Molecular dynamics simulations of wild type and mutant of Pin1 peptidyl-prolyl isomerase

Pin1 catalyses the intrinsically slow process of cis-trans isomerisation and has been identified as a possible drug target in many diseases. Recently, the wild type (WT) and the Cys113Asp mutant of the Pin1 peptidyl-prolyl isomerase (PPIase) domain were determined by nuclear magnetic resonance. In this article, the WT and Cys113Asp mutant of PPIase domain are studied by molecular dynamics simulations. The structural stability analysis shows that the Cys113Asp mutation leads to the higher fluctuation of hydrophobic core in PPIase domain. The intrinsic correlated motions are important for the catalytic function of Pin1, whereas the Cys113Asp mutant system loses pivotal dynamical properties and develops wider conformational states than those in WT system. The intramolecular hydrogen bonds play crucial roles in the structural stability of PPIase domain. The mutated residue Asp113 attracts the side chain of His59 in the Cys113Asp system, which unbalances the internal interactions inside the catalytic tetrad. Meanwhile, the conformational changes of PPIase domain affect the side chain orientations of Lys63 and Arg69, which limit their binding with substrates. The Cys113Asp mutation destabilises the whole binding region of Pin1 PPIase domain, so the catalysis activity is severely reduced. These results are consistent with experimental studies and may help to understand the isomerisation mechanisms of Pin1.     

Involvement of peptidyl-prolyl isomerase Pin1 in the inhibitory effect of fluvastatin on endothelin-1-induced cardiomyocyte hypertrophy

Aims

Cardiac hypertrophy is elicited by endothelin (ET)-1 as well as other neurohumoral factors, hemodynamic overload, and oxidative stress; HMG-CoA reductase inhibitors (statins) were shown to inhibit cardiac hypertrophy partly via the anti-oxidative stress. One of their common intracellular pathways is the phosphorylation cascade of MEK signaling. Pin1 specifically isomerizes the phosphorylated protein with Ser/Thr-Pro bonds and regulates their activity through conformational changes. There is no report whether the Pin1 activation contributes to ET-1-induced cardiomyocyte hypertrophy and whether the Pin1 inactivation contributes to the inhibitory effect of statins. The aim of this study was to reveal these questions.

Main methods

We assessed neonatal rat cardiomyocyte hypertrophy using ET-1 and fluvastatin by the cell surface area, ANP mRNA expression, JNK and c-Jun phosphorylation, and [³H]-leucine incorporation.

Key findings

Fluvastatin inhibited ET-1-induced increase in the cell surface area, ANP expression, and [³H]-leucine incorporation; and it suppressed the signaling cascade from JNK to c-Jun. The phosphorylated Pin1 level, an inactive form, was decreased by ET-1; however, it reached basal level by fluvastatin. Furthermore, Pin1 overexpression clearly elicited cardiomyocyte hypertrophy, which was inhibited by fluvastatin.

Significance

This is the first report that ET-1-induced cardiomyocyte hypertrophy is mediated through the Pin1 activation and that the inhibitory effect of fluvastatin on cardiomyocyte hypertrophy would partly be attributed to the suppression of the Pin1 function. This study firstly suggests that Pin1 determines the size of hypertrophied cardiomyocyte by regulating the activity of phosphorylated molecules and that statins exert their pleiotropic effects partly via Pin1 inactivation.     

Effects of peptidyl-prolyl isomerase 1 depletion in animal models of prion diseases

Pin1 is a peptidyl-prolyl isomerase that induces the cis-trans conversion of specific Ser/Thr-Pro peptide bonds in phosphorylated proteins, leading to conformational changes through which Pin1 regulates protein stability and activity. Since down-regulation of Pin1 has been described in several neurodegenerative disorders, including Alzheimer's Disease (AD), Parkinson's Disease (PD) and Huntington's Disease (HD), we investigated its potential role in prion diseases. Animals generated on wild-type (Pin1^+/+), hemizygous (Pin1^+/?) or knock-out (Pin1^?/?) background for Pin1 were experimentally infected with RML prions. The study indicates that, neither the total depletion nor reduced levels of Pin1 significantly altered the clinical and neuropathological features of the disease.     

Ligand-independent activation of vascular endothelial growth factor receptor 1 by low-density lipoprotein

Elevated serum low-density lipoprotein (LDL) is a risk factor for atherosclerotic disorders. However, prominent atherosclerosis, which has been observed in LDL receptor (LDLR)-knockout mice, has diminished the significance of LDLR as a cause of atherosclerosis, while elaborate studies have focused on the receptors for denatured LDL. Here we report that native LDL (nLDL) activates vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1) but not VEGFR2 through LDLR and is as potent as VEGF in macrophage migration. Binding and co-endocytosis of VEGFR1 and LDLR were enhanced by nLDL, which is concomitant with ubiquitination-mediated degradation of VEGFR1. We propose that LDLR-mediated use of VEGFR1 by nLDL could be a potential therapeutic target in atherosclerotic disorders.     

Hypoxia-induced expression of HIF-1alpha and its target genes in umbilical venous endothelial cells of Tibetans and immigrant Han

The better adaptation of native Tibetans to hypoxia is thought to be partly due to improved umbilical circulation, which results in reduced pre- and postnatal fatalities. We hypothesized that the difference in umbilical circulation between native Tibetans and other high-altitude inhabitants was due to differences in the expression of hypoxia-induced factor (HIF-1) and its target genes vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS). We tested this hypothesis by examining the effect of hypoxia on the expression of HIF-1alpha, VEGF, and iNOS in cultured umbilical venous endothelial cells (UVECs) from native Tibetans and immigrant Hans. UVECs were collected and cultured under hypoxic (0.5% oxygen) or normoxic conditions for 2, 4, 12 and 24 h. The mRNA levels of HIF-1alpha, VEGF, endothelial nitric oxide synthase (eNOS) and iNOS and the protein level of HIF-1alpha were determined with RT-PCR and Western blot analyses, respectively. In both immigrant Han and Tibetans, HIF-1alpha mRNA was constitutively expressed under normoxic condition, and remained constant after hypoxic exposure. In contrast, HIF-1alpha protein was undetectable under normoxic condition, but underwent dynamic changes in response to hypoxia. It was induced at 4 h, peaked at 12 h, and remained elevated at 24 h. Concurrent with the induction of HIF-1alpha protein, the mRNA levels of VEGF and iNOS were also up-regulated whereas that of eNOS was down-regulated. The lack of a hypoxia-related difference in the expression of HIF-1alpha and its target genes suggests that HIF-1alpha does not play a critical role in high altitude adaptation. Alternative mechanisms may be responsible for the better adaptation of native Tibetans.     

Hypoxia-inducible factor 1-alpha and vascular endothelial growth factor in cartilage tumors

Neoangiogenesis has been demonstrated in chondrosarcoma. Anti-angiogenic therapies are being tested in clinical trials for chondrosarcomas. Studies of the underlying mechanisms have been performed almost exclusively in cell lines. We immunostained 20 samples of chondrosarcoma and 20 samples of enchondromas with antibodies against hypoxia-inducible factor 1-alpha (HIF-1-alpha) and vascular endothelial growth factor (VEGF). The immunohistochemical staining of HIF-1-alpha and VEGF were highly correlated. Enchondromas were HIF-1-alpha and VEGF negative, whereas all chondrosarcoma exhibited HIF-1-alpha and VEGF immunostaining. HIF-1-alpha/VEGF double positive cases were almost exclusively chondrosarcomas with a high tumor grade. We suggest that HIF-1-alpha is a marker of malignancy in chondrosarcomas that correlates with tumor neo-angiogenesis. Our findings also suggest that a HIF-1-alpha/VEGF angiogenic pathway may exist in chondrosarcoma in vivo as in other malignant tumors. The inclusion of novel inhibitors to HIF-1-alpha and other factors may optimize anti-angiogenic interventions in chondrosarcoma.     

Regulation of vascular endothelial growth factor signaling by miR-200b

Vascular endothelial growth factor (VEGF) signaling plays an important role in angiogenesis. In the VEGF signaling pathway, the key components are VEGF and its receptors, Flt-1 and KDR. In this study, we show that transfection of synthetic miR-200b reduced protein levels of VEGF, Flt-1, and KDR. In A549 cells, miR-200b targeted the predicted binding sites in the 3′-untranslated region (3′-UTR) of VEGF, Flt-1, and KDR as revealed by a luciferase reporter assay. When transfected with miR-200b, the ability of HUVECs to form a capillary tube on Matrigel and VEGF-induced phosphorylation of ERK1/2 were significantly reduced. Taken together, these results suggest that miR-200b negatively regulates VEGF signaling by targeting VEGF and its receptors and that miR-200b may have therapeutic potential as an angiogenesis inhibitor.     

Porcine placental immunoexpression of vascular endothelial growth factor,placenta growth factor,Flt-1 and Flk-1

Porcine embryo mortalities cause economic losses. Development of the placental vascular bed is required for successful gestation and postnatal survival. We studied the temporal and spatial distributions of vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and their receptors, Flt-1 and Flk-1. We used crossbred swine placental tissues from 30, 60, 80, 90 and 114 (term) days of gestation. Both VEGF and PlGF were present during gestation. At early pregnancy and at term, VEGF probably acts through Flt-1; during intermediate periods, its function is mediated by Flk-1. By day 90, factors other than members of VEGF family appear to be involved.     

The peptidyl-prolyl isomerase Pin1 regulates granulocyte-macrophage colony-stimulating factor mRNA stability in T lymphocytes

Cytokine production is associated with both the normal and pathologic inflammatory response to injury. Previous studies have shown that the immunosuppressants cyclosporin A or FK506, which interact with the peptidyl-propyl isomerases cyclophilin A and FK506-binding protein (FKBP12), respectively, block cytokine expression. A third member of the peptidyl-propyl isomerase family, Pin1 is expressed by immune and other cells. Pin1 has been implicated in cell cycle progression, is overexpressed in human tumors, and may rescue neurons from tau-associated degeneration. However, the role of Pin1 in the immune system remains largely unknown. In this study, we analyze the role of Pin1 in GM-CSF expression by human PBMC and CD4+ lymphocytes. We show that Pin1 isomerase activity is necessary for activation-dependent, GM-CSF mRNA stabilization, accumulation, and protein secretion, but not non-AU-rich elements containing cytokine mRNAs, including TGF-beta and IL-4. Mechanistically, Pin1 mediated the association of the AU-rich element-binding protein, AUF1, with GM-CSF mRNA, which determined the rate of decay by the exosome.     

Regulation of Pin1 peptidyl-prolyl cis/trans isomerase activity by its WW binding module on a multi-phosphorylated peptide of Tau protein

The WW module of the peptidyl-prolyl cis/trans isomerase Pin1 targets specifically phosphorylated proteins involved in the cell cycle through the recognition of phospho-Thr(Ser)-Pro motifs. When the microtubule-associated Tau protein becomes hyperphosphorylated, it equally becomes a substrate for Pin1, with two recognition sites described around the phosphorylated Thr212 and Thr231. The Pin1 WW domain binds both sites with moderate affinity, but only the Thr212-Pro213 bond is isomerized by the catalytic domain of Pin1. We show here that, in a peptide carrying a single recognition site, the WW module increases significantly the enzymatic isomerase activity of Pin1. However, with addition of a second recognition motif, the affinity of both the WW and catalytic domain for the substrate increases, but the isomerization efficacy decreases. We therefore conclude that the WW domain can act as a negative regulator of enzymatic activity when multiple phosphorylation is present, thereby suggesting a subtle mechanism of its functional regulation.     

Induction of three-dimensional assembly and increase in apoptosis of human endothelial cells by simulated microgravity: Impact of vascular endothelial growth factor

Endothelial cells play a crucial role in the pathogenesis of many diseases and are highly sensitive to low gravity conditions. Using a three-dimensional random positioning machine (clinostat) we investigated effects of simulated weightlessness on the human EA.hy926 cell line (4, 12, 24, 48 and 72 h) and addressed the impact of exposure to VEGF (10 ng/ml). Simulated microgravity resulted in an increase in extracellular matrix proteins (ECMP) and altered cytoskeletal components such as microtubules (alpha-tubulin) and intermediate filaments (cytokeratin). Within the initial 4 h, both simulated microgravity and VEGF, alone, enhanced the expression of ECMP (collagen type I, fibronectin, osteopontin, laminin) and flk-1 protein. Synergistic effects between microgravity and VEGF were not seen. After 12 h, microgravity further enhanced all proteins mentioned above. Moreover, clinorotated endothelial cells showed morphological and biochemical signs of apoptosis after 4 h, which were further increased after 72 h. VEGF significantly attenuated apoptosis as demonstrated by DAPI staining, TUNEL flow cytometry and electron microscopy. Caspase-3, Bax, Fas, and 85-kDa apoptosis-related cleavage fragments were clearly reduced by VEGF. After 72 h, most surviving endothelial cells had assembled to three-dimensional tubular structures. Simulated weightlessness induced apoptosis and increased the amount of ECMP. VEGF develops a cell-protective influence on endothelial cells exposed to simulated microgravity.     

Functionally important residues in the peptidyl-prolyl isomerase Pin1 revealed by unigenic evolution

Pin1 is a phosphorylation-dependent member of the parvulin family of peptidyl-prolyl isomerases exhibiting functional conservation between yeast and man. To perform an unbiased analysis of the regions of Pin1 essential for its functions, we generated libraries of randomly mutated forms of the human Pin1 cDNA and identified functional Pin1 alleles by their ability to complement the Pin1 homolog Ess1 in Saccharomyces cerevisiae. We isolated an extensive collection of functional mutant Pin1 clones harboring a total of 356 amino acid substitutions. Surprisingly, many residues previously thought to be critical in Pin1 were found to be altered in this collection of functional mutants. In fact, only 17 residues were completely conserved in these mutants and in Pin1 sequences from other eukaryotic organisms, with only two of these conserved residues located within the WW domain of Pin1. Examination of invariant residues provided new insights regarding a phosphate-binding loop that distinguishes a phosphorylation-dependent peptidyl-prolyl isomerase such as Pin1 from other parvulins. In addition, these studies led to an investigation of residues involved in catalysis including C113 that was previously implicated as the catalytic nucleophile. We demonstrate that substitution of C113 with D does not compromise Pin1 function in vivo nor does this substitution abolish catalytic activity in purified recombinant Pin1. These findings are consistent with the prospect that the function of residue 113 may not be that of a nucleophile, thus raising questions about the model of nucleophilic catalysis. Accordingly, an alternative catalytic mechanism for Pin1 is postulated.     

Cloning of a cancer cell-producing hepatocyte growth factor, vascular endothelial growth factor, and interleukin-8 from gastric cancer cells

A cell colony (IM95m) that produces hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), and interleukin-8 (IL-8) was cloned from gastric cancer cells (IM95 cell line). In culture medium, the highest levels of HGF, VEGF, and IL-8 were about 1.1, 0.9, and 0.17 ng/ml culture medium at 3 d from 10(5) cells. IM95m may be useful in elucidating the role of tumor cells in angiogenesis.     

Regulation of retinal capillary cells by basic fibroblast growth factor, vascular endothelial growth factor, and hypoxia

Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) feature prominently in retinal neovascular diseases. Although the role of VEGF in retinal angiogenesis is well established, the importance of bFGF in this process requires further clarification. This study was undertaken to investigate the responses of retinal capillary cells (endothelial cells and pericytes) to bFGF under hypoxic conditions, as well as the potentially synergistic effects of bFGF and VEGF on the proliferation and cord formation of retinal endothelial cells. Cell proliferation was determined by cell number and by 3H-thymidine incorporation. Cord formation was assessed in three-dimensional gels of collagen type I. VEGF and bFGF increased 3H-thymidine incorporation by both cell types, an effect that was more pronounced in a hypoxic environment. Moreover, the proliferation of pericytes was stimulated to a greater extent by bFGF relative to VEGF. Endothelial migration in collagen gels, however, was induced more effectively by VEGF than by bFGF. A synergistic effect of VEGF and bFGF on cell invasion was observed in the collagen gel assay. VEGF and bFGF each augment proliferation of these cells, especially under hypoxia. We thus propose that these two cytokines have a synergistic effect at several stages of angiogenesis in the retina.