RESUMEN
Abstract Caspases play important roles in cell death, differentiation, and proliferation. Due to their high homology, especially of the active site, specific targeting of a particular caspase using substrate analogues is very difficult. Although commercially available small molecules based on peptides are lacking high specificity due to overlapping cleavage motives between different caspases, they are often used as specific tools. We have selected designed ankyrin repeat proteins (DARPins) against human caspases 1-9 and identified high-affinity binders for the targeted caspases, except for caspase 4. Besides previously reported caspase-specific DARPins, we generated novel DARPins (D1.73, D5.15, D6.11, D8.1, D8.4, and D9.2) and confirmed specificity for caspases 1, 5, 6, and 8 using a subset of caspase family members. In addition, we solved the crystal structure of caspase 8 in complex with DARPin D8.4. This binder interacts with non-conserved residues on the large subunit, thereby explaining its specificity. Structural analysis of this and other previously published crystal structures of caspase/DARPin complexes depicts two general binding areas either involving active site forming loops or a surface area laterally at the large subunit of the enzyme. Both surface areas involve non-conserved surface residues of caspases.
Asunto(s)
Repetición de Anquirina , Caspasas/efectos de los fármacos , Proteínas/genética , Proteínas/farmacología , Caspasa 8/química , Cromatografía en Gel , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Ribosomas , Resonancia por Plasmón de SuperficieRESUMEN
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.
Asunto(s)
Caspasa 3/metabolismo , Caspasa 7/metabolismo , Inhibidores de Caspasas/farmacología , Proteínas Nucleares/farmacología , Repetición de Anquirina , Apoptosis/efectos de los fármacos , Caspasa 3/química , Caspasa 7/química , Inhibidores de Caspasas/química , Células HeLa , Humanos , Modelos Moleculares , Imagen Molecular , Proteínas Nucleares/química , Biblioteca de Péptidos , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/farmacologíaRESUMEN
Dysregulation of apoptosis is associated with several human diseases. The main apoptotic mediators are caspases, which propagate death signals to downstream targets. Executioner caspase-3 is responsible for the majority of cleavage events and its therapeutic potential is of high interest with to date several available active site peptide inhibitors. These molecules inhibit caspase-3, but also homologous caspases. Here, we describe caspase-3 specific inhibitors D3.4 and D3.8, which have been selected from a library of designed ankyrin repeat proteins (DARPins). The crystal structures of D3.4 and mutants thereof show how high specificity and inhibition is achieved. They also show similarities in the binding mode with that of the natural caspase inhibitor XIAP (X-linked inhibitor of apoptosis). The kinetic data reveal a competitive inhibition mechanism. D3.4 is specific for caspase-3 and does not bind the highly homologous caspase-7. D3.4 therefore is an excellent tool to define the precise role of caspase-3 in the various apoptotic pathways.
Asunto(s)
Repetición de Anquirina , Caspasa 3/química , Inhibidores de Caspasas/química , Proteínas/química , Secuencia de Aminoácidos , Unión Competitiva , Caspasa 6/química , Caspasa 7/química , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Imitación Molecular , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
A number of strategies and protocols for the expression, purification and kinetic characterization of human caspases are described in the literature. We have systematically revised these protocols and present comprehensive optimized expression and purification protocols for caspase-1 to -9 as well as improved assay conditions for their reproducible kinetic characterization. Our studies on active site titration revealed that the reproducibility is strongly affected by the presence of DTT in the assay buffer. Furthermore, we observed that not all caspases show a linear relationship between enzymatic activity and protein concentration, which explains the discrepancy between published values of specific activities from different laboratories. Our broad kinetic analysis allows the conclusion that the dependency of caspase activities on protein concentration is an effect of concentration-dependent dimerization, which can also be influenced by kosmotropic salts. The protocol recommendations as an outcome of this work will yield higher reproducibility regarding expression and purification of human caspases and contribute to standardization of enzyme kinetic data.
Asunto(s)
Caspasas/genética , Caspasas/metabolismo , Clonación Molecular/métodos , Caspasas/química , Caspasas/aislamiento & purificación , Dominio Catalítico , Cromatografía en Gel/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Escherichia coli/genética , Expresión Génica , Humanos , Cinética , Replegamiento Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
One of the most common ways to demonstrate a direct protein-protein interaction in vitro is the glutathione-S-transferse (GST)-pulldown. Here we report the detailed characterization of a putative interaction between two transcription factor proteins, GATA-1 and Krüppel-like factor 3 (KLF3/BKLF) that show robust interactions in GST-pulldown experiments. Attempts to map the interaction interface of GATA-1 on KLF3 using a mutagenic screening approach did not yield a contiguous binding face on KLF3, suggesting that the interaction might be non-specific. NMR experiments showed that the proteins do not interact at protein concentrations of 50-100 µM. Rather, the GST tag can cause part of KLF3 to misfold. In addition to misfolding, the fact that both proteins are DNA-binding domains appears to introduce binding artifacts (possibly nucleic acid bridging) that cannot be resolved by the addition of nucleases or ethidium bromide (EtBr). This study emphasizes the need for caution in relying on GST-pulldown results and related methods, without convincing confirmation from different approaches.
Asunto(s)
Factor de Transcripción GATA1/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Animales , Ratones , Resonancia Magnética Nuclear Biomolecular , Unión ProteicaRESUMEN
Polyoxometalates (POMs) are interesting biomedical agents due to their versatile anticancer and antiviral properties, such as remarkable anti-HIV activity. Although POMs are tunable and easily accessible inorganic drug prototypes in principle, their full potential can only be tapped by enhancing their biocompatibility, for example, through organic functionalization. We have therefore investigated the HIV-1 protease inhibition potential of functionalized Keggin- and Dawson-type POMs with organic side chains. Their inhibitory performance was furthermore compared to other POM types, and the buffer dependence of the results is discussed. In addition, chemical shift mapping NMR experiments were performed to exclude POM-substrate interactions. Whereas the introduction of organic side chains into POMs is a promising approach in principle, the influence of secondary effects on the reaction system also merits detailed investigation.