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1.
EMBO J ; 40(22): e108125, 2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34617326

RESUMO

Mutations in VAV1, a gene that encodes a multifunctional protein important for lymphocytes, are found at different frequencies in peripheral T-cell lymphoma (PTCL), non-small cell lung cancer, and other tumors. However, their pathobiological significance remains unsettled. After cataloguing 51 cancer-associated VAV1 mutations, we show here that they can be classified in five subtypes according to functional impact on the three main VAV1 signaling branches, GEF-dependent activation of RAC1, GEF-independent adaptor-like, and tumor suppressor functions. These mutations target new and previously established regulatory layers of the protein, leading to quantitative and qualitative changes in VAV1 signaling output. We also demonstrate that the most frequent VAV1 mutant subtype drives PTCL formation in mice. This process requires the concurrent engagement of two downstream signaling branches that promote the chronic activation and transformation of follicular helper T cells. Collectively, these data reveal the genetic constraints associated with the lymphomagenic potential of VAV1 mutant subsets, similarities with other PTCL driver genes, and potential therapeutic vulnerabilities.


Assuntos
Linfócitos T CD4-Positivos/patologia , Linfoma de Células T Periférico/genética , Mutação , Proteínas Proto-Oncogênicas c-vav/genética , Animais , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD4-Positivos/transplante , Células COS , Proliferação de Células/genética , Chlorocebus aethiops , Humanos , Células Jurkat , Linfoma de Células T Periférico/patologia , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-vav/química , Proteínas Proto-Oncogênicas c-vav/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
2.
Proc Natl Acad Sci U S A ; 114(4): 764-769, 2017 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-28062691

RESUMO

Peripheral T-cell lymphomas (PTCLs) are a heterogeneous group of non-Hodgkin lymphomas frequently associated with poor prognosis and for which genetic mechanisms of transformation remain incompletely understood. Using RNA sequencing and targeted sequencing, here we identify a recurrent in-frame deletion (VAV1 Δ778-786) generated by a focal deletion-driven alternative splicing mechanism as well as novel VAV1 gene fusions (VAV1-THAP4, VAV1-MYO1F, and VAV1-S100A7) in PTCL. Mechanistically these genetic lesions result in increased activation of VAV1 catalytic-dependent (MAPK, JNK) and non-catalytic-dependent (nuclear factor of activated T cells, NFAT) VAV1 effector pathways. These results support a driver oncogenic role for VAV1 signaling in the pathogenesis of PTCL.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Guanina/metabolismo , Linfoma de Células T Periférico/genética , Mutação/genética , Proteínas Proto-Oncogênicas c-vav/genética , Translocação Genética/genética , Processamento Alternativo/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Humanos , Células Jurkat , Deleção de Sequência/genética
3.
Eur J Immunol ; 46(10): 2376-2387, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27405273

RESUMO

Lymphocyte migration, which is essential for effective immune responses, belongs to the so-called amoeboid migration. The lymphocyte migration is up to 100 times faster than between mesenchymal and epithelial cell types. Migrating lymphocytes are highly polarized in three well-defined structural and functional zones: uropod, medial zone, and leading edge (LE). The actiomyosin-dependent driving force moves forward the uropod, whereas massive actin rearrangements protruding the cell membrane are observed at the LE. These actin rearrangements resemble those observed at the immunological synapse driven by clathrin, a protein normally involved in endocytic processes. Here, we used cell lines as well as primary lymphocytes to demonstrate that clathrin and clathrin adaptors colocalize with actin at the LE of migrating lymphocytes, but not in other cellular zones that accumulate both clathrin and actin. Moreover, clathrin and clathrin adaptors, including Hrs, the clathrin adaptor for multivesicular bodies, drive local actin accumulation at the LE. Clathrin recruitment at the LE resulted necessary for a complete cell polarization and further lymphocyte migration in both 2D and 3D migration models. Therefore, clathrin, including the clathrin population associated to internal vesicles, controls lymphocyte migration by regulating actin rearrangements occurring at the LE.


Assuntos
Actinas/metabolismo , Movimento Celular , Clatrina/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Fosfoproteínas/metabolismo , Linfócitos T/fisiologia , Movimento Celular/genética , Polaridade Celular , Clatrina/genética , Humanos , Sinapses Imunológicas , Células Jurkat , Transporte Proteico , RNA Interferente Pequeno/genética , Vesículas Transportadoras/metabolismo
4.
J Cell Sci ; 124(Pt 5): 820-30, 2011 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-21321329

RESUMO

Antigen-specific cognate interaction of T lymphocytes with antigen-presenting cells (APCs) drives major morphological and functional changes in T cells, including actin rearrangements at the immune synapse (IS) formed at the cell-cell contact area. Here we show, using cell lines as well as primary cells, that clathrin, a protein involved in endocytic processes, drives actin accumulation at the IS. Clathrin is recruited towards the IS with parallel kinetics to that of actin. Knockdown of clathrin prevents accumulation of actin and proteins involved in actin polymerization, such as dynamin-2, the Arp2/3 complex and CD2AP at the IS. The clathrin pool involved in actin accumulation at the IS is linked to multivesicular bodies that polarize to the cell-cell contact zone, but not to plasma membrane or Golgi complex. These data underscore the role of clathrin as a platform for the recruitment of proteins that promote actin polymerization at the interface of T cells and APCs.


Assuntos
Actinas/metabolismo , Clatrina/metabolismo , Endossomos/metabolismo , Sinapses Imunológicas/metabolismo , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Polaridade Celular , Células Cultivadas , Clatrina/genética , Dinamina II/genética , Dinamina II/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Sinapses Imunológicas/ultraestrutura , Células Jurkat , Fosfoproteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologia
5.
Blood ; 115(21): 4174-84, 2010 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-20231423

RESUMO

During antigen recognition by T cells, membrane receptors and cytoskeletal molecules form a specialized structure at the T cell-antigen-presenting cell junction called the immune synapse (IS). We report a role for the scaffolding protein A-kinase anchoring protein-450 (AKAP450), a member of the A-kinase anchoring protein family, in IS formation and T-cell signaling in antigen- and superantigen-dependent T-cell activation. Suppression of AKAP450 by overexpression of a dominant-negative form or siRNA knockdown disrupted the positioning and conformational activation of lymphocyte function-associated antigen 1 at the IS and impaired associated signaling events, including phosphorylation of phospholipase C-gamma1 and protein kinase C-. AKAP450 was also required for correct activation and phosphorylation of CD3, LAT, and Vav1, key T-cell receptor-activated intracellular signaling molecules. Consistently, antigen-triggered reorientation of the microtubule-organizing center at the IS and interleukin-2 secretion were diminished in AKAP450-disrupted T cells. These results indicate key roles for AKAP450 in the organization and activation of receptor molecules at the IS during T-cell signaling events.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Complexo CD3/metabolismo , Proteínas do Citoesqueleto/metabolismo , Integrinas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas de Ancoragem à Quinase A/antagonistas & inibidores , Proteínas de Ancoragem à Quinase A/genética , Apresentação de Antígeno , Linfócitos B/imunologia , Linfócitos B/metabolismo , Sequência de Bases , Linhagem Celular , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Humanos , Sinapses Imunológicas/imunologia , Sinapses Imunológicas/metabolismo , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/metabolismo , Centro Organizador dos Microtúbulos/imunologia , Centro Organizador dos Microtúbulos/metabolismo , RNA Interferente Pequeno/genética , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo
6.
Mol Oncol ; 16(19): 3533-3553, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35895495

RESUMO

Mutations in the VAV1 guanine nucleotide exchange factor 1 have been recently found in peripheral T cell lymphoma and nonsmall-cell lung cancer (NSCLC). To understand their pathogenic potential, we generated a gene-edited mouse model that expresses a VAV1 mutant protein that recapitulates the signalling alterations present in the VAV1 mutant subclass most frequently found in tumours. We could not detect any overt tumourigenic process in those mice. However, the concurrent elimination of the Trp53 tumour suppressor gene in them drives T cell lymphomagenesis. This process represents an exacerbation of the normal functions that wild-type VAV1 plays in follicular helper T cells. We also found that, in combination with the Kras oncogene, the VAV1 mutant version favours progression of NSCLC. These data indicate that VAV1 mutations play critical, although highly cell-type-specific, roles in tumourigenesis. They also indicate that such functions are contingent on the mutational landscape of the tumours involved.


Assuntos
Neoplasias , Proteínas Proto-Oncogênicas c-vav , Animais , Edição de Genes , Camundongos , Proteínas Mutantes/metabolismo , Mutação/genética , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
7.
Cell Rep ; 38(11): 110522, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-35294890

RESUMO

A missense change in RRAS2 (Gln72 to Leu), analogous to the Gln61-to-Leu mutation of RAS oncoproteins, has been identified as a long-tail hotspot mutation in cancer and Noonan syndrome. However, the relevance of this mutation for in vivo tumorigenesis remains understudied. Here we show, using an inducible knockin mouse model, that R-Ras2Q72L triggers rapid development of a wide spectrum of tumors when somatically expressed in adult tissues. These tumors show limited overlap with those originated by classical Ras oncogenes. R-Ras2Q72L-driven tumors can be classified into different subtypes according to therapeutic susceptibility. Importantly, the most relevant R-Ras2Q72L-driven tumors are dependent on mTORC1 but independent of phosphatidylinositol 3-kinase-, MEK-, and Ral guanosine diphosphate (GDP) dissociation stimulator. This pharmacological vulnerability is due to the extensive rewiring by R-Ras2Q72L of pathways that orthogonally stimulate mTORC1 signaling. These findings demonstrate that RRAS2Q72L is a bona fide oncogenic driver and unveil therapeutic strategies for patients with cancer and Noonan syndrome bearing RRAS2 mutations.


Assuntos
Proteínas Monoméricas de Ligação ao GTP , Síndrome de Noonan , Animais , Carcinogênese/genética , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina , Proteínas de Membrana , Camundongos , Proteínas Monoméricas de Ligação ao GTP/genética , Mutação/genética , Oncogenes
8.
Biology (Basel) ; 10(9)2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34571765

RESUMO

Guanosine nucleotide exchange factors (GEFs) are responsible for catalyzing the transition of small GTPases from the inactive (GDP-bound) to the active (GTP-bound) states. RHO GEFs, including VAV proteins, play essential signaling roles in a wide variety of fundamental cellular processes and in human diseases. Although the most widespread archetype in the field is that RHO GEFs exert proactive functions in cancer, recent studies in mice and humans are providing new insights into the in vivo function of these proteins in cancer. These results suggest a more complex scenario where the role of GEFs is not so clearly defined. For example, VAV1 can unexpectedly play non-catalytic tumor suppressor functions in T-cell acute lymphoblastic leukemia (T-ALL) by controlling the levels of the active form of NOTCH1 (ICN1). This review focuses on emerging work unveiling tumor suppressor roles for these proteins that should prompt a reevaluation of the role of VAV GEF family in tumor biology.

9.
Biomedicines ; 9(6)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205377

RESUMO

Genetic evidence suggests that three members of the VAV family (VAV1, VAV2 and VAV3) of signal transduction proteins could play important roles in rheumatoid arthritis. However, it is not known currently whether the inhibition of these proteins protects against this disease and, if so, the number of family members that must be eliminated to get a therapeutic impact. To address this issue, we have used a collection of single and compound Vav family knockout mice in experimental models for antigen-dependent (methylated bovine serum albumin injections) and neutrophil-dependent (Zymosan A injections) rheumatoid arthritis in mice. We show here that the specific elimination of Vav1 is sufficient to block the development of antigen-induced arthritis. This protection is likely associated with the roles of this Vav family member in the development and selection of immature T cells within the thymus as well as in the subsequent proliferation and differentiation of effector T cells. By contrast, we have found that depletion of Vav2 reduces the number of neutrophils present in the joints of Zymosan A-treated mice. Despite this, the elimination of Vav2 does not protect against the joint degeneration triggered by this experimental model. These findings indicate that Vav1 is the most important pharmacological target within this family, although its main role is limited to the protection against antigen-induced rheumatoid arthritis. They also indicate that the three Vav family proteins do not play redundant roles in these pathobiological processes.

10.
Small GTPases ; 11(4): 233-239, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-29313423

RESUMO

Rho GDP/GTP exchange factors (GEFs), the enzymes that trigger the stimulation of Rho GTPases during cell signaling, are widely deemed as potential therapeutic targets owing to their protumorigenic functions. However, the sparse use of animal models has precluded a full understanding of their pathophysiological roles at the organismal level. In a recent article in Cancer Cell, we have reported that the Vav1 GEF unexpectedly acts as a tumor suppressor by mediating the noncatalytic nucleation of cytoplasmic complexes between the E3 ubiquitin ligase Cbl-b and the active Notch1 intracellular domain (ICN1). These complexes favor the ubiquitinylation-mediated degradation of ICN1 in the proteosome and, therefore, the dampening of ICN1 signals in cells. The elimination of Vav1 in mice exacerbates ICN1 signaling in specific thymocyte subpopulations and, in collaboration with ancillary mutations, prompts the development of ICN1-driven T cell acute lymphoblastic leukemia (T-ALL). This new Vav1-dependent pathway antagonizes the fitness of T-ALL of the TLX+ clinical subtype in humans. As a result, VAV1 is found recurrently silenced in both TLX+ T-ALL cell lines and patients. These results call for an overall reevaluation of Rho GEF function in cancer.


Assuntos
Guanosina/metabolismo , Neoplasias/metabolismo , Nucleotídeos/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Humanos , Neoplasias/patologia
11.
Front Cell Dev Biol ; 8: 240, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32351958

RESUMO

In the last years, the development of new drugs in oncology has evolved notably. In particular, drug development has shifted from empirical screening of active cytotoxic compounds to molecularly targeted drugs blocking specific biologic pathways that drive cancer progression and metastasis. Using a rational design approach, our group has developed 1A-116 as a promising Rac1 inhibitor, with antitumoral and antimetastatic effects in several types of cancer. Rac1 is over activated in a wide range of tumor types and and it is one of the most studied proteins of the Rho GTPase family. Its role in actin cytoskeleton reorganization has effects on endocytosis, vesicular trafficking, cell cycle progression and cellular migration. In this context, the regulatory activity of Rac1 affects several key processes in the course of the cancer including invasion and metastasis. The purpose of this preclinical study was to focus on the mode of action of 1A-116, conducting an interdisciplinary approach with in silico bioinformatics tools and in vitro assays. Here, we demonstrate that the tryptophan 56 residue is necessary for the inhibitory effects of 1A-116 since this compound interferes with protein-protein interactions (PPI) of Rac1GTPase involving several GEF activators. 1A-116 is also able to inhibit the oncogenic Rac1P29S mutant protein, one of the oncogenic drivers found in sun-exposed melanoma. It also inhibits numerous Rac1-regulated cellular processes such as membrane ruffling and lamellipodia formation. These results deepen our knowledge of 1A-116 inhibition of Rac1 and its biological impact on cancer progression. They also represent a good example of how in silico analyses represent a valuable approach for drug development.

12.
Oncogene ; 39(28): 5098-5111, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32528129

RESUMO

The current paradigm holds that the inhibition of Rho guanosine nucleotide exchange factors (GEFs), the enzymes that stimulate Rho GTPases, can be a valuable therapeutic strategy to treat Rho-dependent tumors. However, formal validation of this idea using in vivo models is still missing. In this context, it is worth remembering that many Rho GEFs can mediate both catalysis-dependent and independent responses, thus raising the possibility that the inhibition of their catalytic activities might not be sufficient per se to block tumorigenic processes. On the other hand, the inhibition of these enzymes can trigger collateral side effects that could preclude the practical implementation of anti-GEF therapies. To address those issues, we have generated mouse models to mimic the effect of the systemic application of an inhibitor for the catalytic activity of the Rho GEF Vav2 at the organismal level. Our results indicate that lowering the catalytic activity of Vav2 below specific thresholds is sufficient to block skin tumor initiation, promotion, and progression. They also reveal that the negative side effects typically induced by the loss of Vav2 can be bypassed depending on the overall level of Vav2 inhibition achieved in vivo. These data underscore the pros and cons of anti-Rho GEF therapies for cancer treatment. They also support the idea that Vav2 could represent a viable drug target.


Assuntos
Proteínas Proto-Oncogênicas c-vav/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/genética , Animais , Biocatálise , Células COS , Carcinogênese/genética , Chlorocebus aethiops , Cricetinae , Humanos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Proteínas Proto-Oncogênicas c-vav/química , Proteínas Proto-Oncogênicas c-vav/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
13.
Nat Commun ; 11(1): 4788, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963234

RESUMO

Regenerative proliferation capacity and poor differentiation are histological features usually linked to poor prognosis in head and neck squamous cell carcinoma (hnSCC). However, the pathways that regulate them remain ill-characterized. Here, we show that those traits can be triggered by the RHO GTPase activator VAV2 in keratinocytes present in the skin and oral mucosa. VAV2 is also required to maintain those traits in hnSCC patient-derived cells. This function, which is both catalysis- and RHO GTPase-dependent, is mediated by c-Myc- and YAP/TAZ-dependent transcriptomal programs associated with regenerative proliferation and cell undifferentiation, respectively. High levels of VAV2 transcripts and VAV2-regulated gene signatures are both associated with poor hnSCC patient prognosis. These results unveil a druggable pathway linked to the malignancy of specific SCC subtypes.


Assuntos
Proliferação de Células , Neoplasias de Cabeça e Pescoço/metabolismo , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Animais , Diferenciação Celular , Modelos Animais de Doenças , Epiderme/metabolismo , Epiderme/patologia , GTP Fosfo-Hidrolases , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Hiperplasia/patologia , Queratinócitos/patologia , Camundongos , Camundongos Knockout , Mucosa/metabolismo , Prognóstico , RNA Mensageiro/metabolismo , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Transcriptoma
14.
Oncogene ; 38(2): 209-227, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30087437

RESUMO

The bidirectional regulation of epithelial-mesenchymal transitions (EMT) is key in tumorigenesis. Rho GTPases regulate this process via canonical pathways that impinge on the stability of cell-to-cell contacts, cytoskeletal dynamics, and cell invasiveness. Here, we report that the Rho GTPase activators Vav2 and Vav3 utilize a new Rac1-dependent and miR-200c-dependent mechanism that maintains the epithelial state by limiting the abundance of the Zeb2 transcriptional repressor in breast cancer cells. In parallel, Vav proteins engage a mir-200c-independent expression prometastatic program that maintains epithelial cell traits only under 3D culture conditions. Consistent with this, the depletion of endogenous Vav proteins triggers mesenchymal features in epithelioid breast cancer cells. Conversely, the ectopic expression of an active version of Vav2 promotes mesenchymal-epithelial transitions using E-cadherin-dependent and independent mechanisms depending on the mesenchymal breast cancer cell line used. In silico analyses suggest that the negative Vav anti-EMT pathway is operative in luminal breast tumors. Gene signatures from the Vav-associated proepithelial and prometastatic programs have prognostic value in breast cancer patients.


Assuntos
Neoplasias da Mama/patologia , MicroRNAs/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Xenoenxertos , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-vav/genética
15.
Mol Cell Oncol ; 5(3): e1432257, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30250888

RESUMO

RHO GDP/GTP exchange factors, including VAV1, are considered key protumorigenic factors. Against this paradigm, we have found that VAV1 plays tumor suppressor roles by buffering NOTCH1 signals in thymocytes. The silencing of this pathway contributes to the pathogenesis of T cell acute lymphoblastic leukemia of the early cortical, TLX+ subtype.

16.
Cancer Cell ; 32(5): 608-623.e9, 2017 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-29136506

RESUMO

Rho guanine exchange factors (GEFs), the enzymes that stimulate Rho GTPases, are deemed as potential therapeutic targets owing to their protumorigenic functions. However, the understanding of the spectrum of their pathobiological roles in tumors is still very limited. We report here that the GEF Vav1 unexpectedly possesses tumor-suppressor functions in immature T cells. This function entails the noncatalytic nucleation of complexes between the ubiquitin ligase Cbl-b and the intracellular domain of Notch1 (ICN1) that favors ICN1 ubiquitinylation and degradation. Ablation of Vav1 promotes ICN1 signaling and the development of T cell acute lymphoblastic leukemia (T-ALL). The downregulation of Vav1 is essential for the pathogenesis of human T-ALL of the TLX+ clinical subtype, further underscoring the suppressor role of this pathway.


Assuntos
Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proteínas Proto-Oncogênicas c-vav/genética , Linfócitos T/metabolismo , Proteínas Supressoras de Tumor/genética , Animais , Western Blotting , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Células Jurkat , Camundongos Knockout , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Proteínas Proto-Oncogênicas c-cbl/genética , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Transdução de Sinais/genética , Linfócitos T/patologia , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/metabolismo
17.
Mol Biol Cell ; 26(16): 2895-912, 2015 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-26108624

RESUMO

Coronin 1A (Coro1A) is involved in cytoskeletal and signaling events, including the regulation of Rac1 GTPase- and myosin II-dependent pathways. Mutations that generate truncated or unstable Coro1A proteins cause immunodeficiencies in both humans and rodents. However, in the case of the peripheral T-cell-deficient (Ptcd) mouse strain, the immunodeficiency is caused by a Glu-26-Lys mutation that targets a surface-exposed residue unlikely to affect the intramolecular architecture and stability of the protein. Here we report that this mutation induces pleiotropic effects in Coro1A protein, including the exacerbation of Coro1A-dependent actin-binding and -bundling activities; the formation of large meshworks of Coro1A(E26K)-decorated filaments endowed with unusual organizational, functional, and staining properties; and the elimination of Coro1A functions associated with both Rac1 and myosin II signaling. By contrast, it does not affect the ability of Coro1A to stimulate the nuclear factor of activated T-cells (NF-AT). Coro1A(E26K) is not a dominant-negative mutant, indicating that its pathological effects are derived from the inability to rescue the complete loss of the wild-type counterpart in cells. These results indicate that Coro1A(E26K) behaves as either a recessive gain-of-function or loss-of-function mutant protein, depending on signaling context and presence of the wild-type counterpart in cells.


Assuntos
Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Animais , Células COS , Técnicas de Cultura de Células , Chlorocebus aethiops , Citoesqueleto/genética , Citoesqueleto/metabolismo , Células HEK293 , Humanos , Camundongos , Proteínas dos Microfilamentos/imunologia , Mutação , Transdução de Sinais
18.
Mol Cell Biol ; 35(20): 3528-46, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26240279

RESUMO

Hypertension-associated cardiorenal diseases represent one of the heaviest burdens for current health systems. In addition to hemodynamic damage, recent results have revealed that hematopoietic cells contribute to the development of these diseases by generating proinflammatory and profibrotic environments in the heart and kidney. However, the cell subtypes involved remain poorly characterized. Here we report that CD39(+) regulatory T (TREG) cells utilize an immunosuppression-independent mechanism to counteract renal and possibly cardiac damage during angiotensin II (AngII)-dependent hypertension. This mechanism relies on the direct apoptosis of tissue-resident neutrophils by the ecto-ATP diphosphohydrolase activity of CD39. In agreement with this, experimental and genetic alterations in TREG/TH cell ratios have a direct impact on tissue-resident neutrophil numbers, cardiomyocyte hypertrophy, cardiorenal fibrosis, and, to a lesser extent, arterial pressure elevation during AngII-driven hypertension. These results indicate that TREG cells constitute a first protective barrier against hypertension-driven tissue fibrosis and, in addition, suggest new therapeutic avenues to prevent hypertension-linked cardiorenal diseases.


Assuntos
Hipertensão/complicações , Insuficiência Renal/imunologia , Linfócitos T Reguladores/fisiologia , Angiotensina II/fisiologia , Animais , Antígenos CD/metabolismo , Apoptose , Apirase/metabolismo , Células Cultivadas , Fibrose , Hipertensão/imunologia , Tolerância Imunológica , Rim/imunologia , Rim/metabolismo , Rim/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Neutrófilos/fisiologia , Proteínas Proto-Oncogênicas c-vav/genética , Proteínas Proto-Oncogênicas c-vav/metabolismo , Insuficiência Renal/etiologia , Linfócitos T Auxiliares-Indutores/fisiologia
19.
Mol Cell Biol ; 34(8): 1412-26, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24492963

RESUMO

The recruitment of leukocytes to sites of inflammation is crucial for a functional immune response. In the present work, we explored the role of mitochondria in lymphocyte adhesion, polarity, and migration. We show that during adhesion to the activated endothelium under physiological flow conditions, lymphocyte mitochondria redistribute to the adhesion zone together with the microtubule-organizing center (MTOC) in an integrin-dependent manner. Mitochondrial redistribution and efficient lymphocyte adhesion to the endothelium require the function of Miro-1, an adaptor molecule that couples mitochondria to microtubules. Our data demonstrate that Miro-1 associates with the dynein complex. Moreover, mitochondria accumulate around the MTOC in response to the chemokine CXCL12/SDF-1α; this redistribution is regulated by Miro-1. CXCL12-dependent cell polarization and migration are reduced in Miro-1-silenced cells, due to impaired myosin II activation at the cell uropod and diminished actin polymerization. These data point to a key role of Miro-1 in the control of lymphocyte adhesion and migration through the regulation of mitochondrial redistribution.


Assuntos
Polaridade Celular/fisiologia , Quimiocina CXCL12/metabolismo , Dineínas/metabolismo , Linfócitos/metabolismo , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Movimento Celular/fisiologia , Polaridade Celular/imunologia , Citoesqueleto/metabolismo , Dineínas/genética , Inativação Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Integrinas/imunologia , Integrinas/metabolismo , Linfócitos/citologia , Microtúbulos/imunologia , Mitocôndrias/imunologia , Transdução de Sinais/imunologia
20.
J Cell Biol ; 182(5): 951-62, 2008 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-18779373

RESUMO

The translocation of the microtubule-organizing center (MTOC) toward the nascent immune synapse (IS) is an early step in lymphocyte activation initiated by T cell receptor (TCR) signaling. The molecular mechanisms that control the physical movement of the lymphocyte MTOC remain largely unknown. We have studied the role of the dynein-dynactin complex, a microtubule-based molecular motor, in the process of T cell activation during T cell antigen-presenting cell cognate immune interactions. Impairment of dynein-dynactin complex activity, either by overexpressing the p50-dynamitin component of dynactin to disrupt the complex or by knocking down dynein heavy chain expression to prevent its formation, inhibited MTOC translocation after TCR antigen priming. This resulted in a strong reduction in the phosphorylation of molecules such as zeta chain-associated protein kinase 70 (ZAP70), linker of activated T cells (LAT), and Vav1; prevented the supply of molecules to the IS from intracellular pools, resulting in a disorganized and dysfunctional IS architecture; and impaired interleukin-2 production. Together, these data reveal MTOC translocation as an important mechanism underlying IS formation and sustained T cell signaling.


Assuntos
Centro Organizador dos Microtúbulos/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Apresentadoras de Antígenos/imunologia , Transporte Biológico/imunologia , Complexo CD3/imunologia , Linhagem Celular , Complexo Dinactina , Dineínas/antagonistas & inibidores , Dineínas/metabolismo , Proteínas de Fluorescência Verde/análise , Humanos , Interleucina-2/metabolismo , Células Jurkat , Ativação Linfocitária , Antígeno-1 Associado à Função Linfocitária/imunologia , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/ultraestrutura , Fosforilação , Subunidades Proteicas/imunologia , Interferência de RNA , Receptores de Antígenos de Linfócitos T/metabolismo
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