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1.
Proc Natl Acad Sci U S A ; 112(13): 4044-9, 2015 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-25775525

RESUMO

Dendritic cells (DCs) are heterogeneous, comprising subsets with functional specializations that play distinct roles in immunity as well as immunopathology. We investigated the molecular control of cell survival of two main DC subsets: plasmacytoid DCs (pDCs) and conventional DCs (cDCs) and their dependence on individual antiapoptotic BCL-2 family members. Compared with cDCs, pDCs had higher expression of BCL-2, lower A1, and similar levels of MCL-1 and BCL-XL. Transgenic overexpression of BCL-2 increased the pDC pool size in vivo with only minor impact on cDCs. With a view to immune intervention, we tested BCL-2 inhibitors and found that ABT-199 (the BCL-2 specific inhibitor) selectively killed pDCs but not cDCs. Conversely, genetic knockdown of A1 profoundly reduced the proportion of cDCs but not pDCs. We also found that conditional ablation of MCL-1 significantly reduced the size of both DC populations in mice and impeded DC-mediated immune responses. Thus, we revealed that the two DC types have different cell survival requirements. The molecular basis of survival of different DC subsets thus advocates the antagonism of selective BCL-2 family members for treating diseases pertaining to distinct DC subsets.


Assuntos
Apoptose , Células Dendríticas/citologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Separação Celular , Sobrevivência Celular , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Transdução de Sinais , Baço/imunologia , Baço/metabolismo , Linfócitos T/citologia , Transgenes , Proteína bcl-X/metabolismo
2.
Cell Rep ; 41(5): 111571, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36323262

RESUMO

The nucleolar surveillance pathway monitors nucleolar integrity and responds to nucleolar stress by mediating binding of ribosomal proteins to MDM2, resulting in p53 accumulation. Inappropriate pathway activation is implicated in the pathogenesis of ribosomopathies, while drugs selectively activating the pathway are in trials for cancer. Despite this, the molecular mechanism(s) regulating this process are poorly understood. Using genome-wide loss-of-function screens, we demonstrate the ribosome biogenesis axis as the most potent class of genes whose disruption stabilizes p53. Mechanistically, we identify genes critical for regulation of this pathway, including HEATR3. By selectively disabling the nucleolar surveillance pathway, we demonstrate that it is essential for the ability of all nuclear-acting stresses, including DNA damage, to induce p53 accumulation. Our data support a paradigm whereby the nucleolar surveillance pathway is the central integrator of stresses that regulate nuclear p53 abundance, ensuring that ribosome biogenesis is hardwired to cellular proliferative capacity.


Assuntos
Proteínas Proto-Oncogênicas c-mdm2 , Proteína Supressora de Tumor p53 , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais/genética , Nucléolo Celular/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo
3.
J Biol Chem ; 285(28): 21214-8, 2010 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-20489211

RESUMO

gp130 is the shared signal-transducing receptor subunit for the large and important family of interleukin 6-like cytokines. Previous x-ray structures of ligand-receptor complexes of this family lack the three membrane-proximal domains that are essential for signal transduction. Here we report the crystal structure of the entire extracellular portion of human gp130 (domains 1-6, D1-D6) at 3.6 A resolution, in an unliganded form, as well as a higher resolution structure of the membrane-proximal fibronectin type III domains (D4-D6) at 1.9 A. This represents the first atomic resolution structure of the complete ectodomain of any "tall" cytokine receptor. These structures show that other than a reorientation of the D1 domain, there is little structural change in gp130 upon ligand binding. They also reveal that the interface between the D4 and D5 domains forms an acute bend in the gp130 structure. Key residues at this interface are highly conserved across the entire tall receptor family, suggesting that this acute bend may be a common feature of these receptors. Importantly, this geometry positions the C termini of the membrane-proximal fibronectin type III domains of the tall cytokine receptors in close proximity within the transmembrane complex, favorable for receptor-associated Janus kinases to trans-phosphorylate and activate each other.


Assuntos
Interleucina-6/química , Moléculas de Adesão de Célula Nervosa/química , Contactinas , Cristalografia por Raios X/métodos , Citocinas/metabolismo , Dimerização , Fibronectinas/química , Humanos , Ligantes , Conformação Molecular , Fosforilação , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Transdução de Sinais , Relação Estrutura-Atividade
4.
Assay Drug Dev Technol ; 16(6): 320-332, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30148664

RESUMO

The nucleolus is a dynamic subnuclear compartment that has a number of different functions, but its primary role is to coordinate the production and assembly of ribosomes. For well over 100 years, pathologists have used changes in nucleolar number and size to stage diseases such as cancer. New information about the nucleolus' broader role within the cell is leading to the development of drugs which directly target its structure as therapies for disease. Traditionally, it has been difficult to develop high-throughput image analysis pipelines to measure nucleolar changes due to the broad range of morphologies observed. In this study, we describe a simple high-content image analysis algorithm using Harmony software (PerkinElmer), with a PhenoLOGIC™ machine-learning component, that can measure and classify three different nucleolar morphologies based on nucleolin and fibrillarin staining ("normal," "peri-nucleolar rings" and "dispersed"). We have utilized this algorithm to determine the changes in these classes of nucleolar morphologies over time with drugs known to alter nucleolar structure. This approach could be further adapted to include other parameters required for the identification of new therapies that directly target the nucleolus.


Assuntos
Nucléolo Celular/patologia , Ensaios de Triagem em Larga Escala , Células A549 , Algoritmos , Nucléolo Celular/metabolismo , Humanos , Aprendizado de Máquina , Estresse Oxidativo , Software , Células Tumorais Cultivadas
5.
J Histochem Cytochem ; 54(5): 487-501, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16399995

RESUMO

The LIM kinase family includes two proteins: LIMK1 and LIMK2. These proteins have identical genomic structure and overall amino acid identity of 50%. Both proteins regulate actin polymerization via phosphorylation and inactivation of the actin depolymerizing factors ADF/cofilin. Although the function of endogenous LIMK1 is well established, little is known about the function of the endogenous LIMK2 protein. To understand the specific role of endogenous LIMK2 protein, we examined its expression in embryonic and adult mice using a rat monoclonal antibody, which recognizes specifically the PDZ domain of LIMK2 but not that of LIMK1. Immunoblotting and immunoprecipitation analyses of mouse tissues and human and mouse cell lines revealed widespread expression of the 75-kDa LIMK2 protein. Immunofluorescence analysis demonstrated that the cellular localization of LIMK2 is different from that of LIMK1. LIMK2 protein is found in the cytoplasm localized to punctae and is not enriched within focal adhesions like LIMK1. Immunohistochemical studies revealed that LIMK2 is widely expressed in embryonic and adult mouse tissues and that its expression pattern is similar to that of LIMK1 except in the testes. We have also demonstrated that endogenous LIMK1 and LIMK2 form heterodimers, and that LIMK2 does not always interact with the same proteins as LIMK1.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas Quinases/biossíntese , Animais , Anticorpos Monoclonais , Especificidade de Anticorpos , Linhagem Celular , Chlorocebus aethiops , Proteínas de Ligação a DNA/imunologia , Complexo de Golgi/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Quinases Lim , Camundongos , Especificidade de Órgãos , Proteínas Quinases/imunologia , Proteínas Serina-Treonina Quinases
6.
Sci Rep ; 6: 25060, 2016 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-27112985

RESUMO

Plasmacytoid dendritic cells (pDCs) play an important role in immunity to certain pathogens and immunopathology in some autoimmune diseases. They are thought to have a longer lifespan than conventional DCs (cDCs), largely based on a slower rate of BrdU labeling by splenic pDCs. Here we demonstrated that pDC expansion and therefore BrdU labeling by pDCs occurs in bone marrow (BM). The rate of labeling was similar between BM pDCs and spleen cDCs. Therefore, slower BrdU labeling of spleen pDCs likely reflects the "migration time" (∼2 days) for BrdU labeled pDCs to traffic to the spleen, not necessarily reflecting longer life span. Tracking the decay of differentiated DCs showed that splenic pDCs and cDCs decayed at a similar rate. We suggest that spleen pDCs have a shorter in vivo lifespan than estimated utilizing some of the previous approaches. Nevertheless, pDC lifespan varies between mouse strains. pDCs from lupus-prone NZB mice survived longer than C57BL/6 pDCs. We also demonstrated that activation either positively or negatively impacted on the survival of pDCs via different cell-death mechanisms. Thus, pDCs are also short-lived. However, the pDC lifespan is regulated by genetic and environmental factors that may have pathological consequence.


Assuntos
Células da Medula Óssea/citologia , Células Dendríticas/citologia , Baço/citologia , Animais , Células da Medula Óssea/metabolismo , Bromodesoxiuridina/metabolismo , Diferenciação Celular , Movimento Celular , Sobrevivência Celular , Células Cultivadas , Células Dendríticas/metabolismo , Camundongos , Baço/metabolismo
7.
J Mol Biol ; 427(10): 1934-48, 2015 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-25765764

RESUMO

We have expressed and purified three soluble fragments of the human LRIG1-ECD (extracellular domain): the LRIG1-LRR (leucine-rich repeat) domain, the LRIG1-3Ig (immunoglobulin-like) domain, and the LRIG1-LRR-1Ig fragment using baculovirus vectors in insect cells. The two LRIG1 domains crystallised so that we have been able to determine the three-dimensional structures at 2.3Å resolution. We developed a three-dimensional structure for the LRIG1-ECD using homology modelling based on the LINGO-1 structure. The LRIG1-LRR domain and the LRIG1-LRR-1Ig fragment are monomers in solution, whereas the LRIG1-3Ig domain appears to be dimeric. We could not detect any binding of the LRIG1 domains or the LRIG1-LRR-1Ig fragment to the EGF receptor (EGFR), either in solution using biosensor analysis or when the EGFR was expressed on the cell surface. The FLAG-tagged LRIG1-LRR-1Ig fragment binds weakly to colon cancer cells regardless of the presence of EGFRs. Similarly, neither the soluble LRIG1-LRR nor the LRIG1-3Ig domains nor the full-length LRIG1 co-expressed in HEK293 cells inhibited ligand-stimulated activation of cell-surface EGFR.


Assuntos
Receptores ErbB/química , Receptores ErbB/metabolismo , Matriz Extracelular/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Técnicas Biossensoriais , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Cristalografia por Raios X , Células HEK293 , Humanos , Proteínas de Repetições Ricas em Leucina , Ligantes , Microscopia de Fluorescência , Modelos Moleculares , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas/química , Proteínas/metabolismo , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Células Tumorais Cultivadas
8.
PLoS One ; 6(5): e19665, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21611124

RESUMO

Female-controlled contraception/HIV prevention is critical to address health issues associated with gender inequality. Therefore, a contraceptive which can be administered in tandem with a microbicide to inhibit sexually transmitted infections, is desirable. Uterine leukemia inhibitory factor (LIF) is obligatory for blastocyst implantation in mice and associated with infertility in women. We aimed to determine whether a PEGylated LIF inhibitor (PEGLA) was an effective contraceptive following vaginal delivery and to identify non-uterine targets of PEGLA in mice.Vaginally-applied (125)I-PEGLA accumulated in blood more slowly (30 min vs 10 min) and showed reduced tissue and blood retention (24 h vs 96 h) compared to intraperitoneal injection in mice. Vaginally-applied PEGLA blocked implantation. PEGLA administered by intraperitoneal injection inhibited bone remodelling whereas vaginally-applied PEGLA had no effect on bone. Further, PEGLA had no effect in an animal model of multiple sclerosis, experimental auto-immune encephalomyelitis, suggesting PEGLA cannot target the central nervous system.Vaginally-administered PEGLA is a promising non-hormonal contraceptive, one which could be delivered alone, or in tandem with a microbicide. Vaginal application reduced the total dose of PEGLA required to block implantation and eliminated the systemic effect on bone, showing the vagina is a promising site of administration for larger drugs which target organs within the reproductive tract.


Assuntos
Osso e Ossos/efeitos dos fármacos , Implantação do Embrião/efeitos dos fármacos , Fator Inibidor de Leucemia/antagonistas & inibidores , Polietilenoglicóis/administração & dosagem , Polietilenoglicóis/farmacologia , Administração Intravaginal , Animais , Remodelação Óssea/efeitos dos fármacos , Osso e Ossos/fisiologia , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Encefalomielite Autoimune Experimental/patologia , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Feminino , Fertilidade/efeitos dos fármacos , Meia-Vida , Injeções Intraperitoneais , Radioisótopos do Iodo , Fator Inibidor de Leucemia/administração & dosagem , Fator Inibidor de Leucemia/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transporte Proteico/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Útero/efeitos dos fármacos , Útero/metabolismo
9.
Exp Cell Res ; 313(20): 4091-106, 2007 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-18028908

RESUMO

LIM kinase 1 (LIMK1) is a key regulator of actin dynamics as it phosphorylates and inactivates cofilin, an actin-depolymerizing factor. LIMK1 activity is also required for microtubule disassembly in endothelial cells. A search for LIMK1-interacting proteins identified p25alpha, a phosphoprotein that promotes tubulin polymerization. We found that p25 is phosphorylated by LIMK1 on serine residues in vitro and in cells. Immunoblotting analysis revealed that p25 is not a brain specific protein as previously reported, but is expressed in all mouse tissues. Immunofluorescence analysis demonstrated that endogenous p25 is co-localized with microtubules and is also found in the nucleus. Down-regulation of p25 by siRNA decreased microtubule levels while its overexpression in stable NIH-3T3 cell lines increased cell size and levels of stable tubulin. Bacterially expressed unphosphorylated p25 promotes microtubule assembly in vitro; however, when phosphorylated in cells, p25 lost its ability to assemble microtubule. Our results represent a surprising connection between the tubulin and the actin cytoskeleton mediated by LIMK1. We propose that the LIMK1 phosphorylation of p25 blocks p25 activity, thus promoting microtubule disassembly.


Assuntos
Quinases Lim/metabolismo , Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Tamanho Celular , Regulação para Baixo , Células HeLa , Humanos , Imuno-Histoquímica , Quinases Lim/química , Camundongos , Modelos Biológicos , Células NIH 3T3 , Especificidade de Órgãos , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Ovinos , Frações Subcelulares/metabolismo , Especificidade por Substrato , Tubulina (Proteína)/metabolismo
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