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Olivier Kocher - One of the best experts on this subject based on the ideXlab platform.
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noncanonical role of the pdz4 domain of the adaptor protein pdzk1 in the regulation of the hepatic high density lipoprotein receptor scavenger receptor class b type i sr bi
Journal of Biological Chemistry, 2013Co-Authors: Kosuke Tsukamoto, Rinku Pal, Kathleen Daniels, Monty Krieger, Thomas E Wales, Ren Sheng, Wonhwa Cho, Walter F Stafford, John R Engen, Olivier KocherAbstract:Abstract The four PDZ (PDZ1-PDZ4) domain-containing-adaptor protein PDZK1 controls the expression, localization and function of the HDL receptor SR-BI in hepatocytes in vivo via a PDZ4-dependent mechanism involving the binding of SR-BI′s cytoplasmic carboxy terminus to the canonical Peptide binding sites of the PDZ1 or PDZ3 domains (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1) or Target Peptide binding-negative (PDZ4[G389P]) mutants of PDZK1, we found that neither PDZ2 nor PDZ3, nor the canonical Target Peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell-surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement for PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, Surface Plasmon Resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1′s regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical Target Peptide binding and non-canonical (Peptide binding independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity.
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noncanonical role of the pdz4 domain of the adaptor protein pdzk1 in the regulation of the hepatic high density lipoprotein receptor scavenger receptor class b type i sr bi
Journal of Biological Chemistry, 2013Co-Authors: Kosuke Tsukamoto, Rinku Pal, Kathleen Daniels, Monty Krieger, Thomas E Wales, Ren Sheng, Wonhwa Cho, Walter F Stafford, John R Engen, Olivier KocherAbstract:The four PDZ (PDZ1 to PDZ4) domain-containing adaptor protein PDZK1 controls the expression, localization, and function of the HDL receptor scavenger receptor class B, type I (SR-BI), in hepatocytes in vivo. This control depends on both the PDZ4 domain and the binding of SR-BI's cytoplasmic C terminus to the canonical Peptide-binding sites of either the PDZ1 or PDZ3 domain (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1), or Target Peptide binding-negative (PDZ4(G389P)) mutants of PDZK1, we found that neither PDZ2 nor PDZ3 nor the canonical Target Peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement of PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, surface plasmon resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1's regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical Target Peptide binding and noncanonical (Peptide binding-independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity. Background: PDZK1 (four PDZ domains) regulates the hepatic HDL receptor SR-BI. Results: PDZK1's PDZ2 and PDZ3 domains are not required, whereas PDZ4 is, possibly because PDZ4 mediates membrane binding. Conclusion: Regulation of SR-BI via PDZK1's PDZ domains is complex. Significance: Combined canonical (Target Peptide binding) and noncanonical (Peptide binding-independent) PDZ domain functions can result in optimal activity of a PDZ domain-containing adaptor protein.
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identification of the pdz3 domain of the adaptor protein pdzk1 as a second physiologically functional binding site for the c terminus of the high density lipoprotein receptor scavenger receptor class b type i
Journal of Biological Chemistry, 2011Co-Authors: Olivier Kocher, Gabriel Birrane, Ayce Yesilaltay, Sharon Shechter, Rinku Pal, Kathleen Daniels, Monty KriegerAbstract:Abstract The normal expression, cell surface localization, and function of the murine high density lipoprotein receptor scavenger receptor class B type I (SR-BI) in hepatocytes in vivo, and thus normal lipoprotein metabolism, depend on its four PDZ domain (PDZ1–PDZ4) containing cytoplasmic adaptor protein PDZK1. Previous studies showed that the C terminus of SR-BI (“Target Peptide”) binds directly to PDZ1 and influences hepatic SR-BI protein expression. Unexpectedly an inactivating mutation in PDZ1 (Tyr20 → Ala) only partially, rather than completely, suppresses the ability of PDZK1 to control hepatic SR-BI. We used isothermal titration calorimetry to show that PDZ3, but not PDZ2 or PDZ4, can also bind the Target Peptide (Kd = 37.0 μm), albeit with ∼10-fold lower affinity than PDZ1. This binding is abrogated by a Tyr253 → Ala substitution. Comparison of the 1.5-A resolution crystal structure of PDZ3 with its bound Target Peptide (505QEAKL509) to that of Peptide-bound PDZ1 indicated fewer Target Peptide stabilizing atomic interactions (hydrogen bonds and hydrophobic interactions) in PDZ3. A double (Tyr20 → Ala (PDZ1) + Tyr253 → Ala (PDZ3)) substitution abrogated all Target Peptide binding to PDZK1. In vivo hepatic expression of a singly substituted (Tyr253 → Ala (PDZ3)) PDZK1 transgene (Tg) was able to correct all of the SR-BI-related defects in PDZK1 knock-out mice, whereas the doubly substituted [Tyr20 → Ala (PDZ1) + Tyr253 → Ala (PDZ3)]Tg was unable to correct these defects. Thus, we conclude that PDZK1-mediated control of hepatic SR-BI requires direct binding of the SR-BI C terminus to either the PDZ1 or PDZ3 domains, and that binding to both domains simultaneously is not required for PDZK1 control of hepatic SR-BI.
Monty Krieger - One of the best experts on this subject based on the ideXlab platform.
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noncanonical role of the pdz4 domain of the adaptor protein pdzk1 in the regulation of the hepatic high density lipoprotein receptor scavenger receptor class b type i sr bi
Journal of Biological Chemistry, 2013Co-Authors: Kosuke Tsukamoto, Rinku Pal, Kathleen Daniels, Monty Krieger, Thomas E Wales, Ren Sheng, Wonhwa Cho, Walter F Stafford, John R Engen, Olivier KocherAbstract:Abstract The four PDZ (PDZ1-PDZ4) domain-containing-adaptor protein PDZK1 controls the expression, localization and function of the HDL receptor SR-BI in hepatocytes in vivo via a PDZ4-dependent mechanism involving the binding of SR-BI′s cytoplasmic carboxy terminus to the canonical Peptide binding sites of the PDZ1 or PDZ3 domains (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1) or Target Peptide binding-negative (PDZ4[G389P]) mutants of PDZK1, we found that neither PDZ2 nor PDZ3, nor the canonical Target Peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell-surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement for PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, Surface Plasmon Resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1′s regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical Target Peptide binding and non-canonical (Peptide binding independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity.
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noncanonical role of the pdz4 domain of the adaptor protein pdzk1 in the regulation of the hepatic high density lipoprotein receptor scavenger receptor class b type i sr bi
Journal of Biological Chemistry, 2013Co-Authors: Kosuke Tsukamoto, Rinku Pal, Kathleen Daniels, Monty Krieger, Thomas E Wales, Ren Sheng, Wonhwa Cho, Walter F Stafford, John R Engen, Olivier KocherAbstract:The four PDZ (PDZ1 to PDZ4) domain-containing adaptor protein PDZK1 controls the expression, localization, and function of the HDL receptor scavenger receptor class B, type I (SR-BI), in hepatocytes in vivo. This control depends on both the PDZ4 domain and the binding of SR-BI's cytoplasmic C terminus to the canonical Peptide-binding sites of either the PDZ1 or PDZ3 domain (no binding to PDZ2 or PDZ4). Using transgenic mice expressing in the liver domain deletion (ΔPDZ2 or ΔPDZ3), domain replacement (PDZ2→1), or Target Peptide binding-negative (PDZ4(G389P)) mutants of PDZK1, we found that neither PDZ2 nor PDZ3 nor the canonical Target Peptide binding activity of PDZ4 were necessary for hepatic SR-BI regulatory activity. Immunohistochemical studies established that the localization of PDZK1 on hepatocyte cell surface membranes in vivo is dependent on its PDZ4 domain and the presence of SR-BI. Analytical ultracentrifugation and hydrogen deuterium exchange mass spectrometry suggested that the requirement of PDZ4 for localization and SR-BI regulation is not due to PDZ4-mediated oligomerization or induction of conformational changes in the PDZ123 portion of PDZK1. However, surface plasmon resonance analysis showed that PDZ4, but not the other PDZ domains, can bind vesicles that mimic the plasma membrane. Thus, PDZ4 may potentiate PDZK1's regulation of SR-BI by promoting its lipid-mediated attachment to the cytoplasmic membrane. Our results show that not all of the PDZ domains of a multi-PDZ domain-containing adaptor protein are required for its biological activities and that both canonical Target Peptide binding and noncanonical (Peptide binding-independent) capacities of PDZ domains may be employed by a single such adaptor for optimal in vivo activity. Background: PDZK1 (four PDZ domains) regulates the hepatic HDL receptor SR-BI. Results: PDZK1's PDZ2 and PDZ3 domains are not required, whereas PDZ4 is, possibly because PDZ4 mediates membrane binding. Conclusion: Regulation of SR-BI via PDZK1's PDZ domains is complex. Significance: Combined canonical (Target Peptide binding) and noncanonical (Peptide binding-independent) PDZ domain functions can result in optimal activity of a PDZ domain-containing adaptor protein.
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identification of the pdz3 domain of the adaptor protein pdzk1 as a second physiologically functional binding site for the c terminus of the high density lipoprotein receptor scavenger receptor class b type i
Journal of Biological Chemistry, 2011Co-Authors: Olivier Kocher, Gabriel Birrane, Ayce Yesilaltay, Sharon Shechter, Rinku Pal, Kathleen Daniels, Monty KriegerAbstract:Abstract The normal expression, cell surface localization, and function of the murine high density lipoprotein receptor scavenger receptor class B type I (SR-BI) in hepatocytes in vivo, and thus normal lipoprotein metabolism, depend on its four PDZ domain (PDZ1–PDZ4) containing cytoplasmic adaptor protein PDZK1. Previous studies showed that the C terminus of SR-BI (“Target Peptide”) binds directly to PDZ1 and influences hepatic SR-BI protein expression. Unexpectedly an inactivating mutation in PDZ1 (Tyr20 → Ala) only partially, rather than completely, suppresses the ability of PDZK1 to control hepatic SR-BI. We used isothermal titration calorimetry to show that PDZ3, but not PDZ2 or PDZ4, can also bind the Target Peptide (Kd = 37.0 μm), albeit with ∼10-fold lower affinity than PDZ1. This binding is abrogated by a Tyr253 → Ala substitution. Comparison of the 1.5-A resolution crystal structure of PDZ3 with its bound Target Peptide (505QEAKL509) to that of Peptide-bound PDZ1 indicated fewer Target Peptide stabilizing atomic interactions (hydrogen bonds and hydrophobic interactions) in PDZ3. A double (Tyr20 → Ala (PDZ1) + Tyr253 → Ala (PDZ3)) substitution abrogated all Target Peptide binding to PDZK1. In vivo hepatic expression of a singly substituted (Tyr253 → Ala (PDZ3)) PDZK1 transgene (Tg) was able to correct all of the SR-BI-related defects in PDZK1 knock-out mice, whereas the doubly substituted [Tyr20 → Ala (PDZ1) + Tyr253 → Ala (PDZ3)]Tg was unable to correct these defects. Thus, we conclude that PDZK1-mediated control of hepatic SR-BI requires direct binding of the SR-BI C terminus to either the PDZ1 or PDZ3 domains, and that binding to both domains simultaneously is not required for PDZK1 control of hepatic SR-BI.
Hermann-josef Thierse - One of the best experts on this subject based on the ideXlab platform.
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proteomic allergen Peptide protein interaction assay for the identification of human skin sensitizers
Toxicology in Vitro, 2013Co-Authors: Lisa Dietz, Sven Kinzebach, Stefanie Ohnesorge, Bastian Franke, Irina Goette, Hermann-josef Thierse, Dieter KoeniggresselAbstract:Modification of proteins by skin sensitizers is a pivotal step in T cell mediated allergic contact dermatitis (ACD). In this process small reactive chemicals interact covalently or non-covalently with cellular or extracellular skin self-proteins or self-Peptides to become recognized by the human immune system. Aiming to develop a novel non-animal in vitro test system for predicting sensitization potential of small reactive chemicals in human skin the allergen-Peptide/protein interaction assay (APIA) has been developed. By applying modern proteomic technologies together with a Target Peptide containing all amino acids, the assay permits the profiling of all amino acid specific allergen-Peptide interactions. Moreover, potentially crucial allergen-specific Cys-modifications are qualitatively monitored by mass spectrometry and confirmed by a dual Peptide approach. Assay conditions chosen mimic the distinct human epidermal reactivity compartments of the skin surface (pH 5.5), stratum basale (pH 6.8), and typical physiological conditions (pH 7.4). An extreme as well as a moderate human contact sensitizer produced Cys-specific mass shifts, whereas a skin irritant did not. Our data indicate that MALDI-MS based and skin-related in vitro technology platforms - like the APIA - are promising tools in developing alternative non-animal allergen assays. This will assist in chemical classification and next generation risk assessment strategies, including REACH and experimental immunotoxicology.
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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modular Peptide binding from a comparison of natural binders to designed armadillo repeat proteins
Journal of Structural Biology, 2014Co-Authors: Christian Reichen, Simon Hansen, Andreas PlückthunAbstract:Several binding scaffolds that are not based on immunoglobulins have been designed as alternatives to traditional monoclonal antibodies. Many of them have been developed to bind to folded proteins, yet cellular networks for signaling and protein trafficking often depend on binding to unfolded regions of proteins. This type of binding can thus be well described as a Peptide-protein interaction. In this review, we compare different Peptide-binding scaffolds, highlighting that armadillo repeat proteins (ArmRP) offer an attractive modular system, as they bind a stretch of extended Peptide in a repeat-wise manner. Instead of generating each new binding molecule by an independent selection, preselected repeats - each complementary to a piece of the Target Peptide - could be designed and assembled on demand into a new protein, which then binds the prescribed complete Peptide. Stacked armadillo repeats (ArmR), each typically consisting of 42 amino acids arranged in three α-helices, build an elongated superhelical structure which enables binding of Peptides in extended conformation. A consensus-based design approach, complemented with molecular dynamics simulations and rational engineering, resulted in well-expressed monomeric proteins with high stability. Peptide binders were selected and several structures were determined, forming the basis for the future development of modular Peptide-binding scaffolds.
Christian Reichen - 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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computationally designed armadillo repeat proteins for modular Peptide recognition
Journal of Molecular Biology, 2016Co-Authors: Christian Reichen, Simon Hansen, Chaithanya Madhurantakam, Fabio Parmeggiani, Patrick Ernst, Cristina Forzani, Annemarie Honegger, Sarel J Fleishman, Ting Zhou, Christina EwaldAbstract:Armadillo repeat proteins (ArmRPs) recognize their Target Peptide in extended conformation and bind, in a first approximation, two residues per repeat. Thus, they may form the basis for building a modular system, in which each repeat is complementary to a piece of the Target Peptide. Accordingly, preselected repeats could be assembled into specific binding proteins on demand and thereby avoid the traditional generation of every new binding molecule by an independent selection from a library. Stacked armadillo repeats, each consisting of 42 aa arranged in three α-helices, build an elongated superhelical structure. Here, we analyzed the curvature variations in natural ArmRPs and identified a repeat pair from yeast importin-α as having the optimal curvature geometry that is complementary to a Peptide over its whole length. We employed a symmetric in silico design to obtain a uniform sequence for a stackable repeat while maintaining the desired curvature geometry. Computationally designed ArmRPs (dArmRPs) had to be stabilized by mutations to remove regions of higher flexibility, which were identified by molecular dynamics simulations in explicit solvent. Using an N-capping repeat from the consensus-design approach, two different crystal structures of dArmRP were determined. Although the experimental structures of dArmRP deviated from the designed curvature, the insertion of the most conserved binding pockets of natural ArmRPs onto the surface of dArmRPs resulted in binders against the expected Peptide with low nanomolar affinities, similar to the binders from the consensus-design series.
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modular Peptide binding from a comparison of natural binders to designed armadillo repeat proteins
Journal of Structural Biology, 2014Co-Authors: Christian Reichen, Simon Hansen, Andreas PlückthunAbstract:Several binding scaffolds that are not based on immunoglobulins have been designed as alternatives to traditional monoclonal antibodies. Many of them have been developed to bind to folded proteins, yet cellular networks for signaling and protein trafficking often depend on binding to unfolded regions of proteins. This type of binding can thus be well described as a Peptide-protein interaction. In this review, we compare different Peptide-binding scaffolds, highlighting that armadillo repeat proteins (ArmRP) offer an attractive modular system, as they bind a stretch of extended Peptide in a repeat-wise manner. Instead of generating each new binding molecule by an independent selection, preselected repeats - each complementary to a piece of the Target Peptide - could be designed and assembled on demand into a new protein, which then binds the prescribed complete Peptide. Stacked armadillo repeats (ArmR), each typically consisting of 42 amino acids arranged in three α-helices, build an elongated superhelical structure which enables binding of Peptides in extended conformation. A consensus-based design approach, complemented with molecular dynamics simulations and rational engineering, resulted in well-expressed monomeric proteins with high stability. Peptide binders were selected and several structures were determined, forming the basis for the future development of modular Peptide-binding scaffolds.
