RESUMO
A series of novel 3,6-di-substituted or 3-substituted pyrazolo[1,5-a]pyrimidines were prepared via a microwave-assisted approach that generated a broad array of derivatives in good yields (20-93%, ave. = 59%). The straightforward synthesis involved sequential treatment of commercially-available acetonitrile derivatives with DMF-dimethylacetal (120 °C, 20 min), followed by treatment with NH2NH2·HBr (120 °C, 20 min), and 1,1,3,3-tetramethoxypropane or 2-aryl-substituted malondialdehdyes (120 °C, 20 min). Compounds were screened for antimitotic activities against MCF7 breast cancer and/or A2780 ovarian cancer cell lines in vitro. The most active compounds exhibited EC50 values ranging from 0.5 to 4.3 µM, with the 3-(4-(trifluoromethyl)phenyl)-6-[4-(2-(piperidin-1-yl)ethoxy]phenyl analogue (34e) and the 3-(2-fluorophenyl)-6-[4-(2-(4-methylpiperizin-1-yl)ethoxy]phenyl analogue (35a) being two to three fold more active than Compound C (Dorsomorphin) in A2780 and MCF7 assays, respectively. Importantly, a monosubstituted 3-(benzothiazol-2-yl) derivative (13) was equipotent with the more synthetically challenging 3,6-disubstituted derivatives (34a-e and 35a-e), and exhibited a promising and unique selectivity profile when screened against a panel consisting of 403 protein kinases (Kinomescan™ selectivity score = 0.005, Kd = 0.55 ± 0.055 µM and 0.410 ± 0.20 µM for JAK1 JH2 pseudokinase and VPS34, respectively).
Assuntos
Classe III de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Janus Quinase 1/antagonistas & inibidores , Pirimidinas/síntese química , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Despite their clinical importance, drug resistance remains problematic for microtubule targeting drugs. D4-9-31, a novel microtubule destabilizing agent, has pharmacology that suggests it can overcome common resistance mechanisms and has been shown to remain efficacious in cell and animal models with acquired taxane resistance. To better understand resistance mechanisms and the breadth of cross-resistance with D4-9-31, this study examines the A2780 ovarian cancer cell line as it develops acquired resistance with continuous exposure to D4-9-31. Analyzing cellular responses to D4-9-31 reveals that D4-9-31 resistance is associated with increased mitochondrial respiration, but no cross-resistance to other microtubule targeting agents is observed. Sequencing of transcripts of parental cells and resistant counterparts reveals mutations and altered expression of microtubule-associated genes, but not in genes commonly associated with resistance to microtubule targeting drugs. Additionally, our findings suggest distinct mechanisms drive short- and long-term drug resistance.
Assuntos
Amidas/uso terapêutico , Microtúbulos/efeitos dos fármacos , Polimerização/efeitos dos fármacos , Piridinas/uso terapêutico , Pirimidinas/uso terapêutico , Amidas/farmacologia , Humanos , Piridinas/farmacologia , Pirimidinas/farmacologiaRESUMO
Microtubule-targeting agents are important tools in cancer treatment. Generating novel microtubule targeting agents with novel pharmacology could dramatically expand the utility of this class of drugs. Here we characterize the pharmacology of recently described small molecule microtubule polymerization inhibitors. Pharmacokinetic experiments show oral bioavailability through gastric absorption. In vitro assays designed to predict absorption, distribution, metabolism, and excretion (ADME) and safety reveal a scaffold that is metabolically stable, evades P-glycoprotein, does not inhibit CYP enzymes, occurs as a significant free fraction in serum, and has exceptionally high cellular permeability. Together with in vivo efficacy models, pharmacology supports further development as a treatment for solid tumors.
Assuntos
Amidas/farmacologia , Microtúbulos/efeitos dos fármacos , Piridinas/farmacologia , Pirimidinas/farmacologia , Amidas/síntese química , Amidas/química , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cães , Relação Dose-Resposta a Droga , Humanos , Camundongos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Microtúbulos/metabolismo , Estrutura Molecular , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/patologia , Polimerização/efeitos dos fármacos , Piridinas/síntese química , Piridinas/química , Pirimidinas/síntese química , Pirimidinas/química , Ratos , Relação Estrutura-AtividadeRESUMO
Epithelial cells can be triggered to actively detach from epithelial tissues and become solitary, migratory and invasive. This process occurs repeatedly in development, where it is termed epithelial-mesenchymal transition (EMT), and can be recapitulated as epithelial scattering in cell culture models. Detachment of cell-cell junctions involves changes in contractile forces, actin cytoskeletal organization, changes in cell-substrate adhesion properties, surface presentation of cell-cell adhesion molecules, and gene expression. That these cellular processes affect each other and share molecular components creates difficulties in generating hypotheses and designing experiments to understand the mechanics of epithelial scattering. Computational modeling is proving a powerful too in such instances. Here we develop a cellular automaton to reveal insights into how cells rupture epithelial cell-cell junctions during scattering. The model is optimized for realistic and stable recapitulation of behavior of single cells, then for realistic simulation of multiple cells forming epithelial colonies. With a workable model of epithelial cell behavior, we then alter model parameters and assess whether we can realistically mimic epithelial scattering. Adjusting model parameters to recapitulate epithelial scattering reveals that induction of cell migration is the major driver of epithelial scattering.
Assuntos
Membrana Celular/metabolismo , Movimento Celular , Células Epiteliais/citologia , Animais , Fenômenos Biomecânicos , Adesão Celular , Transição Epitelial-Mesenquimal , Humanos , Junções Intercelulares/metabolismo , Modelos BiológicosRESUMO
Stimulation of cultured epithelial cells with scatter factor/hepatocyte growth factor (HGF) results in individual cells detaching and assuming a migratory and invasive phenotype. Epithelial scattering recapitulates cancer progression and studies have implicated HGF signaling as a driver of cancer metastasis. Inhibitors of HGF signaling have been proposed to act as anti-cancer agents. We previously screened a small molecule library for compounds that block HGF-induced epithelial scattering. Most hits identified in this screen exhibit anti-mitotic properties. Here we assess the biological mechanism of a compound that blocks HGF-induced scattering with limited anti-mitotic activity. Analogs of this compound have one of two distinct activities: inhibiting either cell migration or cell proliferation with cell cycle arrest in G2/M. Each activity bears unique structure-activity relationships. The mechanism of action of anti-mitotic compounds is by inhibition of microtubule polymerization; these compounds entropically and enthalpically bind tubulin in the colchicine binding site, generating a conformational change in the tubulin dimer.
Assuntos
Amidas/farmacologia , Antineoplásicos/farmacologia , Células Epiteliais/efeitos dos fármacos , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Piridinas/farmacologia , Pirimidinas/farmacologia , Amidas/síntese química , Amidas/química , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Células Epiteliais/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Células MCF-7 , Estrutura Molecular , Piridinas/síntese química , Piridinas/química , Pirimidinas/síntese química , Pirimidinas/química , Relação Estrutura-AtividadeRESUMO
CONTEXT: Kalanchoe pinnata (Lam.) Pers. (Crassulaceae) is a succulent plant that is known for its traditional antivirus and antibacterial usage. OBJECTIVE: This work examines two compounds identified from the K. pinnata plant for their antivirus activity against human alphaherpesvirus (HHV) 1 and 2 and vaccinia virus (VACV). MATERIALS AND METHODS: Compounds KPB-100 and KPB-200 were isolated using HPLC and were identified using NMR and MS. Both compounds were tested in plaque reduction assay of HHV-2 wild type (WT) and VACV. Both compounds were then tested in virus spread inhibition and virus yield reduction (VYR) assays of VACV. KPB-100 was further tested in viral cytopathic effect (CPE) inhibition assay of HHV-2 TK-mutant and VYR assay of HHV-1 WT. RESULTS: KPB-100 and KPB-200 inhibited HHV-2 at IC50 values of 2.5 and 2.9 µg/mL, respectively, and VACV at IC50 values of 3.1 and 7.4 µg/mL, respectively, in plaque reduction assays. In virus spread inhibition assay of VACV KPB-100 and KPB-200 yielded IC50 values of 1.63 and 13.2 µg/mL, respectively, and KPB-100 showed a nearly 2-log reduction in virus in VYR assay of VACV at 20 µg/mL. Finally, KPB-100 inhibited HHV-2 TK- at an IC50 value of 4.5 µg/mL in CPE inhibition assay and HHV-1 at an IC90 of 3.0 µg/mL in VYR assay. DISCUSSION AND CONCLUSION: Both compounds are promising targets for synthetic optimization and in vivo study. KPB-100 in particular showed strong inhibition of all viruses tested.
Assuntos
Antivirais/farmacologia , Kalanchoe/química , Extratos Vegetais/farmacologia , Antivirais/administração & dosagem , Antivirais/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Efeito Citopatogênico Viral/efeitos dos fármacos , Células HeLa , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 2/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Extratos Vegetais/administração & dosagem , Extratos Vegetais/isolamento & purificação , Vaccinia virus/efeitos dos fármacosRESUMO
Vasodilator-stimulated phosphoprotein (VASP) and Zyxin are interacting proteins involved in cellular adhesion and motility. PKA phosphorylates VASP at serine 157, regulating VASP cellular functions. VASP interacts with ABL and is a substrate of the BCR-ABL oncoprotein. The presence of BCR-ABL protein drives oncogenesis in patients with chronic myeloid leukemia (CML) due to a constitutive activation of tyrosine kinase activity. However, the function of VASP and Zyxin in BCR-ABL pathway and the role of VASP in CML cells remain unknown. In vitro experiments using K562 cells showed the involvement of VASP in BCR-ABL signaling. VASP and Zyxin inhibition decreased the expression of anti-apoptotic proteins, BCL2 and BCL-XL. Imatinib induced an increase in phosphorylation at Ser157 of VASP and decreased VASP and BCR-ABL interaction. VASP did not interact with Zyxin in K562 cells; however, after Imatinib treatment, this interaction was restored. Corroborating our data, we demonstrated the absence of phosphorylation at Ser157 in VASP in the bone marrow of CML patients, in contrast to healthy donors. Phosphorylation of VASP on Ser157 was restored in Imatinib responsive patients though not in the resistant patients. Therefore, we herein identified a possible role of VASP in CML pathogenesis, through the regulation of BCR-ABL effector proteins or the absence of phosphorylation at Ser157 in VASP.
Assuntos
Benzamidas/farmacologia , Moléculas de Adesão Celular/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Piperazinas/farmacologia , Pirimidinas/farmacologia , Zixina/metabolismo , Apoptose/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Proliferação de Células/efeitos dos fármacos , Células Clonais , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Células K562 , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína bcl-X/metabolismoRESUMO
Epithelial scattering occurs when cells disassemble cell-cell junctions, allowing individual epithelial cells to act in a solitary manner. Epithelial scattering occurs frequently in development, where it accompanies epithelial-mesenchymal transitions and is required for individual cells to migrate and invade. While migration and invasion have received extensive research focus, how cell-cell junctions are detached remains poorly understood. An open debate has been whether disruption of cell-cell interactions occurs by remodeling of cell-cell adhesions, increased traction forces through cell substrate adhesions, or some combination of both processes. Here we seek to examine how changes in adhesion and contractility are coupled to drive detachment of individual epithelial cells during hepatocyte growth factor (HGF)/scatter factor-induced EMT. We find that HGF signaling does not alter the strength of cell-cell adhesion between cells in suspension, suggesting that changes in cell-cell adhesion strength might not accompany epithelial scattering. Instead, cell-substrate adhesion seems to play a bigger role, as cell-substrate adhesions are stronger in cells treated with HGF and since rapid scattering in cells treated with HGF and TGFß is associated with a dramatic increase in focal adhesions. Increases in the pliability of the substratum, reducing cells ability to generate traction on the substrate, alter cells׳ ability to scatter. Further consistent with changes in substrate adhesion being required for cell-cell detachment during EMT, scattering is impaired in cells expressing both active and inactive RhoA mutants, though in different ways. In addition to its roles in driving assembly of both stress fibers and focal adhesions, RhoA also generates myosin-based contractility in cells. We therefore sought to examine how RhoA-dependent contractility contributes to cell-cell detachment. Inhibition of Rho kinase or myosin II induces the same effect on cells, namely an inhibition of cell scattering following HGF treatment. Interestingly, restoration of myosin-based contractility in blebbistatin-treated cells results in cell scattering, including global actin rearrangements. Scattering is reminiscent of HGF-induced epithelial scattering without a concomitant increase in cell migration or decrease in adhesion strength. This scattering is dependent on RhoA, as blebbistatin-induced scattering is reduced in cells expressing dominant-negative RhoA mutants. This suggests that induction of myosin-based cellular contractility may be sufficient for cell-cell detachment during epithelial scattering.
Assuntos
Movimento Celular/fisiologia , Células Epiteliais/fisiologia , Actinas/metabolismo , Animais , Adesão Celular/fisiologia , Linhagem Celular , Cães , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/fisiologia , Adesões Focais/fisiologia , Fator de Crescimento de Hepatócito/fisiologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Junções Intercelulares/fisiologia , Mutação , Miosina Tipo II/metabolismo , Transdução de Sinais , Fibras de Estresse/fisiologia , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Cell-cell adhesions and the cytoskeletons play important and coordinated roles in cell biology, including cell differentiation, development, and migration. Adhesion and cytoskeletal dynamics are regulated by Rho-GTPases. ARHGAP21 is a negative regulator of Rho-GTPases, particularly Cdc42. Here we assess the function of ARHGAP21 in cell-cell adhesion, cell migration, and scattering. We find that ARHGAP21 is localized in the nucleus, cytoplasm, or perinuclear region but is transiently redistributed to cell-cell junctions 4 h after initiation of cell-cell adhesion. ARHGAP21 interacts with Cdc42, and decreased Cdc42 activity coincides with the appearance of ARHGAP21 at the cell-cell junctions. Cells lacking ARHGAP21 expression show weaker cell-cell adhesions, increased cell migration, and a diminished ability to undergo hepatocyte growth factor-induced epithelial-mesenchymal transition (EMT). In addition, ARHGAP21 interacts with α-tubulin, and it is essential for α-tubulin acetylation in EMT. Our findings indicate that ARHGAP21 is a Rho-GAP involved in cell-cell junction remodeling and that ARHGAP21 affects migration and EMT through α-tubulin interaction and acetylation.
Assuntos
Transição Epitelial-Mesenquimal , Epitélio/metabolismo , Proteínas Ativadoras de GTPase/fisiologia , Tubulina (Proteína)/metabolismo , Acetilação , Animais , Adesão Celular , Comunicação Celular , Linhagem Celular Tumoral , Movimento Celular , Cães , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Células Madin Darby de Rim Canino , Metástase Neoplásica , Interferência de RNA , Fatores de Tempo , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
Hepatocyte growth factor (HGF) signaling drives epithelial cells to scatter by breaking cell-cell adhesions and causing them to migrate as solitary cells, a process that parallels epithelial-mesenchymal transition. HGF binds and activates the c-met receptor tyrosine kinase, but downstream signaling required for scattering remains poorly defined. We have applied a chemical biology approach to identify components of HGF signaling that are required for scattering in an in vitro model system. This approach yields a number of small molecules that block HGF-induced scattering, including a calcium channel blocker. We show that HGF stimulation results in sudden and transient increases in ion channel influxes at the plasma membrane. Although multiple channels occur in the membranes of our model system, we find that TrpC6 is specifically required for HGF-induced scattering. We further demonstrate that HGF-induced ion influxes through TrpC6 channels coincide with a transient increase in nuclear factor of activated T-cells (NFAT)-dependent gene transcription and that NFAT-dependent gene transcription is required for HGF-induced cell scattering.
Assuntos
Membrana Celular/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Íons/metabolismo , Fatores de Transcrição NFATC/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Transcrição Gênica , Actinas/metabolismo , Animais , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Cães , Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Células Madin Darby de Rim Canino , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Canais de Cátion TRPC/metabolismo , Canais de Cátion TRPV/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Cell-cell and cell-substrate adhesions are sites of dramatic actin rearrangements and where actin-membrane connections are tightly regulated. Zyxin-VASP complexes localize to sites of cell-cell and cell-substrate adhesion and function to regulate actin dynamics and actin-membrane connections at these sites. To accomplish these functions, zyxin recruits VASP to cellular sites via proline-rich binding sites near zyxin's amino terminus. While the prevailing thought has been that zyxin simply acts as a scaffold protein for VASP binding, the identification of a LIM domain-VASP interaction could complicate this view. Here we assess how zyxin-VASP binding through both the proline rich motifs and the LIM domains alters specific VASP functions. We find that neither individual interaction alters VASP's actin regulatory activities. In contrast, however, we find that full-length zyxin dramatically reduces VASP-mediated actin bundling and actin assembly. Taken together, these results suggest a model where zyxin-VASP complexes occur in complex organizations with suppressed actin regulatory activity.
Assuntos
Actinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Zixina/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/genética , Sítios de Ligação , Adesão Celular , Moléculas de Adesão Celular/química , Comunicação Celular , Células Eucarióticas/química , Células Eucarióticas/metabolismo , Humanos , Proteínas dos Microfilamentos/química , Fosfoproteínas/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Zixina/químicaRESUMO
HGF signaling induces epithelial cells to disassemble cadherin-based adhesion and increase cell motility and invasion, a process termed epithelial-mesenchymal transition (EMT). EMT plays a major role in cancer metastasis, allowing individual cells to detach from the primary tumor, invade local tissue, and colonize distant tissues with new tumors. While invasion of vascular and lymphatic networks is the predominant route of metastasis, nerves also can act as networks for dissemination of cancer cell to distant sites in a process termed perineual invasion (PNI). Signaling between nerves and invasive cancer cells remains poorly understood, as does cellular decision making that selects the specific route of invasion. Here we examine how HGF signaling contributes to PNI using reductionist culture model systems. We find that TGFß, produced by PC12 cells, enhances scattering in response to HGF stimulation, increasing both cell-cell junction disassembly and cell migration. Further, gradients of TGFß induce migratory mesenchymal cells to undergo chemotaxis towards the source of TGFß. Interestingly, VEGF suppresses TGFß-induced enhancement of scattering. These results have broad implications for how combinatorial growth factor signaling contributes to cancer metastasis, suggesting that VEGF and TGFß might modulate HGF signaling to influence route selection during cancer progression.
Assuntos
Células Epiteliais/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fator de Crescimento de Hepatócito/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Animais , Movimento Celular , Metástase Neoplásica , Células PC12 , Ratos , Transdução de Sinais , Fator de Crescimento Transformador beta/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Vasodilator-stimulated phosphoprotein (VASP) is an actin regulatory protein that functions in adhesion and migration. In epithelial cells, VASP participates in cell-cell adhesion. At the molecular level, VASP drives actin bundling and polymerization. VASP activity is primarily regulated by phosphorylation. Three physiologically relevant phosphorylation sites significantly reduce actin regulatory activity and are targeted by several kinases, most notable Abl and protein kinases A and G (PKA and PKG). AMP-dependent kinase (AMPK) is best characterized as a cellular sensor of ATP depletion, but also alters actin dynamics in epithelial cells and participates in cell polarity pathways downstream of LKB1. While little is known about how AMPK direct changes in actin dynamics, AMPK has been shown to phosphorylate VASP at one of these three well-characterized PKA/PKG phosphorylation sites. Here we show that phosphorylation of VASP by AMPK occurs at a novel site, serine 322, and that phosphorylation at this site alters actin filament binding. We also show that inhibition of AMPK activity results in the accumulation of VASP at cell-cell adhesions and a concomitant increase in cell-cell adhesion.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Actinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Adesão Celular , Moléculas de Adesão Celular/genética , Linhagem Celular , Cães , Camundongos , Proteínas dos Microfilamentos/genética , Dados de Sequência Molecular , Fosfoproteínas/genética , Fosforilação , Serina/genéticaRESUMO
Zyxin is an actin regulatory protein that is concentrated at sites of actin-membrane association, particularly cell junctions. Zyxin participates in actin dynamics by binding VASP, an interaction that occurs via proline-rich N-terminal ActA repeats. An intramolecular association of the N-terminal LIM domains at or near the ActA repeats can prevent VASP and other binding partners from binding full-length zyxin. Such a head-tail interaction likely accounts for how zyxin function in actin dynamics, cell adhesion, and cell migration can be regulated by the cell. Since zyxin binding to several partners, via the LIM domains, requires phosphorylation, it seems likely that zyxin phosphorylation might alter the head-tail interaction and, thus, zyxin activity. Here we show that zyxin point mutants at a known phosphorylation site, serine 142, alter the ability of a zyxin fragment to directly bind a separate zyxin LIM domains fragment protein. Further, expression of the zyxin phosphomimetic mutant results in increased localization to cell-cell contacts of MDCK cells and generates a cellular phenotype, namely inability to disassemble cell-cell contacts, precisely like that produced by expression of zyxin mutants that lack the entire regulatory LIM domain region. These data suggest that zyxin phosphorylation at serine 142 results in release of the head-tail interaction, changing zyxin activity at cell-cell contacts.
Assuntos
Actinas/metabolismo , Proteínas do Citoesqueleto/metabolismo , Serina/metabolismo , Animais , Adesão Celular , Proteínas do Citoesqueleto/genética , Cães , Fosforilação , Mutação Puntual , Estrutura Terciária de Proteína , Serina/genéticaRESUMO
Cell-cell junction remodeling is associated with dramatic actin reorganizations. Several actin regulatory systems have been implicated in actin remodeling events as cell-cell contacts are assembled and disassembled, including zyxin/LPP-VASP complexes. These complexes facilitate strong cell-cell adhesion by maintaining actin-membrane connections. It has been proposed that zyxin and LPP localize to cell-cell junctions via a well-defined interaction with alpha-actinin. This was recently confirmed for LPP, but zyxin localization at cell-cell contacts occurs independently of alpha-actinin binding. Here we seek to map the zyxin sequence responsible for localization to cell-cell contacts and identify the protein that docks zyxin at this cellular location. Previous results have shown that a zyxin fragment excluding the alpha-actin binding site and the LIM domains (amino acids 51-392) can independently localize to cell-cell contacts. Here, expression of smaller zyxin fragments show that zyxin localization requires amino acids 230-280. A yeast-two-hybrid screen, using the central region of zyxin as bait, resulted in the identification of the cell-cell adhesion receptor nectin-4 as a zyxin binding partner. Further demonstrating zyxin-nectin interactions, zyxin binds the intracellular domain of nectin-2 in vitro. Depletion of nectin-2 from L cells expressing E-cadherin results in a loss of zyxin localization to cell-cell contacts, demonstrating that the zyxin-nectin interaction plays a critical role in zyxin targeting to these sites.
Assuntos
Moléculas de Adesão Celular/metabolismo , Zixina/metabolismo , Animais , Adesão Celular , Moléculas de Adesão Celular/genética , Linhagem Celular , Cães , Humanos , Nectinas , Estrutura Terciária de Proteína , Técnicas do Sistema de Duplo-Híbrido , Zixina/química , Zixina/genéticaRESUMO
Tryptase is a serine protease that is released from mast cells during allergic responses. Tryptase inhibitors are being explored as treatments for allergic inflammation in the skin and respiratory system, most notably asthma. Here we report direct tryptase inhibition by natural product compounds. Candidate inhibitors were identified by computational screening of a large (98,000 compounds) virtual library of natural product compounds for tryptase enzymatic site binding. Biochemical assays were used to validate the predicted anti-tryptase activity in vitro, revealing a high (four out of six) success rate for predicting binding using the computational docking model. We further assess tryptase inhibition by a biflavonoid scaffold, whose structure-activity relationship is partially defined by assessing the potency of structurally similar analogs.
Assuntos
Biflavonoides/farmacologia , Produtos Biológicos/farmacologia , Triptases/antagonistas & inibidores , Biflavonoides/química , Produtos Biológicos/química , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Triptases/metabolismoRESUMO
Development is punctuated by morphogenetic rearrangements of epithelial tissues, including detachment of motile cells during epithelial-mesenchymal transition (EMT). Dramatic actin rearrangements occur as cell-cell junctions are dismantled and cells become independently motile during EMT. Characterizing dynamic actin rearrangements and identifying actin machinery driving these rearrangements is essential for understanding basic mechanisms of cell-cell junction remodeling. Using immunofluorescence and live cell imaging of scattering MDCK cells we examine dynamic actin rearrangement events during EMT and demonstrate that zyxin-VASP complexes mediate linkage of dynamic medial actin networks to adherens junction (AJ) membranes. A functional analysis of zyxin in EMT reveals its role in regulating disruption of actin membrane linkages at cell-cell junctions, altering cells' ability to fully detach and migrate independently during EMT. Expression of a constitutively active zyxin mutant results in persistent actin-membrane linkages and cell migration without loss of cell-cell adhesion. We propose zyxin functions in morphogenetic rearrangements, maintaining collective migration by transducing individual cells' movements through AJs, thus preventing the dissociation of individual migratory cells.
Assuntos
Actinas/metabolismo , Junções Aderentes/metabolismo , Movimento Celular , Transdiferenciação Celular , Proteínas do Citoesqueleto/metabolismo , Células Epiteliais/metabolismo , Mesoderma/metabolismo , Animais , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Forma Celular , Proteínas do Citoesqueleto/genética , Cães , Imunofluorescência , Fator de Crescimento de Hepatócito/metabolismo , Mesoderma/citologia , Proteínas dos Microfilamentos/metabolismo , Microscopia de Fluorescência , Microscopia de Vídeo , Mutação , Fosfoproteínas/metabolismo , Interferência de RNA , Fatores de TempoRESUMO
Cadherin-dependent epithelial cell-cell adhesion is thought to be regulated by Rho family small GTPases and PI 3-kinase, but the mechanisms involved are poorly understood. Using time-lapse microscopy and quantitative image analysis, we show that cell-cell contact in MDCK epithelial cells coincides with a spatio-temporal reorganization of plasma membrane Rac1 and lamellipodia from noncontacting to contacting surfaces. Within contacts, Rac1 and lamellipodia transiently concentrate at newest sites, but decrease at older, stabilized sites. Significantly, Rac1 mutants alter kinetics of cell-cell adhesion and strengthening, but not the eventual generation of cell-cell contacts. Products of PI 3-kinase activity also accumulate dynamically at contacts, but are not essential for either initiation or development of cell-cell adhesion. These results define a role for Rac1 in regulating the rates of initiation and strengthening of cell-cell adhesion.
Assuntos
Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Compartimento Celular/fisiologia , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Pseudópodes/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Bioensaio , Movimento Celular/fisiologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , Proteínas de Fluorescência Verde , Humanos , Indicadores e Reagentes , Proteínas Luminescentes , Microscopia de Vídeo , Mutação/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Pseudópodes/ultraestrutura , Fatores de Tempo , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Zyxin is an adhesion protein that regulates actin assembly by binding to VASP family members through N-terminal proline-rich motifs. Evidence suggests that zyxin's C-terminal LIM domains function as a negative regulator of zyxin-VASP complexes. Zyxin LIM domains access to binding partners is negatively regulated by an unknown mechanism. One possibility is that zyxin LIM domains mediate a head-tail interaction, blocking interactions with other proteins. Such a mechanism might prevent both zyxin-VASP complexes activity and LIM domain access. In this report, the effect of LIM domains on zyxin-VASP complex assembly is defined. We find that zyxin LIM domains associate with zyxin's VASP binding sites, preventing zyxin from binding to PKA-phosphorylated VASP. Unphosphorylated VASP overcomes the head-tail interaction, a result of a direct interaction with the LIM domain region. Zyxin, like a growing number of actin regulators, is controlled by intramolecular interactions.
Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Glicoproteínas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Moléculas de Adesão Celular/química , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Proteínas dos Microfilamentos/química , Fosfoproteínas/química , Estrutura Terciária de Proteína , ZixinaRESUMO
The actin cytoskeleton plays a role in cell-cell adhesion but its specific function is not clear. Actin might anchor cadherins or drive membrane protrusions in order to facilitate cell-cell adhesion. Using a mathematical model of the forces involved in cadherin-based adhesion, we investigate its possible functions. The immersed boundary method is used to model the cell membrane and cortex with cadherin binding forces added as linear springs. The simulations indicate that cells in suspension can develop normal cell-cell contacts without actin-based cadherin anchoring or membrane protrusions. The cadherins can be fixed in the membrane or free to move, and the end results are similar. For adherent cells, simulations suggest that the actin cytoskeleton must play an active role for the cells to establish cell-cell contact regions similar to those observed in vitro.