntos
Proteínas Supressoras da Sinalização de Citocina/química , Ubiquitina-Proteína Ligases/química , Cristalografia por Raios X , Humanos , Fosfotirosina/química , Polimorfismo de Nucleotídeo Único , Conformação Proteica , Receptores da Eritropoetina/química , Receptores da Somatotropina/química , Alinhamento de Sequência , Especificidade por Substrato , Proteínas Supressoras da Sinalização de Citocina/genética , UbiquitinaçãoRESUMO
Tyrosine phosphorylation plays a major role in regulating cell signaling pathways governing diverse biological functions such as proliferation and differentiation. Systemically mapping phosphotyrosine (pTyr) sites is the key to understanding molecular mechanisms underlining pTyr-dependent signaling. Although mass spectrometry-based technologies have been widely used for pTyr site profiling and quantification, their applications are often hindered by the poor efficiency in current multistep enrichment procedures for inherently low abundance pTyr peptides, especially under physiological conditions. Taking advantage of the sequence-independent high affinity of SH2 superbinder toward pTyr residues, we have developed a simplified one-step pTyr peptide enrichment method that uses immobilized SH2 superbinder for unbiased and robust enrichment of endogenous pTyr peptides from biological samples. By eliminating the prerequisite global phosphopeptide enrichment step in our previously developed two-step method, we minimized sample loss and improved peptide capture efficiency. Applying this method to Jurkat cells at resting state, where the tyrosine phosphorylation level is low, both the number of identified pTyr peptides and sites are increased by three folds compared to the two-step method. Specifically, we were able to identify 511 nonredundant pTyr peptides, corresponding to 403 high confidence pTyr sites, from Jurkat cells with high level technical reproducibility (Pearson's correlation coefficient as high as 0.94). Further applying this method to two human breast cancer cell lines, BT474 and HCC1954, before and after EGF stimulation, we demonstrated that this approach could be a powerful tool for illustrating pTyr-dependent signaling network controlling cellular behaviors such as drug resistance.