Targeting the Lys48 Linked Ubiquitin Chain using Random non-standard Peptides Integrated Discovery (RaPID)

The ubiquitin (Ub) signal has been demonstrated to play a crucial role in various cellular processes such as cell cycle regulation, DNA damage repair and transcription. It is therefore not surprising that there is a great interest in targeting various components involved in the Ub pathway such as deubiquitinating enzymes (DUBs) and 26S proteasome with the aim of producing novel drugs against several diseases. A great promising and challenging approach to target the Ub system in cancer therapy is to alter the polyUb recognition by the proteasome. Except, Ubistatin A, which is the first inhibitor of proteasome-dependent protein degradation, there is no much progress in this field due to the lack of efficient methods for the preparation of polyUb and modified polyUb chains. In our lab, we have developed tools to synthesize stable homogenous Ub chains with a defined length and linkage, for example, biotin Lys48-linked di-/tetraUb. These analogues have enabled us to use Random Non-Standard Peptides Integrated Discovery (RaPID) method to discover novel cyclic peptides that specifically bind Lys48-linked di-/tetra-Ub chains. We determined the binding affinity coefficient (Kd) using surface plasmon resonance and found that these cyclic peptides bind in the nanomolar range to the chains. The mode of binding to the tetraUb chain was determined by nuclear magnetic resonance (NMR). We also examined of the effect of the newly-synthetized peptides in cellulo and demonstrated that these cyclic peptides are cell-permeable and interfere with the recognition and subsequent processing of the Ub-chains by (1) DUBs, protecting the chains from hydrolysis; and (2) the 26S proteasome, rendering the bound substrates less prone for degradation. In addition, the effect on degradation was demonstrated via radioactive total proteolysis measurements, as well as via model substrate, endogenic bona fide substrates of the UPS, live-cell proteasome activity probe and effect on Ub-conjugates.