Selective inhibition of acylpeptide hydrolase in SAOS-2 osteosarcoma cells: is this enzyme a viable anticancer target?

Serine hydrolases play crucial roles in many physiological and pathophysiological processes and a panel of these enzymes are targets of approved drugs. Despite this, most of the human serine hydrolases remain poorly characterized with respect to their biological functions and substrates and only a limited number of in vivo active inhibitors have been so far identified. Acylpeptide hydrolase (APEH) is a member of the prolyl-oligopeptidase class, with a unique substrate specificity, that has been suggested to have a potential oncogenic role. In this study, a set of peptides was rationally designed from the lead compound SsCEI 4 and in vitro screened for APEH inhibition. Out of these molecules, a dodecapeptide named Ala 3 showed the best inhibitory effects and it was chosen as a candidate for investigating the anti-cancer effects induced by inhibition of APEH in SAOS-2 cell lines. The results clearly demonstrated that Ala 3 markedly reduced cell viability via deregulation of the APEH-proteasome system. Furthermore, flow cytometric analysis revealed that Ala 3 anti-proliferative effects were closely related to the activation of a caspase-dependent apoptotic pathway. Our findings provide further evidence that APEH can play a crucial role in the pathogenesis of cancer, shedding new light on the great potential of this enzyme as an attractive target for the diagnosis and the quest for selective cancer therapies.

Oxidized Substrates of APEH as a Tool to Study the Endoprotease Activity of the Enzyme.

APEH is a ubiquitous and cytosolic serine protease belonging to the prolyl oligopeptidase (POP) family, playing a critical role in the processes of degradation of proteins through both exo- and endopeptidase events. Endopeptidase activity has been associated with protein oxidation; however, the actual mechanisms have yet to be elucidated. We show that a synthetic fragment of GDF11 spanning the region 48-64 acquires sensitivity to the endopeptidase activity of APEH only when the methionines are transformed into the corresponding sulphoxide derivatives. The data suggest that the presence of sulphoxide-modified methionines is an important prerequisite for the substrates to be processed by APEH and that the residue is crucial for switching the enzyme activity from exo- to endoprotease. The cleavage occurs on residues placed on the C-terminal side of Met(O), with an efficiency depending on the methionine adjacent residues, which thereby may play a crucial role in driving and modulating APEH endoprotease activity.

APEH Inhibition Affects Osteosarcoma Cell Viability via Downregulation of the Proteasome.

The proteasome is a multienzymatic complex that controls the half-life of the majority of intracellular proteins, including those involved in apoptosis and cell-cycle progression. Recently, proteasome inhibition has been shown to be an effective anticancer strategy, although its downregulation is often accompanied by severe undesired side effects. We previously reported that the inhibition of acylpeptide hydrolase (APEH) by the peptide SsCEI 4 can significantly affect the proteasome activity in A375 melanoma or Caco-2 adenocarcinoma cell lines, thus shedding new light on therapeutic strategies based on downstream regulation of proteasome functions. In this work, we investigated the functional correlation between APEH and proteasome in a panel of cancer cell lines, and evaluated the cell proliferation upon SsCEI 4-treatments. Results revealed that SsCEI 4 triggered a proliferative arrest specifically in osteosarcoma U2OS cells via downregulation of the APEH-proteasome system, with the accumulation of the typical hallmarks of proteasome: NF-κB, p21(Waf1), and polyubiquitinylated proteins. We found that the SsCEI 4 anti-proliferative effect involved a senescence-like growth arrest without noticeable cytotoxicity. These findings represent an important step toward understanding the mechanism(s) underlying the APEH-mediated downregulation of proteasome in order to design new molecules able to efficiently regulate the proteasome system for alternative therapeutic strategies.

In Vitro Assays for the Bifunctional Acylpeptide Hydrolase (APEH) Enzyme from Antarctic Fish.

The bifunctional enzyme acylpeptide hydrolase (APEH) is involved in important metabolic processes both as an exopeptidase and as an endopeptidase. Hence, the growing interest in the study of this protein and the need to set up in vitro assays for its characterization. This chapter describes two in vitro assays able to detect the activities of APEH, one for the exopeptidase activity and one for the endopeptidase activity. In particular, these assays have been set up on the two APEH isoforms from Antarctic fish, characterized by a distinct functionality and marked exo- and endopeptidase activities.

Acyl Peptide Enzyme Hydrolase (APEH) activity is inhibited by lipid metabolites and peroxidation products.

Acyl Peptide Enzyme Hydrolase (APEH) activity is decreased in certain diseases but the mechanism and impact behind this loss in activity is not well understood. We hypothesized that lipid metabolites and lipid peroxidation products produced in inflammatory diseases may bind to and inhibit APEH activity. In vitro studies carried out in mammalian cell lysates, as well as with purified APEH protein, support our hypothesis that cellular lipid metabolites and lipid peroxidation products significantly decrease APEH activity. Enzymatic assays and molecular docking in silico analysis suggest that larger lipid metabolites are the best APEH inhibitors. APEH activity was measured in vivo in mice exposed to chronic e-cigarette vapor, as e-cigarettes are known to increase reactive oxygen species and lipid peroxidation products. In support of our in vitro findings, APEH activity in our mouse model demonstrates decreased APEH activity in the brains of mice exposed to e-cigarette vapor. These results provide a novel mechanism by which APEH activity may be inhibited in disease states. Furthermore, APEH inhibition may contribute to disease development and progression in pathologies associated with redox imbalances and can potentially act as biomarker for oxidative stress in disease.

Low Erythrocyte Levels of Proteasome and Acyl-Peptide Hydrolase (APEH) Activities in Alzheimer's Disease: A Sign of Defective Proteostasis?

Alzheimer's disease (AD) is a progressive, multifactorial neurodegenerative disorder that is the main cause of dementia. To date, there are no definitive diagnostic tests that can predict or assess onset and progression of the disease. Blood biomarkers for AD are being sought for many years but their identification remains a challenging task. In this study, we investigated the potential relationship between AD and levels of acyl-peptide hydrolase (APEH) and proteasome in erythrocyte samples of 52 participants (26 AD and 26 cognitively healthy controls). A statistically significant decrease in proteasome and exopeptidase/endopeptidase APEH activities was found in AD samples compared to those of healthy controls. Moreover, in contrast to what was observed for proteasome transcripts, APEH activities reduction in AD patients was unrelated to its gene expression levels, suggesting the occurrence of posttranslational modifications or the expression of endogenous inhibitors that might impair enzyme activity. These preliminary data further support a relationship between the APEH-proteasome system and AD molecular players, providing the first evidence of its potential use as a novel blood-based indicator for the routine detection of AD.

Influence of acylpeptide hydrolase polymorphisms on valproic acid level in Chinese epilepsy patients.

BACKGROUND: Concomitant use of meropenem (MEPM) can dramatically decrease valproic acid (VPA) plasma level. It is accepted that inhibition in acylpeptide hydrolase (APEH) activity by MEPM coadministration was the trigger of this drug-drug interaction. AIM: To investigate the influence of APEH genetic polymorphisms on VPA plasma concentration in Chinese epilepsy patients. PATIENTS & METHODS: Urinary VPA-d6 ß-D-glucuronide concentration was determined in 19 patients with VPA treatment alone (n = 10) or concomitant use with MEPM (n = 9). A retrospective study was performed on 149 epilepsy patients to investigate the influence of APEH polymorphisms rs3816877 and rs1131095 on adjusted plasma VPA concentration (CVPA) at steady-state. RESULTS: Urinary VPA-d6 ß-D-glucuronide (VPA-G) concentration was increased significantly in patients with MEPM coadministration. The CVPA of patients carrying the APEH rs3816877 C/C genotype was significantly higher than that of C/T carriers, and the difference was still obvious when stratified by UGT2B7 rs7668258 polymorphism. CONCLUSION: APEH polymorphism has significant influence on VPA pharmacokinetics in Chinese population.