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1.
Gan To Kagaku Ryoho ; 50(12): 1307-1310, 2023 Dec.
Artigo em Japonês | MEDLINE | ID: mdl-38247069

RESUMO

Necitumumab enhances antitumor immunity by decreasing the PD-L1 expression; it is expected to improve the prognosis of patients treated with an immune checkpoint inhibitor(ICI)by inhibiting the IL-8 expression. Since the combined effect of necitumumab and PD-L1 inhibitor was confirmed in an in vivo study conducted in transgenic mice, further antitumor effects can be expected by the combined use of necitumumab and ICI.


Assuntos
Anticorpos Monoclonais Humanizados , Inibidores de Checkpoint Imunológico , Animais , Humanos , Camundongos , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Projetos de Pesquisa , Receptores ErbB/imunologia
2.
Mol Cancer Ther ; 19(4): 988-998, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32241872

RESUMO

The CD137 receptor plays a key role in mediating immune response by promoting T cell proliferation, survival, and memory. Effective agonism of CD137 has the potential to reinvigorate potent antitumor immunity either alone or in combination with other immune-checkpoint therapies. In this study, we describe the discovery and characterization of a unique CD137 agonist, 7A5, a fully human IgG1 Fc effector-null monoclonal antibody. The biological properties of 7A5 were investigated through in vitro and in vivo studies. 7A5 binds CD137, and the binding epitope overlaps with the CD137L binding site based on structure. 7A5 engages CD137 receptor and activates NF-κB cell signaling independent of cross-linking or Fc effector function. In addition, T cell activation measured by cytokine IFNγ production is induced by 7A5 in peripheral blood mononuclear cell costimulation assay. Human tumor xenograft mouse models reconstituted with human immune cells were used to determine antitumor activity in vivo. Monotherapy with 7A5 inhibits tumor growth, and this activity is enhanced in combination with a PD-L1 antagonist antibody. Furthermore, the intratumoral immune gene expression signature in response to 7A5 is highly suggestive of enhanced T cell infiltration and activation. Taken together, these results demonstrate 7A5 is a differentiated CD137 agonist antibody with biological properties that warrant its further development as a cancer immunotherapy. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/19/4/988/F1.large.jpg.


Assuntos
Anticorpos Monoclonais/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Ativação Linfocitária/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Apoptose , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Proliferação de Células , Feminino , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , NF-kappa B/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
3.
J Immunother Cancer ; 6(1): 45, 2018 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-29866166

RESUMO

Unfortunately, after publication of this article [1], it was noticed that corrections to the legends of Figs. 1 and 2 were not correctly incorporated. The correct legends can be seen below.

4.
J Immunother Cancer ; 6(1): 31, 2018 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-29712568

RESUMO

BACKGROUND: Modulation of the PD-1/PD-L1 axis through antagonist antibodies that block either receptor or ligand has been shown to reinvigorate the function of tumor-specific T cells and unleash potent anti-tumor immunity, leading to durable objective responses in a subset of patients across multiple tumor types. RESULTS: Here we describe the discovery and preclinical characterization of LY3300054, a fully human IgG1λ monoclonal antibody that binds to human PD-L1 with high affinity and inhibits interactions of PD-L1 with its two cognate receptors PD-1 and CD80. The functional activity of LY3300054 on primary human T cells is evaluated using a series of in vitro T cell functional assays and in vivo models using human-immune reconstituted mice. LY3300054 is shown to induce primary T cell activation in vitro, increase T cell activation in combination with anti-CTLA4 antibody, and to potently enhance anti-tumor alloreactivity in several xenograft mouse tumor models with reconstituted human immune cells. High-content molecular analysis of tumor and peripheral tissues from animals treated with LY3300054 reveals distinct adaptive immune activation signatures, and also previously not described modulation of innate immune pathways. CONCLUSIONS: LY3300054 is currently being evaluated in phase I clinical trials for oncology indications.


Assuntos
Anticorpos Monoclonais/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Imunoglobulina G/imunologia , Neoplasias/imunologia , Animais , Linhagem Celular , Cricetulus , Feminino , Humanos , Macaca fascicularis , Camundongos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
5.
Cell Rep ; 22(11): 2978-2994, 2018 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-29539425

RESUMO

Abemaciclib, an inhibitor of cyclin dependent kinases 4 and 6 (CDK4/6), has recently been approved for the treatment of hormone receptor-positive breast cancer. In this study, we use murine syngeneic tumor models and in vitro assays to investigate the impact of abemaciclib on T cells, the tumor immune microenvironment and the ability to combine with anti-PD-L1 blockade. Abemaciclib monotherapy resulted in tumor growth delay that was associated with an increased T cell inflammatory signature in tumors. Combination with anti-PD-L1 therapy led to complete tumor regressions and immunological memory, accompanied by enhanced antigen presentation, a T cell inflamed phenotype, and enhanced cell cycle control. In vitro, treatment with abemaciclib resulted in increased activation of human T cells and upregulated expression of antigen presentation genes in MCF-7 breast cancer cells. These data collectively support the clinical investigation of the combination of abemaciclib with agents such as anti-PD-L1 that modulate T cell anti-tumor immunity.


Assuntos
Aminopiridinas/uso terapêutico , Benzimidazóis/uso terapêutico , Inibidor de Quinase Dependente de Ciclina p15/uso terapêutico , Inibidor de Quinase Dependente de Ciclina p18/uso terapêutico , Receptor de Morte Celular Programada 1/metabolismo , Aminopiridinas/farmacologia , Benzimidazóis/farmacologia , Inibidor de Quinase Dependente de Ciclina p15/farmacologia , Inibidor de Quinase Dependente de Ciclina p18/farmacologia , Humanos , Microambiente Tumoral
6.
Mol Cancer Ther ; 17(2): 521-531, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29158469

RESUMO

Acquired resistance to cetuximab, an antibody that targets the EGFR, impacts clinical benefit in head and neck, and colorectal cancers. One of the mechanisms of resistance to cetuximab is the acquisition of mutations that map to the cetuximab epitope on EGFR and prevent drug binding. We find that necitumumab, another FDA-approved EGFR antibody, can bind to EGFR that harbors the most common cetuximab-resistant substitution, S468R (or S492R, depending on the amino acid numbering system). We determined an X-ray crystal structure to 2.8 Å resolution of the necitumumab Fab bound to an S468R variant of EGFR domain III. The arginine is accommodated in a large, preexisting cavity in the necitumumab paratope. We predict that this paratope shape will be permissive to other epitope substitutions, and show that necitumumab binds to most cetuximab- and panitumumab-resistant EGFR variants. We find that a simple computational approach can predict with high success which EGFR epitope substitutions abrogate antibody binding. This computational method will be valuable to determine whether necitumumab will bind to EGFR as new epitope resistance variants are identified. This method could also be useful for rapid evaluation of the effect on binding of alterations in other antibody/antigen interfaces. Together, these data suggest that necitumumab may be active in patients who are resistant to cetuximab or panitumumab through EGFR epitope mutation. Furthermore, our analysis leads us to speculate that antibodies with large paratope cavities may be less susceptible to resistance due to mutations mapping to the antigen epitope. Mol Cancer Ther; 17(2); 521-31. ©2017 AACR.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Cetuximab/uso terapêutico , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Cetuximab/farmacologia , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/metabolismo , Humanos
7.
Clin Cancer Res ; 23(18): 5547-5560, 2017 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-28611205

RESUMO

Purpose: To evaluate the antitumor efficacy of cetuximab in combination with LSN3074753, an analog of LY3009120 and pan-RAF inhibitor in 79 colorectal cancer patient-derived xenograft (PDX) models.Experimental Design: Seventy-nine well-characterized colorectal cancer PDX models were employed to conduct a single mouse per treatment group (n = 1) trial.Results: Consistent with clinical results, cetuximab was efficacious in wild-type KRAS and BRAF PDX models, with an overall response rate of 6.3% and disease control rate (DCR) of 20.3%. LSN3074753 was active in a small subset of PDX models that harbored KRAS or BRAF mutations. However, the combination treatment displayed the enhanced antitumor activity with DCR of 35.4%. Statistical analysis revealed that BRAF and KRAS mutations were the best predictors of the combinatorial activity and were significantly associated with synergistic effect with a P value of 0.01 compared with cetuximab alone. In 12 models with BRAF mutations, the combination therapy resulted in a DCR of 41.7%, whereas either monotherapy had a DCR of 8.3%. Among 44 KRAS mutation models, cetuximab or LSN3074753 monotherapy resulted in a DCR of 13.6% or 11.4%, respectively, and the combination therapy increased DCR to 34.1%. Molecular analysis suggests that EGFR activation is a potential feedback and resistant mechanism of pan-RAF inhibition.Conclusions: MAPK and EGFR pathway activations are two major molecular hallmarks of colorectal cancer. This mouse PDX trial recapitulated clinical results of cetuximab. Concurrent EGFR and RAF inhibition demonstrated synergistic antitumor activity for colorectal cancer PDX models with a KRAS or BRAF mutation. Clin Cancer Res; 23(18); 5547-60. ©2017 AACR.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Cetuximab/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Quimioterapia Combinada , Receptores ErbB/metabolismo , Humanos , Ligantes , Camundongos , Compostos de Fenilureia/farmacologia , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas B-raf/metabolismo , Pirimidinas/farmacologia , Taxa de Sobrevida , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
8.
J Thorac Oncol ; 12(2): 383-389, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27464970

RESUMO

INTRODUCTION: Type 1 insulin-like growth factor receptor is deregulated in solid tumors. Cixutumumab, a monoclonal antibody that inhibits the activity of type 1 insulin-like growth factor receptor, was investigated in combination with pemetrexed/cisplatin in the frontline setting. METHODS: In this open-label, phase II study, patients with stage IV nonsquamous NSCLC and a performance status of 0 to 1 were randomized (1:1) to receive 20 mg/kg cixutumumab, 500 mg/m2 pemetrexed, and 75 mg/m2 cisplatin (cixutumumab [n = 87]) or pemetrexed and cisplatin (control [n = 85]). Eligible patients received pemetrexed-based maintenance therapy with cixutumumab (cixutumumab arm) or without it (control arm). The primary end point was progression-free survival. Secondary end points assessed overall survival, objective response rate, and safety. Survival was analyzed by the Kaplan-Meier method and Cox proportional hazard model. Exploratory correlative analyses were also performed. RESULTS: The mean age of the intent-to-treat population (n = 172) was 59 years (range 32-83). Median progression-free survival was 5.45 months with cixutumumab versus 5.22 months in the control (hazard ratio = 1.15, 95% confidence interval: 0.81-1.61; p = 0.44). Median overall survival was 11.33 months with cixutumumab versus 10.38 months in the control (hazard ratio = 0.93, 95% confidence interval: 0.64-1.36). Objective response rate did not differ between treatments (p = 0.338). Grade 3 or 4 hyperglycemia occurred at a higher rate with cixutumumab than in the control (9.4% versus 1.2%). One death possibly related to cixutumumab occurred. CONCLUSIONS: Efficacy was not improved in patients with nonsquamous NSCLC when cixutumumab was added to pemetrexed/cisplatin. Combination therapy was well tolerated and no new safety concerns were reported.


Assuntos
Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Grandes/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Grandes/metabolismo , Carcinoma de Células Grandes/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Cisplatino/administração & dosagem , Feminino , Seguimentos , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Pemetrexede/administração & dosagem , Prognóstico , Taxa de Sobrevida
9.
Clin Cancer Res ; 22(2): 301-9, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26324738

RESUMO

PURPOSE: This phase II trial evaluated the efficacy and safety of cixutumumab, a human anti-insulin-like growth factor receptor 1 (IGF-1R) monoclonal IgG1 antibody, and explored potential biomarkers in postmenopausal women with hormone receptor-positive breast cancer. EXPERIMENTAL DESIGN: Patients with hormone receptor-positive breast cancer that progressed on antiestrogen therapy received (2:1 randomization) cixutumumab 10 mg/kg and the same antiestrogen (arm A) or cixutumumab alone (arm B) every 2 weeks (q2w). Primary endpoint was progression-free survival (PFS); secondary endpoints included overall survival (OS) and safety. Correlative analyses of IGF-1R, total insulin receptor (IR), and IR isoforms A (IR-A) and B (IR-B) expression in tumor tissue were explored. RESULTS: Ninety-three patients were randomized (arm A, n = 62; arm B, n = 31). Median PFS was 2.0 and 3.1 months for arm A and arm B, respectively. Secondary efficacy measures were similar between the arms. Overall, cixutumumab was well tolerated. IGF-1R expression was not associated with clinical outcomes. Regardless of the treatment, lower IR-A, IR-B, and total IR mRNA expression in tumor tissue was significantly associated with longer PFS [IR-A: HR, 2.62 (P = 0.0062); IR-B: HR, 2.21 (P = 0.0202); and total IR: HR, 2.18 (P = 0.0230)] and OS [IR-A: HR, 2.94 (P = 0.0156); IR-B: HR, 2.69 (P = 0.0245); and total IR: HR, 2.72 (P = 0.0231)]. CONCLUSIONS: Cixutumumab (10 mg/kg) with or without antiestrogen q2w had an acceptable safety profile, but no significant clinical efficacy. Patients with low total IR, IR-A, and IR-B mRNA expression levels had significantly longer PFS and OS, independent of the treatment. The prognostic or predictive value of IR as a biomarker for IGF-1R-targeted therapies requires further validation.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Moduladores de Receptor Estrogênico/uso terapêutico , Receptor IGF Tipo 1/antagonistas & inibidores , Adulto , Idoso , Idoso de 80 Anos ou mais , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais Humanizados , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Intervalo Livre de Doença , Feminino , Humanos , Pessoa de Meia-Idade , Pós-Menopausa/efeitos dos fármacos , Pós-Menopausa/metabolismo , Prognóstico , RNA Mensageiro/metabolismo
10.
Mol Cancer Res ; 13(12): 1615-26, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26263910

RESUMO

UNLABELLED: Despite a recent shift away from anti-insulin-like growth factor I receptor (IGF-IR) therapy, this target has been identified as a key player in the resistance mechanisms to various conventional and targeted agents, emphasizing its value as a therapy, provided that it is used in the right patient population. Molecular markers predictive of antitumor activity of IGF-IR inhibitors remain largely unidentified. The aim of this study is to evaluate the impact of insulin receptor (IR) isoforms on the antitumor efficacy of cixutumumab, a humanized mAb against IGF-IR, and to correlate their expression with therapeutic outcome. The data demonstrate that expression of total IR rather than individual IR isoforms inversely correlates with single-agent cixutumumab efficacy in pediatric solid tumor models in vivo. Total IR, IR-A, and IR-B expression adversely affects the outcome of cixutumumab in combination with chemotherapy in patient-derived xenograft models of lung adenocarcinoma. IR-A overexpression in tumor cells confers complete resistance to cixutumumab in vitro and in vivo, whereas IR-B results in a partial resistance. Resistance in IR-B-overexpressing cells is fully reversed by anti-IGF-II antibodies, suggesting that IGF-II is a driver of cixutumumab resistance in this setting. The present study links IR isoforms, IGF-II, and cixutumumab efficacy mechanistically and identifies total IR as a biomarker predictive of intrinsic resistance to anti-IGF-IR antibody. IMPLICATIONS: This study identifies total IR as a biomarker predictive of primary resistance to IGF-IR antibodies and provides a rationale for new clinical trials enriched for patients whose tumors display low IR expression.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Antígenos CD/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/genética , Receptor de Insulina/metabolismo , Anticorpos Monoclonais Humanizados , Antígenos CD/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Células MCF-7 , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor de Insulina/genética , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
11.
MAbs ; 7(5): 931-45, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26073904

RESUMO

Bi-specific antibodies (BsAbs), which can simultaneously block 2 tumor targets, have emerged as promising therapeutic alternatives to combinations of individual monoclonal antibodies. Here, we describe the engineering and development of a novel, human bi-functional antibody-receptor domain fusion molecule with ligand capture (bi-AbCap) through the fusion of the domain 2 of human vascular endothelial growth factor receptor 1 (VEGFR1) to an antibody directed against insulin-like growth factor - type I receptor (IGF-IR). The bi-AbCap possesses excellent stability and developability, and is the result of minimal engineering. Beyond potent neutralizing activities against IGF-IR and VEGF, the bi-AbCap is capable of cross-linking VEGF to IGF-IR, leading to co-internalization and degradation of both targets by tumor cells. In multiple mouse xenograft tumor models, the bi-AbCap improves anti-tumor activity over individual monotherapies. More importantly, it exhibits superior inhibition of tumor growth, compared with the combination of anti-IGF-IR and anti-VEGF therapies, via powerful blockade of both direct tumor cell growth and tumor angiogenesis. The unique "capture-for-degradation" mechanism of the bi-AbCap is informative for the design of next-generation bi-functional anti-cancer therapies directed against independent signaling pathways. The bi-AbCap design represents an alternative approach to the creation of dual-targeting antibody fusion molecules by taking advantage of natural receptor-ligand interactions.


Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Neutralizantes/farmacologia , Receptores de Somatomedina/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacologia , Afinidade de Anticorpos , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Immunoblotting , Imunoprecipitação , Camundongos , Camundongos Nus , Microscopia Confocal , Neoplasias Experimentais/tratamento farmacológico , Estabilidade Proteica , Receptor IGF Tipo 1 , Ressonância de Plasmônio de Superfície , Ensaios Antitumorais Modelo de Xenoenxerto
12.
J Virol ; 86(21): 11558-66, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22896607

RESUMO

Hepatitis C virus (HCV) infection remains a serious public health problem worldwide. Treatments are limited, and no preventive vaccine is available. Toward developing an HCV vaccine, we engineered two recombinant measles viruses (MVs) expressing structural proteins from the prototypic HCV subtype 1a strain H77. One virus directs the synthesis of the HCV capsid (C) protein and envelope glycoproteins (E1 and E2), which fold properly and form a heterodimer. The other virus expresses the E1 and E2 glycoproteins separately, with each one fused to the cytoplasmic tail of the MV fusion protein. Although these hybrid glycoproteins were transported to the plasma membrane, they were not incorporated into MV particles. Immunization of MV-susceptible, genetically modified mice with either vector induced neutralizing antibodies to MV and HCV. A boost with soluble E2 protein enhanced titers of neutralizing antibody against the homologous HCV envelope. In animals primed with MV expressing properly folded HCV C-E1-E2, boosting also induced cross-neutralizating antibodies against two heterologous HCV strains. These results show that recombinant MVs retain the ability to induce MV-specific humoral immunity while also eliciting HCV neutralizing antibodies, and that anti-HCV immunity can be boosted with a single dose of purified E2 protein. The use of MV vectors could have advantages for pediatric HCV vaccination.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Hepacivirus/imunologia , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Animais , Reações Cruzadas , Portadores de Fármacos/administração & dosagem , Vetores Genéticos , Hepacivirus/genética , Vírus do Sarampo/genética , Camundongos , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/genética , Vacinas Virais/administração & dosagem
13.
J Comp Neurol ; 518(20): 4243-60, 2010 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-20878786

RESUMO

In this study, we used electron tomography as well as immunogold labeling to analyze the morphology and distribution of proteins within postsynaptic densities (PSDs) isolated from rats before birth (embryonic day 19) and at postnatal days 2, 21, and 60. Our data provide direct evidence of distinct morphological and compositional differences in PSDs throughout development. Not all PSD components are present at the early stages of development, with a near lack of the scaffolding molecule PSD-95 at E19 and P2. The presence of NR1 and NR2b suggests that PSD-95 is not directly required for clustering of N-methyl-D-aspartic acid (NMDA) receptors in PSDs early in development. α-Actinin is abundant by E19, suggesting that it is a core structural component of the PSD. Both α and ß isoforms of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) are present early on but then rise in labeling density by approximately fourfold by P21. Among all the molecules studied, only calmodulin (CaM) was found in higher abundance early in PSD development and then fell in amount over time. Spatial analysis of the immunogold label shows a nonrandom distribution for all the proteins studied, lending support to the idea that the PSD is systematically assembled in an organized fashion. Morphological data from electron tomography shows that the PSD undergoes major structural changes throughout development.


Assuntos
Densidade Pós-Sináptica , Animais , Biomarcadores/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Proteína 4 Homóloga a Disks-Large , Tomografia com Microscopia Eletrônica/métodos , Feminino , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Densidade Pós-Sináptica/química , Densidade Pós-Sináptica/fisiologia , Densidade Pós-Sináptica/ultraestrutura , Ratos , Ratos Sprague-Dawley
14.
Biochemistry ; 47(40): 10587-99, 2008 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-18795794

RESUMO

Understanding the principles of calmodulin (CaM) activation of target enzymes will help delineate how this seemingly simple molecule can play such a complex role in transducing Ca (2+)-signals to a variety of downstream pathways. In the work reported here, we use biochemical and biophysical tools and a panel of CaM constructs to examine the lobe specific interactions between CaM and CaMKII necessary for the activation and autophosphorylation of the enzyme. Interestingly, the N-terminal lobe of CaM by itself was able to partially activate and allow autophosphorylation of CaMKII while the C-terminal lobe was inactive. When used together, CaMN and CaMC produced maximal CaMKII activation and autophosphorylation. Moreover, CaMNN and CaMCC (chimeras of the two N- or C-terminal lobes) both activated the kinase but with greater K act than for wtCaM. Isothermal titration calorimetry experiments showed the same rank order of affinities of wtCaM > CaMNN > CaMCC as those determined in the activity assay and that the CaM to CaMKII subunit binding ratio was 1:1. Together, our results lead to a proposed sequential mechanism to describe the activation pathway of CaMKII led by binding of the N-lobe followed by the C-lobe. This mechanism contrasts the typical sequential binding mode of CaM with other CaM-dependent enzymes, where the C-lobe of CaM binds first. The consequence of such lobe specific binding mechanisms is discussed in relation to the differential rates of Ca (2+)-binding to each lobe of CaM during intracellular Ca (2+) oscillations.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Calmodulina/metabolismo , Difosfato de Adenosina/farmacologia , Animais , Sítios de Ligação/genética , Cálcio/metabolismo , Calmodulina/química , Calmodulina/genética , Calorimetria , Fluorometria , Modelos Moleculares , Nucleotídeos/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Ratos , Temperatura
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