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Martin A Schwartz - One of the best experts on this subject based on the ideXlab platform.
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spider silk peptide is a compact linear nanospring ideal for intracellular tension sensing
Nano Letters, 2016Co-Authors: Michael D Brenner, Ruobo Zhou, Daniel E Conway, Luca Lanzano, Enrico Gratton, Martin A Schwartz, Taekjip HaAbstract:Recent development and applications of calibrated, fluorescence resonance energy transfer (FRET)-based tension sensors have led to a new understanding of single molecule mechanotransduction in a number of biological systems. To expand the range of accessible forces, we systematically measured FRET versus force trajectories for 25, 40, and 50 Amino Acid peptide Repeats derived from spider silk. Single molecule fluorescence-force spectroscopy showed that the peptides behaved as linear springs instead of the nonlinear behavior expected for a disordered polymer. Our data are consistent with a compact, rodlike structure that measures 0.26 nm per 5 Amino Acid Repeat that can stretch by 500% while maintaining linearity, suggesting that the remarkable elasticity of spider silk proteins may in part derive from the properties of individual chains. We found the shortest peptide to have the widest range of force sensitivity: between 2 pN and 11 pN. Live cell imaging of the three tension sensor constructs inserted int...
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spider silk peptide is a compact linear nanospring ideal for intracellular tension sensing
Nano Letters, 2016Co-Authors: Michael D Brenner, Ruobo Zhou, Daniel E Conway, Luca Lanzano, Enrico Gratton, Martin A SchwartzAbstract:Recent development and applications of calibrated, fluorescence resonance energy transfer (FRET)-based tension sensors have led to a new understanding of single molecule mechanotransduction in a number of biological systems. To expand the range of accessible forces, we systematically measured FRET versus force trajectories for 25, 40, and 50 Amino Acid peptide Repeats derived from spider silk. Single molecule fluorescence-force spectroscopy showed that the peptides behaved as linear springs instead of the nonlinear behavior expected for a disordered polymer. Our data are consistent with a compact, rodlike structure that measures 0.26 nm per 5 Amino Acid Repeat that can stretch by 500% while maintaining linearity, suggesting that the remarkable elasticity of spider silk proteins may in part derive from the properties of individual chains. We found the shortest peptide to have the widest range of force sensitivity: between 2 pN and 11 pN. Live cell imaging of the three tension sensor constructs inserted into vinculin showed similar force values around 2.4 pN. We also provide a lookup table for force versus intracellular FRET for all three constructs.
Bruce S. Klein - One of the best experts on this subject based on the ideXlab platform.
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immunogenicity and protective efficacy of the wi 1 adhesin of blastomyces dermatitidis
Infection and Immunity, 1998Co-Authors: Marcel Wuthrich, Wunling Chang, Bruce S. KleinAbstract:People infected with Blastomyces dermatitidis develop strong immunity to the yeast surface adhesin WI-1, including antibody responses to the adhesive domain, a 25-Amino-Acid Repeat, and cellular responses to the N terminus. We studied the immunogenicity of WI-1 and the ability of anti-WI-1 immune responses to protect against lethal pulmonary infection in mice. WI-1 immunization, given in Freund's adjuvant subcutaneously in two doses 2 weeks apart, evoked delayed hypersensitivity responses in a concentration-dependent manner. Immunized mice also had anti-WI-1 antibody responses, with titers reaching an endpoint dilution of approximately 1:800,000. Anti-WI-1 immunoglobulin G (IgG) antibody subclasses were IgG1 > IgG2b > IgG2a > IgG3, indicating a mixed T helper 1 and T helper 2 immune response. In protection experiments, WI-1 immunization significantly prolonged the survival of C57BL/6 and BALB/c mice compared to controls following intranasal administration of a lethal dose of B. dermatitidis yeasts (Kaplan-Meier survival curve P values of 0.027 to 0.0002) and also protected a proportion of the animals from death due to progressive pulmonary blastomycosis. Taken together, our results suggest that administration of WI-1 raises antibody and cell-mediated immune responses, which enhance resistance against pulmonary infection with B. dermatitidis. Mechanisms of vaccine-induced resistance require further investigation.
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purification and characterization of the major antigen wi 1 from blastomyces dermatitidis yeasts and immunological comparison with a antigen
Infection and Immunity, 1994Co-Authors: Bruce S. Klein, J M JonesAbstract:The lack of well-defined antigens from Blastomyces dermatitidis has hampered the ability to reliably diagnose human infection and study the immunobiology of blastomycosis. We recently discovered a novel surface protein on B. dermatitidis yeasts, designated WI-1, and demonstrated it to be a key antigenic target of humoral and cellular responses during infection. In the present article, we purified and characterized WI-1 and compared it immunologically with the only Blastomyces antigen commercially available, A antigen. WI-1 was purified by high-performance liquid chromatography over a DEAE-cellulose column. It eluted from the column at a point on the salt gradient corresponding to 460 to 490 mM NaCl, reflecting its Acidic pI of approximately equal to 5.2. Purified WI-1 had a molecular mass of 120 kDa and contained a large amount of cysteine (85 residues) and aromatic Amino Acids but undetectable carbohydrate. In contrast, A antigen had a molecular mass of 135 kDa and contained 37% carbohydrate. Immunological comparison of the two antigens showed that, when radiolabeled, WI-1 was more reactive with anti-Blastomyces antisera than A antigen but did not cross-react with anti-Histoplasma antisera. Proteinase digestion of WI-1 eliminated its recognition by anti-WI-1 and anti-Blastomyces antisera. Proteinase treatment of A antigen had no effect on its recognition by anti-Blastomyces or anti-Histoplasma antisera, but periodate treatment abolished recognition by anti-Histoplasma antisera, indicating that the cross-reactive determinant(s) of A antigen is displayed on the accompanying carbohydrate. In further studies, anti-WI-1 antiserum reacted with A antigen and, conversely, anti-A antiserum and monoclonal antibodies (MAbs) reacted with WI-1, indicating a shared determinant on the two antigens. A recombinant 25-Amino-Acid Repeat, recently cloned from WI-1 and found to be the major target of antibody recognition of WI-1, reacted strongly with anti-A antiserum and MAbs. In MAb competition tests, MAbs specific for the 25-residue Repeat abolished binding of anti-A antiserum to A antigen. In antigen inhibition tests, the recombinant Repeat abolished binding of anti-A antiserum to A antigen. These results demonstrate that the Repeat is the major site of antibody recognition of both WI-1 and A antigen and that the recombinant, nonglycosylated peptide could replace either native antigen in formatting better diagnostic tests for blastomycosis. Moreover, they suggest that producing fungal protein antigens as nonglycosylated peptides in a procaryotic expression system may circumvent problems of antigen cross-reactivity that are due to posttranslational modification.
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immunologic recognition of a 25 Amino Acid Repeat arrayed in tandem on a major antigen of blastomyces dermatitidis
Journal of Clinical Investigation, 1993Co-Authors: Bruce S. Klein, Laura H Hogan, J M JonesAbstract:A 120-kD glycoprotein antigen abundantly expressed on Blastomyces dermatitidis yeasts is a target of cellular and humoral immune responses in human infection. To investigate the antigen and immune response more carefully at the molecular level, we screened an expression library from B. dermatitidis to identify clones that encode this antigen, designated WI-1. A 942-bp cDNA was isolated by immunologic screening with polyclonal, rabbit anti-WI-1 antiserum. Northern hybridization analysis showed that the cDNA hybridized to yeast message approximately equal to 3.9 kb. DNA and deduced protein sequence analysis of the clone demonstrated a 25-Amino Acid Repeat arrayed in tandem, present in 4.5 copies near the 5' end, and rich in predicted antigenic epitopes. Further analysis showed strong homology in these tandem Repeats with invasin, an adhesin of Yersiniae. Cloned cDNA was used to express a 30-kD fusion protein strongly recognized in western blots by rabbit anti-WI-1 antiserum, and by sera from all 35 blastomycosis patients studied. The fusion protein product of subcloned cDNA encoding only the tandem Repeat also was strongly recognized in western blots by sera from the 35 blastomycosis patients, but not by sera from 10 histoplasmosis and 5 coccidioidomycosis patients. An antigen-inhibition radioimmunoassay showed that the tandem Repeat alone completely eliminated rabbit and human anti-WI-1 antibody binding to radiolabeled native WI-1. From these results, we conclude that the 25-Amino Acid Repeat of WI-1 displays an immunodominant B cell epitope, and that the carboxyl-terminus of the molecule exhibits an architecture that may promote adhesion of Blastomyces yeasts to host cells or extracellular matrix proteins and ultimately provide a clearer picture of the molecular pathogenesis of blastomycosis.
Paul Polakis - One of the best experts on this subject based on the ideXlab platform.
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a drosophila homolog of the tumor suppressor gene adenomatous polyposis coli down regulates β catenin but its zygotic expression is not essential for the regulation of armadillo
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Shigemi Hayashi, Bonnee Rubinfeld, Brian Souza, Paul Polakis, Eric Wieschaus, Arnold J LevineAbstract:Mutations in the adenomatous polyposis coli gene (which encodes a protein called APC) are associated with the formation of intestinal polyps and colon cancers. To facilitate the functional study of APC we have isolated its Drosophila homolog (D-APC) by screening an expression library with an antibody against human APC. The isolated cDNA encodes a predicted 2416-Amino Acid protein containing significant homology to multiple domains of mammalian APCs. D-APC has seven complete armadillo Repeats with 60% identity to its human homolog, one β-catenin binding site, and up to 7 copies of a 20-Amino Acid Repeat with the average of 50% identity to human APC at Amino Acid level. D-APC, like its human counterpart, also contains a basic domain. Expression of the domain of D-APC homologous to the region required for β-catenin down-regulation resulted in down-regulation of intracellular β-catenin in a mammalian cell line. This same region bound to the Armadillo (Arm) protein, in vitro, the Drosophila homolog of β-catenin. D-APC RNA and protein expression is very low, if detectable at all, during stages when Arm protein accumulates in a striped pattern in the epidermis of the Drosophila embryos. Removing zygotic D-APC expression did not alter Arm protein distribution, and the final cuticle pattern was not affected significantly. As observed in the rodent, high levels of D-APC expression have been detected in the central nervous system, suggesting a role for D-APC in central nervous system formation.
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The APC Protein and E-cadherin Form Similar but Independent Complexes with α-Catenin, β-Catenin, and Plakoglobin
Journal of Biological Chemistry, 1995Co-Authors: Bonnee Rubinfeld, Brian Souza, Iris Albert, Susan Munemitsu, Paul PolakisAbstract:Abstract The tumor suppressor APC protein associates with the cadherin-binding proteins α- and β-catenin. To examine the relationship between cadherin, catenins, and APC, we have tested combinatorial protein-protein interactions in vivo, using a yeast two-hybrid system, and in vitro, using purified proteins. β-Catenin directly binds to APC at high and low affinity sites. α-Catenin cannot directly bind APC but associates with it by binding to β-catenin. Plakoglobin, also known as γ-catenin, directly binds to both APC and α-catenin and also to the APC-β-catenin complex, but not directly to β-catenin. β-Catenin binds to multiple independent regions of APC, some of which include a previously identified consensus motif and others which contain the centrally located 20 Amino Acid Repeat sequences. The APC binding site on β-catenin may be discontinuous since neither the carboxyl- nor Amino-terminal halves of β-catenin will independently associate with APC, although the Amino-terminal half independently binds α-catenin. The catenins bind to APC and E-cadherin in a similar fashion, but APC and E-cadherin do not associate with each other either in the presence or absence of catenins. Thus, APC forms distinct heteromeric complexes containing combinations of α-catenin, β-catenin, and plakoglobin which are independent from the cadherin-catenin complexes.
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the apc protein and e cadherin form similar but independent complexes with alpha catenin beta catenin and plakoglobin
Journal of Biological Chemistry, 1995Co-Authors: Bonnee Rubinfeld, Brian Souza, Iris Albert, Susan Munemitsu, Paul PolakisAbstract:The tumor suppressor APC protein associates with the cadherin-binding proteins alpha- and beta-catenin. To examine the relationship between cadherin, catenins, and APC, we have tested combinatorial protein-protein interactions in vivo, using a yeast two-hybrid system, and in vitro, using purified proteins. beta-Catenin directly binds to APC at high and low affinity sites. alpha-Catenin cannot directly bind APC but associates with it by binding to beta-catenin. Plakoglobin, also known as gamma-catenin, directly binds to both APC and alpha-catenin and also to the APC-beta-catenin complex, but not directly to beta-catenin. beta-Catenin binds to multiple independent regions of APC, some of which include a previously identified consensus motif and others which contain the centrally located 20 Amino Acid Repeat sequences. The APC binding site on beta-catenin may be discontinuous since neither the carboxyl- nor Amino-terminal halves of beta-catenin will independently associate with APC, although the Amino-terminal half independently binds alpha-catenin. The catenins bind to APC and E-cadherin in a similar fashion, but APC and E-cadherin do not associate with each other either in the presence or absence of catenins. Thus, APC forms distinct heteromeric complexes containing combinations of alpha-catenin, beta-catenin, and plakoglobin which are independent from the cadherin-catenin complexes.
Michael D Brenner - One of the best experts on this subject based on the ideXlab platform.
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spider silk peptide is a compact linear nanospring ideal for intracellular tension sensing
Nano Letters, 2016Co-Authors: Michael D Brenner, Ruobo Zhou, Daniel E Conway, Luca Lanzano, Enrico Gratton, Martin A Schwartz, Taekjip HaAbstract:Recent development and applications of calibrated, fluorescence resonance energy transfer (FRET)-based tension sensors have led to a new understanding of single molecule mechanotransduction in a number of biological systems. To expand the range of accessible forces, we systematically measured FRET versus force trajectories for 25, 40, and 50 Amino Acid peptide Repeats derived from spider silk. Single molecule fluorescence-force spectroscopy showed that the peptides behaved as linear springs instead of the nonlinear behavior expected for a disordered polymer. Our data are consistent with a compact, rodlike structure that measures 0.26 nm per 5 Amino Acid Repeat that can stretch by 500% while maintaining linearity, suggesting that the remarkable elasticity of spider silk proteins may in part derive from the properties of individual chains. We found the shortest peptide to have the widest range of force sensitivity: between 2 pN and 11 pN. Live cell imaging of the three tension sensor constructs inserted int...
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spider silk peptide is a compact linear nanospring ideal for intracellular tension sensing
Nano Letters, 2016Co-Authors: Michael D Brenner, Ruobo Zhou, Daniel E Conway, Luca Lanzano, Enrico Gratton, Martin A SchwartzAbstract:Recent development and applications of calibrated, fluorescence resonance energy transfer (FRET)-based tension sensors have led to a new understanding of single molecule mechanotransduction in a number of biological systems. To expand the range of accessible forces, we systematically measured FRET versus force trajectories for 25, 40, and 50 Amino Acid peptide Repeats derived from spider silk. Single molecule fluorescence-force spectroscopy showed that the peptides behaved as linear springs instead of the nonlinear behavior expected for a disordered polymer. Our data are consistent with a compact, rodlike structure that measures 0.26 nm per 5 Amino Acid Repeat that can stretch by 500% while maintaining linearity, suggesting that the remarkable elasticity of spider silk proteins may in part derive from the properties of individual chains. We found the shortest peptide to have the widest range of force sensitivity: between 2 pN and 11 pN. Live cell imaging of the three tension sensor constructs inserted into vinculin showed similar force values around 2.4 pN. We also provide a lookup table for force versus intracellular FRET for all three constructs.
Bonnee Rubinfeld - One of the best experts on this subject based on the ideXlab platform.
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a drosophila homolog of the tumor suppressor gene adenomatous polyposis coli down regulates β catenin but its zygotic expression is not essential for the regulation of armadillo
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Shigemi Hayashi, Bonnee Rubinfeld, Brian Souza, Paul Polakis, Eric Wieschaus, Arnold J LevineAbstract:Mutations in the adenomatous polyposis coli gene (which encodes a protein called APC) are associated with the formation of intestinal polyps and colon cancers. To facilitate the functional study of APC we have isolated its Drosophila homolog (D-APC) by screening an expression library with an antibody against human APC. The isolated cDNA encodes a predicted 2416-Amino Acid protein containing significant homology to multiple domains of mammalian APCs. D-APC has seven complete armadillo Repeats with 60% identity to its human homolog, one β-catenin binding site, and up to 7 copies of a 20-Amino Acid Repeat with the average of 50% identity to human APC at Amino Acid level. D-APC, like its human counterpart, also contains a basic domain. Expression of the domain of D-APC homologous to the region required for β-catenin down-regulation resulted in down-regulation of intracellular β-catenin in a mammalian cell line. This same region bound to the Armadillo (Arm) protein, in vitro, the Drosophila homolog of β-catenin. D-APC RNA and protein expression is very low, if detectable at all, during stages when Arm protein accumulates in a striped pattern in the epidermis of the Drosophila embryos. Removing zygotic D-APC expression did not alter Arm protein distribution, and the final cuticle pattern was not affected significantly. As observed in the rodent, high levels of D-APC expression have been detected in the central nervous system, suggesting a role for D-APC in central nervous system formation.
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The APC Protein and E-cadherin Form Similar but Independent Complexes with α-Catenin, β-Catenin, and Plakoglobin
Journal of Biological Chemistry, 1995Co-Authors: Bonnee Rubinfeld, Brian Souza, Iris Albert, Susan Munemitsu, Paul PolakisAbstract:Abstract The tumor suppressor APC protein associates with the cadherin-binding proteins α- and β-catenin. To examine the relationship between cadherin, catenins, and APC, we have tested combinatorial protein-protein interactions in vivo, using a yeast two-hybrid system, and in vitro, using purified proteins. β-Catenin directly binds to APC at high and low affinity sites. α-Catenin cannot directly bind APC but associates with it by binding to β-catenin. Plakoglobin, also known as γ-catenin, directly binds to both APC and α-catenin and also to the APC-β-catenin complex, but not directly to β-catenin. β-Catenin binds to multiple independent regions of APC, some of which include a previously identified consensus motif and others which contain the centrally located 20 Amino Acid Repeat sequences. The APC binding site on β-catenin may be discontinuous since neither the carboxyl- nor Amino-terminal halves of β-catenin will independently associate with APC, although the Amino-terminal half independently binds α-catenin. The catenins bind to APC and E-cadherin in a similar fashion, but APC and E-cadherin do not associate with each other either in the presence or absence of catenins. Thus, APC forms distinct heteromeric complexes containing combinations of α-catenin, β-catenin, and plakoglobin which are independent from the cadherin-catenin complexes.
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the apc protein and e cadherin form similar but independent complexes with alpha catenin beta catenin and plakoglobin
Journal of Biological Chemistry, 1995Co-Authors: Bonnee Rubinfeld, Brian Souza, Iris Albert, Susan Munemitsu, Paul PolakisAbstract:The tumor suppressor APC protein associates with the cadherin-binding proteins alpha- and beta-catenin. To examine the relationship between cadherin, catenins, and APC, we have tested combinatorial protein-protein interactions in vivo, using a yeast two-hybrid system, and in vitro, using purified proteins. beta-Catenin directly binds to APC at high and low affinity sites. alpha-Catenin cannot directly bind APC but associates with it by binding to beta-catenin. Plakoglobin, also known as gamma-catenin, directly binds to both APC and alpha-catenin and also to the APC-beta-catenin complex, but not directly to beta-catenin. beta-Catenin binds to multiple independent regions of APC, some of which include a previously identified consensus motif and others which contain the centrally located 20 Amino Acid Repeat sequences. The APC binding site on beta-catenin may be discontinuous since neither the carboxyl- nor Amino-terminal halves of beta-catenin will independently associate with APC, although the Amino-terminal half independently binds alpha-catenin. The catenins bind to APC and E-cadherin in a similar fashion, but APC and E-cadherin do not associate with each other either in the presence or absence of catenins. Thus, APC forms distinct heteromeric complexes containing combinations of alpha-catenin, beta-catenin, and plakoglobin which are independent from the cadherin-catenin complexes.