Abstract
Onconase® is a highly cytotoxic amphibian homolog of Ribonuclease A. Here, we describe the construction of circularly permuted Onconase® variants by connecting the N- and C-termini of this enzyme with amino acid residues that are recognized and cleaved by the human immunodeficiency virus protease. Uncleaved circularly permuted Onconase® variants are unusually stable, non-cytotoxic and can internalize in human T-lymphocyte Jurkat cells. The structure, stability and dynamics of an intact and a cleaved circularly permuted Onconase® variant were determined by Nuclear Magnetic Resonance spectroscopy and provide valuable insight into the changes in catalytic efficiency caused by the cleavage. The understanding of the structural environment and the dynamics of the activation process represents a first step toward the development of more effective drugs for the treatment of diseases related to pathogens expressing a specific protease. By taking advantage of the protease’s activity to initiate a cytotoxic cascade, this approach is thought to be less susceptible to known resistance mechanisms.
Highlights
In Nature, zymogens and limited proteolysis are a common means to regulate the activity of proteases and of many other biological processes
Design of Circularly Permuted ONC Variants To aid the design of circularly permuted ONC variants, structural models were created by modification of the atomic coordinates of wild type ONC [20] and submitting a modeling request to the automated homology modeling server SWISS-MODEL
Interaction of ONCFLG with a Substrate Analog To further characterize the active site of ONCFLG, we studied its interaction with an uncleavable substrate analog, d(UGG)3
Summary
In Nature, zymogens and limited proteolysis are a common means to regulate the activity of proteases and of many other biological processes. Raines and co-workers extended this strategy to bovine pancreatic ribonuclease (RNase A) by connecting the amino and carboxyl termini with a peptide segment that caps the active site and acts like a pro-segment of a natural zymogen and inhibits the native ribonucleolytic activity [2,3,4]. Upon cleavage of this linker by a specific protease, near-wild type activity of RNase A is reconstituted. The high cytotoxicity of ONC has been attributed to a quick internalization process [8,9], its ability to evade the cytosolic RI [7,10], an unusually high conformational stability [11,12] and to its ribonucleolytic activity [13,14,15]
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