Abstract
Acylpeptide hydrolase (APEH), one of the four members of the prolyl oligopeptidase class, catalyses the removal of N-acylated amino acids from acetylated peptides and it has been postulated to play a key role in protein degradation machinery. Disruption of protein turnover has been established as an effective strategy to down-regulate the ubiquitin-proteasome system (UPS) and as a promising approach in anticancer therapy.Here, we illustrate a new pathway modulating UPS and proteasome activity through inhibition of APEH. To find novel molecules able to down-regulate APEH activity, we screened a set of synthetic peptides, reproducing the reactive-site loop of a known archaeal inhibitor of APEH (SsCEI), and the conjugated linoleic acid (CLA) isomers. A 12-mer SsCEI peptide and the trans10-cis12 isomer of CLA, were identified as specific APEH inhibitors and their effects on cell-based assays were paralleled by a dose-dependent reduction of proteasome activity and the activation of the pro-apoptotic caspase cascade. Moreover, cell treatment with the individual compounds increased the cytoplasm levels of several classic hallmarks of proteasome inhibition, such as NFkappaB, p21, and misfolded or polyubiquitinylated proteins, and additive effects were observed in cells exposed to a combination of both inhibitors without any cytotoxicity. Remarkably, transfection of human bronchial epithelial cells with APEH siRNA, promoted a marked accumulation of a mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), herein used as a model of misfolded protein typically degraded by UPS. Finally, molecular modeling studies, to gain insights into the APEH inhibition by the trans10-cis12 CLA isomer, were performed.Our study supports a previously unrecognized role of APEH as a negative effector of proteasome activity by an unknown mechanism and opens new perspectives for the development of strategies aimed at modulation of cancer progression.
Highlights
In all living cells, proteolysis is essential in the control of many basic processes, including protein quality control, cell-cycle progression, signal transduction, apoptosis, and gene expression
Over the past decade, several studies have been focused on the development of specific proteasome inhibitors (PIs), which have relevant anticancer effects and on those involving in the repression of nuclear factor-k (NF-k B) signalling, and in the promotion of apoptosis in transformant cells [7,8]
We have investigated the molecular mechanisms that underlie the interrelationship between Acylpeptide hydrolase (APEH) and the proteasome, and their eventual regulation by natural or synthetic compounds including peptides reproducing the reactive site loop (RSL) of an archaeal APEH inhibitor (SsCEI, Sulfolobus solfataricus chymotrypsin-elastase inhibitor) [17], and the c9t11-conjugated linoleic acid (CLA) and t10c12-CLA isomers
Summary
Proteolysis is essential in the control of many basic processes, including protein quality control, cell-cycle progression, signal transduction, apoptosis, and gene expression. One of the major players in the regulation of intracellular proteolysis is the ubiquitin-proteasome system (UPS) [1]. This is a complex enzymatic machine that primarily contributes to the cytoplasmic turnover of a vast majority of proteins in mammalian cells and it is tightly controlled by a number of endogenous regulators. Over the past decade, several studies have been focused on the development of specific proteasome inhibitors (PIs), which have relevant anticancer effects and on those involving in the repression of nuclear factor-k (NF-k B) signalling, and in the promotion of apoptosis in transformant cells [7,8]
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