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1.
Protein Sci ; 30(12): 2373-2384, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34601763

RESUMEN

The catalytic activity of Syk-family tyrosine kinases is regulated by a tandem Src homology 2 module (tSH2 module). In the autoinhibited state, this module adopts a conformation that stabilizes an inactive conformation of the kinase domain. The binding of the tSH2 module to phosphorylated immunoreceptor tyrosine-based activation motifs necessitates a conformational change, thereby relieving kinase inhibition and promoting activation. We determined the crystal structure of the isolated tSH2 module of Syk and find, in contrast to ZAP-70, that its conformation more closely resembles that of the peptide-bound state, rather than the autoinhibited state. Hydrogen-deuterium exchange by mass spectrometry, as well as molecular dynamics simulations, reveal that the dynamics of the tSH2 modules of Syk and ZAP-70 differ, with most of these differences occurring in the C-terminal SH2 domain. Our data suggest that the conformational landscapes of the tSH2 modules in Syk and ZAP-70 have been tuned differently, such that the autoinhibited conformation of the Syk tSH2 module is less stable. This feature of Syk likely contributes to its ability to more readily escape autoinhibition when compared to ZAP-70, consistent with tighter control of downstream signaling pathways in T cells.


Asunto(s)
Simulación de Dinámica Molecular , Quinasa Syk/química , Proteína Tirosina Quinasa ZAP-70/química , Inmunidad Adaptativa , Animales , Linfocitos B/inmunología , Linfocitos B/metabolismo , Sitios de Unión , Evolución Biológica , Clonación Molecular , Cristalografía por Rayos X , Medición de Intercambio de Deuterio , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes , Transducción de Señal , Quinasa Syk/genética , Quinasa Syk/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Proteína Tirosina Quinasa ZAP-70/genética , Proteína Tirosina Quinasa ZAP-70/inmunología
2.
Molecules ; 26(15)2021 Jul 31.
Artículo en Inglés | MEDLINE | ID: mdl-34361816

RESUMEN

Fostamatinib is a prodrug of the active metabolite tamatinib, which is a spleen tyrosine kinase (Syk) inhibitor used in the treatment of primary chronic adult immune thrombocytopenia and rheumatoid arthritis. A highly sensitive, rapid, reliable, and green method was developed and validated using ultra-performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS) for quantification of tamatinib in rat plasma. Ibrutinib was used as internal standard and liquid-liquid extraction was applied using tert-butyl methyl ether. The analyte was separated on an AcquityTM CSH C18 (2.1 mm × 100 mm, 1.7 µm) column using mobile phase consisting of 10 mM ammonium acetate and acetonitrile (10:90) and the flow rate was 0.25 mL/min. Electrospray ionization (ESI) was carried out in positive mode. Quantitation of tamatinib and the IS was performed using multiple reaction monitoring mode with precursor-to-product transitions of m/z 471.1 > 122.0 and m/z 441.1 > 84.0, respectively. The calibration range was 0.1-1000.0 ng/mL and the linearity of the method was ≥0.997. The developed method greenness was investigated. All principal parameters for the method, including linearity, accuracy, precision, recovery, and stability, were within acceptable ranges. Tamatinib pharmacokinetic study in rats was successfully carried out using the developed method.


Asunto(s)
Inhibidores Enzimáticos/farmacocinética , Oxazinas/farmacocinética , Pirazoles/química , Piridinas/farmacocinética , Quinasa Syk/antagonistas & inhibidores , Aminopiridinas , Animales , Cromatografía Liquida , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Humanos , Extracción Líquido-Líquido , Morfolinas , Oxazinas/sangre , Oxazinas/metabolismo , Pirazoles/metabolismo , Pirazoles/farmacocinética , Piridinas/sangre , Piridinas/metabolismo , Pirimidinas , Ratas , Quinasa Syk/química , Espectrometría de Masas en Tándem
3.
J Mol Model ; 27(1): 14, 2021 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-33403456

RESUMEN

Rheumatoid arthritis (RA) is a systemic autoimmune disorder that commonly affects multiple joints of the body. Currently, there is no permanent cure to the disease, but it can be managed with several potent drugs that cause serious side effects on prolonged use. Traditional remedies are considered promising for the treatment of several diseases, particularly chronic conditions, because they have lower side effects compared to synthetic drugs. In folklore, the rhizome of Alpinia calcarata Roscoe (Zingiberaceae) is used as a major ingredient of herbal formulations to treat RA. Phytoconstituents reported in A. calcarata rhizomes are diterpenoids, sesquiterpenoid, flavonoids, phytosterol, and volatile oils. The present study is intended to understand the molecular-level interaction of phytoconstituents present in A. calcarata rhizomes with RA molecular targets using computational approaches. A total of 30 phytoconstituents reported from the plant were used to carry out docking with 36 known targets of RA. Based on the docking results, 4 flavonoids were found to be strongly interacting with the RA targets. Further, molecular dynamics simulation confirmed stable interaction of quercetin with 6 targets (JAK3, SYK, MMP2, TLR8, IRAK1, and JAK1), galangin with 2 targets (IRAK1 and JAK1), and kaempferol (IRAK1) with one target of RA. Moreover, the presence of these three flavonoids was confirmed in the A. calcarata rhizome extract using LC-MS analysis. The computational study suggests that flavonoids present in A. calcarata rhizome may be responsible for RA modulatory activity. Particularly, quercetin and galangin could be potential development candidates for the treatment of RA. Investigation of Alpinia calcarata constituent interactions with molecular targets of rheumatoid arthritis: docking, molecular dynamics, and network approach.


Asunto(s)
Alpinia/química , Artritis Reumatoide/tratamiento farmacológico , Biología Computacional , Flavonoides/farmacología , Fitoquímicos/farmacología , Artritis Reumatoide/metabolismo , Cromatografía Liquida , Flavonoides/análisis , Flavonoides/química , Humanos , Quinasas Asociadas a Receptores de Interleucina-1/química , Quinasas Asociadas a Receptores de Interleucina-1/efectos de los fármacos , Janus Quinasa 1/química , Janus Quinasa 1/efectos de los fármacos , Janus Quinasa 3/química , Janus Quinasa 3/efectos de los fármacos , Quempferoles/química , Quempferoles/farmacología , Espectrometría de Masas , Metaloproteinasa 2 de la Matriz/química , Metaloproteinasa 2 de la Matriz/efectos de los fármacos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Fitoquímicos/análisis , Fitoquímicos/química , Extractos Vegetales/química , Quercetina/química , Quercetina/farmacología , Rizoma/química , Quinasa Syk/química , Quinasa Syk/efectos de los fármacos , Receptor Toll-Like 8/química , Receptor Toll-Like 8/efectos de los fármacos
4.
Int J Mol Sci ; 21(19)2020 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-32977621

RESUMEN

Resistance to antimalarial drugs has spread rapidly over the past few decades. The WHO recommends artemisinin-based combination therapies for the treatment of uncomplicated malaria, but unfortunately these approaches are losing their efficacy in large areas of Southeast Asia. In 2016, artemisinin resistance was confirmed in 5 countries of the Greater Mekong subregion. We focused our study on Syk inhibitors as antimalarial drugs. The Syk protein is present in human erythrocytes, and the membrane of protein band 3 is its major target following activation by oxidant stress. Tyr phosphorylation of band 3 occurs during P. falciparum growth, leading to the release of microparticles containing hemicromes and structural weakening of the host cell membrane, simplifying merozoite reinfection. Syk inhibitors block these events by interacting with the Syk protein's catalytic site. We performed in vitro proteomics and in silico studies and compared the results. In vitro studies were based on treatment of the parasite's cellular cultures with different concentrations of Syk inhibitors, while proteomics studies were focused on the Tyr phosphorylation of band 3 by Syk protein with the same concentrations of drugs. In silico studies were based on different molecular modeling approaches in order to analyze and optimize the ligand-protein interactions and obtain the highest efficacy in vitro. In the presence of Syk inhibitors, we observed a marked decrease of band 3 Tyr phosphorylation according to the increase of the drug's concentration. Our studies could be useful for the structural optimization of these compounds and for the design of novel Syk inhibitors in the future.


Asunto(s)
Antimaláricos , Eritrocitos , Malaria Falciparum , Plasmodium falciparum/crecimiento & desarrollo , Inhibidores de Proteínas Quinasas , Quinasa Syk , Antimaláricos/química , Antimaláricos/farmacología , Relación Dosis-Respuesta a Droga , Eritrocitos/enzimología , Eritrocitos/parasitología , Humanos , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/enzimología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Quinasa Syk/antagonistas & inhibidores , Quinasa Syk/química , Quinasa Syk/metabolismo
5.
Bioorg Med Chem Lett ; 30(19): 127433, 2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-32717371

RESUMEN

Spleen tyrosine kinase (SYK) is a non-receptor cytosolic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signaling, inhibition of SYK has been targeted in a variety of disease areas. Herein, we report the optimization of a series of potent and selective SYK inhibitors, focusing on improving metabolic stability, pharmacokinetics and hERG inhibition. As a result, we identified 30, which exhibited no hERG activity but unfortunately was poorly absorbed in rats and mice. We also identified a SYK chemical probe, 17, which exhibits excellent potency at SYK, and an adequate rodent PK profile to support in vivo efficacy/PD studies.


Asunto(s)
Indazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Quinasa Syk/antagonistas & inhibidores , Animales , Sitios de Unión , Células CACO-2 , Cristalografía por Rayos X , Canal de Potasio ERG1/antagonistas & inhibidores , Humanos , Indazoles/síntesis química , Indazoles/metabolismo , Indazoles/farmacocinética , Ratones , Microsomas Hepáticos/metabolismo , Estructura Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Ratas Wistar , Relación Estructura-Actividad , Quinasa Syk/química , Quinasa Syk/metabolismo
6.
Sci Rep ; 10(1): 439, 2020 01 16.
Artículo en Inglés | MEDLINE | ID: mdl-31949205

RESUMEN

Flavanomarein (FM) is a major natural compound of Coreopsis tinctoria Nutt with protective effects against diabetic nephropathy (DN). In this study, we investigated the effects of FM on epithelial-mesenchymal transition (EMT) in high glucose (HG)-stimulated human proximal tubular epithelial cells (HK-2) and the underlying mechanisms, including both direct targets and downstream signal-related proteins. The influence of FM on EMT marker proteins was evaluated via western blot. Potential target proteins of FM were searched using Discovery Studio 2017 R2. Gene Ontology (GO) analysis was conducted to enrich the proteins within the protein-protein interaction (PPI) network for biological processes. Specific binding of FM to target proteins was examined via molecular dynamics and surface plasmon resonance analyses (SPR). FM promoted the proliferation of HK-2 cells stimulated with HG and inhibited EMT through the Syk/TGF-ß1/Smad signaling pathway. Spleen tyrosine kinase (Syk) was predicted to be the most likely directly interacting protein with FM. Combined therapy with a Syk inhibitor and FM presents significant potential as an effective novel therapeutic strategy for DN.


Asunto(s)
Transición Epitelial-Mesenquimal/efectos de los fármacos , Glucosa/farmacología , Quinasa Syk/metabolismo , Actinas/metabolismo , Cadherinas/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Fibronectinas/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Riñón/citología , Simulación del Acoplamiento Molecular , Conformación Proteica , Transducción de Señal/efectos de los fármacos , Proteínas Smad/metabolismo , Quinasa Syk/química , Factor de Crecimiento Transformador beta1/metabolismo , Vimentina/metabolismo
7.
Front Immunol ; 11: 575085, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33488575

RESUMEN

Leukocyte inflammatory responses require integrin cell-adhesion molecule signaling through spleen tyrosine kinase (Syk), a non-receptor kinase that binds directly to integrin ß-chain cytoplasmic domains. Here, we developed a high-throughput screen to identify small molecule inhibitors of the Syk-integrin cytoplasmic domain interactions. Screening small molecule compound libraries identified the ß-lactam antibiotics cefsulodin and ceftazidime, which inhibited integrin ß-subunit cytoplasmic domain binding to the tandem SH2 domains of Syk (IC50 range, 1.02-4.9 µM). Modeling suggested antagonist binding to Syk outside the pITAM binding site. Ceftazidime inhibited integrin signaling via Syk, including inhibition of adhesion-dependent upregulation of interleukin-1ß and monocyte chemoattractant protein-1, but did not inhibit ITAM-dependent phosphorylation of Syk mediated by FcγRI signaling. Our results demonstrate a novel means to target Syk independent of its kinase and pITAM binding sites such that integrin signaling via this kinase is abrogated but ITAM-dependent signaling remains intact. As integrin signaling through Syk is essential for leukocyte activation, this may represent a novel approach to target inflammation.


Asunto(s)
Antiinflamatorios/farmacología , Cefsulodina/farmacología , Ceftazidima/farmacología , Cadenas beta de Integrinas/efectos de los fármacos , Leucocitos/efectos de los fármacos , Quinasa Syk/antagonistas & inhibidores , Antiinflamatorios/química , Cefsulodina/química , Ceftazidima/química , Ensayos Analíticos de Alto Rendimiento , Humanos , Cadenas beta de Integrinas/química , Cadenas beta de Integrinas/metabolismo , Leucocitos/enzimología , Masculino , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de Señal , Bibliotecas de Moléculas Pequeñas , Quinasa Syk/química , Quinasa Syk/metabolismo , Células THP-1
8.
Molecules ; 25(1)2019 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-31861689

RESUMEN

The antileukemia cancer activity of organic compounds analogous to ellipticine representes a critical endpoint in the understanding of this dramatic disease. A molecular modeling simulation on a dataset of 23 compounds, all of which comply with Lipinski's rules and have a structure analogous to ellipticine, was performed using the quantitative structure activity relationship (QSAR) technique, followed by a detailed docking study on three different proteins significantly involved in this disease (PDB IDs: SYK, PI3K and BTK). As a result, a model with only four descriptors (HOMO, softness, AC1RABAMBID, and TS1KFABMID) was found to be robust enough for prediction of the antileukemia activity of the compounds studied in this work, with an R2 of 0.899 and Q2 of 0.730. A favorable interaction between the compounds and their target proteins was found in all cases; in particular, compounds 9 and 22 showed high activity and binding free energy values of around -10 kcal/mol. Theses compounds were evaluated in detail based on their molecular structure, and some modifications are suggested herein to enhance their biological activity. In particular, compounds 22_1, 22_2, 9_1, and 9_2 are indicated as possible new, potent ellipticine derivatives to be synthesized and biologically tested.


Asunto(s)
Antineoplásicos/síntesis química , Elipticinas/síntesis química , Leucemia/metabolismo , Quinasa Syk/metabolismo , Agammaglobulinemia Tirosina Quinasa/química , Agammaglobulinemia Tirosina Quinasa/metabolismo , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Teoría Funcional de la Densidad , Elipticinas/química , Elipticinas/farmacología , Humanos , Leucemia/tratamiento farmacológico , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Estructura Molecular , Fosfatidilinositol 3-Quinasas/química , Fosfatidilinositol 3-Quinasas/metabolismo , Relación Estructura-Actividad Cuantitativa , Quinasa Syk/química
9.
Sci Rep ; 9(1): 16023, 2019 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-31690758

RESUMEN

The ability of high-resolution NMR spectroscopy to readout the response of molecular interactions at multiple atomic sites presents a unique capability to define thermodynamic equilibrium constants and kinetic rate constants for complex, multiple-step biological interactions. Nonetheless, the extraction of the relevant equilibrium binding and rate constants requires the appropriate analysis of not only a readout that follows the equilibrium concentrations of typical binding titration curves, but also the lineshapes of NMR spectra. To best take advantage of NMR data for characterizing molecular interactions, we developed NmrLineGuru, a software tool with a user-friendly graphical user interface (GUI) to model two-state, three-state, and four-state binding processes. Application of NmrLineGuru is through stand-alone GUIs, with no dependency on other software and no scripted input. NMR spectra can be fitted or simulated starting with user-specified input parameters and a chosen kinetic model. The ability to both simulate and fit NMR spectra provides the user the opportunity to not only determine the binding parameters that best reproduce the measured NMR spectra for the selected kinetic model, but to also query the possibility that alternative models agree with the data. NmrLineGuru is shown to provide an accurate, quantitative analysis of complex molecular interactions.


Asunto(s)
Espectroscopía de Resonancia Magnética , Interfaz Usuario-Computador , Humanos , Cinética , Modelos Biológicos , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Quinasa Syk/química , Quinasa Syk/metabolismo , Dominios Homologos src
10.
J Biol Chem ; 294(19): 7658-7668, 2019 05 10.
Artículo en Inglés | MEDLINE | ID: mdl-30923129

RESUMEN

Spleen tyrosine kinase (SYK) is a signaling node in many immune pathways and comprises two tandem Src homology (SH) 2 domains, an SH2-kinase linker, and a C-terminal tyrosine kinase domain. Two prevalent models of SYK activation exist. The "OR-gate" model contends that SYK can be fully activated by phosphorylation or binding of its SH2 domains to a dual-phosphorylated immune-receptor tyrosine-based activation motif (ppITAM). An alternative model proposes that SYK activation requires ppITAM binding and phosphorylation of the SH2-kinase linker by a SRC family kinase such as LYN proto-oncogene, SRC family tyrosine kinase (LYN). To evaluate these two models, we generated directly comparable unphosphorylated (upSYK) and phosphorylated (pSYK) proteins with or without an N-terminal glutathione S-transferase (GST) tag, resulting in monomeric or obligatory dimeric SYK, respectively. We assessed the ability of a ppITAM peptide and LYN to activate these SYK proteins. The ppITAM peptide strongly activated GST-SYK but was less effective in activating upSYK untagged with GST. LYN alone activated untagged upSYK to a greater extent than did ppITAM, and inclusion of both proteins rapidly and fully activated upSYK. Using immunoblot and phosphoproteomic approaches, we correlated the kinetics and order of site-specific SYK phosphorylation. Our results are consistent with the alternative model, indicating that ppITAM binding primes SYK for rapid LYN-mediated phosphorylation of Tyr-352 and then Tyr-348 of the SH2-kinase linker, which facilitates activation loop phosphorylation and full SYK activation. This gradual activation mechanism may also explain how SYK maintains ligand-independent tonic signaling, important for B-cell development and survival.


Asunto(s)
Modelos Químicos , Quinasa Syk/química , Secuencias de Aminoácidos , Activación Enzimática , Humanos , Fosforilación , Proto-Oncogenes Mas , Quinasa Syk/metabolismo , Dominios Homologos src , Familia-src Quinasas/química , Familia-src Quinasas/metabolismo
11.
Molecules ; 23(12)2018 Nov 28.
Artículo en Inglés | MEDLINE | ID: mdl-30487406

RESUMEN

Spleen tyrosine kinase (Syk) is a critical target protein for treating immunoreceptor signalling-mediated allergies. In this study, a virtual screening of an in-house Chinese medicine database followed by biological assays was carried out to identify novel Syk inhibitors. A molecular docking method was employed to screen for compounds with potential Syk inhibitory activity. Then, an in vitro kinase inhibition assay was performed to verify the Syk inhibitory activity of the virtual screening hits. Subsequently, a ß-hexosaminidase release assay was conducted to evaluate the anti-mast cell degranulation activity of the active compounds. Finally, tanshinone I was confirmed as a Syk inhibitor (IC50 = 1.64 µM) and exhibited anti-mast cell degranulation activity in vitro (IC50 = 2.76 µM). Docking studies showed that Pro455, Gln462, Leu377, and Lys458 were key amino acid residues for Syk inhibitory activity. This study demonstrated that tanshinone I is a Syk inhibitor with mast cell degranulation inhibitory activity. Tanshinone I may be a potential lead compound for developing effective and safe Syk-inhibiting drugs.


Asunto(s)
Bases de Datos Factuales , Mastocitos/enzimología , Medicina Tradicional China , Simulación del Acoplamiento Molecular , Inhibidores de Proteínas Quinasas , Quinasa Syk , Animales , Degranulación de la Célula/efectos de los fármacos , Línea Celular , Mastocitos/patología , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Quinasa Syk/antagonistas & inhibidores , Quinasa Syk/química , Quinasa Syk/metabolismo
12.
Bioorg Med Chem Lett ; 28(21): 3458-3462, 2018 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-30249354
13.
Fish Shellfish Immunol ; 82: 162-172, 2018 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-30114435

RESUMEN

Spleen tyrosine kinase (SYK), a member of non-receptor tyrosine kinase family, plays an important role in immune responses against pathogen infection, which is capable of activating B cells signaling pathway and regulating inflammatory response. In this study, Nile tilapia (Oreochromis niloticus) ortholog (OnSYK) was identified and characterized at expression pattern against bacterial infection, function in B cells activation pathway and inflammatory response. The cDNA of OnSYK ORF contained 1851 bp of nucleotide sequence encoding polypeptides of 616 amino acids. The deduced OnSYK protein was highly homologous to other species SYK, containing two SH2 domains and a TyrKc domain. Spatial mRNA expression analysis revealed that OnSYK had wide tissue distribution and was highly expressed in the liver. After challenge of Streptococcus agalactiae (S. agalactiae) in vivo, mRNA expression of OnSYK was significantly up-regulated in the head kidney, spleen and liver. The up-regulation of OnSYK transcript was also displayed in the head kidney and spleen leukocytes stimulation with S. agalactiae and LPS in vitro, which was confirmed at protein level in the head kidney leukocytes by FACS analysis. In addition, after induction with mouse anti-OnIgM monoclonal antibody in vitro, the expressions of OnSYK and its downstream molecules (OnLYN, OnBLNK and OnAP-1) were significantly up-regulated in the head kidney leukocytes, and pharmacological inhibition of SYK activity with inhibitor (P505-15) significantly attenuated the expressions of OnLYN, OnBLNK and OnAP-1. Moreover, upon LPS challenge, the expressions of OnSYK, OnTNF-α, OnIL-6 and OnAP-1 were also up-regulated in the head kidney monocytes/macrophages. After treatment with SYK inhibitor (BAY 61-3606), the expressions of OnTNF-α, OnIL-6 and OnAP-1 were inhibited in the LPS-challenged head kidney monocytes/macrophages. Taken together, the results of this study indicated that OnSYK, playing potential roles in BCR signaling and inflammatory response, was likely to get involved in host defense against bacterial infection in Nile tilapia.


Asunto(s)
Cíclidos/genética , Cíclidos/inmunología , Enfermedades de los Peces/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Quinasa Syk/genética , Quinasa Syk/inmunología , Secuencia de Aminoácidos , Animales , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Perfilación de la Expresión Génica/veterinaria , Filogenia , Alineación de Secuencia/veterinaria , Infecciones Estreptocócicas/veterinaria , Streptococcus agalactiae/fisiología , Quinasa Syk/química
14.
Protein Sci ; 27(10): 1780-1796, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30051939

RESUMEN

Spleen tyrosine kinase (Syk) is an essential player in immune signaling through its ability to couple multiple classes of membrane immunoreceptors to intracellular signaling pathways. Ligand binding leads to the recruitment of Syk to a phosphorylated cytoplasmic region of the receptors called ITAM. Syk binds to ITAM with high-affinity (nanomolar Kd ) via its tandem pair of SH2 domains. The affinity between Syk and ITAM is allosterically regulated by phosphorylation at Y130 in a linker connecting the tandem SH2 domains; when Y130 is phosphorylated, the binding affinity decreases (micromolar Kd ). Previous equilibrium binding studies attribute the increase in the binding free energy to an intra-molecular binding (isomerization) step of the tandem SH2 and ITAM, but a physical basis for the increased free energy is unknown. Here, we provide evidence that Y130 phosphorylation imposes an entropy penalty to isomerization, but surprisingly, has negligible effect on the SH2 binding interactions with ITAM and thus on the binding enthalpy. An analysis of NMR chemical shift differences characterized conformational effects of ITAM binding, and binding thermodynamics were measured from isothermal titration calorimetry. Together the data support a previously unknown mechanism for the basis of regulating protein-protein interactions through protein phosphorylation. The decreased affinity for Syk association with immune receptor ITAMs by Y130 phosphorylation is an allosteric mechanism driven by an increased entropy penalty, likely contributed by conformational disorder in the SH2-SH2 inter-domain structure, while SH2-ITAM binding contacts are not affected, and binding enthalpy is unchanged.


Asunto(s)
Entropía , Quinasa Syk/metabolismo , Humanos , Motivo de Activación del Inmunorreceptor Basado en Tirosina , Modelos Moleculares , Fosforilación , Quinasa Syk/química
15.
J Biol Chem ; 293(13): 4591-4602, 2018 03 30.
Artículo en Inglés | MEDLINE | ID: mdl-29440271

RESUMEN

Spleen tyrosine kinase (Syk) is involved in cellular adhesion and also in the activation and development of hematopoietic cells. Syk activation induced by genomic rearrangement has been linked to certain T-cell lymphomas, and Syk inhibitors have been shown to prolong survival of patients with B-cell lineage malignancies. Syk is activated either by its interaction with a double-phosphorylated immunoreceptor tyrosine-based activation motif (pITAM), which induces rearrangements in the Syk structure, or by the phosphorylation of specific tyrosine residues. In addition to its immunoreceptor function, Syk is activated downstream of integrin pathways, and integrins bind to the same region in Syk as does pITAM. However, it is unknown whether integrins and pITAM use the same mechanism to activate Syk. Here, using purified Syk protein and fluorescence-based enzyme assay we investigated whether interaction of the integrin ß3 cytoplasmic domain with the Syk regulatory domain causes changes in Syk activity similar to those induced by pITAM peptides. We observed no direct Syk activation by soluble integrin peptide, and integrin did not compete with pITAM-induced activation even though at high concentrations, the integrin cytoplasmic domain peptide competed with Syk's substrate. However, clustered integrin peptides induced Syk activation, presumably via a transphosphorylation mechanism. Moreover, the clustered integrins also activated a Syk variant in which tyrosines were replaced with phenylalanine (Y348F/Y352F), indicating that clustered integrin-induced Syk activation involved other phosphorylation sites. In conclusion, integrin cytoplasmic domains do not directly induce Syk conformational changes and do not activate Syk via the same mechanism as pITAM.


Asunto(s)
Integrinas/química , Péptidos/química , Quinasa Syk/química , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Activación Enzimática , Humanos , Integrinas/genética , Integrinas/metabolismo , Mutación Missense , Péptidos/genética , Péptidos/metabolismo , Fosforilación , Dominios Proteicos , Quinasa Syk/genética , Quinasa Syk/metabolismo
16.
Sci Rep ; 7(1): 11480, 2017 09 13.
Artículo en Inglés | MEDLINE | ID: mdl-28904407

RESUMEN

The adaptor protein c-Abl SH3 domain binding protein-2 (3BP2) is tyrosine phosphorylated by Syk in response to cross-linking of antigen receptors, which in turn activates various immune responses. Recently, a study using the mouse model of cherubism, a dominant inherited disorder caused by mutations in the gene encoding 3BP2, showed that 3BP2 is involved in the regulation of phagocytosis mediated by Fc receptor for IgG (FcγR) in macrophages. However, the molecular mechanisms underlying 3BP2-mediated regulation of phagocytosis and the physiological relevance of 3BP2 tyrosine phosphorylation remains elusive. In this study, we established various gene knockout U937 cell lines using the CRISPR/Cas9 system and found that 3BP2 is rapidly tyrosine phosphorylated by Syk in response to cross-linking of FcγRI. Depletion of 3BP2 caused significant reduction in the Fc receptor γ chain (FcRγ)-mediated phagocytosis in addition to the FcγRI-mediated induction of chemokine mRNA for IL-8, CCL3L3 and CCL4L2. Syk-dependent tyrosine phosphorylation of 3BP2 was required for overcoming these defects. Finally, we found that the PH and SH2 domains play important roles on FcγRI-mediated tyrosine phosphorylation of 3BP2 in HL-60 cells. Taken together, these results indicate that Syk-dependent tyrosine phosphorylation of 3BP2 is required for optimal FcRγ-mediated phagocytosis and chemokine expression.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Quimiocinas/genética , Regulación de la Expresión Génica , Fagocitosis , Receptores Fc/metabolismo , Quinasa Syk/metabolismo , Tirosina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Quimiocinas/metabolismo , Humanos , Mutación , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores Fc/química , Receptores Fc/genética , Quinasa Syk/química , Quinasa Syk/genética , Células U937 , Dominios Homologos src
17.
Mol Cell Biol ; 37(21)2017 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-28760774

RESUMEN

The nonreceptor tyrosine kinase Syk, a central regulator of immune cell differentiation and activation, is a promising drug target for treatment of leukemia and allergic and inflammatory diseases. The clinical failure of Syk inhibitors underscores the importance of understanding the regulation of Syk function and activity. A series of previous studies emphasized the importance of three C-terminal tyrosines in Syk for kinase activity regulation, as docking sites for downstream effector molecules, and for Ca2+ mobilization. Here, we investigated the roles of these C-terminal tyrosines in the mouse. Surprisingly, expression of a triple tyrosine-to-phenylalanine human Syk mutant, SYK(Y3F), was not associated with discernible signaling defects either in reconstituted DT40 cells or in B or mast cells from mice expressing SYK(Y3F) instead of wild-type Syk. Remarkably, lymphocyte differentiation, calcium mobilization, and 2,4,6-trinitrophenyl (TNP)-specific immune responses were unperturbed in SYK(Y3F) mice. These results emphasize the capacity of immune cells to compensate for specific molecular defects, likely using redundant intermolecular interactions, and highlight the importance of in vivo analyses for understanding cellular signaling mechanisms.


Asunto(s)
Linfocitos B/metabolismo , Mastocitos/metabolismo , Mutación , Quinasa Syk/genética , Quinasa Syk/metabolismo , Animales , Linfocitos B/citología , Diferenciación Celular , Línea Celular , Técnicas de Sustitución del Gen , Humanos , Ratones , Fenilalanina/genética , Transducción de Señal , Quinasa Syk/química , Tirosina/genética
18.
Bioconjug Chem ; 28(9): 2384-2392, 2017 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-28767218

RESUMEN

Members of the Syk family of tyrosine kinases arrange Src homology 2 (SH2) domains in tandem to allow the firm binding of immunoreceptor tyrosine-based interaction motifs (ITAMs). While the advantages provided by the bivalency enhanced interactions are evident, the impact on binding specificity is less-clear. For example, the spleen tyrosine kinase (Syk) and the ζ-chain-associated protein kinase (ZAP-70) recognize the consensus sequence pYXXI/L(X)6-8 pYXXI/L with near-identical nanomolar affinity. The nondiscriminatory recognition, on the one hand, poses a specificity challenge for the design of subtype selective protein binders and, on the other hand, raises the question as to how differential activation of Syk and ZAP-70 is ensured when both kinases are co-expressed. Herein, we identified the criteria for the design of binders that specifically address either the Syk or the Zap-70 tSH2 domain. Our approach is based on DNA-programmed spatial screening. Tyrosine-phosphorylated peptides containing the pYXXI/L motif were attached to oligonucleotides and aligned in tandem on a DNA template by means of nucleic acid hybridization. The distance between the pYXXI/L motifs and the orientation of strands were varied. The exploration exposed remarkably different recognition characteristics. While Syk tSH2 has a rather broad substrate scope, ZAP-70 tSH2 required a proximal arrangement of the phosphotyrosine ligands in defined strand orientation. The spatial screen led to the design of mutually selective, DNA-free binders, which discriminate Zap-70 and Syk tSH2 by 1 order of magnitude in affinity.


Asunto(s)
Péptidos/metabolismo , Fosfotirosina/metabolismo , Quinasa Syk/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , ADN/química , ADN/metabolismo , Humanos , Modelos Moleculares , Péptidos/química , Fosfotirosina/química , Unión Proteica , Especificidad por Sustrato , Quinasa Syk/química , Proteína Tirosina Quinasa ZAP-70/química , Dominios Homologos src
19.
BMC Immunol ; 18(1): 37, 2017 07 17.
Artículo en Inglés | MEDLINE | ID: mdl-28716125

RESUMEN

BACKGROUND: Epidemiological evidence and animal models suggest that exposure to low and non-neurotoxic concentrations of mercury may contribute to idiosyncratic autoimmune disease. Since defects in function and signaling in B cells are often associated with autoimmunity, we investigated whether mercury exposure might alter B cell responsiveness to self-antigens by interfering with B cell receptor (BCR) signal transduction. In this study we determined the effects of mercury on the protein tyrosine kinase SYK, a critical protein involved in regulation of the BCR signaling pathway. METHODS: Phosphorylation sites of murine SYK were mapped before and after treatment of WEHI cell cultures with mercury, or with anti-IgM antibody (positive control) or pervanadate (a potent phosphatase inhibitor). Phosphopeptides were enriched by either titanium dioxide chromatography or anti-phosphotyrosine immunoaffinity, and analyzed by liquid chromatography-mass spectrometry. Select SYK phosphosite cluster regions were profiled for responsiveness to treatments using multiple reaction monitoring (MRM) methodology. RESULTS: A total of 23 phosphosites were identified with high probability in endogenous SYK, including 19 tyrosine and 4 serine residues. For 10 of these sites phosphorylation levels were increased following BCR activation. Using MRM to profile changes in phosphorylation status we found that 4 cluster regions, encompassing 8 phosphosites, were activated by mercury and differentially responsive to all 3 treatments. Phosphorylation of tyrosine-342 and -346 residues were most sensitive to mercury exposure. This cluster is known to propagate normal BCR signal transduction by recruiting adaptor proteins such as PLC-γ and Vav-1 to SYK during formation of the BCR signalosome. CONCLUSIONS: Our data shows that mercury alters the phosphorylation status of SYK on tyrosine sites known to have a role in promoting BCR signals. Considering the importance of SYK in the BCR signaling pathway, these data suggest that mercury can alter BCR signaling in B cells, which might affect B cell responsiveness to self-antigen and have implications with respect to autoimmunity and autoimmune disease.


Asunto(s)
Linfocitos B/efectos de los fármacos , Linfocitos B/enzimología , Mercurio/toxicidad , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Quinasa Syk/metabolismo , Animales , Anticuerpos Antiidiotipos/farmacología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Ratones , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Receptores de Antígenos de Linfocitos B/agonistas , Receptores de Antígenos de Linfocitos B/efectos de los fármacos , Receptores de Antígenos de Linfocitos B/metabolismo , Quinasa Syk/antagonistas & inhibidores , Quinasa Syk/química , Espectrometría de Masas en Tándem , Tirosina/metabolismo , Vanadatos/farmacología
20.
Sci Rep ; 7: 39766, 2017 01 05.
Artículo en Inglés | MEDLINE | ID: mdl-28054556

RESUMEN

Spleen Tyrosine Kinase (Syk) plays a crucial role in immune cell signalling and its altered expression or activation are involved in several cancers. Syk activity relies on its phosphorylation status and its multiple phosphorylation sites predict several Syk conformations. In this report, we characterized Syk structural changes according to its phosphorylation/activation status by Surface Enhanced Raman Spectroscopy (SERS). Unphosphorylated/inactive and phosphorylated/active Syk forms were produced into two expression systems with different phosphorylation capability. Syk forms were then analysed by SERS that was carried out in liquid condition on a lithographically designed gold nanocylinders array. Our study demonstrated that SERS signatures of the two Syk forms were drastically distinct, indicating structural modifications related to their phosphorylation status. By comparison with the atomic structure of the unphosphorylated Syk, the SERS peak assignments of the phosphorylated Syk nearest gold nanostructures revealed a differential interaction with the gold surface. We finally described a model for Syk conformational variations according to its phosphorylation status. In conclusion, SERS is an efficient technical approach for studying in vitro protein conformational changes and might be a powerful tool to determine protein functions in tumour cells.


Asunto(s)
Sistema Inmunológico/fisiología , Conformación Proteica , Quinasa Syk/metabolismo , Regulación Alostérica , Sitio Alostérico/genética , Humanos , Fosforilación , Ingeniería de Proteínas , Transducción de Señal , Espectrometría Raman/métodos , Quinasa Syk/química , Quinasa Syk/genética
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