Abstract
The specific intracellular inhibition of protein activity at the protein level is a highly valuable tool for the validation or modulation of cellular processes. We demonstrate here the use of designed ankyrin repeat proteins (DARPins) as tailor-made intracellular proteinase inhibitors. Site-specific proteolytic processing plays a critical role in the regulation of many biological processes, ranging from basic cellular functions to the propagation of viruses. The NIa(pro) proteinase of tobacco etch virus, a major plant pathogen, can be functionally expressed in Escherichia coli without harming the bacterium. To identify inhibitors of this proteinase, we first selected binders to it from combinatorial libraries of DARPins and tested this pool with a novel in vivo screen for proteinase inhibition. For this purpose, a hybrid protein consisting of the omega subunit of E. coli RNA polymerase was covalently fused to a DNA-binding protein, the lambdacI repressor, containing an NIa(pro) cleavage site in the linker between the two proteins. Thus, this transcriptional activator is inactivated by site-specific proteolytic cleavage, and inhibitors of this cleavage can be identified by the reconstitution of transcription of a reporter gene. Following this two-step approach of selection and screening, we could rapidly isolate NIa(pro) proteinase inhibitors active inside the cell from highly diverse combinatorial DARPin libraries. These findings underline the great potential of DARPins for modulation of protein functionality in the intracellular space. In addition, our novel genetic screen can help to select and identify tailor-made proteinase inhibitors based on other protein scaffolds or even on low molecular weight compounds.
Highlights
Different functions of protein variants originating from the same gene [2]
Even though there are natural intracellular proteinase inhibitors controlling many of the processes mentioned above [10], they have not been used as scaffolds for deriving new specificities up to now
We previously reported the generation of designed ankyrin repeat proteins (DARPins)3 as specific binding molecules, and we showed that they can be selected for intracellular enzyme inhibition, demonstrated for a bacterial kinase [15,16,17], but it was unclear whether they would contain the properties required for proteinase inhibition
Summary
Different functions of protein variants originating from the same gene [2]. the effect mediated by RNA interference is often only weak, especially if the cellular stability of the protein of interest is high, as only the de novo synthesis is (partially) inhibited. Intracellular proteinases are important regulators of signal transduction, RNA transcription, cell cycle progression, apoptosis, and development [6, 7] and many other processes, and they are used by viruses in the processing of polyprotein precursors [8] To elucidate their function early in the discovery process, specific small molecule inhibitors will usually not be available, and a rapid approach to generate specific inhibitors that function inside the cell would be very valuable. Even though there are natural intracellular proteinase inhibitors controlling many of the processes mentioned above [10], they have not been used as scaffolds for deriving new specificities up to now Another approach would be to use scaffolds that are not derived from proteinase inhibitors for this purpose. The challenge is to achieve selective binding without cleavage or by maintaining a stable complex between proteinase and inhibitor even after cleavage of the latter
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
