Interplay between Structure and Charge as a Key to Allosteric Modulation of Human 20S Proteasome by the Basic Fragment of HIV-1 Tat Protein

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.

New Peptide-Based Pharmacophore Activates 20S Proteasome

“…This goal was accomplished by generating structural derivatives of the TAT1 peptide that contained turn-inducing moieties and the preserved key basic residues (Table 1). Our previous structural studies of the TAT1 peptide suggested a strong preference toward formation of two β-like turns in positions 4,5 and 8,9 [22,24] ( Figure 1B, C). Substitutions of residues in TAT1 with alanines (Ala-walking) confirmed the significance of the putative turn regions for core proteasome activation ( Figure 2).…”

“…The whole Tat protein not only inhibits the core proteasome but also competes with the PA28/REG activator [21]. As mentioned above, we found before that TAT1 inhibits the artificially activated 20S core in vitro, but noted that it can activate the naturally latent core [22][23][24]. The goal of our design was to enhance the proteasome activation and improve the peptide's stability while preserving BBB penetrance.…”

“…Synthesis and properties (1) have been described in [22], (2)- (5) and (9) have been described in [24]. Synthesis and purification of (6) and (7) followed the procedures described in [24]. All the peptides have been purified to at least 99% of purity.…”

“…(B) Scheme of HIV-1 Tat protein, with the domain required for transactivation [26,27] shaded blue and with the short fragment designated as TAT1 marked. (C) Our previously reported molecular dynamics simulation of TAT1, suggesting a potential for formation of two turns (modified from [22,24].…”

“…Among many intracellular effects, the HIV-1 Tat protein inhibits the core proteasome [21]. In our previous studies, we noted that short peptide fragments of HIV-1 Tat displayed peculiar in vitro properties: they inhibited detergent-treated core particle but mildly activated the latent core [22][23][24]. However, treatment with sodium dodecyl sulfate detergent, although convenient for in vitro assays, yields mildly denatured proteasome and, under these far-from-physiological conditions, likely with destroyed natural allosteric routes [10].…”