ABSTRACT
Caspases play important roles during apoptosis, inflammation and proliferation. The high homology among family members makes selective targeting of individual caspases difficult, which is necessary to precisely define the role of these enzymes. We have selected caspase-7-specific binders from a library of DARPins (designed ankyrin repeat proteins). The DARPins D7.18 and D7.43 bind specifically to procaspase 7 and active caspase 7, but not to other members of the family. Binding of the DARPins does not affect the active enzyme, but interferes with its activation by other caspases. The crystal structure of the caspase 7-D7.18 complex elucidates the high selectivity and the mode of inhibition. Combining these caspase-7-specific DARPins with the previously reported caspase-3-inhibitory DARPin D3.4S76R reduces the activity of caspase 3 and 7 in double-transfected HeLa cells during apoptosis. In addition, these cells showed less susceptibility to TRAIL (tumour-necrosis-factor-related apoptosis-inducing ligand)-induced apoptosis in living cell experiments. D7.18 and D7.43 are therefore novel tools for in vitro studies on procaspase 7 activation as well as for clarifying the role of its activation in different cellular processes. If applied in combination with D3.4S76R, they represent an excellent instrument to increase our understanding of these enzymes during various cellular processes.
Subject(s)
Caspase 3/metabolism , Caspase 7/metabolism , Caspase Inhibitors/pharmacology , Nuclear Proteins/pharmacology , Ankyrin Repeat , Apoptosis/drug effects , Caspase 3/chemistry , Caspase 7/chemistry , Caspase Inhibitors/chemistry , HeLa Cells , Humans , Models, Molecular , Molecular Imaging , Nuclear Proteins/chemistry , Peptide Library , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , TNF-Related Apoptosis-Inducing Ligand/pharmacologyABSTRACT
Biochemical and detailed structural information of human ether-a-go-go-related gene (hERG) potassium channels are scarce but are a prerequisite to understand the unwanted interactions of hERG with drugs and the effect of mutations that lead to long QT syndrome. Despite the huge interest in hERG, to our knowledge, procedures that provide a purified, functional, and tetrameric hERG channel are not available. Here, we describe hybrid hERG molecules, termed chimeric hERG channels, in which the N-terminal Per-Arnt-Sim (PAS) domain is deleted and the C-terminal C-linker as well as the cyclic nucleotide binding domain (CNBD) portion is replaced by an artificial tetramerization domain. These chimeric hERG channels can be overexpressed in HEK cells, solubilized in detergent, and purified as tetramers. When expressed in Xenopus laevis oocytes, the chimeric channels exhibit efficient trafficking to the cell surface, whereas a hERG construct lacking the PAS and C-linker/CNBD domains is retained in the cytoplasm. The chimeric hERG channels retain essential hERG functions such as voltage-dependent gating and inhibition by astemizole and the scorpion toxin BeKm-1. The chimeric channels are thus powerful tools for helping to understand the contribution of the cytoplasmic hERG domains to the gating process and are suitable for in vitro biochemical and structural studies.
Subject(s)
Ether-A-Go-Go Potassium Channels/metabolism , Recombinant Fusion Proteins/metabolism , Animals , Binding Sites , Cell Membrane/enzymology , Cell Membrane/metabolism , Cells, Cultured , Cytoplasm/enzymology , Cytoplasm/metabolism , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/chemistry , Ether-A-Go-Go Potassium Channels/genetics , Ether-A-Go-Go Potassium Channels/isolation & purification , HEK293 Cells , Humans , Membrane Potentials , Nucleotides, Cyclic/metabolism , Oocytes/cytology , Oocytes/enzymology , Oocytes/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/isolation & purification , Peptide Fragments/metabolism , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Stability , Protein Transport , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Solubility , Xenopus laevisABSTRACT
The human ether-a-go-go related gene (hERG) potassium channel plays a major role in the repolarization of the cardiac action potential. Inhibition of the hERG function by mutations or a wide variety of pharmaceutical compounds cause long QT syndrome and lead to potentially lethal arrhythmias. For detailed insights into the structural and biochemical background of hERG function and drug binding, the purification of recombinant protein is essential. Because the hERG channel is a challenging protein to purify, fast and easy techniques to evaluate different expression, solubilization and purification conditions are of primary importance. Here, we describe the generation of a set of 12 monoclonal antibodies against hERG. Beside their suitability in western blot, immunoprecipitation and immunostaining, these antibodies were used to establish a sandwich ELISA for the detection and relative quantification of hERG in different expression systems. Furthermore, a Fab fragment was used in fluorescence size exclusion chromatography to determine the oligomeric state of hERG after solubilization. These new tools can be used for a fast and efficient screening of expression, solubilization and purification conditions.