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1.
Nature ; 528(7580): 127-31, 2015 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-26580007

RESUMO

Prevailing dogma holds that cell-cell communication through Notch ligands and receptors determines binary cell fate decisions during progenitor cell divisions, with differentiated lineages remaining fixed. Mucociliary clearance in mammalian respiratory airways depends on secretory cells (club and goblet) and ciliated cells to produce and transport mucus. During development or repair, the closely related Jagged ligands (JAG1 and JAG2) induce Notch signalling to determine the fate of these lineages as they descend from a common proliferating progenitor. In contrast to such situations in which cell fate decisions are made in rapidly dividing populations, cells of the homeostatic adult airway epithelium are long-lived, and little is known about the role of active Notch signalling under such conditions. To disrupt Jagged signalling acutely in adult mammals, here we generate antibody antagonists that selectively target each Jagged paralogue, and determine a crystal structure that explains selectivity. We show that acute Jagged blockade induces a rapid and near-complete loss of club cells, with a concomitant gain in ciliated cells, under homeostatic conditions without increased cell death or division. Fate analyses demonstrate a direct conversion of club cells to ciliated cells without proliferation, meeting a conservative definition of direct transdifferentiation. Jagged inhibition also reversed goblet cell metaplasia in a preclinical asthma model, providing a therapeutic foundation. Our discovery that Jagged antagonism relieves a blockade of cell-to-cell conversion unveils unexpected plasticity, and establishes a model for Notch regulation of transdifferentiation.


Assuntos
Anticorpos/uso terapêutico , Transdiferenciação Celular , Pulmão/citologia , Pulmão/metabolismo , Receptores Notch/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Asma/tratamento farmacológico , Asma/metabolismo , Asma/patologia , Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Rastreamento de Células , Transdiferenciação Celular/efeitos dos fármacos , Cílios/metabolismo , Modelos Animais de Doenças , Feminino , Células Caliciformes/citologia , Células Caliciformes/efeitos dos fármacos , Células Caliciformes/patologia , Homeostase/efeitos dos fármacos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Proteína Jagged-2 , Ligantes , Pulmão/efeitos dos fármacos , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Proteínas Serrate-Jagged , Transdução de Sinais/efeitos dos fármacos
2.
J Biol Chem ; 287(19): 15837-50, 2012 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-22396535

RESUMO

Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ∼22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed.


Assuntos
Evolução Molecular , Glicoproteínas de Membrana/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Receptores Imunológicos/metabolismo , Antígeno 12E7 , Sequência de Aminoácidos , Animais , Antígenos CD/química , Antígenos CD/genética , Antígenos CD/metabolismo , Arginina/química , Arginina/genética , Arginina/metabolismo , Sítios de Ligação/genética , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Colectinas/química , Colectinas/genética , Colectinas/metabolismo , Sequência Conservada/genética , Células HEK293 , Humanos , Ligantes , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Dados de Sequência Molecular , Ácido N-Acetilneuramínico/química , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Receptores Imunológicos/química , Receptores Imunológicos/genética , Receptores Depuradores/química , Receptores Depuradores/genética , Receptores Depuradores/metabolismo , Homologia de Sequência de Aminoácidos , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico , Células Vero
3.
Nat Cell Biol ; 14(1): 61-72, 2011 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-22179047

RESUMO

Using RNAi screening, proteomics, cell biological and mouse genetics approaches, we have identified a complex of nine proteins, seven of which are disrupted in human ciliopathies. A transmembrane component, TMEM231, localizes to the basal body before and independently of intraflagellar transport in a Septin 2 (Sept2)-regulated fashion. The localizations of TMEM231, B9D1 (B9 domain-containing protein 1) and CC2D2A (coiled-coil and C2 domain-containing protein 2A) at the transition zone are dependent on one another and on Sept2. Disruption of the complex in vitro causes a reduction in cilia formation and a loss of signalling receptors from the remaining cilia. Mouse knockouts of B9D1 and TMEM231 have identical defects in Sonic hedgehog (Shh) signalling and ciliogenesis. Strikingly, disruption of the complex increases the rate of diffusion into the ciliary membrane and the amount of plasma-membrane protein in the cilia. The complex that we have described is essential for normal cilia function and acts as a diffusion barrier to maintain the cilia membrane as a compartmentalized signalling organelle.


Assuntos
Cílios/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Animais , Linhagem Celular , Citoplasma/metabolismo , Proteínas do Citoesqueleto , Células-Tronco Embrionárias/metabolismo , Técnicas de Inativação de Genes , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Proteínas/metabolismo , Septinas/metabolismo , Transdução de Sinais
4.
J Am Chem Soc ; 129(43): 13153-9, 2007 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-17915868

RESUMO

Chemical protein synthesis is important for dissecting the molecular basis of protein function. Here we advance its scope by demonstrating the significant improvement of the multifaceted pharmaceutical profile of small proteins exclusively via a chemical-based approach. The focus of this work centered on CCL-5 (RANTES) derivatives with potent anti-HIV activity. The overall chemical strategy involved a combination of coded and noncoded amino acid mutagenesis, peptide backbone engineering, and site-specific polymer attachment. The ability to alter specific protein residues, as well as precise control of the position and type of polymer attachment, allows for the exploration of specific molecular designs and resulted in novel CCL-5 analogues with significant differences in their respective biochemical and pharmaceutical properties. Using this approach, the complex-interplay of variables contributing to the noncovalent self-association (aggregation) state, CCR-5 specificity, in vivo elimination half-life, and anti-HIV activity of CCL-5-based protein analogues could be empirically evaluated via total chemical synthesis. This work has led to the identification of potent (sub-nanomolar) anti-HIV proteins with significantly improved pharmaceutical profiles, and illustrates the increasing value of protein chemical synthesis in contemporary therapeutic discovery. These antiviral molecules provide a novel mechanism of action for the development of a new generation of anti-HIV therapeutics which are still desperately needed.


Assuntos
Fármacos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , Proteínas do Vírus da Imunodeficiência Humana/imunologia , Aminoácidos/química , Animais , Fármacos Anti-HIV/farmacocinética , Cromatografia em Gel , Masculino , Modelos Moleculares , Estrutura Molecular , Polímeros/química , Ratos , Receptores Acoplados a Proteínas G/metabolismo
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