Witkowska, Julia; Karpowicz, Przemyslaw; Gaczynska, Maria; Osmulski, Pawel A.; Jankowska, Elzbieta

Journal of Peptide Science (2014), 20(8), 649-656 CODEN: JPSIEI; ISSN: 1075-2617. English.

The proteasome is a 'proteolytic factory' that constitutes an essential part of the ubiquitin-proteasome pathway. The involvement of proteasome in regulation of all major aspects of cellular physiol. makes it an attractive drug target. So far, only inhibitors of the proteasome entered the clinic as anti-cancer drugs. However, proteasome regulators may also be useful for treatment of inflammatory and neurodegenerative diseases. We established in our previous studies that the peptide Tat2, comprising the basic domain of HIV-1 Tat protein (R49KKRRQRR56, supplemented with Q66DPI69 fragment) inhibits the 20S proteasome in a noncompetitive manner. Mechanism of Tat2 likely involves allosteric regulation because it competes with the proteasome natural 11S activator for binding to the enzyme noncatalytic subunits. In this study, we performed alanine walking coupled with biol. activity measurements and FTIR and CD spectroscopy to dissect the contribution of Tat2 charge and conformation to its ability to influence proteasome peptidase activity. In solution, Tat2 and most of its analogs with a single Ala substitution preferentially adopted a conformation containing PPII/turn structural motifs. Replacing either Asp10 or two or more adjacent Arg/Lys residues induced a random coil conformation, probably by disrupting ionic interactions responsible for stabilization of the peptides ordered structure. The random coil Tat2 analogs lost their ability to activate the latent 20S proteasome. In contrast, inhibitory properties of the peptides more significantly depended on their pos. charge. The data provide valuable clues for the future optimization of the Tat2-based proteasome regulators. Copyright .COPYRGT. 2014 European Peptide Society and John Wiley & Sons, Ltd.