Science.gov

Sample records for peptidase iv inhibition

  1. Generation of dipeptidyl peptidase-IV-inhibiting peptides from β-lactoglobulin secreted by Lactococcus lactis.

    PubMed

    Shigemori, Suguru; Oshiro, Kazushi; Wang, Pengfei; Yamamoto, Yoshinari; Wang, Yeqin; Sato, Takashi; Uyeno, Yutaka; Shimosato, Takeshi

    2014-01-01

    Previous studies showed that hydrolysates of β-lactoglobulin (BLG) prepared using gastrointestinal proteases strongly inhibit dipeptidyl peptidase-IV (DPP-IV) activity in vitro. In this study, we developed a BLG-secreting Lactococcus lactis strain as a delivery vehicle and in situ expression system. Interestingly, trypsin-digested recombinant BLG from L. lactis inhibited DPP-IV activity, suggesting that BLG-secreting L. lactis may be useful in the treatment of type 2 diabetes mellitus. PMID:25157356

  2. Inhibition of dipeptidyl peptidase IV by fluoroolefin-containing N-peptidyl-O-hydroxylamine peptidomimetics

    PubMed Central

    Lin, Jian; Toscano, Paul J.; Welch, John T.

    1998-01-01

    Dipeptidyl peptidase IV (EC 3.4.14.5; DPP IV), also known as the leukocyte differentiation antigen CD26 when found as an extracellular membrane-bound proline specific serine protease, cleaves a dipeptide from the N terminus of a polypeptide chain containing a proline residue in the penultimate position. Here we report that known (Z)-Ala-ψ[CF=C]-Pro dipeptide isosteres 1 and 2, which contain O-acylhydroxylamines, were isolated as diastereomeric pairs u-1, l-1, and l-2. The effect of each diastereomeric pair as an inhibitor of human placental dipeptidyl peptidase DPP IV has been examined. The inhibition of DPP IV by these compounds is rapid and efficient. The diastereomeric pair u-1 exhibits very potent inhibitory activity with a Ki of 188 nM. Fluoroolefin containing N-peptidyl-O-hydroxylamine peptidomimetics, by virtue of their inhibitory potency and stability, are superior to N-peptidyl-O-hydroxylamine inhibitors derived from an Ala-Pro dipeptide. PMID:9826645

  3. Dipeptidyl Peptidase IV Inhibition Exerts Renoprotective Effects in Rats with Established Heart Failure.

    PubMed

    Arruda-Junior, Daniel F; Martins, Flavia L; Dariolli, Rafael; Jensen, Leonardo; Antonio, Ednei L; Dos Santos, Leonardo; Tucci, Paulo J F; Girardi, Adriana C C

    2016-01-01

    Circulating dipeptidyl peptidase IV (DPPIV) activity is associated with worse cardiovascular outcomes in humans and experimental heart failure (HF) models, suggesting that DPPIV may play a role in the pathophysiology of this syndrome. Renal dysfunction is one of the key features of HF, but it remains to be determined whether DPPIV inhibitors are capable of improving cardiorenal function after the onset of HF. Therefore, the present study aimed to test the hypothesis that DPPIV inhibition by vildagliptin improves renal water and salt handling and exerts anti-proteinuric effects in rats with established HF. To this end, male Wistar rats were subjected to left ventricle (LV) radiofrequency ablation or sham operation. Six weeks after surgery, radiofrequency-ablated rats who developed HF were randomly divided into two groups and treated for 4 weeks with vildagliptin (120 mg/kg/day) or vehicle by oral gavage. Echocardiography was performed before (pretreatment) and at the end of treatment (post-treatment) to evaluate cardiac function. The fractional area change (FAC) increased (34 ± 5 vs. 45 ± 3%, p < 0.05), and the isovolumic relaxation time decreased (33 ± 2 vs. 27 ± 1 ms; p < 0.05) in HF rats treated with vildagliptin (post-treatment vs. pretreatment). On the other hand, cardiac dysfunction deteriorated further in vehicle-treated HF rats. Renal function was impaired in vehicle-treated HF rats as evidenced by fluid retention, low glomerular filtration rate (GFR) and high levels of urinary protein excretion. Vildagliptin treatment restored urinary flow, GFR, urinary sodium and urinary protein excretion to sham levels. Restoration of renal function in HF rats by DPPIV inhibition was associated with increased active glucagon-like peptide-1 (GLP-1) serum concentration, reduced DPPIV activity and increased activity of protein kinase A in the renal cortex. Furthermore, the anti-proteinuric effect of vildagliptin treatment in rats with established HF was associated with

  4. Dipeptidyl Peptidase IV Inhibition Exerts Renoprotective Effects in Rats with Established Heart Failure

    PubMed Central

    Arruda-Junior, Daniel F.; Martins, Flavia L.; Dariolli, Rafael; Jensen, Leonardo; Antonio, Ednei L.; dos Santos, Leonardo; Tucci, Paulo J. F.; Girardi, Adriana C. C.

    2016-01-01

    Circulating dipeptidyl peptidase IV (DPPIV) activity is associated with worse cardiovascular outcomes in humans and experimental heart failure (HF) models, suggesting that DPPIV may play a role in the pathophysiology of this syndrome. Renal dysfunction is one of the key features of HF, but it remains to be determined whether DPPIV inhibitors are capable of improving cardiorenal function after the onset of HF. Therefore, the present study aimed to test the hypothesis that DPPIV inhibition by vildagliptin improves renal water and salt handling and exerts anti-proteinuric effects in rats with established HF. To this end, male Wistar rats were subjected to left ventricle (LV) radiofrequency ablation or sham operation. Six weeks after surgery, radiofrequency-ablated rats who developed HF were randomly divided into two groups and treated for 4 weeks with vildagliptin (120 mg/kg/day) or vehicle by oral gavage. Echocardiography was performed before (pretreatment) and at the end of treatment (post-treatment) to evaluate cardiac function. The fractional area change (FAC) increased (34 ± 5 vs. 45 ± 3%, p < 0.05), and the isovolumic relaxation time decreased (33 ± 2 vs. 27 ± 1 ms; p < 0.05) in HF rats treated with vildagliptin (post-treatment vs. pretreatment). On the other hand, cardiac dysfunction deteriorated further in vehicle-treated HF rats. Renal function was impaired in vehicle-treated HF rats as evidenced by fluid retention, low glomerular filtration rate (GFR) and high levels of urinary protein excretion. Vildagliptin treatment restored urinary flow, GFR, urinary sodium and urinary protein excretion to sham levels. Restoration of renal function in HF rats by DPPIV inhibition was associated with increased active glucagon-like peptide-1 (GLP-1) serum concentration, reduced DPPIV activity and increased activity of protein kinase A in the renal cortex. Furthermore, the anti-proteinuric effect of vildagliptin treatment in rats with established HF was associated with

  5. Bioactive compounds from culinary herbs inhibit a molecular target for type 2 diabetes management, dipeptidyl peptidase IV.

    PubMed

    Bower, Allyson M; Real Hernandez, Luis M; Berhow, Mark A; de Mejia, Elvira Gonzalez

    2014-07-01

    Greek oregano (Origanum vulgare), marjoram (Origanum majorana), rosemary (Rosmarinus officinalis), and Mexican oregano (Lippia graveolens) are concentrated sources of bioactive compounds. The aims were to characterize and examine extracts from greenhouse-grown or commercially purchased herbs for their ability to inhibit dipeptidyl peptidase IV (DPP-IV) and protein tyrosine phosphatase 1B (PTP1B), enzymes that play a role in insulin secretion and insulin signaling, respectively. Greenhouse herbs contained more polyphenols (302.7-430.1 μg of gallic acid equivalents/mg of dry weight of extract (DWE)) and flavonoids (370.1-661.4 μg of rutin equivalents/mg of DWE) compared to the equivalent commercial herbs. Greenhouse rosemary, Mexican oregano, and marjoram extracts were the best inhibitors of DPP-IV (IC₅₀=16, 29, and 59 μM, respectively). Commercial rosemary, Mexican oregano, and marjoram were the best inhibitors of PTP1B (32.4-40.9% at 500 μM). The phytochemicals eriodictyol, naringenin, hispidulin, cirsimaritin, and carnosol were identified by LC-ESI-MS as being present in greenhouse-grown Mexican oregano and rosemary. Computational modeling indicated that hispidulin, carnosol, and eriodictyol would have the best binding affinities for DPP-IV. Biochemically, the best inhibitors of DPP-IV were cirsimaritin (IC₅₀=0.43±0.07 μM), hispidulin (IC₅₀=0.49±0.06 μM), and naringenin (IC₅₀=2.5±0.29 μM). Overall, herbs contain several flavonoids that inhibit DPP-IV and should be investigated further regarding their potential in diabetes management. PMID:24881464

  6. The dipeptidyl peptidase IV inhibitors vildagliptin and K-579 inhibit a phospholipase C: a case of promiscuous scaffolds in proteins

    PubMed Central

    Dutta, Mouparna; Ghosh, Anindya S.; Oda, Masataka; Venkatramani, Ravindra; Rao, Basuthkar J.; Dandekar, Abhaya M.; Goñi, Félix M.

    2015-01-01

    The long term side effects of any newly introduced drug is a subject of intense research, and often raging controversies. One such example is the dipeptidyl peptidase-IV (DPP4) inhibitor used for treating type 2 diabetes, which is inconclusively implicated in increased susceptibility to acute pancreatitis. Previously, based on a computational analysis of the spatial and electrostatic properties of active site residues, we have demonstrated that phosphoinositide-specific phospholipase C (PI-PLC) from Bacillus cereus is a prolyl peptidase using in vivo experiments. In the current work, we first report the inhibition of the native activity of PI-PLC by two DPP4 inhibitors - vildagliptin (LAF-237) and K-579. While vildagliptin inhibited PI-PLC at micromolar concentrations, K-579 was a potent inhibitor even at nanomolar concentrations. Subsequently, we queried a comprehensive, non-redundant set of 5000 human proteins (50% similarity cutoff) with known structures using serine protease (SPASE) motifs derived from trypsin and DPP4. A pancreatic lipase and a gastric lipase are among the proteins that are identified as proteins having promiscuous SPASE scaffolds that could interact with DPP4 inhibitors. The presence of such scaffolds in human lipases is expected since they share the same catalytic mechanism with PI-PLC. However our methodology also detects other proteins, often with a completely different enzymatic mechanism, that have significantly congruent domains with the SPASE motifs. The reported elevated levels of serum lipase, although contested, could be rationalized by inhibition of lipases reported here. In an effort to further our understanding of the spatial and electrostatic basis of DPP4 inhibitors, we have also done a comprehensive analysis of all 76 known DPP4 structures liganded to inhibitors till date. Also, the methodology presented here can be easily adopted for other drugs, and provide the first line of filtering in the identification of pathways that

  7. Dipeptidyl peptidase IV inhibition potentiates amino acid- and bile acid-induced bicarbonate secretion in rat duodenum.

    PubMed

    Inoue, Takuya; Wang, Joon-Ho; Higashiyama, Masaaki; Rudenkyy, Sergiy; Higuchi, Kazuhide; Guth, Paul H; Engel, Eli; Kaunitz, Jonathan D; Akiba, Yasutada

    2012-10-01

    Intestinal endocrine cells release gut hormones, including glucagon-like peptides (GLPs), in response to luminal nutrients. Luminal L-glutamate (L-Glu) and 5'-inosine monophosphate (IMP) synergistically increases duodenal HCO3- secretion via GLP-2 release. Since L cells express the bile acid receptor TGR5 and dipeptidyl peptidase (DPP) IV rapidly degrades GLPs, we hypothesized that luminal amino acids or bile acids stimulate duodenal HCO3- secretion via GLP-2 release, which is enhanced by DPPIV inhibition. We measured HCO3- secretion with pH and CO2 electrodes using a perfused rat duodenal loop under isoflurane anesthesia. L-Glu (10 mM) and IMP (0.1 mM) were luminally coperfused with or without luminal perfusion (0.1 mM) or intravenous (iv) injection (3 μmol/kg) of the DPPIV inhibitor NVP728. The loop was also perfused with a selective TGR5 agonist betulinic acid (BTA, 10 μM) or the non-bile acid type TGR5 agonist 3-(2-chlorophenyl)-N-(4-chlorophenyl)-N,5-dimethylisoxazole-4-carboxamide (CCDC; 10 μM). DPPIV activity visualized by use of the fluorogenic substrate was present on the duodenal brush border and submucosal layer, both abolished by the incubation with NVP728 (0.1 mM). An iv injection of NVP728 enhanced L-Glu/IMP-induced HCO3- secretion, whereas luminal perfusion of NVP728 had no effect. BTA or CCDC had little effect on HCO3- secretion, whereas NVP728 iv markedly enhanced BTA- or CCDC-induced HCO3- secretion, the effects inhibited by a GLP-2 receptor antagonist. Coperfusion of the TGR5 agonist enhanced L-Glu/IMP-induced HCO3- secretion with the enhanced GLP-2 release, suggesting that TGR5 activation amplifies nutrient sensing signals. DPPIV inhibition potentiated luminal L-Glu/IMP-induced and TGR5 agonist-induced HCO3- secretion via a GLP-2 pathway, suggesting that the modulation of the local concentration of the endogenous secretagogue GLP-2 by luminal compounds and DPPIV inhibition helps regulate protective duodenal HCO3- secretion. PMID:22821947

  8. Different modes of dipeptidyl peptidase IV (CD26) inhibition by oligopeptides derived from the N-terminus of HIV-1 Tat indicate at least two inhibitor binding sites.

    PubMed

    Lorey, Susan; Stöckel-Maschek, Angela; Faust, Jürgen; Brandt, Wolfgang; Stiebitz, Beate; Gorrell, Mark D; Kähne, Thilo; Mrestani-Klaus, Carmen; Wrenger, Sabine; Reinhold, Dirk; Ansorge, Siegfried; Neubert, Klaus

    2003-05-01

    Dipeptidyl peptidase IV (DP IV, CD26) plays an essential role in the activation and proliferation of lymphocytes, which is shown by the immunosuppressive effects of synthetic DP IV inhibitors. Similarly, both human immunodeficiency virus-1 (HIV-1) Tat protein and the N-terminal peptide Tat(1-9) inhibit DP IV activity and T cell proliferation. Therefore, the N-terminal amino acid sequence of HIV-1 Tat is important for the inhibition of DP IV. Recently, we characterized the thromboxane A2 receptor peptide TXA2-R(1-9), bearing the N-terminal MWP sequence motif, as a potent DP IV inhibitor possibly playing a functional role during antigen presentation by inhibiting T cell-expressed DP IV [Wrenger, S., Faust, J., Mrestani-Klaus, C., Fengler, A., Stöckel-Maschek, A., Lorey, S., Kähne, T., Brandt, W., Neubert, K., Ansorge, S. & Reinhold, D. (2000) J. Biol. Chem.275, 22180-22186]. Here, we demonstrate that amino acid substitutions at different positions of Tat(1-9) can result in a change of the inhibition type. Certain Tat(1-9)-related peptides are found to be competitive, and others linear mixed-type or parabolic mixed-type inhibitors indicating different inhibitor binding sites on DP IV, at the active site and out of the active site. The parabolic mixed-type mechanism, attributed to both non-mutually exclusive inhibitor binding sites of the enzyme, is described in detail. From the kinetic investigations and molecular modeling experiments, possible interactions of the oligopeptides with specified amino acids of DP IV are suggested. These findings give new insights for the development of more potent and specific peptide-based DP IV inhibitors. Such inhibitors could be useful for the treatment of autoimmune and inflammatory diseases. PMID:12752434

  9. Improvement of blood glucose levels and obesity in mice given aronia juice by inhibition of dipeptidyl peptidase IV and α-glucosidase.

    PubMed

    Yamane, Takuya; Kozuka, Miyuki; Konda, Daisuke; Nakano, Yoshihisa; Nakagaki, Takenori; Ohkubo, Iwao; Ariga, Hiroyoshi

    2016-05-01

    Aronia berries have many potential effects on health. Previous human studies have shown that aronia juice may be useful for treatment of obesity disorders. Recently, we have reported that aronia juice has an inhibitory effect on dipeptidyl peptidase (DPP IV) activity and that the DPP IV inhibitor in aronia juice was identified as cyanidin 3,5-diglucoside. In this study, we found that body weights and blood glucose levels were reduced in diabetes model KK-Ay mice given aronia juice. We also found that weights of white adipose tissues were reduced in KK-Ay mice given aronia juice. Furthermore, levels of DPP IV activity in the serum and liver from KK-Ay mice were lower than those in the serum and liver from C57BL/6JmsSlc mice. Interestingly, although levels of DPP IV activity were not changed in the serum and liver from aronia-juice-administered KK-Ay mice, levels of DPP IV activity were increased in those from aronia-juice-administered C57BL/6JmsSlc mice. Furthermore, α-glucosidase activity was inhibited in the upper region of the small intestine from aronia-juice-administered KK-Ay mice but not in the lower region. Inhibition of α-glucosidase activity in the upper portion of the small intestine induced a reduction of glucose-dependent insulinotropic polypeptide (GIP) level. The results suggest that DPP IV activity in diabetic mice is inhibited by aronia juice, that the GIP level in the upper region of the small intestine is reduced by inhibition of α-glucosidase activity and that weights of adipose tissues are reduced by aronia juice. PMID:27133429

  10. Molecular cloning and biochemical characterization of Xaa-Pro dipeptidyl-peptidase from Streptococcus mutans and its inhibition by anti-human DPP IV drugs.

    PubMed

    De, Arpan; Lupidi, Giulio; Petrelli, Dezemona; Vitali, Luca A

    2016-05-01

    Streptococcus mutans harbours an intracellular, human DPP IV-analogous enzyme Xaa-Pro dipeptidyl-peptidase (EC 3.4.14.11). According to previous reports, an extracellular isozyme in S. gordonii and S. suis has been associated with virulence. Speculating that even an intracellular form may aid in virulence of S. mutans, we have tried to purify, characterize and evaluate enzyme inhibition by specific inhibitors. The native enzyme was partially purified by ion-exchange and gel filtration chromatography. Owing to low yield, the enzyme was overexpressed in Lactococcus lactis and purified by affinity chromatography. The recombinant enzyme (rSm-XPDAP) had a specific activity of 1070 U mg(-1), while the Vmax and Km were 7 μM min(-1) and 89 ± 7 μM (n = 3), respectively. The serine protease inhibitor phenylmethylsulphonyl fluoride and a DPP IV-specific inhibitor diprotin A proved to be active against rSm-XPDAP. As a novel approach, the evaluation of the effect of anti-human DPP IV (AHD) drugs on rSm-XPDAP activity found saxagliptin to be effective to some extent (Ki = 129 ± 16 μM), which may lead to the synthesis and development of a new class of antimicrobial agents. PMID:27010012

  11. Separation of L-Pro-DL-boroPro into its component diastereomers and kinetic analysis of their inhibition of dipeptidyl peptidase IV. A new method for the analysis of slow, tight-binding inhibition.

    PubMed

    Gutheil, W G; Bachovchin, W W

    1993-08-31

    The potent dipeptidyl peptidase IV (DP IV) inhibitor [1-(2-pyrrolidinylcarbonyl)-2-pyrrolidinyl]boronic acid (L-Pro-DL-boroPro) [Flentke, G. R., Munoz, E., Huber, B. T., Plaut, A. G., Kettner, C. A., & Bachovchin, W. W. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1556-1559] was fractionated into its component L-L and L-D diastereomers by C18 HPLC, and the binding of the purified diastereomers to DP IV was analyzed. Inhibition kinetics confirms that the L-L diastereomer is a potent inhibitor of DP IV, having a Ki of 16 pM. The L-D isomer binds at least 1000-fold more weakly than the L-L, if it binds at all, as the approximately 200-fold weaker inhibition observed for the purified L-D isomer is shown here to be due entirely to the presence of a small amount (0.59%) of the L-L diastereomer contaminating the L-D preparation. The instability of Pro-boroPro, together with its very high affinity for DP IV and the time dependence of the inhibition, makes a rigorous kinetic analysis of its binding to DP IV difficult. Here we have developed a method which takes advantage of the slow rate at which the inhibitor dissociates from the enzyme. The method involves preincubating the enzyme and the inhibitor without substrate and then assaying the free enzyme by the addition of substrate and following its hydrolysis for a period of time which is short relative to the dissociation rate of the inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8103356

  12. Identification of dipeptidyl peptidase IV inhibitors: virtual screening, synthesis and biological evaluation.

    PubMed

    Xing, Junhao; Li, Qing; Zhang, Shengping; Liu, Haomiao; Zhao, Leilei; Cheng, Haibo; Zhang, Yuan; Zhou, Jinpei; Zhang, Huibin

    2014-09-01

    Inhibition of dipeptidyl peptidase IV is an important approach for the treatment of type-2 diabetes. In this study, we reported a multistage virtual screening workflow that integrated 3D pharmacophore models, structural consensus docking, and molecular mechanics/generalized Born surface area binding energy calculation to identify novel dipeptidyl peptidase IV inhibitors. After screening our in-house database, two hit compounds, HWL-405 and HWL-892, having persistent high performance in all stages of virtual screening were identified. These two hit compounds together with several analogs were synthesized and evaluated for in vitro inhibition of dipeptidyl peptidase IV. The experimental data indicated that most designed compounds exhibited significant dipeptidyl peptidase IV inhibitory activity. Among them, compounds 35f displayed the greatest potency against dipeptidyl peptidase IV in vitro with the IC50 value of 78 nm. In an oral glucose tolerance test in normal male Kunming mice, compound 35f reduced blood glucose excursion in a dose-dependent manner. PMID:24674599

  13. Identification and characterization of a dipeptidyl peptidase IV inhibitor from aronia juice

    SciTech Connect

    Kozuka, Miyuki; Yamane, Takuya; Nakano, Yoshihisa; Nakagaki, Takenori; Ohkubo, Iwao; Ariga, Hiroyoshi

    2015-09-25

    Aronia berries have many potential effects on health, including an antioxidant effect, effect for antimutagenesis, hepatoprotection and cardioprotection, an antidiabetic effect and inhibition of cancer cell proliferation. Previous human studies have shown that aronia juice may be useful for treatment of obesity disorders. In this study, we found that aronia juice has an inhibitory effect against dipeptidyl peptidase IV (DPP IV) (EC 3.4.14.5). DPP IV is a peptidase that cleaves the N-terminal region of incretins such as glucagon-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). Inactivation of incretins by DPP IV induces reduction of insulin secretion. Furthermore, we identified that cyanidin 3, 5-diglucoside as the DPP IV inhibitor in aronia juice. DPP IV was inhibited more strongly by cyanidin 3, 5-diglucoside than by cyanidin and cyanidin 3-glucoside. The results suggest that DPP IV is inhibited by cyanidin 3, 5-diglucoside present in aronia juice. The antidiabetic effect of aronia juice may be mediated through DPP IV inhibition by cyanidin 3, 5-diglucoside. - Highlights: • DPP IV activity is inhibited by aronia juice. • DPP IV inhibitor is cyanidin 3, 5-diglucoside in aronia juice. • DPP IV is inhibited by cyanidin 3, 5-diglucoside more than cyanidin and cyanidin 3-glucoside.

  14. Chronic inhibition of circulating dipeptidyl peptidase IV by FE 999011 delays the occurrence of diabetes in male zucker diabetic fatty rats.

    PubMed

    Sudre, Béatrice; Broqua, Pierre; White, Richard B; Ashworth, Doreen; Evans, D Michael; Haigh, Robert; Junien, Jean-Louis; Aubert, Michel L

    2002-05-01

    Acute suppression of dipeptidyl peptidase IV (DPP-IV) activity improves glucose tolerance in the Zucker fatty rat, a rodent model of impaired glucose tolerance, through stabilization of glucagon-like peptide (GLP)-1. This study describes the effects of a new and potent DPP-IV inhibitor, FE 999011, which is able to suppress plasma DPP-IV activity for 12 h after a single oral administration. In the Zucker fatty rat, FE 999011 dose-dependently attenuated glucose excursion during an oral glucose tolerance test and increased GLP-1 (7-36) release in response to intraduodenal glucose. Chronic treatment with FE 999011 (10 mg/kg, twice a day for 7 days) improved glucose tolerance, as suggested by a decrease in the insulin-to-glucose ratio. In the Zucker diabetic fatty (ZDF) rat, a rodent model of type 2 diabetes, chronic treatment with FE 999011 (10 mg/kg per os, once or twice a day) postponed the development of diabetes, with the twice-a-day treatment delaying the onset of hyperglycemia by 21 days. In addition, treatment with FE 999011 stabilized food and water intake to prediabetic levels and reduced hypertriglyceridemia while preventing the rise in circulating free fatty acids. At the end of treatment, basal plasma GLP-1 levels were increased, and pancreatic gene expression for GLP-1 receptor was significantly upregulated. This study demonstrates that DPP-IV inhibitors such as FE 999011 could be of clinical value to delay the progression from impaired glucose tolerance to type 2 diabetes. PMID:11978643

  15. Identification and characterization of a dipeptidyl peptidase IV inhibitor from aronia juice.

    PubMed

    Kozuka, Miyuki; Yamane, Takuya; Nakano, Yoshihisa; Nakagaki, Takenori; Ohkubo, Iwao; Ariga, Hiroyoshi

    2015-09-25

    Aronia berries have many potential effects on health, including an antioxidant effect, effect for antimutagenesis, hepatoprotection and cardioprotection, an antidiabetic effect and inhibition of cancer cell proliferation. Previous human studies have shown that aronia juice may be useful for treatment of obesity disorders. In this study, we found that aronia juice has an inhibitory effect against dipeptidyl peptidase IV (DPP IV) (EC 3.4.14.5). DPP IV is a peptidase that cleaves the N-terminal region of incretins such as glucagon-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1). Inactivation of incretins by DPP IV induces reduction of insulin secretion. Furthermore, we identified that cyanidin 3, 5-diglucoside as the DPP IV inhibitor in aronia juice. DPP IV was inhibited more strongly by cyanidin 3, 5-diglucoside than by cyanidin and cyanidin 3-glucoside. The results suggest that DPP IV is inhibited by cyanidin 3, 5-diglucoside present in aronia juice. The antidiabetic effect of aronia juice may be mediated through DPP IV inhibition by cyanidin 3, 5-diglucoside. PMID:26296465

  16. Dipeptidyl Peptidase IV-Inhibitory Peptides Derived from Silver Carp (Hypophthalmichthys molitrix Val.) Proteins.

    PubMed

    Zhang, Ying; Chen, Ran; Chen, Xiling; Zeng, Zhu; Ma, Huiqin; Chen, Shangwu

    2016-02-01

    The dipeptidyl peptidase IV (DPP-IV)-inhibitory bioactivity of silver carp protein (SCP) hydrolysates were investigated, and their containing efficacious DPP-IV-inhibitory peptides were explored by in silico hydrolysis analysis, peptide separation combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification, and chemical synthesis. SCP hydrolysates generated by six proteases all showed efficient DPP-IV-inhibitory activities, and Neutrase-generated hydrolysates had the greatest DPP-IV inhibition (IC50 of 1.12 mg/mL). In silico Neutrase hydrolysis revealed hundreds of fragments released from myosin, actin, and collagen of SCPs, which include different Pro-motif peptides but only three reported peptidic DPP-IV inhibitors with moderate or weak bioactivity. In addition, three new DPP-IV-inhibitory peptides were identified using LC-MS/MS; in particular, LPIIDI and APGPAGP showed high DPP-IV-inhibitory activity with IC50 of 105.44 and 229.14 μM, respectively, and behaved in competitive/non-competitive mixed-type DPP-IV inhibition mode. The results indicate that the SCP-derived DPP-IV-inhibitory peptides could be potential functional ingredients in the diabetic diet. PMID:26758401

  17. An inhibitory antibody against dipeptidyl peptidase IV improves glucose tolerance in vivo.

    PubMed

    Tang, Jie; Majeti, Jiangwen; Sudom, Athena; Xiong, Yumei; Lu, Mei; Liu, Qiang; Higbee, Jared; Zhang, Yi; Wang, Yan; Wang, Wei; Cao, Ping; Xia, Zhen; Johnstone, Sheree; Min, Xiaoshan; Yang, Xiaoping; Shao, Hui; Yu, Timothy; Sharkov, Nik; Walker, Nigel; Tu, Hua; Shen, Wenyan; Wang, Zhulun

    2013-01-11

    Dipeptidyl peptidase IV (DPP-IV) degrades the incretin hormone glucagon-like peptide 1 (GLP-1). Small molecule DPP-IV inhibitors have been used as treatments for type 2 diabetes to improve glucose tolerance. However, each of the marketed small molecule drugs has its own limitation in terms of efficacy and side effects. To search for an alternative strategy of inhibiting DPP-IV activity, we generated a panel of tight binding inhibitory mouse monoclonal antibodies (mAbs) against rat DPP-IV. When tested in vitro, these mAbs partially inhibited the GLP-1 cleavage activity of purified enzyme and rat plasma. To understand the partial inhibition, we solved the co-crystal structure of one of the mAb Fabs (Ab1) in complex with rat DPP-IV. Although Ab1 does not bind at the active site, it partially blocks the side opening, which prevents the large substrates such as GLP-1 from accessing the active site, but not small molecules such as sitagliptin. When Ab1 was tested in vivo, it reduced plasma glucose and increased plasma GLP-1 concentration during an oral glucose tolerance test in rats. Together, we demonstrated the feasibility of using mAbs to inhibit DPP-IV activity and to improve glucose tolerance in a diabetic rat model. PMID:23184939

  18. An Inhibitory Antibody against Dipeptidyl Peptidase IV Improves Glucose Tolerance in Vivo

    PubMed Central

    Tang, Jie; Majeti, Jiangwen; Sudom, Athena; Xiong, Yumei; Lu, Mei; Liu, Qiang; Higbee, Jared; Zhang, Yi; Wang, Yan; Wang, Wei; Cao, Ping; Xia, Zhen; Johnstone, Sheree; Min, Xiaoshan; Yang, Xiaoping; Shao, Hui; Yu, Timothy; Sharkov, Nik; Walker, Nigel; Tu, Hua; Shen, Wenyan; Wang, Zhulun

    2013-01-01

    Dipeptidyl peptidase IV (DPP-IV) degrades the incretin hormone glucagon-like peptide 1 (GLP-1). Small molecule DPP-IV inhibitors have been used as treatments for type 2 diabetes to improve glucose tolerance. However, each of the marketed small molecule drugs has its own limitation in terms of efficacy and side effects. To search for an alternative strategy of inhibiting DPP-IV activity, we generated a panel of tight binding inhibitory mouse monoclonal antibodies (mAbs) against rat DPP-IV. When tested in vitro, these mAbs partially inhibited the GLP-1 cleavage activity of purified enzyme and rat plasma. To understand the partial inhibition, we solved the co-crystal structure of one of the mAb Fabs (Ab1) in complex with rat DPP-IV. Although Ab1 does not bind at the active site, it partially blocks the side opening, which prevents the large substrates such as GLP-1 from accessing the active site, but not small molecules such as sitagliptin. When Ab1 was tested in vivo, it reduced plasma glucose and increased plasma GLP-1 concentration during an oral glucose tolerance test in rats. Together, we demonstrated the feasibility of using mAbs to inhibit DPP-IV activity and to improve glucose tolerance in a diabetic rat model. PMID:23184939

  19. Involvement of dipeptidyl peptidase IV in an in vivo immune response.

    PubMed

    Kubota, T; Flentke, G R; Bachovchin, W W; Stollar, B D

    1992-08-01

    Dipeptidyl peptidase IV (DP IV) is a serine protease that selectively cleaves X-Pro dipeptides from polypeptides and proteins. Among blood cells, this enzyme occurs preferentially on the surface of CD4+ T cells and the amount of enzyme activity increases with T cell activation. In previous work, two potent and specific peptidyl-boronic acid inhibitors of DP IV, Ala-boroPro and Pro-boroPro, were synthesized and Pro-boroPro was shown to suppress antigen-specific proliferative responses of T cells in vitro. In this study, we tested the in vivo effects of these inhibitors. Subcutaneous injection of Ala-boroPro or Pro-boroPro into BALB/c mice inhibited DP IV activity in serum and spleen cell suspensions. Repeated injections of more than 10 micrograms of Ala-boroPro or Pro-boroPro at 12 h intervals maintained in vivo DP IV activity at less than 30% of the normal level. Repeated injections of the inhibitors during the primary, secondary or tertiary immune response to bovine serum albumin (BSA) reduced anti-BSA antibody production. Without inhibitor, immunization with BSA was followed by a temporary decrease in serum DP IV activity and then by enhanced serum enzyme activity after several days. These results provide the first direct evidence that DP IV plays an important role in immune response in vivo. PMID:1353423

  20. The effects of dipeptidyl peptidase-4 inhibition on microvascular diabetes complications.

    PubMed

    Avogaro, Angelo; Fadini, Gian Paolo

    2014-10-01

    We performed a review of the literature to determine whether the dipeptidyl peptidase-4 inhibitors (DPP4-I) may have the capability to directly and positively influence diabetic microvascular complications. The literature was scanned to identify experimental and clinical evidence that DPP4-I can ameliorate diabetic microangiopathy. We retrieved articles published between 1 January 1980 and 1 March 2014 in English-language peer-reviewed journals using the following terms: ("diabetes" OR "diabetic") AND ("retinopathy" OR "retinal" OR "nephropathy" OR "renal" OR "albuminuria" OR "microalbuminuria" OR "neuropathy" OR "ulcer" OR "wound" OR "bone marrow"); ("dipeptidyl peptidase-4" OR "dipeptidyl peptidase-IV" OR "DPP-4" OR "DPP-IV"); and ("inhibition" OR "inhibitor"). Experimentally, DPP4-I appears to improve inflammation, endothelial function, blood pressure, lipid metabolism, and bone marrow function. Several experimental studies report direct potential beneficial effects of DPP4-I on all microvascular diabetes-related complications. These drugs have the ability to act either directly or indirectly via improved glucose control, GLP-1 bioavailability, and modifying nonincretin substrates. Although preliminary clinical data support that DPP4-I therapy can protect from microangiopathy, insufficient evidence is available to conclude that this class of drugs directly prevents or decreases microangiopathy in humans independently from improved glucose control. Experimental findings and preliminary clinical data suggest that DPP4-I, in addition to improving metabolic control, have the potential to interfere with the onset and progression of diabetic microangiopathy. Further evidence is needed to confirm these effects in patients with diabetes. PMID:25249673

  1. Colorimetrical rate assay for urinary dipeptidyl peptidase IV (DPPIV) activity using a new substrate.

    PubMed

    Shibuya-Saruta, H; Sugiyama, M; Kasahara, Y

    1995-01-01

    We synthesized a new substrate glycyl-L-proline 3,5-dibromo-4-hydroxyanilide (Gly-Pro-DBAP), for dipeptidyl peptidase IV (DPPIV). Its hydrolysis by DPPIV resulted in the formation of a chromophore, 2,6-dibromophenol-indo-p-xylenol, and its maximal absorption wavelength (600 nm) was longer than that of p-nitroaniline (415 nm) released from conventional substrate, glycyl-L-proline p-nitroanilide (Gly-Pro-pNA). We also established the rate assay for urinary DPPIV activity using Gly-Pro-DBAP. The optimum pH was between 8.5 and 9.0. The apparent Km was 1.1 mmol/1. The detectable range was 2.5-350 U/l. No changes in blank values occurred throughout the enzyme reaction in the optimum pH. Its value was also much lower than Gly-Pro-pNA. CVs for within-run and between-run were 1.1% (n = 10) and 3.0% (n = 10), respectively. Among tested peptidases, only DPPIV could hydrolyze Gly-Pro-DBAP. Among the protease inhibitors, only two, diprotin-A and phenylmethylsulfonyl fluoride (PMSA), could inhibit DPPIV activity. The present method did not interfere with urinary ingredients such as hemoglobin. The correlation between the present (y) and conventional (x) methods is presented by the equation y = 1.121x + 0.096 (r = 0.993). Thus the present method provides practical advantages over the conventional method for routine laboratory use. PMID:7714663

  2. Genetic ablation or pharmacological blockade of dipeptidyl peptidase IV does not impact T cell-dependent immune responses

    PubMed Central

    Vora, Kalpit A; Porter, Gene; Peng, Roche; Cui, Yan; Pryor, Kellyann; Eiermann, George; Zaller, Dennis M

    2009-01-01

    Background Current literature suggests that dipeptidyl peptidase IV (DPP-IV; CD26) plays an essential role in T-dependent immune responses, a role that could have important clinical consequences. To rigorously define the role of DPP-IV in the immune system, we evaluated genetic and pharmacological inhibition of the enzyme on T-dependent immune responses in vivo. Results The DPP-IV null animals mounted robust primary and secondary antibody responses to the T dependent antigens, 4-hydroxy-3-nitrophenylacetyl-ovalbumin (NP-Ova) and 4-hydroxy-3-nitrophenylacetyl-chicken gamma globulin (NP-CGG), which were comparable to wild type mice. Serum levels of antigen specific IgM, IgG1, IgG2a, IgG2b and IgG3 were similar between the two groups of animals. DPP-IV null animals mounted an efficient germinal center reaction by day 10 after antigen stimulation that was comparable to wild type mice. Moreover, the antibodies produced by DPP-IV null animals after repeated antigenic challenge were affinity matured. Similar observations were made using wild type animals treated with a highly selective DPP-IV inhibitor during the entire course of the experiments. T cell recall responses to ovalbumin and MOG peptide, evaluated by measuring proliferation and IL-2 release from cells isolated from draining lymph nodes, were equivalent in DPP-IV null and wild type animals. Furthermore, mice treated with DPP-IV inhibitor had intact T-cell recall responses to MOG peptide. In addition, female DPP-IV null and wild type mice treated with DPP-IV inhibitor exhibited normal and robust in vivo cytotoxic T cell responses after challenge with cells expressing the male H-Y minor histocompatibility antigen. Conclusion These data indicate Selective inhibition of DPP-IV does not impair T dependent immune responses to antigenic challenge. PMID:19358731

  3. Isolation and characterisation of dipeptidyl peptidase IV from Prevotella loescheii ATCC 15930.

    PubMed

    Koreeda, Y; Hayakawa, M; Ikemi, T; Abiko, Y

    2001-08-01

    A proline-specific dipeptidyl aminopeptidase, dipeptidyl peptidase IV (EC 3.4.14.5), was purified from a cell sonicate soluble fraction of Prevotella loescheii ATCC 15930 by sequential column chromatography. The molecular mass of the native enzyme was estimated as 160 kDa by high-pressure liquid gel filtration column chromatography and unheated sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The subunit molecular mass was 80 kDa when the enzyme was heated to 100 degrees C in the presence of 2-mercaptoethanol before SDS-PAGE, suggesting that the native enzyme consists of two identical subunits and is folded in 2% SDS. The optimum pH, with glycyl-prolyl-4-methyl-coumaryl-7-amide as the substrate, was 8.0; the isoelectric point was 5.2. Purified enzyme showed a strong preference for dipeptide substrates containing proline and, less efficiently, alanine in the P1 position. The enzyme was markedly inhibited by Cd(2+), Zn(2+), Hg(2+), Co(2+), and serine proteinase inhibitor di-isopropylfluorophosphate. PMID:11389867

  4. Identification of Novel Human Dipeptidyl Peptidase-IV Inhibitors of Natural Origin (Part II): In Silico Prediction in Antidiabetic Extracts

    PubMed Central

    Guasch, Laura; Sala, Esther; Ojeda, María José; Valls, Cristina; Bladé, Cinta; Mulero, Miquel; Blay, Mayte; Ardévol, Anna; Garcia-Vallvé, Santiago; Pujadas, Gerard

    2012-01-01

    Background Natural extracts play an important role in traditional medicines for the treatment of diabetes mellitus and are also an essential resource for new drug discovery. Dipeptidyl peptidase IV (DPP-IV) inhibitors are potential candidates for the treatment of type 2 diabetes mellitus, and the effectiveness of certain antidiabetic extracts of natural origin could be, at least partially, explained by the inhibition of DPP-IV. Methodology/Principal Findings Using an initial set of 29,779 natural products that are annotated with their natural source and an experimentally validated virtual screening procedure previously developed in our lab (Guasch et al.; 2012) [1], we have predicted 12 potential DPP-IV inhibitors from 12 different plant extracts that are known to have antidiabetic activity. Seven of these molecules are identical or similar to molecules with described antidiabetic activity (although their role as DPP-IV inhibitors has not been suggested as an explanation for their bioactivity). Therefore, it is plausible that these 12 molecules could be responsible, at least in part, for the antidiabetic activity of these extracts through their inhibitory effect on DPP-IV. In addition, we also identified as potential DPP-IV inhibitors 6 molecules from 6 different plants with no described antidiabetic activity but that share the same genus as plants with known antidiabetic properties. Moreover, none of the 18 molecules that we predicted as DPP-IV inhibitors exhibits chemical similarity with a group of 2,342 known DPP-IV inhibitors. Conclusions/Significance Our study identified 18 potential DPP-IV inhibitors in 18 different plant extracts (12 of these plants have known antidiabetic properties, whereas, for the remaining 6, antidiabetic activity has been reported for other plant species from the same genus). Moreover, none of the 18 molecules exhibits chemical similarity with a large group of known DPP-IV inhibitors. PMID:23028712

  5. Strategies for the release of dipeptidyl peptidase IV (DPP-IV) inhibitory peptides in an enzymatic hydrolyzate of α-lactalbumin.

    PubMed

    Nongonierma, Alice B; Le Maux, Solène; Hamayon, Joël; FitzGerald, Richard J

    2016-08-10

    Bovine α-lactalbumin (α-La) contains numerous dipeptidyl peptidase IV (DPP-IV) inhibitory peptide sequences within its primary structure. In silico analysis indicated that the targeted hydrolysis of α-La with elastase should release DPP-IV inhibitory peptides. An α-La isolate was hydrolysed with elastase under different conditions using an experimental design approach incorporating 3 factors (temperature, pH and enzyme to substrate ratio (E : S) ratio) at 2 levels. The hydrolyzate generated at pH 8.5, 50 °C, E : S 2.0% (w/w) (H9) displayed the highest mean DPP-IV inhibition value at 3.1 mg mL(-1) of 75.8 ± 3.7% and had a half maximal DPP-IV inhibitory concentration (IC50) value of 1.20 ± 0.12 mg mL(-1). Five α-La-derived DPP-IV inhibitory peptides (GY, GL, GI, NY and WL) predicted to be released in silico were identified by liquid-chromatography tandem mass spectrometry (LC-MS/MS) within H9 and its simulated gastrointestinal digestion (SGID) sample. This preliminary study demonstrated the benefit of using a targeted approach combined with an experimental design in the generation of dietary protein hydrolyzates with DPP-IV inhibitory properties. PMID:27410260

  6. The contributions of dipeptidyl peptidase IV to inflammation in heart failure.

    PubMed

    de Almeida Salles, Thiago; Zogbi, Camila; de Lima, Thais Martins; de Godoi Carneiro, Camila; Garcez, Alexandre Teles; Barbeiro, Hermes Vieira; Antonio, Ednei Luiz; Dos Santos, Leonardo; da Costa Pereira, Alexandre; Tucci, Paulo José Ferreira; de Paula Faria, Daniele; Soriano, Francisco Garcia; Girardi, Adriana Castello Costa

    2016-06-01

    Circulating dipeptidyl peptidase IV (DPPIV) activity correlates with cardiac dysfunction in humans and experimental heart failure (HF) models. Similarly, inflammatory markers are associated with poorer outcomes in HF patients. However, the contributions of DPPIV to inflammation in HF remain elusive. Therefore, this study aimed to investigate whether the cardioprotective effects of DPPIV inhibition after myocardial injury are accompanied by reduced cardiac inflammation, whether circulating DPPIV activity correlates with the levels of systemic inflammatory markers in HF patients, and whether leukocytes and/or splenocytes may be one of the sources of circulating DPPIV in HF. Experimental HF was induced in male Wistar rats by left ventricular myocardial injury after radiofrequency catheter ablation. The rats were divided into three groups: sham, HF, and HF + DPPIV inhibitor (sitagliptin). Six weeks after surgery, cardiac function, perfusion and inflammatory status were evaluated. Sitagliptin treatment improved cardiac function and perfusion, reduced macrophage infiltration, and diminished the levels of inflammatory biomarkers including TNF-α, IL-1β, and CCL2. In HF patients, serum DPPIV activity correlated with CCL2, suggesting that leukocytes may be the source of circulating DPPIV in HF. Unexpectedly, DPPIV release was higher in splenocytes from HF rats and similar in HF circulating mononuclear cells compared with those from sham, suggesting an organ-specific modulation of DPPIV in HF. Collectively, our data provide new evidence that the cardioprotective effects of DPPIV inhibition in HF may be due to suppression of inflammatory cytokines. Moreover, they suggest that a vicious circle between DPPIV and inflammation may contribute to HF development and progression. PMID:27199127

  7. Inhibition of dipeptidyl peptidase 8/9 impairs preadipocyte differentiation

    PubMed Central

    Han, Ruijun; Wang, Xinying; Bachovchin, William; Zukowska, Zofia; Osborn, John W.

    2015-01-01

    Adipocytes are the primary cells in adipose tissue, and adipocyte dysfunction causes lipodystrophy, obesity and diabetes. The dipeptidyl peptidase (DPP) 4 family includes four enzymes, DPP4, DPP8, DPP9 and fibroblast activation protein (FAP). DPP4 family inhibitors have been used for the treatment of type 2 diabetes patients, but their role in adipocyte formation are poorly understood. Here we demonstrate that the DPP8/9 selective inhibitor 1G244 blocks adipogenesis in preadipocyte 3T3-L1 and 3T3-F422A, while DPP4 and FAP inhibitors have no effect. In addition, knockdown of DPP8 or DPP9 significantly impairs adipocyte differentiation in preadipocytes. We further uncovered that blocking the expression or activities of DPP8 and DPP9 attenuates PPARγ2 induction during preadipocyte differentiation. Addition of PPARγ agonist thiazolidinediones (TZDs), or ectopic expression of PPARγ2, is able to rescue the adipogenic defect caused by DPP8/9 inhibition in preadipocytes. These results indicate the importance of DPP8 and DPP9 on adipogenesis. PMID:26242871

  8. Inhibition of dipeptidyl peptidase 8/9 impairs preadipocyte differentiation.

    PubMed

    Han, Ruijun; Wang, Xinying; Bachovchin, William; Zukowska, Zofia; Osborn, John W

    2015-01-01

    Adipocytes are the primary cells in adipose tissue, and adipocyte dysfunction causes lipodystrophy, obesity and diabetes. The dipeptidyl peptidase (DPP) 4 family includes four enzymes, DPP4, DPP8, DPP9 and fibroblast activation protein (FAP). DPP4 family inhibitors have been used for the treatment of type 2 diabetes patients, but their role in adipocyte formation are poorly understood. Here we demonstrate that the DPP8/9 selective inhibitor 1G244 blocks adipogenesis in preadipocyte 3T3-L1 and 3T3-F422A, while DPP4 and FAP inhibitors have no effect. In addition, knockdown of DPP8 or DPP9 significantly impairs adipocyte differentiation in preadipocytes. We further uncovered that blocking the expression or activities of DPP8 and DPP9 attenuates PPARγ2 induction during preadipocyte differentiation. Addition of PPARγ agonist thiazolidinediones (TZDs), or ectopic expression of PPARγ2, is able to rescue the adipogenic defect caused by DPP8/9 inhibition in preadipocytes. These results indicate the importance of DPP8 and DPP9 on adipogenesis. PMID:26242871

  9. Peptide Array on Cellulose Support—A Screening Tool to Identify Peptides with Dipeptidyl-Peptidase IV Inhibitory Activity within the Sequence of α-Lactalbumin

    PubMed Central

    Lacroix, Isabelle M. E.; Li-Chan, Eunice C. Y.

    2014-01-01

    The inhibition of the enzyme dipeptidyl-peptidase IV (DPP-IV) is an effective pharmacotherapeutic approach for the management of type 2 diabetes. Recent findings have suggested that dietary proteins, including bovine α-lactalbumin, could be precursors of peptides able to inhibit DPP-IV. However, information on the location of active peptide sequences within the proteins is far from being comprehensive. Moreover, the traditional approach to identify bioactive peptides from foods can be tedious and long. Therefore, the objective of this study was to use peptide arrays to screen α-lactalbumin-derived peptides for their interaction with DPP-IV. Deca-peptides spanning the entire α-lactalbumin sequence, with a frame shift of 1 amino acid between successive sequences, were synthesized on cellulose membranes using “SPOT” technology, and their binding to and inhibition of DPP-IV was studied. Among the 114 α-lactalbumin-derived decamers investigated, the peptides 60WCKDDQNPHS69 (αKi = 76 µM), 105LAHKALCSEK114 (Ki = 217 µM) and 110LCSEKLDQWL119 (Ki = 217 µM) were among the strongest DPP-IV inhibitors. While the SPOT- and traditionally-synthesized peptides showed consistent trends in DPP-IV inhibitory activity, the cellulose-bound peptides’ binding behavior was not correlated to their ability to inhibit the enzyme. This research showed, for the first time, that peptide arrays are useful screening tools to identify DPP-IV inhibitory peptides from dietary proteins. PMID:25402645

  10. Anti-α-glucosidase and Anti-dipeptidyl Peptidase-IV Activities of Extracts and Purified Compounds from Vitis thunbergii var. taiwaniana.

    PubMed

    Lin, Yin-Shiou; Chen, Chiy-Rong; Wu, Wei-Hau; Wen, Chi-Luan; Chang, Chi-I; Hou, Wen-Chi

    2015-07-22

    Ethanol extracts (Et) from the stem (S) and leaf (L) of Vitis thunbergii var. taiwaniana (VTT) were used to investigate yeast α-glucosidase and porcine kidney dipeptidyl peptidase-IV (DPP-IV) inhibitory activities. Both VTT-Et showed complete α-glucosidase inhibition at 0.1 mg/mL; VTT-S-Et and VTT-L-Et showed 26 and 11% DPP-IV inhibition, respectively, at 0.5 mg/mL. The VTT-Et interventions (20 and 50 mg/kg) resulted in improvements in impaired glucose tolerance of diet-induced obese rats. (+)-Hopeaphenol, (+)-vitisin A, and (-)-vitisin B were isolated from the ethyl acetate fractions of S-Et and showed yeast α-glucosidase inhibition (IC50 = 18.30, 1.22, and 1.02 μM) and porcine kidney DPP-IV inhibition (IC50 = 401, 90.75, and 15.3 μM) compared to acarbose (6.39 mM) and sitagliptin (47.35 nM), respectively. Both (+)-vitisin A and (-)-vitisin B showed mixed noncompetitive inhibition against yeast α-glucosidase and porcine kidney DPP-IV, respectively. These results proposed that VTT extracts might through inhibitions against α-glucosidase and DPP-IV improve the impaired glucose tolerance in diet-induced obese rats. PMID:26138774

  11. The influence of age on intestinal dipeptidyl peptidase IV (DPP IV/CD26), disaccharidases, and alkaline phosphatase enzyme activity in C57BL/6 mice.

    PubMed

    Detel, Dijana; Baticic, Lara; Varljen, Jadranka

    2008-01-01

    The objective of this study was to determine and describe the age-related changes in intestinal brush border membrane enzyme activities that occur in C57Bl/6 mice. Specifically, jejunal, duodenal, and ileal dipeptidyl peptidase IV/CD26, disaccharidase (lactase, sucrase, and maltase), and alkaline phosphatase activities were determined. A significant correlation between analyzed intestinal brush border membrane enzyme activities and animal age was found. Our study revealed that intestinal dipeptidyl peptidase IV/CD26, lactase, sucrase, maltase, and alkaline phosphatase activities decline significantly with age (p < .05). Nevertheless, the horizontal (duodenum to ileum) enzyme activity patterns are not affected by age. PMID:18189167

  12. Potential Role of Dipeptidyl Peptidase IV in the Pathophysiology of Heart Failure

    PubMed Central

    Salles, Thiago A.; dos Santos, Leonardo; Barauna, Valério G.; Girardi, Adriana C. C.

    2015-01-01

    Dipeptidyl peptidase IV (DPPIV) is a widely expressed multifunctional serine peptidase that exists as a membrane-anchored cell surface protein or in a soluble form in the plasma and other body fluids. Numerous substrates are cleaved at the penultimate amino acid by DPPIV, including glucagon-like peptide-1 (GLP-1), brain natriuretic peptide (BNP) and stromal cell-derived factor-1 (SDF-α), all of which play important roles in the cardiovascular system. In this regard, recent reports have documented that circulating DPPIV activity correlates with poorer cardiovascular outcomes in human and experimental heart failure (HF). Moreover, emerging evidence indicates that DPPIV inhibitors exert cardioprotective and renoprotective actions in a variety of experimental models of cardiac dysfunction. On the other hand, conflicting results have been found when translating these promising findings from preclinical animal models to clinical therapy. In this review, we discuss how DPPIV might be involved in the cardio-renal axis in HF. In addition, the potential role for DPPIV inhibitors in ameliorating heart disease is revised, focusing on the effects of the main DPPIV substrates on cardiac remodeling and renal handling of salt and water. PMID:25690036

  13. Synthesis and characterization of constrained peptidomimetic dipeptidyl peptidase IV inhibitors: amino-lactam boroalanines.

    PubMed

    Lai, Jack H; Wu, Wengen; Zhou, Yuhong; Maw, Hlaing H; Liu, Yuxin; Milo, Lawrence J; Poplawski, Sarah E; Henry, Gillian D; Sudmeier, James L; Sanford, David G; Bachovchin, William W

    2007-05-17

    We describe here the epimerization-free synthesis and characterization of a new class of conformationally constrained lactam aminoboronic acid inhibitors of dipeptidyl peptidase IV (DPP IV; E.C. 3.4.14.5). These compounds have the advantage that they cannot undergo the pH-dependent cyclization prevalent in most dipeptidyl boronic acids that attenuates their potency at physiological pH. For example, D-3-amino-1-[L-1-boronic-ethyl]-pyrrolidine-2-one (amino-D-lactam-L-boroAla), one of the best lactam inhibitors of DPP IV, is several orders of magnitude less potent than L-Ala-L-boroPro, as measured by Ki values (2.3 nM vs 30 pM, respectively). At physiological pH, however, it is actually more potent than L-Ala-L-boroPro, as measured by IC50 values (4.2 nM vs 1400 nM), owing to the absence of the potency-attenuating cyclization. In an interesting and at first sight surprising reversal of the relationship between stereochemistry and potency observed with the conformationally unrestrained Xaa-boroPro class of inhibitors, the L-L diastereomers of the lactams are orders of magnitude less effective than the D-L lactams. However, this interesting reversal and the unexpected potency of the D-L lactams as DPP IV inhibitors can be understood in structural terms, which is explained and discussed here. PMID:17458948

  14. Multiple-dose pharmacokinetics and pharmacodynamics of evogliptin (DA-1229), a novel dipeptidyl peptidase IV inhibitor, in healthy volunteers

    PubMed Central

    Gu, Namyi; Park, Min Kyu; Kim, Tae-Eun; Bahng, Mi Young; Lim, Kyoung Soo; Cho, Sang-Heon; Yoon, Seo Hyun; Cho, Joo-Youn; Jang, In-Jin; Yu, Kyung-Sang

    2014-01-01

    Purpose Evogliptin (DA-1229) is a novel, potent, and selective dipeptidyl peptidase IV (DPP-IV) inhibitor in clinical development for the treatment of type 2 diabetes mellitus. This study aimed to investigate the pharmacokinetic and pharmacodynamic profiles and tolerability of evogliptin after repeated oral administration in healthy subjects. Patients and methods A block-randomized, double-blind, placebo-controlled, multiple-dose, dose-escalation study was performed in a total of 30 subjects. Repeated once-daily doses of 5, 10, or 20 mg evogliptin or the same doses of placebo were orally administered to ten subjects in each dosage group for 10 days. Subjects in each group were randomized to receive evogliptin or placebo with a ratio of 8:2. Pharmacokinetics of evogliptin were evaluated, with its concentrations in serial plasma and urine samples collected following the first and last administrations. DPP-IV activity and glucagon-like peptide-1, glucose, and insulin levels were quantified to evaluate evogliptin’s pharmacodynamics on the first and last dosing days. Results All participants completed the study without any serious or severe adverse event. The evogliptin plasma concentration reached its peak within 4–5 hours and decreased relatively slowly, with a terminal elimination half-life of 33–39 hours. Repeated administration resulted in a 1.4- to 1.5-fold accumulation. Evogliptin’s systemic exposure and inhibition of plasma DPP-IV activity increased in a dose-dependent manner. Inhibition of DPP-IV activity >80% was sustained over 24 hours in all evogliptin dose groups and provided an increase in postprandial active glucagon-like peptide-1 levels by 1.5- to 2.4-fold. Postprandial glucose levels in the evogliptin-treated groups were reduced 20%–35% compared to placebo, but were not accompanied by increased insulin levels. Conclusion Repeated administration of evogliptin in healthy subjects was well tolerated and exhibited linear pharmacokinetics within

  15. Synthesized Peptides from Yam Dioscorin Hydrolysis in Silico Exhibit Dipeptidyl Peptidase-IV Inhibitory Activities and Oral Glucose Tolerance Improvements in Normal Mice.

    PubMed

    Lin, Yin-Shiou; Han, Chuan-Hsiao; Lin, Shyr-Yi; Hou, Wen-Chi

    2016-08-24

    RRDY, RL, and DPF were the top 3 of 21 peptides for inhibitions against dipeptidyl peptidase-IV (DPP-IV) from the pepsin hydrolysis of yam dioscorin in silico and were further investigated in a proof-of-concept study in normal ICR mice for regulating glucose metabolism by the oral glucose tolerance test (OGTT). The sample or sitagliptin (positive control) was orally administered by a feeding gauge; 30 min later, the glucose loads (2.5 g/kg) were performed. RRDY, yam dioscorin, or sitagliptin preload, but not DPF, lowered the area under the curve (AUC0-120) of blood glucose and DPP-IV activity and elevated the AUC0-120 of blood insulin, which showed significant differences compared to control (P < 0.05 or 0.001). These results suggested that RRDY and yam dioscorin might be beneficial in glycemic control in normal mice and need further investigations in diabetic animal models. PMID:27499387

  16. Methotrexate and Cyclosporine Treatments Modify the Activities of Dipeptidyl Peptidase IV and Prolyl Oligopeptidase in Murine Macrophages

    PubMed Central

    Olivo, R. A.; Nascimento, N. G.; Teixeira, C. F. P.; Silveira, P. F.

    2008-01-01

    Analysis of the effects of cyclosporine A (25–28 mgkg−1) and/or methotrexate (0.1 mgkg−1) treatments on dipeptidyl peptidase IV (DPPIV) and prolyl oligopeptidase (POP) activities and on algesic response in two distinct status of murine macrophages (Mφs) was undertaken. In resident Mφs, DPPIV and POP were affected by neither individual nor combined treatments. In thioglycolate-elicited Mφs, methotrexate increased DPPIV (99–110%) and POP (60%), while cyclosporine inhibited POP (21%). Combined treatment with both drugs promoted a rise (51–84%) of both enzyme activities. Only cyclosporine decreased (42%) the tolerance to algesic stimulus. Methotrexate was revealed to exert prevalent action over that of cyclosporine on proinflammatory Mφ POP. The opposite effects of methotrexate and cyclosporine on POP activity might influence the availability of the nociceptive mediators bradykinin and substance P in proinflammatory Mφs. The exacerbated response to thermally induced algesia observed in cyclosporine-treated animals could be related to upregulation of those mediators. PMID:18354729

  17. Production of feather hydrolysates with antioxidant, angiotensin-I converting enzyme- and dipeptidyl peptidase-IV-inhibitory activities.

    PubMed

    Fontoura, Roberta; Daroit, Daniel J; Correa, Ana P F; Meira, Stela M M; Mosquera, Mauricio; Brandelli, Adriano

    2014-09-25

    The antioxidant and antihypertensive activities of feather hydrolysates obtained with the bacterium Chryseobacterium sp. kr6 were investigated. Keratin hydrolysates were produced with different concentrations of thermally denatured feathers (10-75 g l(-1)) and initial pH values (6.0-9.0). Soluble proteins accumulated in high amounts in media with 50 and 75 g l(-1) of feathers, reaching values of 18.5 and 22 mg ml(-1), respectively, after 48 hours of cultivation. In media with 50 g l(-1) of feathers, initial pH had minimal effect after 48 hours. Maximal protease production was observed after 24 hours of cultivation, and feather concentration and initial pH values showed no significant effect on enzyme yields at this time. Feather hydrolysates displayed in vitro antioxidant properties, and optimal antioxidant activities were observed in cultures with 50 g l(-1) feathers, at initial pH 8.0, after 48 hours growth at 30°C. Also, feather hydrolysates were demonstrated to inhibit the angiotesin I-converting enzyme by 65% and dipeptidyl peptidase-IV by 44%. The bioconversion of an abundant agroindustrial waste such as chicken feathers can be utilized as a strategy to obtain hydrolysates with antioxidant and antihypertensive activities. Feather hydrolysates might be employed as supplements in animal feed, and also as a potential source of bioactive molecules for feed, food and drug development. PMID:25038398

  18. Dipeptidyl peptidase IV dependent water-soluble prodrugs of highly lipophilic bicyclic nucleoside analogues.

    PubMed

    Diez-Torrubia, Alberto; Balzarini, Jan; Andrei, Graciela; Snoeck, Robert; De Meester, Ingrid; Camarasa, María-José; Velázquez, Sonsoles

    2011-03-24

    We present the first report of the application of the dipeptidyl peptidase IV (DPPIV/CD26) based prodrug approach to hydroxy-containing drug derivatives. In particular, we applied this strategy to the highly lipophilic antiviral drug family of bicyclic furanopyrimidine nucleoside analogues (BCNA) in order to improve their physicochemical and pharmacokinetic properties. Our stability data demonstrated that the prodrugs efficiently release the parent BCNA drug upon selective conversion by purified DPPIV/CD26 and by soluble DPPIV/CD26 present in bovine, murine, and human serum. Vildagliptin, a specific inhibitor of DPPIV/CD26, was able to completely block the hydrolysis of the prodrugs in the presence of purified DPPIV/CD26 human, murine, and bovine serum. Several novel prodrugs showed remarkable increases in water solubility (up to more than 3 orders of magnitude) compared to the poorly soluble parent drug. We also demonstrated a markedly enhanced oral bioavailability of the prodrugs versus the parent drug in mice. PMID:21332170

  19. Structure-activity relationships of boronic acid inhibitors of dipeptidyl peptidase IV. 1. Variation of the P2 position of Xaa-boroPro dipeptides.

    PubMed

    Coutts, S J; Kelly, T A; Snow, R J; Kennedy, C A; Barton, R W; Adams, J; Krolikowski, D A; Freeman, D M; Campbell, S J; Ksiazek, J F; Bachovchin, W W

    1996-05-10

    A series of prolineboronic acid (boroPro) containing dipeptides were synthesized and assayed for their ability to inhibit the serine protease dipeptidyl peptidase IV (DPPIV). Inhibitory activity, which requires the (R)-stereoisomer of boroPro in the P1 position, appears to tolerate a variety of L-amino acids in the P2 position. Substitution at the P2 position which is not tolerated include the D-amino acids, alpha,alpha-disubstituted amino acids, and glycine. Specificity against DPPII and proline specific endopeptidase is reported. A correlation between the ability to inhibit DPPIV in cell culture and in the human mixed lymphocyte reaction is demonstrated. A synthesis of prolineboronic acid is reported as well as conditions for generating the fully unprotected boronic acid dipeptides in either their cyclic or acyclic forms. PMID:8642568

  20. Dipeptidyl-peptidase IV inhibitory activity of peptides derived from tuna cooking juice hydrolysates.

    PubMed

    Huang, Shih-Li; Jao, Chia-Ling; Ho, Kit-Pan; Hsu, Kuo-Chiang

    2012-05-01

    The in vitro DPP-IV inhibitory activity of isolated peptides from of tuna cooking juice hydrolyzed by Protease XXIII (PR) and orientase (OR) was determined. The results showed that the peptide fractions with the molecular weight over 1,422 Da possessed the greatest DPP-IV inhibitory activity. The amino acid sequences of the three peptides isolated from PR and OR hydrolysates were identified by MALDI-TOF/TOF MS/MS, and they were Pro-Gly-Val-Gly-Gly-Pro-Leu-Gly-Pro-Ile-Gly-Pro-Cys-Tyr-Glu (1412.7 Da), Cys-Ala-Tyr-Gln-Trp-Gln-Arg-Pro-Val-Asp-Arg-Ile-Arg (1690.8 Da) and Pro-Ala-Cys-Gly-Gly-Phe-Try-Ile-Ser-Gly-Arg-Pro-Gly (1304.6 Da), while they showed the dose-dependent inhibition effect of DPP-IV with IC(50) values of 116.1, 78.0 and 96.4 μM, respectively. In vitro simulated gastrointestinal digestion retained or even improved the DPP-IV inhibitory activities of the three peptides. The results suggest that tuna cooking juice would be a good precursor of DPP-IV inhibitor, and the DPP-IV inhibitory peptides can successfully passed through the digestive tract. PMID:22450467

  1. Dipeptidyl Peptidase IV as a Potential Target for Selective Prodrug Activation and Chemotherapeutic Action in Cancers

    PubMed Central

    2015-01-01

    The efficacy of chemotherapeutic drugs is often offset by severe side effects attributable to poor selectivity and toxicity to normal cells. Recently, the enzyme dipeptidyl peptidase IV (DPPIV) was considered as a potential target for the delivery of chemotherapeutic drugs. The purpose of this study was to investigate the feasibility of targeting chemotherapeutic drugs to DPPIV as a strategy to enhance their specificity. The expression profile of DPPIV was obtained for seven cancer cell lines using DNA microarray data from the DTP database, and was validated by RT-PCR. A prodrug was then synthesized by linking the cytotoxic drug melphalan to a proline-glycine dipeptide moiety, followed by hydrolysis studies in the seven cell lines with a standard substrate, as well as the glycyl-prolyl-melphalan (GP-Mel). Lastly, cell proliferation studies were carried out to demonstrate enzyme-dependent activation of the candidate prodrug. The relative RT-PCR expression levels of DPPIV in the cancer cell lines exhibited linear correlation with U95Av2 Affymetrix data (r2 = 0.94), and with specific activity of a standard substrate, glycine-proline-p-nitroanilide (r2 = 0.96). The significantly higher antiproliferative activity of GP-Mel in Caco-2 cells (GI50 = 261 μM) compared to that in SK-MEL-5 cells (GI50 = 807 μM) was consistent with the 9-fold higher specific activity of the prodrug in Caco-2 cells (5.14 pmol/min/μg protein) compared to SK-MEL-5 cells (0.68 pmol/min/μg protein) and with DPPIV expression levels in these cells. Our results demonstrate the great potential to exploit DPPIV as a prodrug activating enzyme for efficient chemotherapeutic drug targeting. PMID:25365774

  2. Trelagliptin (SYR-472, Zafatek), Novel Once-Weekly Treatment for Type 2 Diabetes, Inhibits Dipeptidyl Peptidase-4 (DPP-4) via a Non-Covalent Mechanism

    PubMed Central

    Jennings, Andy; Kamran, Ruhi; Ueno, Hikaru; Nishigaki, Nobuhiro; Kosaka, Takuo; Tani, Akiyoshi; Sano, Hiroki; Kinugawa, Yoshinobu; Koumura, Emiko; Shi, Lihong; Takeuchi, Koji

    2016-01-01

    Trelagliptin (SYR-472), a novel dipeptidyl peptidase-4 inhibitor, shows sustained efficacy by once-weekly dosing in type 2 diabetes patients. In this study, we characterized in vitro properties of trelagliptin, which exhibited approximately 4- and 12-fold more potent inhibition against human dipeptidyl peptidase-4 than alogliptin and sitagliptin, respectively, and >10,000-fold selectivity over related proteases including dipeptidyl peptidase-8 and dipeptidyl peptidase-9. Kinetic analysis revealed reversible, competitive and slow-binding inhibition of dipeptidyl peptidase-4 by trelagliptin (t1/2 for dissociation ≈ 30 minutes). X-ray diffraction data indicated a non-covalent interaction between dipeptidyl peptidase and trelagliptin. Taken together, potent dipeptidyl peptidase inhibition may partially contribute to sustained efficacy of trelagliptin. PMID:27328054

  3. Trelagliptin (SYR-472, Zafatek), Novel Once-Weekly Treatment for Type 2 Diabetes, Inhibits Dipeptidyl Peptidase-4 (DPP-4) via a Non-Covalent Mechanism.

    PubMed

    Grimshaw, Charles E; Jennings, Andy; Kamran, Ruhi; Ueno, Hikaru; Nishigaki, Nobuhiro; Kosaka, Takuo; Tani, Akiyoshi; Sano, Hiroki; Kinugawa, Yoshinobu; Koumura, Emiko; Shi, Lihong; Takeuchi, Koji

    2016-01-01

    Trelagliptin (SYR-472), a novel dipeptidyl peptidase-4 inhibitor, shows sustained efficacy by once-weekly dosing in type 2 diabetes patients. In this study, we characterized in vitro properties of trelagliptin, which exhibited approximately 4- and 12-fold more potent inhibition against human dipeptidyl peptidase-4 than alogliptin and sitagliptin, respectively, and >10,000-fold selectivity over related proteases including dipeptidyl peptidase-8 and dipeptidyl peptidase-9. Kinetic analysis revealed reversible, competitive and slow-binding inhibition of dipeptidyl peptidase-4 by trelagliptin (t1/2 for dissociation ≈ 30 minutes). X-ray diffraction data indicated a non-covalent interaction between dipeptidyl peptidase and trelagliptin. Taken together, potent dipeptidyl peptidase inhibition may partially contribute to sustained efficacy of trelagliptin. PMID:27328054

  4. Trelagliptin (SYR-472, Zafatek), novel once-weekly treatment for type 2 diabetes, inhibits dipeptidyl peptidase-4 (DPP-4) via a non-covalent mechanism

    DOE PAGESBeta

    Grimshaw, Charles E.; Jennings, Andy; Kamran, Ruhi; Ueno, Hikaru; Nishigaki, Nobuhiro; Kosaka, Takuo; Tani, Akiyoshi; Sano, Hiroki; Kinugawa, Yoshinobu; Koumura, Emiko; et al

    2016-06-21

    Trelagliptin (SYR-472), a novel dipeptidyl peptidase-4 inhibitor, shows sustained efficacy by once-weekly dosing in type 2 diabetes patients. In this study, we characterized in vitro properties of trelagliptin, which exhibited approximately 4-and 12-fold more potent inhibition against human dipeptidyl peptidase-4 than alogliptin and sitagliptin, respectively, and >10,000-fold selectivity over related proteases including dipeptidyl peptidase-8 and dipeptidyl peptidase-9. Kinetic analysis revealed reversible, competitive and slow-binding inhibition of dipeptidyl peptidase-4 by trelagliptin (t1/2 for dissociation ≈ 30 minutes). X-ray diffraction data indicated a non-covalent interaction between dipeptidyl peptidase and trelagliptin. Altogether, potent dipeptidyl peptidase inhibitionmay partially contribute to sustained efficacy of trelagliptin.

  5. Prognostic significance of the combined expression of neutral endopeptidase and dipeptidyl peptidase IV in intrahepatic cholangiocarcinoma patients after surgery resection

    PubMed Central

    Zhu, Jianyong; Guo, XiaoDong; Qiu, Baoan; Li, Zhiyan; Xia, Nianxin; Yang, Yingxiang; Liu, Peng

    2014-01-01

    Aim The aim of this study was to investigate the relationship between the expression of neutral endopeptidase (NEP) and dipeptidyl peptidase IV (DPP IV) proteins, and the clinical significance of the two proteins in patients with intrahepatic cholangiocarcinomas (IHCC). Methods Expression patterns and subcellular localizations of NEP and DPP IV proteins in 186 primary IHCC and 60 noncancerous liver tissue specimens were detected by immunohistochemistry. Results Both the expression of NEP and DPP IV proteins in IHCC tissues were significantly higher than those in noncancerous liver tissues (both P<0.001). Of 186 patients with IHCC, 128 (68.82%) highly expressed both NEP and DPP IV proteins. In addition, the coexpression of NEP and DPP IV proteins was significantly associated with advanced tumor stage (P=0.009), positive lymph node metastasis (P=0.016) and distant metastasis (P=0.013), and the presence of recurrence (P=0.027). Moreover, Kaplan–Meier analysis showed that IHCC patients with high NEP expression, high DPP IV expression, and combined overexpression of NEP and DPP IV proteins all had poorer overall survival and early recurrence after surgery. Furthermore, Cox analysis suggested that NEP expression, DPP IV expression, and combined expression of NEP and DPP IV proteins were all independent prognostic markers for overall survival and recurrence-free survival in patients with IHCC. Conclusion Our data suggest, for the first time, that both the expression of NEP and DPP IV proteins may be upregulated in human IHCC tissues and the combined expression of NEP and DPP IV proteins may play important roles in progression and prognosis of patients with IHCC. PMID:24570591

  6. Effects of addition of a dipeptidyl peptidase IV inhibitor to metformin on sirolimus-induced diabetes mellitus.

    PubMed

    Jin, Long; Lim, Sun Woo; Jin, Jian; Chung, Byung Ha; Yang, Chul Woo

    2016-08-01

    The guideline for the management of new-onset diabetes after transplantation recommends metformin (MET) as a first-line drug, and addition of a second-line drug is needed to better control of hyperglycemia. We tested the effect of addition of a dipeptidyl peptidase IV (DPP IV) inhibitor to MET on sirolimus (SRL)-induced diabetes mellitus (DM). In animal model of SRL-induced DM, MET treatment improved pancreatic islet function (blood glucose level and insulin secretion) and attenuated oxidative stress and apoptotic cell death. Addition of a DPP IV inhibitor to MET improved these parameters more than MET alone. An in vitro study showed that SRL treatment increased pancreas beta cell death and production of reactive oxygen species (ROS), and pretreatment of ROS inhibitor, or p38MAPK inhibitor effectively decreased SRL-induced islet cell death. Exendin-4 (EXD), a substrate of DPP IV or MET significantly improved cell viability and decreased ROS production compared with SRL treatment, and combined treatment with the 2 drugs improved both parameters. At the subcellular level, impaired mitochondrial respiration by SRL were partially improved by MET or EXD and much improved further after addition of EXD to MET. Our data suggest that addition of a DPP IV inhibitor to MET decreases SRL-induced oxidative stress and improves mitochondrial respiration. This finding provides a rationale for the combined use of a DPP IV inhibitor and MET in treating SRL-induced DM. PMID:27059001

  7. A general method for making peptide therapeutics resistant to serine protease degradation: application to dipeptidyl peptidase IV substrates.

    PubMed

    Heard, Kathryn R; Wu, Wengen; Li, Youhua; Zhao, Peng; Woznica, Iwona; Lai, Jack H; Beinborn, Martin; Sanford, David G; Dimare, Matthew T; Chiluwal, Amrita K; Peters, Diane E; Whicher, Danielle; Sudmeier, James L; Bachovchin, William W

    2013-11-14

    Bioactive peptides have evolved to optimally fulfill specific biological functions, a fact which has long attracted attention for their use as therapeutic agents. While there have been some recent commercial successes fostered in part by advances in large-scale peptide synthesis, development of peptides as therapeutic agents has been significantly impeded by their inherent susceptibility to protease degradation in the bloodstream. Here we report that incorporation of specially designed amino acid analogues at the P1' position, directly C-terminal of the enzyme cleavage site, renders peptides, including glucagon-like peptide-1 (7-36) amide (GLP-1) and six other examples, highly resistant to serine protease degradation without significant alteration of their biological activity. We demonstrate the applicability of the method to a variety of proteases, including dipeptidyl peptidase IV (DPP IV), dipeptidyl peptidase 8 (DPP8), fibroblast activation protein α (FAPα), α-lytic protease (αLP), trypsin, and chymotrypsin. In summary, the "P1' modification" represents a simple, general, and highly adaptable method of generating enzymatically stable peptide-based therapeutics. PMID:24044354

  8. Expression of CD26 and its association with dipeptidyl peptidase IV activity in lymphocytes of type 2 diabetes patients.

    PubMed

    Bellé, Luziane Potrich; Bitencourt, Paula Eliete Rodrigues; de Bona, Karine Santos; Zanette, Régis Adriel; Moresco, Rafael Noal; Moretto, Maria Beatriz

    2011-11-01

    Immune response and inflammation were suggested to play certain roles in the development and complications of type 2 diabetes mellitus. The main objective of this study was to investigate the CD26 expression and its relationship with adenosine deaminase (ADA), dipeptidyl peptidase IV (DPP-IV), γ-glutamyltransferase (GGT), and N-acetyl-β-glucosaminidase (NAG) activities in lymphocytes of type 2 diabetics (T2DM) patients. These parameters were assessed in 25 T2DM patients and 20 control subjects. We observed a decrease in CD26 expression and a significant increase in the ADA activity in T2DM patients when compared with control subjects. There were no differences between activities of DPP-IV, NAG, and GGT in lymphocytes of T2DM patients and control subjects. Meanwhile, a significant negative correlation was observed between CD26 expression and DPP-IV activity in lymphocytes of T2DM patients. Moreover, a positive correlation was found between DPPIV and ADA activities. The results suggest that the reduction of CD26 expression may be associated in the regulation of DPP-IV in T2DM patients. PMID:21614532

  9. Inhibition of kallikrein-related peptidases by the serine protease inhibitor of Kazal-type 6.

    PubMed

    Kantyka, Tomasz; Fischer, Jan; Wu, Zhihong; Declercq, Wim; Reiss, Karina; Schröder, Jens-Michael; Meyer-Hoffert, Ulf

    2011-06-01

    Kallikrein-related peptidases (KLKs) are a group of serine proteases, expressed in several tissues. Their activity is regulated by inhibitors including members of the serine protease of Kazal-type (SPINK) family. Recently, we discovered that SPINK6 is expressed in human skin and inhibits KLK5, KLK7, KLK14 but not KLK8. In this study we tested whether SPINK6 inhibits other members of the KLK family and caspase-14. Using chromogenic substrates, SPINK6 exhibited inhibitory activity against KLK12 and KLK13 with K(i) around 1nM, KLK4 with K(i)=27.3nM, KLK6 with K(i)=140nM, caspase-14 with a K(i) approximating 1μM and no activity against KLK1, KLK3 and KLK11. Taken together, SPINK6 is a potent inhibitor of distinct KLKs members. PMID:21439340

  10. In vivo modulation of CD26 (dipeptidyl peptidase IV) in the mouse: effects of polyreactive and monoreactive antibodies.

    PubMed

    Yamaguchi, N; Plant, C; Biancone, L; Bachovchin, W; McCluskey, R; Andres, G

    1996-10-15

    We previously reported that intravenous injections in rabbits or guinea pigs of divalent antibodies to purified protein or carbohydrate antigens located mainly on endothelial cells induce acute pulmonary edema, which is often lethal. Surviving animals develop resistance to the injurious effect of subsequent injection of antibodies (adaptation), associated with shedding of antigen-antibody complexes from endothelial cells. In the present study, we investigated and compared in mice the effects of 3-day multiple injections of two different rabbit antibody (IgG) preparations against antigens expressed mainly at the surface of epithelial cells. The first preparation contained antibodies to a single transmembrane protein, CD26 (dipeptidyl peptidase IV [DPP IV]) (monoreactive anti-DPP IV IgG); the second contained antibodies against multiple antigens of the renal tubular brush border (BB), including DPP IV (polyreactive anti-BB IgG). Both IgG preparations caused loss of DPP IV from the organs studied, as shown by reduction in enzyme activity in tissue homogenates and by immunofluorescence microscopy, which showed loss of DPP IV from cell surface. However, the monoreactive anti-DPP IV IgG induced considerably greater reduction than polyreactive anti-BB IgG. Loss of DPP IV from the cell surface probably occurred by shedding of immune complexes into vascular and extravascular fluids, including bile and urine. The results may have relevance to hyperacute rejection of xenografts, as from pigs to primates. Since human natural antibodies that bind to porcine cells are polyreactive, a new prophylactic strategy for hyperacute rejection might be based on down-regulation of the major xenogeneic antigen, alpha-galactosyl, by injecting donor animals with monoreactive alpha-galactosyl antibodies before transplantation. PMID:8878393

  11. Arrabidaea chica Hexanic Extract Induces Mitochondrion Damage and Peptidase Inhibition on Leishmania spp.

    PubMed Central

    Rodrigues, Igor A.; Azevedo, Mariana M. B.; Chaves, Francisco C. M.; Alviano, Celuta S.; Alviano, Daniela S.; Vermelho, Alane B.

    2014-01-01

    Currently available leishmaniasis treatments are limited due to severe side effects. Arrabidaea chica is a medicinal plant used in Brazil against several diseases. In this study, we investigated the effects of 5 fractions obtained from the crude hexanic extract of A. chica against Leishmania amazonensis and L. infantum, as well as on the interaction of these parasites with host cells. Promastigotes were treated with several concentrations of the fractions obtained from A. chica for determination of their minimum inhibitory concentration (MIC). In addition, the effect of the most active fraction (B2) on parasite's ultrastructure was analyzed by transmission electron microscopy. To evaluate the inhibitory activity of B2 fraction on Leishmania peptidases, parasites lysates were treated with the inhibitory and subinhibitory concentrations of the B2 fraction. The minimum inhibitory concentration of B2 fraction was 37.2 and 18.6 μg/mL for L. amazonensis and L. infantum, respectively. Important ultrastructural alterations as mitochondrial swelling with loss of matrix content and the presence of vesicles inside this organelle were observed in treated parasites. Moreover, B2 fraction was able to completely inhibit the peptidase activity of promastigotes at pH 5.5. The results presented here further support the use of A. chica as an interesting source of antileishmanial agents. PMID:24818162

  12. Dipeptide boronic acid inhibitors of dipeptidyl peptidase IV: determinants of potency and in vivo efficacy and safety.

    PubMed

    Connolly, Beth A; Sanford, David G; Chiluwal, Amrita K; Healey, Sarah E; Peters, Diane E; Dimare, Matthew T; Wu, Wengen; Liu, Yuxin; Maw, Hlaing; Zhou, Yuhong; Li, Youhua; Jin, Zhiping; Sudmeier, James L; Lai, Jack H; Bachovchin, William W

    2008-10-01

    Dipeptidyl peptidase IV (DPP-IV; E.C. 3.4.14.5), a serine protease that degrades the incretin hormones GLP-1 and GIP, is now a validated target for the treatment of type 2 diabetes. Dipeptide boronic acids, among the first, and still among the most potent DPP-IV inhibitors known, suffer from a concern over their safety. Here we evaluate the potency, in vivo efficacy, and safety of a selected set of these inhibitors. The adverse effects induced by boronic acid-based DPP-IV inhibitors are essentially limited to what has been observed previously for non-boronic acid inhibitors and attributed to cross-reactivity with DPP8/9. While consistent with the DPP8/9 hypothesis, they are also consistent with cross-reactivity with some other intracellular target. The results further show that the potency of simple dipeptide boronic acid-based inhibitors can be combined with selectivity against DPP8/9 in vivo to produce agents with a relatively wide therapeutic index (>500) in rodents. PMID:18783201

  13. Differential Inhibition of Signal Peptide Peptidase Family Members by Established γ-Secretase Inhibitors

    PubMed Central

    Ran, Yong; Ladd, Gabriela Z.; Ceballos-Diaz, Carolina; Jung, Joo In; Greenbaum, Doron; Felsenstein, Kevin M.; Golde, Todd E.

    2015-01-01

    The signal peptide peptidases (SPPs) are biomedically important proteases implicated as therapeutic targets for hepatitis C (human SPP, (hSPP)), plasmodium (Plasmodium SPP (pSPP)), and B-cell immunomodulation and neoplasia (signal peptide peptidase like 2a, (SPPL2a)). To date, no drug-like, selective inhibitors have been reported. We use a recombinant substrate based on the amino-terminus of BRI2 fused to amyloid β 1-25 (Aβ1-25) (FBA) to develop facile, cost-effective SPP/SPPL protease assays. Co-transfection of expression plasmids expressing the FBA substrate with SPP/SPPLs were conducted to evaluate cleavage, which was monitored by ELISA, Western Blot and immunoprecipitation/MALDI-TOF Mass spectrometry (IP/MS). No cleavage is detected in the absence of SPP/SPPL overexpression. Multiple γ-secretase inhibitors (GSIs) and (Z-LL)2 ketone differentially inhibited SPP/SPPL activity; for example, IC50 of LY-411,575 varied from 51±79 nM (on SPPL2a) to 5499±122 nM (on SPPL2b), while Compound E showed inhibition only on hSPP with IC50 of 1465±93 nM. Data generated were predictive of effects observed for endogenous SPPL2a cleavage of CD74 in a murine B-Cell line. Thus, it is possible to differentially inhibit SPP family members. These SPP/SPPL cleavage assays will expedite the search for selective inhibitors. The data also reinforce similarities between SPP family member cleavage and cleavage catalyzed by γ-secretase. PMID:26046535

  14. Effects of dipeptidyl peptidase IV inhibitor sitagliptin on immunological parameters of lymphocytes in intact animals and animals with experimental autoimmune process.

    PubMed

    Robinson, M V; Mel'nikova, E V; Trufakin, V A

    2014-11-01

    The effects of dipeptidyl peptidase IV inhibitor sitagliptin on immunological parameters were studied in animals with experimental autoimmune process. The effects of the drug administered in preventive (before manifestation of autoimmune processes) and therapeutic (after manifestation of autoimmune process) modes were studied. PMID:25408522

  15. Whey proteins as source of dipeptidyl dipeptidase IV (dipeptidyl peptidase-4) inhibitors.

    PubMed

    Tulipano, Giovanni; Sibilia, Valeria; Caroli, Anna Maria; Cocchi, Daniela

    2011-04-01

    Preclinical and clinical studies suggest that whey proteins can reduce postprandial glucose levels and stimulate insulin release in healthy subjects and in subjects with type 2 diabetes by reducing dipeptidyl peptidase-4 (DPP-4) activity in the proximal bowel and hence increasing intact incretin levels. Our aim was to identify DPP-4 inhibitors among short peptides occurring in hydrolysates of β-lactoglobulin, the major whey protein found in the milk of ruminants. We proved that the bioactive peptide Ile-Pro-Ala can be regarded as a moderate DPP-4 inhibitor. PMID:21256171

  16. Dipeptidyl peptidase IV activity and/or structure homologs: Contributing factors in the pathogenesis of rheumatoid arthritis?

    PubMed Central

    Sedo, Aleksi; Duke-Cohan, Jonathan S; Balaziova, Eva; Sedova, Liliana R

    2005-01-01

    Several of the proinflammatory peptides involved in rheumatoid arthritis pathogenesis, including peptides induced downstream of tumor necrosis factor-α as well as the monocyte/T cell-attracting chemokines RANTES and stromal cell-derived factor (SDF)-1α and the neuropeptides vasoactive intestinal peptide (VIP) and substance P, have their biological half-lives controlled by dipeptidyl peptidase IV (DPPIV). Proteolysis by DPPIV regulates not only the half-life but also receptor preference and downstream signaling. In this article, we examine the role of DPPIV homologs, including CD26, the canonical DPPIV, and their substrates in the pathogenesis of rheumatoid arthritis. The differing specific activities of the DPPIV family members and their differential inhibitor response provide new insights into therapeutic design. PMID:16277701

  17. Inhibition of dipeptidyl peptidase 4 regulates microvascular endothelial growth induced by inflammatory cytokines

    SciTech Connect

    Takasawa, Wataru; Ohnuma, Kei; Hatano, Ryo; Endo, Yuko; Dang, Nam H.

    2010-10-08

    Research highlights: {yields} TNF-{alpha} or IL-1{beta} induces EC proliferation with reduction of CD26 expression. {yields} CD26 siRNA or DPP-4 inhibition enhances TNF-{alpha} or IL-1{beta}-induced EC proliferation. {yields} Loss of CD26/DPP-4 enhances aortic sprouting induced by TNF-{alpha} or IL-1{beta}. {yields} Capillary formation induced by TNF-{alpha} or IL-1{beta} is enahced in the CD26{sup -/-} mice. -- Abstract: CD26/DPP-4 is abundantly expressed on capillary of inflamed lesion as well as effector T cells. Recently, CD26/dipeptidyl peptidase 4 (DPP-4) inhibition has been used as a novel oral therapeutic approach for patients with type 2 diabetes. While accumulating data indicate that vascular inflammation is a key feature of both micro- and macro-vascular complications in diabetes, the direct role of CD26/DPP-4 in endothelial biology is to be elucidated. We herein showed that proinflammatory cytokines such as tumor necrosis factor or interleukin-1 reduce expression of CD26 on microvascular endothelial cells, and that genetical or pharmacological inhibition of CD26/DPP-4 enhances endothelial growth both in vitro and in vivo. With DPP-4 inhibitors being used widely in the treatment of type 2 diabetes, our data strongly suggest that DPP-4 inhibition plays a pivotal role in endothelial growth and may have a potential role in the recovery of local circulation following diabetic vascular complications.

  18. Dipeptidyl-peptidase IV (DPP-IV) inhibitor delays tolerance to anxiolytic effect of ethanol and withdrawal-induced anxiety in rats.

    PubMed

    Sharma, Ajaykumar N; Pise, Ashish; Sharma, Jay N; Shukla, Praveen

    2015-06-01

    Dipeptidyl-peptidase IV (DPP-IV) is an enzyme responsible for the metabolism of endogenous gut-derived hormone, glucagon-like peptide-1 (GLP-1). DPP-IV is known for its role in energy homeostasis and pharmacological blockade of this enzyme is a recently approved clinical strategy for the management of type II diabetes. Accumulating evidences suggest that enzyme DPP-IV can affect spectrum of central nervous system (CNS) functions. However, little is known about the role of this enzyme in ethanol-mediated neurobehavioral complications. The objective of the present study was to examine the impact of DPP-IV inhibitor, sitagliptin on the development of tolerance to anxiolytic effect of ethanol and anxiety associated with ethanol withdrawal in rats. A dose-response study revealed that sitaglitpin (20 mg/kg, p.o.) per se exhibit anxiolytic effect in the elevated plus maze (EPM) test in rats. Tolerance to anxiolytic effect of ethanol (2 g/kg, i.p.; 8 % w/v) was observed from 7(th) day of ethanol-diet (6 % v/v) consumption. In contrast, tolerance to anxiolytic effect of ethanol was delayed in rats that were treated daily with sitagliptin (20 mg/kg, p.o.) as tolerance was observed from 13(th)day since commencement of ethanol-diet consumption. Discontinuation of rats from ethanol-diet after 15-days of ethanol consumption resulted in withdrawal anxiety between 8 h and 12 h post-abstinence. However, rats on 15-day ethanol-diet with concomitant sitagliptin (20 mg/kg, p.o.) treatment exhibited delay in appearance (24 h post-withdrawal) of withdrawal anxiety. In summary, DPP-IV inhibitors may prove as an attractive research strategy against ethanol tolerance and dependence. PMID:25129124

  19. Dopamine D3 receptor inhibits the ubiquitin-specific peptidase 48 to promote NHE3 degradation

    PubMed Central

    Armando, Ines; Villar, Van Anthony M.; Jones, John E.; Lee, Hewang; Wang, Xiaoyan; Asico, Laureano D.; Yu, Peiying; Yang, Jian; Escano, Crisanto S.; Pascua-Crusan, Annabelle M.; Felder, Robin A.; Jose, Pedro A.

    2014-01-01

    The dopamine D3 receptor (D3R) is crucial in the regulation of blood pressure and sodium balance, in that Drd3 gene ablation in mice results in hypertension and failure to excrete a dietary salt load. The mechanism responsible for the renal sodium retention in these mice is largely unknown. We now offer and describe a novel mechanism by which D3R decreases sodium transport in the long term by inhibiting the deubiquitinylating activity of ubiquitin-specific peptidase 48 (USP48), thereby promoting Na+-H+ exchanger (NHE)-3 degradation. We found that stimulation with the D3R-specific agonist PD128907 (1 μM, 30 min) promoted the interaction and colocalization among D3R, NHE3, and USP48; inhibited USP48 activity (−35±6%, vs. vehicle), resulting in increased ubiquitinylated NHE3 (+140±10%); and decreased NHE3 expression (−50±9%) in human renal proximal tubule cells (hRPTCs). USP48 silencing decreased NHE3's half-life (USP48 siRNA t1/2=6.1 h vs. vehicle t1/2=12.9 h), whereas overexpression of USP48 increased NHE3 half-life (t1/2=21.8 h), indicating that USP48 protects NHE3 from degradation via deubiquitinylation. USP48 accounted for ∼30% of the total deubiquitinylating activity in these cells. Extending our studies in vivo, we found that pharmacologic blockade of D3R via the D3R-specific antagonist GR103691 (1 μg/kg/min, 4 d) in C57Bl/6J mice increased renal NHE3 expression (+310±15%, vs. vehicle), whereas an innovative kidney-restricted Usp48 silencing via siRNA (3 μg/d, 7 d) increased ubiquitinylated NHE3 (+250±30%, vs. controls), decreased total NHE3 (−23±2%), and lowered blood pressure (−24±2 mm Hg), compared with that in control mice that received either the vehicle or nonsilencing siRNA. Our data demonstrate a crucial role for the dynamic interaction between D3R and USP48 in the regulation of NHE3 expression and function.—Armando, I., Villar, V. A. M., Jones J. E., Lee, H., Wang, X., Asico L. D., Yu, P., Yang, J., Escano, C. S. Jr., Pascua

  20. Type IV prepilin peptidase gene of Neisseria gonorrhoeae MS11: presence of a related gene in other piliated and nonpiliated Neisseria strains.

    PubMed Central

    Dupuy, B; Pugsley, A P

    1994-01-01

    The assembly of type IV pili in Neisseria gonorrhoeae is a complex process likely to require the products of many genes. One of these is the enzyme prepilin peptidase, which cleaves and then N methylates the precursor pilin subunits prior to their assembly into pili. We have used a PCR amplification strategy to clone the N. gonorrhoeae prepilin peptidase gene, pilDNg. A single copy of the gene is shown to be present in the chromosome. Its product promotes correct cleavage of the gonococcal prepillin in Escherichia coli cells carrying both the prepilin peptidase gene and the pilin structural gene. PilDNg also cleaves prePulG, a type IV pilin-like protein of Klebsiella oxytoca. Moreover, PilDNg complements a mutation in the gene coding for the prepilin peptidase-like protein of K. oxytoca, pulO, partially restoring PulG-PulO-dependent extracellular secretion of the enzyme pullulanase. Finally, we show that genes homologous to pilDNg are present and expressed in a variety of species in the genus Neisseria, including some commensal strains. Images PMID:7906688

  1. 4-Substituted boro-proline dipeptides: synthesis, characterization, and dipeptidyl peptidase IV, 8, and 9 activities.

    PubMed

    Wu, Wengen; Liu, Yuxin; Milo, Lawrence J; Shu, Ying; Zhao, Peng; Li, Youhua; Woznica, Iwona; Yu, Gengli; Sanford, David G; Zhou, Yuhong; Poplawski, Sarah E; Connolly, Beth A; Sudmeier, James L; Bachovchin, William W; Lai, Jack H

    2012-09-01

    The boroProline-based dipeptidyl boronic acids were among the first DPP-IV inhibitors identified, and remain the most potent known. We introduced various substitutions at the 4-position of the boroProline ring regioselectively and stereoselectively, and incorporated these aminoboronic acids into a series of 4-substituted boroPro-based dipeptides. Among these dipeptidyl boronic acids, Arg-(4S)-boroHyp (4q) was the most potent inhibitor of DPP-IV, DPP8 and DPP9, while (4S)-Hyp-(4R)-boroHyp (4o) exhibited the most selectivity for DPP-IV over DPP8 and DPP9. PMID:22853995

  2. Dipeptidyl-peptidase 4 Inhibition: Linking Metabolic Control to Cardiovascular Protection

    PubMed Central

    Avogaro, Angelo; de Kreutzenberg, Saula; Fadini, Gianpaolo

    2014-01-01

    Dipeptidyl peptidases 4 (DPP4) inhibitors are a new class of oral anti-hyperglycemic drugs for the treatment of type 2 diabetes (T2DM). They are also called “incretins” because they act by inhibiting the degradation of endogenous incretin hormones, in particular GLP-1, that mediates their main metabolic effects. DPP4 is an ubiquitous protease that regulates not only glucose and lipid metabolism, but also exhibits several systemic effects at different site levels. DPP4 inhibition improves endothelial function, reduces the pro-oxidative and the pro-inflammatory state, and exerts renal effects. These actions are mediated by different DPP4 ligands, such as cytokines, growth factors, neuotransmitters etc. Clinical and experimental studies have demonstrated that DPP4 inhibitors are efficient in protecting cardiac, renal and vascular systems, through antiatherosclerotic and vasculoprotective mechanisms. For these reasons DDP4 inhibitors are thought to be “cardiovascular protective” as well as anti-diabetic drugs. Clinical trials aimed to demonstrate the efficacy of DPP4 inhibitors in reducing cardiovascular events, independent of their anti-hyperglycemic action, are ongoing. These trials will also give necessary information on their safety. PMID:23844811

  3. Does estradiol have an impact on the dipeptidyl peptidase IV enzyme activity of the Prevotella intermedia group bacteria?

    PubMed

    Fteita, Dareen; Könönen, Eija; Gürsoy, Mervi; Söderling, Eva; Gürsoy, Ulvi Kahraman

    2015-12-01

    Initiation and development of pregnancy-associated gingivitis is seemingly related to the microbial shift towards specific gram-negative anaerobes in subgingival biofilms. It is known that Prevotella intermedia sensu lato is able to use estradiol as an alternative source of growth instead of vitamin K. The aim of the present study was to investigate the impact of estradiol on the bacterial dipeptidyl peptidase IV (DPPIV) enzyme activity in vitro as a virulent factor of the Prevotella intermedia group bacteria, namely P. intermedia, Prevotella nigrescens, Prevotella pallens, and Prevotella aurantiaca. In all experiments, 2 strains of each Prevotella species were used. Bacteria were incubated with the concentrations of 0, 30, 90, and 120 nmol/L of estradiol and were allowed to build biofilms at an air-solid interface. DPPIV activities of biofilms were measured kinetically during 20 min using a fluorometric assay. The enzyme activity was later related to the amount of protein produced by the same biofilm, reflecting the biofilm mass. Estradiol significantly increased DPPIV activities of the 8 Prevotella strains in a strain- and dose-dependent manner. In conclusion, our in vitro experiments indicate that estradiol regulates the DPPIV enzyme activity of P. intermedia, P. nigrescens, P. pallens, and P. aurantiaca strains differently. Our results may, at least partly, explain the role of estradiol to elicit a virulent state which contributes to the pathogenesis of pregnancy-related gingivitis. PMID:26386229

  4. Design, synthesis and biological evaluation of hetero-aromatic moieties substituted pyrrole-2-carbonitrile derivatives as dipeptidyl peptidase IV inhibitors.

    PubMed

    Ji, Xun; Su, Mingbo; Wang, Jiang; Deng, Guanghui; Deng, Sisi; Li, Zeng; Tang, Chunlan; Li, Jingya; Li, Jia; Zhao, Linxiang; Jiang, Hualiang; Liu, Hong

    2014-03-21

    A series of novel hetero-aromatic moieties substituted α-amino pyrrole-2-carbonitrile derivatives was designed and synthesized based on structure-activity relationships (SARs) of pyrrole-2-carbonitrile inhibitors. All compounds demonstrated good dipeptidyl peptidase IV (DPP4) inhibitory activities (IC50 = 0.004-113.6 μM). Moreover, compounds 6h (IC50 = 0.004 μM) and 6n (IC50 = 0.01 μM) showed excellent inhibitory activities against DPP4, good selectivity (compound 6h, selective ratio: DPP8/DPP4 = 450.0; DPP9/DPP4 = 375.0; compound 6n, selective ratio: DPP8/DPP4 = 470.0; DPP9/DPP4 = 750.0) and good efficacy in an oral glucose tolerance test in ICR mice. Furthermore, compounds 6h and 6n demonstrated moderate PK properties (compound 6h, F% = 37.8%, t1/2 = 1.45 h; compound 6n, F% = 16.8%, t1/2 = 3.64 h). PMID:24531224

  5. Natural dipeptidyl peptidase-IV inhibitor mangiferin mitigates diabetes- and metabolic syndrome-induced changes in experimental rats

    PubMed Central

    Suman, Rajesh Kumar; Mohanty, Ipseeta Ray; Maheshwari, Ujwala; Borde, Manjusha K; Deshmukh, YA

    2016-01-01

    Background Mangiferin (MNG) is known to possess antidiabetic and antioxidant activity. However, there is no experimental evidence presently available in the literature with regard to its ameliorating effects on diabetes mellitus coexisting with metabolic syndrome. Objective The present study was designed to evaluate the protective effects of MNG on various components of metabolic syndrome with diabetes as an essential component. Material and methods Adult Wistar rats were fed high-fat diets for 10 weeks and challenged with streptozotocin (40 mg/kg) at week three (high-fat diabetic control group). After the confirmation of metabolic syndrome in the setting of diabetes, MNG 40 mg/kg was orally fed to these rats from the fourth to tenth week. Results The treatment with MNG showed beneficial effects on various components of metabolic syndrome, such as reduced dyslipidemia (decreased triglyceride, total cholesterol, low-density lipoprotein cholesterol, and increased high-density lipoprotein cholesterol) and diabetes mellitus (reduced blood glucose and glycosylated hemoglobin). In addition, an increase in serum insulin, C-peptide, and homeostasis model assessment-β and a reduction in homeostasis model assessment of insulin resistance-IR were observed in MNG-treated group compared with high-fat diabetic control group. MNG was also found to be cardioprotective (reduction in creatine phosphokinase-MB levels, atherogenic index, high-sensitivity C-reactive protein). Reduction in serum dipeptidyl peptidase–IV levels in the MNG-treated group correlated with improvement in insulin resistance and enhanced β-cell function. Conclusion The present study has demonstrated antidiabetic, hypolipidemic, and cardioprotective effects of MNG in the setting of diabetes with metabolic syndrome. Thus, MNG has the potential to be developed as a natural alternative to synthetic dipeptidyl peptidase-IV inhibitors beneficial in this comorbid condition. PMID:27621658

  6. Effects of Dipeptidyl Peptidase-4 Inhibition with MK-0431 on Syngeneic Mouse Islet Transplantation

    PubMed Central

    Juang, Jyuhn-Huarng; Kuo, Chien-Hung; Liu, Ying-Hsiu; Chang, Han-Ying; Chen, Chiung-Tong

    2014-01-01

    Dipeptidyl peptidase (DPP)-4 inhibitors increase circulating levels of glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide which may promote β-cell proliferation and survival. This study tested if DPP-4 inhibition with MK-0431 is beneficial for diabetic mice syngeneically transplanted with a marginal number of islets. We syngeneically transplanted 150 C57BL/6 mouse islets under the kidney capsule of each streptozotocin-diabetic mouse and then treated recipients with (n = 21) or without (n = 17) MK-0431 (30 mg/kg/day, po) for 6 weeks. After islet transplantation, blood glucose levels decreased in both MK-0431-treated and control groups. However, the blood glucose and area under the curve of the intraperitoneal glucose tolerance test at 2, 4, and 6 weeks were not significantly different between MK-0431-treated mice and controls. During 6 weeks, both groups exhibited increased body weights over time. However, the weight between two groups did not differ throughout the study period. At 6 weeks after transplantation, the graft beta-cell mass (0.024 ± 0.005 versus 0.023 ± 0.007 mg, P = 0.8793) and insulin content (140 ± 48 versus 231 ± 63 ng, P = 0.2939) were comparable in the MK-0431-treated group and controls. Our results indicate posttransplant DPP-4 inhibition with MK-0431 in the diabetic recipient with a marginal number of islets is not beneficial to transplantation outcome or islet grafts. PMID:25165473

  7. Effects of Inhibiting Dipeptidyl Peptidase-4 (DPP4) in Cows with Subclinical Ketosis

    PubMed Central

    Schulz, Kirsten; Frahm, Jana; Kersten, Susanne; Meyer, Ulrich; Rehage, Jürgen; Piechotta, Marion; Meyerholz, Maria; Breves, Gerhard; Reiche, Dania; Sauerwein, Helga; Dänicke, Sven

    2015-01-01

    The inhibition of dipeptidyl peptidase-4 (DPP4) via specific inhibitors is known to result in improved glucose tolerance and insulin sensitivity and decreased accumulation of hepatic fat in type II diabetic human patients. The metabolic situation of dairy cows can easily be compared to the status of human diabetes and non-alcoholic fatty liver. For both, insulin sensitivity is reduced, while hepatic fat accumulation increases, characterized by high levels of non-esterified fatty acids (NEFA) and ketone bodies.Therefore, in the present study, a DPP4 inhibitor was employed (BI 14332) for the first time in cows. In a first investigation BI 14332 treatment (intravenous injection at dosages of up to 3 mg/kg body weight) was well tolerated in healthy lactating pluriparous cows (n = 6) with a significant inhibition of DPP4 in plasma and liver. Further testing included primi- and pluriparous lactating cows suffering from subclinical ketosis (β-hydroxybutyrate concentrations in serum > 1.2 mM; n = 12). The intension was to offer effects of DPP4 inhibition during comprehensive lipomobilisation and hepatosteatosis. The cows of subclinical ketosis were evenly allocated to either the treatment group (daily injections, 0.3 mg BI 14332/kg body weight, 7 days) or the control group. Under condition of subclinical ketosis, the impact of DPP4 inhibition via BI 14332 was less, as in particular β-hydroxybutyrate and the hepatic lipid content remained unaffected, but NEFA and triglyceride concentrations were decreased after treatment. Owing to lower NEFA, the revised quantitative insulin sensitivity check index (surrogate marker for insulin sensitivity) increased. Therefore, a positive influence on energy metabolism might be quite possible. Minor impacts on immune-modulating variables were limited to the lymphocyte CD4+/CD8+ ratio for which a trend to decreased values in treated versus control animals was noted. In sum, the DPP4 inhibition in cows did not affect glycaemic control like

  8. Reduction of food intake by central administration of cholecystokinin octapeptide in the rat is dependent upon inhibition of brain peptidases.

    PubMed Central

    Griesbacher, T.; Leighton, G. E.; Hill, R. G.; Hughes, J.

    1989-01-01

    1. The effects of intracerebroventricular (i.c.v.) injections of cholecystokinin-octapeptide (CCK-8) and caerulein, an amphibian decapeptide structurally related to CCK-8, are inconsistent in the rat. We have therefore investigated the possibility that enzymatic degradation could be responsible for the lack of activity of CCK-8 seen in some studies on food intake. 2. Injections of CCK-8 at doses of 2.5 nmol and 25 nmol into the lateral cerebral ventricle of rats did not reduce the intake of a highly palatable diet whereas injections of the same doses of caerulein reduced food intake potently and dose-dependently. 3. Co-administration of CCK-8 with a combination of the peptidase inhibitors bestatin (70 nmol), captopril (100 nmol) and thiorphan (120 nmol) resulted in an inhibition of feeding similar to that seen after the injection of caerulein alone. The peptidase inhibitors alone did not affect food intake. 4. When caerulein was injected i.c.v. in combination with bestatin, captopril and thiorphan the effect of caerulein was potentiated, suggesting that enzymatic breakdown of caerulein does occur. 5. It is concluded that the effect of centrally administered CCK-8 on food intake is dependent on the activity of cleaving enzymes in the brain. It is emphasized that the action of brain peptidases is a major factor which has to be considered when investigating the role of peptides in the central nervous system. PMID:2647203

  9. Isolation and Identification of Dipeptidyl Peptidase IV-Inhibitory Peptides from Trypsin/Chymotrypsin-Treated Goat Milk Casein Hydrolysates by 2D-TLC and LC-MS/MS.

    PubMed

    Zhang, Ying; Chen, Ran; Ma, Huiqin; Chen, Shangwu

    2015-10-14

    New dipeptidyl peptidase IV (DPP-IV)-inhibitory peptides from trypsin/chymotrypsin-treated goat milk casein hydrolysates were isolated and identified by two-dimensional silica thin-layer chromatography (2D-TLC) combined to nano LC-MS/MS. 2D-TLC with chloroform/methanol/25% ammonia (2:2:1) and n-butanol/acetic acid/water (4:1:1) as the first- and second-dimension eluents, respectively, in analytical and semipreparative scales, was set up and verified by reversed-phase high-performance liquid chromatography (RP-HPLC) to be feasible and efficient to separate the hydrolysates. Five new DPP-IV-inhibitory peptides, four relatively large oligopeptides (MHQPPQPL, SPTVMFPPQSVL, VMFPPQSVL, and INNQFLPYPY), and AWPQYL were identified, and INNQFLPYPY showed a notable IC50 value of 40.08 μM as an uncompetitive inhibitor. Interactive effects on DPP-IV inhibition were also observed among separated fractions and pure synthetic peptide mixtures with concentration-dependent activity. The study gives new insights into goat casein hydrolysates with identified DPP-IV-inhibitory peptides efficiently isolated by 2D-TLC, which provides a simple and cost-efficient separation process and is compatible with liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification. PMID:26323964

  10. Behavioral effects of neuropeptide Y in F344 rat substrains with a reduced dipeptidyl-peptidase IV activity.

    PubMed

    Karl, Tim; Hoffmann, Torsten; Pabst, Reinhard; von Hörsten, Stephan

    2003-07-01

    Dipeptidyl-peptidase IV (DPPIV/CD26) is involved in several physiological functions by cleavage of dipeptides with a Xaa-Pro or Xaa-Ala sequence of regulatory peptides such as neuropeptide Y (NPY). Cleavage of NPY by DPPIV results in NPY(3-36), which lacks affinity for the Y(1) but not for other NPY receptor subtypes. Among other effects, the NPY Y(1) receptor mediates anxiolytic-like effects of NPY. In previous studies with F344 rat substrains lacking endogenous DPPIV-like activity we found a reduced behavioral stress response, which might be due to a differential degradation of NPY. Here we tested this hypothesis and administered intracerebroventricularly two different doses of NPY (0.0, 0.2, 1.0 nmol) in mutant and wildtype-like F344 substrains. NPY dose-dependently stimulated food intake and feeding motivation, decreased motor activity in the plus maze and social interaction test, and exerted anxiolytic-like effects. More important for the present hypothesis, NPY administration was found to be more potent in the DPPIV-negative substrains in exerting anxiolytic-like effects (increased social interaction time in the social interaction test) and sedative-like effects (decreased motor activity in the elevated plus maze). These data demonstrate for the first time a differential potency of NPY in DPPIV-deficient rats and suggest a changed receptor-specificity of NPY, which may result from a differential degradation of NPY in this genetic model of DPPIV deficiency. Overall, these results provide direct evidence that NPY-mediated effects in the central nervous system are modulated by DPPIV-like enzymatic activity. PMID:12957230

  11. NAAG peptidase inhibition in the periaqueductal gray and rostral ventromedial medulla reduces flinching in the formalin model of inflammation

    PubMed Central

    2012-01-01

    Background Metabotropic glutamate receptors (mGluRs) have been identified as significant analgesic targets. Systemic treatments with inhibitors of the enzymes that inactivate the peptide transmitter N-acetylaspartylglutamate (NAAG), an mGluR3 agonist, have an analgesia-like effect in rat models of inflammatory and neuropathic pain. The goal of this study was to begin defining locations within the central pain pathway at which NAAG activation of its receptor mediates this effect. Results NAAG immunoreactivity was found in neurons in two brain regions that mediate nociceptive processing, the periaqueductal gray (PAG) and the rostral ventromedial medulla (RVM). Microinjection of the NAAG peptidase inhibitor ZJ43 into the PAG contralateral, but not ipsilateral, to the formalin injected footpad reduced the rapid and slow phases of the nociceptive response in a dose-dependent manner. ZJ43 injected into the RVM also reduced the rapid and slow phase of the response. The group II mGluR antagonist LY341495 blocked these effects of ZJ43 on the PAG and RVM. NAAG peptidase inhibition in the PAG and RVM did not affect the thermal withdrawal response in the hot plate test. Footpad inflammation also induced a significant increase in glutamate release in the PAG. Systemic injection of ZJ43 increased NAAG levels in the PAG and RVM and blocked the inflammation-induced increase in glutamate release in the PAG. Conclusion These data demonstrate a behavioral and neurochemical role for NAAG in the PAG and RVM in regulating the spinal motor response to inflammation and that NAAG peptidase inhibition has potential as an approach to treating inflammatory pain via either the ascending (PAG) and/or the descending pain pathways (PAG and RVM) that warrants further study. PMID:22971334

  12. The serine protease, dipeptidyl peptidase IV as a myokine: dietary protein and exercise mimetics as a stimulus for transcription and release.

    PubMed

    Neidert, Leslie E; Mobley, C Brooks; Kephart, Wesley C; Roberts, Michael D; Kluess, Heidi A

    2016-06-01

    Dipeptidyl-peptidase IV (DPP-IV) is an enzyme with numerous roles within the body, mostly related to regulating energy metabolism. DPP-IV is also a myokine, but the stimulus for its release is poorly understood. We investigated the transcription and release of DPP-IV from skeletal muscle in a three-part study using C2C12 myotube cultures, an acute rat exercise and postexercise feeding model, and human feeding or human exercise models. When myotubes were presented with leucine only, hydrolyzed whey protein, or chemicals that cause exercise-related signaling to occur in cell culture, all caused an increase in the mRNA expression of DPP-IV (1.63 to 18.56 fold change, P < 0.05), but only whey protein caused a significant increase in DPP-IV activity in the cell culture media. When rats were fed whey protein concentrate immediately following stimulated muscle contractions, DPP-IV mRNA in both the exercised and nonexercised gastrocnemius muscles significantly increased 2.5- to 3.7-fold (P < 0.05) 3-6 h following the exercise/feeding bout; of note exercise alone or postexercise leucine-only feeding had no significant effect. In humans, plasma and serum DPP-IV activities were not altered by the ingestion of whey protein up to 1 h post consumption, after a 10 min bout of vigorous running, or during the completion of three repeated lower body resistance exercise bouts. Our cell culture and rodent data suggest that whey protein increases DPP-IV mRNA expression and secretion from muscle cells. However, our human data suggest that DPP-IV is not elevated in the bloodstream following acute whey protein ingestion or exercise. PMID:27335432

  13. Inhibition of dipeptidyl aminopeptidase IV (DP-IV) by Xaa-boroPro dipeptides and use of these inhibitors to examine the role of DP-IV in T-cell function.

    PubMed

    Flentke, G R; Munoz, E; Huber, B T; Plaut, A G; Kettner, C A; Bachovchin, W W

    1991-02-15

    Dipeptidyl peptidase IV (DP-IV; dipeptidyl-peptide hydrolase, EC 3.4.14.5) is a serine protease with a specificity for cleaving Xaa-Pro dipeptides from polypeptides and proteins. It is found in a variety of mammalian cells and tissues, including those of lymphoid origin where it is found specifically on the surface of CD4+ T cells. Although the functional significance of this enzyme has not been established, a role in T-cell activation and immune regulation has been proposed. Here we report that Ala-boroPro and Pro-boroPro, where boroPro is the alpha-amino boronic acid analog of proline, are potent and specific inhibitors of DP-IV, having Ki values in the nanomolar range. Blocking the N terminus of Ala-boroPro abolishes the affinity of this inhibitor for DP-IV, while removal of the N-terminal residue, to give boroPro, reduces the affinity for DP-IV by 5 orders of magnitude. The dipeptide boronic acids exhibit slow-binding kinetics, while boroPro does not. We also report here that low concentrations of Pro-boroPro inhibit antigen-induced proliferation and interleukin 2 production in murine T-cell lines but do not inhibit the response of these T cells to the mitogen concanavalin A. These results indicate that DP-IV plays a role in antigen-induced, but not mitogen-induced, activation of T lymphocytes. PMID:1671716

  14. Inhibition of DD-Peptidases by a Specific Trifluoroketone: Crystal Structure of a Complex with the Actinomadura R39 DD-Peptidase†

    PubMed Central

    Dzhekieva, Liudmila; Adediran, S. A.; Herman, Raphael; Kerff, Frédéric; Duez, Colette; Charlier, Paulette; Sauvage, Eric; Pratt, R.F.

    2013-01-01

    Inhibitors of bacterial DD-peptidases represent potential antibiotics. In the search for alternatives to β-lactams, we have investigated a series of compounds designed to generate transition state analogue structures on reaction with DD-peptidases. The compounds contain a combination of a peptidoglycan-mimetic specificity handle and a warhead capable of delivering a tetrahedral anion to the enzyme active site. The latter include a boronic acid, two alcohols, an aldehyde and a trifluoroketone. The compounds were tested against two low molecular mass class C DD-peptidases. As expected from previous observations, the boronic acid was a potent inhibitor, but, rather unexpectedly from precedent, the trifluoroketone [D-α-aminopimelyl-(1,1,1-trifluoro-3-amino)butan-2-one] was also very effective. Taking into account competing hydration, the trifluoroketone was the strongest inhibitor of the Actinomadura R39 DD-peptidase, with a subnanomolar (free ketone) inhibition constant. A crystal structure of the complex between the trifluoroketone and the R39 enzyme showed that a tetrahedral adduct had indeed formed with the active site serine nucleophile. The trifluoroketone moiety, therefore, should be considered along with boronic acids and phosphonates, as a warhead that can be incorporated into new and effective DD-peptidase inhibitors and therefore, perhaps, antibiotics. PMID:23484909

  15. Dipeptidyl peptidase-4 inhibition by Pterocarpus marsupium and Eugenia jambolana ameliorates streptozotocin induced Alzheimer's disease.

    PubMed

    Kosaraju, Jayasankar; Madhunapantula, Subbarao V; Chinni, Santhivardhan; Khatwal, Rizwan Basha; Dubala, Anil; Muthureddy Nataraj, Satish Kumar; Basavan, Duraiswamy

    2014-07-01

    Alzheimer's disease (AD), the most common form of dementia, is characterized by the loss of normal functions of brain cells and neuronal death, ultimately leading to memory loss. Recent accumulating evidences have demonstrated the therapeutic potential of anti-diabetic agents, such as dipeptidyl peptidase-4 (DPP-4) inhibitors, for the treatment of Alzheimer's disease (AD), providing opportunities to explore and test the DPP-4 inhibitors for treating this fatal disease. Prior studies determining the efficacy of Pterocarpus marsupium (PM, Fabaceae) and Eugenia jambolana (EJ, Myrtaceae) extracts for ameliorating type 2 diabetes have demonstrated the DPP-4 inhibitory properties indicating the possibility of using of these extracts even for the treating AD. Therefore, in the present study, the neuroprotective roles of PM and EJ for ameliorating the streptozotocin (STZ) induced AD have been tested in rat model. Experimentally, PM and EJ extracts, at a dose range of 200 and 400mg/kg, were administered orally to STZ induced AD Wistar rats and cognitive evaluation tests were performed using radial arm maze and hole-board apparatus. Following 30 days of treatment with the extracts, a dose- and time-dependent attenuation of AD pathology, as evidenced by decreasing amyloid beta 42, total tau, phosphorylated tau and neuro-inflammation with an increase in glucagon-like peptide-1 (GLP-1) levels was observed. Therefore, PM and EJ extracts contain cognitive enhancers as well as neuroprotective agents against STZ induced AD. PMID:24667360

  16. Substrate complexes of human dipeptidyl peptidase III reveal the mechanism of enzyme inhibition

    PubMed Central

    Kumar, Prashant; Reithofer, Viktoria; Reisinger, Manuel; Wallner, Silvia; Pavkov-Keller, Tea; Macheroux, Peter; Gruber, Karl

    2016-01-01

    Human dipeptidyl-peptidase III (hDPP III) is a zinc-dependent hydrolase cleaving dipeptides off the N-termini of various bioactive peptides. Thus, the enzyme is likely involved in a number of physiological processes such as nociception and is also implicated in several forms of cancer. We present high-resolution crystal structures of hDPP III in complex with opioid peptides (Met-and Leu-enkephalin, endomorphin-2) as well as with angiotensin-II and the peptide inhibitor IVYPW. These structures confirm the previously reported large conformational change of the enzyme upon ligand binding and show that the structure of the closed conformation is independent of the nature of the bound peptide. The overall peptide-binding mode is also conserved ensuring the correct positioning of the scissile peptide bond with respect to the catalytic zinc ion. The structure of the angiotensin-II complex shows, how longer peptides are accommodated in the binding cleft of hDPP III. Differences in the binding modes allow a distinction between real substrates and inhibitory peptides or “slow” substrates. The latter displace a zinc bound water molecule necessitating the energetically much less favoured anhydride mechanism as opposed to the favoured promoted-water mechanism. The structural data also form the necessary framework for the design of specific hDPP III inhibitors. PMID:27025154

  17. Origins of Yersinia pestis Sensitivity to the Arylomycin Antibiotics and the Inhibition of Type I Signal Peptidase

    PubMed Central

    Steed, Danielle B.; Liu, Jian; Wasbrough, Elizabeth; Miller, Lynda; Halasohoris, Stephanie; Miller, Jeremy; Somerville, Brandon; Hershfield, Jeremy R.

    2015-01-01

    Yersinia pestis is the etiologic agent of the plague. Reports of Y. pestis strains that are resistant to each of the currently approved first-line and prophylactic treatments point to the urgent need to develop novel antibiotics with activity against the pathogen. We previously reported that Y. pestis strain KIM6+, unlike most Enterobacteriaceae, is susceptible to the arylomycins, a novel class of natural-product lipopeptide antibiotics that inhibit signal peptidase I (SPase). In this study, we show that the arylomycin activity is conserved against a broad range of Y. pestis strains and confirm that it results from the inhibition of SPase. We next investigated the origins of this unique arylomycin sensitivity and found that it does not result from an increased affinity of the Y. pestis SPase for the antibiotic and that alterations to each component of the Y. pestis lipopolysaccharide—O antigen, core, and lipid A—make at most only a small contribution. Instead, the origins of the sensitivity can be traced to an increased dependence on SPase activity that results from high levels of protein secretion under physiological conditions. These results highlight the potential of targeting protein secretion in cases where there is a heavy reliance on this process and also have implications for the development of the arylomycins as an antibiotic with activity against Y. pestis and potentially other Gram-negative pathogens. PMID:25896690

  18. Kallistatin ameliorates influenza virus pathogenesis by inhibition of kallikrein-related peptidase 1-mediated cleavage of viral hemagglutinin.

    PubMed

    Leu, Chia-Hsing; Yang, Mei-Lin; Chung, Nai-Hui; Huang, Yen-Jang; Su, Yu-Chu; Chen, Yi-Cheng; Lin, Chia-Cheng; Shieh, Gia-Shing; Chang, Meng-Ya; Wang, Shainn-Wei; Chang, Yao; Chao, Julie; Chao, Lee; Wu, Chao-Liang; Shiau, Ai-Li

    2015-09-01

    Proteolytic cleavage of the hemagglutinin (HA) of influenza virus by host trypsin-like proteases is required for viral infectivity. Some serine proteases are capable of cleaving influenza virus HA, whereas some serine protease inhibitors (serpins) inhibit the HA cleavage in various cell types. Kallikrein-related peptidase 1 (KLK1, also known as tissue kallikrein) is a widely distributed serine protease. Kallistatin, a serpin synthesized mainly in the liver and rapidly secreted into the circulation, forms complexes with KLK1 and inhibits its activity. Here, we investigated the roles of KLK1 and kallistatin in influenza virus infection. We show that the levels of KLK1 increased, whereas those of kallistatin decreased, in the lungs of mice during influenza virus infection. KLK1 cleaved H1, H2, and H3 HA molecules and consequently enhanced viral production. In contrast, kallistatin inhibited KLK1-mediated HA cleavage and reduced viral production. Cells transduced with the kallistatin gene secreted kallistatin extracellularly, which rendered them more resistant to influenza virus infection. Furthermore, lentivirus-mediated kallistatin gene delivery protected mice against lethal influenza virus challenge by reducing the viral load, inflammation, and injury in the lung. Taking the data together, we determined that KLK1 and kallistatin contribute to the pathogenesis of influenza virus by affecting the cleavage of the HA peptide and inflammatory responses. This study provides a proof of principle for the potential therapeutic application of kallistatin or other KLK1 inhibitors for influenza. Since proteolytic activation also enhances the infectivity of some other viruses, kallistatin and other kallikrein inhibitors may be explored as antiviral agents against these viruses. PMID:26149981

  19. miR-34b inhibits nasopharyngeal carcinoma cell proliferation by targeting ubiquitin-specific peptidase 22

    PubMed Central

    Xiao, Jianyong; Li, Yingying; Zhang, Wenyin; Jiang, Yanni; Du, Biaoyan; Tan, Yuhui

    2016-01-01

    Objectives This study aimed to investigate the precise role of miR-34b in nasopharyngeal carcinoma (NPC). Materials and methods: The expression of miR-34b and transcription of ubiquitin-specific peptidase 22 (USP22) were examined using quantitative reverse transcription-polymerase chain reaction. Western blot analysis was used to measure the protein expression of USP22. A dualluciferase assay was used to investigate the interaction between miR-34b and USP22. Cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. The cell cycle was analyzed by propidium iodide staining followed by flow cytometry analysis. Results miR-34b was significantly downregulated in NPC tissues and NPC cell lines. Overexpression of miR-34b in NPC SUNE-6-10B cells inhibited cell viability and proliferation. USP22 was highly expressed in NPC cells and promoted cell viability and proliferation. Restoration of USP22 expression could reverse the effect of miR-34b on NPC cell viability and proliferation. Conclusion miR-34b acts as a tumor suppressor in NPC, which is mediated via repression of the oncogene USP22. PMID:27051294

  20. Dipeptidyl peptidase IV (DP IV) activity in serum and on lymphocytes of MRL/Mp-lpr/lpr mice correlates with disease onset.

    PubMed

    Kubota, T; Iizuka, H; Bachovchin, W W; Stollar, B D

    1994-05-01

    DP IV (CD26), a serine protease expressed on activated T cells, participates in immune responses in vivo as well as in vitro. We measured cell surface and serum DP IV in mice of the autoimmune MRL/Mp-lpr/lpr (MRL/l) strain, which is characterized by massive T cell proliferation and production of anti-nuclear autoantibodies. The mass of inguinal lymph nodes correlated with serum DP IV activity. Furthermore, serum DP IV activity increased markedly in parallel with the acceleration of lymph node swelling and anti-nDNA antibody production. Serum DP IV activity in 16-week-old MRL/l mice reached levels up to three higher than those in age-matched MRL/Mp- +/+ mice or BALB/c mice. Immunohistochemical staining and flow cytometric analysis identified DV IV on surfaces of lymphocytes from the enlarged lymph nodes of MRL/l mice. Subcutaneous injection of the mechanism-based inhibitor, Pro-boroPro, reduced protease activity in serum and cell suspensions prepared from spleen and lymph nodes, confirming the identity of the enzyme as DP IV. These results indicate that the massively accumulating lymphocytes of MRL/l mice have a property characteristic of activated T cells, although they express little surface CD4 or CD8 and do not produce IL-2. DP IV may participate in the role these cells play in the pathogenesis of MRL/l autoimmune disease. PMID:7910536

  1. Dipeptidyl Peptidase IV Inhibitory Peptides Derived from Oat (Avena sativa L.), Buckwheat (Fagopyrum esculentum), and Highland Barley (Hordeum vulgare trifurcatum (L.) Trofim) Proteins.

    PubMed

    Wang, Feng; Yu, Guoyong; Zhang, Yanyan; Zhang, Bolin; Fan, Junfeng

    2015-11-01

    Peptides released from oat, buckwheat, and highland barley proteins were examined for their in vitro inhibitory effects on dipeptidyl peptidase IV (DPP4), an enzyme that deactivates incretin hormones involved in insulin secretion. All of the hydrolysates exhibited DPP4 inhibitory activities, with IC50 values ranging from 0.13 mg/mL (oat glutelin alcalase digestion) to 8.15 mg/mL (highland barley albumin tryptic digestion). The lowest IC50 values in gastrointestinal, alcalase, and tryptic digestions were 0.99 mg/mL (oat flour), 0.13 mg/mL (oat glutelin), and 1.83 mg/mL (highland barley glutelin). In all, 35 peptides of more than seven residues were identified in the tryptic hydrolysates of oat globulin using liquid chromatography-mass spectroscopy. Peptides LQAFEPLR and EFLLAGNNK were synthesized and their DPP4 inhibitory activities determined. LQAFEPLR showed high in vitro DPP4 inhibitory activity with an IC50 value of 103.5 μM. PMID:26468909

  2. Vildagliptin, a novel dipeptidyl peptidase IV inhibitor, has no pharmacokinetic interactions with the antihypertensive agents amlodipine, valsartan, and ramipril in healthy subjects.

    PubMed

    He, Yan-Ling; Ligueros-Saylan, Monica; Sunkara, Gangadhar; Sabo, Ron; Zhao, Charlie; Wang, Yibin; Campestrini, Joelle; Pommier, Francoise; Dole, Kiran; Marion, Alan; Dole, William P; Howard, Dan

    2008-01-01

    We conducted 3 open-label, multiple-dose, 3-period, randomized, crossover studies in healthy subjects to assess the potential pharmacokinetic interaction between vildagliptin, a novel dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes, and representatives of 3 commonly prescribed antihypertensive drug classes: (1) the calcium channel blocker, amlodipine; (2) the angiotensin receptor blocker, valsartan; and (3) the angiotensin-converting enzyme inhibitor, ramipril. Coadministration of vildagliptin 100 mg with amlodipine 5 mg, valsartan 320 mg, or ramipril 5 mg had no clinically significant effect on the pharmacokinetics of these drugs. The 90% confidence intervals of the geometric mean ratios for area under the plasma concentration-time curve from time zero to 24 hours (AUC0-24h) and maximum plasma concentration (Cmax) for vildagliptin, amlodipine, and ramipril (and its active metabolite, ramiprilat) were contained within the acceptance range for bioequivalence (0.80-1.25). Valsartan AUC0-24h and Cmax increased by 24% and 14%, respectively, following coadministration of vildagliptin, but this was not considered clinically significant. Vildagliptin was generally well tolerated when given alone or in combination with amlodipine, valsartan, or ramipril in healthy subjects at steady state. No adjustment in dosage based on pharmacokinetic considerations is required should vildagliptin be coadministered with amlodipine, valsartan, or ramipril in patients with type 2 diabetes and hypertension. PMID:17986525

  3. Dipeptidyl peptidase-4 inhibition by Saxagliptin prevents inflammation and renal injury by targeting the Nlrp3/ASC inflammasome

    PubMed Central

    Birnbaum, Yochai; Bajaj, Mandeep; Qian, Jinqiao; Ye, Yumei

    2016-01-01

    Background Glucagon-like peptide-1 (GLP-1) receptor activation delays the progression of diabetic nephropathy (DN) in rodents. The NOD-like receptor 3 (Nlrp3) inflammasome plays an important role in DN. Dipeptidyl peptidase-4 inhibitors (DPP4I) inhibit the degradation of endogenous GLP-1 and various other active substances. We assessed whether DPP4I attenuates diabetes-induced activation of the inflammasome and progression of DN in mice with type 2 diabetes mellitus (T2DM) and type 1 diabetes mellitus (T1DM). Methods BTBR (T2DM), Akita (T1DM) and their matched non-diabetic control (wild-type (WT)) mice received 8-week treatment with Saxagliptin (Saxa) or vehicle. Results Kidney weight and kidney/body weight ratio increased in the BTBR and Akita mice compared to their WT mice. Saxa attenuated these changes in the BTBR, but not in the Akita mice and had no effect in the WT mice. Serum blood urea nitrogen and creatinine significantly increased in the BTBR and Akita mice. Saxa attenuated the increase in the BTBR and Akita mice. Saxa improved glycemic control in the BTBR mice, but had no effect on glucose levels in the Akita and WT mice. Serum C reactive protein, tumor necrosis factor α (TNFα), interleukin (IL)-1β, IL-6 and IL-18 were significantly higher in the BTBR and Akita mice than in the WT mice. Saxa attenuated the increase in the BTBR and Akita mice. Kidney and adipose protein levels of apoptosis-associated speck-like protein 1, NLRP3, TNFα and Caspase-1 were higher in the BTBR and Akita mice than in the WT mice. Saxa reduced the levels in both types of diabetic mice. Conclusions Saxa attenuated diabetes-induced activation of the inflammasome and progression of DN. As Saxa did not affect glucose levels in the Akita mice, these effects are independent of glucose lowering. PMID:27547413

  4. Genetic Deletion or Pharmacological Inhibition of Dipeptidyl Peptidase-4 Improves Cardiovascular Outcomes After Myocardial Infarction in Mice

    PubMed Central

    Sauvé, Meghan; Ban, Kiwon; Momen, M. Abdul; Zhou, Yu-Qing; Henkelman, R. Mark; Husain, Mansoor; Drucker, Daniel J.

    2010-01-01

    OBJECTIVE Glucagon-like peptide-1 (7-36)amide (GLP-1) is cleaved by dipeptidyl peptidase-4 (DPP-4) to GLP-1 (9-36)amide. We examined whether chemical inhibition or genetic elimination of DPP-4 activity affects cardiovascular function in normoglycemic and diabetic mice after experimental myocardial infarction. RESEARCH DESIGN AND METHODS Cardiac structure and function was assessed by hemodynamic monitoring and echocardiography in DPP-4 knockout (Dpp4−/−) mice versus wild-type (Dpp4+/+) littermate controls and after left anterior descending (LAD) coronary artery ligation–induced myocardial infarction (MI). Effects of sustained DPP-4 inhibition with sitagliptin versus treatment with metformin were ascertained after experimental MI in a high-fat diet–streptozotocin model of murine diabetes. Functional recovery from ischemia-reperfusion (I/R) injury was measured in isolated hearts from Dpp4−/− versus Dpp4+/+ littermates and from normoglycemic wild-type (WT) mice treated with sitagliptin or metformin. Cardioprotective signaling in the murine heart was examined by RT-PCR and Western blot analyses. RESULTS Dpp4−/− mice exhibited normal indexes of cardiac structure and function. Survival post-MI was modestly improved in normoglycemic Dpp4−/− mice. Increased cardiac expression of phosphorylated AKT (pAKT), pGSK3β, and atrial natriuretic peptide (ANP) was detected in the nonischemic Dpp4−/− heart, and HO-1, ANP, and pGSK3β proteins were induced in nonischemic hearts from diabetic mice treated with sitagliptin or metformin. Sitagliptin and metformin treatment of wild-type diabetic mice reduced mortality after myocardial infarction. Sitagliptin improved functional recovery after I/R injury ex vivo in WT mice with similar protection from I/R injury also manifest in hearts from Dpp4−/− versus Dpp4+/+ mice. CONCLUSIONS Genetic disruption or chemical inhibition of DPP-4 does not impair cardiovascular function in the normoglycemic or diabetic mouse

  5. Recent Advances in Dipeptidyl-Peptidase-4 Inhibition Therapy: Lessons from the Bench and Clinical Trials

    PubMed Central

    Zhong, Jixin; Gong, Quan; Goud, Aditya; Srinivasamaharaj, Srividya; Rajagopalan, Sanjay

    2015-01-01

    DPP4 inhibitors (DPP4i) are a class of newly developed antidiabetic drugs which preserve incretin hormones and promote postprandial insulin secretion. Although the cardiovascular effect of DPP4 inhibition has been substantially studied, the exact role of DPP4 in cardiovascular disease especially in humans remains elusive. Previous small studies and meta-analyses have suggested a benefit in both surrogate outcomes and cardiovascular events for these agents. However, there was growing evidence in recent years questioning the cardioprotective effect of DPP4i. Further, a signal of heart failure hospitalization in a recent large scale clinical trial SAVOR-TIMI 53 has called into question the safety of these agents and their utility in the treatment of cardiovascular disease. In this review, we will revisit the physiologic function of DPP4 and discuss its role in cardiometabolic disease based on recent experimental and clinical studies. PMID:26075284

  6. Remediation of undesirable secondary interactions encountered in hydrophilic interaction chromatography during development of a quantitative LC-MS/MS assay for a dipeptidyl peptidase IV (DPP-IV) inhibitor in monkey serum.

    PubMed

    Kadar, Eugene P; Wujcik, Chad E

    2009-02-15

    PF-00734200 (3,3-Difluoropyrrolidin-1-yl)-((2S,4S)-4-(4-(pyrimidin-2-yl) piperazin-1-yl)pyrrolidin-2-yl)methanone) is an inhibitor of dipeptidyl peptidase IV (DPP-IV) for the treatment of diabetic complications and other disorders. A sensitive and selective LC-MS/MS assay capable of quantifying PF-00734200 in monkey serum was required to support regulated safety studies. Due to the polar nature of this compound and for ease of sample processing, hydrophilic interaction chromatography (HILIC) was identified as an ideal assay technique. During the initial phase of method development significant peak tailing was observed. The effects of polar organic modifier percentage, buffer concentration, column particle size, and flow rate were assessed to determine the final optimal conditions. PF-00734200 demonstrated a strong dependence on buffer concentration with respect to height equivalent to a theoretical plate (HETP), capacity factor (k'), and tailing factor (T). Improvements in chromatography were observed with increasing buffer concentration due to reduction of electrostatic secondary interactions with ionized silanols. A plot of logk' versus percentage organic modifier at an elevated buffer concentration, produced a linear fit with a correlation coefficient of 0.996, indicating that the primary chromatographic retention mechanism was partitioning. A LC-MS/MS assay was successfully developed and validated for GLP bioanalysis of PF-00734200 in monkey serum utilizing the optimized HILIC conditions. Additionally, carryover was effectively minimized through fortification of ethylene glycol to the sample extract. PMID:19162567

  7. Design, synthesis and biological evaluation of 4-fluoropyrrolidine-2-carbonitrile and octahydrocyclopenta[b]pyrrole-2-carbonitrile derivatives as dipeptidyl peptidase IV inhibitors.

    PubMed

    Ji, Xun; Xia, Chunmei; Wang, Jiang; Su, Mingbo; Zhang, Lei; Dong, Tiancheng; Li, Zeng; Wan, Xia; Li, Jingya; Li, Jia; Zhao, Linxiang; Gao, Zhaobing; Jiang, Hualiang; Liu, Hong

    2014-10-30

    Based on the previous work in our group and the principle of computer-aided drug design, a series of novel β-amino pyrrole-2-carbonitrile derivatives was designed and synthesized. Compounds 8l and 9l were efficacious and selective DPP4 inhibitors resulting in decreased blood glucose in vivo. Compound 8l had moderate DPP4 inhibitory activity (IC50 = 0.05 μM) and good oral bioavailability (F = 53.2%). Compound 9l showed excellent DPP4 inhibitory activity (IC50 = 0.01 μM), good selectivity (selective ratio: DPP8/DPP4 = 898.00; DPP9/DPP4 = 566.00) against related peptidases, and good efficacy in an oral glucose tolerance tests in ICR mice and moderate PK profiles (F = 22.8%, t1/2 = 2.74 h). Moreover, compound 9l did not block hERG channel and exhibited no inhibition of liver metabolic enzymes such as CYP2C9. PMID:25164763

  8. Expression of trophinin and dipeptidyl peptidase IV in endometrial co-culture in the presence of an embryo: A comparative immunocytochemical study.

    PubMed

    Dolanbay, Elif Gelenli; Yardimoglu, Melda; Yalcinkaya, Ender; Yazir, Yusufhan; Aksoy, Ayca; Karaoz, Erdal; Caliskan, Eray

    2016-05-01

    Recurrent implantation failure leads to a reduced pregnancy rate. The expression patterns of trophinin and dipeptidyl peptidase IV (CD26) indicate the involvement of embryo implantation and early placental development. The purpose of the present study was to evaluate endometrial co‑culture cells in the presence of embryo with trophinin and CD26 immunofluorescence staining. Patients with recurrent implantation failure were enrolled in the present study. The patients were aged between 26 and 36 years. Co‑cultures were prepared from endometrial biopsies for each patient. Controlled ovarian hyperstimulation was performed on each of the patients. Certain embryos were maintained in a conventional culture environment (n=80), and others in an endometrial co‑culture environment (n=25). Following embryo transfer, the co‑culture cells were examined under an inverted wide‑field fluorescence microscope. The ratio of a successful pregnancy was 0.38 in the present study (n=5/13 pregnancies). The average age of the successful group (28±3.54 years) was younger compared with the unsuccessful (32.67±2.81) group (P≤0.05). The number of trophinin (+) endometrial cells in the presence of an embryo was significantly lower (P=0.046) in the successful group on the first day. No significant difference between the groups was observed in terms of the number of CD26 (+) cells on the first to the fourth days (P≤0.05). Trophinin and CD26 immunostaining is important in the early period of pregnancy, and it will be beneficial in terms of providing the deficit of conventional culture medium in performed studies with the endometrial co‑culture medium. The co‑culture may be important, particularly in the early period, in patients with recurrent implantation failure in terms of enabling a connection between the cells belonging to the endometrium and the embryo. PMID:27035766

  9. Effect of a Dipeptidyl Peptidase-IV Inhibitor, Des-Fluoro-Sitagliptin, on Neointimal Formation after Balloon Injury in Rats

    PubMed Central

    Lim, Soo; Choi, Sung Hee; Shin, Hayley; Cho, Bong Jun; Park, Ho Seon; Ahn, Byung Yong; Kang, Seon Mee; Yoon, Ji Won; Jang, Hak Chul; Kim, Young-Bum; Park, Kyong Soo

    2012-01-01

    Background Recently, it has been suggested that enhancement of incretin effect improves cardiac function. We investigated the effect of a DPP-IV inhibitor, des-fluoro-sitagliptin, in reducing occurrence of restenosis in carotid artery in response to balloon injury and the related mechanisms. Methods and Findings Otsuka Long-Evans Tokushima Fatty rats were grouped into four: control (normal saline) and sitagliptin 100, 250 and 500 mg/kg per day (n = 10 per group). Sitagliptin or normal saline were given orally from 1 week before to 2 weeks after carotid injury. After 3 weeks of treatment, sitagliptin treatment caused a significant and dose-dependent reduction in intima-media ratio (IMR) in obese diabetic rats. This effect was accompanied by improved glucose homeostasis, decreased circulating levels of high-sensitivity C-reactive protein (hsCRP) and increased adiponectin level. Moreover, decreased IMR was correlated significantly with reduced hsCRP, tumor necrosis factor-α and monocyte chemoattractant protein-1 levels and plasminogen activator inhibitor-1 activity. In vitro evidence with vascular smooth muscle cells (VSMCs) demonstrated that proliferation and migration were decreased significantly after sitagliptin treatment. In addition, sitagliptin increased caspase-3 activity and decreased monocyte adhesion and NFκB activation in VSMCs. Conclusions Sitagliptin has protective properties against restenosis after carotid injury and therapeutic implications for treating macrovascular complications of diabetes. PMID:22493727

  10. Comparison of cysteine peptidase activities in Trichobilharzia regenti and Schistosoma mansoni cercariae.

    PubMed

    Kasný, M; Mikes, L; Dalton, J P; Mountford, A P; Horák, P

    2007-10-01

    Cercariae of the bird schistosome Trichobilharzia regenti and of the human schistosome Schistosoma mansoni employ proteases to invade the skin of their definitive hosts. To investigate whether a similar proteolytic mechanism is used by both species, cercarial extracts of T. regenti and S. mansoni were biochemically characterized, with the primary focus on cysteine peptidases. A similar pattern of cysteine peptidase activities was detected by zymography of cercarial extracts and their chromatographic fractions from T. regenti and S. mansoni. The greatest peptidase activity was recorded in both species against the fluorogenic peptide substrate Z-Phe-Arg-AMC, commonly used to detect cathepsins B and L, and was markedly inhibited (> 96%) by Z-Phe-Ala-CHN2 at pH 4.5. Cysteine peptidases of 33 kDa and 33-34 kDa were identified in extracts of T. regenti and S. mansoni cercariae employing a biotinylated Clan CA cysteine peptidase-specific inhibitor (DCG-04). Finally, cercarial extracts from both T. regenti and S. mansoni were able to degrade native substrates present in skin (collagen II and IV, keratin) at physiological pH suggesting that cysteine peptidases are important in the pentration of host skin. PMID:17517170

  11. Kinetics and sequence specificity of processing of prepilin by PilD, the type IV leader peptidase of Pseudomonas aeruginosa.

    PubMed Central

    Strom, M S; Lory, S

    1992-01-01

    PilD, originally isolated as an essential component for the biogenesis of the type IV pili of Pseudomonas aeruginosa, is a unique endopeptidase responsible for processing the precursors of the P. aeruginosa pilin subunits. It is also required for the cleavage of the leader peptides from the Pdd proteins, which are essential components of an extracellular secretion pathway specific for the export of a number of P. aeruginosa hydrolytic enzymes and toxins. Substrates for PilD are initially synthesized with short, i.e., 6- to 8-amino-acid-long, leader peptides with a net basic charge and share a high degree of amino acid homology through the first 16 to 30 residues at the amino terminus. In addition, they all have a phenylalanine residue at the +1 site relative to the cleavage site, which is N methylated prior to assembly into the oligomeric structures. In this study, the kinetics of leader peptide cleavage from the precursor of the P. aeruginosa pilin subunit by PilD was determined in vitro. The rates of cleavage were compared for purified enzyme and substrate as well as for enzyme and substrate contained within total membranes extracted from P. aeruginosa strains overexpressing the cloned pilD or pilA genes. Optimal conditions were obtained only when both PilD and substrate were contained within total membranes. PilD catalysis of P. aeruginosa prepilin followed normal Michaelis-Menten kinetics, with a measured apparent Km of approximately 650 microM, and a kcat of 180 min-1. The kinetics of PilD processing of another type IV pilin precursor, that from Neisseria gonorrhoeae with a 7-amino-acid-long leader peptide, were essentially the same as that measured for wild-type P. aeruginosa prepilin. Quite different results were obtained for a number of prepilin substrates containing substitutions at the conserved phenylalanine at the +1 position relative to the cleavage site, which were previously shown to be well tolerated in vivo. Substitutions of methionine, serine, and

  12. Therapeutic effects of the dipeptidyl peptidase-IV inhibitor, sitagliptin, on non-alcoholic steatohepatitis in FLS-ob/ob male mice.

    PubMed

    Onoyama, Takumi; Koda, Masahiko; Okamoto, Toshiaki; Kishina, Manabu; Matono, Tomomitsu; Sugihara, Takaaki; Murawaki, Yoshikazu

    2015-11-01

    Non-alcoholic steatohepatitis is characterized by hepatic fat accumulation, inflammation and varying degrees of fibrosis. The dipeptidyl peptidase‑IV enzyme is important in glucose metabolism, as well as lipid accumulation, extracellular matrix metabolism and immune stimulation. Furthermore, the enzyme activity of dipeptidyl peptidase‑IV is known to be increased in non‑alcoholic steatohepatitis. Therefore, dipeptidyl peptidase‑IV inhibitors are potential therapeutic agents for non‑alcoholic steatohepatitis. The present study assessed the therapeutic effects of sitagliptin, a dipeptidyl peptidase‑IV inhibitor, on non‑alcoholic steatohepatitis using fatty liver Shionogi‑ob/ob male mice. Sitagliptin (2 mg/kg/day; n=10) or placebo (control; n=10) was orally administered to fatty liver Shionogi‑ob/ob mice for 12 weeks, and hepatic steatosis, fibrosis, inflammation and oxidative stress were assessed in comparison with the controls. Sitagliptin administration reduced body weight and blood glucose levels, and improved hepatic fibrosis. It also inhibited the gene expression levels of fatty acid synthase, transforming growth factor‑β1, tissue inhibitor of metalloproteinases‑1, procollagen‑type 1, tumor necrosis factor‑α, monocyte chemoattractant protein‑1 and enhanced peroxisome proliferator activated receptor‑α. Furthermore, a marked attenuation of hepatic stellate cell activation and Kupffer cells was observed in the sitagliptin group. A decrease in oxidative stress and apoptosis was also observed. Sitagliptin attenuated the progression of hepatic fibrosis by improving lipid metabolism, inflammation and oxidative stress in non-alcoholic steatohepatitis. PMID:26397061

  13. Serum Levels of Soluble CD26/Dipeptidyl Peptidase-IV in Type 2 Diabetes Mellitus and Its Association with Metabolic Syndrome and Therapy with Antidiabetic Agents in Malaysian Subjects

    PubMed Central

    Ahmed, Radwan H.; Huri, Hasniza Zaman; Al-Hamodi, Zaid; Salem, Sameer D.; Muniandy, Sekaran

    2015-01-01

    Background A soluble form of CD26/dipeptidyl peptidase-IV (sCD26/DPP-IV) induces DPP-IV enzymatic activity that degrades incretin. We investigated fasting serum levels of sCD26/DPP-IV and active glucagon-like peptide-1 (GLP-1) in Malaysian patients with type 2 diabetes mellitus (T2DM) with and without metabolic syndrome (MetS), as well as the associations between sCD26/DPP-IV levels, MetS, and antidiabetic therapy. Methods We assessed sCD26/DPP-IV levels, active GLP-1 levels, body mass index (BMI), glucose, insulin, A1c, glucose homeostasis indices, and lipid profiles in 549 Malaysian subjects (including 257 T2DM patients with MetS, 57 T2DM patients without MetS, 71 non-diabetics with MetS, and 164 control subjects without diabetes or metabolic syndrome). Results Fasting serum levels of sCD26/DPP-IV were significantly higher in T2DM patients with and without MetS than in normal subjects. Likewise, sCD26/DPP-IV levels were significantly higher in patients with T2DM and MetS than in non-diabetic patients with MetS. However, active GLP-1 levels were significantly lower in T2DM patients both with and without MetS than in normal subjects. In T2DM subjects, sCD26/DPP-IV levels were associated with significantly higher A1c levels, but were significantly lower in patients using monotherapy with metformin. In addition, no significant differences in sCD26/DPP-IV levels were found between diabetic subjects with and without MetS. Furthermore, sCD26/DPP-IV levels were negatively correlated with active GLP-1 levels in T2DM patients both with and without MetS. In normal subjects, sCD26/DPP-IV levels were associated with increased BMI, cholesterol, and LDL-cholesterol (LDL-c) levels. Conclusion Serum sCD26/DPP-IV levels increased in T2DM subjects with and without MetS. Active GLP-1 levels decreased in T2DM patients both with and without MetS. In addition, sCD26/DPP-IV levels were associated with Alc levels and negatively correlated with active GLP-1 levels. Moreover, metformin

  14. Teduglutide (ALX-0600), a dipeptidyl peptidase IV resistant glucagon-like peptide 2 analogue, improves intestinal function in short bowel syndrome patients

    PubMed Central

    Jeppesen, P B; Sanguinetti, E L; Buchman, A; Howard, L; Scolapio, J S; Ziegler, T R; Gregory, J; Tappenden, K A; Holst, J; Mortensen, P B

    2005-01-01

    Background and aims: Glucagon-like peptide 2 (GLP-2) may improve intestinal absorption in short bowel syndrome (SBS) patients with an end jejunostomy. Teduglutide (ALX-0600), a dipeptidyl peptidase IV resistant GLP-2 analogue, prolongs the intestinotrophic properties of GLP-2 in animal models. The safety and effect of teduglutide were investigated in SBS patients with and without a colon in continuity. Methods: Teduglutide was given subcutaneously for 21 days once or twice daily to 16 SBS patients in the per protocol investigational group, 10 with end jejunostomy (doses of 0.03 (n = 2), 0.10 (n = 5), or 0.15 (n = 3) mg/kg/day), one with <50% colon in continuity (dose 0.03 mg/kg/day), and five with ≥50% colon in continuity (dose 0.10 mg/kg/day). Nutrient balance studies, D-xylose tests, and intestinal mucosa biopsies were performed at baseline, on the last three days of treatment, and after three weeks of follow up. Pre-study fasting native GLP-2 levels were determined for the five patients with ≥50% colon in continuity. Results: Pooled across groups and compared with baseline, teduglutide increased absolute (+743 (477) g/day; p<0.001) and relative (+22 (16)%; p<0.001) wet weight absorption, urine weight (+555 (485) g/day; p<0.001), and urine sodium excretion (+53 (40) mmol/day; p<0.001). Teduglutide decreased faecal wet weight (−711 (734) g/day; p = 0.001) and faecal energy excretion (−808 (1453) kJ/day (−193 (347) kcal/day); p = 0.040). In SBS patients with end jejunostomy, teduglutide significantly increased villus height (+38 (45)%; p = 0.030), crypt depth (+22 (18)%; p = 0.010), and mitotic index (+115 (108)%; p = 0.010). Crypt depth and mitotic index did not change in colonic biopsies from SBS patients with colon in continuity. The most common side effects were enlargement of the stoma nipple and mild lower leg oedema. The improvements in intestinal absorption and decreases in faecal excretion noted after treatment had

  15. Peptidases and peptidase inhibitors in gut of caterpillars and in the latex of their host plants.

    PubMed

    Ramos, Márcio V; Pereira, Danielle A; Souza, Diego P; Silva, Maria-Lídia S; Alencar, Luciana M R; Sousa, Jeanlex S; Queiroz, Juliany-Fátima N; Freitas, Cleverson D T

    2015-01-01

    Studies investigating the resistance-susceptibility of crop insects to proteins found in latex fluids have been reported. However, latex-bearing plants also host insects. In this study, the gut proteolytic system of Pseudosphinx tetrio, which feeds on Plumeria rubra leaves, was characterized and further challenged against the latex proteolytic system of its own host plant and those of other latex-bearing plants. The gut proteolytic system of Danaus plexippus (monarch) and the latex proteolytic system of its host plant (Calotropis procera) were also studied. The latex proteins underwent extensive hydrolysis when mixed with the corresponding gut homogenates of the hosted insects. The gut homogenates partially digested the latex proteins of foreign plants. The fifth instar of D. plexippus that were fed diets containing foreign latex developed as well as those individuals who were fed diets containing latex proteins from their host plant. In vitro assays detected serine and cysteine peptidase inhibitors in both the gut homogenates and the latex fluids. Curiously, the peptidase inhibitors of caterpillars did not inhibit the latex peptidases of their host plants. However, the peptidase inhibitors of laticifer origin inhibited the proteolysis of gut homogenates. In vivo analyses of the peritrophic membrane proteins of D. plexippus demonstrate resistance against latex peptidases. Only discrete changes were observed when the peritrophic membrane was directly treated with purified latex peptidases in vitro. This study concludes that peptidase inhibitors are involved in the defensive systems of both caterpillars and their host plants. Although latex peptidase inhibitors inhibit gut peptidases (in vitro), the ability of gut peptidases to digest latex proteins (in vivo) regardless of their origin seems to be important in governing the resistance-susceptibility of caterpillars. PMID:25246317

  16. Effect of dipeptidyl peptidase-4 inhibition on circadian blood pressure during the development of salt-dependent hypertension in rats.

    PubMed

    Sufiun, Abu; Rafiq, Kazi; Fujisawa, Yoshihide; Rahman, Asadur; Mori, Hirohito; Nakano, Daisuke; Kobori, Hiroyuki; Ohmori, Koji; Masaki, Tsutomu; Kohno, Masakazu; Nishiyama, Akira

    2015-04-01

    A growing body of evidence has indicated that dipeptidyl peptidase-4 (DPP-4) inhibitors have antihypertensive effects. Here, we aim to examine the effect of vildagliptin, a DPP-4-specific inhibitor, on blood pressure and its circadian-dipping pattern during the development of salt-dependent hypertension in Dahl salt-sensitive (DSS) rats. DSS rats were treated with a high-salt diet (8% NaCl) plus vehicle or vildagliptin (3 or 10 mg kg(-1) twice daily by oral gavage) for 7 days. Blood pressure was measured by the telemetry system. High-salt diet for 7 days significantly increased the mean arterial pressure (MAP), systolic blood pressure (SBP) and were also associated with an extreme dipping pattern of blood pressure in DSS rats. Treatment with vildagliptin dose-dependently decreased plasma DPP-4 activity, increased plasma glucagon-like peptide 1 (GLP-1) levels and attenuated the development of salt-induced hypertension. Furthermore, vildagliptin significantly increased urine sodium excretion and normalized the dipping pattern of blood pressure. In contrast, intracerebroventricular infusion of vildagliptin (50, 500 or 2500 μg) did not alter MAP and heart rate in DSS rats. These data suggest that salt-dependent hypertension initially develops with an extreme blood pressure dipping pattern. The DPP-4 inhibitor, vildagliptin, may elicit beneficial antihypertensive effects, including the improvement of abnormal circadian blood pressure pattern, by enhancing urinary sodium excretion. PMID:25588850

  17. Effect of dipeptidyl peptidase-4 inhibition on circadian blood pressure during the development of salt-dependent hypertension in rats

    PubMed Central

    Sufiun, Abu; Rafiq, Kazi; Fujisawa, Yoshihide; Rahman, Asadur; Mori, Hirohito; Nakano, Daisuke; Kobori, Hiroyuki; Ohmori, Koji; Masaki, Tsutomu; Kohno, Masakazu; Nishiyama, Akira

    2015-01-01

    A growing body of evidence has indicated that dipeptidyl peptidase-4 (DPP-4) inhibitors have antihypertensive effects. Here, we aim to examine the effect of vildagliptin, a DPP-4-specific inhibitor, on blood pressure and its circadian-dipping pattern during the development of salt-dependent hypertension in Dahl salt-sensitive (DSS) rats. DSS rats were treated with a high-salt diet (8% NaCl) plus vehicle or vildagliptin (3 or 10 mg kg−1 twice daily by oral gavage) for 7 days. Blood pressure was measured by the telemetry system. High-salt diet for 7 days significantly increased the mean arterial pressure (MAP), systolic blood pressure (SBP) and were also associated with an extreme dipping pattern of blood pressure in DSS rats. Treatment with vildagliptin dose-dependently decreased plasma DPP-4 activity, increased plasma glucagon-like peptide 1 (GLP-1) levels and attenuated the development of salt-induced hypertension. Furthermore, vildagliptin significantly increased urine sodium excretion and normalized the dipping pattern of blood pressure. In contrast, intracerebroventricular infusion of vildagliptin (50, 500 or 2500 μg) did not alter MAP and heart rate in DSS rats. These data suggest that salt-dependent hypertension initially develops with an extreme blood pressure dipping pattern. The DPP-4 inhibitor, vildagliptin, may elicit beneficial antihypertensive effects, including the improvement of abnormal circadian blood pressure pattern, by enhancing urinary sodium excretion. PMID:25588850

  18. Dipeptidyl peptidase-4 inhibition by gemigliptin prevents abnormal vascular remodeling via NF-E2-related factor 2 activation.

    PubMed

    Choi, Seung Hee; Park, Sungmi; Oh, Chang Joo; Leem, Jaechan; Park, Keun-Gyu; Lee, In-Kyu

    2015-10-01

    Dipeptidyl peptidase-4 (DPP-4) inhibitors exert a potent anti-hyperglycemic effect and reduce cardiovascular risk in type 2 diabetic patients. Several studies have shown that DPP-4 inhibitors including sitagliptin have beneficial effects in atherosclerosis and cardiac infarction involving reactive oxygen species. Here, we show that gemigliptin can directly attenuate the abnormal proliferation and migration of vascular smooth muscle cells (VSMCs) via enhanced NF-E2-related factor 2 (Nrf2) activity. Gemigliptin dramatically prevented ligation injury-induced neointimal hyperplasia in mouse carotid arteries. Likewise, the proliferation of primary VSMCs was significantly attenuated by gemigliptin in a dose-dependent manner consistent with a decrease in phospho-Rb, resulting in G1 cell cycle arrest. We found that gemigliptin enhanced Nrf2 activity not only by mRNA expression, but also by increasing Keap1 proteosomal degradation by p62, leading to the induction of Nrf2 target genes such as HO-1 and NQO1. The anti-proliferative role of gemigliptin disappeared with DPP-4 siRNA knockdown, indicating that the endogenous DPP-4 in VSMCs contributed to the effect of gemigliptin. In addition, gemigliptin diminished TNF-α-mediated cell adhesion molecules such as MCP-1 and VCAM-1 and reduced MMP2 activity in VSMCs. Taken together, our data indicate that gemigliptin exerts a preventative effect on the proliferation and migration of VSMCs via Nrf2. PMID:26187356

  19. Dipeptidyl peptidase-4 inhibition in diabetic rats leads to activation of the transcription factor CREB in β-cells.

    PubMed

    Pugazhenthi, Subbiah; Qin, LiMei; Bouchard, Ron

    2015-05-15

    Incretin therapies are effective in controlling blood glucose levels in type 2 diabetic patients by improving the survival and function of β-cells. They include dipeptidyl peptidase-4 (DPP-4) inhibitors and long-acting glucagon-like peptide-1 (GLP-1) analogs. We have previously reported that GLP-1 enhances the survival of cultured human islets by activation of the transcription factor CREB. To test the in vivo relevance of these findings, we examined the effects of alogliptin, a DPP-4 inhibitor, in Zucker Diabetic rats, a model for type 2 diabetes. The plasma levels of GLP-1 increased in alogliptin-treated diabetic rats leading to normoglycemia. Pancreatic islets of untreated diabetic rats were characterized by decreased immunostaining for insulin and PDX-1. Elevation of GLP-1 in treated diabetic rats resulted in the improved survival of β-cells. Dual immunofluorescent staining showed phosphorylation/activation of CREB in insulin-positive β-cells of islets. This led to increases in the levels of CREB targets including Bcl-2, an antiapoptotic mitochondrial protein, BIRC3, a caspase inhibitor and IRS-2, an adapter protein needed for insulin signaling. Findings from this study suggest potential activation of cytoprotective CREB by GLP-1 in pancreatic β-cells of diabetic patients undergoing incretin-based therapies. PMID:25720341

  20. Biocatalytic ammonolysis of (5S)-4,5-dihydro-1H-pyrrole-1,5-dicarboxylic acid, 1-(1,1-dimethylethyl)-5-ethyl ester: preparation of an intermediate to the dipeptidyl peptidase IV inhibitor Saxagliptin.

    PubMed

    Gill, Iqbal; Patel, Ramesh

    2006-02-01

    An efficient biocatalytic method has been developed for the conversion of (5S)-4,5-dihydro-1H-pyrrole-1,5-dicarboxylic acid, 1-(1,1-dimethylethyl)-5-ethyl ester (1) into the corresponding amide (5S)-5-aminocarbonyl-4,5-dihydro-1H-pyrrole-1-carboxylic acid, 1-(1,1-dimethylethyl)ester (2), which is a critical intermediate in the synthesis of the dipeptidyl peptidase IV (DPP4) inhibitor Saxagliptin (3). Candida antartica lipase B mediates ammonolysis of the ester with ammonium carbamate as ammonia donor to yield up to 71% of the amide. The inclusion of Ascarite and calcium chloride as adsorbents for carbon dioxide and ethanol byproducts, respectively, increases the yield to 98%, thereby offering an efficient and practical alternative to chemical routes which yield 57-64%. PMID:16257208

  1. Metabolism and excretion of the once-daily human glucagon-like peptide-1 analog liraglutide in healthy male subjects and its in vitro degradation by dipeptidyl peptidase IV and neutral endopeptidase.

    PubMed

    Malm-Erjefält, Monika; Bjørnsdottir, Inga; Vanggaard, Jan; Helleberg, Hans; Larsen, Uffe; Oosterhuis, Berend; van Lier, Jan Jaap; Zdravkovic, Milan; Olsen, Anette K

    2010-11-01

    Liraglutide is a novel once-daily human glucagon-like peptide (GLP)-1 analog in clinical use for the treatment of type 2 diabetes. To study metabolism and excretion of [(3)H]liraglutide, a single subcutaneous dose of 0.75 mg/14.2 MBq was given to healthy males. The recovered radioactivity in blood, urine, and feces was measured, and metabolites were profiled. In addition, [(3)H]liraglutide and [(3)H]GLP-1(7-37) were incubated in vitro with dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase (NEP) to compare the metabolite profiles and characterize the degradation products of liraglutide. The exposure of radioactivity in plasma (area under the concentration-time curve from 2 to 24 h) was represented by liraglutide (≥89%) and two minor metabolites (totaling ≤11%). Similarly to GLP-1, liraglutide was cleaved in vitro by DPP-IV in the Ala8-Glu9 position of the N terminus and degraded by NEP into several metabolites. The chromatographic retention time of DPP-IV-truncated liraglutide correlated well with the primary human plasma metabolite [GLP-1(9-37)], and some of the NEP degradation products eluted very close to both plasma metabolites. Three minor metabolites totaling 6 and 5% of the administered radioactivity were excreted in urine and feces, respectively, but no liraglutide was detected. In conclusion, liraglutide is metabolized in vitro by DPP-IV and NEP in a manner similar to that of native GLP-1, although at a much slower rate. The metabolite profiles suggest that both DPP-IV and NEP are also involved in the in vivo degradation of liraglutide. The lack of intact liraglutide excreted in urine and feces and the low levels of metabolites in plasma indicate that liraglutide is completely degraded within the body. PMID:20709939

  2. Design and synthesis of 4-(2,4,5-trifluorophenyl)butane-1,3-diamines as dipeptidyl peptidase IV inhibitors.

    PubMed

    Zhu, Linrong; Li, Yuanyuan; Qiu, Ling; Su, Mingbo; Wang, Xin; Xia, Chunmei; Qu, Yi; Li, Jingya; Li, Jia; Xiong, Bing; Shen, Jingkang

    2013-07-01

    The worldwide prevalence of diabetes has spurred numerous studies on the development of new antidiabetic medicines. As a result, dipeptidyl peptidase IV (DPP4) has been recognized as a validated target. In our efforts to discover new DPP4 inhibitors, we analyzed the complexed structures of DPP4 available in Protein Data Bank and designed a series of triazole compounds. After enzyme activity assays and crystallographic verification of the binding interaction patterns, we found that the triazole compounds can inhibit DPP4 with micromolar IC50 values. Liver microsome stability and cytochrome P450 metabolic tests were performed on this series, revealing undesirable pharmacokinetic profiles for the triazole compounds. To overcome this liability, we substituted the triazole ring with an amide or urea group to produce a new series of DPP4 inhibitors. Based on its enzyme activity, metabolic stability, and selectivity over DPP8 and DPP9, we selected compound 21 r for further study of its in vivo effects in mice using an oral glucose tolerance test (OGTT). The results show that 21 r has efficacy similar to that of sitagliptin at a dose of 3 mg kg(-1) . The crystal structure of 21 r bound to DPP4 also reveals that the trifluoromethyl group is directed toward a subpocket different from the subsite bound by sitagliptin, providing clues for the design of new DPP4 inhibitors. PMID:23671024

  3. (2R)-4-Oxo-4[3-(Trifluoromethyl)-5,6-diihydro:1,2,4}triazolo[4,3-a}pyrazin-7(8H)-y1]-1-(2,4,5-trifluorophenyl)butan-2-amine: A Potent, Orally Active Dipeptidyl Peptidase IV Inhibitor for the Treatment of Type 2 Diabetes

    SciTech Connect

    Kim, D.; Wang, L.; Beconi, M.; Eiermann, G.; Fisher, M.; He, H.; Hickey, G.; Kowalchick, Jennifer; Leiting, Barbara; Lyons, K.; Marsilio, F.; McCann, F.; Patel, R.; Petrov, A.; Scapin, G.; Patel, S.; Roy, R.; Wu, J.; Wyvratt, M.; Zhang, B.; Zhu, L.; Thornberry, N.; Weber, A.

    2010-11-10

    A novel series of {beta}-amino amides incorporating fused heterocycles, i.e., triazolopiperazines, were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-IV) for the treatment of type 2 diabetes. (2R)-4-Oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine (1) is a potent, orally active DPP-IV inhibitor (IC{sub 50} = 18 nM) with excellent selectivity over other proline-selective peptidases, oral bioavailability in preclinical species, and in vivo efficacy in animal models. MK-0431, the phosphate salt of compound 1, was selected for development as a potential new treatment for type 2 diabetes.

  4. Bioactive compounds from culinary herbs inhibit a molecular target for type 2 diabetes management, dipeptidyl peptidase IV

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Greek oregano (Origanum vulgare), marjoram (Origanum majorana), rosemary (Rosmarinus officinalis) and Mexican oregano (Lippia graveolens) are concentrated sources of bioactive compounds. The aims of this study were to characterize extracts from greenhouse grown or commercially purchased herbs for th...

  5. The Evaluation of Dipeptidyl Peptidase (DPP)-IV, α-Glucosidase and Angiotensin Converting Enzyme (ACE) Inhibitory Activities of Whey Proteins Hydrolyzed with Serine Protease Isolated from Asian Pumpkin (Cucurbita ficifolia).

    PubMed

    Konrad, Babij; Anna, Dąbrowska; Marek, Szołtysik; Marta, Pokora; Aleksandra, Zambrowicz; Józefa, Chrzanowska

    2014-01-01

    In the present study, whey protein concentrate (WPC-80) and β-lactoglobulin were hydrolyzed with a noncommercial serine protease isolated from Asian pumpkin (Cucurbita ficifolia). Hydrolysates were further fractionated by ultrafiltration using membranes with cut-offs equal 3 and 10 kDa. Peptide fractions of molecular weight lower than 3 and 3-10 kDa were further subjected to the RP-HPLC. Separated preparations were investigated for their potential as the natural inhibitors of dipeptidyl peptidase (DPP-IV), α-glucosidase and angiotensin converting enzyme (ACE). WPC-80 hydrolysate showed higher inhibitory activities against the three tested enzymes than β-lactoglobulin hydrolysate. Especially high biological activities were exhibited by peptide fractions of molecular weight lower than 3 kDa, with ACE IC50 <0.64 mg/mL and DPP-IV IC50 <0.55 mg/mL. This study suggests that peptides generated from whey proteins may support postprandial glycemia regulation and blood pressure maintenance, and could be used as functional food ingredients in the diet of patients with type 2 diabetes. PMID:25364320

  6. Evolutionary families of peptidase inhibitors.

    PubMed Central

    Rawlings, Neil D; Tolle, Dominic P; Barrett, Alan J

    2004-01-01

    The proteins that inhibit peptidases are of great importance in medicine and biotechnology, but there has never been a comprehensive system of classification for them. Some of the terminology currently in use is potentially confusing. In the hope of facilitating the exchange, storage and retrieval of information about this important group of proteins, we now describe a system wherein the inhibitor units of the peptidase inhibitors are assigned to 48 families on the basis of similarities detectable at the level of amino acid sequence. Then, on the basis of three-dimensional structures, 31 of the families are assigned to 26 clans. A simple system of nomenclature is introduced for reference to each clan, family and inhibitor. We briefly discuss the specificities and mechanisms of the interactions of the inhibitors in the various families with their target enzymes. The system of families and clans of inhibitors described has been implemented in the MEROPS peptidase database (http://merops.sanger.ac.uk/), and this will provide a mechanism for updating it as new information becomes available. PMID:14705960

  7. Astragaloside IV inhibits NF- κ B activation and inflammatory gene expression in LPS-treated mice.

    PubMed

    Zhang, Wei-Jian; Frei, Balz

    2015-01-01

    In this study we investigated the role of astragaloside IV (AS-IV), one of the major active constituents purified from the Chinese medicinal herb Astragalus membranaceus, in LPS-induced acute inflammatory responses in mice in vivo and examined possible underlying mechanisms. Mice were assigned to four groups: vehicle-treated control animals; AS-IV-treated animals (10 mg/kg b.w. AS-IV daily i.p. injection for 6 days); LPS-treated animals; and AS-IV plus LPS-treated animals. We found that AS-IV treatment significantly inhibited LPS-induced increases in serum levels of MCP-1 and TNF by 82% and 49%, respectively. AS-IV also inhibited LPS-induced upregulation of inflammatory gene expression in different organs. Lung mRNA levels of cellular adhesion molecules, MCP-1, TNFα, IL-6, and TLR4 were significantly attenuated, and lung neutrophil infiltration and activation were strongly inhibited, as reflected by decreased myeloperoxidase content, when the mice were pretreated with AS-IV. Similar results were observed in heart, aorta, kidney, and liver. Furthermore, AS-IV significantly suppressed LPS-induced NF-κB and AP-1 DNA-binding activities in lung and heart. In conclusion, our data provide new in vivo evidence that AS-IV effectively inhibits LPS-induced acute inflammatory responses by modulating NF-κB and AP-1 signaling pathways. Our results suggest that AS-IV may be useful for the prevention or treatment of inflammatory diseases. PMID:25960613

  8. NPY1-36 and PYY1-36 activate cardiac fibroblasts: an effect enhanced by genetic hypertension and inhibition of dipeptidyl peptidase 4.

    PubMed

    Zhu, Xiao; Gillespie, Delbert G; Jackson, Edwin K

    2015-11-01

    Cardiac sympathetic nerves release neuropeptide Y (NPY)1-36, and peptide YY (PYY)1-36 is a circulating peptide; therefore, these PP-fold peptides could affect cardiac fibroblasts (CFs). We examined the effects of NPY1-36 and PYY1-36 on the proliferation of and collagen production ([(3)H]proline incorporation) by CFs isolated from Wistar-Kyoto (WKY) normotensive rats and spontaneously hypertensive rats (SHRs). Experiments were performed with and without sitagliptin, an inhibitor of dipeptidyl peptidase 4 [DPP4; an ectoenzyme that metabolizes NPY1-36 and PYY1-36 (Y1 receptor agonists) to NPY3-36 and PYY3-36 (inactive at Y1 receptors), respectively]. NPY1-36 and PYY1-36, but not NPY3-36 or PYY3-36, stimulated proliferation of CFs, and these effects were more potent than ANG II, enhanced by sitagliptin, blocked by BIBP3226 (Y1 receptor antagonist), and greater in SHR CFs. SHR CF membranes expressed more receptor for activated C kinase (RACK)1 [which scaffolds the Gi/phospholipase C (PLC)/PKC pathway] compared with WKY CF membranes. RACK1 knockdown (short hairpin RNA) and inhibition of Gi (pertussis toxin), PLC (U73122), and PKC (GF109203X) blocked the proliferative effects of NPY1-36. NPY1-36 and PYY1-36 stimulated collagen production more potently than did ANG II, and this was enhanced by sitagliptin and greater in SHR CFs. In conclusion, 1) NPY1-36 and PYY1-36, via the Y1 receptor/Gi/PLC/PKC pathway, activate CFs, and this pathway is enhanced in SHR CFs due to increased localization of RACK1 in membranes; and 2) DPP4 inhibition enhances the effects of NPY1-36 and PYY1-36 on CFs, likely by inhibiting the metabolism of NPY1-36 and PYY1-36. The implications are that endogenous NPY1-36 and PYY1-36 could adversely affect cardiac structure/function by activating CFs, and this may be exacerbated in genetic hypertension and by DPP4 inhibitors. PMID:26371160

  9. Evolutionary families of peptidases.

    PubMed Central

    Rawlings, N D; Barrett, A J

    1993-01-01

    The available amino acid sequences of peptidases have been examined, and the enzymes have been allocated to evolutionary families. Some of the families can be grouped together in 'clans' that show signs of distant relationship, but nevertheless, it appears that there may be as many as 60 evolutionary lines of peptidases with separate origins. Some of these contain members with quite diverse peptidase activities, and yet there are some striking examples of convergence. We suggest that the classification by families could be used as an extension of the current classification by catalytic type. PMID:8439290

  10. MEROPS: the peptidase database

    PubMed Central

    Rawlings, Neil D.; Barrett, Alan J.

    2000-01-01

    Important additions have been made to the MEROPS database (http://www.bi.bbsrc.ac.uk/Merops/Merops.htm ). These include sequence alignments and cladograms for many of the families of peptidases, and these have proved very helpful in the difficult task of distinguishing the sequences of peptidases that are simply species variants of already known enzymes from those that represent novel enzymes. PMID:10592261

  11. In silico, in vitro and in vivo analyses of dipeptidyl peptidase IV inhibitory activity and the antidiabetic effect of sodium caseinate hydrolysate.

    PubMed

    Hsieh, Cheng-Hong; Wang, Tzu-Yuan; Hung, Chuan-Chuan; Jao, Chia-Ling; Hsieh, You-Liang; Wu, Si-Xian; Hsu, Kuo-Chiang

    2016-02-01

    The frequency (A), a novel in silico parameter, was developed by calculating the ratio of the number of truncated peptides with Xaa-proline and Xaa-alanine to all peptide fragments from a protein hydrolyzed with a specific protease. The highest in vitro DPP-IV inhibitory activity (72.7%) was observed in the hydrolysate of sodium caseinate by bromelain (Cas/BRO), and the constituent proteins of bovine casein also had relatively high A values (0.10-0.17) with BRO hydrolysis. 1CBR (the <1 kDa fraction of Cas/BRO) showed the greatest in vitro DPP-IV inhibitory activity of 77.5% and was used for in vivo test by high-fat diet-fed and low-dose streptozotocin-induced diabetic rats. The daily administration of 1CBR for 6 weeks was effective to improve glycaemic control in diabetic rats. The results indicate that the novel in silico method has the potential as a screening tool to predict dietary proteins to generate DPP-IV inhibitory and antidiabetic peptides. PMID:26796955

  12. Injury and differentiation following inhibition of mitochondrial respiratory chain complex IV in rat oligodendrocytes

    PubMed Central

    Ziabreva, Iryna; Campbell, Graham; Rist, Julia; Zambonin, Jessica; Rorbach, Joanna; Wydro, Mateusz M; Lassmann, Hans; Franklin, Robin J M; Mahad, Don

    2010-01-01

    Oligodendrocyte lineage cells are susceptible to a variety of insults including hypoxia, excitotoxicity, and reactive oxygen species. Demyelination is a well-recognized feature of several CNS disorders including multiple sclerosis, white matter strokes, progressive multifocal leukoencephalopathy, and disorders due to mitochondrial DNA mutations. Although mitochondria have been implicated in the demise of oligodendrocyte lineage cells, the consequences of mitochondrial respiratory chain defects have not been examined. We determine the in vitro impact of established inhibitors of mitochondrial respiratory chain complex IV or cytochrome c oxidase on oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes as well as on differentiation capacity of OPCs from P0 rat. Injury to mature oligodendrocytes following complex IV inhibition was significantly greater than to OPCs, judged by cell detachment and mitochondrial membrane potential (MMP) changes, although viability of cells that remained attached was not compromised. Active mitochondria were abundant in processes of differentiated oligodendrocytes and MMP was significantly greater in differentiated oligodendrocytes than OPCs. MMP dissipated following complex IV inhibition in oligodendrocytes. Furthermore, complex IV inhibition impaired process formation within oligodendrocyte lineage cells. Injury to and impaired process formation of oligodendrocytes following complex IV inhibition has potentially important implications for the pathogenesis and repair of CNS myelin disorders. © 2010 Wiley-Liss, Inc. PMID:20665559

  13. Effects of N-acetylaspartylglutamate (NAAG) peptidase inhibition on release of glutamate and dopamine in prefrontal cortex and nucleus accumbens in phencyclidine model of schizophrenia.

    PubMed

    Zuo, Daiying; Bzdega, Tomasz; Olszewski, Rafal T; Moffett, John R; Neale, Joseph H

    2012-06-22

    The "glutamate" theory of schizophrenia emerged from the observation that phencyclidine (PCP), an open channel antagonist of the NMDA subtype of glutamate receptor, induces schizophrenia-like behaviors in humans. PCP also induces a complex set of behaviors in animal models of this disorder. PCP also increases glutamate and dopamine release in the medial prefrontal cortex and nucleus accumbens, brain regions associated with expression of psychosis. Increased motor activation is among the PCP-induced behaviors that have been widely validated as models for the characterization of new antipsychotic drugs. The peptide transmitter N-acetylaspartylglutamate (NAAG) activates a group II metabotropic receptor, mGluR3. Polymorphisms in this receptor have been associated with schizophrenia. Inhibitors of glutamate carboxypeptidase II, an enzyme that inactivates NAAG following synaptic release, reduce several behaviors induced by PCP in animal models. This research tested the hypothesis that two structurally distinct NAAG peptidase inhibitors, ZJ43 and 2-(phosphonomethyl)pentane-1,5-dioic acid, would elevate levels of synaptically released NAAG and reduce PCP-induced increases in glutamate and dopamine levels in the medial prefrontal cortex and nucleus accumbens. NAAG-like immunoreactivity was found in neurons and presumptive synaptic endings in both regions. These peptidase inhibitors reduced the motor activation effects of PCP while elevating extracellular NAAG levels. They also blocked PCP-induced increases in glutamate but not dopamine or its metabolites. The mGluR2/3 antagonist LY341495 blocked these behavioral and neurochemical effects of the peptidase inhibitors. The data reported here provide a foundation for assessment of the neurochemical mechanism through which NAAG achieves its antipsychotic-like behavioral effects and support the conclusion NAAG peptidase inhibitors warrant further study as a novel antipsychotic therapy aimed at mGluR3. PMID:22570482

  14. Effects of N-Acetylaspartylglutamate (NAAG) Peptidase Inhibition on Release of Glutamate and Dopamine in Prefrontal Cortex and Nucleus Accumbens in Phencyclidine Model of Schizophrenia*

    PubMed Central

    Zuo, Daiying; Bzdega, Tomasz; Olszewski, Rafal T.; Moffett, John R.; Neale, Joseph H.

    2012-01-01

    The “glutamate” theory of schizophrenia emerged from the observation that phencyclidine (PCP), an open channel antagonist of the NMDA subtype of glutamate receptor, induces schizophrenia-like behaviors in humans. PCP also induces a complex set of behaviors in animal models of this disorder. PCP also increases glutamate and dopamine release in the medial prefrontal cortex and nucleus accumbens, brain regions associated with expression of psychosis. Increased motor activation is among the PCP-induced behaviors that have been widely validated as models for the characterization of new antipsychotic drugs. The peptide transmitter N-acetylaspartylglutamate (NAAG) activates a group II metabotropic receptor, mGluR3. Polymorphisms in this receptor have been associated with schizophrenia. Inhibitors of glutamate carboxypeptidase II, an enzyme that inactivates NAAG following synaptic release, reduce several behaviors induced by PCP in animal models. This research tested the hypothesis that two structurally distinct NAAG peptidase inhibitors, ZJ43 and 2-(phosphonomethyl)pentane-1,5-dioic acid, would elevate levels of synaptically released NAAG and reduce PCP-induced increases in glutamate and dopamine levels in the medial prefrontal cortex and nucleus accumbens. NAAG-like immunoreactivity was found in neurons and presumptive synaptic endings in both regions. These peptidase inhibitors reduced the motor activation effects of PCP while elevating extracellular NAAG levels. They also blocked PCP-induced increases in glutamate but not dopamine or its metabolites. The mGluR2/3 antagonist LY341495 blocked these behavioral and neurochemical effects of the peptidase inhibitors. The data reported here provide a foundation for assessment of the neurochemical mechanism through which NAAG achieves its antipsychotic-like behavioral effects and support the conclusion NAAG peptidase inhibitors warrant further study as a novel antipsychotic therapy aimed at mGluR3. PMID:22570482

  15. Evolution of the Thermopsin Peptidase Family (A5)

    PubMed Central

    Rawlings, Neil D.

    2013-01-01

    Thermopsin is a peptidase from Sulfolobus acidocaldarius that is active at low pH and high temperature. From reversible inhibition with pepstatin, thermopsin is thought to be an aspartic peptidase. It is a member of the only family of peptidases to be restricted entirely to the archaea, namely peptidase family A5. Evolution within this family has been mapped, using a taxonomic tree based on the known classification of archaea. Homologues are found only in archaeans that are both hyperthermophiles and acidophiles, and this implies lateral transfer of genes between archaea, because species with homologues are not necessarily closely related. Despite the remarkable stability and activity in extreme conditions, no tertiary structure has been solved for any member of the family, and the catalytic mechanism is unknown. Putative catalytic residues have been predicted here by examination of aligned sequences. PMID:24312173

  16. Localization of Peptidases in Lactococci

    PubMed Central

    Tan, Paris S. T.; Chapot-Chartier, Marie-Pierre; Pos, Klaas M.; Rousseau, Micheline; Boquien, Clair-Yves; Gripon, Jean-Claude; Konings, Wil N.

    1992-01-01

    The localization of two aminopeptidases, an X-prolyl-dipeptidyl aminopeptidase, an endopeptidase, and a tripeptidase in Lactococcus lactis was studied. Polyclonal antibodies raised against each purified peptidase are specific and do not cross-react with other peptidases. Experiments were performed by immunoblotting after cell fractionation and by electron microscopy of immunogold-labeled peptidases. All peptidases were found to be intracellular. However, immunogold studies showed a peripheral labeling of the X-prolyl-dipeptidyl aminopeptidase, the tripeptidase, and the endopeptidase. This peripheral location was further supported by the detection of these three enzymes in cell membrane fractions in which none of the two aminopeptidases was present. Images PMID:16348629

  17. [Description of an acidic peptidase, insensitive to classical inhibitors, in protein extracts of Trypanosoma cruzi, from a rural area of Venezuela, where Chagas disease is endemic].

    PubMed

    Zambrano, Edgar Armando; de la Cruz, Henry Samuel; Coita, Blanca Elena

    2013-09-01

    Through two peptidase assay methods, one in liquid-phase and another, in gel-phase (gel zymography), an acid peptidase was detected in protein crude extracts of epimastigotes of Trypanosoma cruzi, from a rural area of Venezuela where Chagas disease is endemic. The peptidase shows activity at a pH range between 2.0 and 2.9. Under the experimental conditions described, the acid peptidase was insensitive to usual concentrations of peptidase inhibitors of the types: serine, cysteine, aspartic and metallopeptidases. Nevertheless, like porcine pepsin at pH 2.9, the peptidase was inhibited in the presence of 5mM DTT. PMID:24354241

  18. Type IV traffic ATPase TrwD as molecular target to inhibit bacterial conjugation.

    PubMed

    Ripoll-Rozada, Jorge; García-Cazorla, Yolanda; Getino, María; Machón, Cristina; Sanabria-Ríos, David; de la Cruz, Fernando; Cabezón, Elena; Arechaga, Ignacio

    2016-06-01

    Bacterial conjugation is the main mechanism responsible for the dissemination of antibiotic resistance genes. Hence, the search for specific conjugation inhibitors is paramount in the fight against the spread of these genes. In this pursuit, unsaturated fatty acids have been found to specifically inhibit bacterial conjugation. Despite the growing interest on these compounds, their mode of action and their specific target remain unknown. Here, we identified TrwD, a Type IV secretion traffic ATPase, as the molecular target for fatty acid-mediated inhibition of conjugation. Moreover, 2-alkynoic fatty acids, which are also potent inhibitors of bacterial conjugation, are also powerful inhibitors of the ATPase activity of TrwD. Characterization of the kinetic parameters of ATPase inhibition has led us to identify the catalytic mechanism by which fatty acids exert their activity. These results open a new avenue for the rational design of inhibitors of bacterial conjugation in the fight against the dissemination of antibiotic resistance genes. PMID:26915347

  19. Evolutionary lines of cysteine peptidases.

    PubMed

    Barrett, A J; Rawlings, N D

    2001-05-01

    The proteolytic enzymes that depend upon a cysteine residue for activity have come from at least seven different evolutionary origins, each of which has produced a group of cysteine peptidases with distinctive structures and properties. We show here that the characteristic molecular topologies of the peptidases in each evolutionary line can be seen not only in their three-dimensional structures, but commonly also in the two-dimensional structures. Clan CA contains the families of papain (C1), calpain (C2), streptopain (C10) and the ubiquitin-specific peptidases (C12, C19), as well as many families of viral cysteine endopeptidases. Clan CD contains the families of clostripain (C11), gingipain R (C25), legumain (C13), caspase-1 (C14) and separin (C50). These enzymes have specificities dominated by the interactions of the S1 subsite. Clan CE contains the families of adenain (C5) from adenoviruses, the eukaryotic Ulp1 protease (C48) and the bacterial YopJ proteases (C55). Clan CF contains only pyroglutamyl peptidase I (C15). The picornains (C3) in clan PA have probably evolved from serine peptidases, which still form the majority of enzymes in the clan. The cysteine peptidase activities in clans PB and CH are autolytic only. In conclusion, we suggest that although almost all the cysteine peptidases depend for activity on catalytic dyads of cysteine and histidine, it is worth noting some important differences that they have inherited from their distant ancestral peptidases. PMID:11517925

  20. Astragaloside IV Inhibits NF-κB Activation and Inflammatory Gene Expression in LPS-Treated Mice

    PubMed Central

    Zhang, Wei-Jian; Frei, Balz

    2015-01-01

    In this study we investigated the role of astragaloside IV (AS-IV), one of the major active constituents purified from the Chinese medicinal herb Astragalus membranaceus, in LPS-induced acute inflammatory responses in mice in vivo and examined possible underlying mechanisms. Mice were assigned to four groups: vehicle-treated control animals; AS-IV-treated animals (10 mg/kg b.w. AS-IV daily i.p. injection for 6 days); LPS-treated animals; and AS-IV plus LPS-treated animals. We found that AS-IV treatment significantly inhibited LPS-induced increases in serum levels of MCP-1 and TNF by 82% and 49%, respectively. AS-IV also inhibited LPS-induced upregulation of inflammatory gene expression in different organs. Lung mRNA levels of cellular adhesion molecules, MCP-1, TNFα, IL-6, and TLR4 were significantly attenuated, and lung neutrophil infiltration and activation were strongly inhibited, as reflected by decreased myeloperoxidase content, when the mice were pretreated with AS-IV. Similar results were observed in heart, aorta, kidney, and liver. Furthermore, AS-IV significantly suppressed LPS-induced NF-κB and AP-1 DNA-binding activities in lung and heart. In conclusion, our data provide new in vivo evidence that AS-IV effectively inhibits LPS-induced acute inflammatory responses by modulating NF-κB and AP-1 signaling pathways. Our results suggest that AS-IV may be useful for the prevention or treatment of inflammatory diseases. PMID:25960613

  1. Streptococcus pneumoniae DNA Gyrase and Topoisomerase IV: Overexpression, Purification, and Differential Inhibition by Fluoroquinolones

    PubMed Central

    Pan, Xiao-Su; Fisher, L. Mark

    1999-01-01

    Streptococcus pneumoniae gyrA and gyrB genes specifying the DNA gyrase subunits have been cloned into pET plasmid vectors under the control of an inducible T7 promoter and have been separately expressed in Escherichia coli. Soluble 97-kDa GyrA and 72-kDa GyrB proteins bearing polyhistidine tags at their respective C-terminal and N-terminal ends were purified to apparent homogeneity by one-step nickel chelate column chromatography and were free of host E. coli topoisomerase activity. Equimolar amounts of the gyrase subunits reconstituted ATP-dependent DNA supercoiling with comparable activity to gyrase of E. coli and Staphylococcus aureus. In parallel, S. pneumoniae topoisomerase IV ParC and ParE subunits were similarly expressed in E. coli, purified to near homogeneity as 93- and 73-kDa proteins, and shown to generate efficient ATP-dependent DNA relaxation and DNA decatenation activities. Using the purified enzymes, we examined the inhibitory effects of three paradigm fluoroquinolones—ciprofloxacin, sparfloxacin, and clinafloxacin—which previous genetic studies with S. pneumoniae suggested act preferentially through topoisomerase IV, through gyrase, and through both enzymes, respectively. Surprisingly, all three quinolones were more active in inhibiting purified topoisomerase IV than gyrase, with clinafloxacin showing the greatest inhibitory potency. Moreover, the tested agents were at least 25-fold more effective in stabilizing a cleavable complex (the relevant cytotoxic lesion) with topoisomerase IV than with gyrase, with clinafloxacin some 10- to 32-fold more potent against either enzyme, in line with its superior activity against S. pneumoniae. The uniform target preference of the three fluoroquinolones for topoisomerase IV in vitro is in apparent contrast to the genetic data. We interpret these results in terms of a model for bacterial killing by quinolones in which cellular factors can modulate the effects of target affinity to determine the cytotoxic

  2. Inhibition of inflammasome activation by Coxiella burnetii type IV secretion system effector IcaA

    PubMed Central

    Cunha, Larissa D.; Ribeiro, Juliana M.; Fernandes, Talita D.; Massis, Liliana M.; Khoo, Chen Ai; Moffatt, Jennifer H.; Newton, Hayley J.; Roy, Craig R.; Zamboni, Dario S.

    2015-01-01

    Coxiella burnetii is a highly infectious bacterium that promotes its own replication in macrophages by inhibiting several host cell responses. Here, we show that C. burnetii inhibits caspase-1 activation in primary mouse macrophages. By using co-infection experiments, we determine that the infection of macrophages with C. burnetii inhibits the caspase-11-mediated non-canonical activation of the NLRP3 inflammasome induced by subsequent infection with Escherichia coli or Legionella pneumophila. Genetic screening using flagellin mutants of L. pneumophila as a surrogate host, reveals a novel C. burnetii gene (IcaA) involved in the inhibition of caspase activation. Expression of IcaA in L. pneumophila inhibited the caspase-11 activation in macrophages. Moreover, icaA- mutants of C. burnetii failed to suppress the caspase-11-mediated inflammasome activation induced by L. pneumophila. Our data reveal IcaA as a novel C. burnetii effector protein that is secreted by the Dot/Icm type IV secretion system and interferes with the caspase-11-induced, non-canonical activation of the inflammasome. PMID:26687278

  3. In Vivo Effects of Bradykinin B2 Receptor Agonists with Varying Susceptibility to Peptidases

    PubMed Central

    Jean, Mélissa; Gera, Lajos; Charest-Morin, Xavier; Marceau, François; Bachelard, Hélène

    2016-01-01

    We reported evidence of bradykinin (BK) regeneration from C-terminal extended BK sequences that behave as peptidase-activated B2 receptor (B2R) agonists. Further to these in vitro studies, we carried out in vivo experiments to verify hemodynamic effects of BK analogs exhibiting variable susceptibility toward vascular and blood plasma peptidases. Rats were anesthetized and instrumented to record blood pressure and heart rate responses to bolus intravenous (i.v.) injection of increasing doses of BK, B-9972 (D-Arg-[Hyp3,Igl5,Oic7,Igl8]-BK), BK-Arg, BK-His-Leu or BK-Ala-Pro, in the absence or presence of specific inhibitors. In some experiments, pulsed Doppler flow probes measured hindquarter Doppler shift in response to i.v. injections of kinins. BK caused rapid, transient and dose-related hypotensive effects. These effects were potentiated ∼15-fold by the angiotensin converting enzyme (ACE) inhibitor, enalaprilat, but extensively inhibited by icatibant (a B2R antagonist) and not influenced by the Arg-carboxypeptidase (CP) inhibitor (Plummer’s inhibitor). The hypotensive responses elicited by the peptidase-resistant B2R agonist, B-9972, were not affected by enalaprilat, but were inhibited by icatibant. The hypotensive responses to BK-Arg were abolished by pre-treatment with either the Arg-CP inhibitor or icatibant, pharmacologically evidencing BK regeneration. The hypotensive effects of BK-His-Leu and BK-Ala-Pro, previously reported as ACE-activated substrates, were abolished by icatibant, but not by enalaprilat. In vivo regeneration of BK from these two C-terminally extended analogs with no affinity for the B2R must follow alternative cleavage rules involving unidentified carboxypeptidase(s) when ACE is blocked. The transient hypotensive responses to BK and three tested analogs coincided with concomitant vasodilation (increased Doppler shift signal). Together, these results provide in vivo evidence that interesting hypotensive and vasodilator effects can be

  4. A phage protein that inhibits the bacterial ATPase required for type IV pilus assembly.

    PubMed

    Chung, In-Young; Jang, Hye-Jeong; Bae, Hee-Won; Cho, You-Hee

    2014-08-01

    Type IV pili (TFPs) are required for bacterial twitching motility and for phage infection in the opportunistic human pathogen Pseudomonas aeruginosa. Here we describe a phage-encoded protein, D3112 protein gp05 (hereafter referred to as Tip, representing twitching inhibitory protein), whose expression is necessary and sufficient to mediate the inhibition of twitching motility. Tip interacts with and blocks the activity of bacterial-encoded PilB, the TFP assembly/extension ATPase, at an internal 40-aa region unique to PilB. Tip expression results in the loss of surface piliation. Based on these observations and the fact that many P. aeruginosa phages require TFPs for infection, Tip-mediated twitching inhibition may represent a generalized strategy for superinfection exclusion. Moreover, because TFPs are required for full virulence, PilB may be an attractive target for the development of novel antiinfectives. PMID:25049409

  5. Astragaloside IV inhibits microglia activation via glucocorticoid receptor mediated signaling pathway

    PubMed Central

    Liu, Hong-Shuai; Shi, Hai-Lian; Huang, Fei; Peterson, Karin E.; Wu, Hui; Lan, Yun-Yi; Zhang, Bei-Bei; He, Yi-Xin; Woods, Tyson; Du, Min; Wu, Xiao-Jun; Wang, Zheng-Tao

    2016-01-01

    Inhibition of microglia activation may provide therapeutic treatment for many neurodegenerative diseases. Astragaloside IV (ASI) with anti-inflammatory properties has been tested as a therapeutic drug in clinical trials of China. However, the mechanism of ASI inhibiting neuroinflammation is unknown. In this study, we showed that ASI inhibited microglia activation both in vivo and in vitro. It could enhance glucocorticoid receptor (GR)-luciferase activity and facilitate GR nuclear translocation in microglial cells. Molecular docking and TR-FRET GR competitive binding experiments demonstrated that ASI could bind to GR in spite of relative low affinity. Meanwhile, ASI modulated GR-mediated signaling pathway, including dephosphorylation of PI3K, Akt, I κB and NF κB, therefore, decreased downstream production of proinflammatory mediators. Suppression of microglial BV-2 activation by ASI was abrogated by GR inhibitor, RU486 or GR siRNA. Similarly, RU486 counteracted the alleviative effect of ASI on microgliosis and neuronal injury in vivo. Our findings demonstrated that ASI inhibited microglia activation at least partially by activating the glucocorticoid pathway, suggesting its possible therapeutic potential for neuroinflammation in neurological diseases. PMID:26750705

  6. NAAG peptidase inhibition reduces locomotor activity and some stereotypes in the PCP model of schizophrenia via group II mGluR.

    PubMed

    Olszewski, Rafal T; Bukhari, Noreen; Zhou, Jia; Kozikowski, Alan P; Wroblewski, Jarda T; Shamimi-Noori, Susan; Wroblewska, Barbara; Bzdega, Tomasz; Vicini, Stefano; Barton, Franca B; Neale, Joseph H

    2004-05-01

    Phencyclidine (PCP) administration elicits positive and negative symptoms that resemble those of schizophrenia and is widely accepted as a model for the study of this human disorder. Group II metabotropic glutamate receptor (mGluR) agonists have been reported to reduce the behavioral and neurochemical effects of PCP. The peptide neurotransmitter, N-acetylaspartylglutamate (NAAG), is a selective group II agonist. We synthesized and characterized a urea-based NAAG analogue, ZJ43. This novel compound is a potent inhibitor of enzymes, glutamate carboxypeptidase II (K(i) = 0.8 nM) and III (K(i) = 23 nM) that deactivate NAAG following synaptic release. ZJ43 (100 microM) does not directly interact with NMDA receptors or metabotropic glutamate receptors. Administration of ZJ43 significantly reduced PCP-induced motor activation, falling while walking, stereotypic circling behavior, and head movements. To test the hypothesis that this effect of ZJ43 was mediated by increasing the activation of mGluR3 via increased levels of extracellular NAAG, the group II mGluR selective antagonist LY341495 was co-administered with ZJ43 prior to PCP treatment. This antagonist completely reversed the effects of ZJ43. Additionally, LY341495 alone increased PCP-induced motor activity and head movements suggesting that normal levels of NAAG act to moderate the effect of PCP on motor activation via a group II mGluR. These data support the view that NAAG peptidase inhibitors may represent a new therapeutic approach to some of the components of schizophrenia that are modeled by PCP. PMID:15140187

  7. AS-IV protects against kidney IRI through inhibition of NF-κB activity and PUMA upregulation

    PubMed Central

    Xin, Yan; Li, Gang; Liu, Hongxiu; Ai, Dengbin

    2015-01-01

    Objective: To determine and explore the effect of Astragalus saponin IV (AS-IV) on ischemia/reperfusion (IR)-induced renal injury and its mechanisms. Methods: Experimental model of renal I/R was induced in rats by bilateral renal artery clamp for 45 min followed by reperfusion of 6 h. Rats were divided into three groups: ① sham ② IRI ③ IRI/AS-IV. In IRI/AS-IV groups, AS-IV was orally administered once a day to rats at 2 mg·kg-1·d-1 for 7 days prior to ischemia. At 6 h after reperfusion, the inflammatory cytokines and renal function was assessed and NF-κB activity and PUMA expression was detected. Apoptotic cells was detected by TUNEL assay. Results: AS-IV significantly decreased serum and tissue levels of IL-6 and TNF-α, and reduced apoptotic cell counts and histological damage. AS-IV down-regulated the phosphorylation of p65 subunit of NF-κB (NF-κB p65) and PUMA expression, and the NF-κB activity compared to the I/R groups. Conclusions: AS-IV provided protection against IRI-induced renal injury by reducing apoptosis and inflammation through inhibition of NF-κB activity and PUMA expression. AS-IV pre-treatment ameliorated tubular damage and suppressed the NF-κB p65 expression. PMID:26770431

  8. Angiotensin IV protects cardiac reperfusion injury by inhibiting apoptosis and inflammation via AT4R in rats.

    PubMed

    Park, Byung Mun; Cha, Seung Ah; Lee, Sun Hwa; Kim, Suhn Hee

    2016-05-01

    Angiotensin IV (Ang IV) is formed by aminopeptidase N from Ang III by removing the first N-terminal amino acid. Previously, we reported that Ang III has some cardioprotective effects against global ischemia in Langendorff heart. However, it is not clear whether Ang IV has cardioprotective effects. The aim of the present study was to evaluate the effect of Ang IV on myocardial ischemia-reperfusion (I/R) injury in rats. Before ischemia, male Sprague-Dawley rats received Ang IV (1mg/kg/day) for 3 days. Anesthetized rats were subjected to 45min of ischemia by ligation of left anterior descending coronary artery followed by reperfusion and then, sacrificed 1 day or 1 week after reperfusion. Plasma creatine kinase (CK) and lactate dehydrogenase (LDH) concentrations, and infarct size were measured. Quantitative analysis of apoptotic and inflammatory proteins in ventricles were performed using Western blotting. Pretreatment with Ang IV attenuated I/R-induced increases in plasma CK and LDH levels, and infarct size, which were blunted by Ang IV receptor (AT4R) antagonist and but not by antagonist for AT1R, AT2R, or Mas receptor. I/R increased Bax, caspase-3 and caspase-9 protein levels, and decreased Bcl-2 protein level in ventricles, which were blunted by Ang IV. I/R-induced increases in TNF-α, MMP-9, and VCAM-1 protein levels in ventricles were also blunted by Ang IV. Ang IV increased the phosphorylation of Akt and mTOR. These effects were attenuated by co-treatment with AT4R antagonist or inhibitors of downstream signaling pathway. Myocardial dysfunction after reperfusion was improved by Ang IV. These results suggest that Ang IV has cardioprotective effect against I/R injury by inhibiting apoptosis via AT4R and PI3K-Akt-mTOR pathway. PMID:27038740

  9. Heterogeneous production of metallo-type peptidases in parasites belonging to the family Trypanosomatidae.

    PubMed

    dos Santos, André Luis Souza; Soares, Rosangela Maria de Araújo; Alviano, Celuta Sales; Kneipp, Lucimar Ferreira

    2008-05-01

    Proteolytic enzymes play a central role in the physiology of all living organisms, participating in several metabolic pathways and in different phases of parasite-host interactions. We have identified cell-associated peptidase activities in 33 distinct flagellates, including representatives of almost all known trypanosomatid genera parasitizing insects (Herpetomonas, Crithidia, Leishmania, Trypanosoma, Leptomonas, Phytomonas, Blastocrithidia and Endotrypanum) as well as the biflagellate kinetoplastid Bodo, by using SDS-PAGE containing gelatin as co-polymerized substrate and proteolytic inhibitors. Under the alkaline pH (9.0) conditions employed, all the flagellates presented at least one peptidase, with the exception of Crithidia acanthocephali and Phytomonas serpens, which did not display any detectable proteolytic enzyme activity. All the proteolytic activities were completely inhibited by 1,10-phenanthroline, a zinc-chelating agent, putatively identifying these activities as metallo-type peptidases. EDTA and EGTA, two other metallopeptidase inhibitors, E-64 (a cysteine peptidase inhibitor), pepstatin A (an aspartyl peptidase inhibitor) and PMSF (a serine peptidase inhibitor) did not interfere with the metallopeptidase activities detected in the studied trypanosomatids. Conversely, Bodo-derived peptidases were resistant to 1,10-phenanthroline and only partially inhibited by EDTA, showing a distinct inhibition profile. Together, our data demonstrated great heterogeneity of expression of metallopeptidases in a wide range of parasites belonging to the family Trypanosomatidae. PMID:17942292

  10. Protective effects of astragaloside IV against amyloid beta1-42 neurotoxicity by inhibiting the mitochondrial permeability transition pore opening.

    PubMed

    Sun, Qinru; Jia, Ning; Wang, Weixi; Jin, Hui; Xu, Jiehua; Hu, Haitao

    2014-01-01

    Mitochondrial dysfunction caused by amyloid β-peptide (Aβ) plays an important role in the pathogenesis of Alzheimer disease (AD). Substantial evidence has indicated that the mitochondrial permeability transition pore (mPTP) opening is involved in Aβ-induced neuronal death and reactive oxygen species (ROS) generation. Astragaloside IV (AS-IV), one of the major active constituents of Astragalus membranaceus, has been reported as an effective anti-oxidant for treating neurodegenerative diseases. However, the molecular mechanisms still need to be clarified. In this study, we investigated whether AS-IV could prevent Aβ1-42-induced neurotoxicity in SK-N-SH cells via inhibiting the mPTP opening. The results showed that pretreatment of AS-IV significantly increased the viability of neuronal cells, reduced apoptosis, decreased the generation of intracellular reactive oxygen species (ROS) and decreased mitochondrial superoxide in the presence of Aβ1-42. In addition, pretreatment of AS-IV inhibited the mPTP opening, rescued mitochondrial membrane potential (ΔΨm), enhanced ATP generation, improved the activity of cytochrome c oxidase and blocked cytochrome c release from mitochondria in Aβ1-42 rich milieu. Moreover, pretreatment of AS-IV reduced the expression of Bax and cleaved caspase-3 and increased the expression of Bcl-2 in an Aβ1-42 rich environment. These data indicate that AS-IV prevents Aβ1-42-induced SK-N-SH cell apoptosis via inhibiting the mPTP opening and ROS generation. These results provide novel insights of AS-IV for the prevention and treatment of neurodegenerative disorders such as AD. PMID:24905226

  11. Inhibition of Ubiquitin-specific Peptidase 8 Suppresses Adrenocorticotropic Hormone Production and Tumorous Corticotroph Cell Growth in AtT20 Cells

    PubMed Central

    Jian, Fang-Fang; Li, Yun-Feng; Chen, Yu-Fan; Jiang, Hong; Chen, Xiao; Zheng, Li-Li; Zhao, Yao; Wang, Wei-Qing; Ning, Guang; Bian, Liu-Guan; Sun, Qing-Fang

    2016-01-01

    Background: Two recent whole-exome sequencing researches identifying somatic mutations in the ubiquitin-specific protease 8 (USP8) gene in pituitary corticotroph adenomas provide exciting advances in this field. These mutations drive increased epidermal growth factor receptor (EGFR) signaling and promote adrenocorticotropic hormone (ACTH) production. This study was to investigate whether the inhibition of USP8 activity could be a strategy for the treatment of Cushing's disease (CD). Methods: The anticancer effect of USP8 inhibitor was determined by testing cell viability, colony formation, apoptosis, and ACTH secretion. The immunoblotting and quantitative reverse transcription polymerase chain reaction were conducted to explore the signaling pathway by USP8 inhibition. Results: Inhibition of USP8-induced degradation of receptor tyrosine kinases including EGFR, EGFR-2 (ERBB2), and Met leading to a suppression of AtT20 cell growth and ACTH secretion. Moreover, treatment with USP8 inhibitor markedly induced AtT20 cells apoptosis. Conclusions: Inhibition of USP8 activity could be an effective strategy for CD. It might provide a novel pharmacological approach for the treatment of CD. PMID:27569239

  12. Peptidase activities in Saccharomyces cerevisiae.

    PubMed Central

    Rose, B; Becker, J M; Naider, F

    1979-01-01

    At least four distinct aminopeptidase activities and a single dipeptidase activity were found in cell extracts of a leucine-lysine auxotroph of Saccharomyces cerevisiae. The assay for peptidase activity involved polyacrylamide gel electrophoresis followed by an enzyme-coupled activity staining procedure. The aminopeptidases had largely overlapping specificities but could be distinguished from one another by their electrophoretic mobilities and activities toward different peptide substrates. Substrates tested included both free and blocked di- and tripeptides and amino acid derivatives. Images PMID:378955

  13. HOW IMMUNE PEPTIDASES CHANGE SPECIFICITY

    PubMed Central

    Raymond, Wilfred W.; Trivedi, Neil N.; Makarova, Anastasia; Ray, Manisha; Craik, Charles S.; Caughey, George H.

    2014-01-01

    Cathepsin G is a major secreted serine peptidase of neutrophils and mast cells. Studies in Ctsg-null mice suggest that cathepsin G supports antimicrobial defenses but can injure host tissues. The human enzyme has unusual “Janus-faced” ability to cleave peptides at basic (tryptic) as well as aromatic (chymotryptic) sites. Tryptic activity has been attributed to acidic Glu226 in the primary specificity pocket and underlies proposed important functions such as activation of pro-urokinase. However, most mammals, including mice, substitute Ala for Glu226, suggesting that human tryptic activity may be anomalous. To test this hypothesis, human cathepsin G was compared with mouse wild type and humanized active site mutants, revealing that mouse primary specificity is markedly narrower than that of human cathepsin G, with much greater Tyr activity and selectivity and near absence of tryptic activity. It also differs from human in resisting tryptic peptidase inhibitors (e.g., aprotinin), while favoring angiotensin destruction at Tyr4 over activation at Phe8. Ala226Glu mutants of mouse cathepsin G acquire tryptic activity and human ability to activate pro-urokinase. Phylogenetic analysis reveals that the Ala226Glu missense mutation appearing in primates 31–43 million years ago represented an apparently unprecedented way to create tryptic activity in a serine peptidase. We propose that tryptic activity is not an attribute of ancestral mammalian cathepsin G, which was primarily chymotryptic, and that primate-selective broadening of specificity opposed the general trend of increased specialization by immune peptidases and allowed acquisition of new functions. PMID:20889553

  14. Extracellular peptidases from Deinococcus radiodurans.

    PubMed

    Dalmaso, Gabriel Z L; Lage, Claudia A S; Mazotto, Ana Maria; Dias, Edilma Paraguai de Souza; Caldas, Lucio Ayres; Ferreira, Davis; Vermelho, Alane B

    2015-09-01

    The extremophile Deinococcus radiodurans wild type R1 produces peptidases (metallo- and serine-) in TGY medium and in the media supplemented with human hair (HMY) and chicken feathers (FMY). Enzymatic screening on agar plates revealed peptidase activity. In TGY medium metallopeptidases were detected corresponding to a molecular mass range of 300-85 kDa (gelatinases); 280-130 (caseinases) and a 300 and a 170 kDa (keratinases); and a gelatinolytic serine peptidase (75 kDa). In HMY medium after 144 h, D. radiodurans produced keratinase (290 U/ml), gelatinase (619 U/ml) and sulfite (26 µg/ml). TGY medium produced higher proteolytic activity: 950 U/ml of gelatinolytic (24 h); 470 U/ml of keratinolytic (24 h) and 110 U/ml of caseinolytic (72 h). In the FMY medium, we found gelatinolytic (317 U/ml), keratinolytic (43 U/ml) and caseinolytic (85 U/ml) activities. The sulfite had a maximum release at 48 h (8.1 µg/ml). Enzymography analysis revealed that the keratinases degraded keratin after 24 h of reaction. The addition of sodium sulfite (1.0 %) improved the keratin degradation. Environmental Scanning Electron microscopy revealed alterations such as damage and holes in the hair fiber cuticle after D. radiodurans growth. This work presents for the first time D. radiodurans as a new keratinolytic microorganism. PMID:26216108

  15. Regulation of the Epithelial Na+ Channel by Peptidases

    PubMed Central

    Planès, Carole; Caughey, George H.

    2008-01-01

    Recent investigations point to an important role for peptidases in regulating transcellular ion transport by the epithelial Na+ channel, ENaC. Several peptidases, including furins and proteasomal hydrolases, modulate ENaC maturation and disposal. More idiosyncratically, apical Na+ transport by ENaC in polarized epithelia of kidney, airway, and gut is stimulated constitutively by one or more trypsin-family serine peptidases, as revealed by inhibition of amiloride-sensitive Na+ transport by broad-spectrum antipeptidases, including aprotinin and bikunin/SPINT2. In vitro, the transporting activity of aprotinin-suppressed ENaC can be restored by exposure to trypsin. The prototypical channel-activating peptidase (CAP) is a type 1 membrane-anchored tryptic peptidase first identified in Xenopus kidney cells. Frog CAP1 strongly upregulates Na+ transport when coexpressed with ENaC in oocytes. The amphibian enzyme's apparent mammalian orthologue is prostasin, otherwise known as CAP1, which is coexpressed with ENaC in a variety of epithelia. In airway cells, prostasin is the major basal regulator of ENaC activity, as suggested by inhibition and knockdown experiments. Other candidate regulators of mature ENaC include CAP2/TMPRSS4 and CAP3/matriptase (also known as membrane-type serine protease 1/ST14). Mammalian CAPs are potential targets for treatment of ENaC-mediated Na+ hyperabsorption by the airway in cystic fibrosis (CF) and by the kidney in hypertension. CAPs can be important for mammalian development, as indicated by embryonic lethality in mice with null mutations of CAP1/prostasin. Mice with selectively knocked out expression of CAP1/prostasin in the epidermis and mice with globally knocked out expression of CAP3/matriptase exhibit phenotypically similar defects in skin barrier function and neonatal death from dehydration. In rats, transgenic overexpression of human prostasin disturbs salt balance and causes hypertension. Thus, several converging lines of evidence

  16. (3,3-Difluoro-pyrrolidin-1-yl)-[(2S,4S)-(4-(4-pyrimidin-2-yl-piperazin-1-yl)-pyrrolidin-2-yl]-methanone: A potent, selective, orally active dipeptidyl peptidase IV inhibitor

    SciTech Connect

    Ammirati, Mark J.; Andrews, Kim M.; Boyer, David D.; Brodeur, Anne M.; Danley, Dennis E.; Doran, Shawn D.; Hulin, Bernard; Liu, Shenping; McPherson, R. Kirk; Orena, Stephen J.; Parker, Janice C.; Polivkova, Jana; Qiu, Xiayang; Soglia, Carolyn B.; Treadway, Judith L.; VanVolkenburg, Maria A.; Wilder, Donald C.; Piotrowski, David W.; Pfizer

    2010-10-01

    A series of 4-substituted proline amides was synthesized and evaluated as inhibitors of dipeptidyl pepdidase IV for the treatment of type 2 diabetes. (3,3-Difluoro-pyrrolidin-1-yl)-[(2S,4S)-(4-(4-pyrimidin-2-yl-piperazin-1-yl)-pyrrolidin-2-yl]-methanone (5) emerged as a potent (IC{sub 50} = 13 nM) and selective compound, with high oral bioavailability in preclinical species and low plasma protein binding. Compound 5, PF-00734200, was selected for development as a potential new treatment for type 2 diabetes.

  17. Fluorometric assay using naphthylamide substrates for assessing novel venom peptidase activities.

    PubMed

    Gasparello-Clemente, Elaine; Silveira, Paulo Flávio

    2002-11-01

    In the present study we examined the feasibility of using the fluorometry of naphthylamine derivatives for revealing peptidase activities in venoms of the snakes Bothrops jararaca, Bothrops alternatus, Bothrops atrox, Bothrops moojeni, Bothrops insularis, Crotalus durissus terrificus and Bitis arietans, of the scorpions Tityus serrulatus and Tityus bahiensis, and of the spiders Phoneutria nigriventer and Loxosceles intermedia. Neutral aminopeptidase (APN) and prolyl-dipeptidyl aminopeptidase IV (DPP IV) activities were presented in all snake venoms, with the highest levels in B. alternatus. Although all examined peptidase activities showed relatively low levels in arthropod venoms, basic aminopeptidase (APB) activity from P. nigriventer venom was the exception. Compared to the other peptidase activities, relatively high levels of acid aminopeptidase (APA) activity were restricted to B. arietans venom. B. arietans also exhibited a prominent content of APB activity which was lower in other venoms. Relatively low prolyl endopeptidase and proline iminopeptidase activities were, respectively, detectable only in T. bahiensis and B. insularis. Pyroglutamate aminopeptidase activity was undetectable in all venoms. All examined peptidase activities were undetectable in T. serrulatus venom. In this study, the specificities of a diverse array of peptidase activities from representative venoms were demonstrated for the first time, with a description of their distribution which may contribute to guiding further investigations. The expressive difference between snake and arthropod venoms was indicated by APN and DPP IV activities while APA and APB activities distinguished the venom of B. arietans from those of Brazilian snakes. The data reflected the relatively uniform qualitative distribution of the peptidase activities investigated, together with their unequal quantitative distribution, indicating the evolutionary divergence in the processing of peptides in these different

  18. Low micromolar concentrations of the superoxide probe MitoSOX uncouple neural mitochondria and inhibit complex IV.

    PubMed

    Roelofs, Brian A; Ge, Shealinna X; Studlack, Paige E; Polster, Brian M

    2015-09-01

    MitoSOX Red is a fluorescent probe used for the detection of mitochondrial reactive oxygen species by live cell imaging. The lipophilic, positively charged triphenylphosphonium moiety within MitoSOX concentrates the superoxide-sensitive dihydroethidium conjugate within the mitochondrial matrix. Here we investigated whether common MitoSOX imaging protocols influence mitochondrial bioenergetic function in primary rat cortical neurons and microglial cell lines. MitoSOX dose-dependently uncoupled neuronal respiration, whether present continuously in the assay medium or washed following a ten minute loading protocol. Concentrations of 5-10μM MitoSOX caused severe loss of ATP synthesis-linked respiration. Redistribution of MitoSOX to the cytoplasm and nucleus occurred concomitant to mitochondrial uncoupling. MitoSOX also dose-dependently decreased the maximal respiration rate and this impairment could not be rescued by delivery of a complex IV specific substrate, revealing complex IV inhibition. As in neurons, loading microglial cells with MitoSOX at low micromolar concentrations resulted in uncoupled mitochondria with reduced respiratory capacity whereas submicromolar MitoSOX had no adverse effects. The MitoSOX parent compound dihydroethidium also caused mitochondrial uncoupling and respiratory inhibition at low micromolar concentrations. However, these effects were abrogated by pre-incubating dihydroethidium with cation exchange beads to remove positively charged oxidation products, which would otherwise by sequestered by polarized mitochondria. Collectively, our results suggest that the matrix accumulation of MitoSOX or dihydroethidium oxidation products causes mitochondrial uncoupling and inhibition of complex IV. Because MitoSOX is inherently capable of causing severe mitochondrial dysfunction with the potential to alter superoxide production, its use therefore requires careful optimization in imaging protocols. PMID:26057935

  19. Antimicrobial peptide inhibition of fungalysin proteases that target plant type 19 Family IV defense chitinases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cereal crops and other plants produce secreted seed chitinases that reduce pathogenic infection, most likely by targeting the fungal chitinous cell wall. We have shown that corn (Zea mays) produces three GH family 19, plant class IV chitinases, that help in protecting the plant against Fusarium and ...

  20. The Inhibition Of Pb(IV) Oxide Formation In Chlorinated Water By Orthophosphate

    EPA Science Inventory

    Historically, understanding lead solubility and its control in drinking water has been based on Pb(II) chemistry. Unfortunately, there is very little information available regarding the nature of Pb(IV) oxides in finished drinking water and water distribution systems, and the co...

  1. An Essential Signal Peptide Peptidase Identified in an RNAi Screen of Serine Peptidases of Trypanosoma brucei

    PubMed Central

    Moss, Catherine X.; Brown, Elaine; Hamilton, Alana; Van der Veken, Pieter; Augustyns, Koen; Mottram, Jeremy C.

    2015-01-01

    The serine peptidases of Trypanosoma brucei have been viewed as potential drug targets. In particular, the S9 prolyl oligopeptidase subfamily is thought to be a good avenue for drug discovery. This is based on the finding that some S9 peptidases are secreted and active in the mammalian bloodstream, and that they are a class of enzyme against which drugs have successfully been developed. We collated a list of all serine peptidases in T. brucei, identifying 20 serine peptidase genes, of which nine are S9 peptidases. We screened all 20 serine peptidases by RNAi to determine which, if any, are essential for bloodstream form T. brucei survival. All S9 serine peptidases were dispensable for parasite survival in vitro, even when pairs of similar genes, coding for oligopeptidase B or prolyl oligopeptidase, were targeted simultaneously. We also found no effect on parasite survival in an animal host when the S9 peptidases oligopeptidase B, prolyl oligopeptidase or dipeptidyl peptidase 8 were targeted. The only serine peptidase to emerge from the RNAi screen as essential was a putative type-I signal peptide peptidase (SPP1). This gene was essential for parasite survival both in vitro and in vivo. The growth defect conferred by RNAi depletion of SPP1 was rescued by expression of a functional peptidase from an RNAi resistant SPP1 gene. However, expression of catalytically inactive SPP1 was unable to rescue cells from the SPP1 depleted phenotype, demonstrating that SPP1 serine peptidase activity is necessary for T. brucei survival. PMID:25816352

  2. Aspartic Peptidases of Human Pathogenic Trypanosomatids: Perspectives and Trends for Chemotherapy

    PubMed Central

    Santos, L.O.; Garcia-Gomes, A.S.; Catanho, M.; Sodré, C.L.; Santos, A.L.S.; Branquinha, M.H.; d’Avila-Levy, C.M.

    2013-01-01

    Aspartic peptidases are proteolytic enzymes present in many organisms like vertebrates, plants, fungi, protozoa and in some retroviruses such as human immunodeficiency virus (HIV). These enzymes are involved in important metabolic processes in microorganisms/virus and play major roles in infectious diseases. Although few studies have been performed in order to identify and characterize aspartic peptidase in trypanosomatids, which include the etiologic agents of leishmaniasis, Chagas’ disease and sleeping sickness, some beneficial properties of aspartic peptidase inhibitors have been described on fundamental biological events of these pathogenic agents. In this context, aspartic peptidase inhibitors (PIs) used in the current chemotherapy against HIV (e.g., amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) were able to inhibit the aspartic peptidase activity produced by different species of Leishmania. Moreover, the treatment of Leishmania promastigotes with HIV PIs induced several perturbations on the parasite homeostasis, including loss of the motility and arrest of proliferation/growth. The HIV PIs also induced an increase in the level of reactive oxygen species and the appearance of irreversible morphological alterations, triggering parasite death pathways such as programed cell death (apoptosis) and uncontrolled autophagy. The blockage of physiological parasite events as well as the induction of death pathways culminated in its incapacity to adhere, survive and escape of phagocytic cells. Collectively, these results support the data showing that parasites treated with HIV PIs have a significant reduction in the ability to cause in vivo infection. Similarly, the treatment of Trypanosoma cruzi cells with pepstatin A showed a significant inhibition on both aspartic peptidase activity and growth as well as promoted several and irreversible morphological changes. These studies indicate that aspartic peptidases can be promising targets in

  3. DPP IV inhibitor blocks mescaline-induced scratching and amphetamine-induced hyperactivity in mice.

    PubMed

    Lautar, Susan L; Rojas, Camilo; Slusher, Barbara S; Wozniak, Krystyna M; Wu, Ying; Thomas, Ajit G; Waldon, Daniel; Li, William; Ferraris, Dana; Belyakov, Sergei

    2005-06-28

    Dipeptidyl peptidase IV (DPP IV) is a ubiquitous membrane-bound enzyme that cleaves the two N-terminal amino acids from peptides with a proline or alanine residue in the second position from the amino end. Potential substrates for DPP IV include several neuropeptides, suggesting a role for DPP IV in neurological processes. We have developed a potent DPP IV inhibitor (IC50 = 30 nM), 1-(2-amino-3-methyl-butyryl)-azetidine-2-carbonitrile (AMAC), which has shown efficacy in two established models of psychosis: mescaline-induced scratching and amphetamine-induced hyperactivity. In the mescaline-induced scratching model, AMAC treatment before mescaline administration reduced the number of scratching paroxysms by 68% (P < 0.01). The compound showed a dose-dependent effect, inhibiting significantly at 6, 20 and 60 mg/kg (37%, 39% and 68%, respectively). In the amphetamine-induced hyperactivity model, 50 and 60 mg/kg AMAC, given before injection of amphetamine, significantly reduced hyper-locomotion by 65% and 76%, respectively. Additionally, AMAC showed no significant activity in binding assays for 20 receptors thought to be involved in the pathology of schizophrenia, including dopamine, serotonin and glutamate. A structurally similar analog, 1-(2-dimethylamino-3-methyl-butyryl)-azetidine-2-carbonitrile (DAMAC), that does not inhibit DPP IV, was inactive in both models. Taken together, these data suggest that the antipsychotic effects of AMAC are the result of DPP IV inhibition. PMID:15925329

  4. Inhibiting albumin glycation attenuates dysregulation of VEGFR-1 and collagen IV subchain production and the development of renal insufficiency.

    PubMed

    Cohen, Margo P; Lautenslager, Gregory T; Hud, Elizabeth; Shea, Elizabeth; Wang, Amy; Chen, Sheldon; Shearman, Clyde W

    2007-02-01

    Glomerular cells in culture respond to albumin containing Amadori glucose adducts (the principal serum glycated protein), with activation of protein kinase C-beta(1), increased expression of transforming growth factor (TGF)-beta1, the TGF-beta type II signaling receptor, and the extracellular matrix proteins alpha(1)(IV) collagen and fibronectin and with decreased production of the podocyte protein nephrin. Decreasing the burden of glycated albumin in diabetic db/db mice significantly reduces glomerular overexpression of TGF-beta1 mRNA, restores glomerular nephrin immunofluorescence, and lessens proteinuria, mesangial expansion, renal extracellular matrix protein production, and increased glomerular vascular endothelial growth factor (VEGF) immunostaining. In the present study, db/db mice were treated with a small molecule, designated 23CPPA, that inhibits the nonenzymatic condensation of glucose with the albumin protein to evaluate whether increased glycated albumin influences the production of VEGF receptors (VEGFRs) and type IV collagen subchains and ameliorates the development of renal insufficiency. Renal levels of VEGF and VEGFR-1 proteins and serum creatinine concentrations were significantly higher and renal levels of alpha(3)(IV) collagen and nephrin proteins and endogenous creatinine clearance values were significantly lower in control diabetic than in age-matched nondiabetic (db/m) mice. These changes were significantly attenuated in db/db littermate mice treated from 9 to 18 wk of age with 23CPPA. The findings indicate that inhibiting excess nonenzymatic glycation of serum albumin improves renal molecular biology abnormalities and protects against the development of renal insufficiency in the db/db mouse. PMID:17018845

  5. Inhibition of Elastin Peptide-Mediated Angiogenic Signaling Mechanism(s) in Choroidal Endothelial Cells by the α6(IV)NC1 Collagen Fragment

    PubMed Central

    Gunda, Venugopal; Verma, Raj Kumar; Sudhakar, Yakkanti Akul

    2013-01-01

    Purpose. The inhibitory effects and mechanism(s) of type IV collagen α-6 chain–derived noncollagenous domain (α6[IV]NC1 or hexastatin) on elastin-derived peptide (EDP)–activated choroidal endothelial cell migration, kinase signaling, and membrane type 1 metalloproteinase (MT1-MMP) activation are explored. Methods. Mouse choroidal endothelial cells (MCECs) were incubated in media with soluble EDPs (kappa elastin, mouse elastin, and Val-Gly-Val-Ala-Pro-Gly [VGVAPG] hexapeptide) for different time intervals with or without α6(IV)NC1. The MCECs proliferation, migration, tube formation, MT1-MMP expression, and angiogenic signaling were analyzed in cells subjected to EDP and α6(IV)NC1 treatments. The MCECs also were subjected to EDPs, and specific inhibitors for evaluation of focal adhesion kinase (FAK) and protein kinase B (Akt) phosphorylation. Results. Kappa elastin, mouse elastin, and VGVAPG enhanced the migration, without affecting the proliferation of MCECs. The α6(IV)NC1 inhibited survival and EDP-activated migration of MCECs. The EDP-activated MCEC tube formation on matrigel also was inhibited by α6(IV)NC1. Further, EDP-activated MT1-MMP expression and FAK/phosphoinositide-3-kinase (PI-3K)/mammalian target of rapamycin (mToR)/Akt phosphorylation in MCECs, were reduced by α6(IV)NC1. The EDP-induced FAK and Akt phosphorylation was blocked by FAK- and Akt-specific inhibitors. Conclusions. The EDPs and α6(IV)NC1 are identified to exhibit opposing effects on MCEC angiogenic behavior and signaling. The α6(IV)NC1 inhibited cell survival, EDP-mediated migration, MT1-MMP expression and, FAK/PI-3K/mToR/Akt phosphorylation in MCECs. This work demonstrates α6(IV)NC1 as a prospective endogenous molecule for the treatment of diseases involving choroidal neovascularization in the eye. PMID:24194191

  6. Evaluation of the catalytic specificity, biochemical properties, and milk clotting abilities of an aspartic peptidase from Rhizomucor miehei.

    PubMed

    da Silva, Ronivaldo Rodrigues; Souto, Tatiane Beltramini; de Oliveira, Tássio Brito; de Oliveira, Lilian Caroline Gonçalves; Karcher, Daniel; Juliano, Maria Aparecida; Juliano, Luiz; de Oliveira, Arthur H C; Rodrigues, André; Rosa, Jose C; Cabral, Hamilton

    2016-08-01

    In this study, we detail the specificity of an aspartic peptidase from Rhizomucor miehei and evaluate the effects of this peptidase on clotting milk using the peptide sequence of k-casein (Abz-LSFMAIQ-EDDnp) and milk powder. Molecular mass of the peptidase was estimated at 37 kDa, and optimum activity was achieved at pH 5.5 and 55 °C. The peptidase was stable at pH values ranging from 3 to 5 and temperatures of up 45 °C for 60 min. Dramatic reductions in proteolytic activity were observed with exposure to sodium dodecyl sulfate, and aluminum and copper (II) chloride. Peptidase was inhibited by pepstatin A, and mass spectrometry analysis identified four peptide fragments (TWSISYGDGSSASGILAK, ASNGGGGEYIFGGYDSTK, GSLTTVPIDNSR, and GWWGITVDRA), similar to rhizopuspepsin. The analysis of catalytic specificity showed that the coagulant activity of the peptidase was higher than the proteolytic activity and that there was a preference for aromatic, basic, and nonpolar amino acids, particularly methionine, with specific cleavage of the peptide bond between phenylalanine and methionine. Thus, this peptidase may function as an important alternative enzyme in milk clotting during the preparation of cheese. PMID:27165660

  7. Cathepsin K: a unique collagenolytic cysteine peptidase.

    PubMed

    Novinec, Marko; Lenarčič, Brigita

    2013-09-01

    Cathepsin K has emerged as a promising target for the treatment of osteoporosis in recent years. Initially identified as a papain-like cysteine peptidase expressed in high levels in osteoclasts, the important role of this enzyme in bone metabolism was highlighted by the finding that mutations in the CTSK gene cause the rare recessive disorder pycnodysostosis, which is characterized by severe bone anomalies. At the molecular level, the physiological role of cathepsin K is reflected by its unique cleavage pattern of type I collagen molecules, which is fundamentally different from that of other endogenous collagenases. Several cathepsin K inhibitors have been developed to reduce the excessive bone matrix degradation associated with osteoporosis, with the frontrunner odanacatib about to successfully conclude Phase 3 clinical trials. Apart from osteoclasts, cathepsin K is expressed in different cell types throughout the body and is involved in processes of adipogenesis, thyroxine liberation and peptide hormone regulation. Elevated activity of cathepsin K has been associated with arthritis, atherosclerosis, obesity, schizophrenia, and tumor metastasis. Accordingly, its activity is tightly regulated via multiple mechanisms, including competitive inhibition by endogenous macromolecular inhibitors and allosteric regulation by glycosaminoglycans. This review provides a state-of-the-art description of the activity of cathepsin K at the molecular level, its biological functions and the mechanisms involved in its regulation. PMID:23629523

  8. A novel arctigenin-containing latex glove prevents latex allergy by inhibiting type I/IV allergic reactions.

    PubMed

    Wang, Yong-Xin; Xue, Dan-Ting; Liu, Meng; Zhou, Zheng-Min; Shang, Jing

    2016-03-01

    The present study aimed at developing a natural compound with anti-allergic effect and stability under latex glove manufacturing conditions and investigating whether its anti-allergic effect is maintained after its addition into the latex. The effects of nine natural compounds on growth of the RBL-2H3 cells and mouse primary spleen lymphocytes were determined using MTT assay. The compounds included glycyrrhizin, osthole, tetrandrine, tea polyphenol, catechin, arctigenin, oleanolic acid, baicalin and oxymatrine. An ELISA assay was used for the in vitro anti-type I/IV allergy screening; in this process β-hexosaminidase, histamine, and IL-4 released from RBL-2H3 cell lines and IFN-γ and IL-2 released from mouse primary spleen lymphocytes were taken as screening indices. The physical stability of eight natural compounds and the dissolubility of arctigenin, selected based on the in vitro pharnacodynamaic screening and the stability evaluation, were detected by HPLC. The in vivo pharmacodynamic confirmation of arctigenin and final latex product was evaluated with a passive cutaneous anaphylaxis (PCA) model and an allergen-specific skin response model. Nine natural compounds showed minor growth inhibition on RBL-2H3 cells and mouse primary spleen lymphocytes. Baicalin and arctigenin had the best anti-type I and IV allergic effects among the natural compounds based on the in vitro pharmacodynamic screening. Arctigenin and catechin had the best physical stability under different manufacturing conditions. Arctigenin was the selected for further evaluation and proven to have anti-type I and IV allergic effects in vivo in a dose-dependent manner. The final product of the arctigenin-containing latex glove had anti-type I and IV allergic effects in vivo which were mainly attributed to arctigenin as proved from the dissolubility results. Arctigenin showed anti-type I and IV allergic effects in vitro and in vivo, with a good stability under latex glove manufacturing conditions

  9. Structural Basis for Specificity of Propeptide-Enzyme Interaction in Barley C1A Cysteine Peptidases

    PubMed Central

    Cambra, Inés; Hernández, David; Diaz, Isabel; Martinez, Manuel

    2012-01-01

    C1A cysteine peptidases are synthesized as inactive proenzymes. Activation takes place by proteolysis cleaving off the inhibitory propeptide. The inhibitory capacity of propeptides from barley cathepsin L and B-like peptidases towards commercial and barley cathepsins has been characterized. Differences in selectivity have been found for propeptides from L-cathepsins against their cognate and non cognate enzymes. Besides, the propeptide from barley cathepsin B was not able to inhibit bovine cathepsin B. Modelling of their three-dimensional structures suggests that most propeptide inhibitory properties can be explained from the interaction between the propeptide and the mature cathepsin structures. Their potential use as biotechnological tools is discussed. PMID:22615948

  10. Kinetics of Extracellular Peptidases in Sediments of the White Oak River, NC, USA

    NASA Astrophysics Data System (ADS)

    Steen, A. D.; Kevorkian, R. T.; Alperin, M. J.; Lloyd, K. G.

    2013-12-01

    Recent molecular work has shed light on the mechanisms underlying organoheterotrophy in the marine subsurface, including production of extracellular peptidases by deeply-branching Archaea. Here we present measurements of the potential activity (Vmax) and half-saturation constants (Km) for six extracellular peptidase substrates in sediments from 0 to 83 cm deep in the White Oak River estuary, NC, USA. Potential activities at 83 cm were on average 12% of the values at the surface, but because surface Vmax values were several orders of magnitude greater than comparable values from surface seawater, the deep activities were still substantial. Km values did not display a clear trend with depth. Activities consistent with leucyl aminopeptidase were higher than any other extracellular peptidase, but there was no clear division in activities between endopeptidases (which cleave bonds in the interior of proteins) versus aminopeptidases (which cleave N-terminal amino acids). Competitive inhibition experiments will reveal the extent to which the activities we measured reflect the distinct enzymes. We will also present model-based estimates of organic carbon mineralization rates based on methane and sulfate profiles in order to assess the relative importance of extracellular peptidases as a means to acquire organic carbon in the subsurface. Saturation curves for 5 peptidase substrates at the surface and 83 cm in the White Oak River.

  11. Dipeptidyl peptidase 4 – an important digestive peptidase in Tenebrio molitor larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Dipeptidyl peptidase 4 (DPP 4) is a proline specific serine peptidase that plays an important role in different regulatory processes in mammals. In this report, we isolated and characterized a unique secreted digestive DPP 4 from the anterior midgut of a stored product pest, Tenebrio molitor larvae ...

  12. ASP4000, a slow-binding dipeptidyl peptidase 4 inhibitor, has antihyperglycemic activity of long duration in Zucker fatty rats.

    PubMed

    Tanaka-Amino, Keiko; Matsumoto, Kazumi; Hatakeyama, Yoshifumi; Takakura, Shoji; Mutoh, Seitaro

    2010-03-01

    ASP4000 ((2S)-1-{[(1R,3S,4S,6R)-6-hydroxy-2-azabicyclo[2.2.1]hept-3-yl]carbonyl}-2-pyrrolidinecarbonitrile hydrochloride) is a novel, potent and selective dipeptidyl peptidase 4 (DPP IV, EC 3.4.14.5) inhibitor (Keiko Tanaka-Amino et al. in Eur J pharmacol 59:444-449, 2008). The aim of the present study was to characterize the kinetic profile of and identify the long duration effect of the antihyperglycemic activity of ASP4000. ASP4000 was found to inhibit human recombinant DPP4 activity with a K(i) of 1.05 nM, a k(on) value of 22.3 x 10(5) M(-1) s(-1), and a k (off) of 2.35 x 10(-3) M(-1) s(-1), with higher affinity than that of vildagliptin. The kinetic studies indicate that both the formation and dissociation of ASP4000/DPP4 complex were faster than those of vildagliptin, and that ASP4000 slow-bindingly inhibits DPP4 with a different mode of inhibition than vildagliptin. In addition, ASP4000 augmented the insulin response and ameliorated the glucose excursion during the oral glucose tolerance test in Zucker fatty rats at 4 h post dosing. ASP4000 is expected to be a promising, long duration DPP4 inhibitor for type 2 diabetes. PMID:19238312

  13. Tetrathiomolybdate inhibits mitochondrial complex IV and mediates degradation of hypoxia-inducible factor-1α in cancer cells

    PubMed Central

    Kwang Kim, Kyu; Abelman, Sarah; Yano, Naohiro; Ribeiro, Jennifer R.; Singh, Rakesh K.; Tipping, Marla; Moore, Richard G.

    2015-01-01

    Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that triggers adaptive responses upon low oxygen conditions and plays a crucial role in cancer metabolism and therapy resistance. Tetrathiomolybdate (TM), a therapy option for copper overload disorder, has also been shown to be capable of limiting tumor angiogenesis, although its underlying mechanism remains unclear. Using ovarian and endometrial cancer cell lines, we observed that TM downregulates HIF-1α protein levels and HIF-transcriptional targets involved in tumor angiogenesis and glycolysis, but did not affect HIF-1α protein synthesis. TM-mediated HIF-1α downregulation was suppressed when HIF-prolyl hydroxylase activity was pharmacologically inhibited using deferoxamine or dimethyloxaloylglycine, and also when the oxygen-dependent degradation domains of HIF-1α, which are responsible for the interaction with HIF-prolyl hydroxylase, were deleted. These findings suggest that TM causes HIF-1α downregulation in a HIF-prolyl hydroxylase-dependent manner. Our studies showed that TM inhibits the activity of the copper-dependent mitochondrial complex IV and reduces mitochondrial respiration, thereby possibly increasing oxygen availability, which is crucial for HIF-prolyl hydroxylase activity. Pimonidazole staining also showed that TM elevates oxygen tension in hypoxic cells. Our studies provide mechanistic evidence for TM-mediated HIF-1α regulation and suggest its therapeutic potential as a method of blocking angiogenesis in ovarian and endometrial tumors. PMID:26469226

  14. Hydrolysis of wheat gluten by combining peptidases of Flammulina velutipes and electrodialysis.

    PubMed

    Giesler, Lucienne; Linke, Diana; Rabe, Swen; Appel, Daniel; Berger, Ralf Günter

    2013-09-11

    Wheat gluten hydrolysis, used to generate seasonings, was studied using peptidases from Flammulina velutipes or commercial Flavourzyme. L-amino acids were added in a range from 0.5 to 75.0 mM, and L-isoleucine, L-leucine, L-valine, and L-phenylalanine were identified as the strongest inhibitors for both enzyme mixtures. L-serine inhibited Flammulina velutipes peptidases only, while L-histidine and L-glutamine inhibited Flavourzyme peptidases only. To reduce product inhibition by released L-amino acids, electrodialysis was explored. An increase of the degree of hydrolysis of up to 60% for Flammulina velutipes peptidases and 31% for Flavourzyme compared to that for the best control batch was observed after applying an electrodialysis unit equipped with an ultrafiltration membrane for two times 1 h during the 20 h of hydrolysis. The total transfer of free L-amino acids into the concentrate reached 25-30% per hour. Peptides passed the membrane less easily, although the nominal cutoff was 4 kDa. PMID:23947566

  15. Investigation of DNA binding, DNA photocleavage, topoisomerase I inhibition and antioxidant activities of water soluble titanium(IV) phthalocyanine compounds.

    PubMed

    Özel, Arzu; Barut, Burak; Demirbaş, Ümit; Biyiklioglu, Zekeriya

    2016-04-01

    The binding mode of water soluble peripherally tetra-substituted titanium(IV) phthalocyanine (Pc) compounds Pc1, Pc2 and Pc3 with calf thymus (CT) DNA was investigated by using UV-Vis spectroscopy and thermal denaturation studies in this work. The results of DNA binding constants (Kb) and the changes in the thermal denaturation profile of DNA with the addition of Pc compounds indicated that Pc1, Pc2 and Pc3 are able to bind to CT-DNA with different binding affinities. DNA photocleavage studies of Pc compounds were performed in the absence and presence of oxidizing agents such as hydrogen peroxide (H2O2), ascorbic acid (AA) and 2-mercaptoethanol (ME) using the agarose gel electrophoresis method at irradiation 650nm. According to the results of electrophoresis studies, Pc1, Pc2 and Pc3 cleaved of supercoiled pBR322 DNA via photocleavage pathway. The Pc1, Pc2 and Pc3 compounds were examined for topoisomerase I inhibition by measuring the relaxation of supercoiled pBR322 DNA. The all of Pc compounds inhibited topoisomerase I at 20μM concentration. A series of antioxidant assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, superoxide radical scavenging (SOD) assay and metal chelating effect assay were performed for Pc1, Pc2 and Pc3 compounds. The results of antioxidant assays indicated that Pc1, Pc2 and Pc3 compounds have remarkable superoxide radical scavenging activities, moderate 2,2-diphenyl-1-picrylhydrazyl activities and metal chelating effect activities. All the experimental studies showed that Pc1, Pc2 and Pc3 compounds bind to CT-DNA via minor groove binding, cleave of supercoiled pBR322 DNA via photocleavage pathway, inhibit topoisomerase I and have remarkable superoxide radical scavenging activities. Thanks to these properties the Pc1, Pc2 and Pc3 compounds are suitable agents for photo dynamic therapy. PMID:26882290

  16. Aminopiperidine-Fused Imidazoles as Dipeptidyl Peptidase-IV Inhibitors

    SciTech Connect

    Edmondson, S.; Mastracchio, A; Cox, J; Eiermann, G; He, H; Lyons, K; Patel, R; Patel, S; Petrov, A; et. al.

    2009-01-01

    A new series of DPP-4 inhibitors derived from piperidine-fused benzimidazoles and imidazopyridines is described. Optimization of this class of DPP-4 inhibitors led to the discovery of imidazopyridine 34. The potency, selectivity, cross-species DMPK profiles, and in vivo efficacy of 34 is reported.

  17. Astragaloside IV suppresses transforming growth factor-β1 induced fibrosis of cultured mouse renal fibroblasts via inhibition of the MAPK and NF-κB signaling pathways

    SciTech Connect

    Che, Xiajing; Wang, Qin; Xie, Yuanyuan; Xu, Weijia; Shao, Xinghua; Mou, Shan Ni, Zhaohui

    2015-09-04

    Renal fibrosis, a progressive process characterized by the accumulation of extracellular matrix (ECM) leading to organ dysfunction, is a characteristic of chronic kidney diseases. Among fibrogenic factors known to regulate the renal fibrotic process, transforming growth factor-β (TGF-β) plays a central role. In the present study, we examined the effect of Astragaloside IV (AS-IV), a component of the traditional Chinese medicinal plant Astragalus membranaceus, on the processes associated with renal fibrosis in cultured mouse renal fibroblasts treated with TGF-β1. RT-PCR, western blotting, immunofluorescence staining and collagen assays showed that AS-IV suppressed TGF-β1 induced fibroblast proliferation, transdifferentiation, and ECM production in a dose-dependent manner. Examination of the underlying mechanisms showed that the effect of AS-IV on the inhibition of fibroblast differentiation and ECM formation were mediated by its modulation of the activity of the MAPK and NF-κB signaling pathways. Taken together, our results indicate that AS-IV alleviates renal interstitial fibrosis via a mechanism involving the MAPK and NF-κB signaling pathways and demonstrate the therapeutic potential of AS-IV for the treatment of chronic kidney diseases. - Highlights: • AS-IV suppressed TGF-β1 induced renal fibroblast proliferation. • AS-IV suppressed TGF-β1 induced renal fibroblast transdifferentiation. • AS-IV suppressed TGF-β1 induced ECM production. • AS-IV alleviates renal fibrosis via the MAPK and NF-κB signaling pathways.

  18. Peptidase activity of beta-lactamases.

    PubMed Central

    Rhazi, N; Galleni, M; Page, M I; Frère, J M

    1999-01-01

    Although beta-lactamases have generally been considered as being devoid of peptidase activity, a low but significant hydrolysis of various N-acylated dipeptides was observed with representatives of each class of beta-lactamases. The kcat/Km values were below 0.1 M(-1). s(-1), but the enzyme rate enhancement factors were in the range 5000-20000 for the best substrates. Not unexpectedly, the best 'peptidase' was the class C beta-lactamase of Enterobacter cloacae P99, but, more surprisingly, the activity was always higher with the phenylacetyl- and benzoyl-d-Ala-d-Ala dipeptides than with the diacetyl- and alpha-acetyl-l-Lys-d-Ala-d-Ala tripeptides, which are the preferred substrates of the low-molecular-mass, soluble dd-peptidases. A comparison between the beta-lactamases and dd-peptidases showed that it might be as difficult for a dd-peptidase to open the beta-lactam ring as it is for the beta-lactamases to hydrolyse the peptides, an observation which can be explained by geometric and stereoelectronic considerations. PMID:10393100

  19. Sequential processing of hepatitis C virus core protein by host cell signal peptidase and signal peptide peptidase: a reassessment.

    PubMed

    Pène, V; Hernandez, C; Vauloup-Fellous, C; Garaud-Aunis, J; Rosenberg, A R

    2009-10-01

    Hepatitis C virus (HCV) core protein is believed to play critical roles in the virus morphogenesis and pathogenesis. In HCV polyprotein, core protein terminates with a signal peptide followed by E1 envelope protein. It has remained unclear whether cleavage by host cell signal peptidase (SP) at the core-E1 junction to generate the complete form of core protein, which is anchored in the endoplasmic reticulum membrane, is absolutely required for cleavage within the signal peptide by host cell signal peptide peptidase (SPP) to liberate the mature form of core protein, which is then free for trafficking to lipid droplets. In this study, the possible sources of disagreement in published reports have been examined, and we conclude that a product generated upon inhibition of SP-catalysed cleavage at the core-E1 junction in heterologous expression systems was incorrectly identified as mature core protein. Moreover, inhibition of this cleavage in the most relevant model of human hepatoma cells replicating a full-length HCV genome was shown to abolish interaction of core protein with lipid droplets and production of infectious progeny virus. These results firmly establish that SPP-catalysed liberation of mature core protein is absolutely dependent on prior cleavage by SP at the correct core-E1 site to generate the complete form of core protein, consistent with this obligatory order of processing playing a role in HCV infectious cycle. PMID:19281487

  20. Astragaloside IV prevents damage to human mesangial cells through the inhibition of the NADPH oxidase/ROS/Akt/NF‑κB pathway under high glucose conditions.

    PubMed

    Sun, Li; Li, Weiping; Li, Weizu; Xiong, Li; Li, Guiping; Ma, Rong

    2014-07-01

    Glomerular hypertrophy and hyperfiltration are the two major pathological characteristics of the early stages of diabetic nephropathy (DN), which are respectively related to mesangial cell (MC) proliferation and a decrease in calcium influx conducted by canonical transient receptor potential cation channel 6 (TRPC6). The marked increase in the production of reactive oxygen species (ROS) induced by hyperglycemia is the main sponsor of multiple pathological pathways in DN. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an important source of ROS production in MCs. Astragaloside IV (AS‑IV) is an active ingredient of Radix Astragali which has a potent antioxidative effect. In this study, we aimed to investigate whether high glucose (HG)‑induced NADPH oxidase activation and ROS production contribute to MC proliferation and the downregulation of TRPC6 expression; we also wished to determine the effects of AS‑IV on MCs under HG conditions. Using a human glomerular mesangial cell line, we found that treatment with AS‑IV for 48 h markedly attenuated HG‑induced proliferation and the hypertrophy of MCs in a dose‑dependent manner. The intracellular ROS level was also markedly reduced following treatment with AS‑IV. In addition, the enhanced activity of NADPH oxidase and the expression level of NADPH oxidase 4 (Nox4) protein were decreased. Treatment with AS‑IV also inhibited the phosphorylation level of Akt and IκBα in the MCs. In addition, TRPC6 protein expression and the intracellular free calcium concentration were also markedly reduced following treatment with AS‑IV under HG conditions. These results suggest that AS‑IV inhibits HG‑induced mesangial cell proliferation and glomerular contractile dysfunction through the NADPH oxidase/ROS/Akt/nuclear factor‑κB (NF‑κB) pathway, providing a new perspective for the clinical treatment of DN. PMID:24718766

  1. Apolipoprotein A-IV inhibits AgRP/NPY neurons and activates POMC neurons in the arcuate nucleus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Apolipoprotein A-IV (apoA-IV) in the brain potently suppresses food intake. However the mechanisms underlying its anorexigenic effects remain to be identified. We first examined the effects of apoA-IV on cellular activities in hypothalamic neurons that co-express agouti-related peptide (AgRP) and ne...

  2. The Dipeptidyl Peptidase Family, Prolyl Oligopeptidase, and Prolyl Carboxypeptidase in the Immune System and Inflammatory Disease, Including Atherosclerosis

    PubMed Central

    Waumans, Yannick; Baerts, Lesley; Kehoe, Kaat; Lambeir, Anne-Marie; De Meester, Ingrid

    2015-01-01

    Research from over the past 20 years has implicated dipeptidyl peptidase (DPP) IV and its family members in many processes and different pathologies of the immune system. Most research has been focused on either DPPIV or just a few of its family members. It is, however, essential to consider the entire DPP family when discussing any one of its members. There is a substantial overlap between family members in their substrate specificity, inhibitors, and functions. In this review, we provide a comprehensive discussion on the role of prolyl-specific peptidases DPPIV, FAP, DPP8, DPP9, dipeptidyl peptidase II, prolyl carboxypeptidase, and prolyl oligopeptidase in the immune system and its diseases. We highlight possible therapeutic targets for the prevention and treatment of atherosclerosis, a condition that lies at the frontier between inflammation and cardiovascular disease. PMID:26300881

  3. NAAG peptidase inhibitors and deletion of NAAG peptidase gene enhance memory in novel object recognition test.

    PubMed

    Janczura, Karolina J; Olszewski, Rafal T; Bzdega, Tomasz; Bacich, Dean J; Heston, Warren D; Neale, Joseph H

    2013-02-15

    The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) is inactivated by the extracellular enzyme glutamate carboxypeptidase II. Inhibitors of this enzyme reverse dizocilpine (MK-801)-induced impairment of short-term memory in the novel object recognition test. The objective of this study was to test the hypothesis that NAAG peptidase inhibition enhances long-term (24h delay) memory of C57BL mice. These mice and mice in which glutamate carboxypeptidase II had been knocked out were presented with two identical objects to explore for 10min on day 1 and tested with one of these familiar objects and one novel object on day 2. Memory was assessed as the degree to which the mice recalled the familiar object and explored the novel object to a greater extent on day 2. Uninjected mice or mice injected with saline prior to the acquisition session on day 1 demonstrated a lack of memory of the acquisition experience by exploring the familiar and novel objects to the same extent on day 2. Mice treated with glutamate carboxypeptidase II inhibitors ZJ43 or 2-PMPA prior to the acquisition trial explored the novel object significantly more time than the familiar object on day 2. Consistent with these results, mice in which glutamate carboxypeptidase II had been knocked out distinguished the novel from the familiar object on day 2 while their heterozygous colony mates did not. Inhibition of glutamate carboxypeptidase II enhances recognition memory, a therapeutic action that might be useful in treatment of memory deficits related to age and neurological disorders. PMID:23200894

  4. PsoP1, a milk-clotting aspartic peptidase from the basidiomycete fungus Piptoporus soloniensis.

    PubMed

    El-Baky, Hassan Abd; Linke, Diana; Nimtz, Manfred; Berger, Ralf Günter

    2011-09-28

    The first enzyme of the basidiomycete Piptoporus soloniensis, a peptidase (PsoP1), was characterized after isolation from submerged cultures, purification by fractional precipitation, and preparative native-polyarylamide gel electrophoresis (PAGE). The native molecular mass of PsoP1 was 38 kDa with an isoelectric point of 3.9. Similar to chymosin from milk calves, PsoP1 showed a maximum milk-clotting activity (MCA) at 35-40 °C and was most stable at pH 6 and below 40 °C. The complete inhibition by pepstatin A identified this enzyme as an aspartic peptidase. Electrospray ionization-tandem MS showed an amino acid partial sequence that was more homologous to mammalian milk clotting peptidases than to the chymosin substitute from a fungal species, such as the Zygomycete Mucor miehei. According to sodium dodecyl sulfate-PAGE patterns, the peptidase cleaved κ-casein in a way similar to chymosin and hydrolyzed β-casein slowly, as it would be expected from an efficient chymosin substitute. PMID:21888369

  5. Microwave assisted synthesis of novel acridine-acetazolamide conjugates and investigation of their inhibition effects on human carbonic anhydrase isoforms hCA I, II, IV and VII.

    PubMed

    Ulus, Ramazan; Aday, Burak; Tanç, Muhammet; Supuran, Claudiu T; Kaya, Muharrem

    2016-08-15

    4-Amino-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)benzamide was condensed with cyclic-1,3-diketones (dimedone and cyclohexane-1,3-dione) and aromatic aldehydes under microwave irradiation, leading to a series of acridine-acetazolamide conjugates. The new compounds were investigated as inhibitors of carbonic anhydrases (CA, EC 4.2.1.1), and more precisely cytosolic isoforms hCA I, II, VII and membrane-bound one hCA IV. All investigated isoforms were inhibited in low micromolar and nanomolar range by the new compounds. hCA IV and VII were inhibited with KIs in the range of 29.7-708.8nM (hCA IV), and of 1.3-90.7nM (hCA VII). For hCA I and II the KIs were in the range of 6.7-335.2nM (hCA I) and of 0.5-55.4nM (hCA II). The structure-activity relationships (SAR) for the inhibition of these isoforms with the acridine-acetazolamide conjugates reported here were delineated. PMID:27298005

  6. Production and partial characterization of serine and metallo peptidases secreted by Aspergillus fumigatus Fresenius in submerged and solid state fermentation.

    PubMed

    da Silva, Ronivaldo Rodrigues; de Freitas Cabral, Tatiana Pereira; Rodrigues, André; Cabral, Hamilton

    2013-01-01

    Enzyme production varies in different fermentation systems. Enzyme expression in different fermentation systems yields important information for improving our understanding of enzymatic production induction. Comparative studies between solid-state fermentation (SSF) using agro-industrial waste wheat bran and submerged fermentation (SmF) using synthetic media were carried out to determinate the best parameters for peptidase production by the fungus Aspergillus fumigatus Fresen. Variables tested include: the concentration of carbon and protein nitrogen sources, the size of the inoculum, the pH of the media, temperature, and the length of the fermentation process. The best peptidase production during SSF was obtained after 96 hours using wheat bran at 30 °C with an inoculum of 1 × 10(6) spores and yielded 1500 active units (U/mL). The best peptidase production using SmF was obtained after periods of 72 and 96 hours of fermentation in media containing 0.5% and 0.25% of casein, respectively, at a pH of 6.0 and at 30 °C and yielded 40 U/mL. We also found examples of catabolite repression of peptidase production under SmF conditions. Biochemical characterization of the peptidases produced by both fermentative processes showed optimum activity at pH 8.0 and 50 °C, and also showed that their proteolytic activity is modulated by surfactants. The enzymatic inhibition profile using phenylmethylsulfonyl fluoride (PMSF) in SmF and SSF indicated that both fermentative processes produced a serine peptidase. Additionally, the inhibitory effect of the ethylene-diaminetetraacetic acid (EDTA) chelating agent on the peptidase produced by SmF indicated that this fermentative process also produced a metallopeptidase. PMID:24159310

  7. Tripeptidyl Peptidase II Mediates Levels of Nuclear Phosphorylated ERK1 and ERK2*

    PubMed Central

    Wiemhoefer, Anne; Stargardt, Anita; van der Linden, Wouter A.; Renner, Maria C.; van Kesteren, Ronald E.; Stap, Jan; Raspe, Marcel A.; Tomkinson, Birgitta; Kessels, Helmut W.; Ovaa, Huib; Overkleeft, Herman S.; Florea, Bogdan; Reits, Eric A.

    2015-01-01

    Tripeptidyl peptidase II (TPP2) is a serine peptidase involved in various biological processes, including antigen processing, cell growth, DNA repair, and neuropeptide mediated signaling. The underlying mechanisms of how a peptidase can influence this multitude of processes still remain unknown. We identified rapid proteomic changes in neuroblastoma cells following selective TPP2 inhibition using the known reversible inhibitor butabindide, as well as a new, more potent, and irreversible peptide phosphonate inhibitor. Our data show that TPP2 inhibition indirectly but rapidly decreases the levels of active, di-phosphorylated extracellular signal-regulated kinase 1 (ERK1) and ERK2 in the nucleus, thereby down-regulating signal transduction downstream of growth factors and mitogenic stimuli. We conclude that TPP2 mediates many important cellular functions by controlling ERK1 and ERK2 phosphorylation. For instance, we show that TPP2 inhibition of neurons in the hippocampus leads to an excessive strengthening of synapses, indicating that TPP2 activity is crucial for normal brain function. PMID:26041847

  8. Local administration of N-acetylaspartylglutamate (NAAG) peptidase inhibitors is analgesic in peripheral pain in rats.

    PubMed

    Yamamoto, Tatsuo; Saito, Osamu; Aoe, Tomohiko; Bartolozzi, Alessandra; Sarva, Jayaprakash; Zhou, Jia; Kozikowski, Alan; Wroblewska, Barbara; Bzdega, Tomasz; Neale, Joseph H

    2007-01-01

    The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) selectively activates group II metabotropic glutamate receptors (mGluRs). Systemic administration of inhibitors of the enzymes that inactivate NAAG results in decreased pain responses in rat models of inflammatory and neuropathic pain. These effects are blocked by a group II mGluR antagonist. This research tested the hypothesis that some analgesic effects of NAAG peptidase inhibition are mediated by NAAG acting on sensory neurite mGluRs at the site of inflammation. Group II mGluR agonists, SLx-3095-1, NAAG and APDC, or NAAG peptidase inhibitors, ZJ-43 and 2-PMPA, injected into the rat footpad reduced pain responses in carrageenan or formalin models. The analgesic effects of SLx-3095-1, APDC, ZJ-43, 2-PMPA and NAAG were blocked by co-injection of LY341495, a selective group II mGluR antagonist. Injection of group II mGluR agonists, NAAG or the peptidase inhibitors into the contralateral rat footpad had no effect on pain perception in the injected paw. At 10-100 microm ZJ-43 and 2-PMPA demonstrated no consistent agonist activity at mGluR2 or mGluR3. Consistent with the conclusion that peripherally administered NAAG peptidase inhibitors increase the activation of mGluR3 by NAAG that is released from peripheral sensory neurites, we found that the tissue average concentration of NAAG in the unstimulated rat hind paw was about 6 microm. These data extend our understanding of the role of this peptide in sensory neurons and reveal the potential for treatment of inflammatory pain via local application of NAAG peptidase inhibitors at doses that may have little or no central nervous system effects. PMID:17241276

  9. The astrocyte surface NAAG receptor and NAAG peptidase signaling complex as a therapeutic target.

    PubMed

    Baslow, Morris H

    2008-06-01

    There is evidence that schizophrenia and other neuropathies may involve malfunction of a unique N-acetylaspartylglutamate (NAAG) receptor and its associated NAAG peptidase, a receptor and enzyme found together on the astrocyte surface. NAAG is a peptide neurotransmitter released by stimulated neurons and specifically targeted to the group II metabotropic glutamate receptor 3 (mGlu(3)), activation of which initiates astrocyte Ca(2+) waves responsible for astrocyte-astrocyte and astrocyte-vascular system signaling and induction of vascular hyperemic responses that increase energy supplies to stimulated neurons. In this review, it is hypothesized that the receptor and enzyme exist as a cytostructural unit on the astrocyte surface, and the nature of this proposed mGlu(3)-NAAG peptidase complex is considered in terms of its physiological signaling role, and of the effect of drugs on this role. The mGlu(3) receptor has been the target of extrinsic antagonists and agonists that mimic NAAG structure and compete with natural NAAG for the receptor site. NAAG metabolism has also been the target of extrinsic NAAG-like substances that inhibit NAAG peptidase, competing with NAAG for the enzyme active site. Several drugs that affect the mGlu(3) receptor or NAAG peptidase have reached a stage of human testing. Two are agonists of the mGlu(3) receptor, and another is an NAAG peptidase inhibitor. These substances appear to have potential for treating schizophrenia and other cognitive neuropathies by interfering with a homeostatic NAAG activated neuron-astrocyte-vascular energy supply system. PMID:18596989

  10. Combination of the dipeptidyl peptidase IV inhibitor LAF237 [(S)-1-[(3-hydroxy-1-adamantyl)ammo]acetyl-2-cyanopyrrolidine] with the angiotensin II type 1 receptor antagonist valsartan [N-(1-oxopentyl)-N-[[2'-(1H-tetrazol-5-yl)-[1,1'-biphenyl]-4-yl]methyl]-L-valine] enhances pancreatic islet morphology and function in a mouse model of type 2 diabetes.

    PubMed

    Cheng, Qianni; Law, Pui Ki; de Gasparo, Marc; Leung, Po Sing

    2008-12-01

    LAF237 [(S)-1-[(3-hydroxy-1-adamantyl)ammo]acetyl-2-cyanopyrrolidine] is an inhibitor of dipeptidyl peptidase IV that delays the degradation of glucagon-like peptide-1 (GLP-1). Valsartan [N-(1-oxopentyl)-N-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-l-valine] is an antagonist of the angiotensin II type 1 receptor (AT1R) that reduces the incidence of type 2 diabetes mellitus. LAF237 and valsartan act on a common target through separate pathways to improve pancreatic islet cell function. We hypothesize that the combination of these two drugs acts in a synergistic or additive manner on islet function and structure. To test this hypothesis, we performed in vitro and in vivo studies. To measure the acute effect of the treatment, pancreatic islets of db/db mice were isolated and stimulated in vitro with glucose in the presence of valsartan (1 microM) and exendin-4 (100 nM), a GLP-1 receptor agonist. Combination treatment with valsartan and exendin-4 significantly enhanced glucose-stimulated insulin secretion from isolated islets. For studies of chronic effect, db/db mice received LAF237 (1 mg/kg/day) and/or valsartan (10 mg/kg/day). Islet cell reactive oxygen species (ROS), proliferation, apoptosis, fibrosis, beta-cell area, and glucose homeostasis were evaluated after 8 weeks of treatment, which showed that combination treatment resulted in a significant increase in pancreatic islet beta-cell area compared with monotherapy. This beneficial effect correlated with an increase in beta-cell proliferation and a decrease in ROS-induced islet apoptosis and fibrosis. These in vitro and in vivo data indicate that combination treatment with LAF237 and valsartan has significant beneficial additive effects on pancreatic beta-cell structure and function compared with their respective monotherapeutic effects. PMID:18787107

  11. Carbonic Anhydrase Inhibitors. Part 551 Metal Complexes of 1,3,4-Thiadiazole-2-Sulfonamide Derivatives: In Vitro Inhibition Studies With Carbonic Anhydrase Isozymes I, II and IV

    PubMed Central

    Scozzafava, Andrea; Briganti, Fabrizio; Ilies, Marc A.; Jitianu, Andrei

    1998-01-01

    Coordination compounds of 5-chloroacetamido-1,3,4-thiadiazole-2-sulfonamide (Hcaz) with V(IV), Cr(lll), Fe(ll), Co(ll), Ni(ll) and Cu(ll) have been prepared and characterized by standard procedures (spectroscopic, magnetic, EPR, thermogravimetric and conductimetric measurements). Some of these compounds showed very good in vitro inhibitory properties against three physiologically relevant carbonic anhydrase (CA)isozymes, i.e., CA I, II, and IV. The differences between these isozymes in susceptibility to inhibition by these metal complexes is discussed in relationship to the characteristic features of their active sites, and is rationalized in terms useful for developing isozyme-specific CA inhibitors. PMID:18475829

  12. Interactions of “Bora-Penicilloates” with Serine β-Lactamases and DD-Peptidases

    PubMed Central

    2015-01-01

    Specific boronic acids are generally powerful tetrahedral intermediate/transition state analogue inhibitors of serine amidohydrolases. This group of enzymes includes bacterial β-lactamases and DD-peptidases where there has been considerable development of boronic acid inhibitors. This paper describes the synthesis, determination of the inhibitory activity, and analysis of the results from two α-(2-thiazolidinyl) boronic acids that are closer analogues of particular tetrahedral intermediates involved in β-lactamase and DD-peptidase catalysis than those previously described. One of them, 2-[1-(dihydroxyboranyl)(2-phenylacetamido)methyl]-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid, is a direct analogue of the deacylation tetrahedral intermediates of these enzymes. These compounds are micromolar inhibitors of class C β-lactamases but, very unexpectedly, not inhibitors of class A β-lactamases. We rationalize the latter result on the basis of a new mechanism of boronic acid inhibition of the class A enzymes. A stable inhibitory complex is not accessible because of the instability of an intermediate on its pathway of formation. The new boronic acids also do not inhibit bacterial DD-peptidases (penicillin-binding proteins). This result strongly supports a central feature of a previously proposed mechanism of action of β-lactam antibiotics, where deacylation of β-lactam-derived acyl-enzymes is not possible because of unfavorable steric interactions. PMID:25302576

  13. A phytopathogenic cysteine peptidase from latex of wild rubber vine Cryptostegia grandiflora.

    PubMed

    Ramos, M V; Souza, D P; Gomes, M T R; Freitas, C D T; Carvalho, C P S; Júnior, P A V R; Salas, C E

    2014-04-01

    A 24,118 Da (MALDI-TOF) cysteine peptidase (EC 3.4.22.16) was purified from the latex extract of Cryptostegia grandiflora by two chromatographic steps involving ion exchange and Reverse-phase HPLC. The purified protein (Cg24-I) exhibits a single band profile following reduced SDS-PAGE and a unique amino terminal sequence, 1VPASIDWREKGTVL14, that is similar to other plant cysteine peptidases. Cg24-I displayed optimal proteolytic activity at pH 8.0 with 25 mM phosphate buffer. The proteolytic activity was inhibited with 0.2 mM E-64 and 1 mM iodoacetamide and was stimulated with 1 mM DTT. Cg24-I fully inhibited spore germination of phytopathogenic fungi Fusarium solani at a dose of 28.1 μg/mL. Its toxicity involves the membrane permeabilization of spores as probed by propidium iodide uptake. These results show that latex peptidases are part of the plant's defensive strategy against phytopathogens and that they most likely act synergistically with other recognized defensive proteins. PMID:24596120

  14. A novel allosteric mechanism in the cysteine peptidase cathepsin K discovered by computational methods

    NASA Astrophysics Data System (ADS)

    Novinec, Marko; Korenč, Matevž; Caflisch, Amedeo; Ranganathan, Rama; Lenarčič, Brigita; Baici, Antonio

    2014-02-01

    Allosteric modifiers have the potential to fine-tune enzyme activity. Therefore, targeting allosteric sites is gaining increasing recognition as a strategy in drug design. Here we report the use of computational methods for the discovery of the first small-molecule allosteric inhibitor of the collagenolytic cysteine peptidase cathepsin K, a major target for the treatment of osteoporosis. The molecule NSC13345 is identified by high-throughput docking of compound libraries to surface sites on the peptidase that are connected to the active site by an evolutionarily conserved network of residues (protein sector). The crystal structure of the complex shows that NSC13345 binds to a novel allosteric site on cathepsin K. The compound acts as a hyperbolic mixed modifier in the presence of a synthetic substrate, it completely inhibits collagen degradation and has good selectivity for cathepsin K over related enzymes. Altogether, these properties qualify our methodology and NSC13345 as promising candidates for allosteric drug design.

  15. Antibacterial Activity of and Resistance to Small Molecule Inhibitors of the ClpP Peptidase

    PubMed Central

    Compton, Corey L.; Schmitz, Karl R.; Sauer, Robert T.; Sello, Jason K.

    2014-01-01

    There is rapidly mounting evidence that intracellular proteases in bacteria are compelling targets for antibacterial drugs. Multiple reports suggest that the human pathogen Mycobacterium tuberculosis and other actinobacteria may be particularly sensitive to small molecules that perturb the activities of self-compartmentalized peptidases, which catalyze intracellular protein turnover as components of ATP-dependent proteolytic machines. Here, we report chemical syntheses and evaluations of structurally diverse β-lactones, which have a privileged structure for selective, suicide inhibition of the self-compartmentalized ClpP peptidase. β-lactones with certain substituents on the α- and β-carbons were found to be toxic to M. tuberculosis. Using an affinity-labeled analog of a bioactive β-lactone in a series of chemical proteomic experiments, we selectively captured the ClpP1P2 peptidase from live cultures of two different actinobacteria that are related to M. tuberculosis. Importantly, we found that the growth inhibitory β-lactones also inactivate the M. tuberculosis ClpP1P2 peptidase in vitro via formation of a covalent adduct at the ClpP2 catalytic serine. Given the potent antibacterial activity of these compounds and their medicinal potential, we sought to identify innate mechanisms of resistance. Using a genome mining strategy, we identified a genetic determinant of β-lactone resistance in Streptomyces coelicolor, a non-pathogenic relative of M. tuberculosis. Collectively, these findings validate the potential of ClpP inhibition as a strategy in antibacterial drug development and define a mechanism by which bacteria could resist the toxic effects of ClpP inhibitors. PMID:24047344

  16. Differential expression of cysteine peptidase genes in the inner integument and endosperm of developing seeds of Jatropha curcas L. (Euphorbiaceae).

    PubMed

    Rocha, Antônio J; Soares, Emanoella L; Costa, José H; Costa, Washington L G; Soares, Arlete A; Nogueira, Fábio C S; Domont, Gilberto B; Campos, Francisco A P

    2013-12-01

    In several plant tissues, programmed cell death (PCD) is mediated by the combined action of cysteine peptidases, namely KDEL-tailed cysteine peptidases (KDEL-CysEP) and vacuolar processing enzymes (VPE). Here, we performed a search of the draft genome of Jatropha curcas L. (Euphorbiaceae) and identified 2 genes for KDEL-CysEP (Jc-CysEP1 and Jc-CysEP2) and 3 genes for VPE (Jc-βVPE, Jc-γVPE and Jc-δVPE) and determined the expression patterns of these genes by RT-qPCR in integument and cellular endosperm of seeds collected at seven different developmental stages. We were able to demonstrate that the expression of Jc-CysEP1, Jc-CysEP2, Jc-βVPE and Jc-γVPE proceeded rapidly from Stage IV, with Jc-CysEP2 displaying the highest relative expression; expression of Jc-δVPE could not be detected in any of the tissues/developmental stages analyzed. Additionally, we showed that the expression pattern of these peptidases correlates with anatomical changes in integument and cellular endosperm, thus suggesting a role for both classes of peptidases in PCD and in protein processing, both of which occur simultaneously in each of these tissues. PMID:24157205

  17. Cognitive-enhancing effects of angiotensin IV

    PubMed Central

    Gard, Paul R

    2008-01-01

    Angiotensin IV is a derivative of the potent vasoconstrictor angiotensin II and it has been shown to enhance acquisition, consolidation and recall in animal models of learning and memory when administered centrally or peripherally. Whether changes in angiotensin IV activity underlie the cognitive effects of those cardiovascular drugs designed to disrupt the peripheral renin-angiotensin system in humans remains undetermined, but angiotensin IV appears to be a worthy candidate for consideration in drug development programmes. The mechanism of action of angiotensin IV is still debated, although its AT4 receptor has been convincingly identified as being insulin-regulated amino peptidase, which is also known as oxytocinase and placental leucine aminopeptidase. It is speculated that angiotensin IV may interact with insulin-regulated amino peptidase to enhance neuronal glucose uptake, prevent metabolism of other neuroactive peptides, induce changes in extracellular matrix molecules, or induce release of acetylcholine and/or dopamine. All of these things may be responsible for the beneficial effects on cognition, but none of them are yet proven. Importantly, strain differences in murine responses to angiotensin IV suggest that some individuals may benefit from drugs targeted to the AT4 receptor whilst others may be refractory. At present it thus appears that those individuals with the poorest baseline cognition may receive greatest benefit, but possible genetic differences in responses to angiotensin IV cannot be ruled-out. PMID:19090988

  18. Oral and IV dosages of doxorubicin-methotrexate loaded- nanoparticles inhibit progression of oral cancer by down- regulation of matrix Methaloproteinase 2 expression in vivo.

    PubMed

    Abbasi, Mehran Mesgari; Jahanban-Esfahlan, Rana; Monfaredan, Amir; Seidi, Khaled; Hamishehkar, Hamed; Khiavi, Monir Moradzadeh

    2014-01-01

    Oral cancer is one of the most common and lethal cancers in the world. Combination chemotherapy coupled with nanoparticle drug delivery holds substantial promise in cancer therapy. This study aimed to evaluate the efficacy and safety of two dosages of our novel pH and temperature sensitive doxorubicin-methotrexate-loaded nanoparticles (DOX-MTX NPs) with attention to the MMP-2 mRNA profile in a 4-nitroquinoline-1-oxide induced oral squamous cell carcinoma (OSCC) model in the rat. Our results showed that both IV and oral dosages of DOX-MTX NP caused significant decrease in mRNA levels of MMP-2 compared to the untreated group (p<0.003). Surprisingly, MMP-2 mRNA was not affected in DOX treated compared to cancer group (p>0.05). Our results indicated that IV dosage of MTX-DOX is more effective than free DOX (12 fold) in inhibiting the activity of MMP-2 in OSCCs (P<0.001). Furthermore, MMP-2 mRNA expression in the DOX-MTX treated group showed a significant relation with histopathological changes (P=0.011). Compared to the untreated cancer group, we observed no pathological changes and neither a significant alteration in MMP-2 amount in either of healthy controls that were treated with oral and IV dosages of DOX-MTX NPs whilst cancer group showed a high level of MMP-2 expression compared to healthy controls (p<0.001).Taking together our results indicate that DOX- MTX NPs is a safe chemotherapeutic nanodrug that its oral and IV forms possess potent anti-cancer properties on aggressive tumors like OSCC, possibly by affecting the expression of genes that drive tumor invasion and metastasis. PMID:25605162

  19. Blockade of N-acetylaspartylglutamate peptidases: a novel protective strategy for brain injuries and neurological disorders.

    PubMed

    Zhong, Chunlong; Luo, Qizhong; Jiang, Jiyao

    2014-12-01

    The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) is reported to suppress glutamate release mainly through selective activation of presynaptic Group II metabotropic glutamate receptor subtype 3 (mGluR3). Therefore, strategies of inhibition of NAAG peptidases and subsequent NAAG hydrolysis to elevate levels of NAAG could reduce glutamate release under pathological conditions and be neuroprotective by attenuating excitotoxic cell injury. A series of potent inhibitors of NAAG peptidases has been synthesized and demonstrated efficacy in experimental models of ischemic-hypoxic brain injury, traumatic brain injury, inflammatory pain, diabetic neuropathy, amyotrophic lateral sclerosis and phencyclidine-induced schizophrenia-like behaviors. The excessive glutamatergic transmission has been implicated in all of these neurological disorders. Thus, blockade of NAAG peptidases may augment an endogenous protective mechanism and afford neuroprotection in the brain. This review aims to summarize and provide insight into the current understanding of the novel neuroprotective strategy based on limiting glutamate excitotoxicity for a wide variety of brain injuries and neurological disorders. PMID:24494725

  20. Effects of NAAG peptidase inhibitor 2-PMPA in model chronic pain - relation to brain concentration.

    PubMed

    Nagel, Jens; Belozertseva, Irina; Greco, Sergio; Kashkin, Vladimir; Malyshkin, Andrey; Jirgensons, Aigars; Shekunova, Elena; Eilbacher, Bernd; Bespalov, Anton; Danysz, Wojciech

    2006-12-01

    N-acetylated-alpha-linked-acidic peptidase (NAAG peptidase) converts N-acetyl-aspartyl-glutamate (NAAG, mGluR3 agonist) into N-acetyl-aspartate and glutamate. The NAAG peptidase inhibitor 2-PMPA (2-(phosphonomethyl)pentanedioic acid) had neuroprotective activity in an animal model of stroke and anti-allodynic activity in CCI model despite its uncertain ability to penetrate the blood-brain barrier. The NAAG concentration in brain ECF under basal conditions and its alteration in relation to the brain ECF concentration of 2-PMPA is unclear. We therefore assessed those brain concentrations after i.p. administration of 2-PMPA, using in vivo microdialysis combined with LC/MS/MS analysis. Administration of 2-PMPA (50mg/kg) produced a mean peak concentration of 2-PMPA of 29.66+/-8.1microM. This concentration is about 100,000 fold more than is needed for inhibition of NAAG peptidase, and indicates very good penetration to the brain. Application of 2-PMPA was followed by a linear increase of NAAG-concentration reaching a maximum of 2.89+/-0.42microM at the end of microdialysis. However, during the time the anti-allodynic effects of 2-PMPA were observed, the NAAG concentration in the ECF did not reach levels which are likely to have an impact on any known target. It appears therefore that the observed behavioural effects of 2-PMPA may not be mediated by NAAG nor, in turn, by mGluR3 receptors. PMID:16926034

  1. Histoplasma capsulatum Encodes a Dipeptidyl Peptidase Active against the Mammalian Immunoregulatory Peptide, Substance P

    PubMed Central

    Cooper, Kendal G.; Zarnowski, Robert; Woods, Jon P.

    2009-01-01

    The pathogenic fungus Histoplasma capsulatum secretes dipeptidyl peptidase (Dpp) IV enzyme activity and has two putative DPPIV homologs (HcDPPIVA and HcDPPIVB). We previously showed that HcDPPIVB is the gene responsible for the majority of secreted DppIV activity in H. capsulatum culture supernatant, while we could not detect any functional contribution from HcDPPIVA. In order to determine whether HcDPPIVA encodes a functional DppIV enzyme, we expressed HcDPPIVA in Pichia pastoris and purified the recombinant protein. The recombinant enzyme cleaved synthetic DppIV substrates and had similar biochemical properties to other described DppIV enzymes, with temperature and pH optima of 42°C and 8, respectively. Recombinant HcDppIVA cleaved the host immunoregulatory peptide substance P, indicating the enzyme has the potential to affect the immune response during infection. Expression of HcDPPIVA under heterologous regulatory sequences in H. capsulatum resulted in increased secreted DppIV activity, indicating that the encoded protein can be expressed and secreted by its native organism. However, HcDPPIVA was not required for virulence in a murine model of histoplasmosis. This work reports a fungal enzyme that can function to cleave the immunomodulatory host peptide substance P. PMID:19384411

  2. Dual control mechanism for heme oxygenase: tin(IV)-protoporphyrin potently inhibits enzyme activity while markedly increasing content of enzyme protein in liver.

    PubMed Central

    Sardana, M K; Kappas, A

    1987-01-01

    Tin(IV)-protoporphyrin (Sn-protoporphyrin) potently inhibits heme degradation to bile pigments in vitro and in vivo, a property that confers upon this synthetic compound the ability to suppress a variety of experimentally induced and naturally occurring forms of jaundice in animals and humans. Utilizing rat liver heme oxygenase purified to homogeneity together with appropriate immunoquantitation techniques, we have demonstrated that Sn-protoporphyrin possesses the additional property of potently inducing the synthesis of heme oxygenase protein in liver cells while, concurrently, completely inhibiting the activity of the newly formed enzyme. Substitution of tin for the central iron atom of heme thus leads to the formation of a synthetic heme analogue that regulates heme oxygenase by a dual mechanism, which involves competitive inhibition of the enzyme for the natural substrate heme and simultaneous enhancement of new enzyme synthesis. Cobaltic(III)-protoporphyrin (Co-protoporphyrin) also inhibits heme oxygenase activity in vitro, but unlike Sn-protoporphyrin it greatly enhances the activity of the enzyme in the whole animal. Co-protoporphyrin also acts as an in vivo inhibitor of heme oxygenase; however, its inducing effect on heme oxygenase synthesis is so pronounced as to prevail in vivo over its inhibitory effect on the enzyme. These studies show that certain synthetic heme analogues possess the ability to simultaneously inhibit as well as induce the enzyme heme oxygenase in liver. The net balance between these two actions, as reflected in the rate of heme oxidation activity in the whole animal, appears to be influenced by the nature of the central metal atom of the synthetic metalloporphyrin. Images PMID:3470805

  3. NAAG peptidase inhibitors block cognitive deficit induced by MK-801 and motor activation induced by d-amphetamine in animal models of schizophrenia.

    PubMed

    Olszewski, R T; Janczura, K J; Ball, S R; Madore, J C; Lavin, K M; Lee, J C-M; Lee, M J; Der, E K; Hark, T J; Farago, P R; Profaci, C P; Bzdega, T; Neale, J H

    2012-01-01

    The most widely validated animal models of the positive, negative and cognitive symptoms of schizophrenia involve administration of d-amphetamine or the open channel NMDA receptor blockers, dizocilpine (MK-801), phencyclidine (PCP) and ketamine. The drug ZJ43 potently inhibits glutamate carboxypeptidase II (GCPII), an enzyme that inactivates the peptide transmitter N-acetylaspartylglutamate (NAAG) and reduces positive and negative behaviors induced by PCP in several of these models. NAAG is an agonist at the metabotropic glutamate receptor 3 (mGluR3). Polymorphisms in this receptor have been associated with expression of schizophrenia. This study aimed to determine whether two different NAAG peptidase inhibitors are effective in dopamine models, whether their efficacy was eliminated in GCPII knockout mice and whether the efficacy of these inhibitors extended to MK-801-induced cognitive deficits as assessed using the novel object recognition test. ZJ43 blocked motor activation when given before or after d-amphetamine treatment. (R,S)-2-phosphono-methylpentanedioic acid (2-PMPA), another potent NAAG peptidase inhibitor, also reduced motor activation induced by PCP or d-amphetamine. 2-PMPA was not effective in GCPII knockout mice. ZJ43 and 2-PMPA also blocked MK-801-induced deficits in novel object recognition when given before, but not after, the acquisition trial. The group II mGluR antagonist LY341495 blocked the effects of NAAG peptidase inhibition in these studies. 2-PMPA was more potent than ZJ43 in a test of NAAG peptidase inhibition in vivo. By bridging the dopamine and glutamate theories of schizophrenia with two structurally different NAAG peptidase inhibitors and demonstrating their efficacy in blocking MK-801-induced memory deficits, these data advance the concept that NAAG peptidase inhibition represents a potentially novel antipsychotic therapy. PMID:22850437

  4. Sugarcane Serine Peptidase Inhibitors, Serine Peptidases, and Clp Protease System Subunits Associated with Sugarcane Borer (Diatraea saccharalis) Herbivory and Wounding.

    PubMed

    Medeiros, Ane H; Mingossi, Fabiana B; Dias, Renata O; Franco, Flávia P; Vicentini, Renato; Mello, Marcia O; Moura, Daniel S; Silva-Filho, Marcio C

    2016-01-01

    Sugarcane's (Saccharum spp.) response to Diatraea saccharalis (F.) (Lepidoptera: (Crambidae) herbivory was investigated using a macroarray spotted with 248 sugarcane Expressed Sequence Tags (ESTs) encoding serine peptidase inhibitors, serine peptidases. and Clp protease system subunits. Our results showed that after nine hours of herbivory, 13 sugarcane genes were upregulated and nine were downregulated. Among the upregulated genes, nine were similar to serine peptidase inhibitors and four were similar to Bowman-Birk Inhibitors (BBIs). Phylogenetic analysis revealed that these sequences belong to a phylogenetic group of sugarcane BBIs that are potentially involved in plant defense against insect predation. The remaining four upregulated genes included serine peptidases and one homolog to the Arabidopsis AAA+ chaperone subunit ClpD, which is a member of the Clp protease system. Among the downregulated genes, five were homologous to serine peptidases and four were homologous to Arabidopsis Clp subunits (three homologous to Clp AAA+ chaperones and one to a ClpP-related ClpR subunit). Although the roles of serine peptidase inhibitors in plant defenses against herbivory have been extensively investigated, the roles of plant serine peptidases and the Clp protease system represent a new and underexplored field of study. The up- and downregulated D. saccharalis genes presented in this study may be candidate genes for the further investigation of the sugarcane response to herbivory. PMID:27598134

  5. Dipeptidyl peptidase inhibitors as new drugs for the treatment of type 2 diabetes.

    PubMed

    Mest, H-J; Mentlein, R

    2005-04-01

    Inhibitors of the regulatory protease dipeptidyl peptidase-IV (DPP-IV) are currently under development in preclinical and clinical studies (several pharmaceutical companies, now in Phase III) as potential drugs for the treatment of type 2 diabetes. Their development is based on the observation that DPP-IV rapidly inactivates the incretin hormone glucagon-like peptide-1 (GLP-1), which is released postprandially from the gut and increases insulin secretion. DPP-IV inhibitors stabilise endogenous GLP-1 at physiological concentrations, and induce insulin secretion in a glucose-dependent manner; therefore, they do not demonstrate any hypoglycaemic effects. Furthermore, they are orally bioavailable. In addition to their ability to protect GLP-1 against degradation, DPP-IV inhibitors also stabilise other incretins, including gastric inhibitory peptide and pituitary adenylate cyclase-activating peptide. They also reduce the antagonistic and desensitising effects of the fragments formed by truncation of the incretins. In clinical studies, when used for the treatment of diabetes over a 1-year period, DPP-IV inhibitors show improved efficacy over time. This finding can be explained by a GLP-1-induced increase in the number of beta cells. Potential risks associated with DPP-IV inhibitors include the prolongation of the action of other peptide hormones, neuropeptides and chemokines cleaved by the protease, and their interaction with DPP-IV-related proteases. Based on their mode of action, DPP-IV inhibitors seem to be of particular value in early forms of type 2 diabetes, either alone or in combination with other types of oral agents. PMID:15770466

  6. Identification of dipeptidyl peptidase III in human neutrophils.

    PubMed

    Hashimoto, J; Yamamoto, Y; Kurosawa, H; Nishimura, K; Hazato, T

    2000-07-01

    We have found activity of dipeptidyl peptidase (DPP) III, one of the most important enkephalin-degrading enzymes in the central nervous system, in human neutrophils. HPLC analysis of the peptide fragments produced by treatment of leucine-enkephalin with isolated neutrophils in the presence of inhibitors of other enkephalin-degrading enzymes revealed that the enzyme in human neutrophils cleaved dipeptides from the NH(2) terminus of leucine-enkephalin, suggesting the presence of DPPIII activity in human neutrophils. Using a specific synthesized substrate and proteinase inhibitors, it was found that the neutrophils have 19.2 +/- 3.6 microM/h/5 x 10(6) cells of beta-naphthylamine for the enzyme. It was also confirmed that spinorphin and tynorphin, both reported to inhibit the activities of enkephalin-degrading enzymes, had potent inhibitory activities (IC(50): 4.0 and 0.029 microg/ml, respectively) against the enzyme. The presence of DPPIII activity in human neutrophils suggests that the biologically active peptides which are associated with enkephalin play a physiological role in regulating enkephalin or inflammatory mechanisms in peripheral tissues. PMID:10873616

  7. Synthesis and biological evaluation of azobicyclo[3.3.0] octane derivatives as dipeptidyl peptidase 4 inhibitors for the treatment of type 2 diabetes.

    PubMed

    Cho, Tang Peng; Long, Yang Fang; Gang, Lin Zhi; Yang, Wang; Jun, Lu He; Yuan, Shen Guang; Hong, Fu Jian; Lin, Wang; Liang, Guan Dong; Lei, Zhang; Jing, Luo Jing; Shen, Gong Ai; Hong, She Gao; Dan, Wang; Ying, Feng; Ke, Yan Pang; Ying, Leng; Jun, Feng; Tai, Mong Xian

    2010-06-15

    A series of novel azobicyclo[3.3.0]octane derivatives were synthesized and evaluated as dipeptidyl peptidase 4 (DPP-4) inhibitors. The effort resulted in the discovery of inhibitor 2a, which exhibited excellent efficacies in an oral glucose tolerance test. Introduction of methyl group (2j) could prolong the inhibition of serum DPP-4 activity. PMID:20488702

  8. 1,10-phenanthroline inhibits the metallopeptidase secreted by Phialophora verrucosa and modulates its growth, morphology and differentiation.

    PubMed

    Granato, Marcela Queiroz; Massapust, Priscila de Araújo; Rozental, Sonia; Alviano, Celuta Sales; dos Santos, André Luis Souza; Kneipp, Lucimar Ferreira

    2015-04-01

    Phialophora verrucosa is one of the etiologic agents of chromoblastomycosis, a fungal infection that affects cutaneous and subcutaneous tissues. This disease is chronic, recurrent and difficult to treat. Several studies have shown that secreted peptidases by fungi are associated with important pathophysiological processes. Herein, we have identified and partially characterized the peptidase activity secreted by P. verrucosa conidial cells. Using human serum albumin as substrate, the best hydrolysis profile was detected at extreme acidic pH (3.0) and at 37 °C. The enzymatic activity was completely blocked by classical metallopeptidase inhibitors/chelating agents as 1,10-phenanthroline and EGTA. Zinc ions stimulated the metallo-type peptidase activity in a dose-dependent manner. Several proteinaceous substrates were cleaved, in different extension, by the P. verrucosa metallopeptidase activity, including immunoglobulin G, fibrinogen, collagen types I and IV, fibronectin, laminin and keratin; however, mucin and hemoglobin were not susceptible to proteolysis. As metallopeptidases participate in different cellular metabolic pathways in fungal cells, we also tested the influence of 1,10-phenanthroline and EGTA on P. verrucosa development. Contrarily to EGTA, 1,10-phenanthroline inhibited the fungal viability (MIC 0.8 µg/ml), showing fungistatic effect, and induced profound morphological alterations as visualized by transmission electron microscopy. In addition, 1,10-phenanthroline arrested the filamentation process in P. verrucosa. Our results corroborate the supposition that metallopeptidase inhibitors/chelating agents have potential to control crucial biological events in fungal agents of chromoblastomycosis. PMID:25502596

  9. Cysteine peptidases from Phytomonas serpens: biochemical and immunological approaches.

    PubMed

    Elias, Camila G R; Aor, Ana Carolina; Valle, Roberta S; d'Avila-Levy, Claudia M; Branquinha, Marta H; Santos, André L S

    2009-12-01

    Phytomonas serpens, a phytoflagellate trypanosomatid, shares common antigens with Trypanosoma cruzi. In the present work, we compared the hydrolytic capability of cysteine peptidases in both trypanosomatids. Trypanosoma cruzi epimastigotes presented a 10-fold higher efficiency in hydrolyzing the cysteine peptidase substrate Z-Phe-Arg-AMC than P. serpens promastigotes. Moreover, two weak cysteine-type gelatinolytic activities were detected in P. serpens, while a strong 50-kDa cysteine peptidase was observed in T. cruzi. Cysteine peptidase activities were detected at twofold higher levels in the cytoplasmic fraction when compared with the membrane-rich or the content released from P. serpens. The cysteine peptidase secreted by P. serpens cleaved several proteinaceous substrates. Corroborating these findings, the cellular distribution of the cruzipain-like molecules in P. serpens was attested through immunocytochemistry analysis. Gold particles were observed in all cellular compartments, including the cytoplasm, plasma membrane, flagellum, flagellar membrane and flagellar pocket. Interestingly, some gold particles were visualized free in the flagellar pocket, suggesting the release of the cruzipain-like molecule. The antigenic properties of the cruzipain-like molecules of P. serpens were also analyzed. Interestingly, sera from chagasic patients recognized both cellular and extracellular antigens of P. serpens, including the cruzipain-like molecule. These results point to the use of P. serpens antigens, especially the cruzipain-like cysteine-peptidases, as an alternative vaccination approach to T. cruzi infection. PMID:19780820

  10. Camel milk attenuates the biochemical and morphological features of diabetic nephropathy: inhibition of Smad1 and collagen type IV synthesis.

    PubMed

    Korish, Aida A; Abdel Gader, Abdel Galil; Korashy, Hesham M; Al-Drees, Abdul Majeed; Alhaider, Abdulqader A; Arafah, Maha M

    2015-03-01

    Diabetic nephropathy (DN) is a common microvascular complication of diabetes mellitus (DM) that worsens its morbidity and mortality. There is evidence that camel milk (CM) improves the glycemic control in DM but its effect on the renal complications especially the DN remains unclear. Thus the current study aimed to characterize the effects of CM treatment on streptozotocin (STZ)-induced DN. Using STZ-induced diabetes, we investigated the effect of CM treatment on kidney function, proteinuria, renal Smad1, collagen type IV (Col4), blood glucose, insulin resistance (IR), lipid peroxidation, the antioxidant superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH). In addition renal morphology was also examined. The current results showed that rats with untreated diabetes exhibited marked hyperglycemia, IR, high serum urea and creatinine levels, excessive proteinuria, increased renal Smad1 and Col4, glomerular expansion, and extracellular matrix deposition. There was also increased lipid peroxidation products, decreased antioxidant enzyme activity and GSH levels. Camel milk treatment decreased blood glucose, IR, and lipid peroxidation. Superoxide dismutase and CAT expression, CAT activity, and GSH levels were increased. The renoprotective effects of CM were demonstrated by the decreased serum urea and creatinine, proteinuria, Smad1, Col4, and preserved normal tubulo-glomerular morphology. In conclusion, beside its hypoglycemic action, CM attenuates the early changes of DN, decreased renal Smad1 and Col4. This could be attributed to a primary action on the glomerular mesangial cells, or secondarily to the hypoglycemic and antioxidant effects of CM. The protective effects of CM against DN support its use as an adjuvant anti-diabetes therapy. PMID:25617480

  11. Plasma peptidases as prognostic biomarkers in patients with first-episode psychosis.

    PubMed

    Fernández-Atucha, Ainhoa; Echevarría, Enrique; Larrinaga, Gorka; Gil, Javier; Martínez-Cengotitabengoa, Mónica; González-Pinto, Ana M; Irazusta, Jon; Seco, Jesús

    2015-08-15

    The plasma activity of nine aminopeptidases was monitored over a year in first-episode psychotic patients. We observed significant differences in aminopeptidase B (APB), aminopeptidase N (APN) and dipeptidyl peptidase IV (DPPIV), but not in puromycin-sensitive aminopeptidase (PSA), prolyl endopeptidase (PEP), cysteine aminopeptidase (Cys-AP), aspartate aminopeptidase (Asp-AP), glutamate aminopeptidase (Glu) or piroglutamate aminopeptidase (PGI) in these patients compared to controls, and also a progressive increase in plasma activity, correlated to changes in scores on clinical scales, Global Assessment of Functioning scale (GAF) and Hamilton Depression Rating Scale (HDRS), at 1 month of follow-up. At 1 month after diagnosis, the median score obtained by patients on the GAF was negatively associated with the plasma activity of APB and PEP measured at the beginning of the psychotic episode, indicating a role as a negative prognostic factor that can predict psychiatric symptomatology. In the case of HDRS, scores at 1 month after diagnosis were found to be positively associated with the initial plasma activity of DPPIV, APN and PSA, indicating that their initial elevation is a negative prognostic factor that can predict subsequent depressive symptomatology. Taken together, these results suggest a pathophysiological involvement of plasma peptidases and indicate that aminopeptidase activity can predict the course of first-episode psychosis patients, acting as a prognostic indicator. PMID:25997998

  12. AST IV inhibits H₂O₂-induced human umbilical vein endothelial cell apoptosis by suppressing Nox4 expression through the TGF-β1/Smad2 pathway.

    PubMed

    Ma, Yuhong; Li, Weizu; Yin, Yanyan; Li, Weiping

    2015-06-01

    Endothelial cell apoptosis plays an important role in the pathophysiological mechanisms of vascular complications in diabetes mellitus (DM). NADPH oxidase 4 (Nox4)-dependent reactive oxygen species (ROS) aggregation is the main cause of vascular endothelial cell apoptosis. The transforming growth factor-β1 (TGF-β1)/Smad2 signaling pathway is involved in the apoptosis of several types of cells. However, the association between vascular endothelial cell apoptosis and Nox4, and the involvement of the TGF-β1/Smad2 signaling pathway in vascular endothelial cell apoptosis remain unclear. In the present study, we aimed to investigate the role of Nox4-dependent ROS production and to determine the involvement of the TGF-β1/Smad2 signaling pathway in endothelial cell apoptosis induced by oxidative stress which causes vascular injury in DM. We demonstrated that hydrogen peroxide (H2O2) increased Nox4-dependent-ROS aggregation, as well as the expression of TGF-β1, Smad2, Bax and caspase-3, decreased Bcl-2 expression and increased the apoptosis of human umbilical vein endothelial cells (HUVECs). Treatment with diphenyliodonium (DPI), a specific inhibitor of Nox4 or astragaloside IV (AST IV), a monomer located in an extract of astragaloside, decreased Nox4 expression and the levels of ROS, decreased TGF-β1 and Smad2 expression, altered the expression of apoptosis-related genes and decreased the apoptosis of HUVECs. Treatment with LY2109761, a selective inhibitor of the TGF-β1/Smad2 pathway, produced results similar to those of DPI; however, LY2109761 had no effect on Nox4 expression and ROS levels. Taken together, the findings of the present study suggest that H2O2 contributes to HUVEC apoptosis by inducing Nox4-dependent ROS aggregation and activating the TGF-β1/Smad2 signaling pathway. Our data indicate that the protective effects of AST IV against vascular endothelial cell apoptosis in DM are mainly associated with the decrease in Nox4 expression through the TGF-β1

  13. Cardiovascular effects of dipeptidyl peptidase-4 inhibitors

    PubMed Central

    Papagianni, M; Tziomalos, K

    2015-01-01

    Dipeptidyl peptidase-4 (DPP-4) inhibitors are effective glucose-lowering agents that do not increase body weight and are associated with a low risk for hypoglycemia. Also, they appear to exert beneficial effects on other established cardiovascular risk factors, including dyslipidemia and hypertension. Moreover, DPP-4 inhibitors exert antiinflammatory and antioxidant actions, improve endothelial function and reduce urinary albumin excretion. In contrast to these favorable cardiovascular effects, three recent large, randomized, placebo-controlled trials in patients with type 2 diabetes mellitus (T2DM) and established cardiovascular disease or multiple cardiovascular risk factors showed that DPP-4 inhibitors do not affect the risk of myocardial infarction or ischemic stroke and might increase the risk of heart failure. The findings of the former randomized studies highlight the limitations of surrogate markers and show that beneficial effects on cardiovascular risk factors do not necessarily translate into reductions in hard clinical endpoints. Ongoing trials will shed more light on the safety profile of DPP-4 inhibitors and will clarify whether they will improve the cardiovascular outcomes of patients with T2DM. Hippokratia 2015; 19 (3): 195-199. PMID:27418775

  14. Structure and Catalysis of Acylaminoacyl Peptidase

    PubMed Central

    Harmat, Veronika; Domokos, Klarissza; Menyhárd, Dóra K.; Palló, Anna; Szeltner, Zoltán; Szamosi, Ilona; Beke-Somfai, Tamás; Náray-Szabó, Gábor; Polgár, László

    2011-01-01

    Acylaminoacyl peptidase from Aeropyrum pernix is a homodimer that belongs to the prolyl oligopeptidase family. The monomer subunit is composed of one hydrolase and one propeller domain. Previous crystal structure determinations revealed that the propeller domain obstructed the access of substrate to the active site of both subunits. Here we investigated the structure and the kinetics of two mutant enzymes in which the aspartic acid of the catalytic triad was changed to alanine or asparagine. Using different substrates, we have determined the pH dependence of specificity rate constants, the rate-limiting step of catalysis, and the binding of substrates and inhibitors. The catalysis considerably depended both on the kind of mutation and on the nature of the substrate. The results were interpreted in terms of alterations in the position of the catalytic histidine side chain as demonstrated with crystal structure determination of the native and two mutant structures (D524N and D524A). Unexpectedly, in the homodimeric structures, only one subunit displayed the closed form of the enzyme. The other subunit exhibited an open gate to the catalytic site, thus revealing the structural basis that controls the oligopeptidase activity. The open form of the native enzyme displayed the catalytic triad in a distorted, inactive state. The mutations affected the closed, active form of the enzyme, disrupting its catalytic triad. We concluded that the two forms are at equilibrium and the substrates bind by the conformational selection mechanism. PMID:21084296

  15. Navigating the chemical space of dipeptidyl peptidase-4 inhibitors

    PubMed Central

    Shoombuatong, Watshara; Prachayasittikul, Veda; Anuwongcharoen, Nuttapat; Songtawee, Napat; Monnor, Teerawat; Prachayasittikul, Supaluk; Prachayasittikul, Virapong; Nantasenamat, Chanin

    2015-01-01

    This study represents the first large-scale study on the chemical space of inhibitors of dipeptidyl peptidase-4 (DPP4), which is a potential therapeutic protein target for the treatment of diabetes mellitus. Herein, a large set of 2,937 compounds evaluated for their ability to inhibit DPP4 was compiled from the literature. Molecular descriptors were generated from the geometrically optimized low-energy conformers of these compounds at the semiempirical AM1 level. The origins of DPP4 inhibitory activity were elucidated from computed molecular descriptors that accounted for the unique physicochemical properties inherently present in the active and inactive sets of compounds as defined by their respective half maximal inhibitory concentration values of less than 1 μM and greater than 10 μM, respectively. Decision tree analysis revealed the importance of molecular weight, total energy of a molecule, topological polar surface area, lowest unoccupied molecular orbital, and number of hydrogen-bond donors, which correspond to molecular size, energy, surface polarity, electron acceptors, and hydrogen bond donors, respectively. The prediction model was subjected to rigorous independent testing via three external sets. Scaffold and chemical fragment analysis was also performed on these active and inactive sets of compounds to shed light on the distinguishing features of the functional moieties. Docking of representative active DPP4 inhibitors was also performed to unravel key interacting residues. The results of this study are anticipated to be useful in guiding the rational design of novel and robust DPP4 inhibitors for the treatment of diabetes. PMID:26309399

  16. A hepatitis C virus (HCV) internal ribosome entry site (IRES) domain III–IV-targeted aptamer inhibits translation by binding to an apical loop of domain IIId

    PubMed Central

    Kikuchi, Kunio; Umehara, Takuya; Fukuda, Kotaro; Kuno, Atsushi; Hasegawa, Tsunemi; Nishikawa, Satoshi

    2005-01-01

    The hepatitis C virus (HCV) has a positive single-stranded RNA genome, and translation starts within the internal ribosome entry site (IRES) in a cap-independent manner. The IRES is well conserved among HCV subtypes and has a unique structure consisting of four domains. We used an in vitro selection procedure to isolate RNA aptamers capable of binding to the IRES domains III–IV. The aptamers that were obtained shared the consensus sequence ACCCA, which is complementary to the apical loop of domain IIId that is known to be a critical region of IRES-dependent translation. This convergence suggests that domain IIId is preferentially selected in an RNA–RNA interaction. Mutation analysis showed that the aptamer binding was sequence and structure dependent. One of the aptamers inhibited translation both in vitro and in vivo. Our results indicate that domain IIId is a suitable target site for HCV blockage and that rationally designed RNA aptamers have great potential as anti-HCV drugs. PMID:15681618

  17. Porphyromonas gingivalis Virulence Factor Gingipain RgpB Shows a Unique Zymogenic Mechanism for Cysteine Peptidases*

    PubMed Central

    de Diego, Iñaki; Veillard, Florian T.; Guevara, Tibisay; Potempa, Barbara; Sztukowska, Maryta; Potempa, Jan; Gomis-Rüth, F. Xavier

    2013-01-01

    Zymogenicity is a regulatory mechanism that prevents inadequate catalytic activity in the wrong context. It plays a central role in maintaining microbial virulence factors in an inactive form inside the pathogen until secretion. Among these virulence factors is the cysteine peptidase gingipain B (RgpB), which is the major virulence factor secreted by the periodontopathogen Porphyromonas gingivalis that attacks host vasculature and defense proteins. The structure of the complex between soluble mature RgpB, consisting of a catalytic domain and an immunoglobulin superfamily domain, and its 205-residue N-terminal prodomain, the largest structurally characterized to date for a cysteine peptidase, reveals a novel fold for the prodomain that is distantly related to sugar-binding lectins. It attaches laterally to the catalytic domain through a large concave surface. The main determinant for latency is a surface “inhibitory loop,” which approaches the active-site cleft of the enzyme on its non-primed side in a substrate-like manner. It inserts an arginine (Arg126) into the S1 pocket, thus matching the substrate specificity of the enzyme. Downstream of Arg126, the polypeptide leaves the cleft, thereby preventing cleavage. Moreover, the carbonyl group of Arg126 establishes a very strong hydrogen bond with the co-catalytic histidine, His440, pulling it away from the catalytic cysteine, Cys473, and toward Glu381, which probably plays a role in orienting the side chain of His440 during catalysis. The present results provide the structural determinants of zymogenic inhibition of RgpB by way of a novel inhibitory mechanism for peptidases in general and open the field for the design of novel inhibitory strategies in the treatment of human periodontal disease. PMID:23558682

  18. Serpin peptidase inhibitor clade A member 1 is a biomarker of poor prognosis in gastric cancer

    PubMed Central

    Kwon, C H; Park, H J; Lee, J R; Kim, H K; Jeon, T Y; Jo, H-J; Kim, D H; Kim, G H; Park, D Y

    2014-01-01

    Background: In a previous study, we reported that serpin peptidase inhibitor clade A member 1 (serpinA1) is upregulated in Snail-overexpressing gastric cancer. Although serpinA1 has been studied in several types of cancer, little is known about its roles and mechanisms of action. In this study, we examined the role of serpinA1 in the migration and invasion of gastric cancers and determined its underlying mechanism. Methods: Expression levels were assessed by western blot analyses and real-time PCR. Snail binding to serpinA1 promoter was analysed by chromatin immunoprecipitation (ChIP) assays. The roles of serpinA1 were studied using cell invasion and migration assays. In addition, the clinicopathologic and prognostic significance of serpinA1 expression were validated in 400 gastric cancer patients using immunohistochemical analysis. Results: Overexpression of Snail resulted in upregulation of serpinA1 in gastric cancer cell lines, AGS and MKN45, whereas knockdown of Snail inhibited serpinA1 expression. Chromatin immunoprecipitation analysis showed that overexpression of Snail increased Snail recruitment to the serpinA1 promoter. Overexpression of serpinA1 increased the migration and invasion of gastric cancer cells, whereas knockdown of serpinA1 decreased invasion and migration. Moreover, serpinA1 increased mRNA levels and release of metalloproteinase-8 in gastric cancer cells. Serpin peptidase inhibitor clade A member 1 was observed in the cytoplasm of tumour cells and the stroma by immunohistochemistry. Enhanced serpinA1 expression was significantly associated with increased tumour size, advanced T stage, perineural invasion, lymphovascular invasion, lymph node metastases, and shorter overall survival. Conclusions: Serpin peptidase inhibitor clade A member 1 induces the invasion and migration of gastric cancer cells and its expression is associated with the progression of gastric cancer. These results may provide a potential target to prevent invasion and

  19. Design, Synthesis and Biological Evaluation of N4-Sulfonamido-Succinamic, Phthalamic, Acrylic and Benzoyl Acetic Acid Derivatives as Potential DPP IV Inhibitors

    PubMed Central

    Khalaf, Reema Abu; Sheikha, Ghassan Abu; Al-Sha'er, Mahmoud; Taha, Mutasem

    2013-01-01

    As incidence rate of type II diabetes mellitus continues to rise, there is a growing need to identify novel therapeutic agents with improved efficacy and reduced side effects. Dipeptidyl peptidase IV (DPP IV) is a multifunctional protein involved in many physiological processes. It deactivates the natural hypoglycemic incretin hormone effect. Inhibition of this enzyme increases endogenous incretin level, incretin activity and should restore glucose homeostasis in type II diabetic patients making it an attractive target for the development of new antidiabetic drugs. One of the interesting reported anti- DPP IV hits is Gemifloxacin which is used as a lead compound for the development of new DPP IV inhibitors. In the current work, design and synthesis of a series of N4-sulfonamido-succinamic, phthalamic, acrylic and benzoyl acetic acid derivatives was carried out. The synthesized compounds were evaluated for their in vitro anti-DPP IV activity. Some of them have shown reasonable bioactivity, where the most active one 17 was found to have an IC50 of 33.5 μM. PMID:24358058

  20. Trypanosoma brucei has a canonical mitochondrial processing peptidase.

    PubMed

    Desy, Silvia; Schneider, André; Mani, Jan

    2012-10-01

    Most mitochondrial matrix and inner membrane proteins have N-terminal presequences which serve as import signals. After import these presequences are cleaved by the heterodimeric mitochondrial processing peptidase. In the parasitic protozoa Trypanosoma brucei mitochondrial protein import relies on presequences that are much shorter than in other eukaryotes. How they are processed is unknown. The trypansomal genome encodes four open reading frames that are annotated as mitochondrial processing peptidase. Here we show that RNAi-mediated ablation of two of these proteins leads to a growth arrest and a concomitant accumulation of mitochondrial precursor proteins inside mitochondria. Import experiments using isolated mitochondria from RNAi cell lines reveals that both proteins are required for efficient import and processing of the tested precursor protein. Reciprocal immunoprecipitation demonstrates that the proteins interact with each other. In summary these results show that we have identified the two subunits of the trypanosomal mitochondrial processing peptidase. PMID:22841752

  1. Molecular cloning and expression analysis of peptidase genes in the fish-pathogenic scuticociliate Miamiensis avidus

    PubMed Central

    2013-01-01

    Background Parasite peptidases have been actively studied as vaccine candidates or drug targets for prevention or treatment of parasitic diseases because of their important roles for survival and/or invasion in the host. Like other parasites, the facultative histophagous ciliate Miamiensis avidus would possess peptidases that are closely associated with the invasion into the host tissue and survival in the host. Results The 17 genes encoding peptidases, including seven cathepsin-like cysteine peptidases, four serine carboxypeptidases, a eukaryotic aspartyl protease family protein, an ATP-dependent metalloprotease FtsH family protein, three leishmanolysin family proteins and a peptidase family M49 protein were identified from a Miamiensis avidus cDNA library by BLAST X search. Expression of genes encoding two cysteine peptidases, three leishmanolysin-like peptidases and a peptidase family M49 protein was up-regulated in the cell-fed ciliates compared to the starved ciliates. Especially, one cysteine peptidase (MaPro 4) and one leishmanolysin-like peptidase (MaPro 14) were transcribed more than 100-folds in the cell-fed ciliates. Conclusions The genetic information and transcriptional characteristics of the peptidases in the present results would be helpful to elucidate the role of peptidases in the invasion of scuticociliates into their hosts. PMID:23311870

  2. Decoding the Anti-Trypanosoma cruzi Action of HIV Peptidase Inhibitors Using Epimastigotes as a Model

    PubMed Central

    Sangenito, Leandro S.; Menna-Barreto, Rubem F. S.; d′Avila-Levy, Claudia M.; Branquinha, Marta H.

    2014-01-01

    Background Aspartic peptidase inhibitors have shown antimicrobial action against distinct microorganisms. Due to an increase in the occurrence of Chagas' disease/AIDS co-infection, we decided to explore the effects of HIV aspartic peptidase inhibitors (HIV-PIs) on Trypanosoma cruzi, the etiologic agent of Chagas' disease. Methodology and Principal Findings HIV-PIs presented an anti-proliferative action on epimastigotes of T. cruzi clone Dm28c, with IC50 values ranging from 0.6 to 14 µM. The most effective inhibitors, ritonavir, lopinavir and nelfinavir, also had an anti-proliferative effect against different phylogenetic T. cruzi strains. The HIV-PIs induced some morphological alterations in clone Dm28c epimastigotes, as reduced cell size and swollen of the cellular body. Transmission electron microscopy revealed that the flagellar membrane, mitochondrion and reservosomes are the main targets of HIV-PIs in T. cruzi epimastigotes. Curiously, an increase in the epimastigote-into-trypomastigote differentiation process of clone Dm28c was observed, with many of these parasites presenting morphological alterations including the detachment of flagellum from the cell body. The pre-treatment with the most effective HIV-PIs drastically reduced the interaction process between epimastigotes and the invertebrate vector Rhodnius prolixus. It was also noted that HIV-PIs induced an increase in the expression of gp63-like and calpain-related molecules, and decreased the cruzipain expression in epimastigotes as judged by flow cytometry and immunoblotting assays. The hydrolysis of a cathepsin D fluorogenic substrate was inhibited by all HIV-PIs in a dose-dependent manner, showing that the aspartic peptidase could be a possible target to these drugs. Additionally, we verified that ritonavir, lopinavir and nelfinavir reduced drastically the viability of clone Dm28c trypomastigotes, causing many morphological damages. Conclusions and Significance The results contribute to understand

  3. A novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases

    PubMed Central

    Liu, Ningning; Liu, Chunjiao; Li, Xiaofen; Liao, Siyan; Song, Wenbin; Yang, Changshan; Zhao, Chong; Huang, Hongbiao; Guan, Lixia; Zhang, Peiquan; Liu, Shouting; Hua, Xianliang; Chen, Xin; Zhou, Ping; Lan, Xiaoying; Yi, Songgang; Wang, Shunqing; Wang, Xuejun; Dou, Q. Ping; Liu, Jinbao

    2014-01-01

    The successful development of bortezomib-based therapy for treatment of multiple myeloma has established proteasome inhibition as an effective therapeutic strategy, and both 20S proteasome peptidases and 19S deubiquitinases (DUBs) are becoming attractive targets of cancer therapy. It has been reported that metal complexes, such as copper complexes, inhibit tumor proteasome. However, the involved mechanism of action has not been fully characterized. Here we report that (i) copper pyrithione (CuPT), an alternative to tributyltin for antifouling paint biocides, inhibits the ubiquitin-proteasome system (UPS) via targeting both 19S proteasome-specific DUBs and 20S proteolytic peptidases with a mechanism distinct from that of the FDA-approved proteasome inhibitor bortezomib; (ii) CuPT potently inhibits proteasome-specific UCHL5 and USP14 activities; (iii) CuPT inhibits tumor growth in vivo and induces cytotoxicity in vitro and ex vivo. This study uncovers a novel class of dual inhibitors of DUBs and proteasome and suggests a potential clinical strategy for cancer therapy. PMID:24912524

  4. A novel proteasome inhibitor suppresses tumor growth via targeting both 19S proteasome deubiquitinases and 20S proteolytic peptidases.

    PubMed

    Liu, Ningning; Liu, Chunjiao; Li, Xiaofen; Liao, Siyan; Song, Wenbin; Yang, Changshan; Zhao, Chong; Huang, Hongbiao; Guan, Lixia; Zhang, Peiquan; Liu, Shouting; Hua, Xianliang; Chen, Xin; Zhou, Ping; Lan, Xiaoying; Yi, Songgang; Wang, Shunqing; Wang, Xuejun; Dou, Q Ping; Liu, Jinbao

    2014-01-01

    The successful development of bortezomib-based therapy for treatment of multiple myeloma has established proteasome inhibition as an effective therapeutic strategy, and both 20S proteasome peptidases and 19S deubiquitinases (DUBs) are becoming attractive targets of cancer therapy. It has been reported that metal complexes, such as copper complexes, inhibit tumor proteasome. However, the involved mechanism of action has not been fully characterized. Here we report that (i) copper pyrithione (CuPT), an alternative to tributyltin for antifouling paint biocides, inhibits the ubiquitin-proteasome system (UPS) via targeting both 19S proteasome-specific DUBs and 20S proteolytic peptidases with a mechanism distinct from that of the FDA-approved proteasome inhibitor bortezomib; (ii) CuPT potently inhibits proteasome-specific UCHL5 and USP14 activities; (iii) CuPT inhibits tumor growth in vivo and induces cytotoxicity in vitro and ex vivo. This study uncovers a novel class of dual inhibitors of DUBs and proteasome and suggests a potential clinical strategy for cancer therapy. PMID:24912524

  5. Cysteine Peptidase B Regulates Leishmania mexicana Virulence through the Modulation of GP63 Expression

    PubMed Central

    Casgrain, Pierre-André; Martel, Caroline; McMaster, W. Robert; Mottram, Jeremy C.; Olivier, Martin; Descoteaux, Albert

    2016-01-01

    Cysteine peptidases play a central role in the biology of Leishmania. In this work, we sought to further elucidate the mechanism(s) by which the cysteine peptidase CPB contributes to L. mexicana virulence and whether CPB participates in the formation of large communal parasitophorous vacuoles induced by these parasites. We initially examined the impact of L. mexicana infection on the trafficking of VAMP3 and VAMP8, two endocytic SNARE proteins associated with phagolysosome biogenesis and function. Using a CPB-deficient mutant, we found that both VAMP3 and VAMP8 were down-modulated in a CPB-dependent manner. We also discovered that expression of the virulence-associated GPI-anchored metalloprotease GP63 was inhibited in the absence of CPB. Expression of GP63 in the CPB-deficient mutant was sufficient to down-modulate VAMP3 and VAMP8. Similarly, episomal expression of GP63 enabled the CPB-deficient mutant to establish infection in macrophages, induce the formation of large communal parasitophorous vacuoles, and cause lesions in mice. These findings implicate CPB in the regulation of GP63 expression and provide evidence that both GP63 and CPB are key virulence factors in L. mexicana. PMID:27191844

  6. Characterization of atrial natriuretic peptide degradation by cell-surface peptidase activity on endothelial cells

    NASA Technical Reports Server (NTRS)

    Frost, S. J.; Whitson, P. A.

    1993-01-01

    Atrial natriuretic peptide (ANP) is a fluid-regulating peptide hormone that promotes vasorelaxation, natriuresis, and diuresis. The mechanisms for the release of ANP and for its clearance from the circulation play important roles in modulating its biological effects. Recently, we have reported that the cell surface of an endothelial cell line, CPA47, could degrade 125I-ANP in the presence of EDTA. In this study, we have characterized this degradation of 125I-ANP. The kinetics of ANP degradation by the surface of CPA47 cells were first order, with a Km of 320 +/- 60 nM and Vmax of 35 +/- 14 pmol of ANP degraded/10 min/10(5) cells at pH 7.4. ANP is degraded by the surface of CPA47 cells over a broad pH range from 7.0-8.5. Potato carboxypeptidase inhibitor and bestatin inhibited 125I-ANP degradation, suggesting that this degradative activity on the surface of CPA47 cells has exopeptidase characteristics. The selectivity of CPA47 cell-surface degradation of ANP was demonstrated when 125I-ANP degradation was inhibited in the presence of neuropeptide Y and angiotensin I and II but not bradykinin, bombesin, endothelin-1, or substance P. The C-terminal amino acids phe26 and tyr28 were deduced to be important for ANP interaction with the cell-surface peptidase(s) based on comparison of the IC50 of various ANP analogues and other natriuretic peptides for the inhibition of ANP degradation. These data suggest that a newly characterized divalent cation-independent exopeptidase(s) that selectively recognizes ANP and some other vasoactive peptides exists on the surface of endothelial cells.

  7. Dipeptidyl peptidase-4: A key player in chronic liver disease

    PubMed Central

    Itou, Minoru; Kawaguchi, Takumi; Taniguchi, Eitaro; Sata, Michio

    2013-01-01

    Dipeptidyl peptidase-4 (DPP-4) is a membrane-associated peptidase, also known as CD26. DPP-4 has widespread organ distribution throughout the body and exerts pleiotropic effects via its peptidase activity. A representative target peptide is glucagon-like peptide-1, and inactivation of glucagon-like peptide-1 results in the development of glucose intolerance/diabetes mellitus and hepatic steatosis. In addition to its peptidase activity, DPP-4 is known to be associated with immune stimulation, binding to and degradation of extracellular matrix, resistance to anti-cancer agents, and lipid accumulation. The liver expresses DPP-4 to a high degree, and recent accumulating data suggest that DPP-4 is involved in the development of various chronic liver diseases such as hepatitis C virus infection, non-alcoholic fatty liver disease, and hepatocellular carcinoma. Furthermore, DPP-4 occurs in hepatic stem cells and plays a crucial role in hepatic regeneration. In this review, we described the tissue distribution and various biological effects of DPP-4. Then, we discussed the impact of DPP-4 in chronic liver disease and the possible therapeutic effects of a DPP-4 inhibitor. PMID:23613622

  8. Dipeptidyl Peptidase-4 Regulation of SDF-1/CXCR4 Axis: Implications for Cardiovascular Disease

    PubMed Central

    Zhong, Jixin; Rajagopalan, Sanjay

    2015-01-01

    Dipeptidyl peptidase-4 (DPP4) is a ubiquitously expressed protease that regulates diverse number of physiological functions. As a dipeptidase, it exerts its catalytic effects on proteins/peptides with proline, alanine, or serine in the penultimate (P1) amino acid residue from the amino terminus. The evidence to date supports an important effect of DPP4 in catalytic cleavage of incretin peptides and this perhaps represents the main mechanism by which DPP4 inhibition improves glycemic control. DPP4 also plays an important role in the degradation of multiple chemokines of which stromal cell-derived factor-1 (SDF-1, also known as CXCL12) is perhaps an increasingly recognized target, given its importance in processes, such as hematopoiesis, angiogenesis, and stem cell homing. In the current review, we will summarize the importance of DPP4-mediated enzymatic processing of cytokines/chemokines with an emphasis on SDF-1 and resultant implications for cardiovascular physiology and disease. PMID:26441982

  9. Role of dipeptidyl peptidase-4 inhibitors in new-onset diabetes after transplantation

    PubMed Central

    Lim, Sun Woo; Jin, Ji Zhe; Jin, Long; Jin, Jian; Li, Can

    2015-01-01

    Despite strict pre- and post-transplantation screening, the incidence of new-onset diabetes after transplantation (NODAT) remains as high as 60%. This complication affects the risk of cardiovascular events and patient and graft survival rates. Thus, reducing the impact of NODAT could improve overall transplant success. The pathogenesis of NODAT is multifactorial, and both modifiable and nonmodifiable risk factors have been implicated. Monitoring and controlling the blood glucose profile, implementing multidisciplinary care, performing lifestyle modifications, using a modified immunosuppressive regimen, administering anti-metabolite agents, and taking a conventional antidiabetic approach may diminish the incidence of NODAT. In addition to these preventive strategies, inhibition of dipeptidyl peptidase-4 (DPP4) by the gliptin family of drugs has recently gained considerable interest as therapy for type 2 diabetes mellitus and NODAT. This review focuses on the role of DPP4 inhibitors and discusses recent literature regarding management of NODAT. PMID:26552451

  10. Inhibitors of signal peptide peptidase (SPP) affect HSV-1 infectivity in vitro and in vivo

    PubMed Central

    Allen, Sariah J.; Mott, Kevin R.; Ghiasi, Homayon

    2014-01-01

    Recently we have shown that the highly conserved herpes simplex virus glycoprotein K (gK) binds to signal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. In this study we have demonstrated for the first time that inhibitors of SPP, such as L685,458, (Z-LL)2 ketone, aspirin, ibuprofen and DAPT, significantly reduced HSV-1 replication in tissue culture. Inhibition of SPP activity via (Z-LL)2 ketone significantly reduced viral transcripts in the nucleus of infected cells. Finally, when administered during primary infection, (Z-LL)2 ketone inhibitor reduced HSV-1 replication in the eyes of ocularly infected mice. Thus, blocking SPP activity may represent a clinically effective and expedient approach to the reduction of viral replication and the resulting pathology. PMID:24768597

  11. Parasite Cathepsin D-Like Peptidases and Their Relevance as Therapeutic Targets.

    PubMed

    Sojka, Daniel; Hartmann, David; Bartošová-Sojková, Pavla; Dvořák, Jan

    2016-09-01

    Inhibition of aspartic cathepsin D-like peptidases (APDs) has been often discussed as an antiparasite intervention strategy. APDs have been considered as virulence factors of Trypanosoma cruzi and Leishmania spp., and have been demonstrated to have important roles in protein trafficking mechanisms of apicomplexan parasites. APDs also initiate blood digestion as components of multienzyme proteolytic complexes in malaria, platyhelminths, nematodes, and ticks. Increasing DNA and RNA sequencing data indicate that parasites express multiple APD isoenzymes of various functions that can now be specifically evaluated using new functional-genomic and biochemical tools, from which we can further assess the potential of APDs as targets for novel effective intervention strategies against parasitic diseases that still pose an alarming threat to mankind. PMID:27344362

  12. Dipeptidyl Peptidase-4 Regulation of SDF-1/CXCR4 Axis: Implications for Cardiovascular Disease.

    PubMed

    Zhong, Jixin; Rajagopalan, Sanjay

    2015-01-01

    Dipeptidyl peptidase-4 (DPP4) is a ubiquitously expressed protease that regulates diverse number of physiological functions. As a dipeptidase, it exerts its catalytic effects on proteins/peptides with proline, alanine, or serine in the penultimate (P1) amino acid residue from the amino terminus. The evidence to date supports an important effect of DPP4 in catalytic cleavage of incretin peptides and this perhaps represents the main mechanism by which DPP4 inhibition improves glycemic control. DPP4 also plays an important role in the degradation of multiple chemokines of which stromal cell-derived factor-1 (SDF-1, also known as CXCL12) is perhaps an increasingly recognized target, given its importance in processes, such as hematopoiesis, angiogenesis, and stem cell homing. In the current review, we will summarize the importance of DPP4-mediated enzymatic processing of cytokines/chemokines with an emphasis on SDF-1 and resultant implications for cardiovascular physiology and disease. PMID:26441982

  13. Three ileus cases associated with the use of dipeptidyl peptidase-4 inhibitors in diabetic patients.

    PubMed

    Kanasaki, Keizo; Konishi, Kazunori; Hayashi, Ranji; Shiroeda, Hisakazu; Nomura, Tomoe; Nakagawa, Atsushi; Nagai, Takako; Takeda-Watanabe, Ai; Ito, Hiroki; Tsuda, Shin-Ichi; Kitada, Munehiro; Fujii, Mizue; Kanasaki, Megumi; Nishizawa, Makoto; Nakano, Yasuharu; Tomita, Yasuto; Ueda, Nobuhiko; Kosaka, Takeo; Koya, Daisuke

    2013-11-27

    Dipeptidyl peptidase (DPP)-4 inhibitors are a new class of antidiabetic drugs that increase incretin hormone levels to enhance blood sugar level-dependent insulinotropic effects, suppress glucagon action, and reduce bowel motility. These incretin effects are ideal for blood sugar control. However, the safety profile of DPP-4 inhibitors is not yet established. Herein, we present three cases of ileus, considered to be closely related to the use of DPP-4 inhibitors, in diabetic patients. Each of the three patients exhibited some risk of a deficiency in bowel movement; the onset of ileus was within 40 days after strengthened inhibition of DPP-4. The use of a DPP-4 inhibitor could be safe, although the cases presented herein enable us to inform the scientific community to some of the potential adverse effects of the use of DPP-4 inhibitors in select populations. PMID:24843724

  14. Bunyamwera orthobunyavirus glycoprotein precursor is processed by cellular signal peptidase and signal peptide peptidase

    PubMed Central

    Shi, Xiaohong; Botting, Catherine H.; Li, Ping; Niglas, Mark; Brennan, Benjamin; Shirran, Sally L.; Szemiel, Agnieszka M.; Elliott, Richard M.

    2016-01-01

    The M genome segment of Bunyamwera virus (BUNV)—the prototype of both the Bunyaviridae family and the Orthobunyavirus genus—encodes the glycoprotein precursor (GPC) that is proteolytically cleaved to yield two viral structural glycoproteins, Gn and Gc, and a nonstructural protein, NSm. The cleavage mechanism of orthobunyavirus GPCs and the host proteases involved have not been clarified. In this study, we investigated the processing of BUNV GPC and found that both NSm and Gc proteins were cleaved at their own internal signal peptides (SPs), in which NSm domain I functions as SPNSm and NSm domain V as SPGc. Moreover, the domain I was further processed by a host intramembrane-cleaving protease, signal peptide peptidase, and is required for cell fusion activities. Meanwhile, the NSm domain V (SPGc) remains integral to NSm, rendering the NSm topology as a two-membrane-spanning integral membrane protein. We defined the cleavage sites and boundaries between the processed proteins as follows: Gn, from residue 17–312 or nearby residues; NSm, 332–477; and Gc, 478–1433. Our data clarified the mechanism of the precursor cleavage process, which is important for our understanding of viral glycoprotein biogenesis in the genus Orthobunyavirus and thus presents a useful target for intervention strategies. PMID:27439867

  15. The first crystal structure of the peptidase domain of the U32 peptidase family.

    PubMed

    Schacherl, Magdalena; Montada, Angelika A M; Brunstein, Elena; Baumann, Ulrich

    2015-12-01

    The U32 family is a collection of over 2500 annotated peptidases in the MEROPS database with unknown catalytic mechanism. They mainly occur in bacteria and archaea, but a few representatives have also been identified in eukarya. Many of the U32 members have been linked to pathogenicity, such as proteins from Helicobacter and Salmonella. The first crystal structure analysis of a U32 catalytic domain from Methanopyrus kandleri (gene mk0906) reveals a modified (βα)8 TIM-barrel fold with some unique features. The connecting segment between strands β7 and β8 is extended and helix α7 is located on top of the C-terminal end of the barrel body. The protein exhibits a dimeric quaternary structure in which a zinc ion is symmetrically bound by histidine and cysteine side chains from both monomers. These residues reside in conserved sequence motifs. No typical proteolytic motifs are discernible in the three-dimensional structure, and biochemical assays failed to demonstrate proteolytic activity. A tunnel in which an acetate ion is bound is located in the C-terminal part of the β-barrel. Two hydrophobic grooves lead to a tunnel at the C-terminal end of the barrel in which an acetate ion is bound. One of the grooves binds to a Strep-Tag II of another dimer in the crystal lattice. Thus, these grooves may be binding sites for hydrophobic peptides or other ligands. PMID:26627657

  16. Identification of peptidases in highly pathogenic vs. weakly pathogenic Naegleria fowleri amebae.

    PubMed

    Vyas, Ishan K; Jamerson, Melissa; Cabral, Guy A; Marciano-Cabral, Francine

    2015-01-01

    Naegleria fowleri, a free-living ameba, is the causative agent of Primary Amebic Meningoencephalitis. Highly pathogenic mouse-passaged amebae (Mp) and weakly pathogenic axenically grown (Ax) N. fowleri were examined for peptidase activity. Zymography and azocasein peptidase activity assays demonstrated that Mp and Ax N. fowleri exhibited a similar peptidase pattern. Prominent for whole cell lysates, membranes and conditioned medium (CM) from Mp and Ax amebae was the presence of an activity band of approximately 58 kDa that was sensitive to E64, a cysteine peptidase inhibitor. However, axenically grown N. fowleri demonstrated a high level of this peptidase activity in membrane preparations. The inhibitor E64 also reduced peptidase activity in ameba-CM consistent with the presence of secreted cysteine peptidases. Exposure of Mp amebae to E64 reduced their migration through matrigel that was used as an extracellular matrix, suggesting a role for cysteine peptidases in invasion of the central nervous system (CNS). The collective results suggest that the profile of peptidases is not a discriminative marker for distinguishing Mp from Ax N. fowleri. However, the presence of a prominent level of activity for cysteine peptidases in N. fowleri membranes and CM, suggests that these enzymes may serve to facilitate passage of the amebae into the CNS. PMID:25066578

  17. Mosapride, a selective serotonin 5-HT4 receptor agonist, and alogliptin, a selective dipeptidyl peptidase-4 inhibitor, exert synergic effects on plasma active GLP-1 levels and glucose tolerance in mice.

    PubMed

    Nonogaki, Katsunori; Kaji, Takao

    2015-12-01

    Pharmacologic stimulation of serotonin 5-HT4 receptors increased plasma active glucagon-like-peptide-1 (GLP-1) levels independent of feeding, and that pharmacologic stimulation of 5-HT4 receptors and pharmacologic inhibition of dipeptidyl peptidase-4 exerted synergic effects on plasma active GLP-1 levels and glucose tolerance in mice. PMID:26497774

  18. Cysteine Peptidases as Schistosomiasis Vaccines with Inbuilt Adjuvanticity

    PubMed Central

    El Ridi, Rashika; Tallima, Hatem; Selim, Sahar; Donnelly, Sheila; Cotton, Sophie; Gonzales Santana, Bibiana; Dalton, John P.

    2014-01-01

    Schistosomiasis is caused by several worm species of the genus Schistosoma and afflicts up to 600 million people in 74 tropical and sub-tropical countries in the developing world. Present disease control depends on treatment with the only available drug praziquantel. No vaccine exists despite the intense search for molecular candidates and adjuvant formulations over the last three decades. Cysteine peptidases such as papain and Der p 1 are well known environmental allergens that sensitize the immune system driving potent Th2-responses. Recently, we showed that the administration of active papain to mice induced significant protection (P<0.02, 50%) against an experimental challenge infection with Schistosoma mansoni. Since schistosomes express and secrete papain-like cysteine peptidases we reasoned that these could be employed as vaccines with inbuilt adjuvanticity to protect against these parasites. Here we demonstrate that sub-cutaneous injection of functionally active S. mansoni cathepsin B1 (SmCB1), or a cathepsin L from a related parasite Fasciola hepatica (FhCL1), elicits highly significant (P<0.0001) protection (up to 73%) against an experimental challenge worm infection. Protection and reduction in worm egg burden were further increased (up to 83%) when the cysteine peptidases were combined with other S. mansoni vaccine candidates, glyceraldehyde 3-phosphate dehydrogenase (SG3PDH) and peroxiredoxin (PRX-MAP), without the need to add chemical adjuvants. These studies demonstrate the capacity of helminth cysteine peptidases to behave simultaneously as immunogens and adjuvants, and offer an innovative approach towards developing schistosomiasis vaccines PMID:24465551

  19. Dipeptidyl peptidase-4 inhibitor for steroid-induced diabetes

    PubMed Central

    Yanai, Hidekatsu; Masui, Yoshinori; Yoshikawa, Reo; Kunimatsu, Junwa; Kaneko, Hiroshi

    2010-01-01

    The addition of the dipeptidyl peptidase-4 (DDP-4) inhibitor has been reported to achieve greater improvements in glucose metabolism with fewer adverse events compared to increasing the metformin dose in type 2 diabetic patients. We present a patient with steroid-induced diabetes whose blood glucose levels were ameliorated by the use of the DPP-4 inhibitor, showing that the DPP-4 inhibitors may be an effective and safe oral anti-diabetic drug for steroid-induced diabetes. PMID:21537433

  20. Extracellular peptidases of the cereal pathogen Fusarium graminearum

    PubMed Central

    Lowe, Rohan G. T.; McCorkelle, Owen; Bleackley, Mark; Collins, Christine; Faou, Pierre; Mathivanan, Suresh; Anderson, Marilyn

    2015-01-01

    The plant pathogenic fungus Fusarium graminearum (Fgr) creates economic and health risks in cereals agriculture. Fgr causes head blight (or scab) of wheat and stalk rot of corn, reducing yield, degrading grain quality, and polluting downstream food products with mycotoxins. Fungal plant pathogens must secrete proteases to access nutrition and to breakdown the structural protein component of the plant cell wall. Research into the proteolytic activity of Fgr is hindered by the complex nature of the suite of proteases secreted. We used a systems biology approach comprising genome analysis, transcriptomics and label-free quantitative proteomics to characterize the peptidases deployed by Fgr during growth. A combined analysis of published microarray transcriptome datasets revealed seven transcriptional groupings of peptidases based on in vitro growth, in planta growth, and sporulation behaviors. A high resolution mass spectrometry-based proteomics analysis defined the extracellular proteases secreted by F. graminearum. A meta-classification based on sequence characters and transcriptional/translational activity in planta and in vitro provides a platform to develop control strategies that target Fgr peptidases. PMID:26635820

  1. Streptococcal C5a peptidase is a highly specific endopeptidase.

    PubMed Central

    Cleary, P P; Prahbu, U; Dale, J B; Wexler, D E; Handley, J

    1992-01-01

    Compositional analysis of streptococcal C5a peptidase (SCPA) cleavage products from a synthetic peptide corresponding to the 20 C-terminal residues of C5a demonstrated that the target cleavage site is His-Lys rather than Lys-Asp, as previously suggested. A C5a peptide analog with Lys replaced by Gln was also subject to cleavage by SCPA. This confirmed that His-Lys rather than Lys-Asp is the scissile bond. Cleavage at histidine is unusual but is the same as that suggested for a peptidase produced by group B streptococci. Native C5 protein was also resistant to SCPA, suggesting that the His-Lys bond is inaccessible prior to proteolytic cleavage by C5 convertase. These experiments showed that the streptococcal C5a peptidase is highly specific for C5a and suggest that its function is not merely to process protein for metabolic consumption but to act primarily to eliminate this chemotactic signal from inflammatory foci. Images PMID:1452354

  2. Post-injury administration of NAAG peptidase inhibitor prodrug, PGI-02776, in experimental TBI.

    PubMed

    Feng, Jun-Feng; Van, Ken C; Gurkoff, Gene G; Kopriva, Christina; Olszewski, Rafal T; Song, Minsoo; Sun, Shifeng; Xu, Man; Neale, Joseph H; Yuen, Po-Wai; Lowe, David A; Zhou, Jia; Lyeth, Bruce G

    2011-06-13

    Traumatic brain injury (TBI) leads to a rapid and excessive increase in glutamate concentration in the extracellular milieu, which is strongly associated with excitotoxicity and neuronal degeneration. N-acetylaspartylglutamate (NAAG), a prevalent peptide neurotransmitter in the vertebrate nervous system, is released along with glutamate and suppresses glutamate release by actions at pre-synaptic metabotropic glutamate autoreceptors. Extracellular NAAG is hydrolyzed to N-acetylaspartate and glutamate by peptidase activity. In the present study PGI-02776, a newly designed di-ester prodrug of the urea-based NAAG peptidase inhibitor ZJ-43, was tested for neuroprotective potential when administered intraperitoneally 30 min after lateral fluid percussion TBI in the rat. Stereological quantification of hippocampal CA2-3 degenerating neurons at 24 h post injury revealed that 10 mg/kg PGI-02776 significantly decreased the number of degenerating neurons (p<0.05). Both average latency analysis of Morris water maze performance and assessment of 24-hour memory retention revealed significant differences between sham-TBI and TBI-saline. In contrast, no significant difference was found between sham-TBI and PGI-02776 treated groups in either analysis indicating an improvement in cognitive performance with PGI-02776 treatment. Histological analysis on day 16 post-injury revealed significant cell death in injured animals regardless of treatment. In vitro NAAG peptidase inhibition studies demonstrated that the parent compound (ZJ-43) exhibited potent inhibitory activity while the mono-ester (PGI-02749) and di-ester (PGI-02776) prodrug compounds exhibited moderate and weak levels of inhibitory activity, respectively. Pharmacokinetic assays in uninjured animals found that the di-ester (PGI-02776) crossed the blood-brain barrier. PGI-02776 was also readily hydrolyzed to both the mono-ester (PGI-02749) and the parent compound (ZJ-43) in both blood and brain. Overall, these findings

  3. Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures.

    PubMed

    Semashko, Tatiana A; Vorotnikova, Elena A; Sharikova, Valeriya F; Vinokurov, Konstantin S; Smirnova, Yulia A; Dunaevsky, Yakov E; Belozersky, Mikhail A; Oppert, Brenda; Elpidina, Elena N; Filippova, Irina Y

    2014-03-15

    This study describes the design, synthesis, and use of selective peptide substrates for cysteine peptidases of the C1 papain family, important in many biological processes. The structure of the newly synthesized substrates is Glp-Xaa-Ala-Y (where Glp=pyroglutamyl; Xaa=Phe or Val; and Y=pNA [p-nitroanilide], AMC [4-amino-7-methylcoumaride], or AFC [4-amino-7-trifluoromethyl-coumaride]). Substrates were synthesized enzymatically to guarantee selectivity of the reaction and optical purity of the target compounds, simplifying the scheme of synthesis and isolation of products. The hydrolysis of the synthesized substrates was evaluated by C1 cysteine peptidases from different organisms and with different functions, including plant enzymes papain, bromelain, ficin, and mammalian lysosomal cathepsins B and L. The new substrates were selective for C1 cysteine peptidases and were not hydrolyzed by serine, aspartic, or metallo peptidases. We demonstrated an application of the selectivity of the synthesized substrates during the chromatographic separation of a multicomponent set of digestive peptidases from a beetle, Tenebrio molitor. Used in combination with the cysteine peptidase inhibitor E-64, these substrates were able to differentiate cysteine peptidases from peptidases of other classes in midgut extracts from T. molitor larvae and larvae of the genus Tribolium; thus, they are useful in the analysis of complex mixtures containing peptidases from different classes. PMID:24388866

  4. Campylobacter jejuni gene cj0511 encodes a serine peptidase essential for colonisation

    PubMed Central

    Karlyshev, A.V.; Thacker, G.; Jones, M.A.; Clements, M.O.; Wren, B.W.

    2014-01-01

    According to MEROPS peptidase database, Campylobacter species encode 64 predicted peptidases. However, proteolytic properties of only a few of these proteins have been confirmed experimentally. In this study we identified and characterised a Campylobacter jejuni gene cj0511 encoding a novel peptidase. The proteolytic activity associated with this enzyme was demonstrated in cell lysates. Moreover, enzymatic studies conducted with a purified protein confirmed a prediction of it being a serine peptidase. Furthermore, cj0511 mutant was found to be severely attenuated in chicken colonisation model, suggesting a role of the Cj0511 protein in infection. PMID:24918062

  5. Identification of dipeptidyl peptidase 3 as the Angiotensin-(1-7) degrading peptidase in human HK-2 renal epithelial cells.

    PubMed

    Cruz-Diaz, Nildris; Wilson, Bryan A; Pirro, Nancy T; Brosnihan, K Bridget; Marshall, Allyson C; Chappell, Mark C

    2016-09-01

    Angiotensin-(1-7) (Ang-(1-7)) is expressed within the kidney and exhibits renoprotective actions that antagonize the inflammatory, fibrotic and pro-oxidant effects of the Ang II-AT1 receptor axis. We previously identified a peptidase activity from sheep brain, proximal tubules and human HK-2 proximal tubule cells that metabolized Ang-(1-7); thus, the present study isolated and identified the Ang-(1-7) peptidase. Utilizing ion exchange and hydrophobic interaction chromatography, a single 80kDa protein band on SDS-PAGE was purified from HK-2 cells. The 80kDa band was excised, the tryptic digest peptides analyzed by LC-MS and a protein was identified as the enzyme dipeptidyl peptidase 3 (DPP 3, EC: 3.4.14.4). A human DPP 3 antibody identified a single 80kDa band in the purified enzyme preparation identical to recombinant human DPP 3. Both the purified Ang-(1-7) peptidase and DPP 3 exhibited an identical hydrolysis profile of Ang-(1-7) and both activities were abolished by the metallopeptidase inhibitor JMV-390. DPP 3 sequentially hydrolyzed Ang-(1-7) to Ang-(3-7) and rapidly converted Ang-(3-7) to Ang-(5-7). Kinetic analysis revealed that Ang-(3-7) was hydrolyzed at a greater rate than Ang-(1-7) [17.9 vs. 5.5 nmol/min/μg protein], and the Km for Ang-(3-7) was lower than Ang-(1-7) [3 vs. 12μM]. Finally, chronic treatment of the HK-2 cells with 20nM JMV-390 reduced intracellular DPP 3 activity and tended to augment the cellular levels of Ang-(1-7). We conclude that DPP 3 may influence the cellular expression of Ang-(1-7) and potentially reflect a therapeutic target to augment the actions of the peptide. PMID:27315786

  6. NAAG peptidase as a therapeutic target: Potential for regulating the link between glucose metabolism and cognition.

    PubMed

    Baslow, Morris H

    2006-04-01

    There is a new class of CNS drugs, N-acetylaspartylglutamate (NAAG) peptidase inhibitors, that can affect a two-step, neuron-astrocyte/astrocyte-vascular endothelium, signaling mechanism. Using this homeostatic mechanism, activated neurons continuously interact with the vascular system to indicate ongoing requirements for supplies of glucose (Glc) and oxygen needed to maintain cognitive functions. These new drugs impact the first step by inhibiting NAAG peptidase, located on the astrocyte surface, that cleaves glutamate (Glu) from the neuropeptide NAAG after it has docked with the astrocyte surface metabotropic Glu receptor 3 (mGluR3). As a result, this interferes with initiation of the second step, the astrocyte-vascular endothelium signal, activation of which results in a rapid hyperemic response that increases focal availability of energy supplies. Since NAAG is liberated upon each neuron depolarization, its release is linked to the level of neuronal spiking. This insures that its mGluR3 signal function reflects current levels of neuronal stimulation, so that Glc and oxygen can be supplied in a timely manner for metabolic replacement of ATP stocks depleted during the repolarization process. The metabolism of NAAG is very complex, being a component of the only metabolic sequence in the brain requiring three major brain cell types--neurons, astrocytes and oligodendrocytes--for its successful completion. In this review, we describe the unique NAAG tricellular metabolic cycle and survey some reported actions of these new and novel drugs. We also consider their probable site and mode of action and speculate upon their therapeutic potential. PMID:16804566

  7. Plasmodium falciparum signal peptide peptidase cleaves malaria heat shock protein 101 (HSP101). Implications for gametocytogenesis

    SciTech Connect

    Baldwin, Michael; Russo, Crystal; Li, Xuerong; Chishti, Athar H.

    2014-08-08

    Highlights: • PfSPP is an ER resident protease. • PfSPP is expressed both as a monomer and dimer. • The signal peptide of HSP101 is the first known substrate of PfSPP. • Reduced PfSPP activity may significantly affect ER homeostasis. - Abstract: Previously we described the identification of a Plasmodium falciparum signal peptide peptidase (PfSPP) functioning at the blood stage of malaria infection. Our studies also demonstrated that mammalian SPP inhibitors prevent malaria parasite growth at the late-ring/early trophozoite stage of intra-erythrocytic development. Consistent with its role in development, we tested the hypothesis that PfSPP functions at the endoplasmic reticulum of P.falciparum where it cleaves membrane-bound signal peptides generated following the enzyme activity of signal peptidase. The localization of PfSPP to the endoplasmic reticulum was confirmed by immunofluorescence microscopy and immunogold electron microscopy. Biochemical analysis indicated the existence of monomer and dimer forms of PfSPP in the parasite lysate. A comprehensive bioinformatics screen identified several candidate PfSPP substrates in the parasite genome. Using an established transfection based in vivo luminescence assay, malaria heat shock protein 101 (HSP101) was identified as a substrate of PfSPP, and partial inhibition of PfSPP correlated with the emergence of gametocytes. This finding unveils the first known substrate of PfSPP, and provides new perspectives for the function of intra-membrane proteolysis at the erythrocyte stage of malaria parasite life cycle.

  8. Gliptins and their target dipeptidyl peptidase 4: implications for the treatment of vascular disease.

    PubMed

    Remm, Friederike; Franz, Wolfgang-Michael; Brenner, Christoph

    2016-07-01

    Gliptins are accepted as a standard therapy for diabetes mellitus today. By inhibition of the enzyme dipeptidyl peptidase 4 (DPP4), gliptins prolong the GLP1-dependent insulin secretion in the pancreatic β-cells and thus support physiological blood glucose control. Various studies have now raised hope for an additional protective effect of pharmacological DPP4 inhibition in vascular diseases. Besides GLP1, especially, the inhibition of SDF1 cleavage has been shown to depict a relevant mechanism to enhance endothelial regeneration and reduce atherosclerosis progression via the SDF1-CXCR4 axis. Furthermore, several clinical trials have now shown an excellent safety profile of gliptin therapy in cardiovascular risk patients. In this review, we give a comprehensive overview on DPP4-dependent vascular functions and pathophysiological mechanisms with a detailed discussion of the underlying molecular mechanisms. We further analyse the role of pharmacological DPP4 inhibitors and their potential therapeutic impact on endothelial function and regeneration besides their effect during atherosclerosis development. Finally, we discuss presently available data from in vitro and in vivo studies with respect to the results of the recent clinical trials in diabetic and non-diabetic patients. PMID:27533760

  9. Insecticidal effect of Canavalia ensiformis major urease on nymphs of the milkweed bug Oncopeltus fasciatus and characterization of digestive peptidases.

    PubMed

    Defferrari, Marina S; Demartini, Diogo R; Marcelino, Thiago B; Pinto, Paulo M; Carlini, Celia R

    2011-06-01

    Jackbean (Canavalia ensiformis) ureases are entomotoxic upon the release of internal peptides by insect's digestive enzymes. Here we studied the digestive peptidases of Oncopeltus fasciatus (milkweed bug) and its susceptibility to jackbean urease (JBU). O. fasciatus nymphs fed urease showed a mortality rate higher than 80% after two weeks. Homogenates of midguts dissected from fourth instars were used to perform proteolytic activity assays. The homogenates hydrolyzed JBU in vitro, yielding a fragment similar in size to known entomotoxic peptides. The major proteolytic activity at pH 4.0 upon protein substrates was blocked by specific inhibitors of aspartic and cysteine peptidases, but not significantly affected by inhibitors of metallopeptidases or serine peptidases. The optimal activity upon N-Cbz-Phe-Arg-MCA was at pH 5.0, with complete blockage by E-64 in all pH tested. Optimal activity upon Abz-AIAFFSRQ-EDDnp (a substrate for aspartic peptidases) was detected at pH 5.0, with partial inhibition by Pepstatin A in the pH range 2-8. Fluorogenic substrates corresponding to the N- and C-terminal regions flanking a known entomotoxic peptide within urease sequence were also tested. While the midgut homogenate did not hydrolyze the N-terminal peptide, it cleaved the C-terminal peptide maximally at pH 4.0-5.0, and this activity was inhibited by E-64 (10 μM). The midgut homogenate was submitted to ion-exchange chromatography followed by gel filtration. A 22 kDa active fraction was obtained, resolved in SDS-PAGE (12%), the corresponding band was in-gel digested by trypsin, the peptides were analyzed by mass spectrometry, retrieving a cathepsin L protein. The purified cathepsin L was shown to have at least two possible cleavage sites within the urease sequence, and might be able to release a known insecticidal peptide in a single or cascade event. The results suggest that susceptibility of O. fasciatus nymphs to jackbean urease is, like in other insect models, due mostly

  10. Antinociceptive effects of N-acetylaspartylglutamate (NAAG) peptidase inhibitors ZJ-11, ZJ-17 and ZJ-43 in the rat formalin test and in the rat neuropathic pain model.

    PubMed

    Yamamoto, Tatsuo; Hirasawa, Serabi; Wroblewska, Barbara; Grajkowska, Ewa; Zhou, Jia; Kozikowski, Alan; Wroblewski, Jarda; Neale, Joseph H

    2004-07-01

    The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) acts as an agonist at group II metabotropic glutamate receptors (mGluRs). NAAG is inactivated by extracellular peptidase activity yielding glutamate and N-acetylaspartate. We recently developed a series of potent NAAG peptidase inhibitors, including ZJ-11, ZJ-17 and ZJ-43. In the present study, we examined the effects of intrathecally administered ZJ-11 and ZJ-17 and intravenously administered ZJ-11 and ZJ-43 in the rat formalin test (an inflammatory pain model) and in the rat partial sciatic nerve ligation model (a neuropathic pain model). Intrathecal injection of ZJ-11 or ZJ-17 or intravenous injection of ZJ-11 or ZJ-43 suppressed both phases of the agitation behaviour induced by paw formalin injection. Intrathecal and intravenous injection of ZJ-11 suppressed the expression of Fos-like immunoreactivity, induced by paw formalin injection, in laminae I-II in segments L4-L5 of the spinal cord, suggesting an action on sensory spinal transmission. Partial sciatic nerve ligation induced significant mechanical allodynia 7 days after the nerve injury. Intrathecal injection of ZJ-11 or ZJ-17 or intravenous administration of ZJ-11 or ZJ-43 attenuated the level of mechanical allodynia induced by this nerve ligation. These effects of intrathecally or intravenously administered ZJ compounds in both the formalin test and the partial sciatic nerve ligation model were completely antagonized by pretreatment with LY-341495, a highly selective group II mGluR antagonist. Thus, elevation of extracellular NAAG, induced by the inhibition of NAAG peptidase, activates group II mGluRs and produces an analgesic effect in neuropathic and inflammatory and pain models. In contrast, peptidase inhibition did not affect the threshold for withdrawal from a noxious mechanical stimulus or from an acute thermal stimulus in the hotplate test. PMID:15233757

  11. Analysis of the Structural and Molecular Basis of Voltage-sensitive Sodium Channel Inhibition by the Spider Toxin Huwentoxin-IV (μ-TRTX-Hh2a)

    PubMed Central

    Minassian, Natali A.; Gibbs, Alan; Shih, Amy Y.; Liu, Yi; Neff, Robert A.; Sutton, Steven W.; Mirzadegan, Tara; Connor, Judith; Fellows, Ross; Husovsky, Matthew; Nelson, Serena; Hunter, Michael J.; Flinspach, Mack; Wickenden, Alan D.

    2013-01-01

    Voltage-gated sodium channels (VGSCs) are essential to the normal function of the vertebrate nervous system. Aberrant function of VGSCs underlies a variety of disorders, including epilepsy, arrhythmia, and pain. A large number of animal toxins target these ion channels and may have significant therapeutic potential. Most of these toxins, however, have not been characterized in detail. Here, by combining patch clamp electrophysiology and radioligand binding studies with peptide mutagenesis, NMR structure determination, and molecular modeling, we have revealed key molecular determinants of the interaction between the tarantula toxin huwentoxin-IV and two VGSC isoforms, Nav1.7 and Nav1.2. Nine huwentoxin-IV residues (F6A, P11A, D14A, L22A, S25A, W30A, K32A, Y33A, and I35A) were important for block of Nav1.7 and Nav1.2. Importantly, molecular dynamics simulations and NMR studies indicated that folding was normal for several key mutants, suggesting that these amino acids probably make specific interactions with sodium channel residues. Additionally, we identified several amino acids (F6A, K18A, R26A, and K27A) that are involved in isoform-specific VGSC interactions. Our structural and functional data were used to model the docking of huwentoxin-IV into the domain II voltage sensor of Nav1.7. The model predicts that a hydrophobic patch composed of Trp-30 and Phe-6, along with the basic Lys-32 residue, docks into a groove formed by the Nav1.7 S1-S2 and S3-S4 loops. These results provide new insight into the structural and molecular basis of sodium channel block by huwentoxin-IV and may provide a basis for the rational design of toxin-based peptides with improved VGSC potency and/or selectivity. PMID:23760503

  12. Carbonic Anhydrase Inhibitors. Part 461 Inhibition of Carbonic Anhydrase Isozymes I, II and IV With Trifluoromethylsulfonamide Derivatives and Their Zinc(II) and Copper(II) Complexes

    PubMed Central

    Mincione, Giovanna; Scozzafava, Andrea

    1997-01-01

    Reaction of aromatic/heterocyclic sulfonamides containing a free amino group with triflic anhydride afforded compounds possessing trifluoromethanesulfonamido moieties in their molecule. The Zn(II) and Cu(II) complexes of these new sulfonamides were prepared and characterized by standard procedures (elemental analysis, spectroscopic, magnetic, thermogravimetric and conductimetric measurements). The new derivatives showed good inhibitory activity against three isozymes of carbonic anhydrase (CA), i.e., CA I, II and IV. PMID:18475762

  13. Analysis of the structural and molecular basis of voltage-sensitive sodium channel inhibition by the spider toxin huwentoxin-IV (μ-TRTX-Hh2a).

    PubMed

    Minassian, Natali A; Gibbs, Alan; Shih, Amy Y; Liu, Yi; Neff, Robert A; Sutton, Steven W; Mirzadegan, Tara; Connor, Judith; Fellows, Ross; Husovsky, Matthew; Nelson, Serena; Hunter, Michael J; Flinspach, Mack; Wickenden, Alan D

    2013-08-01

    Voltage-gated sodium channels (VGSCs) are essential to the normal function of the vertebrate nervous system. Aberrant function of VGSCs underlies a variety of disorders, including epilepsy, arrhythmia, and pain. A large number of animal toxins target these ion channels and may have significant therapeutic potential. Most of these toxins, however, have not been characterized in detail. Here, by combining patch clamp electrophysiology and radioligand binding studies with peptide mutagenesis, NMR structure determination, and molecular modeling, we have revealed key molecular determinants of the interaction between the tarantula toxin huwentoxin-IV and two VGSC isoforms, Nav1.7 and Nav1.2. Nine huwentoxin-IV residues (F6A, P11A, D14A, L22A, S25A, W30A, K32A, Y33A, and I35A) were important for block of Nav1.7 and Nav1.2. Importantly, molecular dynamics simulations and NMR studies indicated that folding was normal for several key mutants, suggesting that these amino acids probably make specific interactions with sodium channel residues. Additionally, we identified several amino acids (F6A, K18A, R26A, and K27A) that are involved in isoform-specific VGSC interactions. Our structural and functional data were used to model the docking of huwentoxin-IV into the domain II voltage sensor of Nav1.7. The model predicts that a hydrophobic patch composed of Trp-30 and Phe-6, along with the basic Lys-32 residue, docks into a groove formed by the Nav1.7 S1-S2 and S3-S4 loops. These results provide new insight into the structural and molecular basis of sodium channel block by huwentoxin-IV and may provide a basis for the rational design of toxin-based peptides with improved VGSC potency and/or selectivity. PMID:23760503

  14. Phlorizin, an Active Ingredient of Eleutherococcus senticosus, Increases Proliferative Potential of Keratinocytes with Inhibition of MiR135b and Increased Expression of Type IV Collagen

    PubMed Central

    Choi, Hye-Ryung; Nam, Kyung-Mi; Lee, Hyun-Sun; Yang, Seung-Hye; Kim, Young-Soo; Lee, Jongsung; Date, Akira; Toyama, Kazumi; Park, Kyoung-Chan

    2016-01-01

    E. senticosus extract (ESE), known as antioxidant, has diverse pharmacologic effects. It is also used as an antiaging agent for the skin and phlorizin (PZ) is identified as a main ingredient. In this study, the effects of PZ on epidermal stem cells were investigated. Cultured normal human keratinocytes and skin equivalents are used to test whether PZ affects proliferative potential of keratinocytes and how it regulates these effects. Skin equivalents (SEs) were treated with ESE and the results showed that the epidermis became slightly thickened on addition of 0.002% ESE. The staining intensity of p63 as well as proliferating cell nuclear antigen (PCNA) is increased, and integrin α6 was upregulated. Analysis of ESE confirmed that PZ is the main ingredient. When SEs were treated with PZ, similar findings were observed. In particular, the expression of integrin α6, integrin β1, and type IV collagen was increased. Levels of mRNA for type IV collagen were increased and levels of miR135b were downregulated. All these findings suggested that PZ can affect the proliferative potential of epidermal cells in part by microenvironment changes via miR135b downregulation and following increased expression of type IV collagen. PMID:27042261

  15. Phlorizin, an Active Ingredient of Eleutherococcus senticosus, Increases Proliferative Potential of Keratinocytes with Inhibition of MiR135b and Increased Expression of Type IV Collagen.

    PubMed

    Choi, Hye-Ryung; Nam, Kyung-Mi; Lee, Hyun-Sun; Yang, Seung-Hye; Kim, Young-Soo; Lee, Jongsung; Date, Akira; Toyama, Kazumi; Park, Kyoung-Chan

    2016-01-01

    E. senticosus extract (ESE), known as antioxidant, has diverse pharmacologic effects. It is also used as an antiaging agent for the skin and phlorizin (PZ) is identified as a main ingredient. In this study, the effects of PZ on epidermal stem cells were investigated. Cultured normal human keratinocytes and skin equivalents are used to test whether PZ affects proliferative potential of keratinocytes and how it regulates these effects. Skin equivalents (SEs) were treated with ESE and the results showed that the epidermis became slightly thickened on addition of 0.002% ESE. The staining intensity of p63 as well as proliferating cell nuclear antigen (PCNA) is increased, and integrin α6 was upregulated. Analysis of ESE confirmed that PZ is the main ingredient. When SEs were treated with PZ, similar findings were observed. In particular, the expression of integrin α6, integrin β1, and type IV collagen was increased. Levels of mRNA for type IV collagen were increased and levels of miR135b were downregulated. All these findings suggested that PZ can affect the proliferative potential of epidermal cells in part by microenvironment changes via miR135b downregulation and following increased expression of type IV collagen. PMID:27042261

  16. NAAG peptidase inhibitor reduces acute neuronal degeneration and astrocyte damage following lateral fluid percussion TBI in rats.

    PubMed

    Zhong, Chunlong; Zhao, Xueren; Sarva, Jayaprakash; Kozikowski, Alan; Neale, Joseph H; Lyeth, Bruce G

    2005-02-01

    Traumatic brain injury (TBI) produces a rapid and excessive elevation in extracellular glutamate associated with excitotoxicity and secondary brain pathology. The peptide neurotransmitter Nacetylaspartylglutamate (NAAG) suppresses glutamate transmission through selective activation of presynaptic Group II metabotropic glutamate receptor subtype 3 (mGluR3). Thus, inhibition of NAAG peptidase activity and the prolong presence of synaptic NAAG were hypothesized to have significant potential for cellular protection following TBI. In the present study, a novel NAAG peptidase inhibitor, ZJ-43, was used in four different doses (0, 50, 100, or 150 mg/kg). Each dose was repeatedly administered i.p. (n=5/group) by multiple injections at three times (0 time, 8 h, 16 h) after moderate lateral fluid percussion TBI in the rat. An additional group was co-administered ZJ-43 (150 mg/kg) and the Group II mGluR antagonist, LY341495 (1 mg/kg), which was predicted to abolish any protective effects of ZJ-43. Rats were euthanized at 24 h after TBI, and brains were processed with a selective marker for degenerating neurons (Fluoro-Jade B) and a marker for astrocytes (GFAP). Ipsilateral neuronal degeneration and bilateral astrocyte loss in the CA2/3 regions of the hippocampus were quantified using stereological techniques. Compared with vehicle, ZJ-43 significantly reduced the number of the ipsilateral degenerating neurons (p<0.01) with the greatest neuroprotection at the 50 mg/kg dose. Moreover, LY341495 successfully abolished the protective effects of ZJ-43. 50 mg/kg of ZJ-43 also significantly reduced the ipsilateral astrocyte loss (p<0.05). We conclude that the NAAG peptidase inhibitor ZJ-43 is a potential novel strategy to reduce both neuronal and astrocyte damage associated with the glutamate excitotoxicity after TBI. PMID:15716632

  17. NAAG peptidase inhibitor improves motor function and reduces cognitive dysfunction in a model of TBI with secondary hypoxia.

    PubMed

    Gurkoff, Gene G; Feng, Jun-Feng; Van, Ken C; Izadi, Ali; Ghiasvand, Rahil; Shahlaie, Kiarash; Song, Minsoo; Lowe, David A; Zhou, Jia; Lyeth, Bruce G

    2013-06-17

    Immediately following traumatic brain injury (TBI) and TBI with hypoxia, there is a rapid and pathophysiological increase in extracellular glutamate, subsequent neuronal damage and ultimately diminished motor and cognitive function. N-acetyl-aspartyl glutamate (NAAG), a prevalent neuropeptide in the CNS, is co-released with glutamate, binds to the presynaptic group II metabotropic glutamate receptor subtype 3 (mGluR3) and suppresses glutamate release. However, the catalytic enzyme glutamate carboxypeptidase II (GCP II) rapidly hydrolyzes NAAG into NAA and glutamate. Inhibition of the GCP II enzyme with NAAG peptidase inhibitors reduces the concentration of glutamate both by increasing the duration of NAAG activity on mGluR3 and by reducing degradation into NAA and glutamate resulting in reduced cell death in models of TBI and TBI with hypoxia. In the following study, rats were administered the NAAG peptidase inhibitor PGI-02776 (10mg/kg) 30 min following TBI combined with a hypoxic second insult. Over the two weeks following injury, PGI-02776-treated rats had significantly improved motor function as measured by increased duration on the rota-rod and a trend toward improved performance on the beam walk. Furthermore, two weeks post-injury, PGI-02776-treated animals had a significant decrease in latency to find the target platform in the Morris water maze as compared to vehicle-treated animals. These findings demonstrate that the application of NAAG peptidase inhibitors can reduce the deleterious motor and cognitive effects of TBI combined with a second hypoxic insult in the weeks following injury. PMID:23562458

  18. Selective chromogenic and fluorogenic peptide substrates for the assay of cysteine peptidases in complex mixtures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cysteine peptidases are important in many biological processes. In this study, we describe the design, synthesis and use of selective peptide substrates for cysteine peptidases of the C1 papain family. The structure of the proposed substrates can be expressed by the general formula Glp-Xaa-Ala-Y, wh...

  19. Excretion/secretion products from Schistosoma mansoni adults, eggs and schistosomula have unique peptidase specificity profiles.

    PubMed

    Dvořák, Jan; Fajtová, Pavla; Ulrychová, Lenka; Leontovyč, Adrian; Rojo-Arreola, Liliana; Suzuki, Brian M; Horn, Martin; Mareš, Michael; Craik, Charles S; Caffrey, Conor R; O'Donoghue, Anthony J

    2016-03-01

    Schistosomiasis is one of a number of chronic helminth diseases of poverty that severely impact personal and societal well-being and productivity. Peptidases (proteases) are vital to successful parasitism, and can modulate host physiology and immunology. Interference of peptidase action by specific drugs or vaccines can be therapeutically beneficial. To date, research on peptidases in the schistosome parasite has focused on either the functional characterization of individual peptidases or their annotation as part of global genome or transcriptome studies. We were interested in functionally characterizing the complexity of peptidase activity operating at the host-parasite interface, therefore the excretory-secretory products of key developmental stages of Schistosoma mansoni that parasitize the human were examined. Using class specific peptidase inhibitors in combination with a multiplex substrate profiling assay, a number of unique activities derived from endo- and exo-peptidases were revealed in the excretory-secretory products of schistosomula (larval migratory worms), adults and eggs. The data highlight the complexity of the functional degradome for each developmental stage of this parasite and facilitate further enquiry to establish peptidase identity, physiological and immunological function, and utility as drug or vaccine candidates. PMID:26409899

  20. Localization of two post-proline cleaving peptidases in the midgut of Tenebrio molitor larvae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two soluble post-proline cleaving peptidase activities, PPCP1 and PPCP2, were demonstrated in the midgut of Tenebrio molitor larvae with the substrate benzyloxycarbonyl-L-alanyl-L-proline p-nitroanilide. Both activities were serine peptidases. PPCP1 was active in acidic buffers, with maximum activit...

  1. Welding IV.

    ERIC Educational Resources Information Center

    Allegheny County Community Coll., Pittsburgh, PA.

    Instructional objectives and performance requirements are outlined in this course guide for Welding IV, a competency-based course in advanced arc welding offered at the Community College of Allegheny County to provide students with proficiency in: (1) single vee groove welding using code specifications established by the American Welding Society…

  2. IVS Organization

    NASA Technical Reports Server (NTRS)

    2004-01-01

    International VLBI Service (IVS) is an international collaboration of organizations which operate or support Very Long Baseline Interferometry (VLBI) components. The goals are: To provide a service to support geodetic, geophysical and astrometric research and operational activities. To promote research and development activities in all aspects of the geodetic and astrometric VLBI technique. To interact with the community of users of VLBI products and to integrate VLBI into a global Earth observing system.

  3. Leishmania inhibitor of serine peptidase prevents TLR4 activation by neutrophil elastase promoting parasite survival in murine macrophages

    PubMed Central

    Faria, Marilia S.; Reis, Flavia C. G.; Azevedo-Pereira, Ricardo L.; Morrison, Lesley S.; Mottram, Jeremy C.; Lima, Ana Paula C. A.

    2011-01-01

    Leishmania major is a protozoan parasite that causes skin ulcerations in cutaneous leishmaniasis. In the mammalian host, the parasite resides in professional phagocytes and has evolved to avoid killing by macrophages. We identified L. major genes encoding inhibitors of serine peptidases, ISPs, which are orthologues of bacterial ecotins and found that ISP2 inhibits trypsin-fold S1A family peptidases. Here we show that L. major mutants deficient in ISP2 and ISP3 (Δisp2/3) trigger higher phagocytosis by macrophages through a combined action of the complement type-3 receptor (CR3), toll-like receptor 4 (TLR4) and unregulated activity of neutrophil elastase (NE), leading to parasite killing. While all three components are required to mediate enhanced parasite uptake, only TLR4 and NE are necessary to promote parasite killing after infection. We found that the production of superoxide by macrophages in the absence of ISP2 is the main mechanism controlling the intracellular infection. Furthermore, we show that NE modulates macrophage infection in vivo, and that the lack of ISP leads to reduced parasite burdens at later stages of the infection. Our findings support the hypothesis that ISPs function to prevent the activation of TLR4 by NE during the Leishmania-macrophage interaction in order to promote parasite survival and growth. PMID:21098233

  4. Regulatory signals for intestinal amino acid transporters and peptidases

    SciTech Connect

    Ferraris, R.P.; Kwan, W.W.; Diamond, J. )

    1988-08-01

    Dietary protein ultimately regulates many processes involved in protein digestion, but it is often unclear whether proteins themselves, peptides, or amino acids (AAs) are the proximate regulatory signal. Hence the authors compared several processes involved in protein digestion in mice adapted to one of three rations, identical except for containing 54% of either casein, a partial hydrolysate of casein, or a free AA mixture simulating a complete hydrolysate of casein. The authors measured brush-border uptakes of seven AAs that variously serve as substrates for four AA transporters, and brush-border and cytosolic activities of four peptidases. The three rations yielded essentially the same AA uptake rates. Peptidase activities tended to be lower on the AA ration than on the protein ration. In other studies, all three rations yielded the same rates of brush-border peptide uptake; protein is only modestly more effective than AAs at inducing synthesis of pancreatic proteases; and, depending on the animal species, protein is either much less or much more effective than AAs at stimulating release of cholecystokinin and hence of pancreatic enzymes. Thus the regulators of each process involved in protein digestion are not necessarily that process's substrate.

  5. Cadmium delays non-homologous end joining (NHEJ) repair via inhibition of DNA-PKcs phosphorylation and downregulation of XRCC4 and Ligase IV.

    PubMed

    Li, Weiwei; Gu, Xueyan; Zhang, Xiaoning; Kong, Jinxin; Ding, Nan; Qi, Yongmei; Zhang, Yingmei; Wang, Jufang; Huang, Dejun

    2015-09-01

    Although studies have shown that cadmium (Cd) interfered with DNA damage repair (DDR), whether Cd could affect non-homologous end joining (NHEJ) repair remains elusive. To further understand the effect of Cd on DDR, we used X-ray irradiation of Hela cells as an in vitro model system, along with γH2AX and 53BP1 as markers for DNA damage. Results showed that X-ray significantly increased γH2AX and 53BP1 foci in Hela cells (p < 0.01), all of which are characteristic of accrued DNA damage. The number of foci declined rapidly over time (1-8h postirradiation), indicating an initiation of NHEJ process. However, the disappearance of γH2AX and 53BP1 foci was remarkably slowed by Cd pretreatment (p < 0.01), suggesting that Cd reduced the efficiency of NHEJ. To further elucidate the mechanisms of Cd toxicity, several markers of NHEJ pathway including Ku70, DNA-PKcs, XRCC4 and Ligase IV were examined. Our data showed that Cd altered the phosphorylation of DNA-PKcs, and reduced the expression of both XRCC4 and Ligase IV in irradiated cells. These observations are indicative of the impairment of NHEJ-dependent DNA repair pathways. In addition, zinc (Zn) mitigated the effects of Cd on NHEJ, suggesting that the Cd-induced NHEJ alteration may partly result from the displacement of Zn or from an interference with the normal function of Zn-containing proteins by Cd. Our findings provide a new insight into the toxicity of Cd on NHEJ repair and its underlying mechanisms in human cells. PMID:26201248

  6. Astacin Family Metallopeptidases and Serine Peptidase Inhibitors in Spider Digestive Fluid

    PubMed Central

    Foradori, Matthew J.; Tillinghast, Edward K.; Smith, J. Stephen; Townley, Mark A.; Mooney, Robert E.

    2006-01-01

    Digestive fluid of the araneid spider Argiope aurantia is known to contain zinc metallopeptidases. Using anion-exchange chromatography, size-exclusion chromatography, sucrose density gradient centrifugation, and gel electrophoresis, we isolated two lower-molecular-mass peptidases, designated p16 and p18. The N-terminal amino acid sequences of p16 (37 residues) and p18 (20 residues) are 85% identical over the first 20 residues and are most similar to the N-terminal sequences of the fully active form of meprin (β subunits) from several vertebrates (47–52% and 50–60% identical, respectively). Meprin is a peptidase in the astacin (M12A) subfamily of the astacin (M12) family. Additionally, a 66-residue internal sequence obtained from p16 aligns with the conserved astacin subfamily domain. Thus, at least some spider digestive peptidases appear related to astacin of decapod crustaceans. However, important differences between spider and crustacean metallopeptidases with regard to isoelectric point and their susceptibility to hemolymph-borne inhibitors are demonstrated. Anomalous behavior of the lower-molecular-mass Argiope peptidases during certain fractionation procedures indicates that these peptidases may take part in reversible associations with each other or with other proteins. A. aurantia digestive fluid also contains inhibitory activity effective against insect digestive peptidases. Here we present evidence for at least thirteen, heat-stable serine peptidase inhibitors ranging in molecular mass from about 15 to 32 kDa. PMID:16458560

  7. Characterization of papaya peptidase A as an enzyme of extreme basicity.

    PubMed

    Kaarsholm, N C; Schack, P

    1983-01-01

    Papaya peptidase A, a papain-like enzyme, has formerly been shown to contain a larger excess of basic amino acids and to have a higher isoelectric point than any of the other enzymes in the papaya latex. Determinations of the free electrophoretic mobility as a function of pH now establishes the isoelectric point of papaya peptidase A as 11.7 and that of succinylated papaya peptidase A as 3.8. Although the specific activity of the enzyme appears only slightly affected by the succinylation, the accompanying change in the charge/mobility ratio seems to indicate a relatively large conformational change upon succinylation. PMID:6362305

  8. NAAG peptidase inhibitors and their potential for diagnosis and therapy.

    PubMed

    Zhou, Jia; Neale, Joseph H; Pomper, Martin G; Kozikowski, Alan P

    2005-12-01

    Modulation of N-acetyl-L-aspartyl-L-glutamate peptidase activity with small-molecule inhibitors holds promise for a wide variety of diseases that involve glutamatergic transmission, and has implications for the diagnosis and therapy of cancer. This new class of compounds, of which at least one has entered clinical trials and proven to be well tolerated, has demonstrated efficacy in experimental models of pain, schizophrenia, amyotrophic lateral sclerosis, traumatic brain injury and, when appropriately functionalized, can image prostate cancer. Further investigation of these promising drug candidates will be needed to bring them to the marketplace. The recent publication of the X-ray crystal structure for the enzymatic target of these compounds should facilitate the development of other new agents with enhanced activity that could improve both the diagnosis and treatment of neurological disorders. PMID:16341066

  9. Kallikrein-related peptidases (KLKs) and the hallmarks of cancer.

    PubMed

    Filippou, Panagiota S; Karagiannis, George S; Musrap, Natasha; Diamandis, Eleftherios P

    2016-08-01

    The kallikrein-related peptidases (KLKs) represent the largest family of serine proteases within the human genome and are expressed in various tissues. Although they regulate several important physiological functions, KLKs have also been implicated in numerous pathophysiological processes, including cancer. Growing evidence describing the deregulation of KLK expression and secretion, as well as activation in various malignancies, has uncovered their potential as mediators of cancer progression, biomarkers of disease and as candidate therapeutic targets. The diversity of signalling pathways and proteolytic cascades involving KLKs and their downstream targets appears to affect cancer biology through multiple mechanisms, including those related to the hallmarks of cancer. The aim of this review is to provide an update on the importance of KLK-driven molecular pathways in relation to cancer cell traits associated with the hallmarks of cancer and to highlight their potential in personalized therapeutics. PMID:26886390

  10. Lipoprotein effects of incretin analogs and dipeptidyl peptidase 4 inhibitors

    PubMed Central

    Zhong, Jixin; Maiseyeu, Andrei; Rajagopalan, Sanjay

    2015-01-01

    Elevated post-prandial lipoprotein levels are common in patients with type 2 diabetes. Post-prandial lipoprotein alterations in type 2 diabetics are widely believed to drive inflammation and are considered a major risk factor for cardiovascular disease in diabetic patients. The incretins glucagon like peptide-1 (GLP-1) and glucose insulinotropic peptide (GIP) modulate post-prandial lipoproteins through a multitude of pathways that are independent of insulin and weight loss. Evidence from both animal models and humans seems to suggest an important effect on triglyceride rich lipoproteins (Apo48 containing) with little to no effects on other lipoproteins at least in humans. Dipeptidyl peptidase-4 (DPP4) inhibitors also appear to share these effects suggesting an important role for incretins in these effects. In this review, we will summarize lipid modulating effects of incretin analogs and DPP-4 inhibitors in both animal models and human studies and provide an overview of mechanisms responsible for these effects. PMID:26005496