The Iron-Type Nitrile Hydratase Activator Protein Is A GTPase

The Fe-type nitrile hydratase activator protein from Rhodococcus equi TG328-2 (ReNHase TG328-2) was successfully expressed and purified. Sequence analysis and homology modeling suggest that it is a G3E P-loop guanosine triphosphatase (GTPase) within the COG0523 subfamily. Kinetic studies revealed that the Fe-type activator protein is capable of hydrolyzing GTP to GDP with a kcat value of 1.2 × 10−3 s−1 and a Km value of 40 μM in the presence of 5 mM MgCl2 in 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid at a pH of 8.0. The addition of divalent metal ions, such as Co(II), which binds to the ReNHase TG328-2 activator protein with a Kd of 2.9 μM, accelerated the rate of GTP hydrolysis, suggesting that GTP hydrolysis is potentially connected to the proposed metal chaperone function of the ReNHase TG328-2 activator protein. Circular dichroism data reveal a significant conformational change upon the addition of GTP, which may be linked to the interconnectivity of the cofactor binding sites, resulting in an activator protein that can be recognized and can bind to the NHase α-subunit. A combination of these data establishes, for the first time, that the ReNHase TG328-2 activator protein falls into the COG0523 subfamily of G3E P-loop GTPases, many of which play a role in metal homeostasis processes

1α,25-Dihydroxyvitamin D3 Stimulates Activator Protein 1 DNA-Binding Activity by a Phosphatidylinositol 3-Kinase/Ras/MEK/Extracellular Signal Regulated Kinase 1/2 and c-Jun N-Terminal Kinase 1-Dependent Increase in c-Fos, Fra1, and c-Jun Expression in Human Keratinocytes

1α,25-Dihydroxyvitamin D3 added to human keratinocytes increases differentiation through an activation of the transcription factor activator protein 1. We have previously reported that the 1α,25-dihydroxyvitamin D3-induced increase of activator protein 1 DNA binding activity is mediated by a protein kinase C-independent mechanism. The purpose of this study was to investigate further the mechanisms by which 1α,25-dihydroxyvitamin D3 modulates activator protein 1 DNA binding activity in cultured normal human keratinocytes. Western blotting experiments revealed that 1α,25-dihydroxyvitamin D3 caused a rapid and transient activation of the mitogen-activated protein kinases, extracellular signal regulated kinase 1/2 and c-Jun N-terminal kinase 1. 1α,25-Dihydroxyvitamin D3 also enhanced the expression of the activator protein 1 subunits, c-Fos, Fra1, and c-Jun as determined by northern and western blotting. The 1α,25-dihydroxyvitamin D3-induced activator protein 1 DNA binding activity was completely blocked by the MEK inhibitor PD 98059 indicating that the MEK/extracellular signal regulated kinase pathway is involved in the activation of activator protein 1. Transfection experiments showed that 1α,25-dihydroxyvitamin D3 also increased the activator protein 1-dependent transactivation, which was completely blocked by expression of a dominant negative Ras, suggesting that the 1α,25-dihydroxyvitamin D3-induced activator protein 1 activity involves Ras-dependent signaling. Furthermore, preincubation of the keratinocytes with the specific phosphatidylinositol 3-kinase inhibitors, Wortmannin and LY294002, demonstrated that the 1α,25-dihydroxyvitamin D3-induced activation of extracellular signal regulated kinase 1/2 and c-Jun N-terminal kinase 1 required phosphatidylinositol 3-kinase activity. Finally, preincubation of keratinocytes with a polyclonal antibody against the membrane receptor annexin II, blocked the 1α,25-dihydroxyvitamin D3-induced activation of extracellular signal regulated kinase 1/2 and c-Jun N-terminal kinase 1. Taken together, our results indicate that 1α,25-dihydroxyvitamin D3, via binding to the membrane receptor annexin II, induces activation of the phos-phatidylinositol 3-kinase/Ras/MEK/extracellular signal regulated kinase 1/2 and c-Jun N-terminal kinase 1 signal transduction pathway resulting in increased expression of c-Fos, Fra1, and c-Jun, and subsequently increased activator protein 1 DNA binding activity and gene transcription

Targeting the Ets Binding Site of the HER2/neu Promoter with Pyrrole-Imidazole Polyamides

Three DNA binding polyamides (1-3) were synthesized that bind with high affinity (Ka = 8.7·10^9 M^-1 to 1.4·10^10 M^-1) to two 7-base pair sequences overlapping the Ets DNA binding site (EBS; GAGGAA) within the regulatory region of the HER2/neu proximal promoter. As measured by electrophoretic mobility shift assay, polyamides binding to flanking elements upstream (1) or downstream (2 and 3) of the EBS were one to two orders of magnitude more effective than the natural product distamycin at inhibiting formation of complexes between the purified EBS protein, epithelial restricted with serine box (ESX), and the HER2/neu promoter probe. One polyamide, 2, completely blocked Ets-DNA complex formation at 10 nM ligand concentration, whereas formation of activator protein-2-DNA complexes was unaffected at the activator protein-2 binding site immediately upstream of the HER2/neu EBS, even at 100 nM ligand concentration. At equilibrium, polyamide 1 was equally effective at inhibiting Ets/DNA binding when added before or after in vitro formation of protein-promoter complexes, demonstrating its utility to disrupt endogenous Ets-mediated HER2/neu preinitiation complexes. Polyamide 2, the most potent inhibitor of Ets-DNA complex formation by electrophoretic mobility shift assay, was also the most effective inhibitor of HER2/neu promoter-driven transcription measured in a cell-free system using nuclear extract from an ESX- and HER2/neu-overexpressing human breast cancer cell line, SKBR-3

Pyrroloquinoline quinone increases the expression and activity of Sirt1 and -3 genes in HepG2 cells.

Sirtuin (Sirt) 1 and Sirt 3 are nicotinamide adenine dinucleotide ((+))-dependent protein deacetylases that are important to a number of mitochondrial-related functions; thus, identification of sirtuin activators is important. Herein, we hypothesize that pyrroloquinoline quinone (PQQ) can act as a Sirt1/Sirt3 activator. In HepG2 cell cultures, PQQ increased the expression of Sirt1 and Sirt3 gene, protein, and activity levels (P < .05). We also observed a significant increase in nicotinamide phosphoribosyltransferase gene expression (as early as 18 hours) and increased NAD(+) activity at 24 hours. In addition, targets of Sirt1 and Sirt3 (peroxisome proliferator-activated receptor γ coactivator 1α, nuclear respiratory factor 1 and 2, and mitochondrial transcription factor A) were increased at 48 hours. This is the first report that demonstrates PQQ as an activator of Sirt1 and Sirt3 expression and activity, making it an attractive therapeutic agent for the treatment of metabolic diseases and for healthy aging. Based on our study and the available data in vivo, PQQ has the potential to serve as a therapeutic nutraceutical, when enhancing mitochondrial function

Translocation of plasminogen activator inhibitor-1 during serum stimulated growth of mouse embryo fibroblasts

Serum-stimulated mouse embryo fibroblasts specifically secrete two proteins of molecular weights 48,000 and 26,000. The 48 kDa protein showed affinity to concanavalin A and was precipitated by antibody to plasminogen activator inhibitor. Immunoflowcytometry using anti plasminogen activator inhibitor-1 serum indicate the presence of the 48 kDa protein in quiescent cells; this protein was virtually absent in serum-stimulated cells. The presence of the plasminogen activator inhibitor-1 related protein in quiescent cells and its absence in serum-stimulated cells in combination with the observation on the absence of this protein, in the medium of quiescent cells and its presence in the medium of stimulated cells indicate that the 48 kDa protein was transferred from the cells into the medium upon serum-stimulation. The serum-mediated transfer of plasminogen activator inhibitor-1 from the cells into the medium was inhibited by actinomycin-D suggesting that the transfer process required actinomycin-D sensitive events. Treatment of pre-labelled quiescent cells with medium containing 20% fetal calf serum resulted in the gradual transfer of the labelled 48 kDa protein to the extra cellular matrix. These studies indicate that exposure of quiescent cells to fetal calf serum results in the transfer of plasminogen activator inhibitor-1 from the cells to the growth mediumvia extracellular matrix. The translocation of the protease inhibitor from the cells to the matrix and medium may enable the cellular and possibly the membrane proteases to act on growth factors or their receptors thereby initiating the mitogenic response

Regulation of CLC-1 chloride channel biosynthesis by FKBP8 and Hsp90β.

Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90β. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90β inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90β and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90β play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation

Atrial Natriuretic Peptide Induces Mitogen-Activated Protein Kinase Phosphatase-1 in Human Endothelial Cells via Rac1 and NAD(P)H Oxidase/Nox2-Activation

The cardiovascular hormone atrial natriuretic peptide (ANP) exerts anti-inflammatory effects on tumor necrosis factor-α–activated endothelial cells by inducing mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). The underlying mechanisms are as yet unknown. We aimed to elucidate the signaling pathways leading to an induction of MKP-1 by ANP in primary human endothelial cells. By using antioxidants, generation of reactive oxygen species (ROS) was shown to be crucially involved in MKP-1 upregulation. ANP was found to increase ROS formation in cultured cells as well as in the endothelium of intact rat lung vessels. We applied NAD(P)H oxidase (Nox) inhibitors (apocynin and gp91ds-tat) and revealed this enzyme complex to be crucial for superoxide generation and MKP-1 expression. Moreover, by performing Nox2/4 antisense experiments, we identified Nox2 as the critically involved Nox homologue. Pull-down assays and confocal microscopy showed that ANP activates the small Rho-GTPase Rac1. Transfection of a dominant-negative (RacN17) and constitutively active Rac1 mutant (RacV12) indicated that ANP-induced superoxide generation and MKP-1 expression are mediated via Rac1 activation. ANP-evoked production of superoxide was found to activate c-Jun N-terminal kinase (JNK). Using specific inhibitors, we linked ANP-induced JNK activation to MKP-1 expression and excluded an involvement of protein kinase C, extracellular signal-regulated kinase, and p38 MAPK. MKP-1 induction was shown to depend on activation of the transcription factor activator protein-1 (AP-1) by using electrophoretic mobility shift assay and AP-1 decoys. In summary, our work provides insights into the mechanisms by which ANP induces MKP-1 and shows that ANP is a novel endogenous activator of endothelial Rac1 and Nox/Nox2

Protein C activation by an activator purified from the venom of <i>Agkistrodon halys halys</i>

The protein C activator from Agkistrodon halys halys venom was purified 533-fold by ion-exchange chromatography on QAE-Sephadex A-50, affinity chromatography on aprotinin-Sepharose and Mono-Q fast protein liquid chromatography. The purified enzyme is a single chain protein with an apparent molecular weight of 36 000 that activates protein C by proteolytic removal of a small fragment from the heavy chain. The protein C activator exhibited a high amidolytic activity towards the tripeptide substrates D-Pro-Phe-Arg-pNA (S2302) and D-Phe-(pipecolyl)-Arg-pNA (S2238). The activity of the activator was not affected by thiolprotease or metalloprotease inhibitors. The activator was inhibited, however, by benzamidine, Phe-Pro-Arg chloromethyl ketone,p-nitrophenyl p-guanidinobenzoate and soy bean trypsin inhibitor, which classifies the enzyme as a serine protease. The purified protease was capable of activating both human and bovine protein C. Activation of human protein C only occurred at an appreciable rate in a calcium-free reaction medium at low ionic strength. Ca2+ ions inhibited the activation of human protein C with an apparent K(i) of 0.8 mM. Addition of NaCl to the reaction medium also strongly inhibited human protein C activation (50% inhibition at 20 mM NaCl). Kinetic analysis of human protein C activation by the venom activator (in a calcium-free medium) revealed an apparent K(m) for protein C of 0.52 muM and a k(cat) of 0.17 s-1 at 1 = 0.05 (k(cat)/K(m) = 3.3 X 10(5) M-1 s-1). At I = 0.15 rates of human protein C activation became linear with protein C indicating a strong increase in K(m) with increasing ionic strength. Activation of bovine protein C was hardly affected by variation of Ca2+ and NaCl concentrations in the reaction medium. The apparent K(i)s for calcium ion and NaCl inhibition of bovine protein C activation were &gt; 10 mM and 220 mM, respectively. At I = 0.1 and in the absence of Ca2+ ions bovine protein C was activated with a K(m) of 0.056 muM and a k(cat) of 0.24 s-1 (k(cat)/K(m) = 4.3 x 10(6) M-1 s-1). Our data are indicative for a rather large conformational and/or structural difference between human and bovine protein C at physiological ionic strength

Expression pattern of the urokinase-plasminogen activator system in rat DS-sarcoma: Role of oxygenation status and tumour size

The urokinase plasminogen activator system plays a central role in malignant tumour progression. Both tumour hypoxia and enhancement of urokinase plasminogen activator, urokinase plasminogen activator-receptor and plasminogen activator inhibitor type 1 have been identified as adverse prognostic factors. Upregulation of urokinase plasminogen activator or plasminogen activator inhibitor type 1 could present means by which hypoxia influences malignant progression. Therefore, the impact of hypoxia on the expression pattern of the urokinase plasminogen activator system in rat DS-sarcoma in vivo and in vitro was examined. In the in vivo setting, tumour cells were implanted subcutaneously into rats, which were housed under either hypoxia, atmospheric air or hyperoxia. For in vitro studies, DS-sarcoma cells were incubated for 24 h under hypoxia. Urokinase plasminogen activator and urokinase plasminogen activator-receptor expression were analysed by flow cytometry. Urokinase plasminogen activator activity was measured using zymography. Plasminogen activator inhibitor type 1 protein levels in vitro and in vivo were examined with ELISA. PAI-1 mRNA levels were determined by RT–PCR. DS-sarcoma cells express urokinase plasminogen activator, urokinase plasminogen activator-receptor, and plasminogen activator inhibitor type 1 in vitro and in vivo. The urokinase plasminogen activator activity is enhanced in DS-sarcomas compared to normal tissues and rises with increasing tumour volume. The oxygenation level has no impact on the urokinase plasminogen activator activity in cultured DS-sarcoma cells or in solid tumours, although in vitro an increase in plasminogen activator inhibitor type 1 protein and mRNA expression after hypoxic challenge is detectable. The latter plasminogen activator inhibitor type 1 changes were not detectable in vivo. Hypoxia has been demonstrated to contribute to the upregulation of some components of the system in vitro, although this effect was not reproducible in vivo. This may indicate that the serum level of plasminogen activator inhibitor type 1 is not a reliable surrogate marker of tumour hypoxia

Aberrant Polycystin-1 Expression Results in Modification of Activator Protein-1 Activity, whereas Wnt Signaling Remains Unaffected

Polycystin-1, the polycystic kidney disease 1 gene product, has been implicated in several signaling complexes that are known to regulate essential cellular functions. We investigated the role of polycystin-1 in Wnt signaling and activator protein-1 (AP-1) activation. To this aim, a membrane-targeted construct encoding the conserved C-terminal region of mouse polycystin- 1 reported to mediate signal transduction activity was expressed in human embryonic and renal epithelial cells. To ensure specificity and minimal cotransfection effects, we focused our study on the endogenous proteins that actually transduce the signals, -catenin and T-cell factor/lymphoid-enhancing factor for Wnt signaling and (phosphorylated) c-Jun, ATF2, and c-Fos for AP-1. Our data indicate that the C-terminal region of polycystin-1 activates AP-1 by inducing phosphorylation and expression of at least c-Jun and ATF2, whereas c-Fos was not affected. Under our experimental conditions, polycystin-1 did not modulate Wnt signaling. AP-1 activity was aberrant in human autosomal dominant polycystic kidney disease (ADPKD) renal cystic epithelial cells and in renal epithelial cells expressing transgenic full-length polycystin- 1, resulting in decreased Jun-ATF and increased Jun-Fos activity, whereas Wnt signaling remained unaffected. Since our data indicate that aberrant polycystin- 1 expression results in altered AP-1 activity, polycystin-1 may be required for adequate AP-1 activity