Prolyl Oligopeptidase Inhibition Activates Autophagy via Protein Phosphatase 2A
Abstract
Prolyl oligopeptidase (PREP) is a serine protease that has been extensively studied, particularly in the context of neurodegenerative diseases, yet its physiological function has remained unclear. Previous research has revealed that PREP negatively regulates beclin1-mediated macroautophagy (autophagy), and that inhibition of PREP by a small-molecule inhibitor induces clearance of protein aggregates in Parkinson’s disease models. Since autophagy induction has been suggested as a potential therapy for several diseases, this study aimed to further characterize how PREP regulates autophagy. The levels of various kinases and proteins regulating beclin1-autophagy were measured in HEK-293 and SH-SY5Y cell cultures after PREP inhibition, PREP deletion, and PREP overexpression and restoration, with results verified in vivo using PREP knock-out and wild-type mouse tissue where PREP was restored or overexpressed, respectively. The findings indicate that PREP regulates autophagy by interacting with protein phosphatase 2A (PP2A) and its endogenous inhibitor, protein phosphatase methylesterase 1 (PME1), as well as the activator protein phosphatase 2 phosphatase activator (PTPA), thus adjusting its activity and the levels of PP2A in the intracellular pool. PREP inhibition and deletion increased PP2A activity, leading to activation of death-associated protein kinase 1 (DAPK1), beclin1 phosphorylation, and induced autophagy, while PREP overexpression reduced this effect. Lowered activity of PP2A is associated with several neurodegenerative disorders and cancers, and PP2A activators would have enormous potential as drug therapy, but the development of such compounds has been challenging. The concept of PREP inhibition has been proven safe, and therefore, this study supports the further development of PREP inhibitors as PP2A activators.
Introduction
Autophagy-lysosomal and ubiquitin-proteasomal pathways are responsible for the degradation of most damaged proteins and their aggregates. Proteasomes degrade unstable, short-lived proteins, while long-lived proteins and protein aggregates are degraded by macroautophagy (hereafter referred to as autophagy). Changes in autophagy activity are observed in various neurodegenerative disorders, cancer, metabolic, and infectious diseases. In neurodegenerative proteinopathies such as Parkinson’s, Alzheimer’s (AD), and Huntington’s diseases, accumulation and aggregation of toxic proteins are the main causes of pathology. These toxic protein aggregates can either directly or indirectly inhibit protein degradation pathways, further dampening aggregate clearance from affected cells. Additionally, aging negatively affects protein degradation systems and is considered one of the greatest risks for neurodegenerative proteinopathies, which develop over long periods and are mainly diagnosed in the elderly. Transgenic animals that lack essential autophagy proteins (atg5, atg7, beclin1) develop progressive motor deficits, while neuronal cells harbor ubiquitin-positive inclusions, further validating the importance of autophagy in aggregate clearance.
Pharmacological approaches to either upregulate or inhibit autophagy have received significant attention. For example, cancer treatment could benefit from either autophagy inhibition or deletion, depending on the cancer type. In neurodegenerative disorders, autophagy activation would increase protein aggregate clearance, and compounds such as rapamycin have shown effectiveness in reducing behavioral deficits and protein load in animal models of neurodegenerative diseases. However, adverse effects have limited the clinical trials of classical autophagy activators, and therefore, novel autophagy-activating compounds are needed. Recent studies have identified prolyl oligopeptidase (PREP) inhibitors as novel autophagy inducers, demonstrating that beclin1-mediated autophagy can be activated by a small-molecule PREP inhibitor, KYP-2047. Additionally, in alpha-synuclein-based Parkinson’s disease mouse models, PREP inhibition has shown beneficial impacts both in vitro and in vivo, and the concept of PREP inhibition has been shown to be safe in early clinical trials.
PREP is a serine protease, and research on PREP has mainly focused on its enzymatic activity and ability to cleave short proline-containing peptides. The hydrolytic action of PREP was the rationale behind the development of small-molecule PREP inhibitors, but although some beneficial effects were observed in preclinical memory models, their impact on neuropeptide levels in vivo remained unclear. Moreover, the physiological function of PREP has remained elusive, and in vivo evidence of PREP cleaving proline-containing peptides is inconsistent. Direct protein-protein interactions, as previously shown with alpha-synuclein, might be more relevant for PREP-related actions. Given the growing interest in autophagy for drug discovery, the mechanism by which PREP regulates autophagy was investigated. During this research, it was discovered that the small-molecule PREP inhibitor KYP-2047 leads to activation of protein phosphatase 2A (PP2A), and that PREP negatively modulates PP2A activity by regulating interactions between the catalytic subunit of PP2A (PP2Ac) and its regulatory proteins—protein phosphatase methylesterase 1 (PME1) and protein phosphatase 2 phosphatase activator (PTPA).
Materials and Methods
Reagents were obtained from commercial sources unless otherwise specified. The PREP inhibitor KYP-2047 was synthesized specifically for this study. DNA constructs for wild-type and mutant forms of PREP, as well as constructs for PP2Ac, PME1, and PTPA, were prepared using standard molecular biology techniques. Human embryonic kidney (HEK-293) cells, HEK-293 PREP knockout (PREPko) cells, human neuroblastoma SH-SY5Y cells, and SH-SY5Y PREPko cell lines were cultured under appropriate conditions. Generation of SH-SY5Y PREPko cells was performed using CRISPR-Cas9n plasmids, and removal of PREP was verified by activity assay.
HEK-293 cells stably expressing GFP-LC3B-RFP were created via transfection and selection protocols. All cell culture experiments were conducted between passages 3 to 20. Animal procedures complied with European Communities Council Directive 86/609/EEC and were approved by the Finnish National Animal Experiment Board. PREPko mice and wild-type littermates were used, and tissue samples were collected from animals under standard laboratory conditions.
Cell transfections were performed using Lipofectamine 3000. For co-immunoprecipitation studies, cells were plated and transfected as required. For PREP inhibitor experiments, KYP-2047 was used at concentrations based on previous studies. Additional reagents such as okadaic acid, bafilomycin 1A, and rapamycin were used as controls or for specific assays. Autophagic flux was determined using GFP-LC3-RFP and CytoID assays. Western blot analysis was performed using standard protocols, and protein concentrations were normalized. Co-immunoprecipitation and immunocytochemistry were used to study protein-protein interactions and localization. Protein-fragment complementation assays were performed to screen for direct interactions. Data and statistical analyses were conducted using appropriate software and statistical tests, with significance set at p < 0.05. Results PREP inhibition induces autophagic flux. Previous studies demonstrated that LC3BII levels increased in cell cultures when PREP was inhibited by KYP-2047, with further increases observed when KYP-2047 was combined with bafilomycin A1, indicating increased autophagic flux. In this study, both 4 and 24 hour PREP inhibition resulted in significantly increased LC3BII levels in Western blot analysis. PREP knockout HEK-293 cells also exhibited elevated autophagic flux. At basal conditions, these cells showed decreased LC3BII levels, while PREPko mouse cortical homogenates showed significantly elevated LC3BII levels. Autophagic flux assays using bafilomycin A1 confirmed that both PREP inhibition and removal increase autophagic flux. The impact of PREP modifications on beclin1 and bcl2 phosphorylation and their regulators was investigated. Four hours of PREP inhibition in HEK-293 cells increased both normal and phosphorylated Thr119 beclin1 levels, as well as elevated bcl2 levels. Even after 24 hours of PREP inhibition, beclin1, bcl2, and their corresponding phosphorylated forms remained significantly increased. PREP knockout HEK-293 cells had upregulated beclin1 and phosphorylated beclin1 levels but significantly decreased bcl2 levels and elevated phosphorylated bcl2, indicating that PREP regulates beclin1 more than bcl2. Transient PREP deletion by CRISPR-Cas9 elevated phosphorylated beclin1 and bcl2. In PREPko mouse cortex, bcl-xL levels were lowered and phosphorylated Ser62 bcl-xL was upregulated, similar to bcl2/phosphorylated bcl2 in PREPko cells. PREP overexpression in cells reduced phosphorylated beclin1 and bcl2 levels, while restoration of PREP to PREPko cells decreased beclin1 phosphorylation, supporting the importance of PREP in beclin1 regulation. Beclin1 phosphorylation is regulated by death-associated protein kinase 1 (DAPK), while bcl2 is dephosphorylated by c-Jun N-terminal kinase 1 (JNK1). Reduced DAPK Ser308 phosphorylation activates DAPK, and reduced phosphorylated DAPK levels were observed in HEK-293 cells after 4 hours of KYP-2047 treatment. JNK1 phosphorylation was also reduced after 4 hours of PREP inhibition, but this effect was not seen at the 24-hour time point for either DAPK or JNK1. In HEK-293 PREPko cells and mouse cortex, as well as after transient PREP deletion, upregulation in DAPK and JNK1 levels and decrease in their phosphorylation was significant. PREP overexpression in HEK-293 cells decreased DAPK levels but greatly increased phosphorylated JNK1 levels, and restoration of PREP to PREPko cells significantly elevated phosphorylated DAPK, JNK1, and phosphorylated JNK1, emphasizing the role of PREP in regulation of these kinases. Similar changes were observed in SH-SY5Y cells after PREP inhibition or deletion. These findings were further supported by studies in mouse striatal tissue. Since PREP inhibition and deletion led to dephosphorylation of both DAPK and JNK1, the involvement of a phosphatase that regulates both kinases was considered. Previous studies have reported that PP2A can dephosphorylate both DAPK and JNK1. When the levels of the catalytic subunit (PP2Ac) and its inhibitory phosphorylation (Tyr307) were studied, a slight increase in total PP2Ac and decrease in phosphorylated PP2Ac levels were observed after PREP inhibition, indicating PP2A activation. Similar effects were seen in SH-SY5Y cells. Rapid changes in phosphorylation levels after short-term PREP inhibition were not observed, and PP2Ac mRNA levels were not increased at either 4 or 24 hours of PREP inhibition. KYP-2047 and FTY720 treatments both decreased phosphorylated PP2Ac levels, while okadaic acid treatment increased PP2Ac phosphorylation, as expected. The effect of KYP-2047 could partially offset okadaic acid inhibition of PP2Ac. Deletion of PREP from HEK-293 and SH-SY5Y cells decreased phosphorylated PP2Ac levels, but no changes were seen in PP2Ac protein or mRNA levels. PREPko mouse cortical tissue had elevated PP2Ac protein and mRNA levels, but changes were not seen in PP2Ac phosphorylation compared to wild-type littermates. KYP-2047 dose-dependently dephosphorylated PP2A, with maximal effect achieved at 1 µM. The increased autophagy caused by PREP inhibition was found to be dependent on PP2A activation. Further analysis revealed that PREP inhibition or deletion affected protein levels of regulatory subunit B55α, PME1, and PTPA, as well as their distribution. B55α protein levels were upregulated and PME1 levels decreased after PREP inhibition in HEK-293 and SH-SY5Y cells. PME1 levels were decreased after short-term PREP inhibition and after transient PREP deletion, while B55α and PTPA remained unchanged. In PREPko cells and PREPko mouse cortex, B55α protein levels increased relative to control groups. PREP overexpression or restoration reduced B55α and upregulated PME1 levels in cells and reduced B55α levels in PREPko mouse striatum. PTPA protein levels were decreased only by PREP overexpression or restoration. Inactive PREP overexpression or restoration did not cause changes in B55α, PME1, or PTPA levels. Immunocytochemistry established increased colocalization between PP2Ac-PME1 and PP2Ac-PTPA after PREP inhibition or deletion. The robust change in PP2Ac phosphorylation and changes in PME1 and PTPA levels and colocalizations prompted further investigation of interactions between PREP and the PP2Ac complex subunits or regulatory proteins. Co-immunoprecipitation studies demonstrated interactions between PREP and PP2Ac, PME1, and PTPA. Protein-fragment complementation assays confirmed direct interactions, with KYP-2047 treatment significantly increasing interaction between PREP and PME1 and upregulating interaction with PTPA. Catalytically inactive PREP also formed complexes with these proteins, but only specific mutants affected PP2Ac phosphorylation. Mutation in the highly mobile PREP Loop B affected PREP interaction and led to diminished PREP effect on PP2Ac phosphorylation. Discussion This study identifies PREP as a negative regulator and interaction partner of the PP2A complex. The interaction can be modulated by a small-molecule PREP inhibitor, which induces robust dephosphorylation of PP2A, leading to activation of downstream kinase pathways responsible for the induction of beclin-1-dependent autophagy. Deletion of PREP had a similar effect to PREP inhibition, while PREP overexpression or restoration inhibited PP2A both in cell cultures and in vivo, emphasizing the importance of PREP in PP2A regulation. PP2A is a major Ser/Thr phosphatase in mammalian cells and a main regulator for cell cycle and growth. Its lowered activity and changes in subunit distribution can lead to uncontrollable cell proliferation and tumorigenesis, tau hyperphosphorylation leading to Alzheimer’s disease, and aggregation of alpha-synuclein seen in Parkinson’s disease and dementia with Lewy bodies. Activation of PP2A has been suggested as a therapeutic target for several disorders, with multi-target features that might be key in finding disease-modifying therapies for neurodegenerative diseases. Most currently known PP2A activators are targeted at cancer treatment, but their relatively high toxicity could be a deterrent for the treatment of neurodegenerative disorders. The concept of PREP inhibition has been shown to be safe in preclinical studies and clinical trials, and the novel PP2A regulatory mechanism revealed in this study offers a potentially safe target to induce PP2A activity, solving a problem in PP2A-targeted drug discovery. Although PREP inhibition activates critical kinases that can drive cells to apoptosis, the effect is mild and can be overcome by other regulators, preventing cellular death. Elevated bcl2 levels induced by PREP inhibition can partially protect cells from apoptosis. The findings explain several earlier observations about PREP, including its impacts on cell proliferation, changes in activity during ontogenesis, tumors, inflammation, and neurodegeneration. Elevated PREP activity and protein levels are significantly associated with tumors, and PREP inhibition affects the proliferation rate of certain cancer types where PP2A activity changes are observed. The effects arise from G0/G1 arrest, but the mechanism had previously remained unclear. PP2A is a known master regulator for the cell cycle, and the data indicate that elevated PREP protein levels can reduce PP2A activity, contributing to cell division and differentiation during ontogenesis and tumor growth in cancer. However, the factors leading to elevated PREP protein levels and activity in cancer remain unclear. PREP knockout mice have reduced neuronal size and connections, which may arise from overactive PP2A. Several studies have linked elevated PREP to inflammation, and strong JNK1 phosphorylation by active PREP is known to be a main regulator for inflammation and is connected to neurodegeneration. PP2A deficits have also been linked with tau hyperphosphorylation in Alzheimer’s disease, and increased colocalization between phosphorylated PP2Ac (inactive) and hyperphosphorylated tau in tangle-bearing neurons from Alzheimer’s disease patient brains has been observed. Previous work has shown increased PREP activity and colocalization of PREP with tau tangles in post-mortem Alzheimer’s disease brains, and the current findings further support the possibility that PREP has a role in the pathophysiology of Alzheimer’s disease and other tauopathies. Conclusion PREP regulates autophagy by negatively regulating PP2A, and this may be its main physiological function, while its hydrolytic function is secondary. Increased PREP activity, as seen in tumors and Alzheimer’s disease, can lead to reduced PP2A activity, connecting PREP to the pathophysiology of these disorders. The concept of PREP inhibition is considered safe, even in clinical trials, and therefore, PREP inhibition could offer a safe approach for PP242 and autophagy-activating therapy applicable to several diseases.