Abstract

The proteasome is a giant protease responsible for degradation of the majority of cytosolic proteins. Competitive inhibitors of the proteasome are used against aggressive blood cancers. However, broadening the use of proteasome-targeting drugs requires new mechanistic approaches to the enzyme’s inhibition. In our previous studies we described Tat1 peptide, an allosteric inhibitor of the proteasome derived from a fragment of the basic domain of HIV-Tat1 protein. Here, we attempted to dissect the structural determinants of the proteasome inhibition by Tat1. Single- and multiple- alanine walking scans were performed. Tat1 analogs with stabilized beta-turn conformation at positions 4–5 and 8–9, pointed out by the molecular dynamics modeling and the alanine scan, were synthesized. Structure of Tat1 analogs were analyzed by circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopy studies, supplemented by molecular dynamics simulations. Biological activity tests and structural studies revealed that high flexibility and exposed positive charge are hallmarks of Tat1 peptide. Interestingly, stabilization of a beta-turn at the 8–9 position was necessary to significantly improve the inhibitory potency.

Highlights

  • The 26S proteasome, a main component of the ubiquitin-proteasome proteolytic system, consists of the 20S catalytic core and two 19S regulatory particles (RP) attached to both sides of the core

  • An initial single-Ala walking scan did not identify residues that influenced the ability of Tat1 to inhibit the ChT-L peptidase of the 20S core proteasome (CP)

  • The activity of CP treated with 1 μM of these peptides varied by no more than 4% comparing to Tat1 (S1 Fig)

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Summary

Introduction

The 26S proteasome, a main component of the ubiquitin-proteasome proteolytic system, consists of the 20S catalytic core and two 19S regulatory particles (RP) attached to both sides of the core. The 19S RP is responsible for recognition and binding of polyubiquitinylated protein substrates, their deubiquitinylation, unfolding, and translocation to the catalytic chamber [1]. Two other proteasome activators - 11S (PA28/REG) and PA200 do not process polyubiquitinylated proteins. The PA28 αβ/REG/11S activator enhances proteolysis of peptides and unstructured proteins and its physiological role is to activate production of antigenic peptides [2]. PA200 is PLOS ONE | DOI:10.1371/journal.pone.0143038 November 17, 2015

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