Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 12 de 12
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
FASEB J ; 37(6): e22995, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37219526

RESUMO

Immuno-oncology (IO)-based therapies such as checkpoint inhibitors, bi-specific antibodies, and CAR-T-cell therapies have shown significant success in the treatment of several cancer indications. However, these therapies can result in the development of severe adverse events, including cytokine release syndrome (CRS). Currently, there is a paucity of in vivo models that can evaluate dose-response relationships for both tumor control and CRS-related safety issues. We tested an in vivo PBMC humanized mouse model to assess both treatment efficacy against specific tumors and the concurrent cytokine release profiles for individual human donors after treatment with a CD19xCD3 bispecific T-cell engager (BiTE). Using this model, we evaluated tumor burden, T-cell activation, and cytokine release in response to bispecific T-cell-engaging antibody in humanized mice generated with different PBMC donors. The results show that PBMC engrafted NOD-scid Il2rgnull mice lacking expression of mouse MHC class I and II (NSG-MHC-DKO mice) and implanted with a tumor xenograft predict both efficacy for tumor control by CD19xCD3 BiTE and stimulated cytokine release. Moreover, our findings indicate that this PBMC-engrafted model captures variability among donors for tumor control and cytokine release following treatment. Tumor control and cytokine release were reproducible for the same PBMC donor in separate experiments. The PBMC humanized mouse model described here is a sensitive and reproducible platform that identifies specific patient/cancer/therapy combinations for treatment efficacy and development of complications.


Assuntos
Leucócitos Mononucleares , Linfócitos T , Humanos , Animais , Camundongos , Camundongos Endogâmicos NOD , Resultado do Tratamento , Síndrome da Liberação de Citocina , Citocinas , Modelos Animais de Doenças , Camundongos Knockout , Camundongos SCID
2.
J Biol Chem ; 295(10): 3269-3284, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32005669

RESUMO

Nuclear accumulation of the small phosphoprotein integrin cytoplasmic domain-associated protein-1 (ICAP1) results in recruitment of its binding partner, Krev/Rap1 interaction trapped-1 (KRIT1), to the nucleus. KRIT1 loss is the most common cause of cerebral cavernous malformation, a neurovascular dysplasia resulting in dilated, thin-walled vessels that tend to rupture, increasing the risk for hemorrhagic stroke. KRIT1's nuclear roles are unknown, but it is known to function as a scaffolding or adaptor protein at cell-cell junctions and in the cytosol, supporting normal blood vessel integrity and development. As ICAP1 controls KRIT1 subcellular localization, presumably influencing KRIT1 function, in this work, we investigated the signals that regulate ICAP1 and, hence, KRIT1 nuclear localization. ICAP1 contains a nuclear localization signal within an unstructured, N-terminal region that is rich in serine and threonine residues, several of which are reportedly phosphorylated. Using quantitative microscopy, we revealed that phosphorylation-mimicking substitutions at Ser-10, or to a lesser extent at Ser-25, within this N-terminal region inhibit ICAP1 nuclear accumulation. Conversely, phosphorylation-blocking substitutions at these sites enhanced ICAP1 nuclear accumulation. We further demonstrate that p21-activated kinase 4 (PAK4) can phosphorylate ICAP1 at Ser-10 both in vitro and in cultured cells and that active PAK4 inhibits ICAP1 nuclear accumulation in a Ser-10-dependent manner. Finally, we show that ICAP1 phosphorylation controls nuclear localization of the ICAP1-KRIT1 complex. We conclude that serine phosphorylation within the ICAP1 N-terminal region can prevent nuclear ICAP1 accumulation, providing a mechanism that regulates KRIT1 localization and signaling, potentially influencing vascular development.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/metabolismo , Serina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Animais , Células CHO , Domínio Catalítico , Cricetinae , Cricetulus , Humanos , Proteína KRIT1/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Quinases Ativadas por p21/química , Quinases Ativadas por p21/metabolismo
3.
J Biol Chem ; 292(5): 1884-1898, 2017 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-28003363

RESUMO

Binding of ICAP1 (integrin cytoplasmic domain-associated protein-1) to the cytoplasmic tails of ß1 integrins inhibits integrin activation. ICAP1 also binds to KRIT1 (Krev interaction trapped-1), a protein whose loss of function leads to cerebral cavernous malformation, a cerebrovascular dysplasia occurring in up to 0.5% of the population. We previously showed that KRIT1 functions as a switch for ß1 integrin activation by antagonizing ICAP1-mediated inhibition of integrin activation. Here we use overexpression studies, mutagenesis, and flow cytometry to show that ICAP1 contains a functional nuclear localization signal and that nuclear localization impairs the ability of ICAP1 to suppress integrin activation. Moreover, we find that ICAP1 drives the nuclear localization of KRIT1 in a manner dependent upon a direct ICAP1/KRIT1 interaction. Thus, nuclear-cytoplasmic shuttling of ICAP1 influences both integrin activation and KRIT1 localization, presumably impacting nuclear functions of KRIT1.


Assuntos
Núcleo Celular/metabolismo , Integrina beta1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Células CHO , Núcleo Celular/genética , Cricetinae , Cricetulus , Células HEK293 , Humanos , Integrina beta1/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteína KRIT1 , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Proto-Oncogênicas/genética
4.
J Cell Biol ; 208(7): 987-1001, 2015 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-25825518

RESUMO

Mutations in the essential adaptor proteins CCM2 or CCM3 lead to cerebral cavernous malformations (CCM), vascular lesions that most frequently occur in the brain and are strongly associated with hemorrhagic stroke, seizures, and other neurological disorders. CCM2 binds CCM3, but the molecular basis of this interaction, and its functional significance, have not been elucidated. Here, we used x-ray crystallography and structure-guided mutagenesis to show that an α-helical LD-like motif within CCM2 binds the highly conserved "HP1" pocket of the CCM3 focal adhesion targeting (FAT) homology domain. By knocking down CCM2 or CCM3 and rescuing with binding-deficient mutants, we establish that CCM2-CCM3 interactions protect CCM2 and CCM3 proteins from proteasomal degradation and show that both CCM2 and CCM3 are required for normal endothelial cell network formation. However, CCM3 expression in the absence of CCM2 is sufficient to support normal cell growth, revealing complex-independent roles for CCM3.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Transporte/metabolismo , Proliferação de Células/genética , Sistema Nervoso Central/irrigação sanguínea , Proteínas de Membrana/metabolismo , Neovascularização Fisiológica/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/ultraestrutura , Sítios de Ligação , Proteínas de Transporte/genética , Proteínas de Transporte/ultraestrutura , Linhagem Celular , Cristalografia por Raios X , Expressão Gênica , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/ultraestrutura , Mutagênese , Paxilina/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Proteólise , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/ultraestrutura , Interferência de RNA , RNA Interferente Pequeno , Alinhamento de Sequência
5.
J Cell Sci ; 127(Pt 4): 701-7, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24481819

RESUMO

Loss-of-function mutations in genes encoding KRIT1 (also known as CCM1), CCM2 (also known as OSM and malcavernin) or PDCD10 (also known as CCM3) cause cerebral cavernous malformations (CCMs). These abnormalities are characterized by dilated leaky blood vessels, especially in the neurovasculature, that result in increased risk of stroke, focal neurological defects and seizures. The three CCM proteins can exist in a trimeric complex, and each of these essential multi-domain adaptor proteins also interacts with a range of signaling, cytoskeletal and adaptor proteins, presumably accounting for their roles in a range of basic cellular processes including cell adhesion, migration, polarity and apoptosis. In this Cell Science at a Glance article and the accompanying poster, we provide an overview of current models of CCM protein function focusing on how known protein-protein interactions might contribute to cellular phenotypes and highlighting gaps in our current understanding.


Assuntos
Neoplasias do Sistema Nervoso Central/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Permeabilidade Capilar , Proteínas de Transporte/metabolismo , Neoplasias do Sistema Nervoso Central/irrigação sanguínea , Hemangioma Cavernoso do Sistema Nervoso Central/irrigação sanguínea , Humanos , Proteína KRIT1 , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo
6.
Mol Cell ; 49(4): 719-29, 2013 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-23317506

RESUMO

KRIT1 (Krev/Rap1 Interaction Trapped-1) mutations are observed in ∼40% of autosomal-dominant cerebral cavernous malformations (CCMs), a disease occurring in up to 0.5% of the population. We show that KRIT1 functions as a switch for ß1 integrin activation by antagonizing ICAP1 (Integrin Cytoplasmic Associated Protein-1)-mediated modulation of "inside-out" activation. We present cocrystal structures of KRIT1 with ICAP1 and ICAP1 with integrin ß1 cytoplasmic tail to 2.54 and 3.0 Å resolution (the resolutions at which I/σI = 2 are 2.75 and 3.0 Å, respectively). We find that KRIT1 binds ICAP1 by a bidentate surface, that KRIT1 directly competes with integrin ß1 to bind ICAP1, and that KRIT1 antagonizes ICAP1-modulated integrin activation using this site. We also find that KRIT1 contains an N-terminal Nudix domain, in a region previously designated as unstructured. We therefore provide insights to integrin regulation and CCM-associated KRIT1 function.


Assuntos
Integrina beta1/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Membrana/química , Proteínas Associadas aos Microtúbulos/química , Proteínas Proto-Oncogênicas/química , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular Tumoral , Sequência Conservada , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Integrina beta1/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína KRIT1 , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais
7.
J Biol Chem ; 287(39): 32566-77, 2012 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-22869380

RESUMO

ß-Parvin is a cytoplasmic adaptor protein that localizes to focal adhesions where it interacts with integrin-linked kinase and is involved in linking integrin receptors to the cytoskeleton. It has been reported that despite high sequence similarity to α-parvin, ß-parvin does not bind paxillin, suggesting distinct interactions and cellular functions for these two closely related parvins. Here, we reveal that ß-parvin binds directly and specifically to leucine-aspartic acid repeat (LD) motifs in paxillin via its C-terminal calponin homology (CH2) domain. We present the co-crystal structure of ß-parvin CH2 domain in complex with paxillin LD1 motif to 2.9 Å resolution and find that the interaction is similar to that previously observed between α-parvin and paxillin LD1. We also present crystal structures of unbound ß-parvin CH2 domain at 2.1 Å and 2.0 Å resolution that show significant conformational flexibility in the N-terminal α-helix, suggesting an induced fit upon paxillin binding. We find that ß-parvin has specificity for the LD1, LD2, and LD4 motifs of paxillin, with K(D) values determined to 27, 42, and 73 µM, respectively, by surface plasmon resonance. Furthermore, we show that proper localization of ß-parvin to focal adhesions requires both the paxillin and integrin-linked kinase binding sites and that paxillin is important for early targeting of ß-parvin. These studies provide the first molecular details of ß-parvin binding to paxillin and help define the requirements for ß-parvin localization to focal adhesions.


Assuntos
Actinina/química , Adesões Focais/química , Paxilina/química , Actinina/genética , Actinina/metabolismo , Motivos de Aminoácidos , Cristalografia por Raios X , Adesões Focais/genética , Adesões Focais/metabolismo , Humanos , Paxilina/genética , Paxilina/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Sequências Repetitivas de Aminoácidos , Ressonância de Plasmônio de Superfície
8.
J Biol Chem ; 287(26): 22317-27, 2012 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-22577140

RESUMO

Cerebral cavernous malformations (CCMs) affect 0.1-0.5% of the population resulting in leaky vasculature and severe neurological defects. KRIT1 (Krev interaction trapped-1) mutations associate with ∼40% of familial CCMs. KRIT1 is an effector of Ras-related protein 1 (Rap1) GTPase. Rap1 relocalizes KRIT1 from microtubules to cell membranes to impact integrin activation, potentially important for CCM pathology. We report the 1.95 Šco-crystal structure of KRIT1 FERM domain in complex with Rap1. Rap1-KRIT1 interaction encompasses an extended surface, including Rap1 Switch I and II and KRIT1 FERM F1 and F2 lobes. Rap1 binds KRIT1-F1 lobe using a GTPase-ubiquitin-like fold interaction but binds KRIT1-F2 lobe by a novel interaction. Point mutagenesis confirms the interaction. High similarity between KRIT1-F2/F3 and talin is revealed. Additionally, the mechanism for FERM domains acting as GTPase effectors is suggested. Finally, structure-based alignment of each lobe suggests classification of FERM domains as ERM-like and TMFK-like (talin-myosin-FAK-KRIT-like) and that FERM lobes resemble domain "modules."


Assuntos
Regulação da Expressão Gênica , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X/métodos , GTP Fosfo-Hidrolases/metabolismo , Hemangioma Cavernoso do Sistema Nervoso Central/metabolismo , Humanos , Integrinas/metabolismo , Proteína KRIT1 , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Mutação Puntual , Conformação Proteica , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Transdução de Sinais
9.
Methods Mol Biol ; 750: 261-74, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21618097

RESUMO

It is likely that adult epithelial stem cells will be useful in the treatment of diseases, such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa, and alopecias. Additionally, other skin problems such as burn wounds, chronic wounds, and ulcers will benefit from stem cell-related therapies. However, there are many questions that need to be answered before this goal can be realized. The most important of these questions is what regulates the adhesion of stem cells to the niche versus migration to the site of injury. We have started to identify the mechanisms involved in this decision-making process.


Assuntos
Células-Tronco Adultas/citologia , Diferenciação Celular/fisiologia , Movimento Celular , Células Epiteliais/citologia , Folículo Piloso/citologia , Queratinócitos/citologia , Nicho de Células-Tronco/citologia , Adulto , Células-Tronco Adultas/fisiologia , Células-Tronco Adultas/transplante , Animais , Adesão Celular , Células Epiteliais/fisiologia , Células Epiteliais/transplante , Citometria de Fluxo , Folículo Piloso/fisiologia , Humanos , Imuno-Histoquímica , Queratinócitos/fisiologia , Camundongos , Dermatopatias/terapia , Nicho de Células-Tronco/fisiologia , Transplante de Células-Tronco/métodos , Imagem com Lapso de Tempo
10.
Blood ; 113(24): 6172-81, 2009 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-19246562

RESUMO

Mutations in NOTCH1 are frequently detected in patients with T-cell acute lymphoblastic leukemia (T-ALL) and in mouse T-ALL models. Treatment of mouse or human T-ALL cell lines in vitro with gamma-secretase inhibitors (GSIs) results in growth arrest and/or apoptosis. These studies suggest GSIs as potential therapeutic agents in the treatment of T-ALL. To determine whether GSIs have antileukemic activity in vivo, we treated near-end-stage Tal1/Ink4a/Arf+/- leukemic mice with vehicle or with a GSI developed by Merck (MRK-003). We found that GSI treatment significantly extended the survival of leukemic mice compared with vehicle-treated mice. Notch1 target gene expression was repressed and increased numbers of apoptotic cells were observed in the GSI-treated mice, demonstrating that Notch1 inhibition in vivo induces apoptosis. T-ALL cell lines also exhibit PI3K/mTOR pathway activation, indicating that rapamycin may also have therapeutic benefit. When GSIs are administered in combination with rapamycin, mTOR kinase activity is ablated and apoptosis induced. Moreover, GSI and rapamycin treatment inhibits human T-ALL growth and extends survival in a mouse xenograft model. This work supports the idea of targeting NOTCH1 in T-ALL and suggests that inhibition of the mTOR and NOTCH1 pathways may have added efficacy.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Óxidos S-Cíclicos/farmacologia , Modelos Animais de Doenças , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptor Notch1/metabolismo , Tiadiazóis/farmacologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Western Blotting , Proteínas de Transporte/genética , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/fisiologia , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Transgênicos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Proto-Oncogênicas/fisiologia , Receptor Notch1/genética , Transdução de Sinais , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Serina-Treonina Quinases TOR , Células Tumorais Cultivadas
11.
Cell Cycle ; 6(8): 927-30, 2007 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-17404512

RESUMO

The Notch receptor family and its ligands (Delta-like and Jagged) have been found deregulated in several human cancers. We and the Aster/Pear group recently identified c-myc as a direct transcriptional target gene of the Notch1 pathway in T cell acute lymphoblastic leukemia (T-ALL). Although the oncogenic roles of c-Myc and Notch1 are established, a direct link between Notch1 and c-Myc had not been demonstrated. Importantly, our work in mouse tal1 tumor cell lines revealed that leukemic growth/survival remains dependent on the Notch1-c-Myc pathway. Studies by the Efstratiadis group provide genetic evidence that the Notch1-c-Myc pathway also contributes to mouse mammary tumorigenesis. Taken together, these studies demonstrate that Notch1 mediates T cell and epithelial cell transformation at least in part by sustaining c-Myc lev.


Assuntos
Leucemia de Células T/genética , Proteínas Proto-Oncogênicas c-myc/fisiologia , Receptor Notch1/fisiologia , Animais , Redes Reguladoras de Genes , Humanos , Modelos Biológicos , Oncogenes/fisiologia , Proteínas Proto-Oncogênicas c-myc/genética , Receptor Notch1/genética , Transdução de Sinais
12.
Mol Cell Biol ; 26(21): 8022-31, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16954387

RESUMO

Recent work with mouse models and human leukemic samples has shown that gain-of-function mutation(s) in Notch1 is a common genetic event in T-cell acute lymphoblastic leukemia (T-ALL). The Notch1 receptor signals through a gamma-secretase-dependent process that releases intracellular Notch1 from the membrane to the nucleus, where it forms part of a transcriptional activator complex. To identify Notch1 target genes in leukemia, we developed mouse T-cell leukemic lines that express intracellular Notch1 in a doxycycline-dependent manner. Using gene expression profiling and chromatin immunoprecipitation, we identified c-myc as a novel, direct, and critical Notch1 target gene in T-cell leukemia. c-myc mRNA levels are increased in primary mouse T-cell tumors that harbor Notch1 mutations, and Notch1 inhibition decreases c-myc mRNA levels and inhibits leukemic cell growth. Retroviral expression of c-myc, like intracellular Notch1, rescues the growth arrest and apoptosis associated with gamma-secretase inhibitor treatment or Notch1 inhibition. Consistent with these findings, retroviral insertional mutagenesis screening of our T-cell leukemia mouse model revealed common insertions in either notch1 or c-myc genes. These studies define the Notch1 molecular signature in mouse T-ALL and importantly provide mechanistic insight as to how Notch1 contributes to human T-ALL.


Assuntos
Regulação Neoplásica da Expressão Gênica , Leucemia de Células T/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Receptor Notch1/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Animais , Apoptose/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/metabolismo , Perfilação da Expressão Gênica , Humanos , Camundongos , Mutagênese Insercional , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Receptor Notch1/genética , Retroviridae/genética , Retroviridae/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...