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1.
Cell ; 184(20): 5230-5246.e22, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34551315

RESUMO

Although mutations leading to a compromised nuclear envelope cause diseases such as muscular dystrophies or accelerated aging, the consequences of mechanically induced nuclear envelope ruptures are less known. Here, we show that nuclear envelope ruptures induce DNA damage that promotes senescence in non-transformed cells and induces an invasive phenotype in human breast cancer cells. We find that the endoplasmic reticulum (ER)-associated exonuclease TREX1 translocates into the nucleus after nuclear envelope rupture and is required to induce DNA damage. Inside the mammary duct, cellular crowding leads to nuclear envelope ruptures that generate TREX1-dependent DNA damage, thereby driving the progression of in situ carcinoma to the invasive stage. DNA damage and nuclear envelope rupture markers were also enriched at the invasive edge of human tumors. We propose that DNA damage in mechanically challenged nuclei could affect the pathophysiology of crowded tissues by modulating proliferation and extracellular matrix degradation of normal and transformed cells.


Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Dano ao DNA , Exodesoxirribonucleases/metabolismo , Membrana Nuclear/metabolismo , Fosfoproteínas/metabolismo , Animais , Linhagem Celular , Senescência Celular , Colágeno/metabolismo , Progressão da Doença , Feminino , Humanos , Camundongos , Invasividade Neoplásica , Membrana Nuclear/ultraestrutura , Proteólise , Ensaios Antitumorais Modelo de Xenoenxerto
2.
EMBO J ; 35(11): 1160-74, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27154205

RESUMO

The immunological synapse generation and function is the result of a T-cell polarization process that depends on the orchestrated action of the actin and microtubule cytoskeleton and of intracellular vesicle traffic. However, how these events are coordinated is ill defined. Since Rab and Rho families of GTPases control intracellular vesicle traffic and cytoskeleton reorganization, respectively, we investigated their possible interplay. We show here that a significant fraction of Rac1 is associated with Rab11-positive recycling endosomes. Moreover, the Rab11 effector FIP3 controls Rac1 intracellular localization and Rac1 targeting to the immunological synapse. FIP3 regulates, in a Rac1-dependent manner, key morphological events, like T-cell spreading and synapse symmetry. Finally, Rab11-/FIP3-mediated regulation is necessary for T-cell activation leading to cytokine production. Therefore, Rac1 endosomal traffic is key to regulate T-cell activation.


Assuntos
Actinas/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Quinase I-kappa B/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Linhagem Celular , Células Cultivadas , Endossomos/metabolismo , Humanos , Quinase I-kappa B/genética , Sinapses Imunológicas/metabolismo , Interleucina-2/metabolismo , Células Jurkat , RNA Interferente Pequeno/genética
3.
J Immunol ; 198(7): 2967-2978, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28235866

RESUMO

The role of endosomes in receptor signal transduction is a long-standing question, which remains largely unanswered. The T cell Ag receptor and various components of its proximal signaling machinery are associated with distinct endosomal compartments, but how endosomal traffic affects T cell signaling remains ill-defined. In this article, we demonstrate in human T cells that the subcellular localization and function of the protein tyrosine kinase Lck depends on the Rab11 effector FIP3 (Rab11 family interacting protein-3). FIP3 overexpression or silencing and its ability to interact with Rab11 modify Lck subcellular localization and its delivery to the immunological synapse. Importantly, FIP3-dependent Lck localization controls early TCR signaling events, such as tyrosine phosphorylation of TCRζ, ZAP70, and LAT and intracellular calcium concentration, as well as IL-2 gene expression. Interestingly, FIP3 controls both steady-state and poststimulation phosphotyrosine and calcium levels. Finally, our findings indicate that FIP3 modulates TCR-CD3 cell surface expression via the regulation of steady-state Lck-mediated TCRζ phosphorylation, which in turn controls TCRζ protein levels. This may influence long-term T cell activation in response to TCR-CD3 stimulation. Therefore, our data underscore the importance of finely regulated endosomal traffic in TCR signal transduction and T cell activation leading to IL-2 production.


Assuntos
Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Western Blotting , Endossomos/imunologia , Técnicas de Silenciamento de Genes , Humanos , Quinase I-kappa B/imunologia , Sinapses Imunológicas/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Microscopia Confocal , Reação em Cadeia da Polimerase , Transporte Proteico/imunologia , Proteínas rab de Ligação ao GTP/imunologia
4.
PLoS Pathog ; 11(5): e1004838, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25941809

RESUMO

Understanding how a pathogen colonizes and adapts to a new host environment is a primary aim in studying emerging infectious diseases. Adaptive mutations arise among the thousands of variants generated during RNA virus infection, and identifying these variants will shed light onto how changes in tropism and species jumps can occur. Here, we adapted Coxsackie virus B3 to a highly permissive and less permissive environment. Using deep sequencing and bioinformatics, we identified a multi-step adaptive process to adaptation involving residues in the receptor footprints that correlated with receptor availability and with increase in virus fitness in an environment-specific manner. We show that adaptation occurs by selection of a dominant mutation followed by group selection of minority variants that together, confer the fitness increase observed in the population, rather than selection of a single dominant genotype.


Assuntos
Adaptação Biológica/genética , Enterovirus Humano B/genética , Replicação Viral/genética , Adaptação Biológica/imunologia , Linhagem Celular , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação/genética , Fenótipo
5.
J Cell Sci ; 124(Pt 3): 321-7, 2011 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-21224393

RESUMO

The activating immune receptor NKG2D binds to several stress-induced ligands that are structurally different. MHC-class-I-related chain (MIC) A/B molecules have a transmembrane domain, whereas most UL16 binding proteins (ULBPs) are glycosylphosphatidylinositol (GPI)-linked molecules. The significance of this variability in membrane anchors is unclear. Here, we demonstrate that ULBP2, but not ULBP1 or ULBP3, can reach the cell surface without the GPI modification. Several proteins are expressed at the cell surface as both transmembrane and GPI-linked molecules, either via alternative splicing or by the expression of linked genes. However, to our knowledge, ULBP2 is the first single mammalian cDNA that can be expressed as either a transmembrane or a GPI-anchored protein. The rate of maturation and the levels of cell surface expression of the non-GPI-linked form were lower than those of the GPI-linked ULBP2. Nonetheless, non-GPI ULBP2 was recognised by NKG2D and triggered NK cell cytotoxicity. These data show that differences in membrane attachment by NKG2D ligands are more important for regulation of their surface expression than for cytotoxic recognition by NKG2D and emphasise that detailed characterisation of the cell biology of individual NKG2D ligands will be necessary to allow targeted modulation of this system.


Assuntos
Membrana Celular/metabolismo , Proteínas Ligadas por GPI/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Células Matadoras Naturais/metabolismo , Animais , Células CHO , Membrana Celular/imunologia , Cricetinae , Cricetulus , Citotoxicidade Imunológica , Retículo Endoplasmático/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Células Matadoras Naturais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Transporte Proteico
6.
Eur J Immunol ; 41(12): 3667-76, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21928280

RESUMO

MICA and MICB (MHC-class-I-related chain A/B) are transmembrane proteins expressed in pathological conditions that are ligands for NKG2D, an activating receptor found on cytotoxic lymphocytes. The recognition on target cells of NKG2D ligands leads to the activation of lysis and cytokine secretion by NK cells and T cells. Besides being expressed at the cell surface, MICA/B can be released as soluble proteins. Soluble NKG2D ligands downmodulate expression of the NKG2D receptor on lymphocytes, leading to a diminished cytotoxic response. Prior studies suggested that recruitment of MICA/B molecules to cholesterol-enriched microdomains was an important factor regulating the proteolytic release of these molecules. We now show that recruitment of MICA to these microdomains depends on palmitoylation of two cysteine residues that allow MICA molecules to reside in the membrane in the same domains as caveolin-1. Compared with WT molecules, nonpalmitoylated mutant MICA molecules were shed to the supernatant with low efficiency; however, both WT and mutant MICA were able to trigger NK cell cytotoxicity. These data suggest that the presence of NKG2D ligands at the plasma membrane is sufficient to activate cytotoxicity and reflect the need of different ligands to exploit different cellular pathways to reach the cell surface upon different stress situations.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Microdomínios da Membrana/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Animais , Células CHO , Caveolina 1/imunologia , Caveolina 1/metabolismo , Linhagem Celular Transformada , Colesterol/metabolismo , Cricetinae , Cisteína/metabolismo , Citoplasma/metabolismo , Citotoxicidade Imunológica/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ligantes , Lipoilação , Microdomínios da Membrana/imunologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia
7.
J Biol Chem ; 285(12): 8543-51, 2010 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-20080967

RESUMO

Tumor cells release NKG2D ligands to evade NKG2D-mediated immune surveillance. The purpose of our investigation was to explore the cellular mechanisms of release used by various members of the ULBP family. Using biochemical and cellular approaches in both transfectant systems and tumor cell lines, this paper shows that ULBP1, ULBP2, and ULBP3 are released from cells with different kinetics and by distinct mechanisms. Whereas ULBP2 is mainly shed by metalloproteases, ULBP3 is abundantly released as part of membrane vesicles known as exosomes. Interestingly, exosomal ULBP3 protein is much more potent for down-modulation of the NKG2D receptor than soluble ULBP2 protein. This is the first report showing functionally relevant differences in the biochemistry of the three members of the ULBP family and confirms that in depth study of the biochemical features of individual NKG2D ligands will be necessary to understand and manipulate the biology of these proteins for therapy.


Assuntos
Glicosilfosfatidilinositóis/metabolismo , Ligantes , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Animais , Bioquímica/métodos , Células CHO , Linhagem Celular , Cricetinae , Cricetulus , Exossomos/metabolismo , Citometria de Fluxo/métodos , Proteínas Ligadas por GPI , Humanos , Sistema Imunitário , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Cinética , Modelos Biológicos
8.
J Immunol ; 182(8): 4800-8, 2009 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-19342658

RESUMO

Recognition of MHC class I-related chain (MIC) molecules on the surface of target cells by the activating receptor NKG2D leads to their lysis by immune effector cells. Up-regulation of NKG2D ligands is broadly related to stress, although the detailed molecular mechanisms that control the presence of these molecules at the plasma membrane are unclear. To investigate the posttranslational mechanisms that control surface expression of the human NKG2D ligand MICB, we studied the subcellular localization and trafficking of this molecule. We found that in several cellular systems, the expression of MICB molecules on the cell surface is accompanied by an intracellular accumulation of the molecule in the trans-Golgi network and late endosome-related compartments. Surprisingly, MICB has a much shorter half-life at the plasma membrane than MHC molecules and this depends on both recycling to internal compartments and shedding to the extracellular medium. Internalization of MICB depends partially on clathrin, but importantly, the lipid environment of the membrane also plays a crucial role in this process. We suggest that the brief residence of MICB at the plasma membrane modulates, at least in part, the function of this molecule in the immune system.


Assuntos
Membrana Celular/imunologia , Colesterol/imunologia , Clatrina/imunologia , Endocitose/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Linhagem Celular Tumoral , Endossomos/imunologia , Humanos , Fatores de Tempo
9.
J Immunol ; 182(1): 49-53, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19109134

RESUMO

MHC class I-related chain (MIC) A/B are transmembrane proteins expressed in pathological conditions that are ligands for the activating receptor NKG2D found on cytotoxic lymphocytes. Soluble NKG2D ligands are detected in sera of patients suffering from multiple types of cancer where they are associated with reduced levels of receptor expression and compromised function of NK and CTLs. In this study, we report the identification of a metalloproteinase involved in the cleavage process of MIC; inhibition and knockdown of ADAM17/TACE blocks the shedding of these proteins. Strikingly, the recruitment of both enzyme and substrate to detergent-resistant membrane microdomains is crucial for efficient proteolysis. These findings provide a novel insight into the molecular mechanisms of MIC shedding.


Assuntos
Proteínas ADAM/fisiologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas ADAM/antagonistas & inibidores , Proteína ADAM17 , Linhagem Celular , Linhagem Celular Tumoral , Dipeptídeos/farmacologia , Humanos , Hidrólise/efeitos dos fármacos , Microdomínios da Membrana/enzimologia , Fenilalanina/análogos & derivados , Fenilalanina/farmacologia , Solubilidade , Tiofenos/farmacologia
10.
Nat Commun ; 9(1): 2443, 2018 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-29934494

RESUMO

Cancer cells' ability to migrate through constricting pores in the tissue matrix is limited by nuclear stiffness. MT1-MMP contributes to metastasis by widening matrix pores, facilitating confined migration. Here, we show that modulation of matrix pore size or of lamin A expression known to modulate nuclear stiffness directly impinges on levels of MT1-MMP-mediated pericellular collagenolysis by cancer cells. A component of this adaptive response is the centrosome-centered distribution of MT1-MMP intracellular storage compartments ahead of the nucleus. We further show that this response, including invadopodia formation in association with confining matrix fibrils, requires an intact connection between the nucleus and the centrosome via the linker of nucleoskeleton and cytoskeleton (LINC) complex protein nesprin-2 and dynein adaptor Lis1. Our results uncover a digest-on-demand strategy for nuclear translocation through constricted spaces whereby confined migration triggers polarization of MT1-MMP storage compartments and matrix proteolysis in front of the nucleus depending on nucleus-microtubule linkage.


Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Movimento Celular , Metaloproteinase 14 da Matriz/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias/patologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Centrossomo/metabolismo , Humanos , Lamina Tipo A/metabolismo , Invasividade Neoplásica/patologia , Podossomos/metabolismo , Proteólise
11.
Oncogene ; 37(50): 6425-6441, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30065298

RESUMO

Membrane type 1-matrix metalloproteinase (MT1-MMP), a membrane-tethered protease, is key for matrix breakdown during cancer invasion and metastasis. Assembly of branched actin networks by the Arp2/3 complex is required for MT1-MMP traffic and formation of matrix-degradative invadopodia. Contrasting with the well-established role of actin filament branching factor cortactin in invadopodia function during cancer cell invasion, the contribution of coronin-family debranching factors to invadopodia-based matrix remodeling is not known. Here, we investigated the contribution of coronin 1C to the invasive potential of breast cancer cells. We report that expression of coronin 1C is elevated in invasive human breast cancers, correlates positively with MT1-MMP expression in relation with increased metastatic risk and is a new independent prognostic factor in breast cancer. We provide evidence that, akin to cortactin, coronin 1C is required for invadopodia formation and matrix degradation by breast cancer cells lines and for 3D collagen invasion by multicellular spheroids. Using intravital imaging of orthotopic human breast tumor xenografts, we find that coronin 1C accumulates in structures forming in association with collagen fibrils in the tumor microenvironment. Moreover, we establish the role of coronin 1C in the regulation of positioning and trafficking of MT1-MMP-positive endolysosomes. These results identify coronin 1C as a novel player of the multi-faceted mechanism responsible for invadopodia formation, MT1-MMP surface exposure and invasiveness in breast cancer cells.


Assuntos
Metaloproteinase 14 da Matriz/metabolismo , Proteínas dos Microfilamentos/metabolismo , Podossomos/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Animais , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Humanos , Camundongos , Invasividade Neoplásica/patologia , Podossomos/patologia , Transporte Proteico/fisiologia , Esferoides Celulares , Neoplasias de Mama Triplo Negativas/metabolismo
12.
Cell Rep ; 21(1): 181-194, 2017 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-28978472

RESUMO

Adenomatous polyposis coli (APC) is a polarity regulator and tumor suppressor associated with familial adenomatous polyposis and colorectal cancer development. Although extensively studied in epithelial transformation, the effect of APC on T lymphocyte activation remains poorly defined. We found that APC ensures T cell receptor-triggered activation through Nuclear Factor of Activated T cells (NFAT), since APC is necessary for NFAT's nuclear localization in a microtubule-dependent fashion and for NFAT-driven transcription leading to cytokine gene expression. Interestingly, NFAT forms clusters juxtaposed with microtubules. Ultimately, mouse Apc deficiency reduces the presence of NFAT in the nucleus of intestinal regulatory T cells (Tregs) and impairs Treg differentiation and the acquisition of a suppressive phenotype, which is characterized by the production of the anti-inflammatory cytokine IL-10. These findings suggest a dual role for APC mutations in colorectal cancer development, where mutations drive the initiation of epithelial neoplasms and also reduce Treg-mediated suppression of the detrimental inflammation that enhances cancer growth.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Regulação Neoplásica da Expressão Gênica , Microtúbulos/imunologia , Fatores de Transcrição NFATC/genética , Linfócitos T Reguladores/imunologia , Polipose Adenomatosa do Colo/imunologia , Polipose Adenomatosa do Colo/patologia , Proteína da Polipose Adenomatosa do Colo/antagonistas & inibidores , Proteína da Polipose Adenomatosa do Colo/imunologia , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Células HCT116 , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Células Jurkat , Linfonodos/imunologia , Linfonodos/patologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microtúbulos/ultraestrutura , Fatores de Transcrição NFATC/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Linfócitos T Reguladores/patologia
13.
Cancer Res ; 70(2): 481-9, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-20068167

RESUMO

The MHC class I-related chain (MIC) A and MICB ligands for the activating receptor NKG2D can be shed from tumor cells, and the presence of these soluble molecules in sera is related with compromised immune response and progression of disease. Recently, thiol disulphide isomerases and members of the ADAM (a disintegrin and metalloproteinase) gene family were identified as key enzymes in mediating MICA/B shedding from cells. Here, we report shedding of the most frequently expressed MICA allele in human populations (MICA*008) into exosomes, small membrane vesicles that are secreted upon fusion with the plasma membrane. Although similar to other MICA/B molecules in the extracellular domain, the predicted transmembrane and cytoplasmic domains of MICA*008 are quite different, and this difference seemed to be critical for the mode of release from tumor cells. Treatment of natural killer (NK) cells with exosomes containing MICA*008 molecules not only triggered downregulation of NKG2D from the cell surface but also provoked a marked reduction in NK cytotoxicity that is independent of NKG2D ligand expression by the target cell. Our findings reveal a mechanism of NK suppression in cancer that may facilitate immune escape and progression.


Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Células Matadoras Naturais/imunologia , Alelos , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Citotoxicidade Imunológica , Regulação para Baixo , Exossomos/imunologia , Exossomos/metabolismo , Proteínas Ligadas por GPI , Células HeLa , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Microdomínios da Membrana/imunologia , Microdomínios da Membrana/metabolismo
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