Electrostatic Map Of Proteasome \u03b1-Rings Encodes The Design of Allosteric Porphyrin-Based Inhibitors Able To Affect 20S Conformation By Cooperative Binding

Antonio Di Dato,Roberto Paolesse,Marco Persico,Caterina Fattorusso,Alessandra Cunsolo,Manuela Stefanelli,Roberto Purrello,Diego Sbardella,Massimo Coletta,Danilo Milardi,Anna Maria Santoro,Alessandro D’Urso,Grazia R Tundo

doi:10.1038/s41598-017-17008-7

Abstract

The importance of allosteric proteasome inhibition in the treatment of cancer is becoming increasingly evident. Motivated by this urgent therapeutic need, we have recently identified cationic porphyrins as a highly versatile class of molecules able to regulate proteasome activity by interfering with gating mechanisms. In the present study, the mapping of electrostatic contacts bridging the regulatory particles with the α-rings of the human 20S proteasome led us to the identification of (meso-tetrakis(4-N-methylphenyl pyridyl)-porphyrin (pTMPyPP4) as a novel non-competitive inhibitor of human 20S proteasome. pTMPyPP4 inhibition mechanism implies a positive cooperative binding to proteasome, which disappears when a permanently open proteasome mutant (α-3ΔN) is used, supporting the hypothesis that the events associated with allosteric proteasome inhibition by pTMPyPP4 interfere with 20S gating and affect its “open-closed” equilibrium. Therefore, we propose that the spatial distribution of the negatively charged residues responsible for the interaction with regulatory particles at the α-ring surface of human 20S may be exploited as a blueprint for the design of allosteric proteasome regulators.

Highlights

The human proteasome is a supramolecular protein assembly with a key role in the intracellular degradation of proteins
We performed a bioinformatics study to identify the negatively charged residues of the human 20S core particle (CP) involved in ionic interactions with positively charged residues of the regulatory particles (RPs)
The starting hypothesis of this work is that the spatial distribution of electrostatic charges, present at the surface of the α-rings, may guide the design of cationic porphyrins able to dynamically interfere with the proteasome activation mechanism

Summary

Introduction

The human proteasome is a supramolecular protein assembly with a key role in the intracellular degradation of proteins. The obtained results allowed us to map the spatial position of all negatively charged residues of human 20S involved in ionic interactions with PA28α, PA200, and 19S RPs. Interestingly, it resulted a regular arrangement of such residues on the α-ring surface (Fig. 4), disclosing a structural code accounting for the observed role played by ionic interactions in the allosteric regulation of proteasome catalytic activity[17,26].

Results

Conclusion