The Experts below are selected from a list of 21573 Experts worldwide ranked by ideXlab platform
Nora B. Caberoy - One of the best experts on this subject based on the ideXlab platform.
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Galectin-3 is a new MerTK-specific eat-me signal.
Journal of cellular physiology, 2011Co-Authors: Nora B. Caberoy, Gabriela Alvarado, Jo Lawrence BigcasAbstract:Phagocytosis of apoptotic cells and cellular debris is a critical process of maintaining tissue and immune homeostasis. Defects in the phagocytosis process cause autoimmunity and degenerative diseases. Phagocytosis ligands or “eat-me” signals control the initiation of the process by linking apoptotic cells to receptors on phagocyte surface and triggering signaling cascades for cargo engulfment. Eat-me signals are traditionally identified on a case-by-case basis with challenges, and the identification of their cognate receptors is equally daunting. Here we identified galectin-3 (Gal-3) as a new MerTK ligand by an advanced dual Functional Cloning strategy, in which phagocytosis-based Functional Cloning is combined with receptor-based affinity Cloning to directly identify receptor-specific eat-me signal. Gal-3 interaction with MerTK was independently verified by co-immunoprecipitation. Functional analyses showed that Gal-3 stimulates the phagocytosis of apoptotic cells and cellular debris by macrophages and retinal pigment epithelial cells with MerTK activation and autophosphorylation. The Gal-3-mediated phagocytosis was blocked by excessive soluble MerTK extracellular domain and lactose. These results suggest that Gal-3 is a legitimate MerTK-specific eat-me signal. The strategy of dual Functional Cloning with applicability to other phagocytic receptors will facilitate unbiased identification of their unknown ligands and improve our capacity for therapeutic modulation of phagocytic activity and innate immune response.
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Unraveling the Molecular Mystery of Retinal Pigment Epithelium Phagocytosis
Advances in Experimental Medicine and Biology, 2011Co-Authors: Nora B. Caberoy, Wei LiAbstract:Retinal pigment epithelium (RPE) phagocytosis is essential for clearance of shed photoreceptor outer segments (POS) to prevent retinal degeneration. Despite the importance, our understanding of the molecular mechanisms of RPE phagocytosis is relatively limited. Phagocytosis ligands, receptors, and signaling cascades are traditionally identified on case-by-case basis with challenges. It is even more daunting to identify a new signaling pathway in the absence of any molecular probe. We developed a phagocytosis-based Functional Cloning strategy with open reading frame (ORF) phage display and identified tubby-like protein 1 (Tulp1) as a new phagocytosis ligand in an unbiased manner. We used Tulp1 as a molecular probe and characterized the well-known MerTK as the receptor of Tulp1. Tulp1 as a genuine MerTK ligand was independently validated by co-immunoprecipitation, MerTK autophosphorylation and MerTK-dependent intracellular signaling. Moreover, Tulp1 was characterized as a bridging molecule with a C-terminal phagocytosis prey-binding domain. These results suggest that Tulp1 is a genuine RPE phagocytosis ligand and that ORF phage display is a valid technology for unbiased identification of phagocytosis ligands, which will further lead to their receptors and signaling cascades. Thus, this new strategy will facilitate the unbiased mapping of molecular mechanisms of RPE and other phagocytes.
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Identification of tubby and tubby-like protein 1 as eat-me signals by phage display
Experimental Cell Research, 2009Co-Authors: Nora B. Caberoy, Dony Maiguel, Y. Kim, Wei LiAbstract:Abstract Phagocytosis is an important process for the removal of apoptotic cells or cellular debris. Eat-me signals control the initiation of phagocytosis and hold the key for in-depth understanding of its molecular mechanisms. However, because of difficulties to identify unknown eat-me signals, only a limited number of them have been identified and characterized. Using a newly developed Functional Cloning strategy of open reading frame (ORF) phage display, we identified nine putative eat-me signals, including tubby-like protein 1 (Tulp1). This further led to the elucidation of tubby as the second eat-me signal in the same protein family. Both proteins stimulated phagocytosis of retinal pigment epithelium (RPE) cells and macrophages. Tubby-conjugated fluorescent microbeads facilitated RPE phagocytosis. Tubby and Tulp1, but not other family members, enhanced the uptake of membrane vesicles by RPE cells in synergy. Retinal membrane vesicles of Tubby mice and Tulp1−/− mice showed reduced activities for RPE phagocytosis, which were compensated by purified tubby and Tulp1, respectively. These data reveal a novel activity of tubby and Tulp1, and demonstrate that unbiased identification of eat-me signals by the broadly applicable strategy of ORF phage display can provide detailed insights into phagocyte biology.
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Can phage display be used as a tool to Functionally identify endogenous eat-me signals in phagocytosis?
Journal of biomolecular screening, 2009Co-Authors: Nora B. Caberoy, Yixiong ZhouAbstract:Removal of apoptotic cells and cellular debris by phagocytosis is essential for development, tissue homeostasis, and resolution of inflammation. Eat-me signals control the initiation of phagocytosis, holding a key to the understanding of phagocyte biology. Because of a lack of Functional Cloning strategy, eat-me signals are conventionally identified and characterized on a case-by-case basis. The feasibility of Functional Cloning of eat-me signals by phage display is investigated by characterizing the biological behavior of T7 phages displaying 2 well-known eat-me signals: growth arrest-specific gene 6 (Gas6) and milk fat globule-EGF8 (MFG-E8). Gas6-phage binds to all 3 known Gas6 receptors: Mer, Axl, and Tyro3 receptor tyrosine kinases. Gas6-phage and MFG-E8-phage are capable of binding to phagocytes and nonphagocytes. However, both phages stimulate phage uptake only in phagocytes, including macrophages, microglia, and retinal pigment epithelium cells, but not in nonphagocytes. Furthermore, Functional phage selection by phagocytosis in phagocytes enriches both Gas6-phage and MFG-E8-phage, suggesting that phage display can be used as a tool to Functionally identify unknown eat-me signals from phage display cDNA library.
Gary F. Merrill - One of the best experts on this subject based on the ideXlab platform.
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Thioredoxin Reductase-dependent Inhibition of MCB Cell Cycle Box Activity in Saccharomyces cerevisiae
Journal of Biological Chemistry, 1997Co-Authors: André K. Machado, Brian A Morgan, Gary F. MerrillAbstract:Abstract Mlu1 cell cycle box (MCB) elements are found near the start site of yeast genes expressed at G1/S. Basal promoters dependent on the elements for upstream activating sequence activity are inactive in Δswi6 yeast. Yeast were screened for mutations that activated MCB reporter genes in the absence of Swi6. The mutations identified a single complementation group. Functional Cloning revealed the mutations were alleles of theTRR1 gene encoding thioredoxin reductase. Although deletion of TRR1 activated MCB reporter genes, high copy expression did not suppress reporter gene activity. The trr1 mutations strongly (20-fold) stimulated MCB- and SCB (Swi4/Swi6 cell cycle box)-containing reporter genes, but also weakly (3-fold) stimulated reporter genes that lacked these elements. The trr1mutations did not affect the level or periodicity of three endogenous MCB gene mRNAs (TMP1, RNR1, andSWI4). Deletion of thioredoxin genes TRX1 andTRX2 recapitulated the stimulatory effect oftrr1 mutations on MCB reporter gene activity. Conditions expected to oxidize thioredoxin (exposure to H2O2) induced MCB gene expression, whereas conditions expected to conserve thioredoxin (exposure to hydroxyurea) inhibited MCB gene expression. The results suggest that thioredoxin oxidation contributes to MCB element activation and suggest a link between thioredoxin-oxidizing processes such as ribonucleotide reduction and cell cycle-specific gene transcription.
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Thioredoxin Reductase-dependent Inhibition of MCB Cell Cycle Box Activity in Saccharomyces cerevisiae
Journal of Biological Chemistry, 1997Co-Authors: André K. Machado, Brian A Morgan, Gary F. MerrillAbstract:Mlu1 cell cycle box (MCB) elements are found near the start site of yeast genes expressed at G1/S. Basal promoters dependent on the elements for upstream activating sequence activity are inactive in Deltaswi6 yeast. Yeast were screened for mutations that activated MCB reporter genes in the absence of Swi6. The mutations identified a single complementation group. Functional Cloning revealed the mutations were alleles of the TRR1 gene encoding thioredoxin reductase. Although deletion of TRR1 activated MCB reporter genes, high copy expression did not suppress reporter gene activity. The trr1 mutations strongly (20-fold) stimulated MCB- and SCB (Swi4/Swi6 cell cycle box)-containing reporter genes, but also weakly (3-fold) stimulated reporter genes that lacked these elements. The trr1 mutations did not affect the level or periodicity of three endogenous MCB gene mRNAs (TMP1, RNR1, and SWI4). Deletion of thioredoxin genes TRX1 and TRX2 recapitulated the stimulatory effect of trr1 mutations on MCB reporter gene activity. Conditions expected to oxidize thioredoxin (exposure to H2O2) induced MCB gene expression, whereas conditions expected to conserve thioredoxin (exposure to hydroxyurea) inhibited MCB gene expression. The results suggest that thioredoxin oxidation contributes to MCB element activation and suggest a link between thioredoxin-oxidizing processes such as ribonucleotide reduction and cell cycle-specific gene transcription.
Wei Li - One of the best experts on this subject based on the ideXlab platform.
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Unraveling the Molecular Mystery of Retinal Pigment Epithelium Phagocytosis
Advances in Experimental Medicine and Biology, 2011Co-Authors: Nora B. Caberoy, Wei LiAbstract:Retinal pigment epithelium (RPE) phagocytosis is essential for clearance of shed photoreceptor outer segments (POS) to prevent retinal degeneration. Despite the importance, our understanding of the molecular mechanisms of RPE phagocytosis is relatively limited. Phagocytosis ligands, receptors, and signaling cascades are traditionally identified on case-by-case basis with challenges. It is even more daunting to identify a new signaling pathway in the absence of any molecular probe. We developed a phagocytosis-based Functional Cloning strategy with open reading frame (ORF) phage display and identified tubby-like protein 1 (Tulp1) as a new phagocytosis ligand in an unbiased manner. We used Tulp1 as a molecular probe and characterized the well-known MerTK as the receptor of Tulp1. Tulp1 as a genuine MerTK ligand was independently validated by co-immunoprecipitation, MerTK autophosphorylation and MerTK-dependent intracellular signaling. Moreover, Tulp1 was characterized as a bridging molecule with a C-terminal phagocytosis prey-binding domain. These results suggest that Tulp1 is a genuine RPE phagocytosis ligand and that ORF phage display is a valid technology for unbiased identification of phagocytosis ligands, which will further lead to their receptors and signaling cascades. Thus, this new strategy will facilitate the unbiased mapping of molecular mechanisms of RPE and other phagocytes.
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Identification of tubby and tubby-like protein 1 as eat-me signals by phage display
Experimental Cell Research, 2009Co-Authors: Nora B. Caberoy, Dony Maiguel, Y. Kim, Wei LiAbstract:Abstract Phagocytosis is an important process for the removal of apoptotic cells or cellular debris. Eat-me signals control the initiation of phagocytosis and hold the key for in-depth understanding of its molecular mechanisms. However, because of difficulties to identify unknown eat-me signals, only a limited number of them have been identified and characterized. Using a newly developed Functional Cloning strategy of open reading frame (ORF) phage display, we identified nine putative eat-me signals, including tubby-like protein 1 (Tulp1). This further led to the elucidation of tubby as the second eat-me signal in the same protein family. Both proteins stimulated phagocytosis of retinal pigment epithelium (RPE) cells and macrophages. Tubby-conjugated fluorescent microbeads facilitated RPE phagocytosis. Tubby and Tulp1, but not other family members, enhanced the uptake of membrane vesicles by RPE cells in synergy. Retinal membrane vesicles of Tubby mice and Tulp1−/− mice showed reduced activities for RPE phagocytosis, which were compensated by purified tubby and Tulp1, respectively. These data reveal a novel activity of tubby and Tulp1, and demonstrate that unbiased identification of eat-me signals by the broadly applicable strategy of ORF phage display can provide detailed insights into phagocyte biology.
Jean-luc Jestin - One of the best experts on this subject based on the ideXlab platform.
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Functional Cloning by phage display
Biochimie, 2008Co-Authors: Jean-luc JestinAbstract:This review focusses on the isolation of proteins from genomic or cDNA expression products libraries displayed on phage. The use of phage display is highlighted for the characterization of binding proteins with diverse biological functions. Phage display is compared with another strategy, the yeast two-hybrid method. The combination of both strategies is especially powerful to eliminate false positives and to get information on the biochemical functions of proteins.
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A novel strategy for the Functional Cloning of enzymes using filamentous phage display: the case of nucleotidyl transferases
Nucleic acids research, 2002Co-Authors: Erika Brunet, Camille Chauvin, Valérie Choumet, Jean-luc JestinAbstract:In vitro selections for catalytic activity have been designed for the isolation of genes encoding enzymes from libraries of proteins displayed on filamentous phages. The proteins are generally expressed as C-terminal fusions with the N-terminus of the minor coat protein p3 for display on phages. As full-length cDNAs generally contain several stop codons near their 3′ end, this approach cannot be used for their expression on the surface of phages. Here we show that in vitro selection for catalytic activity is compatible with a system for expression of proteins as N-terminal fusions on the surface of bacteriophages. It is highlighted for the Stoffel fragment of Taq DNA polymerase I and makes use of (p3–Jun/Fos–Stoffel fragment) fusions. The efficiency of the selection is measured by an enrichment factor found to be about 55 for a phage polymerase versus a phage not expressing a polymerase. This approach could provide a method for the Functional Cloning of nucleotidyl transferases from cDNA libraries using filamentous phage display.
André K. Machado - One of the best experts on this subject based on the ideXlab platform.
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Thioredoxin Reductase-dependent Inhibition of MCB Cell Cycle Box Activity in Saccharomyces cerevisiae
Journal of Biological Chemistry, 1997Co-Authors: André K. Machado, Brian A Morgan, Gary F. MerrillAbstract:Abstract Mlu1 cell cycle box (MCB) elements are found near the start site of yeast genes expressed at G1/S. Basal promoters dependent on the elements for upstream activating sequence activity are inactive in Δswi6 yeast. Yeast were screened for mutations that activated MCB reporter genes in the absence of Swi6. The mutations identified a single complementation group. Functional Cloning revealed the mutations were alleles of theTRR1 gene encoding thioredoxin reductase. Although deletion of TRR1 activated MCB reporter genes, high copy expression did not suppress reporter gene activity. The trr1 mutations strongly (20-fold) stimulated MCB- and SCB (Swi4/Swi6 cell cycle box)-containing reporter genes, but also weakly (3-fold) stimulated reporter genes that lacked these elements. The trr1mutations did not affect the level or periodicity of three endogenous MCB gene mRNAs (TMP1, RNR1, andSWI4). Deletion of thioredoxin genes TRX1 andTRX2 recapitulated the stimulatory effect oftrr1 mutations on MCB reporter gene activity. Conditions expected to oxidize thioredoxin (exposure to H2O2) induced MCB gene expression, whereas conditions expected to conserve thioredoxin (exposure to hydroxyurea) inhibited MCB gene expression. The results suggest that thioredoxin oxidation contributes to MCB element activation and suggest a link between thioredoxin-oxidizing processes such as ribonucleotide reduction and cell cycle-specific gene transcription.
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Thioredoxin Reductase-dependent Inhibition of MCB Cell Cycle Box Activity in Saccharomyces cerevisiae
Journal of Biological Chemistry, 1997Co-Authors: André K. Machado, Brian A Morgan, Gary F. MerrillAbstract:Mlu1 cell cycle box (MCB) elements are found near the start site of yeast genes expressed at G1/S. Basal promoters dependent on the elements for upstream activating sequence activity are inactive in Deltaswi6 yeast. Yeast were screened for mutations that activated MCB reporter genes in the absence of Swi6. The mutations identified a single complementation group. Functional Cloning revealed the mutations were alleles of the TRR1 gene encoding thioredoxin reductase. Although deletion of TRR1 activated MCB reporter genes, high copy expression did not suppress reporter gene activity. The trr1 mutations strongly (20-fold) stimulated MCB- and SCB (Swi4/Swi6 cell cycle box)-containing reporter genes, but also weakly (3-fold) stimulated reporter genes that lacked these elements. The trr1 mutations did not affect the level or periodicity of three endogenous MCB gene mRNAs (TMP1, RNR1, and SWI4). Deletion of thioredoxin genes TRX1 and TRX2 recapitulated the stimulatory effect of trr1 mutations on MCB reporter gene activity. Conditions expected to oxidize thioredoxin (exposure to H2O2) induced MCB gene expression, whereas conditions expected to conserve thioredoxin (exposure to hydroxyurea) inhibited MCB gene expression. The results suggest that thioredoxin oxidation contributes to MCB element activation and suggest a link between thioredoxin-oxidizing processes such as ribonucleotide reduction and cell cycle-specific gene transcription.
