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1.
Mol Oncol ; 17(10): 1981-1999, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-36912768

RESUMO

Human epidermal growth factor receptor 2 (ErbB2/HER2) is a tyrosine kinase receptor that is overexpressed in 25% of primary human breast cancers, as well as in multiple other cancers. HER2-targeted therapies improved progression-free and overall survival in patients with HER2+ breast cancers. However, associated resistance mechanisms and toxicity highlight the need for new therapeutic approaches for these cancers. We recently established that, in normal cells, HER2 is stabilized in a catalytically repressed state by direct interaction with members of the ezrin/radixin/moesin (ERM) family. In HER2-overexpressing tumors, the low expression of moesin contributes to the aberrant activation of HER2. Through a screen designed to find moesin-mimicking compounds, we identified ebselen oxide. We show that ebselen oxide, and some derivatives, conferred an efficient allosteric inhibition of overexpressed HER2, as well as mutated and truncated oncogenic forms of HER2, which are resistant to current therapies. Ebselen oxide selectively inhibited anchorage-dependent and -independent proliferation of HER2+ cancer cells and showed a significant benefit in combination with current anti-HER2 therapeutic agents. Finally, ebselen oxide significantly blocked HER2+ breast tumor progression in vivo. Collectively, these data provide evidence that ebselen oxide is a newly identified allosteric inhibitor of HER2 to be considered for therapeutic intervention on HER2+ cancers.


Assuntos
Antineoplásicos , Neoplasias da Mama , Humanos , Feminino , Lapatinib/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Receptor ErbB-2/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral
2.
Commun Biol ; 5(1): 800, 2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-35945264

RESUMO

Multidomain kinases use many ways to integrate and process diverse stimuli. Here, we investigated the mechanism by which the protein tyrosine kinase 2-beta (PYK2) functions as a sensor and effector of cellular calcium influx. We show that the linker between the PYK2 kinase and FAT domains (KFL) encompasses an unusual calmodulin (CaM) binding element. PYK2 KFL is disordered and engages CaM through an ensemble of transient binding events. Calcium increases the association by promoting structural changes in CaM that expose auxiliary interaction opportunities. KFL also forms fuzzy dimers, and dimerization is enhanced by CaM binding. As a monomer, however, KFL associates with the PYK2 FERM-kinase fragment. Thus, we identify a mechanism whereby calcium influx can promote PYK2 self-association, and hence kinase-activating trans-autophosphorylation. Collectively, our findings describe a flexible protein module that expands the paradigms for CaM binding and self-association, and their use for controlling kinase activity.


Assuntos
Cálcio , Calmodulina , Cálcio/metabolismo , Calmodulina/metabolismo , Dimerização , Quinase 2 de Adesão Focal/química , Quinase 2 de Adesão Focal/metabolismo , Fosforilação
3.
Cancer Res ; 81(21): 5464-5476, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34493594

RESUMO

Therapies targeting the tyrosine kinase receptor HER2 have significantly improved survival of patients with HER2+ cancer. However, both de novo and acquired resistance remain a challenge, particularly in the brain metastatic setting. Here we report that, unlike other HER tyrosine kinase receptors, HER2 possesses a binding motif in its cytosolic juxtamembrane region that allows interaction with members of the Ezrin/Radixin/Moesin (ERM) family. Under physiologic conditions, this interaction controls the localization of HER2 in ERM-enriched domains and stabilizes HER2 in a catalytically repressed state. In HER2+ breast cancers, low expression of Moesin correlated with increased HER2 expression. Restoring expression of ERM proteins in HER2+ breast cancer cells was sufficient to revert HER2 activation and inhibit HER2-dependent proliferation. A high-throughput assay recapitulating the HER2-ERM interaction allowed for screening of about 1,500 approved drugs. From this screen, we found Zuclopenthixol, an antipsychotic drug that behaved as a Moesin-mimicking compound, because it directly binds the juxtamembrane region of HER2 and specifically inhibits HER2 activation in HER2+ cancers, as well as activation of oncogenic mutated and truncated forms of HER2. Zuclopenthixol efficiently inhibited HER2+ breast tumor progression in vitro and in vivo and, more importantly, showed significant activity on HER2+ brain tumor progression. Collectively, these data reveal a novel class of allosteric HER2 inhibitors, increasing the number of approaches to consider for intervention on HER2+ breast cancers and brain metastases. SIGNIFICANCE: This study demonstrates the functional role of Moesin in maintaining HER2 in a catalytically repressed state and provides novel therapeutic approaches targeting HER2+ breast cancers and brain metastasis using Moesin-mimicking compounds.


Assuntos
Biomimética/métodos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Clopentixol/farmacologia , Regulação Neoplásica da Expressão Gênica , Proteínas dos Microfilamentos/metabolismo , Receptor ErbB-2/antagonistas & inibidores , Regulação Alostérica , Animais , Apoptose , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundário , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Antagonistas de Dopamina/farmacologia , Feminino , Humanos , Camundongos , Camundongos Nus , Proteínas dos Microfilamentos/genética , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Nat Microbiol ; 4(6): 972-984, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30911127

RESUMO

Bacterial virulence factors are attractive targets for the development of therapeutics. Type IV pili, which are associated with a remarkable array of properties including motility, the interaction between bacteria and attachment to biotic and abiotic surfaces, represent particularly appealing virulence factor targets. Type IV pili are present in numerous bacterial species and are critical for their pathogenesis. In this study, we report that trifluoperazine and related phenothiazines block functions associated with Type IV pili in different bacterial pathogens, by affecting piliation within minutes. Using Neisseria meningitidis as a paradigm of Gram-negative bacterial pathogens that require Type IV pili for pathogenesis, we show that piliation is sensitive to altered activity of the Na+ pumping NADH-ubiquinone oxidoreductase (Na+-NQR) complex and that these compounds probably altered the establishment of the sodium gradient. In vivo, these compounds exert a strong protective effect. They reduce meningococcal colonization of the human vessels and prevent subsequent vascular dysfunctions, intravascular coagulation and overwhelming inflammation, the hallmarks of invasive meningococcal infections. Finally, they reduce lethality. This work provides a proof of concept that compounds with activity against bacterial Type IV pili could beneficially participate in the treatment of infections caused by Type IV pilus-expressing bacteria.


Assuntos
Fímbrias Bacterianas/efeitos dos fármacos , Fímbrias Bacterianas/fisiologia , Infecções Meningocócicas/prevenção & controle , Neisseria meningitidis/efeitos dos fármacos , Fatores de Virulência , Animais , Antibacterianos/farmacologia , Vasos Sanguíneos/lesões , Vasos Sanguíneos/microbiologia , Vasos Sanguíneos/patologia , Combinação de Medicamentos , Complexo I de Transporte de Elétrons , Feminino , Fímbrias Bacterianas/genética , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Bactérias Gram-Negativas , Humanos , Camundongos , Neisseria meningitidis/genética , Neisseria meningitidis/crescimento & desenvolvimento , Fenotiazinas/farmacologia , Pele/patologia , Transplante de Pele , ATPase Trocadora de Sódio-Potássio , Trifluoperazina/farmacologia
5.
Nat Commun ; 8: 15764, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28569760

RESUMO

Neisseria meningitidis (meningococcus) is an invasive bacterial pathogen that colonizes human vessels, causing thrombotic lesions and meningitis. Establishment of tight interactions with endothelial cells is crucial for meningococci to resist haemodynamic forces. Two endothelial receptors, CD147 and the ß2-adrenergic receptor (ß2AR), are sequentially engaged by meningococci to adhere and promote signalling events leading to vascular colonization, but their spatiotemporal coordination is unknown. Here we report that CD147 and ß2AR form constitutive hetero-oligomeric complexes. The scaffolding protein α-actinin-4 directly binds to the cytosolic tail of CD147 and governs the assembly of CD147-ß2AR complexes in highly ordered clusters at bacterial adhesion sites. This multimolecular assembly process increases the binding strength of meningococci to endothelial cells under shear stress, and creates molecular platforms for the elongation of membrane protrusions surrounding adherent bacteria. Thus, the specific organization of cellular receptors has major impacts on host-pathogen interaction.


Assuntos
Actinina/metabolismo , Basigina/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Neisseria meningitidis/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Aderência Bacteriana/fisiologia , Basigina/genética , Células Endoteliais/metabolismo , Células Endoteliais/microbiologia , Humanos , Complexos Multiproteicos/metabolismo , Neisseria meningitidis/patogenicidade , Receptores Adrenérgicos beta 2/genética
6.
Clin Rev Allergy Immunol ; 53(1): 78-86, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27628237

RESUMO

On average, 10 % of infertile couples have unexplained infertility. Auto-immune disease (systemic lupus erythematosus, anti-phospholipid syndrome) accounts for a part of these cases. In the last 20 years, aspecific auto-immunity, defined as positivity of auto-antibodies in blood sample without clinical or biological criteria for defined diseases, has been evoked in a subpopulation of infertile women. A systematic review was performed (PUBMED) using the MESH search terms "infertility" and "auto-immunity" or "reproductive technique" or "assisted reproduction" or "in vitro fertilization" and "auto-immunity." We retained clinical and physiopathological studies that were applicable to the clinician in assuming joint management of both infertility associated with serum auto-antibodies in women. Thyroid auto-immunity which affects thyroid function could be a cause of infertility; even in euthyroidia, the presence of anti-thyroperoxydase antibodies and/or thyroglobulin are related to infertility. The presence of anti-phospholipid (APL) and/or anti-nuclear (ANA) antibodies seems to be more frequent in the population of infertile women; serum auto-antibodies are associated with early ovarian failure, itself responsible for fertility disorders. However, there exist few publications on this topic. The methods of dosage, as well as the clinical criteria of unexplained infertility deserve to be standardized to allow a precise response to the question of the role of serum auto-antibodies in these women. The direct pathogenesis of this auto-immunity is unknown, but therapeutic immunomodulators, prescribed on a case-by-case basis, could favor pregnancy even in cases of unexplained primary or secondary infertility.


Assuntos
Autoanticorpos/sangue , Autoanticorpos/imunologia , Infertilidade Feminina/sangue , Infertilidade Feminina/imunologia , Glândulas Suprarrenais/imunologia , Anticorpos Antinucleares/sangue , Anticorpos Antinucleares/imunologia , Anticorpos Antifosfolipídeos/sangue , Anticorpos Antifosfolipídeos/imunologia , Autoimunidade , Endométrio/imunologia , Feminino , Fertilização in vitro , Humanos , Ovário/imunologia , Gravidez , Técnicas de Reprodução Assistida , Trofoblastos/imunologia
7.
Cell Mol Life Sci ; 70(1): 137-52, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22802128

RESUMO

Cytonuclear signaling is essential for long-term alterations of cellular properties. Several pathways involving regulated nuclear accumulation of Ser/Thr kinases have been described but little is known about cytonuclear trafficking of tyrosine kinases. Proline-rich tyrosine kinase 2 (Pyk2) is a cytoplasmic non-receptor tyrosine kinase enriched in neurons and involved in functions ranging from synaptic plasticity to bone resorption, as well as in cancer. We previously showed the Ca(2+)-induced, calcineurin-dependent, nuclear localization of Pyk2. Here, we characterize the molecular mechanisms of Pyk2 cytonuclear localization in transfected PC12 cells. The 700-841 linker region of Pyk2 recapitulates its depolarization-induced nuclear accumulation. This region includes a nuclear export motif regulated by phosphorylation at residue S778, a substrate of cAMP-dependent protein kinase and calcineurin. Nuclear import is controlled by a previously identified sequence in the N-terminal domain and by a novel nuclear targeting signal in the linker region. Regulation of cytonuclear trafficking is independent of Pyk2 activity. The region regulating nuclear localization is absent from the non-neuronal shorter splice isoform of Pyk2. Our results elucidate the mechanisms of Ca(2+)-induced nuclear accumulation of Pyk2. They also suggest that Pyk2 nuclear accumulation is a novel type of signaling response that may contribute to specific long-term adaptations in neurons.


Assuntos
Núcleo Celular/enzimologia , Quinase 2 de Adesão Focal/análise , Quinase 2 de Adesão Focal/metabolismo , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Animais , Calcineurina/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Quinase 2 de Adesão Focal/química , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/metabolismo , Sinais de Exportação Nuclear , Sinais de Localização Nuclear , Células PC12 , Fosforilação , Isoformas de Proteínas/análise , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Processamento de Proteína , Transporte Proteico , Ratos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo
8.
J Cell Sci ; 120(Pt 17): 3034-44, 2007 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-17684059

RESUMO

Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor tyrosine kinase expressed in many cell types and enriched in neurons. PYK2 is a cytoplasmic enzyme activated by increases in cytosolic free Ca(2+) through an unknown mechanism. We report that depolarization or electrical stimulation of hippocampal slices induced a rapid and transient nuclear accumulation of PYK2. Depolarization of cultured neurons or PC12 cells also triggered a Ca(2+)-dependent nuclear accumulation of PYK2, much more pronounced than that induced by blockade of nuclear export with leptomycin B. Src-family kinase activity, PYK2 autophosphorylation and kinase activity were not required for its nuclear translocation. Depolarization induced a slight decrease in PYK2 apparent molecular mass, compatible with a Ca(2+)-activated dephosphorylation. Pretreatment of PC12 cells with inhibitors of calcineurin (protein phosphatase 2B), cyclosporin A and FK506, prevented depolarization-induced nuclear translocation and tyrosine phosphorylation of PYK2. Transfection with dominant-negative and constitutively active calcineurin-A confirmed the role of calcineurin in the regulation of PYK2 tyrosine phosphorylation and nuclear accumulation. Our results show that depolarization independently induces nuclear translocation and tyrosine phosphorylation of PYK2, and that both responses require calcineurin activation. We suggest that PYK2 exerts some of its actions in the nucleus and that the effects of calcineurin inhibitors may involve PYK2 inhibition.


Assuntos
Calcineurina/metabolismo , Núcleo Celular/metabolismo , Quinase 2 de Adesão Focal/metabolismo , Neurônios/metabolismo , Tirosina/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Calcineurina/genética , Cálcio/metabolismo , Células Cultivadas , Estimulação Elétrica , Quinase 2 de Adesão Focal/genética , Hipocampo/citologia , Hipocampo/metabolismo , Masculino , Camundongos , Neurônios/citologia , Células PC12 , Fosforilação , Ratos , Ratos Sprague-Dawley
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