The Experts below are selected from a list of 2616 Experts worldwide ranked by ideXlab platform
Richard J. Simpson - One of the best experts on this subject based on the ideXlab platform.
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solubilisation of the Armadillo Repeat protein β catenin using a zwitterionic detergent allows resolution of phosphorylated forms by 2de
Electrophoresis, 2012Co-Authors: Meredith J Layton, Nicole Church, Maree C Faux, Robert J. A. Goode, Eugene A. Kapp, Antony W. Burgess, Richard J. SimpsonAbstract:β-catenin is a member of the Armadillo Repeat family of proteins and has important functions in cell-cell adhesion and Wnt signalling. Different protein species of β-catenin have been shown to exist in the cell and the relative proportions of these species are altered upon stimulation of cells with Wnt-3a (Gottardi and Gumbiner, 2004). In order to determine whether posttranslational modifications (PTMs) of β-catenin underlie these different protein species, we have used 2DE separation and immunoblotting with an antibody specific for β-catenin. High-resolution separation of differentially modified species of β-catenin in 2DE required the addition of ASB-16, a zwitterionic detergent that can solubilise integral membrane proteins. ASB-16 was also necessary for focussing of other Armadillo Repeat proteins, such as γ-catenin and p120-catenin. 2DE using ASB-16 allowed detection of a previously unreported phosphorylation site in the transcriptionally active form of β-catenin that binds to GST-Tcf in response to Wnt signalling.
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Solubilisation of the Armadillo‐Repeat protein β‐catenin using a zwitterionic detergent allows resolution of phosphorylated forms by 2DE
Electrophoresis, 2012Co-Authors: Meredith J Layton, Nicole Church, Maree C Faux, Robert J. A. Goode, Eugene A. Kapp, Antony W. Burgess, Richard J. SimpsonAbstract:β-catenin is a member of the Armadillo Repeat family of proteins and has important functions in cell-cell adhesion and Wnt signalling. Different protein species of β-catenin have been shown to exist in the cell and the relative proportions of these species are altered upon stimulation of cells with Wnt-3a (Gottardi and Gumbiner, 2004). In order to determine whether posttranslational modifications (PTMs) of β-catenin underlie these different protein species, we have used 2DE separation and immunoblotting with an antibody specific for β-catenin. High-resolution separation of differentially modified species of β-catenin in 2DE required the addition of ASB-16, a zwitterionic detergent that can solubilise integral membrane proteins. ASB-16 was also necessary for focussing of other Armadillo Repeat proteins, such as γ-catenin and p120-catenin. 2DE using ASB-16 allowed detection of a previously unreported phosphorylation site in the transcriptionally active form of β-catenin that binds to GST-Tcf in response to Wnt signalling.
Enhua Wang - One of the best experts on this subject based on the ideXlab platform.
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N-Terminal 1–54 Amino Acid Sequence and Armadillo Repeat Domain Are Indispensable for P120-Catenin Isoform 1A in Regulating E-Cadherin
2016Co-Authors: Yuan Miao, Yang Liu, Guiyang Jiang, Maggie Stoecker, Endi Wang, Enhua WangAbstract:P120-catenin (p120ctn) exerts important roles in regulating E-cadherin and invasiveness in cancer cells. However, the mechanisms by which p120ctn isoforms 1 and 3 modulate E-cadherin expression are poorly understood. In the current study, HBE, H460, SPC and LTE cell lines were used to examine the effects of p120ctn isoforms 1A and 3A on E-cadherin expression and cell invasiveness. E-cadherin was localized on the cell membrane of HBE and H460 cells, while it was confined to the cytoplasm in SPC and LTE cells. Depletion of endogenous p120ctn resulted in reduced E-cadherin expression; however, p120ctn ablation showed opposite effects on invasiveness in the cell lines by decreasing invasiveness in SPC and LTE cells and increasing it in HBE and H460 cells. Restitution of 120ctn isoform 1A restored E-cadherin on the cell membrane and blocked cell invasiveness in H460 and HBE cells, while it restored cytoplasmic E-cadherin and enhanced cell invasiveness in SPC and LTE cells. P120ctn isoform 3A increased the invasiveness in all four cell lines despite the lack of effect on E-cadherin expression, suggesting a regulatory pathway independent of E-cadherin. Moreover, five p120ctn isoform 1A deletion mutants were constructed and expressed in H460 and SPC cells. The results showed that only the M4 mutant, which contains N-terminal 1–54 amino acids and the Armadillo Repeat domain, was functional in regulating E-cadherin and cell invasiveness, as observed in p120ctn isoform 1A. In conclusion, the N-terminal 1–54 amino acid sequence and Armadillo Repeat domain of p120ctn isoform 1A are indispensable for regulating E-cadherin protein. P120ctn isofor
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N-Terminal 1–54 Amino Acid Sequence and Armadillo Repeat Domain Are Indispensable for P120-Catenin Isoform 1A in Regulating E-Cadherin
PloS one, 2012Co-Authors: Yuan Miao, Yang Liu, Guiyang Jiang, Maggie Stoecker, Endi Wang, Enhua WangAbstract:P120-catenin (p120ctn) exerts important roles in regulating E-cadherin and invasiveness in cancer cells. However, the mechanisms by which p120ctn isoforms 1 and 3 modulate E-cadherin expression are poorly understood. In the current study, HBE, H460, SPC and LTE cell lines were used to examine the effects of p120ctn isoforms 1A and 3A on E-cadherin expression and cell invasiveness. E-cadherin was localized on the cell membrane of HBE and H460 cells, while it was confined to the cytoplasm in SPC and LTE cells. Depletion of endogenous p120ctn resulted in reduced E-cadherin expression; however, p120ctn ablation showed opposite effects on invasiveness in the cell lines by decreasing invasiveness in SPC and LTE cells and increasing it in HBE and H460 cells. Restitution of 120ctn isoform 1A restored E-cadherin on the cell membrane and blocked cell invasiveness in H460 and HBE cells, while it restored cytoplasmic E-cadherin and enhanced cell invasiveness in SPC and LTE cells. P120ctn isoform 3A increased the invasiveness in all four cell lines despite the lack of effect on E-cadherin expression, suggesting a regulatory pathway independent of E-cadherin. Moreover, five p120ctn isoform 1A deletion mutants were constructed and expressed in H460 and SPC cells. The results showed that only the M4 mutant, which contains N-terminal 1-54 amino acids and the Armadillo Repeat domain, was functional in regulating E-cadherin and cell invasiveness, as observed in p120ctn isoform 1A. In conclusion, the N-terminal 1-54 amino acid sequence and Armadillo Repeat domain of p120ctn isoform 1A are indispensable for regulating E-cadherin protein. P120ctn isoform 1A exerts opposing effects on cell invasiveness, corresponding to the subcellular localization of E-cadherin.
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n terminal 1 54 amino acid sequence and Armadillo Repeat domain are indispensable for p120 catenin isoform 1a in regulating e cadherin
PLOS ONE, 2012Co-Authors: Yuan Miao, Yang Liu, Guiyang Jiang, Maggie Stoecker, Endi Wang, Enhua WangAbstract:P120-catenin (p120ctn) exerts important roles in regulating E-cadherin and invasiveness in cancer cells. However, the mechanisms by which p120ctn isoforms 1 and 3 modulate E-cadherin expression are poorly understood. In the current study, HBE, H460, SPC and LTE cell lines were used to examine the effects of p120ctn isoforms 1A and 3A on E-cadherin expression and cell invasiveness. E-cadherin was localized on the cell membrane of HBE and H460 cells, while it was confined to the cytoplasm in SPC and LTE cells. Depletion of endogenous p120ctn resulted in reduced E-cadherin expression; however, p120ctn ablation showed opposite effects on invasiveness in the cell lines by decreasing invasiveness in SPC and LTE cells and increasing it in HBE and H460 cells. Restitution of 120ctn isoform 1A restored E-cadherin on the cell membrane and blocked cell invasiveness in H460 and HBE cells, while it restored cytoplasmic E-cadherin and enhanced cell invasiveness in SPC and LTE cells. P120ctn isoform 3A increased the invasiveness in all four cell lines despite the lack of effect on E-cadherin expression, suggesting a regulatory pathway independent of E-cadherin. Moreover, five p120ctn isoform 1A deletion mutants were constructed and expressed in H460 and SPC cells. The results showed that only the M4 mutant, which contains N-terminal 1-54 amino acids and the Armadillo Repeat domain, was functional in regulating E-cadherin and cell invasiveness, as observed in p120ctn isoform 1A. In conclusion, the N-terminal 1-54 amino acid sequence and Armadillo Repeat domain of p120ctn isoform 1A are indispensable for regulating E-cadherin protein. P120ctn isoform 1A exerts opposing effects on cell invasiveness, corresponding to the subcellular localization of E-cadherin.
Peter A Kanetsky - One of the best experts on this subject based on the ideXlab platform.
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chemotherapy refractory testicular germ cell tumor is associated with a variant in Armadillo Repeat gene deleted in velco cardio facial syndrome arvcf
Frontiers in Endocrinology, 2012Co-Authors: Chunkit Fung, David J Vaughn, Nandita Mitra, Stephanie L Ciosek, Saran Vardhanabhuti, Katherine L Nathanson, Peter A KanetskyAbstract:Introduction: There is evidence that inherited genetic variation affects both testicular germ cell tumor (TGCT) treatment outcome and risks of late-complications arising from cisplatin-based chemotherapy. Using a candidate gene approach, we examined associations of three genes involved in the cisplatin metabolism pathway, GSTP1, COMT, and TPMT, with TGCT outcome and cisplatin-induced neurotoxicity. Material and Methods: Our study population includes a subset of patients (n=137) from a genome-wide association study at the University of Pennsylvania that evaluates inherited genetic susceptibility to TGCT. All patients in our study had at least one course of cisplatin-based chemotherapy with at least one year of follow up. A total of 90 markers in GSTP1, COMT and TPMT and their adjacent genomic regions (± 20 kb) were analyzed for associations with refractory TGCT after first course of chemotherapy, progression-free survival (PFS), overall survival (OS), peripheral neuropathy, and ototoxicity. Results: After adjustment for multiple comparisons, one SNP, rs2073743, in the flanking region (± 20 kb) of COMT was associated with refractory TGCT after initial chemotherapy. This SNP lies within the intron region of the Armadillo Repeat gene deleted in Velco-Cardio-Facial syndrome (ARVCF). The G allele of rs2073743 predisposed patients to refractory disease with a relative risk of 2.6 (95% CI 1.1, 6.3; P=0.03). Assuming recessive inheritance, patients with the GG genotype had 22.7 times higher risk (95% CI 3.3, 155.8; P=0.04) of developing refractory disease when compared to those with the GC or CC genotypes. We found no association of our candidate genes with peripheral neuropathy, ototoxicity, PFS and OS. Discussion: This is the first study to suggest that germline genetic variants of ARVCF may affect TGCT outcome. The result of this study is hypothesis generating and should be validated in future studies.
Andreas Plückthun - One of the best experts on this subject based on the ideXlab platform.
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curvature of designed Armadillo Repeat proteins allows modular peptide binding
Journal of Structural Biology, 2018Co-Authors: Simon Hansen, Christian Reichen, Peer R. E. Mittl, Patrick Ernst, Sebastian L B Konig, Christina Ewald, Daniel Nettels, Benjamin Schuler, Andreas PlückthunAbstract:Designed Armadillo Repeat proteins (dArmRPs) were developed to create a modular peptide binding technology where each of the structural Repeats binds two residues of the target peptide. An essential prerequisite for such a technology is a dArmRP geometry that matches the peptide bond length. To this end, we determined a large set (n=27) of dArmRP X-ray structures, of which 12 were previously unpublished, to calculate curvature parameters that define their geometry. Our analysis shows that consensus dArmRPs exhibit curvatures close to the optimal range for modular peptide recognition. Binding of peptide ligands can induce a curvature within the desired range, as confirmed by single-molecule FRET experiments in solution. On the other hand, computationally designed ArmRPs, where side chains have been chosen with the intention to optimally fit into a geometrically optimized backbone, turned out to be more divergent in reality, and thus not suitable for continuous peptide binding. Furthermore, we show that the formation of a crystal lattice can induce small but significant deviations from the curvature adopted in solution, which can interfere with the evaluation of Repeat protein scaffolds when high accuracy is required. This study corroborates the suitability of consensus dArmRPs as a scaffold for the development of modular peptide binders.
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MAX randomisation: designed, non-degenerate saturation mutagenesis of Armadillo Repeat proteins.
2018Co-Authors: Anupama Chembath, Andreas Plückthun, Ben Wagstaffe, Yvonne Stark, Anna V. HineAbstract:Non-degenerate saturation mutagenesis is critical to library composition both in terms of library size and amino acid representation. Unlike conventional methodologies,non-degeneracy permits equal representation of all encoded amino acids and effectively eliminates termination codons from saturated positions. We have previously described both ‘MAX’ randomisation, which saturates physically separated codons and ‘ProxiMAX’ randomisation, which saturates contiguous codons. Both allow the additional advantage of encoding only required amino acids without reference to codon sequence, but their methodologies are quite different. Whilst MAX randomisation employs a manual process of selectional hybridisation between individual oligonucleotides and a conventionally-randomised template, ProxiMAX relies on saturation cycling; Repeated cycles of blunt-ended ligation, Type IIS restriction and PCR amplification. MAX randomisation is thus typically employed in the research laboratory to engineer active sites of enzymes (or α-helices within other proteins), whilst ProxiMAX is now a chiefly commercial, automated process employed in antibody engineering. Here we present the application of MAX randomisation to engineer libraries of Armadillo Repeat Proteins (ArmRPs), α-helical proteins that selectively bind extended peptides. We have utilised the MAX randomisation technique to engineer ArmRPs for the generation of gene libraries encoding multiple Repeat modules, saturating seven positions and encoding between 4 and 18 amino acids within each location, achieving excellent correlation between the design and observed specifications. We also present early developments to extend MAX randomisation into the realm of multiple contiguous codons.
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Structures of designed Armadillo Repeat proteins binding to peptides fused to globular domains.
Protein science : a publication of the Protein Society, 2017Co-Authors: Simon Hansen, Chaithanya Madhurantakam, Peer R. E. Mittl, Jonathan D. Kiefer, Andreas PlückthunAbstract:Designed Armadillo Repeat proteins (dArmRP) are α-helical solenoid Repeat proteins with an extended peptide binding groove that were engineered to develop a generic modular technology for peptide recognition. In this context, the term "peptide" not only denotes a short unstructured chain of amino acids, but also an unstructured region of a protein, as they occur in termini, loops, or linkers between folded domains. Here we report two crystal structures of dArmRPs, in complex with peptides fused either to the N-terminus of Green Fluorescent Protein or to the C-terminus of a phage lambda protein D. These structures demonstrate that dArmRPs bind unfolded peptides in the intended conformation also when they constitute unstructured parts of folded proteins, which greatly expands possible applications of the dArmRP technology. Nonetheless, the structures do not fully reflect the binding behavior in solution, that is, some binding sites remain unoccupied in the crystal and even unexpected peptide residues appear to be bound. We show how these differences can be explained by restrictions of the crystal lattice or the composition of the crystallization solution. This illustrates that crystal structures have to be interpreted with caution when protein-peptide interactions are characterized, and should always be correlated with measurements in solution.
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Structures of designed Armadillo-Repeat proteins show propagation of inter-Repeat interface effects
Acta Crystallographica Section D-biological Crystallography, 2016Co-Authors: Christian Reichen, Simon Hansen, Chaithanya Madhurantakam, Markus G. Grütter, Andreas Plückthun, Peer R. E. MittlAbstract:The Armadillo Repeat serves as a scaffold for the development of modular peptide-recognition modules. In order to develop such a system, three crystal structures of designed Armadillo-Repeat proteins with third-generation N-caps (YIII-type), four or five internal Repeats (M-type) and second-generation C-caps (AII-type) were determined at 1.8 A (His-YIIIM4AII), 2.0 A (His-YIIIM5AII) and 1.95 A (YIIIM5AII) resolution and compared with those of variants with third-generation C-caps. All constructs are full consensus designs in which the internal Repeats have exactly the same sequence, and hence identical conformations of the internal Repeats are expected. The N-cap and internal Repeats M1 to M3 are indeed extremely similar, but the comparison reveals structural differences in internal Repeats M4 and M5 and the C-cap. These differences are caused by long-range effects of the C-cap, contacting molecules in the crystal, and the intrinsic design of the Repeat. Unfortunately, the rigid-body movement of the C-terminal part impairs the regular arrangement of internal Repeats that forms the putative peptide-binding site. The second-generation C-cap improves the packing of buried residues and thereby the stability of the protein. These considerations are useful for future improvements of an Armadillo-Repeat-based peptide-recognition system.
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Crystal structures of designed Armadillo Repeat proteins: implications of construct design and crystallization conditions on overall structure.
Protein science : a publication of the Protein Society, 2014Co-Authors: Christian Reichen, Chaithanya Madhurantakam, Andreas Plückthun, Peer R. E. MittlAbstract:Designed Armadillo Repeat proteins (dArmRP) are promising modular proteins for the engineering of binding molecules that recognize extended polypeptide chains. We determined the structure of a dArmRP containing five internal Repeats and 3rd generation capping Repeats in three different states by X-ray crystallography: without N-terminal His6-tag and in the presence of calcium (YM5A/Ca2+), without N-terminal His6-tag and in the absence of calcium (YM5A), and with N-terminal His6-tag and in the presence of calcium (His-YM5A/Ca2+). All structures show different quaternary structures and superhelical parameters. His-YM5A/Ca2+ forms a crystallographic dimer, which is bridged by the His6-tag, YM5A/Ca2+ forms a domain-swapped tetramer, and only in the absence of calcium and the His6-tag, YM5A forms a monomer. The changes of superhelical parameters are a consequence of calcium binding, because calcium ions interact with negatively charged residues, which can also participate in the modulation of helix dipole moments between adjacent Repeats. These observations are important for further optimizations of dArmRPs and provide a general illustration of how construct design and crystallization conditions can influence the exact structure of the investigated protein.
Yuan Miao - One of the best experts on this subject based on the ideXlab platform.
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N-Terminal 1–54 Amino Acid Sequence and Armadillo Repeat Domain Are Indispensable for P120-Catenin Isoform 1A in Regulating E-Cadherin
2016Co-Authors: Yuan Miao, Yang Liu, Guiyang Jiang, Maggie Stoecker, Endi Wang, Enhua WangAbstract:P120-catenin (p120ctn) exerts important roles in regulating E-cadherin and invasiveness in cancer cells. However, the mechanisms by which p120ctn isoforms 1 and 3 modulate E-cadherin expression are poorly understood. In the current study, HBE, H460, SPC and LTE cell lines were used to examine the effects of p120ctn isoforms 1A and 3A on E-cadherin expression and cell invasiveness. E-cadherin was localized on the cell membrane of HBE and H460 cells, while it was confined to the cytoplasm in SPC and LTE cells. Depletion of endogenous p120ctn resulted in reduced E-cadherin expression; however, p120ctn ablation showed opposite effects on invasiveness in the cell lines by decreasing invasiveness in SPC and LTE cells and increasing it in HBE and H460 cells. Restitution of 120ctn isoform 1A restored E-cadherin on the cell membrane and blocked cell invasiveness in H460 and HBE cells, while it restored cytoplasmic E-cadherin and enhanced cell invasiveness in SPC and LTE cells. P120ctn isoform 3A increased the invasiveness in all four cell lines despite the lack of effect on E-cadherin expression, suggesting a regulatory pathway independent of E-cadherin. Moreover, five p120ctn isoform 1A deletion mutants were constructed and expressed in H460 and SPC cells. The results showed that only the M4 mutant, which contains N-terminal 1–54 amino acids and the Armadillo Repeat domain, was functional in regulating E-cadherin and cell invasiveness, as observed in p120ctn isoform 1A. In conclusion, the N-terminal 1–54 amino acid sequence and Armadillo Repeat domain of p120ctn isoform 1A are indispensable for regulating E-cadherin protein. P120ctn isofor
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N-Terminal 1–54 Amino Acid Sequence and Armadillo Repeat Domain Are Indispensable for P120-Catenin Isoform 1A in Regulating E-Cadherin
PloS one, 2012Co-Authors: Yuan Miao, Yang Liu, Guiyang Jiang, Maggie Stoecker, Endi Wang, Enhua WangAbstract:P120-catenin (p120ctn) exerts important roles in regulating E-cadherin and invasiveness in cancer cells. However, the mechanisms by which p120ctn isoforms 1 and 3 modulate E-cadherin expression are poorly understood. In the current study, HBE, H460, SPC and LTE cell lines were used to examine the effects of p120ctn isoforms 1A and 3A on E-cadherin expression and cell invasiveness. E-cadherin was localized on the cell membrane of HBE and H460 cells, while it was confined to the cytoplasm in SPC and LTE cells. Depletion of endogenous p120ctn resulted in reduced E-cadherin expression; however, p120ctn ablation showed opposite effects on invasiveness in the cell lines by decreasing invasiveness in SPC and LTE cells and increasing it in HBE and H460 cells. Restitution of 120ctn isoform 1A restored E-cadherin on the cell membrane and blocked cell invasiveness in H460 and HBE cells, while it restored cytoplasmic E-cadherin and enhanced cell invasiveness in SPC and LTE cells. P120ctn isoform 3A increased the invasiveness in all four cell lines despite the lack of effect on E-cadherin expression, suggesting a regulatory pathway independent of E-cadherin. Moreover, five p120ctn isoform 1A deletion mutants were constructed and expressed in H460 and SPC cells. The results showed that only the M4 mutant, which contains N-terminal 1-54 amino acids and the Armadillo Repeat domain, was functional in regulating E-cadherin and cell invasiveness, as observed in p120ctn isoform 1A. In conclusion, the N-terminal 1-54 amino acid sequence and Armadillo Repeat domain of p120ctn isoform 1A are indispensable for regulating E-cadherin protein. P120ctn isoform 1A exerts opposing effects on cell invasiveness, corresponding to the subcellular localization of E-cadherin.
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n terminal 1 54 amino acid sequence and Armadillo Repeat domain are indispensable for p120 catenin isoform 1a in regulating e cadherin
PLOS ONE, 2012Co-Authors: Yuan Miao, Yang Liu, Guiyang Jiang, Maggie Stoecker, Endi Wang, Enhua WangAbstract:P120-catenin (p120ctn) exerts important roles in regulating E-cadherin and invasiveness in cancer cells. However, the mechanisms by which p120ctn isoforms 1 and 3 modulate E-cadherin expression are poorly understood. In the current study, HBE, H460, SPC and LTE cell lines were used to examine the effects of p120ctn isoforms 1A and 3A on E-cadherin expression and cell invasiveness. E-cadherin was localized on the cell membrane of HBE and H460 cells, while it was confined to the cytoplasm in SPC and LTE cells. Depletion of endogenous p120ctn resulted in reduced E-cadherin expression; however, p120ctn ablation showed opposite effects on invasiveness in the cell lines by decreasing invasiveness in SPC and LTE cells and increasing it in HBE and H460 cells. Restitution of 120ctn isoform 1A restored E-cadherin on the cell membrane and blocked cell invasiveness in H460 and HBE cells, while it restored cytoplasmic E-cadherin and enhanced cell invasiveness in SPC and LTE cells. P120ctn isoform 3A increased the invasiveness in all four cell lines despite the lack of effect on E-cadherin expression, suggesting a regulatory pathway independent of E-cadherin. Moreover, five p120ctn isoform 1A deletion mutants were constructed and expressed in H460 and SPC cells. The results showed that only the M4 mutant, which contains N-terminal 1-54 amino acids and the Armadillo Repeat domain, was functional in regulating E-cadherin and cell invasiveness, as observed in p120ctn isoform 1A. In conclusion, the N-terminal 1-54 amino acid sequence and Armadillo Repeat domain of p120ctn isoform 1A are indispensable for regulating E-cadherin protein. P120ctn isoform 1A exerts opposing effects on cell invasiveness, corresponding to the subcellular localization of E-cadherin.