Peptide Aptamer-mediated Inhibition of Target Proteins by Sequestration into Aggresomes

Evangelia Tomai,Karin Butz,Claudia Lohrey,Fritz Von Weizsäcker,Hanswalter Zentgraf,Felix Hoppe-Seyler

doi:10.1074/jbc.m604258200

Abstract

Peptide aptamers (PAs) can be employed to block the intracellular function of target proteins. Little is known about the mechanism of PA-mediated protein inhibition. Here, we generated PAs that specifically bound to the duck hepatitis B virus (HBV) core protein. Among them, PA34 strongly blocked duck HBV replication by inhibiting viral capsid formation. We found that PA34 led to a dramatic intracellular redistribution of its target protein into perinuclear inclusion bodies, which exhibit the typical characteristics of aggresomes. As a result, the core protein is efficiently removed from the viral life cycle. Corresponding findings were obtained for bioactive PAs that bind to the HBV core protein or to the human papillomavirus-16 (HPV16) E6 protein, respectively. The observation that PAs induce the specific sequestration of bound proteins into aggresomes defines a novel mechanism as to how this new class of intracellular inhibitors blocks the function of their target proteins.

Highlights

To determine the molecular basis of the inhibitory action of Peptide aptamers (PAs), we generated PAs that bind to the duck hepatitis B virus (DHBV) core protein
Further analyses of bioactive PAs binding to the hepatitis B virus (HBV) core or to the human papillomavirus (HPV) E6 protein indicate that the specific translocation of target proteins into cellular aggresomes represents a general mechanism for the inhibitory action of PAs
Isolation of Peptide Aptamers Binding to the DHBV Core Protein—To identify PAs that bind to the DHBV core protein, a yeast two-hybrid interaction screen was performed, using the N-terminal 180 amino acids of DHBV core as bait

Summary

Introduction

To determine the molecular basis of the inhibitory action of PAs, we generated PAs that bind to the duck hepatitis B virus (DHBV) core protein. We found that PAs efficiently blocked viral replication by coaggregating and translocating the DHBV core protein into cellular aggresomes. The cytoplasm was cleared from soluble core protein This shows that PAs can act as effective antiviral agents through depriving a virus of essential factors for life cycle progression. Further analyses of bioactive PAs binding to the hepatitis B virus (HBV) core or to the human papillomavirus (HPV) E6 protein indicate that the specific translocation of target proteins into cellular aggresomes represents a general mechanism for the inhibitory action of PAs. Peptide aptamers (PAs) are combinatorial protein reagents that are usually selected from randomized expression libraries in yeast, by virtue of their ability to bind to a given target protein under intracellular conditions. PAs have emerged as valuable new tools to study intracellular protein

Methods

Results

Conclusion