Prolyl Isomerase Research Articles

Proteomic analysis and immunodetection of antigens from early developmental stages of Trichinella spiralis

Trichinella spiralis is an ubiquitous parasitic nematode that lives in muscle tissue of many hosts and causes trichinellosis in humans. Numerous efforts have been directed at specific detection of this infection and strategies for its control. TSL-1 and other antigens, mainly from muscle larvae (ML), have been used to induce partial protection in rodents. An improvement in protective immunity may be achieved by using antigens from other parasite stages. Further, identification of other parasite antigens may provide insights into their role in the host-parasite interaction. In this study, T. spiralis antigens from early developmental parasite stages, namely ML and pre-adult (PA) obtained at 6h, 18h and 30h post-infection, were identified by proteomic and mass spectrometry analyses. Our findings showed a differential expression of several proteins with molecular weights in the range of 13–224kDa and pI range of 4.54–9.89. Bioinformatic analyses revealed a wide diversity of functions in the identified proteins, which include structural, antioxidant, actin binding, peptidyl prolyl cis-trans isomerase, motor, hydrolase, ATP binding, magnesium and calcium binding, isomerase and translation elongation factor. This, together with the differential recognition of antigens from these parasite stages by antibodies present in intestinal fluid, in supernatants from intestinal explants, and in serum samples from mice infected with T. spiralis or re-infected with this parasite, provides information that may lead to alternatives in the design of vaccines against this parasite or for modulation of immune responses.

Proteomic analysis of egg white heparin-binding proteins: towards the identification of natural antibacterial molecules.

The chicken egg resists most environmental microbes suggesting that it potentially contains efficient antimicrobial molecules. Considering that some heparin-binding proteins in mammals are antibacterial, we investigated the presence and the antimicrobial activity of heparin-binding proteins from chicken egg white. Mass spectrometry analysis of the proteins recovered after heparin-affinity chromatography, revealed 20 proteins, including known antimicrobial proteins (avidin, lysozyme, TENP, ovalbumin-related protein X and avian bêta-defensin 11). The antibacterial activity of three new egg candidates (vitelline membrane outer layer protein 1, beta-microseminoprotein-like (LOC101750704) and pleiotrophin) was demonstrated against Listeria monocytogenes and/or Salmonella enterica Enteritidis. We showed that all these molecules share the property to inhibit bacterial growth through their heparin-binding domains. However, vitelline membrane outer layer 1 has additional specific structural features that can contribute to its antimicrobial potential. Moreover, we identified potential supplementary effectors of innate immunity including mucin 5B, E-selectin ligand 1, whey acidic protein 3, peptidyl prolyl isomerase B and retinoic acid receptor responder protein 2. These data support the concept of using heparin affinity combined to mass spectrometry to obtain an overview of the various effectors of innate immunity composing biological milieus, and to identify novel antimicrobial candidates of interest in the race for alternatives to antibiotics.

Everolimus induces Met inactivation by disrupting the FKBP12/Met complex.

Inhibition of the mechanistic target of rapamycin (mTOR) is a promising treatment strategy for several cancer types. Rapamycin derivatives such as everolimus are allosteric mTOR inhibitors acting through interaction with the intracellular immunophilin FKBP12, a prolyl isomerase with different cellular functions. Although mTOR inhibitors have significantly improved survival of different cancer patients, resistance and lack of predictive factors of response remain unsolved issues. To elucidate the mechanisms of resistance to everolimus, we evaluated Met activation in everolimus-sensitive/resistant human cancer cells, in vitro and in vivo. Biochemical and computational analyses were performed. Everolimus-resistant cells were xenografted into mice (10/group) and studied for their response to everolimus and Met inhibitors. The statistical significance of the in vitro results was evaluated by Student's t test.Everolimus reduced Met phosphorylation in everolimus-sensitive cells. This event was mediated by the formation of a Met-FKBP12 complex, which in turn is disrupted by everolimus. Aberrant Met activation in everolimus-resistant cells and overexpression of wild-type/mutant Met caused everolimus resistance. Pharmacological inhibition and RNA silencing of Met are effective in condition of everolimus resistance (P<0.01). In mice xenografted with everolimus-resistant cells, the combination of everolimus with the Met inhibitor PHA665752 reduced tumor growth and induced a statistically significant survival advantage (combination vs control P=0.0005).FKBP12 binding is required for full Met activation and everolimus can inhibit Met. Persistent Met activation might sustain everolimus resistance. These results identify a novel everolimus mechanism of action and suggest the development of clinical strategies based on Met inhibitors in everolimus-resistant cancers.

Inhibition of Aβ(1-40) fibril formation by cyclophilins.

Cyclophilins interact directly with the Alzheimer's disease peptide Aβ (amyloid β-peptide) and are therefore involved in the early stages of Alzheimer's disease. Aβ binding to CypD (cyclophilin D) induces dysfunction of human mitochondria. We found that both CypD and CypA suppress invitro fibril formation of Aβ(1-40) at substoichiometric concentrations when present early in the aggregation process. The prototypic inhibitor CsA (cyclosporin A) of both cyclophilins as well as the new water-soluble MM258 derivative prevented this suppression. A SPOT peptide array approach and NMR titration experiments confirmed binding of Aβ(1-40) to the catalytic site of CypD mainly via residues Lys(16)-Glu(22) The peptide Aβ(16-20) representing this section showed submicromolar IC50 values for the peptidyl prolyl cis-trans isomerase activity of CypD and CypA and low-micromolar KD values in ITC experiments. Chemical cross-linking and NMR-detected hydrogen-deuterium exchange experiments revealed a shift in the populations of small Aβ(1-40) oligomers towards the monomeric species, which we investigated in the present study as being the main process of prevention of Aβ fibril formation by cyclophilins.

Effects of Pin1 Loss in Hdh(Q111) Knock-in Mice.

Huntington’s disease (HD) is a fatal, dominantly inherited, neurodegenerative disorder due to a pathological expansion of the CAG repeat in the coding region of the HTT gene. In the quest for understanding the molecular basis of neurodegeneration, we have previously demonstrated that the prolyl isomerase Pin1 plays a crucial role in mediating p53-dependent apoptosis triggered by mutant huntingtin (mHtt) in vitro. To assess the effects of the lack of Pin1 in vivo, we have bred Pin1 knock-out mice with HdhQ111 knock-in mice, a genetically precise model of HD. We show that Pin1 genetic ablation modifies a portion of HdhQ111 phenotypes in a time-dependent fashion. As an early event, Pin1 activity reduces the DNA damage response (DDR). In midlife mice, by taking advantage of next-generation sequencing technology, we show that Pin1 activity modulates a portion of the alterations triggered by mHtt, extending the role of Pin1 to two additional HdhQ111 phenotypes: the unbalance in the “synthesis/concentration of hormones”, as well as the alteration of “Wnt/β-catenin signaling”. In aging animals, Pin1 significantly increases the number of mHtt-positive nuclear inclusions while it reduces gliosis. In summary, this work provides further support for a role of Pin1 in HD pathogenesis.

Crystallizability of an Engineered Monomeric Mutant of FK506-binding Protein from Shewanella sp. SIB1: Preliminary Diffraction Data Analysis

Background and Objective: A 22 kDa FK506-binding protein from a psychrophilic bacterium Shewanella sp. SIB1 (SIB1 FKBP22) is a member of peptidyl prolyl cis-trans isomerase (PPIase). This protein is homodimer with a V-shaped form, consisting of N and C-domains, that are connected through a long α-helix, responsible for dimerization and PPIase activity, respectively. Understanding on structural mechanisms behind the function of SIB1 FKBP22 is limited by unsuccessful attempts on crystallization of the full length SIB1 FKBP22 homodimer due to its flexibility and low stability. Despite the isolated N-domain, with the absence of α-helix and C-domain was successfully crystallized and structurally solved, the comprehensive structural arrangement of SIB1 FKBP22 remains missing. The objective of this study is to construct a crystallizable SIB1 FKBP22 derivatives consisting of N and C-domains with its α-helix that reflect full length of SIB1 FKBP22 and a platform for comprehensive structural analysis. Materials and Methods: A monomeric mutant of SIB1 FKBP22 was constructed by combining two gene fragments encoding Met 8-Ile 205 and Met 1-Ala 60 of the first and second monomer of SIB1 FKBP22, respectively, with three glycine residues. This design yielded the mutant has tandemly repeated N-domain connected to C-domain through a long α-helix hence designated as NNC-FKBP22. Results: The NNC-FKBP22 was monomeric in solution implying that it did not form a V-shaped dimeric structure. The crystallization of NNC-FKBP22 was attempted under sitting-drop vapor diffusion. The crystals were obtained under 1.0 M sodium citrate with 10 mM CHES/sodium hydroxide at pH 9.5. The crystal was in the space group of P212121 with unit cell dimension a = 94.635, b = 92.479 and c = 337.327 A. A complete native data was collected from a rotating anode source to a resolution of 3.5 A at 100 K with an Rmerge value of 25.50. Conclusion: The NNC-FKBP22 was successfully crystallized implying that stabilization of SIB1 FKBP22 under protein engineering platform is promising approach to decipher its atomic structure. Protein engineering technique used in this study would also be applicable for other cold-adapted proteins with high structural flexibility and low stability.

TNFα reduces eNOS activity in endothelial cells through serine 116 phosphorylation and Pin1 binding: Confirmation of a direct, inhibitory interaction of Pin1 with eNOS

Production of NO by the endothelial nitric oxide synthase (eNOS) has a major role in blood pressure control and suppression of atherosclerosis. In a previous study, we presented evidence implicating the Pin1 prolyl isomerase in negative modulation of eNOS activity in bovine aortic endothelial cells (BAECs). Pin1 recognizes phosphoserine/phosphothreonine–proline motifs in target proteins and catalyzes prolyl isomerization at the peptide bond. In the present study, we show, first, with purified proteins, that Pin1 binds to eNOS directly via the Pin1 WW domain. Binding is enhanced by mimicking phosphorylation of eNOS at S116. Interaction of Pin1 with eNOS markedly reduces eNOS enzymatic activity. Second, in BAECs, we show that TNFα induces ERK 1/2-mediated S116 phosphorylation of eNOS, accompanied by Pin1 binding. TNFα treatment of BAECs results in a reduction in NO release from the cells in a manner that depends on the activities of both Pin1 and ERK 1/2. Evidence is also presented that this mechanism of eNOS regulation cannot occur in rat and mouse cells because there is no proline residue in the mouse and rat amino acid sequences adjacent to the putative phosphorylation site. Moreover, we find that phosphorylation of this site is not detectable in mouse eNOS.

Mitochondrial Thioredoxin System as a Modulator of Cyclophilin D Redox State.

The mitochondrial thioredoxin system (NADPH, thioredoxin reductase, thioredoxin) is a major redox regulator. Here we have investigated the redox correlation between this system and the mitochondrial enzyme cyclophilin D. The peptidyl prolyl cis-trans isomerase activity of cyclophilin D was stimulated by the thioredoxin system, while it was decreased by cyclosporin A and the thioredoxin reductase inhibitor auranofin. The redox state of cyclophilin D, thioredoxin 1 and 2 and peroxiredoxin 3 was measured in isolated rat heart mitochondria and in tumor cell lines (CEM-R and HeLa) by redox Western blot analysis upon inhibition of thioredoxin reductase with auranofin, arsenic trioxide, 1-chloro-2,4-dinitrobenzene or after treatment with hydrogen peroxide. A concomitant oxidation of thioredoxin, peroxiredoxin and cyclophilin D was observed, suggesting a redox communication between the thioredoxin system and cyclophilin. This correlation was further confirmed by i) co-immunoprecipitation assay of cyclophilin D with thioredoxin 2 and peroxiredoxin 3, ii) molecular modeling and iii) depleting thioredoxin reductase by siRNA. We conclude that the mitochondrial thioredoxin system controls the redox state of cyclophilin D which, in turn, may act as a regulator of several processes including ROS production and pro-apoptotic factors release.

Pin1-mediated Modification Prolongs the Nuclear Retention of β-Catenin in Wnt3a-induced Osteoblast Differentiation

The canonical Wnt signaling pathway, in which β-catenin nuclear localization is a crucial step, plays an important role in osteoblast differentiation. Pin1, a prolyl isomerase, is also known as a key enzyme in osteogenesis. However, the role of Pin1 in canonical Wnt signal-induced osteoblast differentiation is poorly understood. We found that Pin1 deficiency caused osteopenia and reduction of β-catenin in bone lining cells. Similarly, Pin1 knockdown or treatment with Pin1 inhibitors strongly decreased the nuclear β-catenin level, TOP flash activity, and expression of bone marker genes induced by canonical Wnt activation and vice versa in Pin1 overexpression. Pin1 interacts directly with and isomerizes β-catenin in the nucleus. The isomerized β-catenin could not bind to nuclear adenomatous polyposis coli, which drives β-catenin out of the nucleus for proteasomal degradation, which consequently increases the retention of β-catenin in the nucleus and might explain the decrease of β-catenin ubiquitination. These results indicate that Pin1 could be a critical target to modulate β-catenin-mediated osteogenesis.

Phosphate-Induced Renal Fibrosis Requires the Prolyl Isomerase Pin1.

Tubulo-interstitial fibrosis is a common, destructive endpoint for a variety of kidney diseases. Fibrosis is well correlated with the loss of kidney function in both humans and rodents. The identification of modulators of fibrosis could provide novel therapeutic approaches to reducing disease progression or severity. Here, we show that the peptidyl-prolyl isomerase Pin1 is an important molecular contributor that facilitates renal fibrosis in a well-characterized animal model. While wild-type mice fed a high phosphate diet (HPD) for 8–12 weeks developed calcium deposition, macrophage infiltration and extracellular matrix (ECM) accumulation in the kidney interstitium, Pin1 null mice showed significantly less pathology. The serum Pi in both WT and KO mice were significantly increased by the HPD, but the serum Ca was slightly decreased in KO compared to WT. In addition, both WT and KO HPD mice had less weight gain but exhibited normal organ mass (kidney, lung, spleen, liver and heart). Unexpectedly, renal function was not initially impaired in either genotype irrespective of the HPD. Our results suggest that diet containing high Pi induces rapid renal fibrosis before a significant impact on renal function and that Pin1 plays an important role in the fibrotic process.

Pin1 modulates chemo-resistance by up-regulating FoxM1 and the involvements of Wnt/β-catenin signaling pathway in cervical cancer

The prolyl isomerase Pin1, which is frequently highly expressed in many different cancers, can directly regulate cell proliferation and the cell cycle. However, the role of Pin1 in chemo-resistance remains to be elucidated in cervical cancer. The purpose of the present study was to investigate the role of Pin1 in the chemo-resistance of cervical cancer. The cisplatin resistance was assessed using the MTT assay. Pin1, FoxM1, β-catenin, Cyclin D1, and c-myc expression levels were detected by RT-qPCR or Western blot. The results showed that Pin1 expression displayed a similar expression pattern with the resistance to cisplatin in five cervical cell lines. Knockdown of Pin1 significantly increased the sensitivity to cisplatin in HeLa cells, while Pin1 overexpression decreased the sensitivity to cisplatin in Me180 cells. Knockdown of Pin1 significantly down-regulated FoxM1 expression in HeLa cells, while Pin1 overexpression showed a contrary effect in Me180 cells. Besides, overexpression of Pin1 markedly increased the protein expression of β-catenin and its target genes cyclin D1 and c-myc. FoxM1 siRNA remarkably reversed the promotory effect of pcDNA-Pin1(+) on β-catenin and its target genes cyclin D1 and c-myc in Me180 cells. Furthermore, we also found that FoxM1 siRNA and IWP-2 markedly decreased cell viability, and IWP-2 decreased cell viability to the maximum extent in the Me180 cells co-transfected with pcDNA-Pin1(+) and FoxM1 siRNA. Taken together, these data suggest that Pin1 contributes to cisplatin resistance, partly by up-regulating FoxM1 and Wnt/β-catenin signaling pathway involved in cervical cancer.

Peptidyl Prolyl Isomerase PIN1 Directly Binds to and Stabilizes Hypoxia-Inducible Factor-1α.

Peptidyl prolyl isomerase (PIN1) regulates the functional activity of a subset of phosphoproteins through binding to phosphorylated Ser/Thr-Pro motifs and subsequently isomerization of the phosphorylated bonds. Interestingly, PIN1 is overexpressed in many types of malignancies including breast, prostate, lung and colon cancers. However, its oncogenic functions have not been fully elucidated. Here, we report that PIN1 directly interacts with hypoxia-inducible factor (HIF)-1α in human colon cancer (HCT116) cells. PIN1 binding to HIF-1α occurred in a phosphorylation-dependent manner. We also found that PIN1 interacted with HIF-1α at both exogenous and endogenous levels. Notably, PIN1 binding stabilized the HIF-1α protein, given that their levels were significantly increased under hypoxic conditions. The stabilization of HIF-1α resulted in increased transcriptional activity, consequently upregulating expression of vascular endothelial growth factor, a major contributor to angiogenesis. Silencing of PIN1 or pharmacologic inhibition of its activity abrogated the angiogenesis. By utilizing a bioluminescence imaging technique, we were able to demonstrate that PIN1 inhibition dramatically reduced the tumor volume in a subcutaneous mouse xenograft model and angiogenesis as well as hypoxia-induced transcriptional activity of HIF-1α. These results suggest that PIN1 interacting with HIF-1α is a potential cancer chemopreventive and therapeutic target.

Inhibition of the FKBP family of peptidyl prolyl isomerases induces abortive translocation and degradation of the cellular prion protein.

Prion diseases are fatal neurodegenerative disorders for which there is no effective treatment. Because the cellular prion protein (PrP(C)) is required for propagation of the infectious scrapie form of the protein, one therapeutic strategy is to reduce PrP(C) expression. Recently FK506, an inhibitor of the FKBP family of peptidyl prolyl isomerases, was shown to increase survival in animal models of prion disease, with proposed mechanisms including calcineurin inhibition, induction of autophagy, and reduced PrP(C) expression. We show that FK506 treatment results in a profound reduction in PrP(C) expression due to a defect in the translocation of PrP(C) into the endoplasmic reticulum with subsequent degradation by the proteasome. These phenotypes could be bypassed by replacing the PrP(C) signal sequence with that of prolactin or osteopontin. In mouse cells, depletion of ER luminal FKBP10 was almost as potent as FK506 in attenuating expression of PrP(C). However, this occurred at a later stage, after translocation of PrP(C) into the ER. Both FK506 treatment and FKBP10 depletion were effective in reducing PrP(Sc) propagation in cell models. These findings show the involvement of FKBP proteins at different stages of PrP(C) biogenesis and identify FKBP10 as a potential therapeutic target for the treatment of prion diseases.

Cyclophilin C Participates in the US2-Mediated Degradation of Major Histocompatibility Complex Class I Molecules

Human cytomegalovirus uses a variety of mechanisms to evade immune recognition through major histocompatibility complex class I molecules. One mechanism mediated by the immunoevasin protein US2 causes rapid disposal of newly synthesized class I molecules by the endoplasmic reticulum-associated degradation pathway. Although several components of this degradation pathway have been identified, there are still questions concerning how US2 targets class I molecules for degradation. In this study we identify cyclophilin C, a peptidyl prolyl isomerase of the endoplasmic reticulum, as a component of US2-mediated immune evasion. Cyclophilin C could be co-isolated with US2 and with the class I molecule HLA-A2. Furthermore, it was required at a particular expression level since depletion or overexpression of cyclophilin C impaired the degradation of class I molecules. To better characterize the involvement of cyclophilin C in class I degradation, we used LC-MS/MS to detect US2-interacting proteins that were influenced by cyclophilin C expression levels. We identified malectin, PDIA6, and TMEM33 as proteins that increased in association with US2 upon cyclophilin C knockdown. In subsequent validation all were shown to play a functional role in US2 degradation of class I molecules. This was specific to US2 rather than general ER-associated degradation since depletion of these proteins did not impede the degradation of a misfolded substrate, the null Hong Kong variant of α1-antitrypsin.

Cyclophilin function in Cancer; lessons from virus replication

Cyclophilins belong to a group of proteins that possess peptidyl prolyl isomerase activity and catalyse the cis-trans conversion of proline peptide bonds. Cyclophilin members play important roles in protein folding and as molecular chaperones, in addition to a well-established role as host factors required for completion of the virus life cycle. Members of the cyclophilin family are overexpressed in a range of human malignancies including hepatocellular cancer, pancreatic cancer, nonsmall cell lung cancer, gastric cancer, colorectal cancer and glioblastoma multiforme, however, their precise role in tumourigenesis remains unclear. In recent years, mounting evidence supports a role for prolyl isomerisation during mammalian cell division; a process with striking similarity to plasma membrane remodelling during virus replication. Here, we summarise our current understanding of the role of cyclophilins in cancer. We review the function of cyclophilins during mammalian cell division and during HIV-1 infection, and highlight common processes involving members of the ESCRT and Rab GTPase families.

FK506-Binding Proteins and Their Diverse Functions.

FK506 binding proteins (FKBPs) are a family of highly conserved proteins in eukaryotes. The prototype of this protein family, FKBP12, is the binding partner for immunosuppressive drugs FK506 and rapamycin. FKBP12 functions as a cis/trans peptidyl prolyl isomerase (PPIase) that catalyzes interconversion between prolyl cis/trans conformations. Members of the FKBP family contain one or several PPIase domains, which do not always exhibit PPIase activity yet are all essential for their function. FKBPs are involved in diverse cellular functions including protein folding, cellular signaling, apoptosis and transcription. They elicit their function through direct binding and altering conformation of their target proteins, hence acting as molecular switches. In this review, we provide a general summary for the structures and diverse functions of FKBPs found in mammalian cells.

The Role of Peptidyl Prolyl Isomerases in Aging and Vascular Diseases

Peptidyl prolyl isomerases (PPIases) are proteins belonging to the immunophilin family and are characterised by their cis-trans isomerization activity at the X-Pro peptide bond, in addition to their tetratricopeptide repeat (TPR) domain, important for interaction with the molecular chaperone, Hsp90. Due to this unique structure these proteins are able to facilitate protein-protein interactions which can impact significantly on a range of cellular processes such as cell signalling, differentiation, cell cycle progression, metabolic activity and apoptosis. Malfunction and/or dysregulation of most members of this class of proteins promotes cellular damage and tissue/organ failure, predisposing to ageing and age-related diseases. Many individual genes within the PPIase family are associated with several age-related diseases including cardiovascular diseases (CVDs), atherosclerosis, type II diabetes mellitus (T2D), chronic kidney disease (CDK), neurodegeneration, cancer and age-related macular degeneration (AMD), in addition to the ageing process itself. This review will focus on the different roles of PPIases, and their therapeutic/ biomarker potential in these age-related vascular diseases.

Involvement of FKBP6 in hepatitis C virus replication.

The chaperone system is known to be exploited by viruses for their replication. In the present study, we identified the cochaperone FKBP6 as a host factor required for hepatitis C virus (HCV) replication. FKBP6 is a peptidyl prolyl cis-trans isomerase with three domains of the tetratricopeptide repeat (TPR), but lacks FK-506 binding ability. FKBP6 interacted with HCV nonstructural protein 5A (NS5A) and also formed a complex with FKBP6 itself or FKBP8, which is known to be critical for HCV replication. The Val121 of NS5A and TPR domains of FKBP6 were responsible for the interaction between NS5A and FKBP6. FKBP6 was colocalized with NS5A, FKBP8, and double-stranded RNA in HCV-infected cells. HCV replication was completely suppressed in FKBP6-knockout hepatoma cell lines, while the expression of FKBP6 restored HCV replication in FKBP6-knockout cells. A treatment with the FKBP8 inhibitor N-(N′, N′-dimethylcarboxamidomethyl)cycloheximide impaired the formation of a homo- or hetero-complex consisting of FKBP6 and/or FKBP8, and suppressed HCV replication. HCV infection promoted the expression of FKBP6, but not that of FKBP8, in cultured cells and human liver tissue. These results indicate that FKBP6 is an HCV-induced host factor that supports viral replication in cooperation with NS5A.

Peptidyl prolyl isomerase Pin1-inhibitory activity of D-glutamic and D-aspartic acid derivatives bearing a cyclic aliphatic amine moiety.

Pin1 is a peptidyl prolyl isomerase that specifically catalyzes cis-trans isomerization of phosphorylated Thr/Ser-Pro peptide bonds in substrate proteins and peptides. Pin1 is involved in many important cellular processes, including cancer progression, so it is a potential target of cancer therapy. We designed and synthesized a novel series of Pin1 inhibitors based on a glutamic acid or aspartic acid scaffold bearing an aromatic moiety to provide a hydrophobic surface and a cyclic aliphatic amine moiety with affinity for the proline-binding site of Pin1. Glutamic acid derivatives bearing cycloalkylamino and phenylthiazole groups showed potent Pin1-inhibitory activity comparable with that of known inhibitor VER-1. The results indicate that steric interaction of the cyclic alkyl amine moiety with binding site residues plays a key role in enhancing Pin1-inhibitory activity.

Prolyl isomerase Pin1 regulates cadmium-induced autophagy via ubiquitin-mediated post-translational stabilization of phospho-Ser GSK3αβ in human hepatocellular carcinoma cells

Accumulating evidence suggests that prolyl-isomerase (Pin1) is an important regulator of apoptosis. In the present study, Pin1 was shown to negatively regulate the stabilization of glycogen synthase kinase (GSK) 3αβ serine phosphorylation by inhibiting ubiquitin (Ub)-proteasome degradation, and to induce autophagy following cadmium (Cd) exposure. Cd-induced autophagy in human hepatoma (HepG2) cells has been demonstrated by green fluorescent protein (GFP)-LC3B plasmid DNA transfection, and LC3-II conversion by autophagy inhibitors. Atg5 silencing inhibited Cd-induced apoptosis, indicating that autophagy is involved in cell death. Exposing HepG2 cells to Cd (≤6μM) initially increased p-Ser GSK3αβ, but then resulted in a gradual decrease, which correlated with polyubiquitinated protein levels, which is indicative of protein degradation. However, high Cd concentrations lead to p-Ser GSK3αβ and polyubiquitinated protein accumulation, indicating proteasome impairment. Cd-induced p-Ser GSK3αβ was enhanced by proteasome inhibition (MG132) and ubiquitin deficiency (cyclohexamide), but no ubiquitination of p-Ser GSK3αβ or GSK3αβ was detected. Cd exposure resulted in p-Ser GSK3αβ redistribution from the nucleus into the cytosol, and this along with autophagy, was inhibited by leptomycin B. Cd concentrations <6μM did not alter Pin1; however, Cd≥6μM decreased Pin1. Pin1 overexpression decreased Cd-induced p-Ser GSK3αβ, autophagy, and polyubiquitinated protein levels, while its inhibition reversed the effects. Silencing and overexpression of GSK3αβ had no effect on Cd-induced Pin1 levels. Immunoprecipitation studies showed that Pin1 and p-Ser GSK3αβ interact. Collectively, Pin1 protects cells by inhibiting p-Ser GSK3αβ, and Pin1 decrease upregulates p-Ser GSK3αβ, through the inhibition of Ub-mediated proteasome degradation, and stimulates cell death via autophagy.