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Sample records for kallikrein-related peptidases klks

  1. The solution structure of the kallikrein-related peptidases inhibitor SPINK6

    NARCIS (Netherlands)

    Jung, Sascha; Fischer, Jan; Spudy, Björn; Kerkow, Tim; Sönnichsen, Frank D; Xue, Li; Bonvin, Alexandre M J J; Goettig, Peter; Magdolen, Viktor; Meyer-Hoffert, Ulf; Grötzinger, Joachim

    2016-01-01

    Kallikrein-related peptidases (KLKs) are crucial for epidermal barrier function and are involved in the proteolytic regulation of the desquamation process. Elevated KLK levels were reported in atopic dermatitis. In skin, the proteolytic activity of KLKs is regulated by specific inhibitors of the ser

  2. Quantification of Human Kallikrein-Related Peptidases in Biological Fluids by Multiplatform Targeted Mass Spectrometry Assays.

    Science.gov (United States)

    Karakosta, Theano D; Soosaipillai, Antoninus; Diamandis, Eleftherios P; Batruch, Ihor; Drabovich, Andrei P

    2016-09-01

    Human kallikrein-related peptidases (KLKs) are a group of 15 secreted serine proteases encoded by the largest contiguous cluster of protease genes in the human genome. KLKs are involved in coordination of numerous physiological functions including regulation of blood pressure, neuronal plasticity, skin desquamation, and semen liquefaction, and thus represent promising diagnostic and therapeutic targets. Until now, quantification of KLKs in biological and clinical samples was accomplished by enzyme-linked immunosorbent assays (ELISA). Here, we developed multiplex targeted mass spectrometry assays for the simultaneous quantification of all 15 KLKs. Proteotypic peptides for each KLK were carefully selected based on experimental data and multiplexed in single assays. Performance of assays was evaluated using three different mass spectrometry platforms including triple quadrupole, quadrupole-ion trap, and quadrupole-orbitrap instruments. Heavy isotope-labeled synthetic peptides with a quantifying tag were used for absolute quantification of KLKs in sweat, cervico-vaginal fluid, seminal plasma, and blood serum, with limits of detection ranging from 5 to 500 ng/ml. Analytical performance of assays was evaluated by measuring endogenous KLKs in relevant biological fluids, and results were compared with selected ELISAs. The multiplex targeted proteomic assays were demonstrated to be accurate, reproducible, sensitive, and specific alternatives to antibody-based assays. Finally, KLK4, a highly prostate-specific protein and a speculated biomarker of prostate cancer, was unambiguously detected and quantified by immunoenrichment-SRM assay in seminal plasma and blood serum samples from individuals with confirmed prostate cancer and negative biopsy. Mass spectrometry revealed exclusively the presence of a secreted isoform and thus unequivocally resolved earlier disputes about KLK4 identity in seminal plasma. Measurements of KLK4 in either 41 seminal plasma or 58 blood serum samples

  3. Expression of kallikrein-related peptidase 7 is decreased in prostate cancer

    Directory of Open Access Journals (Sweden)

    Chong-Yu Zhang

    2015-02-01

    Full Text Available Recent evidence suggests that the human kallikrein 7 (KLK7 is differentially regulated in a variety of tumors. The aim of this study was to determine the expression of kallikrein-related peptidase 7 and KLK7 in our large collection of prostate samples. Between August 2000 and December 2012, 116 patients with histologically confirmed prostate cancer (PCa and 92 with benign prostate hyperplasia (BPH were recruited into the study. Using immunohistochemistry, quantitative reverse transcription polymerase chain reaction (RT-PCR and western blot, kallikrein-related peptidase 7 expression in BPH and PCa tissues was determined at the mRNA and protein levels. The relationships between kallikrein-related peptidase 7 mRNA expression and clinicopathological features were analyzed. A total of 64 of 92 (69.57% benign cases showed positive staining for KLK7 and 23 of 116 (19.83% malignant cases showed positive, the difference of KLK7 expression between PCa and BPH was statistically significant (P < 0.001. The expression level of kallikrein-related peptidase 7 mRNA was significantly decreased in PCa tissues compared with that in BPH tissues and normal prostate tissue. Kallikrein-related peptidase 7 mRNA exhibited different expression patterns in terms of localization depending on pathological category of PCa. Similarly, our western immunoblot analyses demonstrated that the protein expression levels of KLK7 was lower in PCa than in BPH tissues and normal prostate tissue. Kallikrein-related peptidase 7 and KLK7 expression are down-regulated in PCa and lower expression of kallikrein-related peptidase 7 closely correlates with higher Gleason score and higher prostate-specific antigen level.

  4. Identification of lympho-epithelial Kazal-type inhibitor 2 in human skin as a kallikrein-related peptidase 5-specific protease inhibitor.

    Directory of Open Access Journals (Sweden)

    Ulf Meyer-Hoffert

    Full Text Available Kallikreins-related peptidases (KLKs are serine proteases and have been implicated in the desquamation process of the skin. Their activity is tightly controlled by epidermal protease inhibitors like the lympho-epithelial Kazal-type inhibitor (LEKTI. Defects of the LEKTI-encoding gene serine protease inhibitor Kazal type (Spink5 lead to the absence of LEKTI and result in the genodermatose Netherton syndrome, which mimics the common skin disease atopic dermatitis. Since many KLKs are expressed in human skin with KLK5 being considered as one of the most important KLKs in skin desquamation, we proposed that more inhibitors are present in human skin. Herein, we purified from human stratum corneum by HPLC techniques a new KLK5-inhibiting peptide encoded by a member of the Spink family, designated as Spink9 located on chromosome 5p33.1. This peptide is highly homologous to LEKTI and was termed LEKTI-2. Recombinant LEKTI-2 inhibited KLK5 but not KLK7, 14 or other serine proteases tested including trypsin, plasmin and thrombin. Spink9 mRNA expression was detected in human skin samples and in cultured keratinocytes. LEKTI-2 immune-expression was focally localized at the stratum granulosum and stratum corneum at palmar and plantar sites in close localization to KLK5. At sites of plantar hyperkeratosis, LEKTI-2 expression was increased. We suggest that LEKTI-2 contributes to the regulation of the desquamation process in human skin by specifically inhibiting KLK5.

  5. Kallikrein-Related Peptidase 5 Contributes to H3N2 Influenza Virus Infection in Human Lungs.

    Science.gov (United States)

    Magnen, Mélia; Gueugnon, Fabien; Guillon, Antoine; Baranek, Thomas; Thibault, Virginie C; Petit-Courty, Agnès; de Veer, Simon J; Harris, Jonathan; Humbles, Alison A; Si-Tahar, Mustapha; Courty, Yves

    2017-08-15

    Hemagglutinin (HA) of influenza virus must be activated by proteolysis before the virus can become infectious. Previous studies indicated that HA cleavage is driven by membrane-bound or extracellular serine proteases in the respiratory tract. However, there is still uncertainty as to which proteases are critical for activating HAs of seasonal influenza A viruses (IAVs) in humans. This study focuses on human KLK1 and KLK5, 2 of the 15 serine proteases known as the kallikrein-related peptidases (KLKs). We find that their mRNA expression in primary human bronchial cells is stimulated by IAV infection. Both enzymes cleaved recombinant HA from several strains of the H1 and/or H3 virus subtype in vitro, but only KLK5 promoted the infectivity of A/Puerto Rico/8/34 (H1N1) and A/Scotland/20/74 (H3N2) virions in MDCK cells. We assessed the ability of treated viruses to initiate influenza in mice. The nasal instillation of only the KLK5-treated virus resulted in weight loss and lethal outcomes. The secretion of this protease in the human lower respiratory tract is enhanced during influenza. Moreover, we show that pretreatment of airway secretions with a KLK5-selective inhibitor significantly reduced the activation of influenza A/Scotland/20/74 virions, providing further evidence of its importance. Differently, increased KLK1 secretion appeared to be associated with the recruitment of inflammatory cells in human airways regardless of the origin of inflammation. Thus, our findings point to the involvement of KLK5 in the proteolytic activation and spread of seasonal influenza viruses in humans.IMPORTANCE Influenza A viruses (IAVs) cause acute infection of the respiratory tract that affects millions of people during seasonal outbreaks every year. Cleavage of the hemagglutinin precursor by host proteases is a critical step in the life cycle of these viruses. Consequently, host proteases that activate HA can be considered promising targets for the development of new antivirals

  6. Kallikrein-related peptidases in cancers of gastrointestinal tract: an inside view of their role and clinical significance.

    Science.gov (United States)

    Linardoutsos, Dimitrios; Gazouli, Maria; Machairas, Anastasios; Bramis, Ioannis; Zografos, Georgios C

    2014-01-01

    Human tissue kallikrein (KLK1) and is related peptidases (KLK2-KLK15) are a family of 15 homologous serine proteases, participating in numerous processes of normal physiology. Considering the irreversible impact of proteases on substrates, the tissue-dependent regulation of KLKs activity becomes crucial for their beneficial role in normal homeostasis. Moreover, KLKs expression is strongly regulated at the transcriptional and post-transcriptional level by steroid hormones and miRNAs, respectively. Deregulation of KLKs expression, secretion and/or activation has been observed in most human malignancies and there is a trend to identify their role in the multi-complex process of cancer development. The identification of extracellular matrix (ECM) proteins, cell-surface receptors, cell-surface adhesion molecules and growth factors among substrates, clearly support the driving role of KLK abnormal expression and function during tumorigenesis and cancer progression. KLKs have also clinical utility in cancer diagnosis and monitoring like KLK 3 (PSA) in prostate cancer. In this review, we tried to summarize the existing literature about the role of KLKs in gastrointestinal cancers as well as to emphasize their clinical significance for patients' prognosis.

  7. Non-combinatorial library screening reveals subsite cooperativity and identifies new high-efficiency substrates for kallikrein-related peptidase 14.

    Science.gov (United States)

    de Veer, Simon J; Swedberg, Joakim E; Parker, Edward A; Harris, Jonathan M

    2012-04-01

    An array of substrates link the tryptic serine protease, kallikrein-related peptidase 14 (KLK14), to physiological functions including desquamation and activation of signaling molecules associated with inflammation and cancer. Recognition of protease cleavage sequences is driven by complementarity between exposed substrate motifs and the physicochemical signature of an enzyme's active site cleft. However, conventional substrate screening methods have generated conflicting subsite profiles for KLK14. This study utilizes a recently developed screening technique, the sparse matrix library, to identify five novel high-efficiency sequences for KLK14. The optimal sequence, YASR, was cleaved with higher efficiency (k(cat)/K(m)=3.81 ± 0.4 × 10(6) M(-1) s(-1)) than favored substrates from positional scanning and phage display by 2- and 10-fold, respectively. Binding site cooperativity was prominent among preferred sequences, which enabled optimal interaction at all subsites as indicated by predictive modeling of KLK14/substrate complexes. These simulations constitute the first molecular dynamics analysis of KLK14 and offer a structural rationale for the divergent subsite preferences evident between KLK14 and closely related KLKs, KLK4 and KLK5. Collectively, these findings highlight the importance of binding site cooperativity in protease substrate recognition, which has implications for discovery of optimal substrates and engineering highly effective protease inhibitors.

  8. Increased anxiety-like behavior in neuropsin (kallikrein-related peptidase 8) gene-deficient mice.

    Science.gov (United States)

    Horii, Yoichiro; Yamasaki, Nobuyuki; Miyakawa, Tsuyoshi; Shiosaka, Sadao

    2008-06-01

    Neuropsin (kallikrein-related peptidase 8) is concentrated in the hippocampus, amygdala, olfactory bulb, and prefrontal cortex. Earlier studies showed that protease deficiency causes a significant impairment of early-phase long-term potentiation in the Schaffer collateral pathway and hippocampus-dependent memory in the Y maze and Morris water maze (Z. Chen et al., 1995; A. Hirata et al., 2001; H. Tamura et al., 2006). In addition to neuropsin's participation in the hippocampal memory, amygdalar and cortical localization of the gene suggests extrahippocampal behavioral function, and the authors therefore examined neuropsin-deficient mice, including tests of sensory motor reflex, open field, light-dark transition, Rota-Rod, elevated plus-maze, hot plate, startle response-prepulse inhibition, Porsolt forced swim, Barnes maze, eight-arm radial maze, and contextual and cued fear conditioning tests. Here, the authors found increased anxiety in neuropsin-deficient mice, suggesting the involvement of this protease in emotional responses. (Copyright) 2008 APA, all rights reserved.

  9. Evaluation of human tissue kallikrein-related peptidases 6 and 10 expression in early gastroesophageal adenocarcinoma.

    Science.gov (United States)

    Grin, Andrea; Samaan, Sara; Tripathi, Monika; Rotondo, Fabio; Kovacs, Kalman; Bassily, Mena N; Yousef, George M

    2015-04-01

    Kallikreins are a family of serine proteases that are linked to malignancy of different body organs with potential clinical utility as tumor markers. In this study, we investigated kallikrein-related peptidase 6 (KLK6) and KLK10 expression in early gastroesophageal junction adenocarcinoma and Barrett esophagus (BE) with and without dysplasia. Immunohistochemistry revealed significantly increased KLK6 expression in early invasive cancer compared with dysplastic (P = .009) and nondysplastic BE (P = .0002). There was a stepwise expression increase from metaplasia to dysplasia and invasive tumors. Significantly higher KLK10 was seen in dysplastic lesions compared with metaplasia but not between dysplastic lesions and invasive cancers. KLK6 staining intensity was increased at the invasive front (P = .006), suggesting its role in tumor invasiveness. Neither KLK6 nor KLK10 was significantly associated with other prognostic markers, including depth of invasion, indicating their potential as independent biomarkers. Our results should be interpreted with caution due to limited sample size. There was a significant correlation between KLK6 and KLK10 expression both at the invasive front and within the main tumor, indicating a collaborative effect. We then compared KLK6 and KLK10 messenger RNA expression between metaplastic and cancerous tissues in an independent data set of esophageal carcinoma from The Cancer Genome Atlas. KLK6 and KLK10 may be useful markers and potential therapeutic targets in gastroesophageal junction tumors.

  10. Kallikrein-related peptidase 8 is expressed in myocardium and induces cardiac hypertrophy

    Science.gov (United States)

    Cao, Buqing; Yu, Qing; Zhao, Wei; Tang, Zhiping; Cong, Binghai; Du, Jiankui; Lu, Jianqiang; Zhu, Xiaoyan; Ni, Xin

    2016-01-01

    The tissue kallikrein-related peptidase family (KLK) is a group of trypsin- and chymotrypsin-like serine proteases that share a similar homology to parent tissue kallikrein (KLK1). KLK1 is identified in heart and has anti-hypertrophic effects. However, whether other KLK family members play a role in regulating cardiac function remains unknown. In the present study, we demonstrated for the first time that KLK8 was expressed in myocardium. KLK8 expression was upregulated in left ventricle of cardiac hypertrophy models. Both intra-cardiac adenovirus-mediated and transgenic-mediated KLK8 overexpression led to cardiac hypertrophy in vivo. In primary neonatal rat cardiomyocytes, KLK8 knockdown inhibited phenylephrine (PE)-induced cardiomyocyte hypertrophy, whereas KLK8 overexpression promoted cardiomyocyte hypertrophy via a serine protease activity-dependent but kinin receptor-independent pathway. KLK8 overexpression increased epidermal growth factor (EGF) production, which was blocked by the inhibitors of serine protease. EGF receptor (EGFR) antagonist and EGFR knockdown reversed the hypertrophy induced by KLK8 overexpression. KLK8-induced cardiomyocyte hypertrophy was also significantly decreased by blocking the protease-activated receptor 1 (PAR1) or PAR2 pathway. Our data suggest that KLK8 may promote cardiomyocyte hypertrophy through EGF signaling- and PARs-dependent but a kinin receptor-independent pathway. It is implied that different KLK family members can subtly regulate cardiac function and remodeling. PMID:26823023

  11. Expression of kallikrein-related peptidase 7 is decreased in prostate cancer

    Institute of Scientific and Technical Information of China (English)

    Chong-Yu Zhang; Yu Zhu; Wen-Bin Rui; Jun Dai; Zhou-Jun Shen

    2015-01-01

    Recent evidence suggests that the human kallikrein 7 (KLK7) is differentially regulated in a variety of tumors. The aim of this study was to determine the expression of kallikrein‑related peptidase 7 and KLK7 in our large collection of prostate samples. Between August 2000 and December 2012, 116 patients with histologically confirmed prostate cancer (PCa) and 92 with benign prostate hyperplasia (BPH) were recruited into the study. Using immunohistochemistry, quantitative reverse transcription polymerase chain reaction (RT‑PCR) and western blot, kallikrein‑related peptidase 7 expression in BPH and PCa tissues was determined at the mRNA and protein levels. The relationships between kallikrein‑related peptidase 7 mRNA expression and clinicopathological features were analyzed. A total of 64 of 92 (69.57%) benign cases showed positive staining for KLK7 and 23 of 116 (19.83%) malignant cases showed positive, the difference of KLK7 expression between PCa and BPH was statistically significant (P < 0.001). The expression level of kallikrein‑related peptidase 7 mRNA was significantly decreased in PCa tissues compared with that in BPH tissues and normal prostate tissue. Kallikrein‑related peptidase 7 mRNA exhibited different expression patterns in terms of localization depending on pathological category of PCa. Similarly, our western immunoblot analyses demonstrated that the protein expression levels of KLK7 was lower in PCa than in BPH tissues and normal prostate tissue. Kallikrein‑related peptidase 7 and KLK7 expression are down‑regulated in PCa and lower expression of kallikrein‑related peptidase 7 closely correlates with higher Gleason score and higher prostate‑specific antigen level.

  12. Rapid elimination kinetics of free PSA or human kallikrein-related peptidase 2 after initiation of gonadotropin-releasing hormone-antagonist treatment of prostate cancer

    DEFF Research Database (Denmark)

    Ulmert, David; Vickers, Andrew J; Scher, Howard I

    2012-01-01

    The utility of conventional prostate-specific antigen (PSA) measurements in blood for monitoring rapid responses to treatment for prostate cancer is limited because of its slow elimination rate. Prior studies have shown that free PSA (fPSA), intact PSA (iPSA) and human kallikrein-related peptidase...... of tPSA, fPSA, iPSA and hK2 after rapid induction of castration with degarelix (Firmagon(®)), a novel GnRH antagonist....

  13. The kallikrein-related peptidase 13 (KLK13) gene is substantially up-regulated after exposure of gastric cancer cells to antineoplastic agents.

    Science.gov (United States)

    Florou, Dimitra; Mavridis, Konstantinos; Scorilas, Andreas

    2012-12-01

    Gastric cancer constitutes one of the most common neoplasms globally. Kallikrein-related peptidases have attracted interest as potential tumor markers and future targets for novel cancer therapeutics. We have recently reported KLK13 clinical importance as a favorable prognostic biomarker for gastric cancer patients' survival. By aiming to explore how the molecular profile of KLK13 is modified in stomach cancer cells treated with antineoplastic drugs, we examined, for the first time, the mRNA alterations of this gene following gastric cancer cells' exposure to the prominent chemotherapeutic substances epirubicin, oxaliplatin, or methotrexate. The antiproliferative effects of these agents, on AGS cells' growth, were determined by the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide and trypan blue assays. Total RNA, isolated from the harvested cells, was reverse-transcribed to cDNA. KLK13 levels were quantified via real-time PCR using the SYBR Green chemistry. The relative changes of KLK13 expression were calculated with the comparative C (t) (2(-ddCt)) method. Distinct KLK13 profiles resulted from AGS cells' incubation with epirubicin or methotrexate for 24, 36, and 48 h. KLK13 expression increased in a time-dependent manner up to 5.70 times (for epirubicin) or 5.76 times (for methotrexate) at 48 h compared with the corresponding untreated cells. According to our results, KLK13 expression is implicated in the molecular pathways that are triggered after administration of anticancer agents on gastric cancer cells. Moreover, our data support the possibility that KLK13 may be exploited as a future molecular predictor of gastric cancer cells' response to chemotherapy.

  14. Micro-RNA analysis of renal biopsies in human lupus nephritis demonstrates up-regulated miR-422a driving reduction of kallikrein-related peptidase 4.

    Science.gov (United States)

    Krasoudaki, Eleni; Banos, Aggelos; Stagakis, Elias; Loupasakis, Konstantinos; Drakos, Elias; Sinatkas, Vaios; Zampoulaki, Amalia; Papagianni, Aikaterini; Iliopoulos, Dimitrios; Boumpas, Dimitrios T; Bertsias, George K

    2016-10-01

    Aberrancies in gene expression in immune effector cells and in end-organs are implicated in lupus pathogenesis. To gain insights into the mechanisms of tissue injury, we profiled the expression of micro-RNAs in inflammatory kidney lesions of human lupus nephritis (LN). Kidney specimens were from patients with active proliferative, membranous or mixed LN and unaffected control tissue. Micro-RNAs were quantified by TaqMan Low Density Arrays. Bioinformatics was employed to predict gene targets, gene networks and perturbed signaling pathways. Results were validated by transfection studies (luciferase assay, real-time PCR) and in murine LN. Protein expression was determined by immunoblotting and immunohistochemistry. Twenty-four micro-RNAs were dysregulated (9 up-regulated, 15 down-regulated) in human LN compared with control renal tissue. Their predicted gene targets participated in pathways associated with TGF-β, kinases, NF-κB, HNF4A, Wnt/β-catenin, STAT3 and IL-4. miR-422a showed the highest upregulation (17-fold) in active LN and correlated with fibrinoid necrosis lesions (β = 0.63, P = 0.002). In transfection studies, miR-422a was found to directly target kallikrein-related peptidase 4 (KLK4) mRNA. Concordantly, KLK4 mRNA was significantly reduced in the kidneys of human and murine LN and correlated inversely with miR-422a levels. Immunohistochemistry confirmed reduced KLK4 protein expression in renal mesangial and tubular epithelial cells in human and murine LN. KLK4, a serine esterase with putative renoprotective properties, is down-regulated by miR-422a in LN kidney suggesting that, in addition to immune activation, local factors may be implicated in the disease. © The Author 2015. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

  15. Long distance contribution to the KL-KS mass difference

    CERN Document Server

    Christ, N H; Sachrajda, C T; Soni, A; Yu, J

    2012-01-01

    We develop and demonstrate techniques needed to compute the long distance contribution to the $K_{L}$-$K_{S}$ mass difference, $\\Delta M_K$, in lattice QCD and carry out a first, exploratory calculation of this fundamental quantity. The calculation is performed on 2+1 flavor, domain wall fermion, $16^3\\times32$ configurations with a 421 MeV pion mass. We include only current-current operators and drop all disconnected and double penguin diagrams. The short distance part of the mass difference in a 2+1 flavor calculation contains a quadratic divergence cut off by the lattice spacing. Here, this quadratic divergence is eliminated through the GIM mechanism by introducing a valence charm quark. The inclusion of the charm quark makes the complete calculation accessible to lattice methods provided the discretization errors associated with the charm quark can be controlled. The long distance effects are discussed for each parity channel separately. While we can see a clear signal in the parity odd channel, the signa...

  16. Activity of human kallikrein-related peptidase 6 (KLK6) on substrates containing sequences of basic amino acids. Is it a processing protease?

    Science.gov (United States)

    Silva, Roberta N; Oliveira, Lilian C G; Parise, Carolina B; Oliveira, Juliana R; Severino, Beatrice; Corvino, Angela; di Vaio, Paola; Temussi, Piero A; Caliendo, Giuseppe; Santagada, Vincenzo; Juliano, Luiz; Juliano, Maria A

    2017-05-01

    Human kallikrein 6 (KLK6) is highly expressed in the central nervous system and with elevated level in demyelinating disease. KLK6 has a very restricted specificity for arginine (R) and hydrolyses myelin basic protein, protein activator receptors and human ionotropic glutamate receptor subunits. Here we report a previously unreported activity of KLK6 on peptides containing clusters of basic amino acids, as in synthetic fluorogenic peptidyl-Arg-7-amino-4-carbamoylmethylcoumarin (peptidyl-ACC) peptides and FRET peptides in the format of Abz-peptidyl-Q-EDDnp (where Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-(2,4-dinitrophenyl) ethylenediamine), in which pairs or sequences of basic amino acids (R or K) were introduced. Surprisingly, KLK6 hydrolyzed the fluorogenic peptides Bz-A-R(↓)R-ACC and Z-R(↓)R-MCA between the two R groups, resulting in non-fluorescent products. FRET peptides containing furin processing sequences of human MMP-14, nerve growth factor (NGF), Neurotrophin-3 (NT-3) and Neurotrophin-4 (NT-4) were cleaved by KLK6 at the same position expected by furin. Finally, KLK6 cleaved FRET peptides derived from human proenkephalin after the KR, the more frequent basic residues flanking enkephalins in human proenkephalin sequence. This result suggests the ability of KLK6 to release enkephalin from proenkephalin precursors and resembles furin a canonical processing proteolytic enzyme. Molecular models of peptides were built into the KLK6 structure and the marked preference of the cut between the two R of the examined peptides was related to the extended conformation of the substrates. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. LOCALIZATION OF PEPTIDASES IN LACTOCOCCI

    NARCIS (Netherlands)

    TAN, PST; CHAPOTCHARTIER, MP; POS, KM; ROUSSEAU, M; BOQUIEN, CY; GRIPON, JC; KONINGS, WN

    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 b

  18. 组织激肽释放酶家族在胰腺癌中的研究进展%Kallikrein-related peptidases in pancreatic cancer: expression and significance

    Institute of Scientific and Technical Information of China (English)

    郑苏丽; 熊光冰; 曹喆; 杨刚; 张太平

    2016-01-01

    Pancreatic cancer is a one of the most malignant digestive cancer.Because the lack of effective methods for early diagnosis,most patients have been ineligible for surgical resection when diagnosed.Kallikrein family is a group of serine proteases,because of its ability to decompose the extracellular matrix proteins,it may be closely related to the invasion and metastasis of various cancers.And some members of kallikrein family may become cancer diagnostic biomarkers.This paper reviews all the recent articles about kallikrein family study in pancreatic cancer.%胰腺癌是一种恶性程度极高的消化道恶性肿瘤.因其缺乏有效的早期诊断方法,多数患者确诊时已无法进行根治性手术.激肽释放酶家族是一组丝氨酸蛋白酶,因其具有分解细胞外基质蛋白的能力,可能与多种癌症的侵袭和转移密切相关,并有可能成为多种癌症诊断的生物标志物.本文回顾了近年来激肽释放酶基因家族研究中与胰腺癌有关的文章,对激肽释放酶家族成员在胰腺癌中的表达及意义进行综述.

  19. Evolution of the plasma and tissue kallikreins, and their alternative splicing isoforms.

    Directory of Open Access Journals (Sweden)

    Vassiliki Lila Koumandou

    Full Text Available Kallikreins are secreted serine proteases with important roles in human physiology. Human plasma kallikrein, encoded by the KLKB1 gene on locus 4q34-35, functions in the blood coagulation pathway, and in regulating blood pressure. The human tissue kallikrein and kallikrein-related peptidases (KLKs have diverse expression patterns and physiological roles, including cancer-related processes such as cell growth regulation, angiogenesis, invasion, and metastasis. Prostate-specific antigen (PSA, the product of the KLK3 gene, is the most widely used biomarker in clinical practice today. A total of 15 KLKs are encoded by the largest contiguous cluster of protease genes in the human genome (19q13.3-13.4, which makes them ideal for evolutionary analysis of gene duplication events. Previous studies on the evolution of KLKs have traced mammalian homologs as well as a probable early origin of the family in aves, amphibia and reptilia. The aim of this study was to address the evolutionary and functional relationships between tissue KLKs and plasma kallikrein, and to examine the evolution of alternative splicing isoforms. Sequences of plasma and tissue kallikreins and their alternative transcripts were collected from the NCBI and Ensembl databases, and comprehensive phylogenetic analysis was performed by Bayesian as well as maximum likelihood methods. Plasma and tissue kallikreins exhibit high sequence similarity in the trypsin domain (>50%. Phylogenetic analysis indicates an early divergence of KLKB1, which groups closely with plasminogen, chymotrypsin, and complement factor D (CFD, in a monophyletic group distinct from trypsin and the tissue KLKs. Reconstruction of the earliest events leading to the diversification of the tissue KLKs is not well resolved, indicating rapid expansion in mammals. Alternative transcripts of each KLK gene show species-specific divergence, while examination of sequence conservation indicates that many annotated human KLK isoforms

  20. An essential signal peptide peptidase identified in an RNAi screen of serine peptidases of Trypanosoma brucei.

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    Catherine X Moss

    Full Text Available 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.

  1. Dipeptidyl peptidases 8 and 9: specificity and molecular characterization compared with dipeptidyl peptidase IV

    DEFF Research Database (Denmark)

    Bjelke, Jais R; Christensen, Jesper; Nielsen, Per F

    2006-01-01

    of the two proteins can be expressed as active peptidases, which are 882 and 892 amino acids in length for dipeptidyl peptidase 8 and 9 respectively. We show further that the purified proteins are active dimers and that they show similar Michaelis-Menten kinetics and substrate specificity. Both cleave...

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

    Science.gov (United States)

    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 ...

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

    Science.gov (United States)

    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.

  4. Signal Peptidase Enzymology and Substrate Specificity Profiling.

    Science.gov (United States)

    Dalbey, R E; Pei, D; Ekici, Ö D

    2017-01-01

    Signal peptidases are membrane proteases that play crucial roles in the protein transport pathway of bacteria. They cleave off the signal peptide from precursor proteins that are membrane inserted by the SecYEG or Tat translocons. Signal peptide cleavage releases the translocated protein from the inner membrane allowing the protein to be exported to the periplasm, outer membrane, or secreted into the medium. Signal peptidases are very important proteins to study. They are unique serine proteases with a Ser-Lys dyad, catalyze cleavage at the membrane surface, and are promising potential antibacterial drug targets. This chapter will focus on the isolation of signal peptidases and the preprotein substrates, as well as describe a peptide library approach for characterizing the substrate specificity.

  5. Expression, purification and characterization of recombinant human serine proteinase inhibitor Kazal-type 6 (SPINK6) in Pichia pastoris.

    Science.gov (United States)

    Lu, Hairong; Huang, Jinjiang; Li, Guodong; Ge, Kuikui; Wu, Hongyu; Huang, Qingshan

    2012-03-01

    Human serine proteinase inhibitor Kazal-type 6 (SPINK6) belongs to the medically important SPINK family. Malfunctions of SPINK members are linked to many diseases, including pancreatitis, skin barrier defects, and cancer. SPINK6 has been shown to selectively inhibit Kallikrein-related peptidases (KLKs) in human skin. As a SPINK protein, it contains a typical Kazal domain, which requires three intramolecular disulfide bonds for correct folding and activity. Preparation of functional protein is a prerequisite for studying this important human factor. Here, we report the successful generation of tagless SPINK6 using a yeast expression system. The recombinant protein was secreted and purified by cation exchange and size-exclusion chromatography. The protein identity was confirmed by MALDI-TOF MS and N-terminal sequencing. Pichia pastoris-derived recombinant human SPINK6 (rhSPINK6) showed higher inhibitory activity against Kallikrein-related peptidase 14 (KLK14) (K(i)=0.16 nM) than previously reported Escherichia coli-derived rhSPINK6 (K(i)=0.5 nM). This protein also exhibited moderate inhibition of bovine trypsin (K(i)=33 nM), while previous E. coli-derived rhSPINK6 did not. The results indicate that P. pastoris is a better system to generate active rhSPINK6, warranting further studies on this medically important SPINK family candidate.

  6. Type I signal peptidases of Bacillus subtilis

    NARCIS (Netherlands)

    Tjalsma, Harold; Bolhuis, Albert; Bron, Sierd; Jongbloed, Jan; Meijer, Wilfried J.J.; Noback, Michiel; van Roosmalen, Maarten; Venema, Gerhardus; van Dijl, Jan Maarten; Hopsu Havu, VK; Jarvinen, M; Kirschke, H

    1997-01-01

    Bacillus subtilis contains at least three chromosomally-encoded type I signal peptidases (SPases; SipS, SipT, and SipU), which remove signal peptides from secretory proteins. In addition, certain B. subtilis (natto) strains contain plasmid-encoded type I SPases (SipP). The known type I SPases from

  7. Salmonella typhimurium peptidase active on carnosine.

    Science.gov (United States)

    Kirsh, M; Dembinski, D R; Hartman, P E; Miller, C G

    1978-01-01

    Wild-type Salmonella typhimurium can use carnosine (beta-alanyl-L-histidine) as a source of histidine, but carnosine utilization is blocked in particular mutants defective in the constitutive enzyme peptidase D, the product of the pepD gene. Biochemical evidence for assigning carnosinase activity to peptidase D (a broad-specificity dipeptidase) includes: (i) coelution of carnosinase and dipeptidase activity from diethylaminoethyl-cellulose and Bio-Gel P-300 columns; (ii) coelectrophoresis of carnosinase and dipeptidase on polyacrylamide gels; and (iii) inactivation of carnosinase and dipeptidase activities at identical rates at both 4 and 42 degrees C. Genetic evidence indicates that mutations leading to loss of carnosinase activity map at pepD. Several independent pepD mutants have been isolated by different selection procedures, and the patterns of peptide utilization of strains carrying various pepD alleles have been studied. Many pepD mutations lead to the production of partially active peptidase D enzymes with substrate specificities that differ strikingly from those of the wild-type enzyme. The growth yields of carnosinase-deficient strains growing in Difco nutrient broth indicate that carnosine is the major utilizable source of histidine in this medium. PMID:26655

  8. Identification and characterization of a bacterial glutamic peptidase

    Directory of Open Access Journals (Sweden)

    Jensen Kenneth

    2010-12-01

    Full Text Available Abstract Background Glutamic peptidases, from the MEROPS family G1, are a distinct group of peptidases characterized by a catalytic dyad consisting of a glutamate and a glutamine residue, optimal activity at acidic pH and insensitivity towards the microbial derived protease inhibitor, pepstatin. Previously, only glutamic peptidases derived from filamentous fungi have been characterized. Results We report the first characterization of a bacterial glutamic peptidase (pepG1, derived from the thermoacidophilic bacteria Alicyclobacillus sp. DSM 15716. The amino acid sequence identity between pepG1 and known fungal glutamic peptidases is only 24-30% but homology modeling, the presence of the glutamate/glutamine catalytic dyad and a number of highly conserved motifs strongly support the inclusion of pepG1 as a glutamic peptidase. Phylogenetic analysis places pepG1 and other putative bacterial and archaeal glutamic peptidases in a cluster separate from the fungal glutamic peptidases, indicating a divergent and independent evolution of bacterial and fungal glutamic peptidases. Purification of pepG1, heterologously expressed in Bacillus subtilis, was performed using hydrophobic interaction chromatography and ion exchange chromatography. The purified peptidase was characterized with respect to its physical properties. Temperature and pH optimums were found to be 60°C and pH 3-4, in agreement with the values observed for the fungal members of family G1. In addition, pepG1 was found to be pepstatin-insensitive, a characteristic signature of glutamic peptidases. Conclusions Based on the obtained results, we suggest that pepG1 can be added to the MEROPS family G1 as the first characterized bacterial member.

  9. Peptidase Activities of Tripeptidyl Peptidase Ⅰ(TPP Ⅰ): Exopeptidase and Endopeptidase

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The defect of TPP Ⅰ causes a disease, late infantile neuronal ceroid lipofuscinosis(LINCL, CLN2). To investigate the bio-activity of tripeptidyl peptidase Ⅰ(TPP Ⅰ) from rat kidneys, the effects of digestion of angiotensin Ⅱ(Ang Ⅱ) and a synthetic endo-type substrate(Gly1-Lys-Pro-Iie-Pro5-Phe-Phe-Arg-Leu-Lys10) via TPP Ⅰ were analyzed by HPLC and TOF-MS. The data suggest that the degradation rate of Ang Ⅱ can reach 18.2% by the rat TPP Ⅰ and DRV(Asp-Arg-Val) can be released from N-termini of Ang Ⅱ within 16 h. In addition, the synthetic endo-type substrate is cleaved at the same position between Phe6 and Phe7. Accordingly, TPP Ⅰ shows two kinds of peptidase activities. One is a tripeptidyl peptidase activity and the other is a pepstatin insensitive carboxyl endopeptidase activity. Tripeptidyl peptidase activity and pepstatin insensitive carboxyl endopeptidase activity seem to be dual phases of one enzyme, TPP Ⅰ.

  10. The chemistry and enzymology of the type I signal peptidases

    NARCIS (Netherlands)

    Dalbey, RE; Lively, MO; Bron, S; VanDijl, JM

    1997-01-01

    The discovery that proteins exported from the cytoplasm are typically synthesized as larger precursors with cleavable signal peptides has focused interest on the peptidases that remove the signal peptides. Here, we review the membrane-bound peptidases dedicated to the processing of protein precursor

  11. The chemistry and enzymology of the type I signal peptidases

    NARCIS (Netherlands)

    Dalbey, RE; Lively, MO; Bron, S; VanDijl, JM

    The discovery that proteins exported from the cytoplasm are typically synthesized as larger precursors with cleavable signal peptides has focused interest on the peptidases that remove the signal peptides. Here, we review the membrane-bound peptidases dedicated to the processing of protein

  12. Dipeptidyl peptidase 4 - An important digestive peptidase in Tenebrio molitor larvae.

    Science.gov (United States)

    Tereshchenkova, Valeriia F; Goptar, Irina A; Kulemzina, Irina A; Zhuzhikov, Dmitry P; Serebryakova, Marina V; Belozersky, Mikhail A; Dunaevsky, Yakov E; Oppert, Brenda; Filippova, Irina Yu; Elpidina, Elena N

    2016-09-01

    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 (TmDPP 4), with a biological function different than that of the well-studied mammalian DPP 4. The sequence of the purified enzyme was confirmed by mass-spectrometry, and was identical to the translated RNA sequence found in a gut EST database. The purified peptidase was characterized according to its localization in the midgut, and substrate specificity and inhibitor sensitivity were compared with those of human recombinant DPP 4 (rhDPP 4). The T. molitor enzyme was localized mainly in the anterior midgut of the larvae, and 81% of the activity was found in the fraction of soluble gut contents, while human DPP 4 is a membrane enzyme. TmDPP 4 was stable in the pH range 5.0-9.0, with an optimum activity at pH 7.9, similar to human DPP 4. Only specific inhibitors of serine peptidases, diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, suppressed TmDPP 4 activity, and the specific dipeptidyl peptidase inhibitor vildagliptin was most potent. The highest rate of TmDPP 4 hydrolysis was found for the synthetic substrate Arg-Pro-pNA, while Ala-Pro-pNA was a better substrate for rhDPP 4. Related to its function in the insect midgut, TmDPP 4 efficiently hydrolyzed the wheat storage proteins gliadins, which are major dietary proteins of T. molitor.

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

    Science.gov (United States)

    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

  14. Evolution of the thermopsin peptidase family (A5.

    Directory of Open Access Journals (Sweden)

    Neil D Rawlings

    Full Text Available 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.

  15. Targeting Prolyl Peptidases in Triple-Negative Breast Cancer

    Science.gov (United States)

    2017-02-01

    AWARD NUMBER: W81XWH-16-1-0025 TITLE: Targeting Prolyl Peptidases in Triple-Negative Breast Cancer PRINCIPAL INVESTIGATOR: Carl G. Maki, PhD...SUBTITLE Targeting Prolyl Peptidases in Triple-Negative Breast Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-16-1-0025 5c. PROGRAM ELEMENT NUMBER 6...ABSTRACT Triple negative breast cancer (TNBC) is an aggressive sub-type with limited treatment options and poor prognosis. The most life-threatening

  16. Dipeptidyl peptidase expression during experimental colitis in mice

    DEFF Research Database (Denmark)

    Yazbeck, Roger; Sulda, Melanie L; Howarth, Gordon S

    2010-01-01

    We have previously demonstrated that inhibition of dipeptidyl peptidase (DP) activity partially attenuates dextran sulfate sodium (DSS) colitis in mice. The aim of this study was to further investigate the mechanisms of this protection.......We have previously demonstrated that inhibition of dipeptidyl peptidase (DP) activity partially attenuates dextran sulfate sodium (DSS) colitis in mice. The aim of this study was to further investigate the mechanisms of this protection....

  17. Papain-like peptidases: structure, function, and evolution.

    Science.gov (United States)

    Novinec, Marko; Lenarčič, Brigita

    2013-06-01

    Papain-like cysteine peptidases are a diverse family of peptidases found in most known organisms. In eukaryotes, they are divided into multiple evolutionary groups, which can be clearly distinguished on the basis of the structural characteristics of the proenzymes. Most of them are endopeptidases; some, however, evolved into exopeptidases by obtaining additional structural elements that restrict the binding of substrate into the active site. In humans, papain-like peptidases, also called cysteine cathepsins, act both as non-specific hydrolases and as specific processing enzymes. They are involved in numerous physiological processes, such as antigen presentation, extracellular matrix remodeling, and hormone processing. Their activity is tightly regulated and dysregulation of one or more cysteine cathepsins can result in severe pathological conditions, such as cardiovascular diseases and cancer. Other organisms can utilize papain-like peptidases for different purposes and they are often part of host-pathogen interactions. Numerous parasites, such as Plasmodium and flukes, utilize papain-like peptidases for host invasion, whereas plants, in contrast, use these enzymes for host defense. This review presents a state-of-the-art description of the structure and phylogeny of papain-like peptidases as well as an overview of their physiological and pathological functions in humans and in other organisms.

  18. Evolutionary histories of expanded peptidase families in Schistosoma mansoni

    Directory of Open Access Journals (Sweden)

    Larissa Lopes Silva

    2011-11-01

    Full Text Available Schistosoma mansoni is one of the three main causative agents of human schistosomiasis, a major health problem with a vast socio-economic impact. Recent advances in the proteomic analysis of schistosomes have revealed that peptidases are the main virulence factors involved in the pathogenesis of this disease. In this context, evolutionary studies can be applied to identify peptidase families that have been expanded in genomes over time in response to different selection pressures. Using a phylogenomic approach, we searched for expanded endopeptidase families in the S. mansoni predicted proteome with the aim of contributing to the knowledge of such enzymes as potential therapeutic targets. We found three endopeptidase families that comprise leishmanolysins (metallopeptidase M8 family, cercarial elastases (serine peptidase S1 family and cathepsin D proteins (aspartic peptidase A1 family. Our results suggest that the Schistosoma members of these families originated from successive gene duplication events in the parasite lineage after its diversification from other metazoans. Overall, critical residues are conserved among the duplicated genes/proteins. Furthermore, each protein family displays a distinct evolutionary history. Altogether, this work provides an evolutionary view of three S. mansoni peptidase families, which allows for a deeper understanding of the genomic complexity and lineage-specific adaptations potentially related to the parasitic lifestyle.

  19. Altered Peptidase Activities in Thyroid Neoplasia and Hyperplasia

    Directory of Open Access Journals (Sweden)

    Gorka Larrinaga

    2013-01-01

    Full Text Available Background. Papillary thyroid carcinoma (PTC, follicular thyroid adenoma (FTA, and thyroid nodular hyperplasia (TNH are the most frequent diseases of the thyroid gland. Previous studies described the involvement of dipeptidyl-peptidase IV (DPPIV/CD26 in the development of thyroid neoplasia and proposed it as an additional tool in the diagnosis/prognosis of these diseases. However, very little is known about the involvement of other peptidases in neoplastic and hyperplastic processes of this gland. Methods. The catalytic activity of 10 peptidases in a series of 30 PTC, 10 FTA, and 14 TNH was measured fluorimetrically in tumour and nontumour adjacent tissues. Results. The activity of DPPIV/CD26 was markedly higher in PTC than in FTA, TNH, and nontumour tissues. Aspartyl aminopeptidase (AspAP, alanyl aminopeptidase (AlaAP, prolyl endopeptidase, pyroglutamyl peptidase I, and aminopeptidase B activities were significantly increased in thyroid neoplasms when compared to nontumour tissues. AspAP and AlaAP activities were also significantly higher in PTC than in FTA and TNH. Conclusions. These data suggest the involvement of DPPIV/CD26 and some cytosolic peptidases in the neoplastic development of PTC and FTA. Further studies will help to define the possible clinical usefulness of AlaAP and AspAP in the diagnosis/prognosis of thyroid neoplasms.

  20. Vigilin interacts with signal peptide peptidase

    Directory of Open Access Journals (Sweden)

    Lu Stephen Hsueh-Jeng

    2012-05-01

    Full Text Available Abstract Background Signal peptide peptidase (SPP, a member of the presenilin-like intra-membrane cleaving aspartyl protease family, migrates on Blue Native (BN gels as 100 kDa, 200 kDa and 450 kDa species. SPP has recently been implicated in other non-proteolytic functions such as retro-translocation of MHC Class I molecules and binding of misfolded proteins in the endoplasmic reticulum (ER. These high molecular weight SPP complexes might contain additional proteins that regulate the proteolytic activity of SPP or support its non-catalytic functions. Results In this study, an unbiased iTRAQ-labeling mass spectrometry approach was used to identify SPP-interacting proteins. We found that vigilin, a ubiquitous multi-KH domain containing cytoplasmic protein involved in RNA binding and protein translation control, selectively enriched with SPP. Vigilin interacted with SPP and both proteins co-localized in restricted intracellular domains near the ER, biochemically co-fractionated and were part of the same 450 kDa complex on BN gels. However, vigilin does not alter the protease activity of SPP, suggesting that the SPP-vigilin interaction might be involved in the non-proteolytic functions of SPP. Conclusions We have identified and validated vigilin as a novel interacting partner of SPP that could play an important role in the non-proteolytic functions of SPP. This data adds further weight to the idea that intramembrane-cleaving aspartyl proteases, such as presenilin and SPPs, could have other functions besides the proteolysis of short membrane stubs.

  1. P1 peptidase – a mysterious protein of family Potyviridae

    Indian Academy of Sciences (India)

    Jana Rohožková; Milan Navrátil

    2011-03-01

    The Potyviridae family, named after its type member, Potato virus Y (PVY), is the largest of the 65 plant virus groups and families currently recognized. The coding region for P1 peptidase is located at the very beginning of the viral genome of the family Potyviridae. Until recently P1 was thought of as serine peptidase with RNA-binding activity and with possible influence in cell-to-cell viral spreading. This N-terminal protein, among all of the potyviruses, is the most divergent protein: varying in length and in its amino acid sequence. Nevertheless, P1 peptidase in many ways is still a mysterious viral protein. In this review, we would like to offer a comprehensive overview, discussing the proteomic, biochemical and phylogenetic views of the P1 protein.

  2. Plasmodia express two threonine-peptidase complexes during asexual development.

    Science.gov (United States)

    Mordmüller, Benjamin; Fendel, Rolf; Kreidenweiss, Andrea; Gille, Christoph; Hurwitz, Robert; Metzger, Wolfram G; Kun, Jürgen F J; Lamkemeyer, Tobias; Nordheim, Alfred; Kremsner, Peter G

    2006-07-01

    Threonine-peptidases of the T1-family are multi-subunit complexes with broad substrate specificity. In eukaryotes, at least 14 genes encode subunits of the prototypic T1 threonine-peptidase, the proteasome. The proteasome determines the turnover of most proteins and thereby plays a fundamental role in diverse processes such as protein quality control, signal transduction, and cell cycle regulation. While eukaryotes and archaea possess a proteasome, bacteria generally express a second member of the T1-family, the proteasomal predecessor ClpQ/hslV that has a similar structure but is encoded by only one gene. The plasmodial genome is an exception because it encodes proteasomal subunits as well as a ClpQ/hslV-orthologe (Plasmodium falciparum-hslV; PfhslV). Structure, expression, and function of both types of peptidase-complex in P. falciparum are presently unknown. Our aim was to analyze both the coding sequences and derived proteins of both peptidase-complexes because highly specific and potent inhibitors can be designed against this class of enzymes. The proteasome was found expressed throughout the cell cycle, whereas PfhslV was detectable in schizonts and merozoites only. Treatment of P. falciparum with the threonine-peptidase inhibitor epoxomicin blocked two of three catalytically active proteasome subunits. This led to the accumulation of ubiquitinated proteins and, finally, to parasite death. In conclusion, we provide the first functional analysis of plasmodial threonine-peptidase-complexes and identify a lead compound for the development of a novel class of antimalarial drugs.

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

    Science.gov (United States)

    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

  4. Recurrent angioedema associated with pharmacological inhibition of dipeptidyl peptidase IV

    DEFF Research Database (Denmark)

    Hermanrud, Thorbjørn; Bygum, Anette; Rasmussen, Eva Rye

    2017-01-01

    -converting enzyme inhibitors (ACEI-AAE) is well known, but other pharmaceutical agents also affect the degradation of bradykinin and substance P. We present a middle-aged man with recurrent episodes of severe AE of the oral cavity, hypopharynx and larynx due to pharmacological inhibition of dipeptidyl peptidase IV....

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

    Science.gov (United States)

    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.

  6. SIGNAL PEPTIDASE-I OF BACILLUS-SUBTILIS : PATTERNS OF CONSERVED AMINO-ACIDS IN PROKARYOTIC AND EUKARYOTIC TYPE-I SIGNAL PEPTIDASES

    NARCIS (Netherlands)

    van Dijl, Jan Maarten; DEJONG, A; Vehmaanperä, J; VENEMA, G; BRON, S

    Signal peptidases (SPases) remove signal peptides from secretory proteins. The sipS (signal peptidase of subtilis) gene, which encodes an SPase of Bacillus subtilis, was cloned in Escherichia coli and was also found to be active in E. coli. Its overproduction in B. subtilis resulted in increased

  7. Activity of soluble aminopeptidase A and dipeptidyl peptidase IV and membrane-bound aminopeptidase B and pyroglutamyl peptidase I in adenoid hyperplasia, tonsillar hyperplasia and chronic tonsillitis.

    Science.gov (United States)

    Larrinaga, Gorka; Perez, Itxaro; Sanz, Begoña; Irazusta, Amaya; Zarrazquin, Idoia; Sanchez, Clara Elena; Sanchez, Carmen Elena; Rey, Ana Sanchez Del; Zabala, Aitor; Santaolalla, Francisco

    2011-11-01

    To analyze soluble and membrane-bound peptidase activities in the tonsils and adenoids removed from patients with adenoid hyperplasia, tonsillar hyperplasia and chronic tonsillitis. A total of 48 tissue samples from patients undergoing adenoidectomy and tonsillectomy for adenoid hyperplasia, tonsillar hyperplasia or chronic tonsillitis were analyzed. The catalytic activity of a pool of peptidases in the soluble (dipeptidyl peptidase IV, aminopeptidase A, aminopeptidase N and cystinyl aminopeptidase) and membrane-bound (prolyl endopeptidase, aspartyl aminopeptidase, aminopeptidase B and pyroglutamyl peptidase I) fractions was measured fluorometrically. The activity of membrane-bound aminopeptidase B was higher in cases of chronic tonsillitis and adenoid hyperplasia than in tonsillar hyperplasia, p=0.004. Soluble dipeptidyl peptidase IV and membrane-bound pyroglutamyl peptidase I were found to be more active in tissues from male chronic tonsillitis tissues, pchronic tonsillitis, soluble aminopeptidase A was found to have a higher level of activity in tissues from children than those from adults, p=0.005. Our results suggest a potential role of soluble aminopeptidase A, soluble dipeptidyl peptidase IV, membrane-bound aminopeptidase B and membrane-bound pyroglutamyl peptidase I in the pathobiology of adenoid hyperplasia, tonsillar hyperplasia and chronic tonsillitis that is differently regulated as a function of gender. These finfings may modify in the future the clinical approach to these diseases. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  8. Role of inhibitors of serine peptidases in protecting Leishmania donovani against the hydrolytic peptidases of sand fly midgut.

    Science.gov (United States)

    Verma, Sudha; Das, Sushmita; Mandal, Abhishek; Ansari, Md Yousuf; Kumari, Sujata; Mansuri, Rani; Kumar, Ajay; Singh, Ruby; Saini, Savita; Abhishek, Kumar; Kumar, Vijay; Sahoo, Ganesh Chandra; Das, Pradeep

    2017-06-23

    In vector-borne diseases such as leishmaniasis, the sand fly midgut is considered to be an important site for vector-parasite interaction. Digestive enzymes including serine peptidases such as trypsin and chymotrypsin, which are secreted in the midgut are one of the obstacles for Leishmania in establishing a successful infection. The presence of some natural inhibitors of serine peptidases (ISPs) has recently been reported in Leishmania. In the present study, we deciphered the role of these ISPs in the survival of Leishmania donovani in the hostile sand fly midgut environment. In silico and co-immunoprecipitation studies were performed to observe the interaction of L. donovani ISPs with trypsin and chymotrypsin. Zymography and in vitro enzyme assays were carried out to observe the inhibitory effect of purified recombinant ISPs of L. donovani (rLdISPs) on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of ISPs in the amastigote to promastigote transition stages were studied by semi-quantitative RT-PCR and Western blot. The role of LdISP on the survival of ISP overexpressed (OE) and ISP knocked down (KD) Leishmania parasites inside the sand fly gut was investigated by in vitro and in vivo cell viability assays. We identified two ecotin-like genes in L. donovani, LdISP1 and LdISP2. In silico and co-immunoprecipitation results clearly suggest a strong interaction of LdISP molecules with trypsin and chymotrypsin. Zymography and in vitro enzyme assay confirmed the inhibitory effect of rLdISP on trypsin, chymotrypsin and the sand fly midgut peptidases. The expression of LdISP2 was found to be strongly associated with the amastigote to promastigote phase transition. The activities of the digestive enzymes were found to be significantly reduced in the infected sand flies when compared to uninfected. To our knowledge, our study is the first report showing the possible reduction of chymotrypsin activity in L. donovani infected sand flies compared to

  9. Cysteine Peptidases as Schistosomiasis Vaccines with Inbuilt Adjuvanticity

    Science.gov (United States)

    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

  10. Cysteine peptidases as schistosomiasis vaccines with inbuilt adjuvanticity.

    Directory of Open Access Journals (Sweden)

    Rashika El Ridi

    Full Text Available 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.

  11. Extracellular peptidases of the cereal pathogen Fusarium graminearum.

    Directory of Open Access Journals (Sweden)

    Rohan George Thomas Lowe

    2015-11-01

    Full Text Available 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 characterise 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 behaviours. An orbitrap MS/MS proteomics technique defined the extracellular proteases secreted by Fusarium 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.

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

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    A.V. Karlyshev

    2014-01-01

    Full Text Available 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.

  13. Let’s not forget: Peptidases in Alzheimer’s disease

    NARCIS (Netherlands)

    Stargardt, A.

    2014-01-01

    In this thesis, we investigated the role of peptidases in Alzheimer’s disease (AD). AD is characterized by extracellular aggregates consisting of amyloid-β (Aβ) peptides and accumulation of these peptides starts decades before the onset of the first symptoms. We described which peptidases degrade th

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

    Science.gov (United States)

    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...

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

    Science.gov (United States)

    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...

  16. In vitro analysis of signal peptidase and membrane tannslocation activity in yeast microsomal membranes. Kobomaku kakubun wo mochiiru signal peptidase kassei oyobi makutoka kassei sokuteiho no kento

    Energy Technology Data Exchange (ETDEWEB)

    Nakayama, K.; Machida, M.; Jigami, Y. (The National Chemical Laboratory for Industry, Tsukuba (Japan))

    1991-05-29

    Studies have been pursued on proteins produced using microorganisms and cultured cells. The studies aim at facilitating the purification of the protein by making it secrete outside the cell and improving productivity. Signal peptidase is related to the secretion/translocation process of the protein. An in vitro analysis system for activity of yeast signal peptidase is made to elucidate effects of activity of signal peptidase on velocity and amount of secretion of the protein. As a result, in a combination of wheat germ extract and canine pancreatic membranes, the single peptidase activity and the membrane translocation activity are seen in a colibacillus {beta}-lactamase precursor and a yeast {alpha}-factor precursor, respectively. Moreover, in a combination of yeast lysates and yeast microsomal membranes, as the result of measuring the activity with the {alpha}-factor precursor as a substrate, the membrane translocation activity can be detected in this system. 16 refs., 4 figs., 1 tab.

  17. Activity of a peptidase secreted by Phanerochaete chrysosporium depends on lysine to subsite S'1.

    Science.gov (United States)

    da Silva, Ronivaldo Rodrigues; de Oliveira, Lilian Caroline Gonçalves; Juliano, Maria Aparecida; Juliano, Luiz; Rosa, Jose C; Cabral, Hamilton

    2017-01-01

    Peptidases are enzymes that catalyze the rupture of peptide bonds. Catalytic specificity studies of these enzymes have illuminated their modes of action and preferred hydrolysis targets. We describe the biochemical characteristics and catalytic specificity of a lysine-dependent peptidase secreted by the basidiomycete fungus Phanerochaete chrysosporium. We attained 5.7-fold purification of a ∼23-kDa neutral peptidase using size-exclusion (Sephadex G-50 resin) and ion-exchange (Source 15S resin) chromatography. Using the Fluorescence Resonance Energy Transfer substrate Abz-KLRSSKQ-EDDnp, we detected maximal activity at pH 7.0 and 45-55°C. The peptidase retained ∼80% of its enzymatic activity for a wide range of conditions (pH 4-9; temperatures up to 50°C for 1h). The peptidase activity was lowered by the ionic surfactants, sodium dodecyl sulfate and cetyltrimethylammonium bromide; the reducing agent, dithiothreitol; the chaotrope, guanidine; copper (II) ion; and the cysteine peptidase-specific inhibitors, iodoacetic acid and N-ethylmaleimide. The peptidase preferred the basic amino acids K and R and high selectivity on S'1 subsite, exhibiting a condition of lysine-dependence to catalysis on anchoring of this subsite. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. The enteroinsular axis in dipeptidyl peptidase IV-negative rats

    DEFF Research Database (Denmark)

    Pederson, Raymond; Kieffer, T J; Pauly, R

    1996-01-01

    Evidence has accumulated that the incretins glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1(7-36) amide) are degraded and rendered biologically inactive in plasma by the enzyme dipeptidyl peptidase IV (DPIV). A strain of Fischer rats lacking the DPIV enzyme...... with incubation in plasma from DPIV-deficient rats. It was hypothesized that the reduced GIP response and decreased sensitivity of the pancreas to GIP are compensatory mechanisms that maintain insulin and glucose levels within a normal range despite abnormal degradation of GIP. An explanation of the lack...... of effect of the absence of DPIV on the GLP-1(7-36) response to oral glucose and insulinotropic action of this peptide must await further study....

  19. Bacteriophage-Derived Peptidase CHAPK Eliminates and Prevents Staphylococcal Biofilms

    Directory of Open Access Journals (Sweden)

    Mark Fenton

    2013-01-01

    Full Text Available New antibacterial agents are urgently needed for the elimination of biofilm-forming bacteria that are highly resistant to traditional antimicrobial agents. Proliferation of such bacteria can lead to significant economic losses in the agri-food sector. This study demonstrates the potential of the bacteriophage-derived peptidase, CHAPK, as a biocidal agent for the rapid disruption of biofilm-forming staphylococci, commonly associated with bovine mastitis. Purified CHAPK applied to biofilms of Staphylococcus aureus DPC5246 completely eliminated the staphylococcal biofilms within 4 h. In addition, CHAPK was able to prevent biofilm formation by this strain. The CHAPK lysin also reduced S. aureus in a skin decolonization model. Our data demonstrates the potential of CHAPK as a biocidal agent for prevention and treatment of biofilm-associated staphylococcal infections or as a decontaminating agent in the food and healthcare sectors.

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

    Science.gov (United States)

    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.

  1. Inhibition of DD-peptidases by a specific trifluoroketone: crystal structure of a complex with the Actinomadura R39 DD-peptidase.

    Science.gov (United States)

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

    2013-03-26

    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 upon 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 includes 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, we found 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.

  2. Bacillus thuringiensis Cry3Aa protoxin intoxication of Tenebrio molitor induces widespread changes in the expression of serine peptidase transcripts.

    Science.gov (United States)

    Oppert, Brenda; Martynov, Alexander G; Elpidina, Elena N

    2012-09-01

    The yellow mealworm, Tenebrio molitor, is a pest of stored grain products and is sensitive to the Bacillus thuringiensis (Bt) Cry3Aa toxin. As digestive peptidases are a determining factor in Cry toxicity and resistance, we evaluated the expression of peptidase transcripts in the midgut of T. molitor larvae fed either a control or Cry3Aa protoxin diet for 24 h (RNA-Seq), or in larvae exposed to the protoxin for 6, 12, or 24 h (microarrays). Cysteine peptidase transcripts (9) were similar to cathepsins B, L, and K, and their expression did not vary more than 2.5-fold in control and Cry3Aa-treated larvae. Serine peptidase transcripts (48) included trypsin, chymotrypsin and chymotrypsin-like, elastase 1-like, and unclassified serine peptidases, as well as homologs lacking functional amino acids. Highly expressed trypsin and chymotrypsin transcripts were severely repressed, and most serine peptidase transcripts were expressed 2- to 15-fold lower in Cry3Aa-treated larvae. Many serine peptidase and homolog transcripts were found only in control larvae. However, expression of a few serine peptidase transcripts was increased or found only in Cry3Aa-treated larvae. Therefore, Bt intoxication significantly impacted the expression of serine peptidases, potentially important in protoxin processing, while the insect maintained the production of critical digestive cysteine peptidases.

  3. Dipeptidyl peptidase IV inhibitors: a promising new therapeutic approach for the management of type 2 diabetes

    DEFF Research Database (Denmark)

    Deacon, Carolyn F; Holst, Jens J

    2005-01-01

    of appetite. Glucagon-like peptide-1 is, however, extremely rapidly inactivated by the serine peptidase, dipeptidyl peptidase IV, so that the native peptide is not useful clinically. A new approach to utilise the beneficial effects of glucagon-like peptide-1 in the treatment of type 2 diabetes has been......-12 months duration in patients with type 2 diabetes, dipeptidyl peptidase IV inhibitors have proved efficacious, both as monotherapy and when given in combination with metformin. Fasting and postprandial glucose concentrations were reduced, leading to reductions in glycosylated haemoglobin levels, while...... the development of orally active dipeptidyl peptidase IV inhibitors. Preclinical studies have demonstrated that this approach is effective in enhancing endogenous levels of glucagon-like peptide-1, resulting in improved glucose tolerance in glucose-intolerant and diabetic animal models. In recent studies of 3...

  4. Comparison of two pig intestinal brush border peptidases with the corresponding renal enzymes

    DEFF Research Database (Denmark)

    Norén, O; Sjöström, H; Danielsen, Erik Michael;

    1979-01-01

    -glutamyl-transferase was found to be identical to that of the kidney enzyme. The electrophoretic mobilities of dipeptidyl peptidase IV from the two organs differed greatly. The difference was almost abolished by treatment with neuraminidase, suggesting that the variation in mobility was due to different contents of sialic acid......Intestinal dipeptidyl peptidase IV and gamma-glutamyltransferase were compared to the corresponding kidney enzymes with respect to immunological and electrophoretic properties. The influences of selected effectors on the two enzymes were also studied. The two kidney peptidases exhibited...... the reaction of total identity with the corresponding intestinal enzymes in immunodiffusion. Furthermore, the intestinal dipeptidyl peptidase IV and gamma-glutamyl transferase showed the same inhibition patterns as the corresponding kidney enzymes and the acceptor specificity of the intestinal gamma...

  5. Dipeptidyl peptidase IV in two human glioma cell lines

    Directory of Open Access Journals (Sweden)

    A Sedo

    2009-12-01

    Full Text Available There is growing evidence that dipeptidyl peptidase IV [DPP-IV, EC 3.4.14.5] takes part in the metabolism of biologically active peptides participating in the regulation of growth and transformation of glial cells. However, the knowledge on the DPP-IV expression in human glial and glioma cells is still very limited. In this study, using histochemical and biochemical techniques, the DPP-IV activity was demonstrated in two commercially available human glioma cell lines of different transformation degree, as represented by U373 astrocytoma (Grade III and U87 glioblastoma multiforme (Grade IV lines. Higher total activity of the enzyme, as well as its preferential localisation in the plasma membrane, was observed in U87 cells. Compared to U373 population, U87 cells were morphologically more pleiomorphic, they were cycling at lower rate and expressing less Glial Fibrillary Acidic Protein. The data revealed positive correlation between the degree of transformation of cells and activity of DPP-IV. Great difference in expression of this enzyme, together with the phenotypic differences of cells, makes these lines a suitable standard model for further 57 studies of function of this enzyme in human glioma cells.

  6. Dipeptidyl peptidase-4 inhibitors: Novel mechanism of actions

    Directory of Open Access Journals (Sweden)

    Awadhesh Kumar Singh

    2014-01-01

    Full Text Available The pharmacological actions of the glucagon-like peptide-1 receptor agonists (GLP-1RA are largely predictable as they interact directly with GLP-1 receptors on beta cells to mediate their glucose lowering effects by increasing GLP-1 in pharmacological range and not at all dependent upon endogenous GLP-1 secretion. The mechanism of action of dipeptidyl peptidase-4 inhibitors (DPP-4I are relatively less clear although classical mechanism is to inhibit the endogenous GLP-1 metabolism and thereby increasing GLP-1 level in the physiological range. DPP-4I also increase the half-life of GLP-1 to some extent by inhibiting their quick degradation by DPP enzyme ubiquitously present in the body. Interestingly, even with the effective blockade with currently existing DPP-4I, the half-life of GLP-1 only increases from 1 min to 5 min and therefore its residual time in plasma still remains pretty short. Intriguingly, this GLP-1 rise is so modest and so short-lived that it may be difficult to believe that this would sufficiently engage and activate the GLP-1 receptor in beta cell to produce significant insulinotropic effect. However, in clinical trials as well as in real life scenario, the anti-glycemic efficacies seen with DPP-4I are quite satisfactory and sometime very much competitive to GLP-1RA as evident from their head-to-head trials including meta-analysis. This efficacy outcome challenges the "only" GLP-1 dependent mechanism of glucose lowering and provokes an insight that other neuro-endocrine pathway may be playing a second fiddle. This review will collate those emerging concept and put a perspective as to how DPP-4I might be working though other pathway besides direct GLP-1 mediated receptor activation.

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

    Directory of Open Access Journals (Sweden)

    Shoombuatong W

    2015-08-01

    Full Text Available Watshara Shoombuatong,1 Veda Prachayasittikul,1,2 Nuttapat Anuwongcharoen,1 Napat Songtawee,1 Teerawat Monnor,1 Supaluk Prachayasittikul,1 Virapong Prachayasittikul,2 Chanin Nantasenamat1,2 1Center of Data Mining and Biomedical Informatics, 2Department of Clinical Microbiology and Applied Technology, Faculty of Medical Technology, Mahidol University, Bangkok, Thailand Abstract: 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

  8. An Entamoeba cysteine peptidase specifically expressed during encystation.

    Science.gov (United States)

    Ebert, Frank; Bachmann, Anna; Nakada-Tsukui, Kumiko; Hennings, Ina; Drescher, Babette; Nozaki, Tomoyoshi; Tannich, Egbert; Bruchhaus, Iris

    2008-12-01

    Protozoan parasites of the genus Entamoeba possess a considerable number of cysteine peptidases (CPs), the function of most of these molecules for amoeba biology needs to be established. In order to determine whether CPs may play a role during Entamoeba stage conversion from trophozoites into cysts and vice versa, expression of cp genes was analysed in the reptilian parasite Entamoeba invadens, a model organism for studying Entamoeba cyst development. By homology search, 28 papain-like cp genes were identified in public E. invadens genome databases. For eight of these genes the expression profiles during stage conversion was determined. By Northern blot analysis, transcripts for eicp-a9, -b7, -b8 and -c2, respectively, were detected neither in trophozoites or cysts nor at any of the point of times analysed during stage conversion. On the other hand, eicp-a5 is constitutively expressed during all developmental stages, whereas eicp-a3 and eicp-a11, respectively, are trophozoite-specific. Only eicp-b9 was found to be cyst-specific as it is expressed exclusively 18 to 28 h after cyst induction. Cyst-specific expression was confirmed by immunofluorescence microscopy of the corresponding protein EiCP-B9. In immature cysts, the molecule is located in structures that accumulate near the cyst wall, but which are uniformly distributed in mature cysts. The precise function of EiCP-B9 during Entamoeba encystation remains to be determined. However, colocalisation studies with an Entamoeba marker for autophagosomes suggest that EiCP-B9 is not associated with Entamoeba autophagy.

  9. Behavioral characteristics of ubiquitin-specific peptidase 46-deficient mice.

    Directory of Open Access Journals (Sweden)

    Saki Imai

    Full Text Available We have previously identified Usp46, which encodes for ubiquitin-specific peptidase 46, as a quantitative trait gene affecting the immobility time of mice in the tail suspension test (TST and forced swimming test. The mutation that we identified was a 3-bp deletion coding for lysine (Lys 92, and mice with this mutation (MT mice, as well as Usp46 KO mice exhibited shorter TST immobility times. Behavioral pharmacology suggests that the gamma aminobutyric acid A (GABAA receptor is involved in regulating TST immobility time. In order to understand how far Usp46 controls behavioral phenotypes, which could be related to mental disorders in humans, we subjected Usp46 MT and KO mice to multiple behavioral tests, including the open field test, ethanol preference test, ethanol-induced loss of righting reflex test, sucrose preference test, novelty-suppressed feeding test, marble burying test, and novel object recognition test. Although behavioral phenotypes of the Usp46 MT and KO mice were not always identical, deficiency of Usp46 significantly affected performance in all these tests. In the open field test, activity levels were lower in Usp46 KO mice than wild type (WT or MT mice. Both MT and KO mice showed lower ethanol preference and shorter recovery times after ethanol administration. Compared to WT mice, Usp46 MT and KO mice exhibited decreased sucrose preference, took longer latency periods to bite pellets, and buried more marbles in the sucrose preference test, novelty-suppressed feeding test, and marble burying test, respectively. In the novel object recognition test, neither MT nor KO mice showed an increase in exploration of a new object 24 hours after training. These findings indicate that Usp46 regulates a wide range of behavioral phenotypes that might be related to human mental disorders and provides insight into the function of USP46 deubiquitinating enzyme in the neural system.

  10. Active-site residues in the type IV prepilin peptidase homologue PibD from the archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Szabo, Z; Albers, SV; Driessen, AJM

    2006-01-01

    Archaeal preflagellin peptidases and bacterial type IV prepilin peptidases belong to a family of aspartic acid proteases that cleave the leader peptides of precursor proteins with type W prepilin signal sequences. The substrate repertoire of PibD from the crenarchaeon Sulfolobus solfataricus is unus

  11. Active-Site Residues in the Type IV Prepilin Peptidase Homologue PibD from the Archaeon Sulfolobus solfataricus

    NARCIS (Netherlands)

    Szabo, Zalan; Albers, Sonja-Verena; Driessen, Arnold J.M.

    2006-01-01

    Archaeal preflagellin peptidases and bacterial type IV prepilin peptidases belong to a family of aspartic acid proteases that cleave the leader peptides of precursor proteins with type IV prepilin signal sequences. The substrate repertoire of PibD from the crenarchaeon Sulfolobus solfataricus is unu

  12. Proteomic analysis of human skin treated with larval schistosome peptidases reveals distinct invasion strategies among species of blood flukes.

    Directory of Open Access Journals (Sweden)

    Jessica Ingram

    2011-09-01

    Full Text Available Skin invasion is the initial step in infection of the human host by schistosome blood flukes. Schistosome larvae have the remarkable ability to overcome the physical and biochemical barriers present in skin in the absence of any mechanical trauma. While a serine peptidase with activity against insoluble elastin appears to be essential for this process in one species of schistosomes, Schistosoma mansoni, it is unknown whether other schistosome species use the same peptidase to facilitate entry into their hosts.Recent genome sequencing projects, together with a number of biochemical studies, identified alternative peptidases that Schistosoma japonicum or Trichobilharzia regenti could use to facilitate migration through skin. In this study, we used comparative proteomic analysis of human skin treated with purified cercarial elastase, the known invasive peptidase of S. mansoni, or S. mansoni cathespin B2, a close homolog of the putative invasive peptidase of S. japonicum, to identify substrates of either peptidase. Select skin proteins were then confirmed as substrates by in vitro digestion assays.This study demonstrates that an S. mansoni ortholog of the candidate invasive peptidase of S. japonicum and T. regenti, cathepsin B2, is capable of efficiently cleaving many of the same host skin substrates as the invasive serine peptidase of S. mansoni, cercarial elastase. At the same time, identification of unique substrates and the broader species specificity of cathepsin B2 suggest that the cercarial elastase gene family amplified as an adaptation of schistosomes to human hosts.

  13. Oxytocin biotransformation in the rat limbic brain: Characterization of peptidase activities and significance in the formation of oxytocin fragments

    NARCIS (Netherlands)

    Burbach, J.P.H.; Kloet, E.R. de; Wied, D. de

    1980-01-01

    The enzymatic conversion of oxytocin by brain peptidases has been studied. Oxytocin was incubated with synaptosomal plasma membranes (SPM) isolated from the rat brain. Qualitative studies using a microdansylation technique revealed two types of oxytocin converting peptidases, e.g. aminopeptidase and

  14. Cysteine peptidases CPA and CPB are vital for autophagy and differentiation in Leishmania mexicana.

    Science.gov (United States)

    Williams, Roderick A; Tetley, Laurence; Mottram, Jeremy C; Coombs, Graham H

    2006-08-01

    In the past, ultrastructural investigations of Leishmania mexicana amastigotes revealed structures that were tentatively identified as autophagosomes. This study has now provided definitive data that autophagy occurs in the parasite during differentiation both to metacyclic promastigotes and to amastigotes, autophagosomes being particularly numerous during metacyclic to amastigote form transformation. Moreover, the results demonstrate that inhibiting two major lysosomal cysteine peptidases (CPA and CPB) or removing their genes not only interferes with the autophagy pathway but also prevents metacyclogenesis and transformation to amastigotes, thus adding support to the hypothesis that autophagy is required for cell differentiation. The study suggests that L. mexicana CPA and CPB perform similar roles to the aspartic peptidase PEP4 and the serine peptidase PRB1 in Saccharomyces cerevisiae. The results also provide an explanation for why L. mexicana CPA/CPB-deficient mutants transform to amastigotes very poorly and lack virulence in macrophages and mice.

  15. Peptidases compartmentalized to the Ascaris suum intestinal lumen and apical intestinal membrane.

    Directory of Open Access Journals (Sweden)

    Douglas P Jasmer

    2015-01-01

    Full Text Available The nematode intestine is a tissue of interest for developing new methods of therapy and control of parasitic nematodes. However, biological details of intestinal cell functions remain obscure, as do the proteins and molecular functions located on the apical intestinal membrane (AIM, and within the intestinal lumen (IL of nematodes. Accordingly, methods were developed to gain a comprehensive identification of peptidases that function in the intestinal tract of adult female Ascaris suum. Peptidase activity was detected in multiple fractions of the A. suum intestine under pH conditions ranging from 5.0 to 8.0. Peptidase class inhibitors were used to characterize these activities. The fractions included whole lysates, membrane enriched fractions, and physiological- and 4 molar urea-perfusates of the intestinal lumen. Concanavalin A (ConA was confirmed to bind to the AIM, and intestinal proteins affinity isolated on ConA-beads were compared to proteins from membrane and perfusate fractions by mass spectrometry. Twenty-nine predicted peptidases were identified including aspartic, cysteine, and serine peptidases, and an unexpectedly high number (16 of metallopeptidases. Many of these proteins co-localized to multiple fractions, providing independent support for localization to specific intestinal compartments, including the IL and AIM. This unique perfusion model produced the most comprehensive view of likely digestive peptidases that function in these intestinal compartments of A. suum, or any nematode. This model offers a means to directly determine functions of these proteins in the A. suum intestine and, more generally, deduce the wide array functions that exist in these cellular compartments of the nematode intestine.

  16. IDENTIFICATION OF THE POTENTIAL ACTIVE-SITE OF THE SIGNAL PEPTIDASE SIPS OF BACILLUS-SUBTILIS - STRUCTURAL AND FUNCTIONAL SIMILARITIES WITH LEXA-LIKE PROTEASES

    NARCIS (Netherlands)

    VANDIJL, JM; DEJONG, A; VENEMA, G; BRON, S

    1995-01-01

    Signal peptidases remove signal peptides from secretory proteins. By comparing the type I signal peptidase, SipS, of Bacillus subtilis with signal peptidases from prokaryotes, mitochondria, and the endoplasmic reticular membrane, patterns of conserved amino acids were discovered. The conserved resid

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

    Science.gov (United States)

    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

  18. Linagliptin: a novel methylxanthin based approved dipeptidyl peptidase-4 inhibitor.

    Science.gov (United States)

    Agrawal, Ritesh; Jain, Pratima; Dikshit, S N

    2012-06-01

    Chemically, methylxanthine nucleus based Linagliptin (BI-1356, BI-1356-BS) is a dipeptidyl peptidase-IV inhibitor, which has been developed by Boehringer Ingelheim in association with Lilly for the treatment of Type-II Diabetes. Linagliptin was marketed by Lilly under the trade name Tradjenta and Trajenta. Linagliptin was approved as the once-daily dose by USFDA on 2 May 2011, for the treatment of Type-II Diabetes. Linagliptin 5mg once daily dose was approved based on a clinical trial program, which was conducted on approximately 4,000 adults with Type-II Diabetes. Linagliptin demonstrated statistically significant mean difference in HbA1c from placebo of up to 0.72 percent, when it was used monotherapically. In patients, who were not adequately controlled on metformin or metformin plus sulphonylurea, the addition of Linagliptin resulted in a statistically significant mean difference in HbA1c from placebo of -0.6 percent. Linagliptin was observed to produce significant reduction in fasting plasma glucose (FPG) compared to placebo, when used as a monotherapy in combination with metformin, sulfonylurea and/or pioglitazone. Linagliptin demonstrated significant reduction post-prandial glucose (PPG) levels in two hours as compared with placebo in monotherapy as well as in combination with metformin. In vitro assays also anticipated that Linagliptin is a potent DPPIV inhibitor as well as it exhibits good selectivity for DPP-IV as compared with other DPPs. The in-vivo studies also demonstrated same anticipation with respect to Linagliptin. Consequently, increasing the GLP-1 levels so far improved glucose tolerance in both healthy animals. X-ray crystallography anticipates that Linagliptin complexes with human DPPIV enzyme, e.g. butynyl substituent occupies the S1 hydrophobic pocket of the enzyme; the aminopiperidine substituent in the xanthine scaffold occupies the S2 subsite and its primary amine interacts with the key amino acid residues, which involves in the

  19. Non-functional expression of Escherichia coli signal peptidase I in Bacillus subtilis

    NARCIS (Netherlands)

    van Dijl, J M; Jong, de Anne; Smith, H; Bron, Sierd; Venema, G

    The Escherichia coli lep gene, encoding signal peptidase I (SPase I) was provided with Bacillus subtilis transcription/translation signals and expressed in this organism. When present on a low-copy-number plasmid, the amount of E. coli SPase I produced (per mg cell protein) in B. subtilis was half

  20. Detergent-independent in vitro activity of a truncated Bacillus signal peptidase

    NARCIS (Netherlands)

    van Roosmalen, ML; Jongbloed, JDH; de Jong, A; van Eerden, J; Venema, G; Bron, S; van Dijl, JM

    2001-01-01

    The Cram-positive eubacterium Bacillus subtilis contains five chromosomally encoded type I signal peptidases (SPases) for the processing of secretory preproteins. Even though four of these SPases, denoted SipS, SipT, SipU and SipV, are homologous to the unique SPase I of Escherichia coli, they are s

  1. Dipeptidyl peptidase-4 inhibitors in the treatment of type 2 diabetes: A comparative review

    DEFF Research Database (Denmark)

    Deacon, Carolyn F.

    2011-01-01

    The dipeptidyl peptidase (DPP)-4 inhibitors are a new class of antihyperglycaemic agents which were developed for the treatment of type 2 diabetes by rational drug design, based on an understanding of the underlying mechanism of action and knowledge of the structure of the target enzyme. Although...

  2. Association of circulating dipeptidyl-peptidase 4 levels with osteoporotic fracture in postmenopausal women.

    Science.gov (United States)

    Kim, H; Baek, K H; Lee, S-Y; Ahn, S H; Lee, S H; Koh, J-M; Rhee, Y; Kim, C H; Kim, D-Y; Kang, M-I; Kim, B-J; Min, Y-K

    2017-03-01

    Postmenopausal women with osteoporotic fracture (OF) had higher plasma dipeptidyl-peptidase 4 (DPP4) levels than those without. Furthermore, higher plasma DPP4 levels were significantly associated with higher bone turnover and a higher prevalence of OF. These results indicated that DPP4 may be associated with OF by mediating bone turnover rate.

  3. Non-functional expression of Escherichia coli signal peptidase I in Bacillus subtilis

    NARCIS (Netherlands)

    van Dijl, J M; de Jong, A; Smith, H; Bron, S; Venema, G

    1991-01-01

    The Escherichia coli lep gene, encoding signal peptidase I (SPase I) was provided with Bacillus subtilis transcription/translation signals and expressed in this organism. When present on a low-copy-number plasmid, the amount of E. coli SPase I produced (per mg cell protein) in B. subtilis was half t

  4. NONFUNCTIONAL EXPRESSION OF ESCHERICHIA-COLI SIGNAL PEPTIDASE-I IN BACILLUS-SUBTILIS

    NARCIS (Netherlands)

    VANDIJL, JM; DEJONG, A; SMITH, H; BRON, S; VENEMA, G; van Dijl, Jan Maarten

    1991-01-01

    The Escherichia coli lep gene, encoding signal peptidase I (SPase I) was provided with Bacillus subtilis transcription/translation signals and expressed in this organism. When present on a low-copy-number plasmid, the amount of E. coli SPase I produced (per mg cell protein) in B. subtilis was half t

  5. Distinction between major and minor Bacillus signal peptidases based on phylogenetic and structural criteria

    NARCIS (Netherlands)

    van Roosmalen, ML; Jongbloed, JDH; Dubois, JYF; Venema, G; Bron, S; van Dijl, JM

    2001-01-01

    The processing of secretory preproteins by signal peptidases (SPases) is essential for cell viability. As previously shown for Bacillus subtilis, only certain SPases of organisms containing multiple paralogous SPases are essential. This allows a distinction between SPases that are of major and minor

  6. Bioinformatic analysis of the neprilysin (M13 family of peptidases reveals complex evolutionary and functional relationships

    Directory of Open Access Journals (Sweden)

    Pinney John W

    2008-01-01

    Full Text Available Abstract Background The neprilysin (M13 family of endopeptidases are zinc-metalloenzymes, the majority of which are type II integral membrane proteins. The best characterised of this family is neprilysin, which has important roles in inactivating signalling peptides involved in modulating neuronal activity, blood pressure and the immune system. Other family members include the endothelin converting enzymes (ECE-1 and ECE-2, which are responsible for the final step in the synthesis of potent vasoconstrictor endothelins. The ECEs, as well as neprilysin, are considered valuable therapeutic targets for treating cardiovascular disease. Other members of the M13 family have not been functionally characterised, but are also likely to have biological roles regulating peptide signalling. The recent sequencing of animal genomes has greatly increased the number of M13 family members in protein databases, information which can be used to reveal evolutionary relationships and to gain insight into conserved biological roles. Results The phylogenetic analysis successfully resolved vertebrate M13 peptidases into seven classes, one of which appears to be specific to mammals, and insect genes into five functional classes and a series of expansions, which may include inactive peptidases. Nematode genes primarily resolved into groups containing no other taxa, bar the two nematode genes associated with Drosophila DmeNEP1 and DmeNEP4. This analysis reconstructed only one relationship between chordate and invertebrate clusters, that of the ECE sub-group and the DmeNEP3 related genes. Analysis of amino acid utilisation in the active site of M13 peptidases reveals a basis for their biochemical properties. A relatively invariant S1' subsite gives the majority of M13 peptidases their strong preference for hydrophobic residues in P1' position. The greater variation in the S2' subsite may be instrumental in determining the specificity of M13 peptidases for their substrates

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

    Science.gov (United States)

    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.

  8. Extracellular peptidase hunting for improvement of protein production in plant cells and roots

    Directory of Open Access Journals (Sweden)

    Jérôme eLallemand

    2015-02-01

    Full Text Available Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion, in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA and human serum immunoglobulins G (hIgGs. Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing.

  9. Functional analysis of C1 family cysteine peptidases in the larval gut of Tenebrio molitor and Tribolium castaneum

    Science.gov (United States)

    We studied protein digestion the tenebrionids Tenebrio molitor and Tribolium castaneum, pests of stored grains and grain products, to identify potential targets for biopesticide development. Tenebrionid larvae have highly compartmentalized guts, with primarily cysteine peptidases in the acidic anter...

  10. Administration of a dipeptidyl peptidase IV inhibitor enhances the intestinal adaptation in a mouse model of short bowel syndrome

    DEFF Research Database (Denmark)

    Okawada, Manabu; Holst, Jens Juul; Teitelbaum, Daniel H

    2011-01-01

    Glucagon-like peptide-2 induces small intestine mucosal epithelial cell proliferation and may have benefit for patients who suffer from short bowel syndrome. However, glucagon-like peptide-2 is inactivated rapidly in vivo by dipeptidyl peptidase IV. Therefore, we hypothesized that selectively...... inhibiting dipeptidyl peptidase IV would prolong the circulating life of glucagon-like peptide-2 and lead to increased intestinal adaptation after development of short bowel syndrome....

  11. Expression patterns of cysteine peptidase genes across the Tribolium castaneum life cycle provide clues to biological function.

    Science.gov (United States)

    Perkin, Lindsey; Elpidina, Elena N; Oppert, Brenda

    2016-01-01

    The red flour beetle, Tribolium castaneum, is a major agricultural pest responsible for considerable loss of stored grain and cereal products worldwide. T. castaneum larvae have a highly compartmentalized gut, with cysteine peptidases mostly in the acidic anterior part of the midgut that are critical to the early stages of food digestion. In previous studies, we described 26 putative cysteine peptidase genes in T. castaneum (types B, L, O, F, and K) located mostly on chromosomes 3, 7, 8, and 10. In the present study, we hypothesized that specific cysteine peptidase genes could be associated with digestive functions for food processing based on comparison of gene expression profiles in different developmental stages, feeding and non-feeding. RNA-Seq was used to determine the relative expression of cysteine peptidase genes among four major developmental stages (egg, larvae, pupae, and adult) of T. castaneum. We also compared cysteine peptidase genes in T. castaneum to those in other model insects and coleopteran pests. By combining transcriptome expression, phylogenetic comparisons, response to dietary inhibitors, and other existing data, we identified key cysteine peptidases that T. castaneum larvae and adults use for food digestion, and thus new potential targets for biologically-based control products.

  12. DOCKING STUDIES OF CURCUMIN AS A POTENTIAL LEAD COMPOUND TO DEVELOP NOVEL DIPEPTYDYL PEPTIDASE-4 INHIBITORS

    Directory of Open Access Journals (Sweden)

    Enade Perdana Istyastono

    2010-06-01

    Full Text Available Interaction of curcumin to dipeptydyl peptidase-4 (DPP-4 has been studied by employing docking method using Molecular Operating Environment (MOE and AutoDock as the docking software applications. Although MOE can sample more conformational spaces that represent the original interaction poses than AutoDock, both softwares serve as valid and acceptable docking applications to study the interactions of small compound to DPP-4. The calculated free energy of binding (DGbinding results from MOE and AutoDock shows that curcumin is needed to be optimized to reach similar or better DGbinding compare to the reference compound. Curcumin can be considered as a good lead compound in the development of new DPP-4 inhibitor. The results of these studies can serve as an initial effort of the further study.     Keywords: curcumin, docking, molecular operating environment (MOE, AutoDock, dipeptydyl peptidase-4 (DPP-4

  13. Human dipeptidyl peptidase III acts as a post-proline-cleaving enzyme on endomorphins.

    Science.gov (United States)

    Barsun, Marina; Jajcanin, Nina; Vukelić, Bojana; Spoljarić, Jasminka; Abramić, Marija

    2007-03-01

    Dipeptidyl peptidase III (DPP III) is a zinc exopeptidase with an implied role in the mammalian pain-modulatory system owing to its high affinity for enkephalins and localisation in the superficial laminae of the spinal cord dorsal horn. Our study revealed that this human enzyme hydrolyses opioid peptides belonging to three new groups, endomorphins, hemorphins and exorphins. The enzymatic hydrolysis products of endomorphin-1 were separated and quantified by capillary electrophoresis and the kinetic parameters were determined for human DPP III and rat DPP IV. Both peptidases cleave endomorphin-1 at comparable rates, with liberation of the N-terminal Tyr-Pro. This is the first evidence of DPP III acting as an endomorphin-cleaving enzyme.

  14. Hieronymain I, a new cysteine peptidase isolated from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae).

    Science.gov (United States)

    Bruno, Mariela A; Pardo, Marcelo F; Caffini, Néstor O; López, Laura M I

    2003-02-01

    A new peptidase, named hieronymain I, was purified to homogeneity from unripe fruits of Bromelia hieronymi Mez (Bromeliaceae) by acetone fractionation followed by cation exchange chromatography (FPLC) on CM-Sepharose FF. Homogeneity of the enzyme was confirmed by mass spectroscopy (MALDI-TOF), isoelectric focusing, and SDS-PAGE. Hieronymain is a basic peptidase (pI > 9.3) and its molecular mass was 24,066 Da. Maximum proteolytic activity on casein (>90% of maximum activity) was achieved at pH 8.5-9.5. The enzyme was completely inhibited by E-64 and iodoacetic acid and activated by the addition of cysteine; these results strongly suggest that the isolated protease should be included within the cysteine group. The N-terminal sequence of hieronymain (ALPESIDWRAKGAVTEVKRQDG) was compared with 25 plant cysteine proteases that showed more than 50% of identity.

  15. Generation of Dipeptidyl Peptidase-IV-Inhibiting Peptides from β-Lactoglobulin Secreted by Lactococcus lactis

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    Suguru Shigemori

    2014-01-01

    Full Text Available 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.

  16. Peptidases prevent μ-opioid receptor internalization in dorsal horn neurons by endogenously released opioids

    OpenAIRE

    Song, Bingbing; Marvizón, Juan Carlos G.

    2003-01-01

    To evaluate the effect of peptidases on μ-opioid receptor (MOR) activation by endogenous opioids, we measured MOR-1 internalization in rat spinal cord slices. A mixture of inhibitors of aminopeptidases (amastatin), dipeptidyl carboxypeptidase (captopril), and neutral endopeptidase (phosphoramidon) dramatically increased the potencies of Leu-enkephalin and dynorphin A to produce MOR-1 internalization, and also enhanced the effects of Met-enkephalin and α-neoendorphin, but not endomorphins or β...

  17. Inhibition of cysteine peptidase activity in ascitic fluid in pancreatic cancer patients.

    Directory of Open Access Journals (Sweden)

    Adam Skalski

    2011-04-01

    Full Text Available The work's objective is to answer the question whether there is any possibility of activity inhibition of cysteine peptidases inhibitors playing an important role in key processes accompanying cancer formation, including pancreas. There is a justified speculation that specific inhibitors of these enzymes may inhibit development of cancer processes by inhibiting their activity. In vitro studies confirmed that these enzymes in ascitic fluid were inhibited with egg whites inhibitors even to 90% of their original activity.

  18. PCP and MK-801 Induced Behaviors Reduced by NAAG Peptidase Inhibition via Metabotropic Glutamate Receptors

    Science.gov (United States)

    Olszewski, Rafal T.; Wegorzewska, Marta M.; Monteiro, Ana C.; Krolikowski, Kristyn A.; Zhou, Jia; Kozikowski, Alan P.; Long, Katrice; Mastropaolo, John; Deutsch, Stephen I.; Neale, Joseph H.

    2007-01-01

    Background NMDA receptor open channel blockers phencyclidine (PCP) and dizocilpine (MK-801) elicit schizophrenia-like symptoms in humans and in animal models. Group II metabotropic glutamate receptor agonists reverse the behavioral effects of PCP and MK-801 in animal models. N-Acetylaspartylglutamate (NAAG), third most prevalent neurotransmitter in the mammalian nervous system, is a selective group II metabotropic glutamate receptor agonist. We previously reported that ZJ43, a potent inhibitor of the enzymes that inactivate synaptically released NAAG, reduced motor and stereotypic effects of PCP in the rat. Methods To confirm the efficacy of NAAG peptidase inhibition in decreasing motor behaviors induced by PCP and MK-801, ZJ43 was tested in additional schizophrenia models. Results ZJ43 reduced MK-801-induced motor activation in a mouse model that has been used to characterize the efficacy of a wide range of pharmacotherapies for this human disorder. In a second mouse strain, the peptidase inhibitor reduced PCP-induced stereotypic movements. ZJ43 also reduced PCP-induced negative symptoms in a resident-intruder assay. The group II metabotropic glutamate receptor antagonist, LY341495, blocked the effect of NAAG peptidase inhibition in these mouse models of positive and negative PCP- and MK-801-induced behaviors. Additionally, LY341495 alone increased some PCP-induced behaviors suggesting that normal levels of NAAG act to moderate the effect of PCP via a group II mGluR. Conclusion These data support the proposal that NAAG peptidase inhibition and elevation of synaptic NAAG levels represent a new therapeutic approach to treating the positive and negative symptoms of schizophrenia that are modeled by open channel NMDA receptor antagonists. PMID:17597589

  19. How immune peptidases change specificity: cathepsin G gained tryptic function but lost efficiency during primate evolution.

    Science.gov (United States)

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

    2010-11-01

    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 an unusual "Janus-faced" ability to cleave peptides at basic (tryptic) as well as aromatic (chymotryptic) sites. Tryptic activity has been attributed to acidic Glu(226) in the primary specificity pocket and underlies proposed important functions, such as activation of prourokinase. However, most mammals, including mice, substitute Ala(226) for Glu(226), 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 Tyr(4) over activation at Phe(8). Ala(226)Glu mutants of mouse cathepsin G acquire tryptic activity and human ability to activate prourokinase. Phylogenetic analysis reveals that the Ala(226)Glu 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.

  20. Dipeptidyl peptidase 4 inhibition with sitagliptin: a new therapy for type 2 diabetes

    DEFF Research Database (Denmark)

    Deacon, Carolyn F

    2007-01-01

    Sitagliptin is a once-daily, orally active, competitive and fully reversible inhibitor of dipeptidyl peptidase 4, the enzyme that is responsible for the rapid degradation of the incretin hormone glucagon-like peptide-1. It is the first in this new class of antihyperglycaemic agents to gain...... regulatory approval for the treatment of Type 2 diabetes, both as a monotherapy and for use in combination with metformin or a thiazolidinedione. In clinical trials of

  1. A model of tripeptidyl-peptidase I (CLN2, a ubiquitous and highly conserved member of the sedolisin family of serine-carboxyl peptidases

    Directory of Open Access Journals (Sweden)

    Oyama Hiroshi

    2003-11-01

    Full Text Available Abstract Background Tripeptidyl-peptidase I, also known as CLN2, is a member of the family of sedolisins (serine-carboxyl peptidases. In humans, defects in expression of this enzyme lead to a fatal neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis. Similar enzymes have been found in the genomic sequences of several species, but neither systematic analyses of their distribution nor modeling of their structures have been previously attempted. Results We have analyzed the presence of orthologs of human CLN2 in the genomic sequences of a number of eukaryotic species. Enzymes with sequences sharing over 80% identity have been found in the genomes of macaque, mouse, rat, dog, and cow. Closely related, although clearly distinct, enzymes are present in fish (fugu and zebra, as well as in frogs (Xenopus tropicalis. A three-dimensional model of human CLN2 was built based mainly on the homology with Pseudomonas sp. 101 sedolisin. Conclusion CLN2 is very highly conserved and widely distributed among higher organisms and may play an important role in their life cycles. The model presented here indicates a very open and accessible active site that is almost completely conserved among all known CLN2 enzymes. This result is somehow surprising for a tripeptidase where the presence of a more constrained binding pocket was anticipated. This structural model should be useful in the search for the physiological substrates of these enzymes and in the design of more specific inhibitors of CLN2.

  2. Calcium Regulates the Activity and Structural Stability of Tpr, a Bacterial Calpain-like Peptidase.

    Science.gov (United States)

    Staniec, Dominika; Ksiazek, Miroslaw; Thøgersen, Ida B; Enghild, Jan J; Sroka, Aneta; Bryzek, Danuta; Bogyo, Matthew; Abrahamson, Magnus; Potempa, Jan

    2015-11-01

    Porphyromonas gingivalis is a peptide-fermenting asaccharolytic periodontal pathogen. Its genome contains several genes encoding cysteine peptidases other than gingipains. One of these genes (PG1055) encodes a protein called Tpr (thiol protease) that has sequence similarity to cysteine peptidases of the papain and calpain families. In this study we biochemically characterize Tpr. We found that the 55-kDa Tpr inactive zymogen proteolytically processes itself into active forms of 48, 37, and 33 kDa via sequential truncations at the N terminus. These processed molecular forms of Tpr are associated with the bacterial outer membrane where they are likely responsible for the generation of metabolic peptides required for survival of the pathogen. Both autoprocessing and activity were dependent on calcium concentrations >1 mm, consistent with the protein's activity within the intestinal and inflammatory milieus. Calcium also stabilized the Tpr structure and rendered the protein fully resistant to proteolytic degradation by gingipains. Together, our findings suggest that Tpr is an example of a bacterial calpain, a calcium-responsive peptidase that may generate substrates required for the peptide-fermenting metabolism of P. gingivalis. Aside from nutrient generation, Tpr may also be involved in evasion of host immune response through degradation of the antimicrobial peptide LL-37 and complement proteins C3, C4, and C5. Taken together, these results indicate that Tpr likely represents an important pathogenesis factor for P. gingivalis.

  3. Heterologous production of the stain solving peptidase PPP1 from Pleurotus pulmonarius.

    Science.gov (United States)

    Leonhardt, Robin-Hagen; Krings, Ulrich; Berger, Ralf G; Linke, Diana

    2016-05-01

    A novel stain solving subtilisin-like peptidase (PPP1) was identified from the culture supernatant of the agaricomycete Pleurotus pulmonarius. It was purified to homogeneity using a sequence of preparative isoelectric focusing, anion exchange and size exclusion chromatography. Peptides were identified by ab initio sequencing (nLC-ESI-QTOF-MS/MS), characterizing the enzyme as a member of the subtilase family (EC 3.4.21.X). An expression system was established featuring the pPIC9K vector, an alternative Kozak sequence, the codon optimized gene ppp1 gene without the native signal sequence with C-terminal hexa-histidine tag, and Pichia pastoris GS115 as expression host. Intracellular active enzyme was obtained from cultivations in shake flasks and in a five liter bioreactor. With reaction optima of 40 °C and a pH > 8.5, considerable bleaching of pre-stained fabrics (blood, milk and India ink), and the possibility of larger-scale production, the heterologous enzyme is well suitable for detergent applications, especially at lower temperatures as part of a more energy- and cost-efficient washing process. Showing little sequence similarity to other subtilases, this unique peptidase is the first subtilisin-like peptidase from Basidiomycota, which has been functionally produced in Pichia pastoris.

  4. Digestive peptidase evolution in holometabolous insects led to a divergent group of enzymes in Lepidoptera.

    Science.gov (United States)

    Dias, Renata O; Via, Allegra; Brandão, Marcelo M; Tramontano, Anna; Silva-Filho, Marcio C

    2015-03-01

    Trypsins and chymotrypsins are well-studied serine peptidases that cleave peptide bonds at the carboxyl side of basic and hydrophobic L-amino acids, respectively. These enzymes are largely responsible for the digestion of proteins. Three primary processes regulate the activity of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. Here, we present a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects and reveal divergent characteristics of Lepidoptera enzymes in comparison with those of Coleoptera and Diptera. In particular, trypsin subsite S1 was more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2-S4 were more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a lineage-specific trypsin group belonging only to the Noctuidae family. Evidence for facilitated trypsin auto-activation events were also observed in all the insect orders studied, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to their mammal counterparts. Overall, our findings suggest that the need for fast digestion allowed holometabolous insects to evolve divergent groups of peptidases with high auto-activation rates, and highlight that the evolution of trypsins led to a most diverse group of enzymes in Lepidoptera.

  5. Intracerebroventricular administration of N-acetylaspartylglutamate (NAAG peptidase inhibitors is analgesic in inflammatory pain

    Directory of Open Access Journals (Sweden)

    Kozikowski Alan

    2008-08-01

    Full Text Available Abstract Background The peptide neurotransmitter N-Acetylaspartylglutamate (NAAG is the third most prevalent transmitter in the mammalian central nervous system. Local, intrathecal and systemic administration of inhibitors of enzymes that inactivate NAAG decrease responses to inflammatory pain in rat models. Consistent with NAAG's activation of group II metabotropic glutamate receptors, this analgesia is blocked by a group II antagonist. Results This research aimed at determining if analgesia obtained following systemic administration of NAAG peptidase inhibitors is due to NAAG activation of group II mGluRs in brain circuits that mediate perception of inflammatory pain. NAAG and NAAG peptidase inhibitors, ZJ43 and 2-PMPA, were microinjected into a lateral ventricle prior to injection of formalin in the rat footpad. Each treatment reduced the early and late phases of the formalin-induced inflammatory pain response in a dose-dependent manner. The group II mGluR antagonist reversed these analgesic effects consistent with the conclusion that analgesia was mediated by increasing NAAG levels and the peptide's activation of group II receptors. Conclusion These data contribute to proof of the concept that NAAG peptidase inhibition is a novel therapeutic approach to inflammatory pain and that these inhibitors achieve analgesia by elevating synaptic levels of NAAG within pain processing circuits in brain.

  6. Intracerebroventricular administration of N-acetylaspartylglutamate (NAAG) peptidase inhibitors is analgesic in inflammatory pain

    Science.gov (United States)

    Yamamoto, Tatsuo; Kozikowski, Alan; Zhou, Jia; Neale, Joseph H

    2008-01-01

    Background The peptide neurotransmitter N-Acetylaspartylglutamate (NAAG) is the third most prevalent transmitter in the mammalian central nervous system. Local, intrathecal and systemic administration of inhibitors of enzymes that inactivate NAAG decrease responses to inflammatory pain in rat models. Consistent with NAAG's activation of group II metabotropic glutamate receptors, this analgesia is blocked by a group II antagonist. Results This research aimed at determining if analgesia obtained following systemic administration of NAAG peptidase inhibitors is due to NAAG activation of group II mGluRs in brain circuits that mediate perception of inflammatory pain. NAAG and NAAG peptidase inhibitors, ZJ43 and 2-PMPA, were microinjected into a lateral ventricle prior to injection of formalin in the rat footpad. Each treatment reduced the early and late phases of the formalin-induced inflammatory pain response in a dose-dependent manner. The group II mGluR antagonist reversed these analgesic effects consistent with the conclusion that analgesia was mediated by increasing NAAG levels and the peptide's activation of group II receptors. Conclusion These data contribute to proof of the concept that NAAG peptidase inhibition is a novel therapeutic approach to inflammatory pain and that these inhibitors achieve analgesia by elevating synaptic levels of NAAG within pain processing circuits in brain. PMID:18673570

  7. Interactions of "bora-penicilloates" with serine β-lactamases and DD-peptidases.

    Science.gov (United States)

    Dzhekieva, Liudmila; Adediran, S A; Pratt, R F

    2014-10-21

    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.

  8. Characterization of the peptidase activity of recombinant porcine pregnancy-associated glycoprotein-2.

    Science.gov (United States)

    Telugu, Bhanu Prakash V L; Green, Jonathan A

    2008-12-01

    The pregnancy-associated glycoproteins (PAGs) belong to the aspartic peptidase family. They are expressed exclusively in trophoblasts of even-toed ungulates such as swine, cattle, sheep, etc. In pigs, two distinct PAG transcripts (and some variants) have been described. One of the transcripts, porcine PAG-1 (poPAG-1) may not be capable of acting as a peptidase. The second transcript, poPAG-2, possesses a conserved catalytic centre and has been predicted, but not shown, to have proteolytic activity. The thrust of this work was to test such a possibility. PoPAG-2 was expressed as a recombinant protein with an amino-terminal 'FLAG-tag' in a Baculoviral expression system. The expressed proteins were affinity purified by using an anti-FLAG antibody. The purified preparations were then analysed for proteolytic activity against a fluorescent substrate. Porcine PAG-2 had optimal proteolytic activity around pH 3.5. Against this substrate, it had a k(cat)/K(m) of 1.2 microM(-1) s(-1) and was inhibited by the aspartic peptidase inhibitor, pepstatin A, with a K(i) of 12.5 nM. Since the proteolytic activity of PAGs in the pig has now been established, the search for putative substrates to gain insight into the physiological role of PAGs will likely be the focus of future investigations.

  9. Cloning, expression, and chromosomal assignment of the human mitochondrial intermediate peptidase gene (MIPEP).

    Science.gov (United States)

    Chew, A; Buck, E A; Peretz, S; Sirugo, G; Rinaldo, P; Isaya, G

    1997-03-15

    The mitochondrial intermediate peptidase of Saccharomyces cerevisiae (YMIP) is a component of the yeast mitochondrial protein import machinery critically involved in the biogenesis of the oxidative phosphorylation (OXPHOS) system. This leader peptidase removes specific octapeptides from the amino terminus of nuclear-encoded OXPHOS subunits and components of the mitochondrial genetic apparatus. To address the biologic role of the human peptidase [MIPEP gene, HMIP polypeptide], we have initiated its molecular and functional characterization. A full-length cDNA was isolated by screening a human liver library using a rat MIP (RMIP) cDNA as a probe. The encoded protein contained a typical mitochondrial leader peptide and showed 92 and 54% homology to RMIP and YMIP, respectively. A survey of human mitochondrial protein precursors revealed that, similar to YMIP, HMIP is primarily involved in the maturation of OXPHOS-related proteins. Northern analysis showed that the MIPEP gene is differentially expressed in human tissues, with the highest levels of expression in the heart, skeletal muscle, and pancreas, three organ systems that are frequently affected in OXPHOS disorders. Using fluorescence in situ hybridization, the MIPEP locus was assigned to 13q12. This information offers the possibility of testing the potential involvement of HMIP in the pathophysiology of nuclear-driven OXPHOS disorders.

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

    Science.gov (United States)

    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.

  11. Digestive peptidase evolution in holometabolous insects led to a divergent group of enzymes in Lepidoptera

    KAUST Repository

    Dias, Renata O.

    2015-03-01

    © 2015 Elsevier Ltd. Trypsins and chymotrypsins are well-studied serine peptidases that cleave peptide bonds at the carboxyl side of basic and hydrophobic l-amino acids, respectively. These enzymes are largely responsible for the digestion of proteins. Three primary processes regulate the activity of these peptidases: secretion, precursor (zymogen) activation and substrate-binding site recognition. Here, we present a detailed phylogenetic analysis of trypsins and chymotrypsins in three orders of holometabolous insects and reveal divergent characteristics of Lepidoptera enzymes in comparison with those of Coleoptera and Diptera. In particular, trypsin subsite S1 was more hydrophilic in Lepidoptera than in Coleoptera and Diptera, whereas subsites S2-S4 were more hydrophobic, suggesting different substrate preferences. Furthermore, Lepidoptera displayed a lineage-specific trypsin group belonging only to the Noctuidae family. Evidence for facilitated trypsin auto-activation events were also observed in all the insect orders studied, with the characteristic zymogen activation motif complementary to the trypsin active site. In contrast, insect chymotrypsins did not seem to have a peculiar evolutionary history with respect to their mammal counterparts. Overall, our findings suggest that the need for fast digestion allowed holometabolous insects to evolve divergent groups of peptidases with high auto-activation rates, and highlight that the evolution of trypsins led to a most diverse group of enzymes in Lepidoptera.

  12. HIV aspartyl peptidase inhibitors interfere with cellular proliferation, ultrastructure and macrophage infection of Leishmania amazonensis.

    Directory of Open Access Journals (Sweden)

    Lívia O Santos

    Full Text Available BACKGROUND: Leishmania is the etiologic agent of leishmanisais, a protozoan disease whose pathogenic events are not well understood. Current therapy is suboptimal due to toxicity of the available therapeutic agents and the emergence of drug resistance. Compounding these problems is the increase in the number of cases of Leishmania-HIV coinfection, due to the overlap between the AIDS epidemic and leishmaniasis. METHODOLOGY/PRINCIPAL FINDINGS: In the present report, we have investigated the effect of HIV aspartyl peptidase inhibitors (PIs on the Leishmania amazonensis proliferation, ultrastructure, interaction with macrophage cells and expression of classical peptidases which are directly involved in the Leishmania pathogenesis. All the HIV PIs impaired parasite growth in a dose-dependent fashion, especially nelfinavir and lopinavir. HIV PIs treatment caused profound changes in the leishmania ultrastructure as shown by transmission electron microscopy, including cytoplasm shrinking, increase in the number of lipid inclusions and some cells presenting the nucleus closely wrapped by endoplasmic reticulum resembling an autophagic process, as well as chromatin condensation which is suggestive of apoptotic death. The hydrolysis of HIV peptidase substrate by L. amazonensis extract was inhibited by pepstatin and HIV PIs, suggesting that an aspartyl peptidase may be the intracellular target of the inhibitors. The treatment with HIV PIs of either the promastigote forms preceding the interaction with macrophage cells or the amastigote forms inside macrophages drastically reduced the association indexes. Despite all these beneficial effects, the HIV PIs induced an increase in the expression of cysteine peptidase b (cpb and the metallopeptidase gp63, two well-known virulence factors expressed by Leishmania spp. CONCLUSIONS/SIGNIFICANCE: In the face of leishmaniasis/HIV overlap, it is critical to further comprehend the sophisticated interplays among Leishmania

  13. Unleashing the therapeutic potential of human kallikrein-related serine proteases.

    Science.gov (United States)

    Prassas, Ioannis; Eissa, Azza; Poda, Gennadiy; Diamandis, Eleftherios P

    2015-03-01

    Tissue kallikreins are a family of fifteen secreted serine proteases encoded by the largest protease gene cluster in the human genome. In the past decade, substantial progress has been made in characterizing the natural substrates, endogenous inhibitors and in vivo functions of kallikreins, and studies have delineated important pathophysiological roles for these proteases in a variety of tissues. Thus, kallikreins are now considered attractive targets for the development of novel therapeutics for airway, cardiovascular, tooth, brain, skin and neoplastic diseases. In this Review, we discuss recent advances in our understanding of the physiological functions and pathological implications of kallikrein proteases, and highlight progress in the identification of kallikrein inhibitors, which together are bringing us closer to therapeutically targeting kallikreins in selected disease settings.

  14. Cysteine peptidase and its inhibitor activity levels and vitamin E concentration in normal human serum and colorectal carcinomas

    Institute of Scientific and Technical Information of China (English)

    Robert Szwed; Zygmunt Grzebieniak; Yousif Saleh; Godwin Bwire Ekonjo; Maciej Siewinski

    2005-01-01

    AIM: Cysteine peptidase (CP) and its inhibitor (CPI) are a matrix protease that may be associated with colorectal carcinoma invasion and progression, and vitamin E is also a stimulator of the immunological system. Our purpose was to determine the correlation between the expression of cysteine peptidases and their endogenous inhibitors,and the level of vitamin E in sera of patients with colorectal cancer in comparison with healthy individuals.METHODS: The levels of cysteine peptidases and their inhibitors were determined in the sera of patients with primary and metastatic colorectal carcinoma and healthy individuals using fluorogenic substrate, and the level of vitamin E was determined by HPLC.RESULTS: The levels of cysteine peptidases and their inhibitors were significantly higher in the metastatic colorectal cancer patients than that in the healthy controls (P<0.05).The activity of CP increased 2.2-fold, CPI 2.8-fold and vitamin E decreased 3.4-fold in sera of patients with metastasis in comparison with controls. The level of vitamin E in healthy individuals was higher, whereas the activity of cysteine peptidases and their inhibitors associated with complexes was lower than that in patients with cancer of the digestive tract.CONCLUSION: These results suggest that the serum levels of CP and their inhibitors could be an indicator of the prognosis for patients with metastatic colorectal cancer. Vitamin E can be administered prophylactically to prevent digestive tract neoplasmas.

  15. Characterization of a Bacteriophage-Derived Murein Peptidase for Elimination of Antibiotic-Resistant Staphylococcus aureus.

    Science.gov (United States)

    Keary, Ruth; Sanz-Gaitero, Marta; van Raaij, Mark J; O'Mahony, Jim; Fenton, Mark; McAuliffe, Olivia; Hill, Colin; Ross, R Paul; Coffey, Aidan

    2016-01-01

    Staphylococcus aureus is a major cause of infection in humans and animals, causing a wide variety of diseases, from local inflammations to fatal sepsis. The bacterium is commonly multi-drug resistant and thus many front-line antibiotics have been rendered ineffective for treating such infections. Research on murein/peptidoglycan hydrolases, derived from bacterial viruses (bacteriophages), has demonstrated that such proteins are attractive candidates for development as novel antibacterial agents for combatting Gram-positive pathogens. Here we review the research produced to-date on the bacteriophage-derived CHAPK murein peptidase. Initially, we sequenced and annotated the genome of anti-staphylococcal bacteriophage K and cloned the gene for the bacteriophage endolysin, a murein hydrolase which plays a role in cell killing during the bacteriophage life cycle. An highly active domain of the enzyme, a cysteine, histidine-dependent amido hydrolase/peptidase (CHAPK), was cloned, overexpressed in E. coli and purified. This CHAPK enzyme was demonstrated to rapidly lyse several strains of methicillin resistant S. aureus and both disrupted and prevented the formation of a staphylococcal biofilm. The staphylolytic activity of the peptidase was demonstrated in vivo using a mouse model, without adverse effects on the animals. The crystal structure of the enzyme was elucidated, revealing a calcium ion close to the active site. Site-directed mutagenesis indicated that this calcium ion is involved in the catalytic mechanism of the enzyme. The crystal structure of this enzyme is a valuable source of information for efficient engineering of this and similar CHAP-domain-containing proteins. Overall, the data collected to date on CHAPK has demonstrated its strong potential as a novel therapeutic candidate for treatment of staphylococcal infections and has provided us with insight into the fundamental enzymatic mechanisms of CHAP domain-containing peptidoglycan hydrolases.

  16. Peptidases digestivas do peixe bijupirá (Rachycentron canadum) selvagens e cultivados

    OpenAIRE

    Mendes de Santana, Werlayne

    2011-01-01

    Peptidases dos cecos pilóricos de bijupirás selvagens e cultivados (Rachycentron canadum) foram caracterizados utilizando substratos específicos de tripsina e quimotripsina, inibidores e íons metálicos. Além disso, o perfil protéico foi determinado empregando SDS-PAGE e zimogramas. Para tanto pH ótimo e temperatura dos bijupirás selvagens e cultivados foi 7,0-10,0 e 40-60 ° C para a tripsina, como 7,0-9,5 e 40-55 ° C para quimotripsina. A tripsina e quimotripsina foram estáveis ...

  17. Exendin-4, but not dipeptidyl peptidase IV inhibition, increases small intestinal mass in GK rats

    DEFF Research Database (Denmark)

    Simonsen, Lotte; Pilgaard, Sofie; Orskov, Cathrine

    2007-01-01

    Long-term treatment with dipeptidyl peptidase IV inhibitors (DPPIV-I) or glucagon-like peptide (GLP)-1 analogs may potentially affect intestinal growth by down- or upregulating the intestinotrophic hormone GLP-2. This study compared the intestinotrophic effects of 12-wk administration of vehicle,...... GLP-2 receptor. DPPIV-I had no intestinal effects. In conclusion, the continued presence of Ex-4 is necessary to maintain weight loss in GK rats. Effective antihyperglycemic treatment with Ex-4 increases intestinal mass reversibly, whereas DPPIV-I lacks intestinal effects....

  18. Signaling domain of Sonic Hedgehog as cannibalistic calcium-regulated zinc-peptidase.

    Directory of Open Access Journals (Sweden)

    Rocio Rebollido-Rios

    2014-07-01

    Full Text Available Sonic Hedgehog (Shh is a representative of the evolutionary closely related class of Hedgehog proteins that have essential signaling functions in animal development. The N-terminal domain (ShhN is also assigned to the group of LAS proteins (LAS = Lysostaphin type enzymes, D-Ala-D-Ala metalloproteases, Sonic Hedgehog, of which all members harbor a structurally well-defined Zn2+ center; however, it is remarkable that ShhN so far is the only LAS member without proven peptidase activity. Another unique feature of ShhN in the LAS group is a double-Ca2+ center close to the zinc. We have studied the effect of these calcium ions on ShhN structure, dynamics, and interactions. We find that the presence of calcium has a marked impact on ShhN properties, with the two calcium ions having different effects. The more strongly bound calcium ion significantly stabilizes the overall structure. Surprisingly, the binding of the second calcium ion switches the putative catalytic center from a state similar to LAS enzymes to a state that probably is catalytically inactive. We describe in detail the mechanics of the switch, including the effect on substrate co-ordinating residues and on the putative catalytic water molecule. The properties of the putative substrate binding site suggest that ShhN could degrade other ShhN molecules, e.g. by cleavage at highly conserved glycines in ShhN. To test experimentally the stability of ShhN against autodegradation, we compare two ShhN mutants in vitro: (1 a ShhN mutant unable to bind calcium but with putative catalytic center intact, and thus, according to our hypothesis, a constitutively active peptidase, and (2 a mutant carrying additionally mutation E177A, i.e., with the putative catalytically active residue knocked out. The in vitro results are consistent with ShhN being a cannibalistic zinc-peptidase. These experiments also reveal that the peptidase activity depends on pH.

  19. Signaling domain of Sonic Hedgehog as cannibalistic calcium-regulated zinc-peptidase.

    Science.gov (United States)

    Rebollido-Rios, Rocio; Bandari, Shyam; Wilms, Christoph; Jakuschev, Stanislav; Vortkamp, Andrea; Grobe, Kay; Hoffmann, Daniel

    2014-07-01

    Sonic Hedgehog (Shh) is a representative of the evolutionary closely related class of Hedgehog proteins that have essential signaling functions in animal development. The N-terminal domain (ShhN) is also assigned to the group of LAS proteins (LAS = Lysostaphin type enzymes, D-Ala-D-Ala metalloproteases, Sonic Hedgehog), of which all members harbor a structurally well-defined Zn2+ center; however, it is remarkable that ShhN so far is the only LAS member without proven peptidase activity. Another unique feature of ShhN in the LAS group is a double-Ca2+ center close to the zinc. We have studied the effect of these calcium ions on ShhN structure, dynamics, and interactions. We find that the presence of calcium has a marked impact on ShhN properties, with the two calcium ions having different effects. The more strongly bound calcium ion significantly stabilizes the overall structure. Surprisingly, the binding of the second calcium ion switches the putative catalytic center from a state similar to LAS enzymes to a state that probably is catalytically inactive. We describe in detail the mechanics of the switch, including the effect on substrate co-ordinating residues and on the putative catalytic water molecule. The properties of the putative substrate binding site suggest that ShhN could degrade other ShhN molecules, e.g. by cleavage at highly conserved glycines in ShhN. To test experimentally the stability of ShhN against autodegradation, we compare two ShhN mutants in vitro: (1) a ShhN mutant unable to bind calcium but with putative catalytic center intact, and thus, according to our hypothesis, a constitutively active peptidase, and (2) a mutant carrying additionally mutation E177A, i.e., with the putative catalytically active residue knocked out. The in vitro results are consistent with ShhN being a cannibalistic zinc-peptidase. These experiments also reveal that the peptidase activity depends on pH.

  20. Cloning and nucleotide sequence of the Enterobacter aerogenes signal peptidase II (lsp) gene.

    OpenAIRE

    Isaki, L; Kawakami, M; Beers, R; Hom, R; Wu, H.C.

    1990-01-01

    In Escherichia coli, prolipoprotein signal peptidase is encoded by the lsp gene, which is organized into an operon consisting of ileS, lsp, and three open reading frames, designated genes x, orf-149, and orf-316. The Enterobacter aerogenes lsp gene was cloned and expressed in E. coli. The nucleotide sequence of the Enterobacter aerogenes lsp gene and a part of its flanking sequences were determined. A high degree of homology was found between the E. coli ileS-lsp operon and the corresponding ...

  1. Synthesis and structure-activity relationships of potent 4-fluoro-2-cyanopyrrolidine dipeptidyl peptidase IV inhibitors.

    Science.gov (United States)

    Fukushima, Hiroshi; Hiratate, Akira; Takahashi, Masato; Mikami, Ayako; Saito-Hori, Masako; Munetomo, Eiji; Kitano, Kiyokazu; Chonan, Sumi; Saito, Hidetaka; Suzuki, Akio; Takaoka, Yuji; Yamamoto, Koji

    2008-04-01

    Dipeptidyl peptidase IV (DPP-IV) inhibitors are promising antidiabetic drugs, and several drugs are in the developmental stage. We previously reported that the introduction of fluorine to the 4-position of 2-cyanopyrrolidine enhanced the DPP-IV inhibitory effect. In the present report, we examined the structure-activity relationship (SAR) of 2-cyano-4-fluoropyrrolidine with N-substituted glycine at the 1-position. We report the identification of a potent and stable DPP-IV inhibitor (TS-021) with a long-term persistent plasma drug concentration and a potent antihyperglycemic activity.

  2. Inhibition by penem of processing peptidases from cyanobacteria and chloroplast thylakoids.

    Science.gov (United States)

    Barbrook, A C; Packer, J C; Howe, C J

    1996-12-02

    Proteins targeted to the thylakoid lumen of plants and cyanobacteria and the periplasmic space of cyanobacteria are synthesised with N-terminal presequences which are removed following translocation across the membrane. These presequences are thought to direct translocation of the preprotein by a sec-type pathway. Detergent extracts of cyanobacterial and chloroplast membranes contain enzymes which are capable of processing precursors to the mature size. We show that the processing of a range of precursors by both cyanobacterial and chloroplast enzymes is inhibited by the penem SB216357. This is the first report of an inhibitor of these enzymes and indicates that they are type 1 signal peptidases.

  3. Substrate specificity of low-molecular mass bacterial DD-peptidases.

    Science.gov (United States)

    Nemmara, Venkatesh V; Dzhekieva, Liudmila; Sarkar, Kumar Subarno; Adediran, S A; Duez, Colette; Nicholas, Robert A; Pratt, R F

    2011-11-22

    The bacterial DD-peptidases or penicillin-binding proteins (PBPs) catalyze the formation and regulation of cross-links in peptidoglycan biosynthesis. They are classified into two groups, the high-molecular mass (HMM) and low-molecular mass (LMM) enzymes. The latter group, which is subdivided into classes A-C (LMMA, -B, and -C, respectively), is believed to catalyze DD-carboxypeptidase and endopeptidase reactions in vivo. To date, the specificity of their reactions with particular elements of peptidoglycan structure has not, in general, been defined. This paper describes the steady-state kinetics of hydrolysis of a series of specific peptidoglycan-mimetic peptides, representing various elements of stem peptide structure, catalyzed by a range of LMM PBPs (the LMMA enzymes, Escherichia coli PBP5, Neisseria gonorrhoeae PBP4, and Streptococcus pneumoniae PBP3, and the LMMC enzymes, the Actinomadura R39 dd-peptidase, Bacillus subtilis PBP4a, and N. gonorrhoeae PBP3). The R39 enzyme (LMMC), like the previously studied Streptomyces R61 DD-peptidase (LMMB), specifically and rapidly hydrolyzes stem peptide fragments with a free N-terminus. In accord with this result, the crystal structures of the R61 and R39 enzymes display a binding site specific to the stem peptide N-terminus. These are water-soluble enzymes, however, with no known specific function in vivo. On the other hand, soluble versions of the remaining enzymes of those noted above, all of which are likely to be membrane-bound and/or associated in vivo and have been assigned particular roles in cell wall biosynthesis and maintenance, show little or no specificity for peptides containing elements of peptidoglycan structure. Peptidoglycan-mimetic boronate transition-state analogues do inhibit these enzymes but display notable specificity only for the LMMC enzymes, where, unlike peptide substrates, they may be able to effectively induce a specific active site structure. The manner in which LMMA (and HMM) DD-peptidases

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

    Science.gov (United States)

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

    2015-01-01

    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-[Hyp(3),Igl(5),Oic(7),Igl(8)]-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

  5. Dipeptidyl peptidase IV is sorted to the secretory granules in pancreatic islet A-cells

    DEFF Research Database (Denmark)

    Poulsen, Mona Dam; Hansen, Gert Helge; Dabelsteen, Erik

    1993-01-01

    Dipeptidyl peptidase IV (DP IV:EC 3.4.14.5) was localized in endocrine cells of pig pancreas by immunohistochemical and enzyme histochemical methods. Immunolight microscopy with both monoclonal and polyclonal antibodies demonstrated DP IV immunoreactivity in cells located in the peripheral part...... labeling using a monoclonal glucagon antibody as the second primary antibody. These results show that DP IV is sorted to secretory granules in the pig pancreatic islet A-cells. Furthermore, this secretory granule enzyme, as opposed to intestinal brush border DP IV, is suggested to be a soluble protein...

  6. Alogliptin, a potent and selective dipeptidyl peptidase-IV inhibitor for the treatment of type 2 diabetes

    DEFF Research Database (Denmark)

    Deacon, Carolyn F

    2008-01-01

    Takeda San Diego Inc is developing alogliptin, a small-molecule, orally available dipeptidyl peptidase IV (DPP IV) inhibitor, for the potential treatment of type 2 diabetes. In January 2008, Takeda announced that an NDA for alogliptin had been submitted to the FDA.......Takeda San Diego Inc is developing alogliptin, a small-molecule, orally available dipeptidyl peptidase IV (DPP IV) inhibitor, for the potential treatment of type 2 diabetes. In January 2008, Takeda announced that an NDA for alogliptin had been submitted to the FDA....

  7. Induction of protective immune responses against schistosomiasis using functionally active cysteine peptidases

    Science.gov (United States)

    El Ridi, Rashika; Tallima, Hatem; Dalton, John P.; Donnelly, Sheila

    2014-01-01

    Each year schistosomiasis afflicts up to 600 million people in 74 tropical and sub-tropical countries, predominantly in the developing world. Yet we depend on a single drug, praziquantel, for its treatment and control. There is no vaccine available but one is urgently needed especially since praziquantel-resistant parasites are likely to emerge at some time in the future. The disease is caused by several worm species of the genus Schistosoma. These express several classes of papain-like cysteine peptidases, cathepsins B and L, in various tissues but particularly in their gastrodermis where they employ them as digestive enzymes. We have shown that sub-cutaneous injection of recombinant and functionally active Schistosoma mansoni cathepsin B1 (SmCB1), or a cathepsin L from a related parasite Fasciola hepatica (FhCL1), elicits highly significant protection (up to 73%) against an experimental challenge worm infection in murine models of schistosomiasis. The immune modulating properties of this subcutaneous injection can boost protection levels (up to 83%) when combined with other S. mansoni vaccine candidates, glyceraldehyde 3-phosphate dehydrogenase (SG3PDH) and peroxiredoxin (PRX-MAP). Here, we discuss these data in the context of the parasite’s biology and development, and provide putative mechanism by which the native-like cysteine peptidase induce protective immune responses. PMID:24847355

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

    Directory of Open Access Journals (Sweden)

    Thiago A. Salles

    2015-02-01

    Full Text Available 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.

  9. Broadening the Spectrum of β-Lactam Antibiotics through Inhibition of Signal Peptidase Type I

    Science.gov (United States)

    Therien, Alex G.; Huber, Joann L.; Wilson, Kenneth E.; Beaulieu, Patrick; Caron, Alexandre; Claveau, David; Deschamps, Kathleen; Donald, Robert G. K.; Galgoci, Andrew M.; Gallant, Michel; Gu, Xin; Kevin, Nancy J.; Lafleur, Josiane; Leavitt, Penny S.; Lebeau-Jacob, Christian; Lee, Suzy S.; Lin, Molly M.; Michels, Anna A.; Ogawa, Aimie M.; Painter, Ronald E.; Parish, Craig A.; Park, Young-Whan; Benton-Perdomo, Liliana; Petcu, Mihai; Phillips, John W.; Powles, Mary Ann; Skorey, Kathryn I.; Tam, John; Tan, Christopher M.; Young, Katherine; Wong, Simon; Waddell, Sherman T.

    2012-01-01

    The resistance of methicillin-resistant Staphylococcus aureus (MRSA) to all β-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of β-lactams against MRSA, an approach that has led to the discovery of two classes of natural product antibiotics, a cyclic depsipeptide (krisynomycin) and a lipoglycopeptide (actinocarbasin), which potentiate the activity of imipenem against MRSA strain COL. We report here that these imipenem synergists are inhibitors of the bacterial type I signal peptidase SpsB, a serine protease that is required for the secretion of proteins that are exported through the Sec and Tat systems. A synthetic derivative of actinocarbasin, M131, synergized with imipenem both in vitro and in vivo with potent efficacy. The in vitro activity of M131 extends to clinical isolates of MRSA but not to a methicillin-sensitive strain. Synergy is restricted to β-lactam antibiotics and is not observed with other antibiotic classes. We propose that the SpsB inhibitors synergize with β-lactams by preventing the signal peptidase-mediated secretion of proteins required for β-lactam resistance. Combinations of SpsB inhibitors and β-lactams may expand the utility of these widely prescribed antibiotics to treat MRSA infections, analogous to β-lactamase inhibitors which restored the utility of this antibiotic class for the treatment of resistant Gram-negative infections. PMID:22710113

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

    Directory of Open Access Journals (Sweden)

    Igor A. Rodrigues

    2014-01-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    Energy Technology Data Exchange (ETDEWEB)

    Kozuka, Miyuki [Department of Health and Nutrition, Faculty of Human Science, Hokkaido Bunkyo University, Eniwa 061-1449 (Japan); Yamane, Takuya, E-mail: t-yamane@pharm.hokudai.ac.jp [Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812 (Japan); Nakano, Yoshihisa [Center for Research and Development Bioresources, Research Organization for University-Community Collaborations, Osaka Prefecture University, Sakai, Osaka 599-8570 (Japan); Nakagaki, Takenori [Institute of Food Sciences, Nakagaki Consulting Engineer Co., Ltd, Nishi-ku, Sakai 593-8328 (Japan); Ohkubo, Iwao [Department of Nutrition, School of Nursing and Nutrition, Tenshi College, Higashi-ku, Sapporo 065-0013 (Japan); Ariga, Hiroyoshi [Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo 060-0812 (Japan)

    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.

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

    Science.gov (United States)

    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

  14. Induction of protective immune responses against schistosomiasis using functionally active cysteine peptidases

    Directory of Open Access Journals (Sweden)

    Rashika eEl Ridi

    2014-05-01

    Full Text Available Each year schistosomiasis afflicts up to 600 million people in 74 tropical and sub-tropical countries, predominantly in the developing world. Yet we depend on a single drug, praziquantel, for its treatment and control. There is no vaccine available but one is urgently needed especially since praziquantel-resistant parasites are likely to emerge at some time in the future. The disease is caused by several worm species of the genus Schistosoma. These express several classes of papain-like cysteine peptidases, cathepsins B and L, in various tissues but particularly in their gastrodermis where they employ them as digestive enzymes. We have shown that sub-cutaneous injection of recombinant and functionally active S. mansoni cathepsin B1 (SmCB1, or a cathepsin L from a related parasite Fasciola hepatica (FhCL1, elicits highly significant protection (up to 73% against an experimental challenge worm infection in murine models of schistosomiasis. The immune modulating properties of this subcutaneous injection can boost protection levels (up to 83% when combined with other S. mansoni vaccine candidates, glyceraldehyde 3-phosphate dehydrogenase (SG3PDH and peroxiredoxin (PRX-MAP. This cysteine-treatment can also protect mice from S. haematobium infection. Here, we discuss these data in the context of the parasite’s biology and development, and provide putative mechanism by which the native-like cysteine peptidase induce protective immune responses.

  15. The dipeptidyl peptidase-4 inhibitor vildagliptin improves beta-cell function and insulin sensitivity in subjects with impaired fasting glucose

    DEFF Research Database (Denmark)

    Utzschneider, Kristina M; Tong, Jenny; Montgomery, Brenda;

    2007-01-01

    OBJECTIVE: To evaluate the effect of treatment with the dipeptidyl peptidase (DPP)-4 inhibitor vildagliptin on insulin sensitivity and beta-cell function in subjects with impaired fasting glucose (IFG). RESEARCH DESIGN AND METHODS: A total of 22 subjects with IFG (11 female and 11 male, mean +/- SD...

  16. Gene cloning and biochemical characterization of eryngase, a serine aminopeptidase of Pleurotus eryngii belonging to the family S9 peptidases.

    Science.gov (United States)

    Arima, Jiro; Tokai, Shota; Chiba, Masanori; Ichiyanagi, Tsuyoshi; Yabuta, Yukinori; Mori, Nobuhiro; Aimi, Tadanori

    2014-01-01

    Pleurotus eryngii serine aminopeptidase that has peptide bond formation activity, redesignated as eryngase, was cloned and expressed. Eryngase has a family S9 peptidase unit in the C-terminal region having a catalytic triad of Ser, Asp, and His. In the phylogenetic relations among the subfamilies of family S9 peptidase (S9A, prolyl oligopeptidase; S9B, dipeptidyl peptidase; S9C, acylaminoacyl peptidase; S9D, glutamyl endopeptidase), eryngase existed alone in the neighbor of S9C subfamily. Mutation of the active site Ser524 of the eryngase with Ala eliminated its catalytic activity. In contrast, S524C mutant maintained low catalytic activity. Investigation of aminolysis activity using l-Phe-NH2 as a substrate showed that S524C mutant exhibited no hydrolysis reaction but synthesized a small amount of l-Phe-l-Phe-NH2 by the catalysis of aminolysis. In contrast, wild-type eryngase hydrolyzed the product of aminolysis l-Phe-l-Phe-NH2. Results show that the S524C mutant preferentially catalyzed aminolysis when on an l-Phe-NH2 substrate.

  17. The crystal structure of human dipeptidyl peptidase I (cathepsin C) in complex with the inhibitor Gly-Phe-CHN2

    DEFF Research Database (Denmark)

    Mølgaard, Anne; Arnau, Jose; Lauritzen, C.

    2007-01-01

    hDDPI (human dipeptidyl peptidase I) is a lysosomal cysteine protease involved in zymogen activation of granule-associated proteases, including granzymes A and B from cytotoxic T-lymphocytes and natural killer cells, cathepsin G and neutrophil elastase, and mast cell tryptase and chymase...

  18. Preservation of active incretin hormones by inhibition of dipeptidyl peptidase IV suppresses meal-induced incretin secretion in dogs

    DEFF Research Database (Denmark)

    Deacon, C F; Wamberg, S; Bie, P

    2002-01-01

    The incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are degraded by dipeptidyl peptidase IV (DPP IV), thereby losing insulinotropic activity. DPP IV inhibition reduces exogenous GLP-1 degradation, but the extent of endogenous incretin prote...

  19. Expression patterns of cysteine peptidase genes across the Tribolium castaneum life cycle provide clues to biological function

    Science.gov (United States)

    The red flour beetle, Tribolium castaneum, is a major agricultural pest responsible for considerable loss of stored grain and cereal products worldwide. T. castaneum larvae have a highly compartmentalized gut, with cysteine peptidases mostly in the acidic anterior part of the midgut. We have descri...

  20. Measurements of islet function and glucose metabolism with the dipeptidyl peptidase 4 inhibitor vildagliptin in patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Azuma, Koichiro; Rádiková, Zofia; Mancino, Juliet;

    2007-01-01

    OBJECTIVE: Pharmacological inhibition with the dipeptidyl peptidase 4 (DPP-4) inhibitor vildagliptin prolongs the action of endogenously secreted incretin hormones leading to improved glycemic control in patients with type 2 diabetes mellitus (T2DM). We undertook a double-blinded, randomized-orde...

  1. Role of peptidases of the intestinal microflora and prey in temperature adaptations of the digestive system in planktivorous and benthivorous fish.

    Science.gov (United States)

    Kuz'mina, V V; Skvortsova, E G; Shalygin, M V; Kovalenko, K E

    2015-12-01

    Many fish enzymatic systems possess limited adaptations to low temperature; however, little data are available to judge whether enzymes of fish prey and intestinal microbiota can mitigate this deficiency. In this study, the activity of serine peptidases (casein-lytic, mainly trypsin and hemoglobin-lytic, mainly chymotrypsin) of intestinal mucosa, chyme and intestinal microflora in four species of planktivorous (blue bream) and benthivorous (roach, crucian carp, perch) was investigated across a wide temperature range (0-70 °C) to identify adaptations to low temperature. At 0 °C, the relative activity of peptidases of intestinal mucosa (microflora (5-12.6%) is considerably less than that of chyme peptidases (up to 40% of maximal activity). The level of peptidase relative activity in crucian carp intestinal microflora was 45% of maximal activity. The shape of t°-function curves of chyme peptidase also differs in fish from different biotopes. Fish from the littoral group are characterized by a higher degree of adaptation of chyme casein-lytic peptidases to functioning at low temperatures as compared to fish from the pelagic group. The role of intestinal microbiota and prey peptidases in digestive system adaptations of planktivorous and benthivorous fish to low temperatures is discussed.

  2. THE EFFECT OF STANUM (IV AND GERMANІUM (IV COORDINATION COMPOUNDS ON Bacillus thuringiensis var. israelensis ІМV В-7465 PEPTIDASES ACTIVITY

    Directory of Open Access Journals (Sweden)

    Nidialkova N. А.

    2015-08-01

    Full Text Available The purpose of the research was to investigate the influence of stanum (IV and germanium (IV coordination compounds on peptidases 1 and 2 of Bacillus thuringiensis var. israelensis ІМV В-7465 activity. The study of coordination compounds effect on peptidase activity was carried out by complexes with the enzymes incubation and residual activity to the collagen, elastin and fibrin determination. It was revealed the regularity in the influence of different structure complex compounds on peptidases of B. thuringiensis var. israelensis ІМV В-7465 activity. The stanum (IV complexes with salicyloylhydrazones of aromatic aldehydes increase collagenase and elastase activities. Substituents replacement in aldehyde fragment of stanum (IV with isonicotinoylhydrazones of aromatic aldehydes complexes by the less polar ones contributed to increase of elastase activity of both enzymes while an absence of substituents enables to increase peptidase 2 fibrinolytic activity. The complexes of germanіum (IV with isonicotinoylhydrazone of salicylic aldehyde which contain Zn and Co, increased collagenase activity of the peptidase 1 as well as elastase and fibrinolytic activities of the peptidase 2. In general, all tested complexes may be considered as peptidase effectors of B. thuringiensis var. israelensis ІМV В-7465. A difference of complexes effect on activities of the both enzymes is due to the characteristics of the structure of coordination compounds.

  3. A Nonhost Peptidase Inhibitor of ~14 kDa from Butea monosperma (Lam. Taub. Seeds Affects Negatively the Growth and Developmental Physiology of Helicoverpa armigera

    Directory of Open Access Journals (Sweden)

    Prabhash K. Pandey

    2014-01-01

    Full Text Available Helicoverpa armigera is one of the major devastating pests of crop plants. In this context a serine peptidase inhibitor purified from the seeds of Butea monosperma was evaluated for its effect on developmental physiology of H. armigera larvae. B. monosperma peptidase inhibitor on 12% denaturing polyacrylamide gel electrophoresis exhibited a single protein band of ~14 kDa with or without reduction. In vitro studies towards total gut proteolytic enzymes of H. armigera and bovine trypsin indicated measurable inhibitory activity. B. monosperma peptidase inhibitor dose for 50% mortality and weight reduction by 50% were 0.5% w/w and 0.10% w/w, respectively. The IC50 of B. monosperma peptidase inhibitor against total H. armigera gut proteinases activity was 2.0 µg/mL. The larval feeding assays suggested B. monosperma peptidase inhibitor to be toxic as reflected by its retarded growth and development, consequently affecting fertility and fecundity of pest and prolonging the larval-pupal duration of the insect life cycle of H. armigera. Supplementing B. monosperma peptidase inhibitor in artificial diet at 0.1% w/w, both the efficiencies of conversion of ingested as well as digested food were downregulated, whereas approximate digestibility and metabolic cost were enhanced. The efficacy of Butea monosperma peptidase inhibitor against progressive growth and development of H. armigera suggest its usefulness in insect pest management of food crops.

  4. S28 peptidases: lessons from a seemingly 'dysfunctional' family of two

    Directory of Open Access Journals (Sweden)

    Kozarich John W

    2010-06-01

    Full Text Available Abstract A recent paper in BMC Structural Biology reports the crystal structure of human prolylcarboxypeptidase (PRCP, one of the two members of the S28 peptidase family. Comparison of the substrate-binding site of PRCP with that of its family partner, dipeptidyl dipeptidase 7 (DPP7, helps to explain the different enzymatic activities of these structurally similar proteins, and also reveals a novel apparent charge-relay system in PRCP involving the active-site catalytic histidine. See research article: http://www.biomedcentral.com/1472-6807/10/16/ Commentary The S28 serine peptidase family is something of an enzymatic odd couple. While showing low sequence similarity to all proteins except each other, the two known family members appear to be at odds functionally; one, prolylcarboxypeptidase (PRCP, is a carboxypeptidase that cleaves single hydrophobic residues from the carboxyl termini of proteins that end with a Pro-X motif (where X is any hydrophobic amino acid, while the other, human dipeptidyl peptidase (DPP7, is an aminopeptidase that cleaves amino-terminal X-Pro dipeptides. The structural basis of this orthogonal specificity would undoubtedly be interesting, and a recent report in BMC Structural Biology from the Merck Global Structural Biology group (Soisson et al. 1 has now met that expectation. In addition they reveal a new wrinkle to the iconic catalytic triad common to most serine hydrolases. The practical pharmaceutical interest in both these enzymes as potential drug targets is at present speculative. PRCP can inactivate a number of peptide hormones, such as angiotensin II, III and prekallikrein, implicating a role for the enzyme in hypertension, tissue proliferation and smooth-muscle growth. These properties suggest that this enzyme may well be a useful target for hypertension and anti-inflammatory therapy 2. Another (non-S28 family dipeptidyl dipeptidase (DPP4 is a major drug target in type 2 diabetes, and Merck has already

  5. Crystal structure of bacterial cell-surface alginate-binding protein with an M75 peptidase motif

    Energy Technology Data Exchange (ETDEWEB)

    Maruyama, Yukie; Ochiai, Akihito [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Mikami, Bunzo [Laboratory of Applied Structural Biology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Hashimoto, Wataru [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Murata, Kousaku, E-mail: kmurata@kais.kyoto-u.ac.jp [Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2011-02-18

    Research highlights: {yields} Bacterial alginate-binding Algp7 is similar to component EfeO of Fe{sup 2+} transporter. {yields} We determined the crystal structure of Algp7 with a metal-binding motif. {yields} Algp7 consists of two helical bundles formed through duplication of a single bundle. {yields} A deep cleft involved in alginate binding locates around the metal-binding site. {yields} Algp7 may function as a Fe{sup 2+}-chelated alginate-binding protein. -- Abstract: A gram-negative Sphingomonas sp. A1 directly incorporates alginate polysaccharide into the cytoplasm via the cell-surface pit and ABC transporter. A cell-surface alginate-binding protein, Algp7, functions as a concentrator of the polysaccharide in the pit. Based on the primary structure and genetic organization in the bacterial genome, Algp7 was found to be homologous to an M75 peptidase motif-containing EfeO, a component of a ferrous ion transporter. Despite the presence of an M75 peptidase motif with high similarity, the Algp7 protein purified from recombinant Escherichia coli cells was inert on insulin B chain and N-benzoyl-Phe-Val-Arg-p-nitroanilide, both of which are substrates for a typical M75 peptidase, imelysin, from Pseudomonas aeruginosa. The X-ray crystallographic structure of Algp7 was determined at 2.10 A resolution by single-wavelength anomalous diffraction. Although a metal-binding motif, HxxE, conserved in zinc ion-dependent M75 peptidases is also found in Algp7, the crystal structure of Algp7 contains no metal even at the motif. The protein consists of two structurally similar up-and-down helical bundles as the basic scaffold. A deep cleft between the bundles is sufficiently large to accommodate macromolecules such as alginate polysaccharide. This is the first structural report on a bacterial cell-surface alginate-binding protein with an M75 peptidase motif.

  6. Reduced serum dipeptidyl peptidase-IV after metformin and pioglitazone treatments.

    Science.gov (United States)

    Lenhard, James M; Croom, Dallas K; Minnick, Dana T

    2004-11-05

    Dipeptidyl peptidase-IV (DPP-IV) regulates metabolism by degrading incretins involved in nutritional regulation. Metformin and pioglitazone improve insulin sensitivity whereas glyburide promotes insulin secretion. Zucker diabetic rats were treated with these antidiabetic agents for 2 weeks and DPP-IV activity and expression were determined. Serum DPP-IV activity increased whereas tissue activity decreased as the rats aged. Treatment of rats with metformin, pioglitazone, and glyburide did not alter DPP-IV mRNA expression in liver or kidney. Metformin and pioglitazone significantly (PMetformin, pioglitazone, and glyburide had no effect on serum DPP-IV activity in vitro, indicating these are not competitive DPP-IV inhibitors. We propose the in vivo inhibitory effects observed with metformin and pioglitazone on serum DPP-IV activity results from reduced DPP-IV secretion.

  7. Comparative study to predict dipeptidyl peptidase IV inhibitory activity of β-amino amide scaffold

    Directory of Open Access Journals (Sweden)

    S Patil

    2015-01-01

    Full Text Available Comparative study was performed on 34 β-amino amide derivatives as dipeptidyl peptidase IV inhibitors in order to determine their structural requirement to enhance the antidiabetic activities. Hologram quantitative structure activity relationships models utilized specialized fragment fingerprints (hologram length 353 which showed good predictivity with cross-validated q 2 and conventional r 2 values of 0.971 and 0.971, respectively. Models were validated and optimized by a test set of eight compounds and gave satisfactory predictive ability. Hologram quantitative structure activity relationships maps were helpful in prediction of the structural features of the ligands to account for the activity in terms of positively and negatively contributing towards activity. The information obtained from maps could be effectively use as a guiding tool for further structure modifications and synthesis of new potent antidiabetic agents.

  8. Vers l'évolution d'une DD-peptidase en beta-lactamase

    OpenAIRE

    Labarbe, Carole

    2006-01-01

    Les DD-peptidases sont des enzymes impliquées dans la synthèse de la paroi bactérienne. Elles sont aussi appelées "Penicillin Binding Proteins" (PBPs) car elles forment des acyl-enzymes stables avec des antibiotiques de type beta-lactames comme la pénicilline, la stabilité de ces complexes étant à l'origine de l'effet antibiotique. Certaines bactéries sont résistantes aux b-lactames grâce à la production de beta-lactamases, capables d'hydrolyser ces antibiotiques jusqu'à 10E8 fois plus rapide...

  9. Brush border peptidases and arylamidases in the experimental blind loop syndrome of the rat.

    Science.gov (United States)

    Mazzacca, G; Musella, S; Andria, G; D'Agostino, L; Cimino, L; Budillon, G

    1977-10-01

    Peptidase and arylamidase activities were assessed in purified brush borders from jejunum of rats with surgically created blind loops. The blind loop segment and the jejunum proximal and distal to the blind loop were studied. Comparable jejunal segments from control rats were also studied. The blind loop syndrome was documented by presence of macrocytic anemia. Enzyme activities were determined on purified brush borders. In rats with the blind loop syndromes enzymatic activities hydrolizing sucrose, L-Leucyl-beta-naphthylamide, L-lysyl-beta-naphthylamide, alpha-L-glutamyl-beta-naphthylamide, L-phenylalanyl-alanine and L-leucyl-glycine were significantly reduced as compared to controls (P less than 0.001). After a short course of antibiotic therapy enzymatic activities returned to normal. Our findings suggest a reversible intestinal mucosa damage in the rat with blind loop syndrome.

  10. Low expression of angiotensinogen and dipeptidyl peptidase 1 in saliva of patients with proliferative verrucous leukoplakia

    Science.gov (United States)

    Flores, Isadora Luana; Santos-Silva, Alan Roger; Coletta, Ricardo Della; Leme, Adriana Franco Paes; Lopes, Marcio Ajudarte

    2016-01-01

    AIM To elucidate the profile of the salivary proteome. METHODS Unstimulated whole mouth saliva was collected from 30 volunteers [15 proliferative verrucous leukoplakia (PVL) patients and 15 controls] and proteins were submitted for mass spectrometry-based proteomics using the discovery approach, followed by analyses of variance and logistic regression tests. RESULTS A total of two hundred and eighty-three proteins were confidently identified in saliva. By combining two low abundance proteins from the PVL group, angiotensinogen (AGT) and dipeptidyl peptidase 1 (DPP1), a model for group differentiation was built with a concordance index of 94.2%, identifying both proteins as potential etiologic biomarkers for PVL. CONCLUSION This study suggests that both AGT and DPP1 may be involved in developmental mechanisms of PVL. PMID:27900324

  11. Dipeptidyl-peptidase IV activity is correlated with colorectal cancer prognosis.

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    Gorka Larrinaga

    Full Text Available Dipeptidyl-peptidase IV (EC 3.4.14.5 (DPPIV is a serine peptidase involved in cell differentiation, adhesion, immune modulation and apoptosis, functions that control neoplastic transformation. Previous studies have demonstrated altered expression and activity of tissue and circulating DPPIV in several cancers and proposed its potential usefulness for early diagnosis in colorectal cancer (CRC.The activity and mRNA and protein expression of DPPIV was prospectively analyzed in adenocarcinomas, adenomas, uninvolved colorectal mucosa and plasma from 116 CRC patients by fluorimetric, quantitative RT-PCR and immunohistochemical methods. Results were correlated with the most important classic pathological data related to aggressiveness and with 5-year survival rates. Results showed that: 1 mRNA levels and activity of DPPIV increased in colorectal neoplasms (Kruskal-Wallis test, p<0.01; 2 Both adenomas and CRCs displayed positive cytoplasmic immunostaining with luminal membrane reinforcement; 3 Plasmatic DPPIV activity was lower in CRC patients than in healthy subjects (Mann-U test, p<0.01; 4 Plasmatic DPPIV activity was associated with worse overall and disease-free survivals (log-rank p<0.01, Cox analysis p<0.01.1 Up-regulation of DPPIV in colorectal tumors suggests a role for this enzyme in the neoplastic transformation of colorectal tissues. This finding opens the possibility for new therapeutic targets in these patients. 2 Plasmatic DPPIV is an independent prognostic factor in survival of CRC patients. The determination of DPPIV activity levels in the plasma may be a safe, minimally invasive and inexpensive way to define the aggressiveness of CRC in daily practice.

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

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    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.

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

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    Baldwin, Michael; Russo, Crystal; Li, Xuerong [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Chishti, Athar H., E-mail: athar.chishti@tufts.edu [Department of Developmental, Molecular and Chemical Biology, Tufts University School of Medicine, Boston, MA 02111 (United States); Sackler School of Graduate Biomedical Sciences, Programs in Physiology, Pharmacology, and Microbiology, Tufts University School of Medicine, Boston, MA 02111 (United States)

    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.

  14. Effect of molecular weight on the transepithelial transport and peptidase degradation of casein-derived peptides by using Caco-2 cell model.

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    Wang, Bo; Li, Bo

    2017-03-01

    The transepithelial transport routes of casein-derived peptides with different molecular weights (MWs) were investigated using a Caco-2 cell monolayer. The peptidase hydrolysis during transport was also studied. The results indicate that the paracellular route was the main pathway for F1 (1600-1300Da) and F2 (1000-500Da), and the bioavailabilities were 10.66% and 9.54%, respectively. Peptidase hydrolysis results reveal that brush-border peptidases (BBPs) as well as some other peptidases were responsible for peptide degradation in the paracellular route. The maximum hydrolysis rate of the former was 6.91 and 5.59μM Gly/min for the latter. However, PepT1 was involved in the transport of F3 (transport and the maximum hydrolysis rate was 11.4μM Gly/min. Furthermore, we found that the amino acid sequence of di- and tripeptides might affect their bioavailabilities significantly.

  15. Proteolytic profiling and comparative analyses of active trypsin-like serine peptidases in preimaginal stages of Culex quinquefasciatus

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    Borges-Veloso Andre

    2012-06-01

    Full Text Available Abstract Background The mosquito Culex quinquefasciatu s, a widespread insect in tropical and sub-tropical regions of the world, is a vector of multiple arboviruses and parasites, and is considered an important risk to human and veterinary health. Proteolytic enzymes play crucial roles in the insect physiology including the modulation of embryonic development and food digestion. Therefore, these enzymes represent important targets for the development of new control strategies. This study presents zymographic characterization and comparative analysis of the proteolytic activity found in eggs, larval instars and pupae of Culex quinquefasciatus. Methods The proteolytic profiles of eggs, larvae and pupa of Cx. quinquefasciatus were characterized by SDS-PAGE co-polymerized with 0.1% gelatin, according to the pH, temperature and peptidase inhibitor sensitivity. In addition, the proteolytic activities were characterized in solution using 100 μM of the fluorogenic substrate Z-Phe-Arg-AMC. Results Comparison of the proteolytic profiles by substrate-SDS-PAGE from all preimaginal stages of the insect revealed qualitative and quantitative differences in the peptidase expression among eggs, larvae and pupae. Use of specific inhibitors revealed that the proteolytic activity from preimaginal stages is mostly due to trypsin-like serine peptidases that display optimal activity at alkaline pH. In-solution, proteolytic assays of the four larval instars using the fluorogenic substrate Z-Phe-Arg-AMC in the presence or absence of a trypsin-like serine peptidase inhibitor confirmed the results obtained by substrate-SDS-PAGE analysis. The trypsin-like serine peptidases of the four larval instars were functional over a wide range of temperatures, showing activities at 25°C and 65°C, with an optimal activity between 37°C and 50°C. Conclusion The combined use of zymography and in-solution assays, as performed in this study, allowed for a more detailed analysis of the

  16. Profiling of proteolytic enzymes in the gut of the tick Ixodes ricinus reveals an evolutionarily conserved network of aspartic and cysteine peptidases

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    Mareš Michael

    2008-03-01

    Full Text Available Abstract Background Ticks are vectors for a variety of viral, bacterial and parasitic diseases in human and domestic animals. To survive and reproduce ticks feed on host blood, yet our understanding of the intestinal proteolytic machinery used to derive absorbable nutrients from the blood meal is poor. Intestinal digestive processes are limiting factors for pathogen transmission since the tick gut presents the primary site of infection. Moreover, digestive enzymes may find practical application as anti-tick vaccine targets. Results Using the hard tick, Ixodes ricinus, we performed a functional activity scan of the peptidase complement in gut tissue extracts that demonstrated the presence of five types of peptidases of the cysteine and aspartic classes. We followed up with genetic screens of gut-derived cDNA to identify and clone genes encoding the cysteine peptidases cathepsins B, L and C, an asparaginyl endopeptidase (legumain, and the aspartic peptidase, cathepsin D. By RT-PCR, expression of asparaginyl endopeptidase and cathepsins B and D was restricted to gut tissue and to those developmental stages feeding on blood. Conclusion Overall, our results demonstrate the presence of a network of cysteine and aspartic peptidases that conceivably operates to digest host blood proteins in a concerted manner. Significantly, the peptidase components of this digestive network are orthologous to those described in other parasites, including nematodes and flatworms. Accordingly, the present data and those available for other tick species support the notion of an evolutionary conservation of a cysteine/aspartic peptidase system for digestion that includes ticks, but differs from that of insects relying on serine peptidases.

  17. Biochemical properties and evaluation of washing performance in commercial detergent compatibility of two collagenolytic serine peptidases secreted by Aspergillus fischeri and Penicillium citrinum.

    Science.gov (United States)

    Ida, Érika Lika; da Silva, Ronivaldo Rodrigues; de Oliveira, Tássio Brito; Souto, Tatiane Beltramini; Leite, Juliana Abigail; Rodrigues, André; Cabral, Hamilton

    2017-03-16

    Filamentous fungi secrete diverse peptidases with different biochemical properties, which is of considerable importance for application in various commercial sectors. In this study, we describe the isolation of two fungal species collected from the soil of decayed organic matter: Aspergillus fischeri and Penicillium citrinum. In a submerged bioprocess, we observed better peptidase production with the fungus P. citrinum, which reached a peak production at 168 h with 760 U/mL, in comparison with the fungus A. fischeri, which reached a peak production at 72 h with 460 U/mL. In both situations, the fermentative medium contained 0.5% crushed feathers as a source of nitrogen. On performing biochemical characterization, we detected two alkaline serine peptidases: The one secreted by P. citrinum had optimal activity at pH 7.0 and at 45°C, while the one secreted by A. fischeri had optimal activity in pH 6.5-8 and at 55-60°C. Metallic ions were effective in modulating these peptidases; in particular, Cu(2+) promoted negative modulation of both peptidases. The peptidases were stable and functional under conditions of nonionic surfactants, temperatures up to 45°C for 1 h, and incubation over a wide pH range. In addition, it was observed that both peptidases had the capacity to hydrolyze collagen and performed well in removing an egg protein stain when supplemented into a commercial powder detergent; this was especially true for the peptidase from P. citrinum.

  18. Dipeptidyl peptidase Ⅳ(DPP Ⅳ): a novel emerging target for the treatment of type 2 diabetes

    Institute of Scientific and Technical Information of China (English)

    Jing Wu; Yiding Chen; Xiaoli Shi; Wei Gu

    2009-01-01

    The enzyme, dipeptidyl peptidase Ⅳ(DPP Ⅳ), is a novel target for the treatment of type 2 diabetes. Dipeptidyl peptidase Ⅳ inhibition improves the impaired insulin secretion and decrease postprandial concentrations of glucagon by enhancing the incretin hormone levels lucagon-like peptide-1(GLP-1) and glucose-dependent insulinotropic polypeptide/gastric inhibitory polypeptide(GIP). Recently, DPP Ⅳ inhibitors have attracted more and more attention, several of which have entered pre-clinical and clinical trials, and one has received approval for use as an anti-diabetic agent. Among the DPP Ⅳ inhibitors, two leading agents(sitagliptin and vildagliptin) have been shown to be effective in reducing glycosylated hemoglobin(HbAlc) and fasting plasma glucose(FPG) in patients with type 2 diabetes. This review summarizes the evidence supporting DPP Ⅳ inhibitors as potential antidiabetic agents.

  19. Inhibition of Streptococcus pneumoniae penicillin-binding protein 2x and Actinomadura R39 DD-peptidase activities by ceftaroline.

    Science.gov (United States)

    Zervosen, Astrid; Zapun, André; Frère, Jean-Marie

    2013-01-01

    Although the rate of acylation of a penicillin-resistant form of Streptococcus pneumoniae penicillin-binding protein 2x (PBP2x) by ceftaroline is 80-fold lower than that of its penicillin-sensitive counterpart, it remains sufficiently high (k(2)/K = 12,600 M(-1) s(-1)) to explain the sensitivity of the penicillin-resistant strain to this new cephalosporin. Surprisingly, the Actinomadura R39 DD-peptidase is not very sensitive to ceftaroline.

  20. Altered cardiac bradykinin metabolism in experimental diabetes caused by the variations of angiotensin-converting enzyme and other peptidases.

    Science.gov (United States)

    Adam, Albert; Leclair, Patrick; Montpas, Nicolas; Koumbadinga, Gérémy Abdull; Bachelard, Hélène; Marceau, François

    2010-04-01

    The peptidases angiotensin-converting enzyme (ACE) and neutral endopeptidase 24.11 (NEP) mediate most of the kinin catabolism in normal cardiac tissue and are the molecular targets of inhibitory drugs that favorably influence diabetic complications. We studied the variations of those kininases in the myocardium of rats in experimental diabetes. ACE and NEP activities were significantly decreased in heart membranes 4-8weeks post-streptozotocin (STZ) injection. However, insulin-dependent diabetes did not modify significantly bradykinin (BK) half-life (t(1/2)) while the effect of both ACE (enalaprilat) and ACE and NEP (omapatrilat) inhibitors on BK degradation progressively decreased, which may be explained by the upregulation of other unidentified metallopeptidase(s). In vivo insulin treatment restored the activities of both ACE and NEP. ACE and NEP activities were significantly higher in hearts of young Zucker rats than in those of Sprague-Dawley rats. BK t(1/2) and the effects of peptidase inhibitors on t(1/2) varied accordingly. It is concluded that kininase activities are subjected to large and opposite variations in rat cardiac tissue in type I and II diabetes models. A number of tissue or molecular factors may determine these variations, such as remodeling of cardiac tissue, ectoenzyme shedding to the extracellular fluid and the pathologic regulation of peptidase gene expression.

  1. Novel N-substituted aminobenzamide scaffold derivatives targeting the dipeptidyl peptidase-IV enzyme

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    Al-Balas QA

    2014-01-01

    Full Text Available Qosay A Al-Balas,1 Munia F Sowaileh,1 Mohammad A Hassan,1 Amjad M Qandil,1,2 Karem H Alzoubi,3 Nizar M Mhaidat,3 Ammar M Almaaytah,4 Omar F Khabour51Department of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan; 2Pharmaceutical Sciences Department, College of Pharmacy, King Saud bin Abdulaziz University for Health Sciences, Riyadh, Saudi Arabia; 3Department of Clinical Pharmacy, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan; 4Department of Pharmaceutical Technology, Faculty of Pharmacy, Jordan University of Science and Technology, Irbid, Jordan; 5Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, Jordan University of Science and Technology, Irbid, JordanBackground: The dipeptidyl peptidase-IV (DPP-IV enzyme is considered a pivotal target for controlling normal blood sugar levels in the body. Incretins secreted in response to ingestion of meals enhance insulin release to the blood, and DPP-IV inactivates these incretins within a short period and stops their action. Inhibition of this enzyme escalates the action of incretins and induces more insulin to achieve better glucose control in diabetic patients. Thus, inhibition of this enzyme will lead to better control of blood sugar levels.Methods: In this study, computer-aided drug design was used to help establish a novel N-substituted aminobenzamide scaffold as a potential inhibitor of DPP-IV. CDOCKER software available from Discovery Studio 3.5 was used to evaluate a series of designed compounds and assess their mode of binding to the active site of the DPP-IV enzyme. The designed compounds were synthesized and tested against a DPP-IV enzyme kit provided by Enzo Life Sciences. The synthesized compounds were characterized using proton and carbon nuclear magnetic resonance, mass spectrometry, infrared spectroscopy, and determination of melting point.Results: Sixty

  2. In vivo inhibition of dipeptidyl peptidase IV activity by pro-pro-diphenyl-phosphonate (Prodipine).

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    De Meester, I; Belyaev, A; Lambeir, A M; De Meyer, G R; Van Osselaer, N; Haemers, A; Scharpé, S

    1997-07-01

    Dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5), also known as CD26, is a membrane-bound serine protease that cleaves off aminoterminal dipeptides from peptides with a penultimate proline (or, at a much slower rate, a penultimate alanine). Recently, we synthesized and characterized a number of dipeptide-derived diphenylphosphonates. Out of the resulting series of slow-binding irreversible inhibitors of DPP IV, diphenyl 1-(S)-prolylpyrrolidine-2(R,S)-phosphonate hydrochloride (Pro-Pro-diphenylphosphonate or Prodipine) was selected for further study. We investigated the in vivo applicability of Prodipine. Male rabbits weighing 3-4 kg received a single intravenous injection with 10 mg Prodipine or saline. After 1 hr, plasma DPP IV activity had decreased to less than 20% of the preinjection value and remained unchanged during a 24-hr observation period. In a next step, we aimed to study (i) the dose dependency and (ii) the duration of the effect after a single intravenous dose of Prodipine. A profound and long-lasting inhibition of plasma DPP IV activity was observed in the treated animals (1, 5 or 10 mg). It took 5 to 8 days to reach half of the pretreatment DPP IV activity and generally more than 20 days for a complete recovery. Systemic treatment with Prodipine not only led to inhibition of plasma DPP IV activity but also decreased tissue DPP IV activity in circulating mononuclear cells, kidney cortex, thymus, spleen, lung, and liver. No differences in activities of the related peptidases aminopeptidase P (APP, EC 3.4.11.9), prolyl oligopeptidase (PO, EC 3.4.21.26), or aminopeptidase M (mAAP, EC 3.4.11.2) were detected between Prodipine-treated and control rabbits. The in vivo applicability of this chemically stable, irreversible inhibitor of DPP IV opens new possibilities, not only to further unravel the biological functions of this intriguing ectopeptidase, but also to explore this enzyme as a new target in various fields of pharmacological research.

  3. The potential of signal peptide peptidase as a therapeutic target for hepatitis C.

    Science.gov (United States)

    Moriishi, Kohji

    2017-09-01

    Chronic infection with hepatitis C virus (HCV) causes liver steatosis, cirrhosis, metabolic syndrome with inflammation, and eventually leads to hepatocellular carcinoma. HCV core protein is a well-known capsid protein and pathogenic factor related to lipid accumulation, type 2 diabetes mellitus, and carcinogenesis. Cleavage of the C-terminal transmembrane region by signal peptide peptidase (SPP) is required for maturation of the core protein. Areas covered: Herein, this review details the general aspects of the structure, lifecycle, pathogenesis, and maturation of the HCV core protein, the function of SPP, and clinically available direct-acting antivirals (DAAs). SPP is classified into a group of GXGD-type intramembrane proteases including presenilin-1, which is a component of γ-secretase complex. Several SPP inhibitors were previously identified from γ-secretase inhibitors, but have not yet been improved based on specificity to SPP. Finally, the author discusses the potential of SPP inhibitors for hepatitis C therapy. Expert opinion: Currently available DAAs therapies are limited because of different viral genotypes and underlying conditions in each patient. DAA-resistant viruses have also been reported. Development of SPP-selective inhibitors may improve current HCV therapies by decreasing in the emergence of DAA-resistant viruses irrespective of viral genotype.

  4. Crystal Structures of Human Dipeptidyl Peptidase Ⅳ in its Apo and Diprotin B-complexed Forms

    Institute of Scientific and Technical Information of China (English)

    Hajime HIRAMATSU; Kiyoshi KYONO; Atsushi YAMAMOTO; Kazuhiko SAEKI; Hideaki SHIMA; Shigeru SUGIYAMA; Koji INAKA; Ryo SHIMIZU

    2007-01-01

    Dipeptidyl peptidase Ⅳ (DPPIV), which belongs to the prolyl oligopeptidase family of serine proteases, is known to have a variety of regulatory biological functions and has been shown to be implicated in type 2 diabetes. It is therefore important to develop selective human DPPIV (hDPPIV) inhibitors. In this study, we determined the crystal structure of apo hDPPIV at 1.9 (A) resolution. Our high-resolution crystal structure of apo hDPPIV revealed the presence of sodium ion and glycerol molecules at the active site. In order to elucidate the hDPPIV binding mode and substrate specificity, we determined the crystal structure of hDPPIV-diprotin B (Val-Pro-Leu) complex at 2.1 (A) resolution, and clarified the difference in binding mode between diprotin B and diprotin A (Ile-Pro-Ile) into the active site of hDPPIV. Comparison between our crystal structures and the reported apo hDPPIV structures revealed that positively charged functional groups and conserved water molecules contributed to the interaction of ligands with hDPPIV. These results are useful for the design of potent hDPPIV inhibitors.

  5. SVMDLF: A novel R-based Web application for prediction of dipeptidyl peptidase 4 inhibitors.

    Science.gov (United States)

    Chandra, Sharat; Pandey, Jyotsana; Tamrakar, Akhilesh K; Siddiqi, Mohammad Imran

    2017-06-06

    Dipeptidyl peptidase 4 (DPP4) is a well-known target for the antidiabetic drugs. However, currently available DPP4 inhibitor screening assays are costly and labor-intensive. It is important to create a robust in silico method to predict the activity of DPP4 inhibitor for the new lead finding. Here, we introduce an R-based Web application SVMDLF (SVM-based DPP4 Lead Finder) to predict the inhibitor of DPP4, based on support vector machine (SVM) model, predictions of which are confirmed by in vitro biological evaluation. The best model generated by MACCS structure fingerprint gave the Matthews correlation coefficient of 0.87 for the test set and 0.883 for the external test set. We screened Maybridge database consisting approximately 53,000 compounds. For further bioactivity assay, six compounds were shortlisted, and of six hits, three compounds showed significant DPP4 inhibitory activities with IC50 values ranging from 8.01 to 10.73 μm. This application is an OpenCPU server app which is a novel single-page R-based Web application for the DPP4 inhibitor prediction. The SVMDLF is freely available and open to all users at http://svmdlf.net/ocpu/library/dlfsvm/www/ and http://www.cdri.res.in/svmdlf/. © 2017 John Wiley & Sons A/S.

  6. The Effects of Dipeptidyl Peptidase-4 Inhibitors on Cardiovascular Disease Risks in Type 2 Diabetes Mellitus

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    Pegah Yousefzadeh

    2013-01-01

    Full Text Available Objective. To review the current literature investigating the effects of dipeptidyl peptidase-4 (DPP-4 inhibitors on the risk factors of cardiovascular disease (CVD. Methods. We conducted a search of PubMed and MEDLINE database, using the term DPP-4 inhibitor in combination with the following terms: metabolic syndrome, hypertension, dyslipidemia, insulin resistance, obesity, and CVD. We reviewed 100 relevant studies out of 227 articles, excluding single case reports, studies using animal models, and reports not written in English. We included 38 references in this review article. Results. The majority of the recent clinical studies have demonstrated that DPP-4 inhibitors have beneficial effects on cardiovascular (CV system. These agents may have the potential to lower blood pressure, improve lipid profile and endothelial dysfunction, decrease the macrophage-mediated inflammatory response, and prevent myocardial injury. Conclusion. DPP-4 inhibitors have some CV protective effects in type 2 diabetes mellitus (T2DM in addition to their antidiabetic actions. Long-term outcome clinical trials are under way to investigate the effects of the DPP-4 inhibitors on the elevated CV risks in patients with T2DM. Further investigation in a large cohort is warranted to assess the exact mechanisms of CV protective effects of DPP-4 inhibitors.

  7. DIPEPTIDYL PEPTIDASE-4 REGULATION OF SDF-1/CXCR4 AXIS: IMPLICATIONS FOR CARDIOVASCULAR DISEASE

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    Jixin eZhong

    2015-09-01

    Full Text Available Dipeptidyl peptidase-4 (DPP4 is a ubiquitously expressed protease that regulates a diverse number of physiologic 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 such as 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.

  8. Dipeptidyl Peptidase 4: A New Link between Diabetes Mellitus and Atherosclerosis?

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    Wellington Santana da Silva Júnior

    2015-01-01

    Full Text Available Type 2 diabetes mellitus (T2DM has become one of the most prevalent noncommunicable diseases in the past years. It is undoubtedly associated with atherosclerosis and increased risk for cardiovascular diseases. Incretins, which are intestinal peptides secreted during digestion, are able to increase insulin secretion and its impaired function and/or secretion is involved in the pathophysiology of T2DM. Dipeptidyl peptidase 4 (DPP4 is an ubiquitous enzyme that regulates incretins and consequently is related to the pathophysiology of T2DM. DPP4 is mainly secreted by endothelial cells and acts as a regulatory protease for cytokines, chemokines, and neuropeptides involved in inflammation, immunity, and vascular function. In T2DM, the activity of DPP4 seems to be increased and there are a growing number of in vitro and in vivo studies suggesting that this enzyme could be a new link between T2DM and atherosclerosis. Gliptins are a new class of pharmaceutical agents that acts by inhibiting DPP4. Thus, it is expected that gliptin represents a new pharmacological approach not only for reducing glycemic levels in T2DM, but also for the prevention and treatment of atherosclerotic cardiovascular disease in diabetic subjects. We aimed to review the evidences that reinforce the associations between DPP4, atherosclerosis, and T2DM.

  9. The dipeptidyl peptidase-4 inhibitor sitagliptin suppresses mouse colon tumorigenesis in type 2 diabetic mice.

    Science.gov (United States)

    Yorifuji, Naoki; Inoue, Takuya; Iguchi, Munetaka; Fujiwara, Kaori; Kakimoto, Kazuki; Nouda, Sadaharu; Okada, Toshihiko; Kawakami, Ken; Abe, Yosuke; Takeuchi, Toshihisa; Higuchi, Kazuhide

    2016-02-01

    Patients with type 2 diabetes mellitus are known to have an increased risk of colorectal neoplasia. Dipeptidyl peptidase-4 (DPP-4) inhibitors have been used as a new therapeutic tool for type 2 diabetes. Since the substrates for DPP-4 include intestinotrophic hormones and chemokines such as GLP-2 and stromal cell-derived factor-1 (SDF-1), which are associated with tumor progression, DPP-4 inhibitors may increase the risk of colorectal tumors. However, the influence of DPP-4 inhibitors on colorectal neoplasia in patients with type 2 diabetes remains unknown. In the present study, we show that long-term administration of a DPP-4 inhibitor, sitagliptin (STG), suppressed colon carcinogenesis in leptin-deficient (ob/ob) C57BL/6J mice. Colonic mucosal concentrations of glucagon‑like peptide-1 (GLP-1) and GLP-2 were significantly elevated in the ob/ob mice. However, mucosal GLP concentrations and the plasma level of SDF-1 were not affected by the administration of STG. Real‑time PCR analysis revealed that colonic mucosal IL-6 mRNA expression, which was significantly upregulated in the ob/ob mice, was significantly suppressed by the long-term administration of STG. These results suggest that a DPP-4 inhibitor may suppress colon carcinogenesis in mice with type 2 diabetes in a GLP-independent manner. Since DPP-4 has multiple biological functions, further studies analyzing other factors related to colon carcinogenesis are needed.

  10. Early-onset Evans syndrome, immunodeficiency, and premature immunosenescence associated with tripeptidyl-peptidase II deficiency.

    Science.gov (United States)

    Stepensky, Polina; Rensing-Ehl, Anne; Gather, Ruth; Revel-Vilk, Shoshana; Fischer, Ute; Nabhani, Schafiq; Beier, Fabian; Brümmendorf, Tim H; Fuchs, Sebastian; Zenke, Simon; Firat, Elke; Pessach, Vered Molho; Borkhardt, Arndt; Rakhmanov, Mirzokhid; Keller, Bärbel; Warnatz, Klaus; Eibel, Hermann; Niedermann, Gabriele; Elpeleg, Orly; Ehl, Stephan

    2015-01-29

    Autoimmune cytopenia is a frequent manifestation of primary immunodeficiencies. Two siblings presented with Evans syndrome, viral infections, and progressive leukopenia. DNA available from one patient showed a homozygous frameshift mutation in tripeptidyl peptidase II (TPP2) abolishing protein expression. TPP2 is a serine exopeptidase involved in extralysosomal peptide degradation. Its deficiency in mice activates cell death programs and premature senescence. Similar to cells from naïve, uninfected TPP2-deficient mice, patient cells showed increased major histocompatibility complex I expression and most CD8(+) T-cells had a senescent CCR7-CD127(-)CD28(-)CD57(+) phenotype with poor proliferative responses and enhanced staurosporine-induced apoptosis. T-cells showed increased expression of the effector molecules perforin and interferon-γ with high expression of the transcription factor T-bet. Age-associated B-cells with a CD21(-) CD11c(+) phenotype expressing T-bet were increased in humans and mice, combined with antinuclear antibodies. Moreover, markers of senescence were also present in human and murine TPP2-deficient fibroblasts. Telomere lengths were normal in patient fibroblasts and granulocytes, and low normal in lymphocytes, which were compatible with activation of stress-induced rather than replicative senescence programs. TPP2 deficiency is the first primary immunodeficiency linking premature immunosenescence to severe autoimmunity. Determination of senescent lymphocytes should be part of the diagnostic evaluation of children with refractory multilineage cytopenias.

  11. Dipeptidyl peptidase-IV (DPP-IV inhibitory activity of parotid exudate of Bufo melanostictus

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    Allenki Venkatesham

    2009-01-01

    Full Text Available Type 2 diabetes arises as a result of β-cell failure combined with concomitant insulin resistance. Glucagon-like peptide-1 is a gastrointestinal hormone that is released postprandially from the L cells of the gut and exerts a glucose- dependent and direct insulinotropic effect on the pancreatic β cell. Which activate adenylate cyclase and enhances insulin secretion. GLP-1 is rapidly degraded by DPP-IV to GLP-1(9-37 amide following release from gut L cells. GLP-1 directly enhances glucose-dependent insulin secretion via an increase in β-cell cAMP. Dipeptidyl peptidase IV (DPP-IV is a plasma membrane glycoprotein ectopeptidase. In mammals, DPP-IV was widely expressed on the surface of endothelial and epithelial cells and highest levels in humans have been reported to occur in the intestine, bone marrow and kidney. Inhibiting DPP-IV reduces its rapid degradation of GLP-1, increasing circulating levels of the active hormone in vivo and prolonging its beneficial effects. The IC 50 value of parotid exudate was found to be 9.4 μg/ml. The maximum % inhibition (61.8 was showed at a concentration of 12μg/ml. Parotid exudate through inhibition of DPP-IV, improves glucose tolerance and enhances insulin secretion. DPP-IV inhibitors are a novel class of oral hypoglycemic agents with a potential to improve pancreatic beta cell function and the clinical course of type 2 diabetes.

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

    Science.gov (United States)

    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

  13. Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC.

    Science.gov (United States)

    Raj, V Stalin; Mou, Huihui; Smits, Saskia L; Dekkers, Dick H W; Müller, Marcel A; Dijkman, Ronald; Muth, Doreen; Demmers, Jeroen A A; Zaki, Ali; Fouchier, Ron A M; Thiel, Volker; Drosten, Christian; Rottier, Peter J M; Osterhaus, Albert D M E; Bosch, Berend Jan; Haagmans, Bart L

    2013-03-14

    Most human coronaviruses cause mild upper respiratory tract disease but may be associated with more severe pulmonary disease in immunocompromised individuals. However, SARS coronavirus caused severe lower respiratory disease with nearly 10% mortality and evidence of systemic spread. Recently, another coronavirus (human coronavirus-Erasmus Medical Center (hCoV-EMC)) was identified in patients with severe and sometimes lethal lower respiratory tract infection. Viral genome analysis revealed close relatedness to coronaviruses found in bats. Here we identify dipeptidyl peptidase 4 (DPP4; also known as CD26) as a functional receptor for hCoV-EMC. DPP4 specifically co-purified with the receptor-binding S1 domain of the hCoV-EMC spike protein from lysates of susceptible Huh-7 cells. Antibodies directed against DPP4 inhibited hCoV-EMC infection of primary human bronchial epithelial cells and Huh-7 cells. Expression of human and bat (Pipistrellus pipistrellus) DPP4 in non-susceptible COS-7 cells enabled infection by hCoV-EMC. The use of the evolutionarily conserved DPP4 protein from different species as a functional receptor provides clues about the host range potential of hCoV-EMC. In addition, it will contribute critically to our understanding of the pathogenesis and epidemiology of this emerging human coronavirus, and may facilitate the development of intervention strategies.

  14. Endoplasmic reticulum-associated amino-peptidase 1 and rheumatic disease: genetics.

    Science.gov (United States)

    Ombrello, Michael J; Kastner, Daniel L; Remmers, Elaine F

    2015-07-01

    This article will review the genetic evidence implicating ERAP1, which encodes the endoplasmic reticulum-associated amino-peptidase 1, in susceptibility to rheumatic disease. Genetic variants and haplotypes of ERAP1 are associated with AS, psoriasis, and Behçet's disease in people of varying ancestries. In each of these diseases, disease-associated variants of ERAP1 have been shown to interact with disease-associated class I human leukocyte antigen alleles to influence disease risk. Functionally, disease-associated missense variants of ERAP1 concertedly alter ERAP1 enzymatic function, both quantitatively and qualitatively, whereas other disease-associated variants influence ERAP1 expression. Therefore, ERAP1 haplotypes (or allotypes) should be examined as functional units. Biologically, this amounts to an examination of the gene regulation and function of the protein encoded by each allotype. Genetically, the relationship between disease risk and ERAP1 allotypes should be examined to determine whether allotypes or individual variants produce the most parsimonious risk models. Future investigations of ERAP1 should focus on comprehensively characterizing naturally occurring ERAP1 allotypes, examining the enzymatic function and gene expression of each allotype, and identifying specific allotypes that influence disease susceptibility.

  15. Computational Analysis of Gynura bicolor Bioactive Compounds as Dipeptidyl Peptidase-IV Inhibitor.

    Science.gov (United States)

    Rozano, Lina; Abdullah Zawawi, Muhammad Redha; Ahmad, Muhamad Aizuddin; Jaganath, Indu Bala

    2017-01-01

    The inhibition of dipeptidyl peptidase-IV (DPPIV) is a popular route for the treatment of type-2 diabetes. Commercially available gliptin-based drugs such as sitagliptin, anagliptin, linagliptin, saxagliptin, and alogliptin were specifically developed as DPPIV inhibitors for diabetic patients. The use of Gynura bicolor in treating diabetes had been reported in various in vitro experiments. However, an understanding of the inhibitory actions of G. bicolor bioactive compounds on DPPIV is still lacking and this may provide crucial information for the development of more potent and natural sources of DPPIV inhibitors. Evaluation of G. bicolor bioactive compounds for potent DPPIV inhibitors was computationally conducted using Lead IT and iGEMDOCK software, and the best free-binding energy scores for G. bicolor bioactive compounds were evaluated in comparison with the commercial DPPIV inhibitors, sitagliptin, anagliptin, linagliptin, saxagliptin, and alogliptin. Drug-likeness and absorption, distribution, metabolism, and excretion (ADME) analysis were also performed. Based on molecular docking analysis, four of the identified bioactive compounds in G. bicolor, 3-caffeoylquinic acid, 5-O-caffeoylquinic acid, 3,4-dicaffeoylquinic acid, and trans-5-p-coumaroylquinic acid, resulted in lower free-binding energy scores when compared with two of the commercially available gliptin inhibitors. The results revealed that bioactive compounds in G. bicolor are potential natural inhibitors of DPPIV.

  16. Computational Analysis of Gynura bicolor Bioactive Compounds as Dipeptidyl Peptidase-IV Inhibitor

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    Lina Rozano

    2017-01-01

    Full Text Available The inhibition of dipeptidyl peptidase-IV (DPPIV is a popular route for the treatment of type-2 diabetes. Commercially available gliptin-based drugs such as sitagliptin, anagliptin, linagliptin, saxagliptin, and alogliptin were specifically developed as DPPIV inhibitors for diabetic patients. The use of Gynura bicolor in treating diabetes had been reported in various in vitro experiments. However, an understanding of the inhibitory actions of G. bicolor bioactive compounds on DPPIV is still lacking and this may provide crucial information for the development of more potent and natural sources of DPPIV inhibitors. Evaluation of G. bicolor bioactive compounds for potent DPPIV inhibitors was computationally conducted using Lead IT and iGEMDOCK software, and the best free-binding energy scores for G. bicolor bioactive compounds were evaluated in comparison with the commercial DPPIV inhibitors, sitagliptin, anagliptin, linagliptin, saxagliptin, and alogliptin. Drug-likeness and absorption, distribution, metabolism, and excretion (ADME analysis were also performed. Based on molecular docking analysis, four of the identified bioactive compounds in G. bicolor, 3-caffeoylquinic acid, 5-O-caffeoylquinic acid, 3,4-dicaffeoylquinic acid, and trans-5-p-coumaroylquinic acid, resulted in lower free-binding energy scores when compared with two of the commercially available gliptin inhibitors. The results revealed that bioactive compounds in G. bicolor are potential natural inhibitors of DPPIV.

  17. Signal peptide peptidase (SPP) assembles with substrates and misfolded membrane proteins into distinct oligomeric complexes

    Science.gov (United States)

    Schrul, Bianca; Kapp, Katja; Sinning, Irmgard; Dobberstein, Bernhard

    2010-01-01

    SPP (signal peptide peptidase) is an aspartyl intramembrane cleaving protease, which processes a subset of signal peptides, and is linked to the quality control of ER (endoplasmic reticulum) membrane proteins. We analysed SPP interactions with signal peptides and other membrane proteins by co-immunoprecipitation assays. We found that SPP interacts specifically and tightly with a large range of newly synthesized membrane proteins, including signal peptides, preproteins and misfolded membrane proteins, but not with all co-expressed type II membrane proteins. Signal peptides are trapped by the catalytically inactive SPP mutant SPPD/A. Preproteins and misfolded membrane proteins interact with both SPP and the SPPD/A mutant, and are not substrates for SPP-mediated intramembrane proteolysis. Proteins interacting with SPP are found in distinct complexes of different sizes. A signal peptide is mainly trapped in a 200 kDa SPP complex, whereas a preprotein is predominantly found in a 600 kDa SPP complex. A misfolded membrane protein is detected in 200, 400 and 600 kDa SPP complexes. We conclude that SPP not only processes signal peptides, but also collects preproteins and misfolded membrane proteins that are destined for disposal. PMID:20196774

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

    Energy Technology Data Exchange (ETDEWEB)

    Takasawa, Wataru [Division of Clinical Immunology, The Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Ohnuma, Kei [Department of Rheumatology and Allergy, Research Hospital, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Hatano, Ryo; Endo, Yuko [Division of Clinical Immunology, The Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Dang, Nam H. [Division of Hematology/Oncology, University of Florida, 1600 SW Archer Road, Box 100278, Room MSB M410A, Gainesville, FL 32610 (United States); Morimoto, Chikao, E-mail: morimoto@ims.u-tokyo.ac.jp [Division of Clinical Immunology, The Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Department of Rheumatology and Allergy, Research Hospital, The Institute of Medical Science, The University of Tokyo, 4-6-1, Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan)

    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.

  19. C-Peptide Levels Predict the Effectiveness of Dipeptidyl Peptidase-4 Inhibitor Therapy

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    Sevin Demir

    2016-01-01

    Full Text Available Background. Our aim was to define the conditions that affect therapeutic success when dipeptidyl peptidase-4 (DPP-4 inhibitor is added to metformin monotherapy. Materials and Methods. We reviewed the medical records of 56 patients who had received DPP-4 inhibitor as an add-on to metformin monotherapy and evaluated their response in the first year of therapy. Fasting blood glucose (FBG, HbA1c, C-peptide, and weight of the patients were recorded at 3-month intervals during the first year of treatment. Results. Patients who added DPP-4 inhibitor to metformin monotherapy had significant weight loss (P=0.004 and FBG and HbA1c levels were significantly lowered during the first 6 months (both P<0.001. Baseline levels of C-peptide were predictive for success of the treatment (P=0.02, even after correction for confounding factors, for example, age, gender, or BMI (P=0.03. Duration of diabetes was not a predictor of response to treatment (P=0.60. Conclusion. Our study demonstrates that in patients having inadequate glycemic control, the addition of a DPP-4 inhibitor as a second oral agent to metformin monotherapy provides better glycemic control, protects β-cell reserves, and does not cause weight gain. These effects depend on baseline C-peptide levels.

  20. Effect of temperature on the stability of various peptidases during peptide-enriched soy sauce fermentation.

    Science.gov (United States)

    Nakahara, Takeharu; Yamaguchi, Hitomi; Uchida, Riichiro

    2012-03-01

    We previously developed a peptide-enriched soy sauce-like seasoning called Fermented Soybean Seasoning (FSS) with high-temperature fermentation, and we have reported the antihypertensive effects of FSS. Seryl-tyrosine (Ser-Tyr) and glycyl-tyrosine (Gly-Tyr) were identified from FSS as active constituents in the antihypertensive effects. They were found to be particularly enriched in FSS; more so than in regular soy sauce. In the present study, we clarified one of the mechanisms underlying the accumulation of these bioactive peptides during high temperature soy sauce brewing. Crude enzyme extracts were prepared from model soy sauce mash (moromi) fermented at various temperatures. Leucine aminopeptidase-I, II, and seryl-tyrosine hydrolytic activity were found to decrease in the moromi incubated at the fermentation temperature of FSS whereas almost no decrease was observed in that of regular soy sauce. The concentrations of ACE inhibitory peptides, Ser-Tyr and Gly-Tyr, in the moromi incubated at high temperature were revealed to be higher than those at low temperature through quantitative LC-MS/MS analysis. These results suggested that the peptidases responsible for degrading low molecular weight bioactive peptides were inactivated during the high temperature fermentation, thus, these peptides would be likely to remain in the high temperature fermentation.

  1. Ubiquitin-Specific Peptidase USP22 Negatively Regulates the STAT Signaling Pathway by Deubiquitinating SIRT1

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    Ning Ao

    2014-06-01

    Full Text Available Background/Aims: The ubiquitin-specific peptidase USP22 mediates various cellular and organismal processes, such as cell growth, apoptosis, and tumor malignancy. However, the molecular mechanisms that regulate USP22 activity remain poorly understood. Here we identify STAT3 as a new USP22 interactor. Methods:· We used western blotting and RT-PCR to measure key protein, acetylated STAT3, and mRNA levels in HEK293 and colorectal cancer cell lines transfected with expression plasmids or specific siRNAs. Co-immunoprecipitation was used to demonstrate protein-protein interaction and protein complex composition. Results: USP22 overexpression down-regulated STAT3 acetylation by deubiquitinating SIRT1. The three proteins were found to be present in a single protein complex. SiRNA-mediated depletion of endogenous USP22 resulted in SIRT1 destabilization and elevated STAT3 acetylation. Consistent with this finding, USP22 also down-regulated the expression of two known STAT3 target genes, MMP9 and TWIST. Conclusion: We show that USP22 is a new regulator of the SIRT1-STAT3 signaling pathway and report a new mechanistic explanation for cross talk between USP22 and the SIRT1-STAT pathways.

  2. Selective adsorption of plant cysteine peptidases onto TiO2.

    Science.gov (United States)

    Llerena-Suster, C R F; Foresti, M L; Briand, L E; Morcelle, S R

    2009-08-01

    A crude extract rich in plant cysteine peptidases was obtained from the latex of the fruits of Araujia hortorum, a South American climbing plant. The highly concentrated extract was immobilized onto titanium dioxide to produce biocatalysts through a simple adsorption procedure. Absorbance measurement at 280 nm and Bradford's method for protein quantification revealed that the protein content of the crude extract was selectively adsorbed onto the titanium dioxide surface at a very high rate. In 5 min of contact with the support all protein present in the crude extract was selectively withdrawn from the solution, leading to an immobilized biocatalyst with a high protein concentration. Caseinolytic assays indicated that, except for the catalyst obtained with the highest crude amount contacted with the support, all the proteolytic activity present in the crude extract was adsorbed onto TiO(2). The amidasic activity of the immobilized catalysts (Ah/TiO(2)) was tested in the hydrolysis of a synthetic chromogenic substrate (PFLNA) showing partial deactivation with respect to the native enzyme. In amidasic activity assays the ionic strength of the buffer medium showed to be a key feature to consider in order to avoid protease desorption from the support, indicating the importance of electrostatic interactions between the enzymes and TiO(2). Reuse of the produced biocatalysts with PFLNA as substrate revealed that after five successive uses Ah/TiO(2) retained more than 20% of its initial activity.

  3. Dipeptidyl peptidase-4 inhibitors as add-on therapy to insulin: rationale and evidences.

    Science.gov (United States)

    Singh, Awadhesh Kumar; Singh, Ritu

    2016-01-08

    Type 2 diabetes mellitus being a progressive disease will eventually require insulin therapy. While insulin therapy is the ultimate option, many patients still fall short of target glycemic goals. This could, perhaps be due to the fear, unwillingness and practical barriers to insulin intensification. Hypoglycemia, oedema and weight gain is another limitation. Newer therapies with dipeptidyl peptidase-4 (DPP-4) inhibitors and sodium-glucose co-transporter-2 (SGLT-2) inhibitors are exciting options as both classes do not cause hypoglycemia and are either weight neutral or cause weight loss. DPP-4 inhibitors are an appealing option as an add-on therapy to insulin especially in elderly and patients with renal impairment. Moreover, glucose-dependent insulinotropic polypeptide (GIP) mediated augmentation of glucagon by DPP-4 inhibitors could also protect against hypoglycemia. These collective properties make these class a potential add-on candidate to insulin therapy. This article will review the efficacy and safety of DPP-4 inhibitors as an add-on to insulin therapy.

  4. Dipeptidyl-Peptidase IV inhibitors and glycemic control in type 2 Diabetes Mellitus

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    Lokendra Bahadur Sapkota

    2016-03-01

    Full Text Available Background & Objectives: Type 2 diabetes mellitus (T2DM is a progressive disease, characterized by insulin resistance, impaired glucose-induced insulin secretion, inappropriately elevated glucagon concentrations, and hyperglycemia. Many patients cannot obtain satisfactory glycemic control with current therapies. New and more effective agents, targeted not only at treatment, but also at prevention of the disease, its progression, and its associated complications, are, therefore, required. The dipeptidyl peptidase-4 (DPP-4 inhibitors are a newer class of oral drugs for the treatment of T2DM. They inhibit the breakdown of glucagon-like peptide-1 (GLP-1 and glucose-dependent insulinotropic polypeptide (GIP thereby increasing the incretin effect in patients with T2DM. In clinical practice they are associated with significant reductions in HbA1c, no weight gain and a low risk of hypoglycemia. Since incretin response is markedly diminished in Asian populations, these agents can be used to achieve satisfactory glycemic control in Nepalese T2DM patients.JCMS Nepal. 2016;12(1:28-32.

  5. Leishmania major: disruption of signal peptidase type I and its consequences on survival, growth and infectivity.

    Science.gov (United States)

    Taheri, Tahereh; Salmanian, Ali-Hatef; Gholami, Elham; Doustdari, Fatemeh; Zahedifard, Farnaz; Rafati, Sima

    2010-10-01

    Leishmania major (L. major) signal peptidase type I (SPase I) is an endopeptidase encoded by a single-copy gene. In all organisms, SPase I is responsible for removing the signal peptide from secretory pre-proteins and releasing mature proteins to cellular or extra-cellular space. In this study, the role of SPase I in L. major is investigated by gene deletion using homologous recombination (HR). The null mutant of SPase I was not possible to create, suggesting that SPase I is an essential gene for parasite survival. The obtained heterozygote mutant by disrupting one allele of SPase I in L. major showed significantly reduced level of infectivity in bone marrow-derived macrophages. In addition, the heterozygote mutants are unable to cause cutaneous lesion in susceptible BALB/c mice. This is the first report showing that SPase I may have an important role in Leishmania infectivity, e.g. in differentiation and survival of amastigotes. Apparently, the SPase I expression is not essential for in vitro growth of the parasite.

  6. Dipeptidyl peptidase-4 expression in pancreatic tissue from patients with congenital hyperinsulinism.

    Science.gov (United States)

    Rahman, Sofia A; Senniappan, Senthil; Sherif, Maha; Tahir, Sophia; Hussain, Khalid

    2015-01-01

    Congenital hyperinsulinism (CHI) is caused by unregulated insulin release and leads to hyperinsulinaemic-hypoglycaemia (HH). Glucagon like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), peptide YY (PYY) and the enzyme; dipeptidyl peptidase-4 (DPP-4) all regulate appetite and glucose homeostasis. These proteins have been identified as possible contributors to HH but the mechanism remains poorly understood. We aimed to look at the expression pattern of pancreatic DPP-4 in children with focal and diffuse CHI (FCHI and DCHI, respectively). Using immunohistochemistry; we determined DPP-4 expression patterns in the pancreas of CHI patients. DPP-4 was found to be expressed in the pancreatic β, α and δ-cells in and around the focal area. However, it was predominantly co-localised with β-cells in the paediatric tissue samples. Additionally, proliferating β-cells expressed DPP-4 in DCHI, which was absent in the FCHI pancreas. Insulin was found to be present in the exocrine acini and duct cells of the DCHI pancreas suggestive of exocrine to endocrine transdifferentiation. Furthermore, 6 medically-unresponsive DCHI pancreatic samples showed an up-regulation of total pancreatic DPP-4 expression. In conclusion; the expression studies have shown DPP-4 to be altered in HH, however, further work is required to understand the underlying role for this enzyme.

  7. Tyrosine 547 constitutes an essential part of the catalytic mechanism of dipeptidyl peptidase IV.

    Science.gov (United States)

    Bjelke, Jais R; Christensen, Jesper; Branner, Sven; Wagtmann, Nicolai; Olsen, Christina; Kanstrup, Anders B; Rasmussen, Hanne B

    2004-08-13

    Human dipeptidyl peptidase IV (DPP-IV) is a ubiquitously expressed type II transmembrane serine protease. It cleaves the penultimate positioned prolyl bonds at the N terminus of physiologically important peptides such as the incretin hormones glucagon-like peptide 1 and glucose-dependent insulinotropic peptide. In this study, we have characterized different active site mutants. The Y547F mutant as well as the catalytic triad mutants S630A, D708A, and H740L showed less than 1% wild type activity. X-ray crystal structure analysis of the Y547F mutant revealed no overall changes compared with wild type apoDPP-IV, except the ablation of the hydroxyl group of Tyr(547) and a water molecule positioned in close proximity to Tyr(547). To elucidate further the reaction mechanism, we determined the crystal structure of DPP-IV in complex with diisopropyl fluorophosphate, mimicking the tetrahedral intermediate. The kinetic and structural findings of the tyrosine residue are discussed in relation to the catalytic mechanism of DPP-IV and to the inhibitory mechanism of the 2-cyanopyrrolidine class of potent DPP-IV inhibitors, proposing an explanation for the specificity of this class of inhibitors for the S9b family among serine proteases.

  8. Increased Plasma Dipeptidyl Peptidase-4 Activities in Patients with Coronary Artery Disease

    Science.gov (United States)

    Yang, Guang; Li, Yuzi; Cui, Lan; Jiang, Haiying; Li, Xiang; Jin, Chunzi; Jin, Dehao; Zhao, Guangxian; Jin, Jiyong; Sun, Rui; Piao, Limei; Xu, Wenhu; Fang, Chenghu; Lei, Yanna; Yuan, Kuichang; Xuan, Chunhua; Ding, Dazi

    2016-01-01

    Dipeptidyl peptidase-4 (DPP4) is one of the most potent mammalian serine proteases participated in the pathogenesis of subclinical atherosclerosis. Here we investigated whether the plasma soluble form of DPP4 is associated with the prevalence of coronary artery disease (CAD) with and without diabetes mellitus (DM). A cross-sectional study was conducted of 496 aged 26–81 years with (n = 362) and without (n = 134) CAD. Plasma DPP4 activity, high sensitive C-reactive protein (hs-CRP), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein levels were measured. The coronary atherosclerotic plaques were evaluated by coronary angiography. The CAD patients with (n = 84) and without (n = 278) DM had significantly higher DPP4 levels (11.8 ± 3.1 vs. 6.9 ± 3.5 ng/mL, Psyntax scores. A multiple logistic regression analysis demonstrated that plasma DPP4 activity was independent predictor of CAD (odds ratio, 1.56; 95% CI, 1.19–1.73; P<0.01). Our study shows that increased DPP4 activity levels are associated with the presence of CAD and that the plasma DPP4 level serves as a novel biomarker for CAD even without DM. PMID:27654253

  9. The Binding Site of Human Adenosine Deaminase for Cd26/Dipeptidyl Peptidase IV

    Science.gov (United States)

    Richard, Eva; Arredondo-Vega, Francisco X.; Santisteban, Ines; Kelly, Susan J.; Patel, Dhavalkumar D.; Hershfield, Michael S.

    2000-01-01

    Human, but not murine, adenosine deaminase (ADA) forms a complex with the cell membrane protein CD26/dipeptidyl peptidase IV. CD26-bound ADA has been postulated to regulate extracellular adenosine levels and to modulate the costimulatory function of CD26 on T lymphocytes. Absence of ADA–CD26 binding has been implicated in causing severe combined immunodeficiency due to ADA deficiency. Using human–mouse ADA hybrids and ADA point mutants, we have localized the amino acids critical for CD26 binding to the helical segment 126–143. Arg142 in human ADA and Gln142 in mouse ADA largely determine the capacity to bind CD26. Recombinant human ADA bearing the R142Q mutation had normal catalytic activity per molecule, but markedly impaired binding to a CD26+ ADA-deficient human T cell line. Reduced CD26 binding was also found with ADA from red cells and T cells of a healthy individual whose only expressed ADA has the R142Q mutation. Conversely, ADA with the E217K active site mutation, the only ADA expressed by a severely immunodeficient patient, showed normal CD26 binding. These findings argue that ADA binding to CD26 is not essential for immune function in humans. PMID:11067872

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

    Science.gov (United States)

    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. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Staphylococcus aureus type I signal peptidase: essential or not essential, that’s the question

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    Wouter L.W. Hazenbos

    2017-03-01

    Full Text Available Secretion of proteins into the extracellular environment is crucial for the normal physiology and virulence of pathogenic bacteria. Type I signal peptidase (SPase I mediates the final step of bacterial secretion, by cleaving proteins at their signal peptide once they are translocated by the Sec or twin-arginine (Tat translocon. SPase I has long been thought to be essential for viability in multiple bacterial pathogens. Challenging this view, we and others have recently created Staphylococcus aureus bacteria lacking the SPase I SpsB that are viable and able to grow in vitro when over-expressing a native gene cassette encoding for a putative ABC transporter. This transporter apparently compensates for SpsB’s essential function by mediating alternative cleavage of a subset of proteins at a site distinct from the SpsB-cleavage site, leading to SpsB-independent secretion. This alternative secretion system also drives the main mechanism of resistance to an arylomycin-derived SpsB inhibitor, by means of mutations in a putative transcriptional repressor (cro/cI causing over-expression of the ABC transporter. These findings raise multiple interesting biological questions. Unraveling the mechanism of SpsB-independent secretion may provide an interesting twist to the paradigm of bacterial secretion.

  12. Competitive Inhibition of the Endoplasmic Reticulum Signal Peptidase by Non-cleavable Mutant Preprotein Cargos.

    Science.gov (United States)

    Cui, Jingqiu; Chen, Wei; Sun, Jinhong; Guo, Huan; Madley, Rachel; Xiong, Yi; Pan, Xingyi; Wang, Hongliang; Tai, Andrew W; Weiss, Michael A; Arvan, Peter; Liu, Ming

    2015-11-20

    Upon translocation across the endoplasmic reticulum (ER) membrane, secretory proteins are proteolytically processed to remove their signal peptide by signal peptidase (SPase). This process is critical for subsequent folding, intracellular trafficking, and maturation of secretory proteins. Prokaryotic SPase has been shown to be a promising antibiotic target. In contrast, to date, no eukaryotic SPase inhibitors have been reported. Here we report that introducing a proline immediately following the natural signal peptide cleavage site not only blocks preprotein cleavage but also, in trans, impairs the processing and maturation of co-expressed preproteins in the ER. Specifically, we find that a variant preproinsulin, pPI-F25P, is translocated across the ER membrane, where it binds to the catalytic SPase subunit SEC11A, inhibiting SPase activity in a dose-dependent manner. Similar findings were obtained with an analogous variant of preproparathyroid hormone, demonstrating that inhibition of the SPase does not depend strictly on the sequence or structure of the downstream mature protein. We further show that inhibiting SPase in the ER impairs intracellular processing of viral polypeptides and their subsequent maturation. These observations suggest that eukaryotic SPases (including the human ortholog) are, in principle, suitable therapeutic targets for antiviral drug design.

  13. Effect of acute hypoxic shock on the rat brain morphology and tripeptidyl peptidase I activity.

    Science.gov (United States)

    Petrova, Emilia B; Dimitrova, Mashenka B; Ivanov, Ivaylo P; Pavlova, Velichka G; Dimitrova, Stella G; Kadiysky, Dimitar S

    2016-06-01

    Hypoxic events are known to cause substantial damage to the hippocampus, cerebellum and striatum. The impact of hypoxic shock on other brain parts is not sufficiently studied. Recent studies show that tripeptidyl peptidase I (TPPI) activity in fish is altered after a hypoxic stress pointing out at a possible enzyme involvement in response to hypoxia. Similar studies are not performed in mammals. In this work, the effect of sodium nitrite-induced acute hypoxic shock on the rat brain was studied at different post-treatment periods. Morphological changes in cerebral cortex, cerebellum, medulla oblongata, thalamus, mesencephalon and pons were assessed using silver-copper impregnation for neurodegeneration. TPPI activity was biochemically assayed and localized by enzyme histochemistry. Although less vulnerable to oxidative stress, the studied brain areas showed different histopathological changes, such as neuronal loss and tissue vacuolization, dilatation of the smallest capillaries and impairment of neuronal processes. TPPI activity was strictly regulated following the hypoxic stress. It was found to increase 12-24h post-treatment, then decreased followed by a slow process of recovery. The enzyme histochemistry revealed a temporary enzyme deficiency in all types of neurons. These findings indicate a possible involvement of the enzyme in rat brain response to hypoxic stress.

  14. Identification and characterization of dipeptidyl peptidase IV enzyme activity in the American crocodile (Crocodylus acutus).

    Science.gov (United States)

    Merchant, Mark; Mead, Stephanie; McAdon, Charles; McFatter, Justin; Wasilewski, Joe

    2010-07-01

    Serum from the American crocodile was assayed for dipeptidyl peptidase IV (DPP4) activity. We measured the DPP4-mediated hydrolysis of Ala-Pro-AFC. The generation of AFC was dependent on the titer of serum, with significant DPP4 activity (0.20 + or - 0.03 nmol product formed) measured using only 2 microL of crocodile serum, with maximum activity measured using 500 microL of serum. The hydrolysis of substrate was inhibited in a concentration-dependent manner by diprotin A, a specific inhibitor of DPP4 activity, indicating that this activity was due to the presence of DPP4. The crocodile serum DPP4 exhibited classical Michaelis-Menten kinetics, with K(m) and V(max) extrapolated, by double-reciprocal plot, to be 14.7 + or - 1.3 microM and 75.5 + or - 4.3 nmol/min, respectively. Crocodile DPP4 catalyzed the hydrolysis of Ala-Pro-AFC rapidly, with substantial activity measured within 5 min of the addition of substrate. After an initial rapid increase in activity, near maximal activity (7.43 + or - 0.24 nmol product formed) measured at 180 min. Crocodile serum DPP4 activity was temperature-dependent, with steadily increased activity from 5 to 40 degrees C.

  15. Production of leucine amino peptidase in lab scale bioreactors using Streptomyces gedanensis.

    Science.gov (United States)

    Rahulan, Raji; Dhar, Kiran S; Madhavan Nampoothiri, K; Pandey, Ashok

    2011-09-01

    Studies were conducted on the production of leucine amino peptidase (LAP) by Streptomyces gedanensis to ascertain the performance of the process in shake flask, parallel fermenter and 5-L fermenter utilizing soy bean meal as the carbon source. Experiments were conducted to analyze the effects of aeration and agitation rate on cell growth and LAP production. The results unveiled that an agitation rate of 300 rpm, 50% dissolved oxygen (DO) upholding and 0.15 vvm strategies were the optimal for the enzyme production, yielding 22.72 ± 0.11 IU/mL LAP in parallel fermenter which was comparable to flask level (24.65 ± 0.12 IU/mL LAP) fermentation. Further scale-up, in 5-L fermenter showed 50% DO and 1 vvm aeration rate was the best, producing optimum and the production was 20.09 ± 0.06 IU/mL LAP. The information obtained could be useful to design a strategy to improve a large-scale bioreactor cultivation of cells and production of LAP.

  16. An angiotensin-(1-7) peptidase in the kidney cortex, proximal tubules, and human HK-2 epithelial cells that is distinct from insulin-degrading enzyme.

    Science.gov (United States)

    Wilson, Bryan A; Cruz-Diaz, Nildris; Marshall, Allyson C; Pirro, Nancy T; Su, Yixin; Gwathmey, TanYa M; Rose, James C; Chappell, Mark C

    2015-03-15

    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 ANG II. We previously identified an peptidase that preferentially metabolized ANG-(1-7) to ANG-(1-4) in the brain medulla and cerebrospinal fluid (CSF) of sheep (Marshall AC, Pirro NT, Rose JC, Diz DI, Chappell MC. J Neurochem 130: 313-323, 2014); thus the present study established the expression of the peptidase in the kidney. Utilizing a sensitive HPLC-based approach, we demonstrate a peptidase activity that hydrolyzed ANG-(1-7) to ANG-(1-4) in the sheep cortex, isolated tubules, and human HK-2 renal epithelial cells. The peptidase was markedly sensitive to the metallopeptidase inhibitor JMV-390; human HK-2 cells expressed subnanomolar sensitivity (IC50 = 0.5 nM) and the highest specific activity (123 ± 5 fmol·min(-1)·mg(-1)) compared with the tubules (96 ± 12 fmol·min(-1)·mg(-1)) and cortex (107 ± 9 fmol·min(-1)·mg(-1)). The peptidase was purified 41-fold from HK-2 cells; the activity was sensitive to JMV-390, the chelator o-phenanthroline, and the mercury-containing compound p-chloromercuribenzoic acid (PCMB), but not to selective inhibitors against neprilysin, neurolysin and thimet oligopeptidase. Both ANG-(1-7) and its endogenous analog [Ala(1)]-ANG-(1-7) (alamandine) were preferentially hydrolyzed by the peptidase compared with ANG II, [Asp(1)]-ANG II, ANG I, and ANG-(1-12). Although the ANG-(1-7) peptidase and insulin-degrading enzyme (IDE) share similar inhibitor characteristics of a metallothiolendopeptidase, we demonstrate marked differences in substrate specificity, which suggest these peptidases are distinct. We conclude that an ANG-(1-7) peptidase is expressed within the renal proximal tubule and may play a potential role in the renal renin-angiotensin system to regulate ANG-(1-7) tone.

  17. Serpin peptidase inhibitor (SERPINB5) haplotypes are associated with susceptibility to hepatocellular carcinoma

    Science.gov (United States)

    Yang, Shun-Fa; Yeh, Chao-Bin; Chou, Ying-Erh; Lee, Hsiang-Lin; Liu, Yu-Fan

    2016-05-01

    Hepatocellular carcinoma (HCC) represents the second leading cause of cancer-related death worldwide. The serpin peptidase inhibitor SERPINB5 is a tumour-suppressor gene that promotes the development of various cancers in humans. However, whether SERPINB5 gene variants play a role in HCC susceptibility remains unknown. In this study, we genotyped 6 SNPs of the SERPINB5 gene in an independent cohort from a replicate population comprising 302 cases and 590 controls. Additionally, patients who had at least one rs2289520 C allele in SERPINB5 tended to exhibit better liver function than patients with genotype GG (Child-Pugh grade A vs. B or C; P = 0.047). Next, haplotype blocks were reconstructed according to the linkage disequilibrium structure of the SERPINB5 gene. A haplotype “C-C-C” (rs17071138 + rs3744941 + rs8089204) in SERPINB5-correlated promoter showed a significant association with an increased HCC risk (AOR = 1.450 P = 0.031). Haplotypes “T-C-A” and “C-C-C” (rs2289519 + rs2289520 + rs1455555) located in the SERPINB5 coding region had a decreased (AOR = 0.744 P = 0.031) and increased (AOR = 1.981 P = 0.001) HCC risk, respectively. Finally, an additional integrated in silico analysis confirmed that these SNPs affected SERPINB5 expression and protein stability, which significantly correlated with tumour expression and subsequently with tumour development and aggressiveness. Taken together, our findings regarding these biomarkers provide a prediction model for risk assessment.

  18. Obese patients have higher circulating protein levels of dipeptidyl peptidase IV.

    Science.gov (United States)

    Stengel, Andreas; Goebel-Stengel, Miriam; Teuffel, Pauline; Hofmann, Tobias; Buße, Petra; Kobelt, Peter; Rose, Matthias; Klapp, Burghard F

    2014-11-01

    Dipeptidyl peptidase IV (DPPIV) is a protease with broad distribution involved in various homeostatic processes such as immune defense, psychoneuroendocrine functions and nutrition. While DPPIV protein levels were investigated in patients with hyporectic disorders, less is known under conditions of obesity. Therefore, we investigated DPPIV across a broad range of body mass index (BMI). Blood samples from hospitalized patients with normal weight (BMI 18.5-25 kg/m(2)), anorexia nervosa (BMI 50 kg/m(2), n = 15/group) were tested cross-sectionally and DPPIV concentration and total enzyme activity and the DPPIV targets, pancreatic polypeptide (PP) and glucagon-like peptide (GLP-1) were measured. DPPIV protein expression was detected in human plasma indicated by a strong band at the expected size of 110 kDa and another major band at 50 kDa, likely representing a fragment comprised of two heavy chains. Obese patients had higher DPPIV protein levels compared to normal weight and anorexics (+50%, p0.05), while the concentration/activity ratio was higher in obese patients (p<0.05). Plasma PP levels were highest in anorexic patients (∼ 2-fold increase compared to other groups, p<0.05), whereas GLP-1 did not differ among groups (p<0.05). Taken together, circulating DPPIV protein levels depend on body weight with increased levels in obese resulting in an increased concentration/activity ratio. Since DPPIV deactivates food intake-inhibitory hormones like PP, an increased DPPIV concentration/activity ratio might contribute to reduced food intake-inhibitory signaling under conditions of obesity.

  19. Bis-Pyrano Prenyl Isoflavone Improves Glucose Homeostasis by Inhibiting Dipeptidyl Peptidase-4 in Hyperglycemic Rats.

    Science.gov (United States)

    Altenhofen, Delsi; da Luz, Gabrielle; Frederico, Marisa Jádna Silva; Venzke, Dalila; Brich, Mayara; Vigil, Silvana; Fröde, Tania Silvia; Linares, Carlos Eduardo Blanco; Pizzolatti, Moacir Geraldo; Silva, Fátima Regina Mena Barreto

    2017-01-01

    Isoflavones widely distributed in plants prevent diabetes. This study investigated the in vivo and in vitro effect of 3',4'-dihydroxy-6″,6″,6″',6″'-tetramethylbis(pyrano[2″,3″:5,6::2″',3″':7,8]isoflavone (bis-pyrano prenyl isoflavone) on glucose homeostasis in hyperglycemic rats. The ethyl acetate fraction from aerial parts of Polygala molluginifolia that contain isoflavones was assayed on glucose tolerance, on in vitro maltase activity and on protein glycation. The isoflavone bis-pyrano prenyl isolated from this fraction was investigated on glucose homeostasis. The in vivo action of the isoflavone exhibits an anti-hyperglycemic effect by improving glucose tolerance, augmenting the liver glycogen, inhibiting maltase activity, and stimulating glucagon-like peptide-1 (GLP-1) and insulin secretion. The in vitro isoflavone inhibits dipeptidyl peptidase-4 (DPP-4) activity since the glucose tolerance was improved in the presence of the isoflavone as much as sitagliptin, an inhibitor of DPP-4. However, the co-incubation with isoflavone and sitagliptin exhibited an additive anti-hyperglycemic action. The isoflavone increased the GLP-1 faster than the positive hyperglycemic group, which shows that the intestine is a potential target. Thus, to clarify the main site of action in which isoflavone improves glucose balance, the in vitro mechanism of action of this compound was tested in intestine using calcium influx as a trigger for the signal pathways for GLP-1 secretion. The isoflavone stimulates calcium influx in intestine and its mechanism involves voltage-dependent calcium channels, phospholipase C, protein kinase C, and stored calcium contributing for GLP-1 secretion. In conclusion, the isoflavone regulates glycaemia by acting mainly in a serum target, the DPP-4 inhibitor. Furthermore, the long-term effect of isoflavone prevents protein glycation. J. Cell. Biochem. 118: 92-103, 2017. © 2016 Wiley Periodicals, Inc.

  20. Predictive Factors for Efficacy of Dipeptidyl Peptidase-4 Inhibitors in Patients with Type 2 Diabetes Mellitus.

    Science.gov (United States)

    Yagi, Shusuke; Aihara, Ken-Ichi; Akaike, Masashi; Fukuda, Daiju; Salim, Hotimah Masdan; Ishida, Masayoshi; Matsuura, Tomomi; Ise, Takayuki; Yamaguchi, Koji; Iwase, Takashi; Yamada, Hirotsugu; Soeki, Takeshi; Wakatsuki, Tetsuzo; Shimabukuro, Michio; Matsumoto, Toshio; Sata, Masataka

    2015-08-01

    Predictive factors for the efficacy of dipeptidyl peptidase-4 (DPP-4) inhibitors for lowering glycosylated hemoglobin (HbA1c) remain unclear in patients with type 2 diabetes mellitus. The aim of this study is therefore to clarify predictive factors of the efficacy of DPP-4 inhibitors for lowering HbA1c after 12 months of treatment. A total of 191 consecutive type 2 diabetic patients (male sex 55%, mean age, 68.3±35.8 years), who had been treated with DPP-4 inhibitors for 12 months, were enrolled in this study and evaluated retrospectively. After 12 months of DPP-4 inhibitor treatment, random blood glucose level, and HbA1c level, decreased from 167±63 to 151±49 mg/dL (P<0.01), and from 7.5%±1.3% to 6.9%±0.9% (P<0.01) respectively, without severe side effects. Multiple regression analysis showed that predictors of DPP-4 inhibitor treatment efficacy in lowering HbA1c level after 12 months were a decrease in HbA1c level after 3 months of treatment, a high baseline HbA1c level, a low baseline body mass index, and the absence of coronary artery disease. Most suitable candidates for treatment with DPP-4 inhibitors are diabetics who are not obese and do not have coronary artery disease. In addition, long-term efficacy of DPP-4 inhibitors can be predicted by decrement of HbA1c after 3 months of treatment.

  1. Predictive Factors for Efficacy of Dipeptidyl Peptidase-4 Inhibitors in Patients with Type 2 Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    Shusuke Yagi

    2015-08-01

    Full Text Available BackgroundPredictive factors for the efficacy of dipeptidyl peptidase-4 (DPP-4 inhibitors for lowering glycosylated hemoglobin (HbA1c remain unclear in patients with type 2 diabetes mellitus. The aim of this study is therefore to clarify predictive factors of the efficacy of DPP-4 inhibitors for lowering HbA1c after 12 months of treatment.MethodsA total of 191 consecutive type 2 diabetic patients (male sex 55%, mean age, 68.3±35.8 years, who had been treated with DPP-4 inhibitors for 12 months, were enrolled in this study and evaluated retrospectively.ResultsAfter 12 months of DPP-4 inhibitor treatment, random blood glucose level, and HbA1c level, decreased from 167±63 to 151±49 mg/dL (P<0.01, and from 7.5%±1.3% to 6.9%±0.9% (P<0.01 respectively, without severe side effects. Multiple regression analysis showed that predictors of DPP-4 inhibitor treatment efficacy in lowering HbA1c level after 12 months were a decrease in HbA1c level after 3 months of treatment, a high baseline HbA1c level, a low baseline body mass index, and the absence of coronary artery disease.ConclusionMost suitable candidates for treatment with DPP-4 inhibitors are diabetics who are not obese and do not have coronary artery disease. In addition, long-term efficacy of DPP-4 inhibitors can be predicted by decrement of HbA1c after 3 months of treatment.

  2. Dipeptidyl Peptidase-4 Inhibitors, Peripheral Arterial Disease, and Lower Extremity Amputation Risk in Diabetic Patients.

    Science.gov (United States)

    Chang, Chun-Chin; Chen, Yung-Tai; Hsu, Chien-Yi; Su, Yu-Wen; Chiu, Chun-Chih; Leu, Hsin-Bang; Huang, Po-Hsun; Chen, Jaw-Wen; Lin, Shing-Jong

    2017-03-01

    Recent studies have elucidated the vascular protective effects of dipeptidyl peptidase-4 (DPP-4) inhibitors. However, to date, no large-scale studies have been carried out to determine the impact of DPP-4 inhibitors on the occurrence of peripheral arterial disease, and lower extremity amputation risk in patients with type 2 diabetes mellitus. We conducted a retrospective registry analysis using Taiwan's National Health Insurance Research Database to investigate the correlation between the use of DPP-4 inhibitors and risk of peripheral arterial disease in patients with type 2 diabetes mellitus. A total of 82,169 propensity score-matched pairs of DPP-4 inhibitor users and nonusers with type 2 diabetes mellitus were examined for the period 2009 to 2011. The mean age of the study subjects was 58.9 ± 12.0 years, and 54% of subjects were male. During the mean follow-up of 3.0 years (maximum, 4.8 years), a total of 3369 DPP-4 inhibitor users and 3880 DPP-4 inhibitor nonusers were diagnosed with peripheral arterial disease. Compared with nonusers, DPP-4 inhibitor users were associated with a lower risk of peripheral arterial disease (hazard ratio 0.84; 95% confidence interval, 0.80-0.88). Additionally, DPP-4 inhibitor users had a decreased risk of lower-extremity amputation than nonusers (hazard ratio 0.65; 95% confidence interval, 0.54-0.79). The association between use of DPP-4 inhibitors and risk of peripheral arterial disease was also consistent in subgroup analysis. This large-scale nationwide population-based cohort study is the first to demonstrate that treatment with DPP-4 inhibitors is associated with lower risk of peripheral arterial disease occurrence and limb amputation in patients with type 2 diabetes mellitus. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Structure of the nisin leader peptidase NisP revealing a C-terminal autocleavage activity.

    Science.gov (United States)

    Xu, Yueyang; Li, Xin; Li, Ruiqing; Li, Shanshan; Ni, Hongqian; Wang, Hui; Xu, Haijin; Zhou, Weihong; Saris, Per E J; Yang, Wen; Qiao, Mingqiang; Rao, Zihe

    2014-06-01

    Nisin is a widely used antibacterial lantibiotic polypeptide produced by Lactococcus lactis. NisP belongs to the subtilase family and functions in the last step of nisin maturation as the leader-peptide peptidase. Deletion of the nisP gene in LAC71 results in the production of a non-active precursor peptide with the leader peptide unremoved. Here, the 1.1 Å resolution crystal structure of NisP is reported. The structure shows similarity to other subtilases, which can bind varying numbers of Ca atoms. However, no calcium was found in this NisP structure, and the predicted calcium-chelating residues were placed so as to not allow NisP to bind a calcium ion in this conformation. Interestingly, a short peptide corresponding to its own 635-647 sequence was found to bind to the active site of NisP. Biochemical assays and native mass-spectrometric analysis confirmed that NisP possesses an auto-cleavage site between residues Arg647 and Ser648. Further, it was shown that NisP mutated at the auto-cleavage site (R647P/S648P) had full catalytic activity for nisin leader-peptide cleavage, although the C-terminal region of NisP was no longer cleaved. Expressing this mutant in L. lactis LAC71 did not affect the production of nisin but did decrease the proliferation rate of the bacteria, suggesting the biological significance of the C-terminal auto-cleavage of NisP.

  4. Kinetics of reactions of the Actinomadura R39 DD-peptidase with specific substrates.

    Science.gov (United States)

    Adediran, S A; Kumar, Ish; Nagarajan, Rajesh; Sauvage, Eric; Pratt, R F

    2011-01-25

    The Actinomadura R39 DD-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(D-cysteinyl)propanoyl-D-alanyl-D-alanine and 3-(D-cysteinyl)propanoyl-D-alanyl-D-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed DD-carboxypeptidase, DD-transpeptidase, and DD-endopeptidase activities. These results confirm the specificity of the enzyme for a free D-amino acid at the N-terminus of good substrates and indicated a preference for extended D-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a DD-endopeptidase in vivo. pH-rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases.

  5. Dipeptidyl peptidase-4 inhibitors in type 2 diabetes therapy--focus on alogliptin.

    Science.gov (United States)

    Capuano, Annalisa; Sportiello, Liberata; Maiorino, Maria Ida; Rossi, Francesco; Giugliano, Dario; Esposito, Katherine

    2013-01-01

    Type 2 diabetes mellitus is a complex and progressive disease that is showing an apparently unstoppable increase worldwide. Although there is general agreement on the first-line use of metformin in most patients with type 2 diabetes, the ideal drug sequence after metformin failure is an area of increasing uncertainty. New treatment strategies target pancreatic islet dysfunction, in particular gut-derived incretin hormones. Inhibition of the enzyme dipeptidyl peptidase-4 (DPP-4) slows degradation of endogenous glucagon-like peptide-1 (GLP-1) and thereby enhances and prolongs the action of the endogenous incretin hormones. The five available DPP-4 inhibitors, also known as 'gliptins' (sitagliptin, vildagliptin, saxagliptin, linagliptin, alogliptin), are small molecules used orally with similar overall clinical efficacy and safety profiles in patients with type 2 diabetes. The main differences between the five gliptins on the market include: potency, target selectivity, oral bioavailability, long or short half-life, high or low binding to plasma proteins, metabolism, presence of active or inactive metabolites, excretion routes, dosage adjustment for renal and liver insufficiency, and potential drug-drug interactions. On average, treatment with gliptins is expected to produce a mean glycated hemoglobin (HbA1c) decrease of 0.5%-0.8%, with about 40% of diabetic subjects at target for the HbA1c goal pioglitazone, glibenclamide, voglibose, or insulin therapy significantly improves glycemic control compared with placebo in adult or elderly patients with inadequately controlled type 2 diabetes. In the EXAMINE trial, alogliptin is being compared with placebo on cardiovascular outcomes in approximately 5,400 patients with type 2 diabetes. In clinical studies, DPP-4 inhibitors were generally safe and well tolerated. However, there are limited data on their tolerability, due to their relatively recent marketing approval. Alogliptin will be used most when avoidance of

  6. Disposition of vildagliptin, a novel dipeptidyl peptidase 4 inhibitor, in rats and dogs.

    Science.gov (United States)

    He, Handan; Tran, Phi; Yin, Hequn; Smith, Harold; Flood, Dennis; Kramp, Roger; Filipeck, Ron; Fischer, Volker; Howard, Dan

    2009-03-01

    The pharmacokinetics, absorption, metabolism, and excretion of vildagliptin, a potent and orally active inhibitor of dipeptidyl peptidase 4, were evaluated in male rats and dogs. Vildagliptin was rapidly absorbed with peak plasma concentrations occurring between 0.5 and 1.5 h. Moderate to high bioavailability was observed in both species (45-100%). The distribution and elimination half-lives of vildagliptin were short: 0.57 h [82% of area under the plasma drug concentration-time curve (AUC)] and 8.8 h in the rat and 0.05 and 0.89 h (87% of AUC) in the dog, respectively. The volume of distribution was 1.6 and 8.6 l/kg in dogs and rats, respectively, indicating moderate to high tissue distribution. The plasma clearance of vildagliptin was relatively high for the rat (2.9 l/h/kg) and dog (1.3 l/h/kg) compared with their hepatic blood flow. The major circulating components in plasma after an intravenous or oral dose were the parent compound (rat and dog), a carboxylic acid metabolite from the hydrolysis of the amide bond M15.3 (dog), and a carboxylic acid metabolite from the hydrolysis of the cyano moiety M20.7 (rat and dog). After intravenous dosing, urinary excretion of radioactivity (47.6-72.4%) was the major route of elimination for rats and dogs as 18.9 to 21.3% of the dose was excreted into urine as unchanged parent drug. The recovery was good in both species (81-100% of the dose). Vildagliptin was mainly metabolized before excretion in both species. Similar to plasma, the most predominant metabolite in excreta was M20.7 in rats and dogs, and another major metabolite in dogs was M15.3.

  7. Selective inhibition of dipeptidyl peptidase 4 by targeting a substrate-specific secondary binding site.

    Science.gov (United States)

    Kühn-Wache, Kerstin; Bär, Joachim W; Hoffmann, Torsten; Wolf, Raik; Rahfeld, Jens-Ulrich; Demuth, Hans-Ulrich

    2011-03-01

    Dipeptidyl peptidase 4/CD26 (DP4) is a multifunctional serine protease liberating dipeptide from the N-terminus of (oligo)peptides which can modulate the activity of these peptides. The enzyme is involved in physiological processes such as blood glucose homeostasis and immune response. DP4 substrate specificity is characterized in detail using synthetic dipeptide derivatives. The specificity constant k(cat)/K(m) strongly depends on the amino acid in P₁-position for proline, alanine, glycine and serine with 5.0 x 10⁵ M⁻¹ s⁻¹, 1.8 x 10⁴ M⁻¹ s⁻¹, 3.6 x 10² M⁻¹ s⁻¹, 1.1 x 10² M⁻¹ s⁻¹, respectively. By contrast, kinetic investigation of larger peptide substrates yields a different pattern. The specific activity of DP4 for neuropeptide Y (NPY) cleavage comprising a proline in P₁-position is the same range as the k(cat)/K(m) values of NPY derivatives containing alanine or serine in P₁-position with 4 x 10⁵ M⁻¹ s⁻¹, 9.5 x 10⁵ M⁻¹ s⁻¹ and 2.1 x 10⁵ M⁻¹ s⁻¹, respectively. The proposed existence of an additional binding region outside the catalytic center is supported by measurements of peptide substrates with extended chain length. This 'secondary' binding site interaction depends on the amino acid sequence in P₄'-P₈'-position. Interactions with this binding site could be specifically blocked for substrates of the GRF/glucagon peptide family. By contrast, substrates not belonging to this peptide family and dipeptide derivative substrates that only bind to the catalytic center of DP4 were not inhibited. This more selective inhibition approach allows, for the first time, to distinguish between substrate families by substrate-discriminating inhibitors.

  8. Berry and Citrus Phenolic Compounds Inhibit Dipeptidyl Peptidase IV: Implications in Diabetes Management

    Directory of Open Access Journals (Sweden)

    Junfeng Fan

    2013-01-01

    Full Text Available Beneficial health effects of fruits and vegetables in the diet have been attributed to their high flavonoid content. Dipeptidyl peptidase IV (DPP-IV is a serine aminopeptidase that is a novel target for type 2 diabetes therapy due to its incretin hormone regulatory effects. In this study, well-characterized anthocyanins (ANC isolated from berry wine blends and twenty-seven other phenolic compounds commonly present in citrus, berry, grape, and soybean, were individually investigated for their inhibitory effects on DPP-IV by using a luminescence assay and computational modeling. ANC from blueberry-blackberry wine blends strongly inhibited DPP-IV activity (IC50, 0.07 ± 0.02 to >300 μM. Of the twenty-seven phenolics tested, the most potent DPP-IV inhibitors were resveratrol (IC50, 0.6 ± 0.4 nM, luteolin (0.12 ± 0.01 μM, apigenin (0.14 ± 0.02 μM, and flavone (0.17 ± 0.01 μM, with IC50 values lower than diprotin A (4.21 ± 2.01 μM, a reference standard inhibitory compound. Analyses of computational modeling showed that resveratrol and flavone were competitive inhibitors which could dock directly into all three active sites of DPP-IV, while luteolin and apigenin docked in a noncompetitive manner. Hydrogen bonding was the main binding mode of all tested phenolic compounds with DPP-IV. These results indicate that flavonoids, particularly luteolin, apigenin, and flavone, and the stilbenoid resveratrol can act as naturally occurring DPP-IV inhibitors.

  9. Identification and Characterization of Noncovalent Interactions That Drive Binding and Specificity in DD-Peptidases and β-Lactamases.

    Science.gov (United States)

    Hargis, Jacqueline C; Vankayala, Sai Lakshmana; White, Justin K; Woodcock, H Lee

    2014-02-11

    Bacterial resistance to standard (i.e., β-lactam-based) antibiotics has become a global pandemic. Simultaneously, research into the underlying causes of resistance has slowed substantially, although its importance is universally recognized. Key to unraveling critical details is characterization of the noncovalent interactions that govern binding and specificity (DD-peptidases, antibiotic targets, versus β-lactamases, the evolutionarily derived enzymes that play a major role in resistance) and ultimately resistance as a whole. Herein, we describe a detailed investigation that elicits new chemical insights into these underlying intermolecular interactions. Benzylpenicillin and a novel β-lactam peptidomimetic complexed to the Stremptomyces R61 peptidase are examined using an arsenal of computational techniques: MD simulations, QM/MM calculations, charge perturbation analysis, QM/MM orbital analysis, bioinformatics, flexible receptor/flexible ligand docking, and computational ADME predictions. Several key molecular level interactions are identified that not only shed light onto fundamental resistance mechanisms, but also offer explanations for observed specificity. Specifically, an extended π-π network is elucidated that suggests antibacterial resistance has evolved, in part, due to stabilizing aromatic interactions. Additionally, interactions between the protein and peptidomimetic substrate are identified and characterized. Of particular interest is a water-mediated salt bridge between Asp217 and the positively charged N-terminus of the peptidomimetic, revealing an interaction that may significantly contribute to β-lactam specificity. Finally, interaction information is used to suggest modifications to current β-lactam compounds that should both improve binding and specificity in DD-peptidases and their physiochemical properties.

  10. SYNTHESIS AND PROCESSING OF ESCHERICHIA-COLI TEM-BETA-LACTAMASE AND BACILLUS-LICHENIFORMIS ALPHA-AMYLASE IN ESCHERICHIA-COLI : THE ROLE OF SIGNAL PEPTIDASE-I

    NARCIS (Netherlands)

    van Dijl, J M; SMITH, H; BRON, S; VENEMA, G

    A mutant of Escherichia coli, in which signal peptidase I synthesis can be regulated, was constructed. The mutant was used to study the effects of signal peptidase I limitation on the synthesis and efficiency of processing of two proteins: the periplasmic E. coli TEM-beta-lactamase and Bacillus

  11. Efficacy and safety of dipeptidyl peptidase-4 inhibitors as an add-on to insulin treatment in patients with Type 2 diabetes

    DEFF Research Database (Denmark)

    Frandsen, Christian S.; Madsbad, S

    2014-01-01

    diabetes. METHODS: We searched the MEDLINE and PubMed databases to identify all randomized controlled clinical trials evaluating dipeptidyl peptidase-4 inhibitors as an add-on to insulin in patients with Type 2 diabetes, which were selected for review. The abstracts and posters of the recent annual......AIM: To review and discuss the results from the clinical controlled trials comparing a dipeptidyl peptidase-4 inhibitor with placebo treatment as add-on to insulin treatment with respect to changes in HbA1c , weight, fasting plasma glucose, risk of hypoglycaemia and safety in patients with Type 2...... meetings of the American Diabetes Association and European Association for the Study of Diabetes were hand searched, as were the reference lists of articles identified. RESULTS: Adding a dipeptidyl peptidase-4 inhibitor to insulin treatment resulted in a glucose-lowering effect of ~ 6.6-8.7 mmol/mol (0...

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

    Science.gov (United States)

    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

  13. Glucagon-like peptide receptor agonists and dipeptidyl peptidase-4 inhibitors in the treatment of diabetes: a review of clinical trials

    DEFF Research Database (Denmark)

    Madsbad, Sten; Krarup, Thure; Deacon, Carolyn F;

    2008-01-01

    PURPOSE OF REVIEW: To discuss the virtues and shortcomings of the glucagon-like peptide-1 receptor agonists and the dipeptidyl peptidase-4 inhibitors in the treatment of type 2 diabetes. RECENT FINDINGS: The injectable glucagon-like peptide-1 receptor agonists exenatide significantly improves......-acting glucagon-like peptide-1 receptor agonists liraglutide and exenatide long-acting release reduce haemoglobin A1c by about 1.0-2.0% and have fewer gastrointestinal side-effects. The orally available dipeptidyl peptidase-4 inhibitors, that is sitagliptin and vildagliptin reduce haemoglobin A1c by 0...

  14. First results from $\\phi \\to K_{L}K_{S}$ decays with the KLOE detector

    CERN Document Server

    Adinolfi, M; Ambrosino, F; Andryakov, A; Antonelli, A; Antonelli, M; Anulli, F; Bacci, Cesare; Bankamp, A; Barbiellini, Guido; Bellini, F; Bencivenni, G; Bertolucci, Sergio; Bini, C; Bloise, C; Bocci, V; Bossi, F; Branchini, P; Bulychjov, S A; Cabibbo, G; Calcaterra, A; Caloi, R M; Campana, P; Capon, G; Carboni, G; Cardini, A; Casarsa, M; Cataldi, G; Ceradini, F; Cervelli, F; Cevenini, F; Chiefari, G; Ciambrone, P; Conetti, S; De Lucia, E; De Robertis, G; De Sangro, R; De Simone, P; De Zorzi, G; Dell'Agnello, S; Denig, A; Di Domenico, A; Di Donato, C; Di Falco, S; Doria, A; Drago, E; Elia, V; Erriquez, O; Farilla, A; Felici, G; Ferrari, A; Ferrer, M L; Finocchiaro, G; Forti, C; Franceschi, A; Franzini, P; Gao, M L; Gatti, C; Gauzzi, P; Giovannella, S; Golovatyuk, V M; Gorini, E; Grancagnolo, F; Grandegger, W; Graziani, E; Guarnaccia, P; Von Hagel, U; Han, H G; Han, S W; Huang, X; Incagli, M; Ingrosso, L; Jiang, Y Y; Kim, W; Kluge, W; Kulikov, V; Lacava, F; Lanfranchi, G; Lee-Franzini, Juliet; Lomtadze, T A; Luisi, C; Mao Chen Sheng; Martemyanov, M; Martini, A; Matsyuk, M; Mei, W; Merola, L; Messi, R; Miscetti, S; Moalem, A; Moccia, S; Moulson, M; Müller, S; Murtas, F; Napolitano, M; Nedosekin, A A; Panareo, M; Pacciani, L; Pagès, P; Palutan, M; Paoluzi, L; Pasqualucci, E; Passalacqua, L; Passaseo, M; Passeri, A; Patera, V; Petrolo, E; Petrucci, Guido; Picca, D; Pirozzi, G; Pistillo, C; Pollack, M E; Pontecorvo, L; Primavera, M; Ruggieri, F; Santangelo, P; Santovetti, E; Saracino, G; Schamberger, R D; Schwick, C; Sciascia, B; Sciubba, A; Scuri, F; Sfiligoi, I; Shan, J; Silano, P; Spadaro, T; Spagnolo, S; Spiriti, E; Stanescu, C; Tong, G L; Tortora, L; Valente, E; Valente, P; Valeriani, B; Venanzoni, G; Veneziano, Stefano; Wu, Y; Xie, Y G; Zhao, P P; Zhou, Y

    2001-01-01

    The KLOE experiment has collected 2.4 1/pb of integrated luminosity during the commissioning of the DAFNE phi-factory in 1999. The performance of the detector has been studied using phi -> KL KS decays collected during this period, yielding also first measurements of relevant K parameters such as masses and lifetimes. A clean Kshort -> pi+ pi- sample is used to select Klong -> 2pi CP-violating decays and Klong -> Kshort regeneration events in the detector material. Results on the regeneration probability in beryllium-alluminum alloy and carbon-fiber plus aluminum composite are presented.

  15. X-ray crystalline structures of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus, and its cys-free mutant.

    Science.gov (United States)

    Tanaka, H; Chinami, M; Mizushima, T; Ogasahara, K; Ota, M; Tsukihara, T; Yutani, K

    2001-07-01

    In order to elucidate the mechanism of the thermostability of proteins from hyperthermophiles, X-ray crystalline structures of pyrrolidone carboxyl peptidase from a hyperthermophile, Pyrococcus furiosus (PfPCP), and its mutant protein with Ser substituted at Cys142 and Cys188 were determined at 2.2 and 2.7 A resolution, respectively. The obtained structures were compared with those previously reported for pyrrolidone carboxyl peptidases from a hyperthermophilie, Thermococcus litoralis (TlPCP), and from a mesophile, Bacillus amyloliquefaciens (BaPCP). The PfPCP structure is a tetramer of four identical subunits similar to that of the TlPCP and BaPCP. The largest structural changes among the three PCPs were detected in the C-terminal protrusion, which interacts with that of another subunit. A comparison of the three structures indicated that the high stability of PfPCP is caused by increases in hydrophobic interactions and hydrogen bonds, the formation of an intersubunit ion-pair network, and improvement to an ideal conformation. On the basis of the structures of the three proteins, it can be concluded that PfPCP does not have any special factors responsible for its extremely high stability and that the conformational structure of PfPCP is superior in its combination of positive and negative stabilizing factors compared with BaPCP.

  16. Expression of a novel dipeptidyl peptidase 8 (DPP8) transcript variant, DPP8-v3, in human testis

    Institute of Scientific and Technical Information of China (English)

    Hui Zhu; Zuo-Min Zhou; Li Lu; Min Xu; Hui Wang; Jian-Min Li; Jia-Hao Sha

    2005-01-01

    Aim: To investigate the role of a novel dipeptidyl peptidase 8 transcript variant (DPP8-v3) gene in testis development and/or spermatogenesis. Methods: A human testis cDNA microarray was hybridized with mRNA of human adult and fetal testes. Differentially expressed clones were sequenced and characterized and their expression was analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Southern-blot analysis. Results: A new transcript variant of the human dipeptidyl peptidase (DPP8), exhibiting a 5-fold higher expression level in human adult than that in fetal testes, was cloned and was named DPP8 variant 3 (DPP8-v3). The full-length sequence of DPP8-v3was 3,030 bp, encoding a protein of 898 amino acids. Conclusion: DPP8-v3 is a novel human DPP8 transcript variant highly expressed in the adult testis. Similar to DPPⅣ, DPP8-v3 may play a key role in the immunoregulation of testes and accordingly may influence spermatogenesis and male fertility.

  17. Alterations in plasma dipeptidyl peptidase IV enzyme activity in depression and schizophrenia: effects of antidepressants and antipsychotic drugs.

    Science.gov (United States)

    Maes, M; De Meester, I; Scharpe, S; Desnyder, R; Ranjan, R; Meltzer, H Y

    1996-01-01

    Recently, our laboratory reported that the activity of dipeptidyl-peptidase IV (DPP IV) was significantly lower in the peripheral blood of major depressed patients than in normal controls. The present study examines plasma DPP IV activity in 43 major depressed and 13 schizophrenic subjects versus 21 normal controls and the effects of antidepressants and antipsychotic drugs on plasma DPP IV activity. DPP IV activity was significantly lower in major depressed subjects than in normal controls and schizophrenic subjects. There was a trend towards higher DPP IV activity in schizophrenic patients than in normal controls. There were no significant effects of antidepressants or neuroleptics on plasma DPP IV activity in depressed and schizophrenic patients, respectively. There were no significant relationships between plasma DPP IV activity and plasma cortisol or immune-inflammatory markers, such as serum interleukin-6 (IL-6) or soluble IL-2 receptor. A significant and positive correlation was found between plasma DPP IV and prolyl endopeptidase (PEP) enzyme activity in the study group as a whole and in schizophrenic subjects. The results support the hypothesis that lower and higher plasma DPP IV activities are trait markers of major depression and schizophrenia, respectively. It is concluded that alterations in the enzyme activity of peptidases, such as DPP IV and PEP, play a role in the pathophysiology of major depression and schizophrenia.

  18. Acid peptidase activity released from in vitro produced porcine embryos: a candidate marker to predict developmental competence.

    Science.gov (United States)

    Telugu, Bhanu Prakash V L; Spate, Lee; Prather, Randall S; Green, Jonathan A

    2009-04-01

    The ability to efficiently create high quality embryos, competent to produce normal viable offspring in vitro, facilitates diverse technological advancements in animal agriculture and assisted reproduction. Current methods for evaluation of embryos are predominantly based on morphological characteristics which are prone to potential bias of the scorer. Metabolic and genetic markers have also been explored for quality assessment, but they are cost prohibitive or require longer periods of time for evaluation. We hypothesized that secreted enzymes could provide another means of embryo quality assessment. In this report, we provide evidence that medium conditioned by porcine embryos often has proteolytic activity that operates in acidic conditions (acid peptidase activity or APA). The APA could be inhibited by pepstatin A, suggesting that the activity is derived from one or more aspartic peptidases. We also provide evidence that single embryos, incubated for as few as 24 hr, released enough APA that it was possible to measure it accurately at day 5 of culture. We also observed that such activity on day 6 could be positively correlated with advanced developmental stage and embryo quality. In addition, those embryos that were graded identically by morphological evaluations often differed in the amount of APA--with some being significantly higher than the experimental threshold value. Therefore, the APA of embryos might serve as an additional marker for evaluation of embryos.

  19. Gold Nanoparticle-Based Colorimetric and Electrochemical Methods for Dipeptidyl Peptidase-IV Activity Assay and Inhibitor Screening

    Directory of Open Access Journals (Sweden)

    Ning Xia

    2016-10-01

    Full Text Available We presented the colorimetric and electrochemical methods for determination of the dipeptidyl peptidase-IV (DPP-IV activity and screening of its inhibitor using gold nanoparticle (AuNP as the probe. In the colorimetric assay, the substrate peptide with a sequence of Arg-Pro-Arg induced the aggregation and color change of AuNPs, whereas cleavage of the peptide by DPP-IV prevented the aggregation of AuNPs. Furthermore, the aggregation of AuNPs in the solution was easily initiated on a solid/liquid (electrode/electrolyte surface, which induced a decrease in the electron-transfer resistance. However, once the peptide was clipped by DPP-IV, the assembly of AuNPs on electrode surface was prevented. Consequently, a higher electron-transfer resistance was observed. The colorimetric and electrochemical assays allowed for the determination of DPP-IV with the detection limits of 70 μU/mL and 0.55 μU/mL, respectively. Meanwhile, the proposed methods were used to determine DPP-IV inhibitor with satisfactory results. Both the colorimetric and electrochemical methods are simple, rapid and sufficiently sensitive for DPP-IV activity assay and inhibitor screening. The results also demonstrated that the AuNP-based colorimetric assay could be converted into an enhanced surface tethered electrochemical assay with improving sensitivity. The simple detection principle may be extended to the design of other peptidases biosensors with easy manipulation procedures.

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

    Directory of Open Access Journals (Sweden)

    Kirsten Schulz

    Full Text Available 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

  1. Impact of dipeptidyl peptidase-4 inhibitors on serum adiponectin: a meta-analysis.

    Science.gov (United States)

    Liu, Xin; Men, Peng; Wang, Yuhui; Zhai, Suodi; Liu, George

    2016-11-23

    Adiponectin, an adipose-specific protein, is negatively correlated with pro-atherogenic low-density lipoprotein cholesterol (LDL-C) and other cardiovascular risk factors such as insulin resistance. Therefore, low levels of adiponectin are associated with a higher risk for diabetes and cardiovascular disease. Dipeptidyl peptidase-4 inhibitors (DPP4i) have been used for the treatment of type 2 diabetes mellitus (T2DM) as reversible inhibitors through interacting with DPP4 substrate and increase serum incretins such as glucagon-like peptide-1 (GLP-1). The present study aimed to evaluate the effect of DPP4i on serum adiponectin in T2DM patients. The PubMed, Embase, and Cochrane library databases were searched from inception to February 2016. Randomized controlled trials, evaluating the DPP4i (sitagliptin and vildagliptin) versus comparator (placebo or active-comparison), in T2DM patients with duration of ≥ 12 weeks, were identified. Weighted differences in means of adiponectin levels were calculated by using a fixed or random-effects model. Ten randomized controlled trials, including 1,495 subjects, were identified. Compared with placebo, DPP4i (sitagliptin and vildagliptin) treatment significantly elevated adiponectin levels by 0.74 μg/mL (95% confidence interval [CI], 0.45 to 1.03) relative to that using an active-comparison by 0.00 μg/mL (95% CI, -0.57 to 0.56). Compared with active-comparison, vildagliptin treatment increased adiponectin levels by 0.32 μg/mL (95% CI, -0.01 to 0.65), whereas sitagliptin treatment decreased adiponectin levels by -0.24 μg/mL (95% CI, -1.07 to 0.58). Trials examining effects of other DPP4i were not found. Sitagliptin and vildagliptin increased serum adiponectin levels and had no stronger effect than traditional oral antidiabetic drugs. Further trials with larger sample size are needed to confirm the results and investigate the association between serum adiponectin levels and treatment of other DPP-4 inhibitors

  2. Cardiovascular effects of dipeptidyl peptidase-4 inhibitors: from risk factors to clinical outcomes.

    Science.gov (United States)

    Scheen, André J

    2013-05-01

    Dipeptidyl peptidase-4 (DPP-4) inhibitors (gliptins) are oral incretin-based glucose-lowering agents with proven efficacy and safety in the management of type 2 diabetes mellitus (T2DM). In addition, preclinical data and mechanistic studies suggest a possible additional non-glycemic beneficial action on blood vessels and the heart, via both glucagon-like peptide-1-dependent and glucagon-like peptide-1-independent effects. As a matter of fact, DPP-4 inhibitors improve several cardiovascular risk factors: they improve glucose control (mainly by reducing the risk of postprandial hyperglycemia) and are weight neutral; may lower blood pressure somewhat; improve postprandial (and even fasting) lipemia; reduce inflammatory markers; diminish oxidative stress; improve endothelial function; and reduce platelet aggregation in patients with T2DM. In addition, positive effects on the myocardium have been described in patients with ischemic heart disease. Results of post hoc analyses of phase 2/3 controlled trials suggest a possible cardioprotective effect with a trend (sometimes significant) toward lower incidence of major cardiovascular events with sitagliptin, vildagliptin, saxagliptin, linagliptin, or alogliptin compared with placebo or other active glucose-lowering agents. However, the definite relationship between DPP-4 inhibition and better cardiovascular outcomes remains to be proven. Major prospective clinical trials involving various DPP-4 inhibitors with predefined cardiovascular outcomes are under way in patients with T2DM and a high-risk cardiovascular profile: the Sitagliptin Cardiovascular Outcome Study (TECOS) on sitagliptin, the Saxagliptin Assessment of Vascular Outcomes Recorded in Patients With Diabetes Mellitus-Thrombolysis in Myocardial Infarction (SAVOR-TIMI) 53 trial on saxagliptin, the Cardiovascular Outcomes Study of Alogliptin in Subjects With Type 2 Diabetes and Acute Coronary Syndrome (EXAMINE) trial on alogliptin, and the Cardiovascular Outcome

  3. Dipeptidyl peptidase-4 inhibitors in type 2 diabetes therapy – focus on alogliptin

    Directory of Open Access Journals (Sweden)

    Capuano A

    2013-09-01

    Full Text Available Annalisa Capuano,1 Liberata Sportiello,1 Maria Ida Maiorino,2 Francesco Rossi,1 Dario Giugliano,2 Katherine Esposito3 1Department of Experimental Medicine, 2Department of Medical, Surgical, Neurological, Metabolic Sciences, and Geriatrics, 3Department of Clinical and Experimental Medicine and Surgery, Second University of Naples, Naples, Italy Abstract: Type 2 diabetes mellitus is a complex and progressive disease that is showing an apparently unstoppable increase worldwide. Although there is general agreement on the first-line use of metformin in most patients with type 2 diabetes, the ideal drug sequence after metformin failure is an area of increasing uncertainty. New treatment strategies target pancreatic islet dysfunction, in particular gut-derived incretin hormones. Inhibition of the enzyme dipeptidyl peptidase-4 (DPP-4 slows degradation of endogenous glucagon-like peptide-1 (GLP-1 and thereby enhances and prolongs the action of the endogenous incretin hormones. The five available DPP-4 inhibitors, also known as 'gliptins' (sitagliptin, vildagliptin, saxagliptin, linagliptin, alogliptin, are small molecules used orally with similar overall clinical efficacy and safety profiles in patients with type 2 diabetes. The main differences between the five gliptins on the market include: potency, target selectivity, oral bioavailability, long or short half-life, high or low binding to plasma proteins, metabolism, presence of active or inactive metabolites, excretion routes, dosage adjustment for renal and liver insufficiency, and potential drug–drug interactions. On average, treatment with gliptins is expected to produce a mean glycated hemoglobin (HbA1c decrease of 0.5%–0.8%, with about 40% of diabetic subjects at target for the HbA1c goal <7%. There are very few studies comparing DPP-4 inhibitors. Alogliptin as monotherapy or added to metformin, pioglitazone, glibenclamide, voglibose, or insulin therapy significantly improves glycemic control

  4. Dipeptidyl peptidase IV inhibition exerts renoprotective effects in rats with established heart failure

    Directory of Open Access Journals (Sweden)

    Daniel Francisco De Arruda Junior

    2016-07-01

    Full Text Available 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 four 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 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. Recombinant human tripeptidyl peptidase-1 infusion to the monkey CNS: Safety, pharmacokinetics, and distribution

    Energy Technology Data Exchange (ETDEWEB)

    Vuillemenot, Brian R., E-mail: bvuillemenot@bmrn.com [BioMarin Pharmaceutical Inc., Novato, CA (United States); Kennedy, Derek [BioMarin Pharmaceutical Inc., Novato, CA (United States); Reed, Randall P.; Boyd, Robert B. [Northern Biomedical Research, Inc., Muskegon, MI (United States); Butt, Mark T. [Tox Path Specialists, LLC, Hagerstown, MD (United States); Musson, Donald G.; Keve, Steve; Cahayag, Rhea; Tsuruda, Laurie S.; O' Neill, Charles A. [BioMarin Pharmaceutical Inc., Novato, CA (United States)

    2014-05-15

    CLN2 disease is caused by deficiency in tripeptidyl peptidase-1 (TPP1), leading to neurodegeneration and death. The safety, pharmacokinetics (PK), and CNS distribution of recombinant human TPP1 (rhTPP1) were characterized following a single intracerebroventricular (ICV) or intrathecal-lumbar (IT-L) infusion to cynomolgus monkeys. Animals received 0, 5, 14, or 20 mg rhTPP1, ICV, or 14 mg IT-L, in artificial cerebrospinal fluid (aCSF) vehicle. Plasma and CSF were collected for PK analysis. Necropsies occurred at 3, 7, and 14 days post-infusion. CNS tissues were sampled for rhTPP1 distribution. TPP1 infusion was well tolerated and without effect on clinical observations or ECG. A mild increase in CSF white blood cells (WBCs) was detected transiently after ICV infusion. Isolated histological changes related to catheter placement and infusion were observed in ICV treated animals, including vehicle controls. The CSF and plasma exposure profiles were equivalent between animals that received an ICV or IT-L infusion. TPP1 levels peaked at the end of infusion, at which point the enzyme was present in plasma at 0.3% to 0.5% of CSF levels. TPP1 was detected in brain tissues with half-lives of 3–14 days. CNS distribution between ICV and IT-L administration was similar, although ICV resulted in distribution to deep brain structures including the thalamus, midbrain, and striatum. Direct CNS infusion of rhTPP1 was well tolerated with no drug related safety findings. The favorable nonclinical profile of ICV rhTPP1 supports the treatment of CLN2 by direct administration to the CNS. - Highlights: • TPP1 enzyme replacement therapy to the CNS is in development for CLN2 disease. • Toxicology, pharmacokinetics, and CNS distribution were assessed in monkeys. • TPP1 infusion directly to the brain did not result in any safety concerns. • A positive pharmacokinetic and distribution profile resulted from TPP1 infusion. • This study demonstrates the feasibility of ICV administered

  6. Use of dipeptidyl peptidase-4 inhibitors and the reporting of infections: A disproportionality analysis in the World Health Organization VigiBase

    NARCIS (Netherlands)

    M.J.C. Willemen (Marc); A.K. Mantel-Teeuwisse (Aukje); S.M.J.M. Straus (Sabine); L. Meyboom (Leen); T.C.G. Egberts (Toine); H.G.M. Leufkens (Hubert)

    2011-01-01

    textabstractOBJECTIVE - Dipeptidyl peptidase-4 (DPP-4) inhibitors are a new class of antidiabetic drugs. They inactivate incretin hormones but also have many other effects throughout the body, among which are effects on the immune system. This might result in an increased infection risk. This study

  7. Long-term treatment with Sitagliptin, a dipeptidyl peptidase-4 inhibitor, reduces colon carcinogenesis and reactive oxygen species in 1,2-dimethylhydrazine-induced rats

    DEFF Research Database (Denmark)

    Femia, Angelo Pietro; Raimondi, Laura; Maglieri, Giulia

    2013-01-01

    Type 2 diabetes mellitus (T2DM) and insulin resistance (IR) increase colon cancer risk. Antidiabetic drugs stabilizing incretin hormones, such as inhibitors of dipeptidyl peptidase-4 activity (DPP4i), may affect colon carcinogenesis; however, the data remain controversial. Therefore, the authors ...

  8. The dipeptidyl peptidase IV inhibitor vildagliptin suppresses endogenous glucose production and enhances islet function after single-dose administration in type 2 diabetic patients

    DEFF Research Database (Denmark)

    Balas, Bogdan; Baig, Muhammad R; Watson, Catherine;

    2007-01-01

    AIMS/HYPOTHESIS: Vildagliptin is a selective dipeptidyl peptidase IV inhibitor that augments meal-stimulated levels of biologically active glucagon-like peptide-1. Chronic vildagliptin treatment decreases postprandial glucose levels and reduces hemoglobin A1c in type 2 diabetic patients. However,...

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

    OpenAIRE

    Jeppesen, P B; Sanguinetti, E L; A. Buchman; 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.

  10. Conserved serine and histidine residues are critical for activity of the ER-type signal peptidase SipW of Bacillus subtilis

    NARCIS (Netherlands)

    Tjalsma, H; Stover, AG; Driks, A; Venema, G; Bron, S; van Dijl, JM

    2000-01-01

    Type I signal peptidases (SPases) are required for the removal of signal peptides from translocated proteins and, subsequently, release of the mature protein from the trans side of the membrane. Interestingly, prokaryotic (P-type) and endoplasmic reticular (ER-type) SPases are functionally equivalen

  11. Kinetics and stereochemistry of hydrolysis of an N-(phenylacetyl)-α-hydroxyglycine ester catalyzed by serine β-lactamases and DD-peptidases.

    Science.gov (United States)

    Pelto, Ryan B; Pratt, R F

    2012-09-28

    The α-hydroxydepsipeptide 3-carboxyphenyl N-(phenylacetyl)-α-hydroxyglycinate (5) is a quite effective substrate of serine β-lactamases and low molecular mass DD-peptidases. The class C P99 and ampC β-lactamases catalyze the hydrolysis of both enantiomers of 5, although they show a strong preference for one of them. The class A TEM-2 and class D OXA-1 β-lactamases and the Streptomyces R61 and Actinomadura R39 DD-peptidases catalyze hydrolysis of only one enantiomer of at any significant rate. Experiments show that all of the above enzymes strongly prefer the same enantiomer, a surprising result since β-lactamases usually prefer L(S) enantiomers and DD-peptidases D(R). Product analysis, employing peptidylglycine α-amidating lyase, showed that the preferred enantiomer is D(R). Thus, it is the β-lactamases that have switched preference rather than the DD-peptidases. Molecular modeling of the P99 β-lactamase active site suggests that the α-hydroxyl 5 of may interact with conserved Asn and Lys residues. Both α-hydroxy and α-amido substituents on a glycine ester substrate can therefore enhance its productive interaction with the β-lactamase active site, although their effects are not additive; this may also be true for inhibitors.

  12. Requirement of Signal Peptidase ComC and Thiol-Disulfide Oxidoreductase DsbA for Optimal Cell Surface Display of Pseudopilin ComGC in Staphylococcus aureus

    NARCIS (Netherlands)

    van der Kooi-Pol, Magdalena M.; Reilman, Ewoud; Sibbald, Mark J. J. B.; Veenstra-Kyuchukova, Yanka K.; Kouwen, Thijs R. H. M.; Buist, Girbe; van Dijl, Jan Maarten

    2012-01-01

    Staphylococcus aureus is an important Gram-positive bacterial pathogen producing many secreted and cell surface-localized virulence factors. Here we report that the staphylococcal thiol-disulfide oxidoreductase DsbA is essential for stable biogenesis of the ComGC pseudopilin. The signal peptidase

  13. Bacillus subtilis contains four closely related type I signal peptidases with overlapping substrate specificities - Constitutive and temporally controlled expression of different sip genes

    NARCIS (Netherlands)

    Tjalsma, H; Noback, MA; Bron, S; Venema, G; Yamane, K; vanDijl, JM

    1997-01-01

    Most biological membranes contain one or two type I signal peptidases for the removal of signal peptides from secretory precursor proteins. In this respect, the Grampositive bacterium Bacillus subtilis seems to be exceptional, because it contains at least four chromosomally-encoded type I signal pep

  14. Dipeptidyl peptidase-4 inhibitors administered in combination with metformin result in an additive increase in the plasma concentration of active GLP-1

    DEFF Research Database (Denmark)

    Migoya, E M; Bergeron, R; Miller, J L

    2010-01-01

    The aim of the study was to investigate the effects of a dipeptidyl peptidase-4 (DPP-4) inhibitor, of metformin, and of the combination of the two agents, on incretin hormone concentrations. Active and inactive (or total) incretin plasma concentrations, plasma DPP-4 activity, and preproglucagon (...

  15. Association study of ubiquitin-specific peptidase 46 (USP46) with bipolar disorder and schizophrenia in a Japanese population.

    Science.gov (United States)

    Kushima, Itaru; Aleksic, Branko; Ito, Yoshihito; Nakamura, Yukako; Nakamura, Kazuhiko; Mori, Norio; Kikuchi, Mitsuru; Inada, Toshiya; Kunugi, Hiroshi; Nanko, Shinichiro; Kato, Tadafumi; Yoshikawa, Takeo; Ujike, Hiroshi; Suzuki, Michio; Iwata, Nakao; Ozaki, Norio

    2010-03-01

    Recently, ubiquitin-specific peptidase 46 (Usp46) has been identified as a quantitative trait gene responsible for immobility in the tail suspension test and forced swimming test in mice. Mice with 3-bp deletion in Usp46 exhibited loss of 'behavioral despair' under inescapable stresses in addition to abnormalities in circadian behavioral rhythms and the GABAergic system. Considering the face and construct validity as an animal model for bipolar disorder, we explored an association of USP46 and bipolar disorder in a Japanese population. We also examined an association of USP46 and schizophrenia. We found nominal evidence for an association of rs12646800 and schizophrenia. This association was not significant after correction for multiple testing. No significant association was detected for bipolar disorder. In conclusion, our data argue against the presence of any strong genetic susceptibility factors for bipolar disorder or schizophrenia in the region USP46.

  16. Long-term inhibition of dipeptidyl peptidase IV improves glucose tolerance and preserves islet function in mice

    DEFF Research Database (Denmark)

    Reimer, M Kvist; Holst, Jens Juul; Ahrén, B

    2002-01-01

    and such mice rendered glucose-intolerant and insulin-resistant by feeding a high-fat diet. DESIGN: In mice fed a standard diet (11% fat) or a high-fat diet (58% fat), NVP DPP728 (0.12 micromol/g body weight) was administered in the drinking water for an 8 week period. RESULTS: DPPIV inhibition reduced plasma......OBJECTIVES: Inhibitors of the glucagon-like peptide-1 (GLP-1)-degrading enzyme, dipeptidyl peptidase IV (DPPIV), are being explored in the treatment of diabetes. We examined the long-term influence of a selective, orally active inhibitor of DPPIV (NVP DPP728), in normal female C57BL/6J mice...

  17. Synthesis and biological evaluation of novel benzyl-substituted (S)-phenylalanine derivatives as potent dipeptidyl peptidase 4 inhibitors.

    Science.gov (United States)

    Liu, Yang; Si, Meimei; Tang, Li; Shangguan, Shihao; Wu, Haoshu; Li, Jia; Wu, Peng; Ma, Xiaodong; Liu, Tao; Hu, Yongzhou

    2013-09-15

    A series of novel benzyl-substituted (S)-phenylalanine derivatives were synthesized and evaluated for their dipeptidyl peptidase 4 (DPP-4) inhibitory activity and selectivity. It was found that most synthesized target compounds were potent DPP-4 inhibitors with IC50 values in 3.79-25.52 nM, which were significantly superior to that of the marketed drug sitagliptin. Furthermore, the 4-fluorobenzyl substituted phenylalanine derivative 6g not only displayed the potent DPP-4 inhibition with an IC50 value of 3.79 nM, but also showed better selectivity against DPP-4 over other related enzymes including DPP-7, DPP-8, and DPP-9. In an oral glucose tolerance test (OGTT) in normal Sprague Dawley rats, compound 6g reduced blood glucose excursion in a dose-dependent manner.

  18. Entropy-driven binding of opioid peptides induces a large domain motion in human dipeptidyl peptidase III.

    Science.gov (United States)

    Bezerra, Gustavo A; Dobrovetsky, Elena; Viertlmayr, Roland; Dong, Aiping; Binter, Alexandra; Abramic, Marija; Macheroux, Peter; Dhe-Paganon, Sirano; Gruber, Karl

    2012-04-24

    Opioid peptides are involved in various essential physiological processes, most notably nociception. Dipeptidyl peptidase III (DPP III) is one of the most important enkephalin-degrading enzymes associated with the mammalian pain modulatory system. Here we describe the X-ray structures of human DPP III and its complex with the opioid peptide tynorphin, which rationalize the enzyme's substrate specificity and reveal an exceptionally large domain motion upon ligand binding. Microcalorimetric analyses point at an entropy-dominated process, with the release of water molecules from the binding cleft ("entropy reservoir") as the major thermodynamic driving force. Our results provide the basis for the design of specific inhibitors that enable the elucidation of the exact role of DPP III and the exploration of its potential as a target of pain intervention strategies.

  19. Interaction of human dipeptidyl peptidase IV and human immunodeficiency virus type-1 transcription transactivator in Sf9 cells

    Directory of Open Access Journals (Sweden)

    Reutter Werner

    2010-10-01

    Full Text Available Abstract Background Dipeptidyl peptidase IV (DPPIV also known as the T cell activation marker CD26 is a multifunctional protein which is involved in various biological processes. The association of human-DPPIV with components of the human immunodeficiency virus type-1 (HIV1 is well documented and raised some discussions. Several reports implicated the interaction of human-DPPIV with the HIV1 transcription transactivator protein (HIV1-Tat and the inhibition of the dipeptidyl peptidase activity of DPPIV by the HIV1-Tat protein. Furthermore, enzyme kinetic data implied another binding site for the HIV1-Tat other than the active centre of DPPIV. However, the biological significance of this interaction of the HIV1-Tat protein and human-DPPIV has not been studied, yet. Therefore, we focused on the interaction of HIV1-Tat protein with DPPIV and investigated the subsequent biological consequences of this interaction in Spodoptera frugiperda cells, using the BAC-TO-BAC baculovirus system. Results The HIV1-Tat protein (Tat-BRU co-localized and co-immunoprecipitated with human-DPPIV protein, following co-expression in the baculovirus-driven Sf9 cell expression system. Furthermore, tyrosine phosphorylation of DPPIV protein was up-regulated in Tat/DPPIV-co-expressing cells after 72 h culturing and also in DPPIV-expressing Sf9 cells after application of purified recombinant Tat protein. As opposed to the expression of Tat alone, serine phosphorylation of the Tat protein was decreased when co-expressed with human-DPPIV protein. Conclusions We show for the first time that human-DPPIV and HIV1-Tat co-immunoprecipitate. Furthermore, our findings indicate that the interaction of HIV1-Tat and human-DPPIV may be involved in signalling platforms that regulate the biological function of both human-DPPIV and HIV1-Tat.

  20. HIV Aspartic Peptidase Inhibitors Modulate Surface Molecules and Enzyme Activities Involved with Physiopathological Events in Fonsecaea pedrosoi

    Directory of Open Access Journals (Sweden)

    Vanila F. Palmeira

    2017-05-01

    Full Text Available Fonsecaea pedrosoi is the main etiological agent of chromoblastomycosis, a recalcitrant disease that is extremely difficult to treat. Therefore, new chemotherapeutics to combat this fungal infection are urgently needed. Although aspartic peptidase inhibitors (PIs currently used in the treatment of human immunodeficiency virus (HIV have shown anti-F. pedrosoi activity their exact mechanisms of action have not been elucidated. In the present study, we have investigated the effects of four HIV-PIs on crucial virulence attributes expressed by F. pedrosoi conidial cells, including surface molecules and secreted enzymes, both of which are directly involved in the disease development. In all the experiments, conidia were treated with indinavir, nelfinavir, ritonavir and saquinavir (100 μM for 24 h, and then fungal cells were used to evaluate the effects of HIV-PIs on different virulence attributes expressed by F. pedrosoi. In comparison to untreated controls, exposure of F. pedrosoi cells to HIV-PIs caused (i reduction on the conidial granularity; (ii irreversible surface ultrastructural alterations, such as shedding of electron dense and amorphous material from the cell wall, undulations/invaginations of the plasma membrane with and withdrawal of this membrane from the cell wall; (iii a decrease in both mannose-rich glycoconjugates and melanin molecules and an increase in glucosylceramides on the conidial surface; (iv inhibition of ergosterol and lanosterol production; (v reduction in the secretion of aspartic peptidase, esterase and phospholipase; (vi significant reduction in the viability of non-pigmented conidia compared to pigmented ones. In summary, HIV-PIs are efficient drugs with an ability to block crucial biological processes of F. pedrosoi and can be seriously considered as potential compounds for the development of new chromoblastomycosis chemotherapeutics.

  1. A glutathione peroxidase, intracellular peptidases and the TOR complexes regulate peptide transporter PEPT-1 in C. elegans.

    Directory of Open Access Journals (Sweden)

    Jacqueline Benner

    Full Text Available The intestinal peptide transporter PEPT-1 in Caenorhabditis elegans is a rheogenic H(+-dependent carrier responsible for the absorption of di- and tripeptides. Transporter-deficient pept-1(lg601 worms are characterized by impairments in growth, development and reproduction and develop a severe obesity like phenotype. The transport function of PEPT-1 as well as the influx of free fatty acids was shown to be dependent on the membrane potential and on the intracellular pH homeostasis, both of which are regulated by the sodium-proton exchanger NHX-2. Since many membrane proteins commonly function as complexes, there could be proteins that possibly modulate PEPT-1 expression and function. A systematic RNAi screening of 162 genes that are exclusively expressed in the intestine combined with a functional transport assay revealed four genes with homologues existing in mammals as predicted PEPT-1 modulators. While silencing of a glutathione peroxidase surprisingly caused an increase in PEPT-1 transport function, silencing of the ER to Golgi cargo transport protein and of two cytosolic peptidases reduced PEPT-1 transport activity and this even corresponded with lower PEPT-1 protein levels. These modifications of PEPT-1 function by gene silencing of homologous genes were also found to be conserved in the human epithelial cell line Caco-2/TC7 cells. Peptidase inhibition, amino acid supplementation and RNAi silencing of targets of rapamycin (TOR components in C. elegans supports evidence that intracellular peptide hydrolysis and amino acid concentration are a part of a sensing system that controls PEPT-1 expression and function and that involves the TOR complexes TORC1 and TORC2.

  2. Induction of Protective Immune Responses against Schistosomiasis Haematobium in Hamsters and Mice Using Cysteine Peptidase-Based Vaccine

    Directory of Open Access Journals (Sweden)

    Hatem A M Tallima

    2015-03-01

    Full Text Available One of the major lessons we learned from the radiation-attenuated cercariae (RA vaccine studies is that protective immunity against schistosomiasis is dependent on the induction of T helper (Th1/Th2-related immune responses. Since most schistosome larval and adult-worm-derived molecules used for vaccination uniformly induce a polarized Th1 response, it was essential to include a type 2 immune responses-inducing molecule, such as cysteine peptidases, in the vaccine formula. Here we demonstrate that a single subcutaneous injection of Syrian hamsters with 200 microg active papain 1 h before percutaneous exposure to 150 cercariae of Schistosoma haematobium led to highly significant (P 50% in worm burden and worm egg counts in intestine. Immunization of hamsters with 20 microg recombinant glyceraldehyde 3-phosphate dehydrogenase (rSG3PDH and 20 ug 2-cys peroxiredoxin-derived peptide in a multiple antigen peptide construct (PRX MAP together with papain (20 microg/hamster as adjuvant led to considerable (64% protection against challenge S. haematobium infection, similar to the levels reported with irradiated cercariae. Cysteine peptidases-based vaccination was also effective in protecting outbred mice against a percutaneous challenge infection with S. haematobium cercariae. In two experiments, a mixture of Schistosoma mansoni cathepsin B1 (SmCB1 and Fasciola hepatica cathepsin L1 (FhCL1 led to highly significant (P < 0.005 reduction of 70% in challenge S. haematobium worm burden and 60% reduction in liver egg counts. Mice vaccinated with SmCB1/FhCL1/ rSG3PDH mixture and challenged with S. haematobium cercariae three weeks after the second immunization displayed highly significant (P < 0.005 reduction of 72% in challenge worm burden and no eggs in liver of 8-10 mice/group, as compared to unimmunized mice, associated with production of a mixture of type 1 and type 2-related cytokines and antibody responses.

  3. Dipeptidyl peptidase-4 inhibitor MK-626 restores insulin secretion through enhancing autophagy in high fat diet-induced mice.

    Science.gov (United States)

    Liu, Limei; Liu, Jian; Yu, Xiaoxing

    2016-02-12

    Autophagy is cellular machinery for maintenance of β-cell function and mass. The current study aimed to investigate the regulatory effects of MK-626, a dipeptidyl peptidase-4 inhibitor, on insulin secretion through the activation of autophagy in high fat diet-induced obese mice. C57BL/6 mice were fed with a rodent diet containing 45 kcal% fat for 16 weeks to induce obesity and then were received either vehicle or MK-626 (3 mg/kg/day) orally during the final 4 weeks. Mouse islets were isolated. Phosphorylation of serine/threonine-protein kinase mTOR and levels of light chain 3B I (LC3B I), LC3B II, sequestosome-1 (SQSTM1/p62) and autophagy-related protein-7 (Atg7) were examined by Western blotting. Glucagon like-peptide-1 (GLP-1) level and insulin secretion were measured by ELISA. GLP-1 level in plasma was decreased in obese mice, which was elevated by dipeptidyl peptidase-4 inhibitor MK-626. In the islets of obese mice, phosphorylation of mTOR, ratio of LC3B I and LC3B II, and level of p62 were elevated and the expression of Atg7 and insulin secretion were reduced compared to those of C57BL/6 mice. However, such effects were reversed by MK-626. Autophagy activator rapamycin stimulated insulin secretion in obese mice but autophagy inhibitor chloroquine treatment inhibited insulin secretion in obese mice administrated by MK-626. Furthermore, the beneficial effects of MK-626 were inhibited by GLP-1 receptor antagonist exendin 9-39. The present study reveals the activation of autophagy to mediate the anti-diabetic effect of GLP-1.

  4. Dipeptidyl peptidase-4 inhibition and narrow-band ultraviolet-B light in psoriasis (DINUP): study protocol for a randomised controlled trial.

    LENUS (Irish Health Repository)

    Lynch, Maeve

    2016-01-15

    Moderate to severe psoriasis is a systemic inflammatory disease associated with insulin resistance, obesity and type 2 diabetes (T2DM). Sitagliptin is a dipeptidyl peptidase-4 (DPP-4) inhibitor that improves glycaemia and has a marketing authorisation for the treatment of T2DM. Non-immunosuppressive therapies that are effective for psoriasis and its associated comorbidities would be a significant advance in the treatment of this chronic disease.

  5. The P1 and P1' residue specificities of physarolisin I, a serine-carboxyl peptidase from the true slime mold Physarum polycephalum.

    Science.gov (United States)

    Nishii, Wataru; Kubota, Keiko; Takahashi, Kenji

    2009-05-01

    The P1 and P1' residue specificities of physarolisin I were investigated using combinatorial peptide substrates. The results indicated that certain hydrophobic residues and acidic residues are preferred at the P1 position and some hydrophobic residues at the P1' position. This P1 specificity, different from other serine-carboxyl peptidases, appears to be explained partially by the nature of the S1 subsite residues.

  6. Caracterização do mecanismo catalítico da SGP, enzima protótipo das glutâmico peptidases

    OpenAIRE

    Kondo, Marcia Yuri

    2012-01-01

    Proteolytic enzymes are widespread in all organisms and are involved in a wide range of biological roles. There are seven distinct classes of proteases (serine, cysteine, aspartate, metalloproteases, threonine, glutamate and the newly identified asparagine peptide lyases), grouped according to their catalytic mechanism. The glutamic peptidases are carboxylic (acidic) proteases insensitive to pepstatin inhibitor and were annotated as a new group in 2004 (Family G1 Merops). These proteases have...

  7. MIP1, a new yeast gene homologous to the rat mitochondrial intermediate peptidase gene, is required for oxidative metabolism in Saccharomyces cerevisiae.

    OpenAIRE

    Isaya, G; Miklos, D; Rollins, R A

    1994-01-01

    Cleavage of amino-terminal octapeptides, F/L/IXXS/T/GXXXX, by mitochondrial intermediate peptidase (MIP) is typical of many mitochondrial precursor proteins imported to the matrix and the inner membrane. We previously described the molecular characterization of rat liver MIP (RMIP) and indicated a putative homolog in the sequence predicted from gene YCL57w of yeast chromosome III. A new yeast gene, MIP1, has now been isolated by screening a Saccharomyces cerevisiae genomic library with an RMI...

  8. Evidence for cleavage of the metalloprotease Vsm from Vibrio splendidus strain JZ6 by an M20 peptidase (PepT-like protein at low temperature

    Directory of Open Access Journals (Sweden)

    Rui Liu

    2016-10-01

    Full Text Available Metalloprotease Vsm is a major extracellular virulence factor of Vibrio splendidus. The toxicity of Vsm from V. splendidus strain JZ6 has been characterized, and production of this virulence factor proved to be temperature-regulated. The present study provides evidence that two forms (JZE1 and JZE2 of Vsm protein exist in extracellular products (ECPs of strain JZ6, and a significant conversion of these two forms was detected by SDS-PAGE and immunoblotting analyses of samples obtained from cells grown at 4, 10, 16, 20, 24 and 28 ℃. Mass spectroscopy confirmed that JZE1 was composed only of the peptidase_M4 domain of Vsm, and JZE2 contained both the PepSY domain and the peptidase_M4 domain. An M20 peptidase T-like protein (PepTL was screened from the transcriptome data of strain JZ6, which was considered as a crucial molecule to produce the active Vsm (JZE1 by cleavage of the propeptide. Similar to that of Vsm, PepTL mRNA accumulation was highest at 4 ℃ (836.82-fold of that at 28 ℃, decreased with increasing of temperature and reached its lowest level at 28 ℃. Deletion of the gene encoding the PepTL resulted in a mutant strain that did not produce the JZE1 cleavage product. The peptidase activity of PepTL recombinant protein (rPepTL was confirmed by cleaving the Vsm in ECPs with an in vitro degradation reaction. These results demonstrate that PepTL participates in activating Vsm in strain JZ6 by proteolytic cleavage at low temperature.

  9. The dipeptidyl peptidase 4 inhibitor vildagliptin does not accentuate glibenclamide-induced hypoglycemia but reduces glucose-induced glucagon-like peptide 1 and gastric inhibitory polypeptide secretion

    DEFF Research Database (Denmark)

    El-Ouaghlidi, Andrea; Rehring, Erika; Holst, Jens Juul

    2007-01-01

    BACKGROUND/AIMS: Inhibition of dipeptidyl peptidase 4 by vildagliptin enhances the concentrations of the active form of the incretin hormones glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP). The present study asked whether vildagliptin accentuates glibenclamide-induced hy...... of glibenclamide is not accentuated by the coadministration of vildagliptin. This may be explained by a negative feedback regulation of GLP-1 and GIP secretion that limits the degree to which the active incretin levels are enhanced....

  10. Biochemical and milk-clotting properties and mapping of catalytic subsites of an extracellular aspartic peptidase from basidiomycete fungus Phanerochaete chrysosporium.

    Science.gov (United States)

    da Silva, Ronivaldo Rodrigues; de Oliveira, Lilian Caroline Gonçalves; Juliano, Maria Aparecida; Juliano, Luiz; de Oliveira, Arthur H C; Rosa, Jose C; Cabral, Hamilton

    2017-06-15

    For a long time, proteolytic enzymes have been employed as key tools of industrial processes, especially in the dairy industry. In the present work, we used Phanerochaete chrysosporium for biochemical characterization and analysis of catalytic specificity of an aspartic peptidase. Our results revealed an aspartic peptidase with molecular mass ∼38kDa, maximal activity at pH 4.5 and 50°C, and stability above 80% in the pH range of 3-8 and temperature up to 55°C for 1h. In a milk-clotting assay, this peptidase showed maximal milk clotting activity at 60-65°C and maintenance of enzymatic activity above 80% in the presence of 20mM CaCl2. In a specificity assay, we observed stronger restriction of catalysis at the S1 subsite, with a preference for lysine, arginine, leucine, tyrosine, and phenylalanine residues. The restricted proteolysis and milk-clotting potential are attractive properties for the use in cheese production. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Inhibition of bacterial DD-peptidases (penicillin-binding proteins) in membranes and in vivo by peptidoglycan-mimetic boronic acids.

    Science.gov (United States)

    Dzhekieva, Liudmila; Kumar, Ish; Pratt, R F

    2012-04-03

    The DD-peptidases or penicillin-binding proteins (PBPs) catalyze the final steps of bacterial peptidoglycan biosynthesis and are inhibited by the β-lactam antibiotics. There is at present a question of whether the active site structure and activity of these enzymes is the same in the solubilized (truncated) DD-peptidase constructs employed in crystallographic and kinetics studies as in membrane-bound holoenzymes. Recent experiments with peptidoglycan-mimetic boronic acids have suggested that these transition state analogue-generating inhibitors may be able to induce reactive conformations of these enzymes and thus inhibit strongly. We have now, therefore, measured the dissociation constants of peptidoglycan-mimetic boronic acids from Escherichia coli and Bacillus subtilis PBPs in membrane preparations and, in the former case, in vivo, by means of competition experiments with the fluorescent penicillin Bocillin Fl. The experiments showed that the boronic acids bound measurably (K(i) boronic acids, therefore, are unable to induce tightly binding conformations of these enzymes in vivo. There is no evidence from these experiments that DD-peptidase inhibitors are more or less effective in vivo than in homogeneous solution.

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

    Directory of Open Access Journals (Sweden)

    Suman RK

    2016-08-01

    Full Text Available Rajesh Kumar Suman,1 Ipseeta Ray Mohanty,1 Ujwala Maheshwari,2 Manjusha K Borde,1 YA Deshmukh1 1Department of Pharmacology, 2Department of Pathology, MGM Medical College, Kamothe, Navi Mumbai, India 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

  13. Collagenolytic activities of the major secreted cathepsin L peptidases involved in the virulence of the helminth pathogen, Fasciola hepatica.

    Directory of Open Access Journals (Sweden)

    Mark W Robinson

    Full Text Available BACKGROUND: The temporal expression and secretion of distinct members of a family of virulence-associated cathepsin L cysteine peptidases (FhCL correlates with the entry and migration of the helminth pathogen Fasciola hepatica in the host. Thus, infective larvae traversing the gut wall secrete cathepsin L3 (FhCL3, liver migrating juvenile parasites secrete both FhCL1 and FhCL2 while the mature bile duct parasites, which are obligate blood feeders, secrete predominantly FhCL1 but also FhCL2. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that FhCL1, FhCL2 and FhCL3 exhibit differences in their kinetic parameters towards a range of peptide substrates. Uniquely, FhCL2 and FhCL3 readily cleave substrates with Pro in the P2 position and peptide substrates mimicking the repeating Gly-Pro-Xaa motifs that occur within the primary sequence of collagen. FhCL1, FhCL2 and FhCL3 hydrolysed native type I and II collagen at neutral pH but while FhCL1 cleaved only non-collagenous (NC, non-Gly-X-Y domains FhCL2 and FhCL3 exhibited collagenase activity by cleaving at multiple sites within the α1 and α2 triple helix regions (Col domains. Molecular simulations created for FhCL1, FhCL2 and FhCL3 complexed to various seven-residue peptides supports the idea that Trp67 and Tyr67 in the S2 subsite of the active sites of FhCL3 and FhCL2, respectively, are critical to conferring the unique collagenase-like activity to these enzymes by accommodating either Gly or Pro residues at P2 in the substrate. The data also suggests that FhCL3 accommodates hydroxyproline (Hyp-Gly at P3-P2 better than FhCL2 explaining the observed greater ability of FhCL3 to digest type I and II collagens compared to FhCL2 and why these enzymes cleave at different positions within the Col domains. CONCLUSIONS/SIGNIFICANCE: These studies further our understanding of how this helminth parasite regulates peptidase expression to ensure infection, migration and establishment in host tissues.

  14. Application of robotics to steady state enzyme kinetics: analysis of tight-binding inhibitors of dipeptidyl peptidase IV.

    Science.gov (United States)

    Wang, Aiying; Huang, Yanting; Taunk, Prakash; Magnin, David R; Ghosh, Krishnendu; Robertson, James G

    2003-10-15

    Using available commercial robotics and instrumentation, we developed a fully automated and rigorous steady state enzyme kinetic assay for dipeptidyl peptidase IV (DPP IV; E.C. 3.4.14.5). The automated assay was validated with isoleucyl thiazolidide, a potent inhibitor of DPP IV with K(is)=110nM. Signal window analysis indicated that the assay had a 98% probability of detecting an inhibitor yielding 15% inhibition, with a predicted false positive rate of 0.13%. A mechanistic inhibition version of the automated assay was validated with isoleucyl 4-cyanothiazolidide, a very potent inhibitor of DPP IV. Isoleucyl 4-cyanothiazolidide was a competitive inhibitor of purified porcine DPP IV with K(is)=1 nM. Similar K(is) values were obtained for purified rat DPP IV and for DPP IV activity in human plasma from normal and diabetic donors. The pH dependence of K(is) for isoleucyl 4-cyanothiazolidide yielded a bell-shaped profile, with pK(a)=5.0 and pK(b)=7.6. To date, over 100,000 data points have been generated in profiling targeted compound libraries and in the analysis of tight-binding inhibitors of DPP IV. The data also show that robotic analysis is capable of producing full mechanistic inhibition analysis in a timely fashion to support drug discovery.

  15. Novel water-soluble prodrugs of acyclovir cleavable by the dipeptidyl-peptidase IV (DPP IV/CD26) enzyme.

    Science.gov (United States)

    Diez-Torrubia, Alberto; Cabrera, Silvia; de Castro, Sonia; García-Aparicio, Carlos; Mulder, Gwenn; De Meester, Ingrid; Camarasa, María-José; Balzarini, Jan; Velázquez, Sonsoles

    2013-01-01

    We herein report for the first time the successful use of the dipeptidyl peptidase IV (DPPIV/CD26) prodrug approach to guanine derivatives such as the antiviral acyclovir (ACV). The solution- and solid-phase synthesis of the tetrapeptide amide prodrug 3 and the tripeptide ester conjugate 4 of acyclovir are reported. The synthesis of the demanding tetrapeptide amide prodrug of ACV 3 was first established in solution and successfully transferred onto solid support by using Ellman's dihydropyran (DHP) resin. In contrast with the valyl ester prodrug (valacyclovir, VACV), the tetrapeptide amide prodrug 3 and the tripeptide ester conjugate 4 of ACV proved fully stable in PBS. Both prodrugs converted to VACV (for 4) or ACV (for 3) upon exposure to purified DPPIV/CD26 or human or bovine serum. Vildagliptin, a potent inhibitor of DPPIV/CD26 efficiently inhibited the DPPIV/CD26-catalysed hydrolysis reaction. Both amide and ester prodrugs of ACV showed pronounced anti-herpetic activity in cell culture and significantly improved the water solubility in comparison with the parent drug.

  16. Multi-Organ Damage in Human Dipeptidyl Peptidase 4 Transgenic Mice Infected with Middle East Respiratory Syndrome-Coronavirus.

    Directory of Open Access Journals (Sweden)

    Guangyu Zhao

    Full Text Available The Middle East Respiratory Syndrome Coronavirus (MERS-CoV causes severe acute respiratory failure and considerable extrapumonary organ dysfuction with substantial high mortality. For the limited number of autopsy reports, small animal models are urgently needed to study the mechanisms of MERS-CoV infection and pathogenesis of the disease and to evaluate the efficacy of therapeutics against MERS-CoV infection. In this study, we developed a transgenic mouse model globally expressing codon-optimized human dipeptidyl peptidase 4 (hDPP4, the receptor for MERS-CoV. After intranasal inoculation with MERS-CoV, the mice rapidly developed severe pneumonia and multi-organ damage, with viral replication being detected in the lungs on day 5 and in the lungs, kidneys and brains on day 9 post-infection. In addition, the mice exhibited systemic inflammation with mild to severe pneumonia accompanied by the injury of liver, kidney and spleen with neutrophil and macrophage infiltration. Importantly, the mice exhibited symptoms of paralysis with high viral burden and viral positive neurons on day 9. Taken together, this study characterizes the tropism of MERS-CoV upon infection. Importantly, this hDPP4-expressing transgenic mouse model will be applicable for studying the pathogenesis of MERS-CoV infection and investigating the efficacy of vaccines and antiviral agents designed to combat MERS-CoV infection.

  17. Insights into the Hypertensive Effects of Tityus serrulatus Scorpion Venom: Purification of an Angiotensin-Converting Enzyme-Like Peptidase

    Directory of Open Access Journals (Sweden)

    Daniela Cajado-Carvalho

    2016-11-01

    Full Text Available The number of cases of envenomation by scorpions has grown significantly in Brazil since 2007, with the most severe cases being caused by the Tityus serrulatus scorpion. Although envenomed patients mostly suffer neurotoxic manifestations, other symptoms, such as hypertension, cannot be exclusively attributed to neurotoxins. Omics analyses have detected plentiful amounts of metalloproteases in T. serrulatus venom. However, the roles played by these enzymes in envenomation are still unclear. Endeavoring to investigate the functions of scorpion venom proteases, we describe here for the first time an Angiotensin I-Converting Enzyme-like peptidase (ACE-like purified from T. serrulatus venom. The crude venom cleaved natural and fluorescent substrates and these activities were inhibited by captopril. Regarding the serum neutralization, the scorpion antivenom was more effective at blocking the ACE-like activity than arachnid antivenom, although neither completely inhibited the venom cleavage action, even at higher doses. ACE-like was purified from the venom after three chromatographic steps and its identity was confirmed by mass spectrometric and transcriptomic analyses. Bioinformatics analysis showed homology between the ACE-like transcript sequences from Tityus spp. and human testis ACE. These findings advance our understanding of T. serrulatus venom components and may improve treatment of envenomation victims, as ACE-like may contribute to envenomation symptoms, especially the resulting hypertension.

  18. Dipeptidyl Peptidase 4 Inhibition May Facilitate Healing of Chronic Foot Ulcers in Patients with Type 2 Diabetes

    Directory of Open Access Journals (Sweden)

    Raffaele Marfella

    2012-01-01

    Full Text Available The pathophysiology of chronic diabetic ulcers is complex and still incompletely understood, both micro- and macroangiopathy strongly contribute to the development and delayed healing of diabetic wounds, through an impaired tissue feeding and response to ischemia. With adequate treatment, some ulcers may last only weeks; however, many ulcers are difficult to treat and may last months, in certain cases years; 19–35% of ulcers are reported as nonhealing. As no efficient therapy is available, it is a high priority to develop new strategies for treatment of this devastating complication. Because experimental and pathological studies suggest that incretin hormone glucagon-like peptide-1 may improves VEGF generation and promote the upregulation of HIF-1α through a reduction of oxidative stress, the study evaluated the effect of the augmentation of GLP-1, by inhibitors of the dipeptidyl peptidase-4, such as vildagliptin, on angiogenesis process and wound healing in diabetic chronic ulcers. Although elucidation of the pathophysiologic importance of these aspects awaits further confirmations, the present study evidences an additional aspect of how DPP-4 inhibition might contribute to improved ulcer outcome.

  19. Metabolic inactivation of the circadian transmitter, pigment dispersing factor (PDF), by neprilysin-like peptidases in Drosophila.

    Science.gov (United States)

    Isaac, R Elwyn; Johnson, Erik C; Audsley, Neil; Shirras, Alan D

    2007-12-01

    Recent studies have firmly established pigment dispersing factor (PDF), a C-terminally amidated octodecapeptide, as a key neurotransmitter regulating rhythmic circadian locomotory behaviours in adult Drosophila melanogaster. The mechanisms by which PDF functions as a circadian peptide transmitter are not fully understood, however; in particular, nothing is known about the role of extracellular peptidases in terminating PDF signalling at synapses. In this study we show that PDF is susceptible to hydrolysis by neprilysin, an endopeptidase that is enriched in synaptic membranes of mammals and insects. Neprilysin cleaves PDF at the internal Ser7-Leu8 peptide bond to generate PDF1-7 and PDF8-18. Neither of these fragments were able to increase intracellular cAMP levels in HEK293 cells cotransfected with the Drosophila PDF receptor cDNA and a firefly luciferase reporter gene, confirming that such cleavage results in PDF inactivation. The Ser7-Leu8 peptide bond was also the principal cleavage site when PDF was incubated with membranes prepared from heads of adult Drosophila. This endopeptidase activity was inhibited by the neprilysin inhibitors phosphoramidon (IC(50,) 0.15 micromol l(-1)) and thiorphan (IC(50,) 1.2 micromol l(-1)). We propose that cleavage by a member of the Drosophila neprilysin family of endopeptidases is the most likely mechanism for inactivating synaptic PDF and that neprilysin might have an important role in regulating PDF signals within circadian neural circuits.

  20. Production of a novel wheat gluten hydrolysate containing dipeptidyl peptidase-IV inhibitory tripeptides using ginger protease.

    Science.gov (United States)

    Taga, Yuki; Hayashida, Osamu; Kusubata, Masashi; Ogawa-Goto, Kiyoko; Hattori, Shunji

    2017-09-01

    Wheat gluten is a Pro-rich protein complex comprising glutenins and gliadins. Previous studies have reported that oral intake of enzymatic hydrolysates of gluten has beneficial effects, such as suppression of muscle injury and improvement of hepatitis. Here, we utilized ginger protease that preferentially cleaves peptide bonds with Pro at the P2 position to produce a novel type of wheat gluten hydrolysate. Ginger protease efficiently hydrolyzed gluten, particularly under weak acidic conditions, to peptides with an average molecular weight of gluten hydrolysate contained substantial amounts of tripeptides, including Gln-Pro-Gln, Gln-Pro-Gly, Gln-Pro-Phe, Leu-Pro-Gln, and Ser-Pro-Gln (e.g. 40.7 mg/g at pH 5.2). These gluten-derived tripeptides showed high inhibitory activity on dipeptidyl peptidase-IV with IC50 values of 79.8, 70.9, 71.7, 56.7, and 78.9 μM, respectively, suggesting that the novel gluten hydrolysate prepared using ginger protease can be used as a functional food for patients with type 2 diabetes.

  1. Insights into the Hypertensive Effects of Tityus serrulatus Scorpion Venom: Purification of an Angiotensin-Converting Enzyme-Like Peptidase

    Science.gov (United States)

    Cajado-Carvalho, Daniela; Kuniyoshi, Alexandre Kazuo; Duzzi, Bruno; Iwai, Leo Kei; de Oliveira, Úrsula Castro; Junqueira de Azevedo, Inácio de Loiola Meirelles; Kodama, Roberto Tadashi; Portaro, Fernanda Vieira

    2016-01-01

    The number of cases of envenomation by scorpions has grown significantly in Brazil since 2007, with the most severe cases being caused by the Tityus serrulatus scorpion. Although envenomed patients mostly suffer neurotoxic manifestations, other symptoms, such as hypertension, cannot be exclusively attributed to neurotoxins. Omics analyses have detected plentiful amounts of metalloproteases in T. serrulatus venom. However, the roles played by these enzymes in envenomation are still unclear. Endeavoring to investigate the functions of scorpion venom proteases, we describe here for the first time an Angiotensin I-Converting Enzyme-like peptidase (ACE-like) purified from T. serrulatus venom. The crude venom cleaved natural and fluorescent substrates and these activities were inhibited by captopril. Regarding the serum neutralization, the scorpion antivenom was more effective at blocking the ACE-like activity than arachnid antivenom, although neither completely inhibited the venom cleavage action, even at higher doses. ACE-like was purified from the venom after three chromatographic steps and its identity was confirmed by mass spectrometric and transcriptomic analyses. Bioinformatics analysis showed homology between the ACE-like transcript sequences from Tityus spp. and human testis ACE. These findings advance our understanding of T. serrulatus venom components and may improve treatment of envenomation victims, as ACE-like may contribute to envenomation symptoms, especially the resulting hypertension. PMID:27886129

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

    Science.gov (United States)

    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.

  3. Uric acid inhibition of dipeptidyl peptidase IV in vitro is dependent on the intracellular formation of triuret.

    Science.gov (United States)

    Mohandas, Rajesh; Sautina, Laura; Beem, Elaine; Schuler, Anna; Chan, Wai-Yan; Domsic, John; McKenna, Robert; Johnson, Richard J; Segal, Mark S

    2014-08-01

    Uric acid affects endothelial and adipose cell function and has been linked to diseases such as hypertension, metabolic syndrome, and cardiovascular disease. Interestingly uric acid has been shown to increase endothelial progenitor cell (EPC) mobilization, a potential mechanism to repair endothelial injury. Since EPC mobilization is dependent on activity of the enzyme CD26/dipeptidyl peptidase (DPP)IV, we examined the effect uric acid will have on CD26/DPPIV activity. Uric acid inhibited the CD26/DPPIV associated with human umbilical vein endothelial cells but not human recombinant (hr) CD26/DPPIV. However, triuret, a product of uric acid and peroxynitrite, could inhibit cell associated and hrCD26/DPPIV. Increasing or decreasing intracellular peroxynitrite levels enhanced or decreased the ability of uric acid to inhibit cell associated CD26/DPPIV, respectively. Finally, protein modeling demonstrates how triuret can act as a small molecule inhibitor of CD26/DPPIV activity. This is the first time that uric acid or a uric acid reaction product has been shown to affect enzymatic activity and suggests a novel avenue of research in the role of uric acid in the development of clinically important diseases. Published by Elsevier Inc.

  4. Diuretic and Natriuretic Effects of Dipeptidyl Peptidase-4 Inhibitor Teneligliptin: The Contribution of Glucagon-like Peptide-1.

    Science.gov (United States)

    Moroi, Masao; Kubota, Tetsuya

    2015-08-01

    Glucagon-like peptide-1 receptor (GLP-1R) agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors are antidiabetic agents; however, their mechanisms of action are different. GLP-1R and DPP-4 are also expressed in the renal proximal tubular brush border, where they regulate Na reabsorption. We investigated whether the DPP-4 inhibitor, teneligliptin, has diuretic and natriuretic effects and whether these are associated with the stimulation of the GLP-1R in rats. Oral administration of teneligliptin resulted in a reduction of plasma DPP-4 activity over 6 hours, as well as an induction of diuresis and natriuresis. Although teneligliptin did not change the increase in blood glucose levels by glucose loading, percentage of urine volume and Na/K ratio with teneligliptin to vehicle were augmented by glucose loading. Peak levels of plasma GLP-1 did not change after oral administration of teneligliptin when glucose was not loaded but increased at least 2-fold with glucose loading. Furthermore, the natriuretic effect of teneligliptin was inhibited by the GLP-1R antagonist, exendin9-39, whereas the diuresis was not affected. These results suggest that the mechanism of natriuresis was different from that of diuresis, and the natriuresis is associated with the stimulation of GLP-1R. There may be mechanistic differences in DPP-4 inhibition between diuresis and natriuresis.

  5. Fungalysin and dipeptidyl-peptidase gene transcription in Microsporum canis strains isolated from symptomatic and asymptomatic cats.

    Science.gov (United States)

    Mathy, Anne; Baldo, Aline; Schoofs, Laura; Cambier, Ludivine; Defaweux, Valérie; Tabart, Jérémy; Maréchal, Françoise; Symoens, Françoise; Mignon, Bernard

    2010-11-20

    Microsporum canis is the main pathogenic fungus that causes a superficial cutaneous infection called dermatophytosis in domestic carnivores. In cats, M. canis causes symptomatic or asymptomatic infection. Recent conflicting data raise the question of whether the clinical status of the infected cat (symptomatic or asymptomatic) is directly correlated to the proteolytic activity of M. canis strains. Here, the transcription of fungalysin and dipeptidyl-peptidase genes (DPP) of M. canis was compared between four strains isolated from symptomatic and asymptomatic cats during the first steps of the infection process, namely in arthroconidia, during adherence of arthroconidia to corneocytes and during early invasion of the epidermis, using a new ex vivo model made of feline epidermis. There was no detectable transcription of the fungalysin genes in arthroconidia or during the first steps of the infection process for any of the tested strains, suggesting that these proteases play a role later in the infection process. Among DPP, the DPP IV gene was the most frequently transcribed both in arthroconidia and later during infection (adherence and invasion), but no significant differences were observed between M. canis strains isolated from symptomatic and asymptomatic cats. This study shows that the clinical aspect of M. canis feline dermatophytosis depends upon factors relating to the host rather than to the proteolytic activity of the infective fungal strain.

  6. Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

    Energy Technology Data Exchange (ETDEWEB)

    Marcondes, Marcelo F. [Department of Biophysics, Federal University of Sao Paulo, Sao Paulo 04044-020, SP (Brazil); Torquato, Ricardo J.S. [Department of Biochemistry, Federal University of Sao Paulo, Sao Paulo 04044-020, SP (Brazil); Assis, Diego M.; Juliano, Maria A. [Department of Biophysics, Federal University of Sao Paulo, Sao Paulo 04044-020, SP (Brazil); Hayashi, Mirian A.F. [Department of Pharmacology, Federal University of Sao Paulo, Sao Paulo 04044-020, SP (Brazil); Oliveira, Vitor, E-mail: vitor@biofis.epm.br [Department of Biophysics, Federal University of Sao Paulo, Sao Paulo 04044-020, SP (Brazil)

    2010-01-01

    In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6x His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.

  7. In silico screening of novel inhibitors of M17 Leucine Amino Peptidase (LAP) of Plasmodium vivax as therapeutic candidate.

    Science.gov (United States)

    Rout, Subhashree; Mahapatra, Rajani Kanta

    2016-08-01

    M17 LAP (Leucine Amino Peptidase) plays an important role in the hydrolysis of amino acids essential for growth and development of Plasmodium vivax (Pv), the pathogen causing malaria. In this paper a homology model of PvLAP was generated using MODELLER v9.15. From different in-silico methods such as structure based, ligand based and de novo drug designing a total of 90 compounds were selected for docking studies. A final list of 10 compounds was prepared. The study reported the identification of 2-[(3-azaniumyl-2-hydroxy-4-phenylbutanoyl) amino]-4-methylpentanoate as the best inhibitor in terms of docking score and pharmacophoric features. The reliability of the binding mode of the inhibitor is confirmed by molecular dynamics (MD) simulation study with GROMACS software for a simulation time of 20ns in water environment. Finally, in silico ADMET analysis of the inhibitors using MedChem Designer v3 evaluated the drug likeness of the best hits to be considered for industrial pharmaceutical research.

  8. CPR1: a gene encoding a putative signal peptidase that functions in pathogenicity of Colletotrichum graminicola to maize.

    Science.gov (United States)

    Thon, M R; Nuckles, E M; Takach, J E; Vaillancourt, L J

    2002-02-01

    Colletotrichum graminicola causes anthracnose leaf blight and stalk rot of maize. We used restriction-enzyme mediated insertional (REMI) mutagenesis to identify a gene in this fungus that is required for pathogenicity to both stalks and leaves. The predicted polypeptide encoded by this gene, which we have named CPR1, is similar to a family of proteins that comprise one subunit of the eukaryotic microsomal signal peptidase. The nonpathogenic CPR1 REMI mutant contains a plasmid integration in the 3' untranslated region of the gene, 19 bp downstream from the stop codon. The result is a significant reduction in transcript levels in comparison to the wild type, perhaps as a result of increased transcript instability. We were unable to knock out the CPR1 gene, and it may be essential for viability. Microscopic examination of the REMI mutant on maize leaves revealed that it is fully capable of penetrating and colonizing host cells during the initial, biotrophic phases of the disease interaction but, unlike the wild type, it appears to be unable to switch to a necrotrophic mode of growth. We suggest that the CPR1 REMI mutant may be unable to secrete sufficient quantities of degradative enzymes to support that transition. The CPR1 REMI mutant provides us with a useful tool for future studies of the role of fungal protein transport in this important stalk rot disease of maize.

  9. Stringency of bacterial prolipoprotein signal peptidase (LspA) in recognition of signal peptides - Structure-function correlation.

    Science.gov (United States)

    Sangith, Nikhil; Sankaran, Krishnan

    2017-06-24

    Bacterial lipid modification of proteins is an essential post-translational event committed by Phosphatidylglycerol: prolipoprotein diacylglyceryl transferase (Lgt) by catalysing diacyglyceryl transfer from Phosphatidylglycerol to cysteine present in the characteristic 'lipobox' ([LVI] ((-3)) [ASTVI] ((-2)) [GAS] ((-1)) C ((+1))) of prolipoprotein signal peptides. This is then followed by the cleavage of the signal peptide by lipoprotein-specific signal peptidase (LspA). It had been known for long that threonine at the -1 position allows diacylglyceryl modification by Lgt, but not signal peptide cleavage by LspA. We have addressed this unexplained stringency by computational analysis of the recently published 3D structure of LspA with its competitive inhibitor as well as transition state analogue, globomycin using PyMoL viewing tool and VADAR (Volume, Area, Dihedral Angle Reporter) web server. The propensity to form hydrogen bond (2.9a) between the hydroxyl group of threonine (not possible with serine) and the NH of the lipid-modified cysteine, possible only in the transition state, will prevent the protonation of NH of the leaving peptide and therefore its cleavage. This knowledge could be useful for designing inhibitors of this essential pathway in bacteria or for engineering LspA. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Mammalian mitochondrial intermediate peptidase: Structure/function analysis of a new homologue from Schizophyllum commune and relationship to thimet oligopeptidases

    Energy Technology Data Exchange (ETDEWEB)

    Isaya, G.; Sakati, W.R.; Rollins, R.A. [Yale Univ. School of Medicine, New Haven, CT (United States)] [and others

    1995-08-10

    Mitochondrial intermediate peptidase (MIP) is a component of the mitochondrial protein import machinery required for maturation of nuclear-encoded precursor proteins targeted to the mitochondrial matrix or inner membrane. We previously characterized this enzyme in rat (RMIP) and Saccharomyces cerevisiae (YMIP) and showed that MIP activity is essential for mitochondrial function in yeast. We have now defined the structure of a new MIP homologue (SMIP) from the basidiomycete fungus Schizophyllum commune. SMIP includes 4 exons of 523, 486, 660, and 629 bp separated by 3 short introns. The predicted SMIP, YMIP, and RMIP sequences share 31-37% identity and 54-57% similarity over 700 amino acids. When SMIP and RMIP were expressed in a yeast mip1{Delta} mutant, they were both able to rescue the respiratory-deficient phenotype caused by genetic inactivation of YMIP, indicating that the function of this enzyme is conserved in eukaryotes. Moreover, the MIP sequences show 20-24% identity and 40-47% similarity to a family of oligopeptidases from bacteria, yeast, and mammals. MIP and these proteins are characterized by a highly conserved motif, F-H-E-X-G-H-(X){sub 12}-G-(X){sub 5}-D-(X){sub 2}-E-X-P-S-(X){sub 3}-E-X, centered around a zinc-binding site and appear to represent a new family of genes associated with proteolytic processing in the mitochondrial and cytosolic compartments. 48 refs., 8 figs.

  11. Mammalian mitochondrial intermediate peptidase: structure/function analysis of a new homologue from Schizophyllum commune and relationship to thimet oligopeptidases.

    Science.gov (United States)

    Isaya, G; Sakati, W R; Rollins, R A; Shen, G P; Hanson, L C; Ullrich, R C; Novotny, C P

    1995-08-10

    Mitochondrial intermediate peptidase (MIP) is a component of the mitochondrial protein import machinery required for maturation of nuclear-encoded precursor proteins targeted to the mitochondrial matrix or inner membrane. We previously characterized this enzyme in rat (RMIP) and Saccharomyces cerevisiae (YMIP) and showed that MIP activity is essential for mitochondrial function in yeast. We have now defined the structure of a new MIP homologue (SMIP) from the basidiomycete fungus Schizophyllum commune. SMIP includes 4 exons of 523, 486, 660, and 629 bp separated by 3 short introns. The predicted SMIP, YMIP, and RMIP sequences share 31-37% identity and 54-57% similarity over 700 amino acids. When SMIP and RMIP were expressed in a yeast mip1 delta mutant, they were both able to rescue the respiratory-deficient phenotype caused by genetic inactivation of YMIP, indicating that the function of this enzyme is conserved in eukaryotes. Moreover, the MIP sequences show 20-24% identity and 40-47% similarity to a family of oligopeptidases from bacteria, yeast, and mammals. MIP and these proteins are characterized by a highly conserved motif, F-H-E-X-G-H-(X)2-H-(X)12-G-(X)5-D-(X)2-E-X-P-S-(X)3-E-X, centered around a zinc-binding site and appear to represent a new family of genes associated with proteolytic processing in the mitochondrial and cytosolic compartments.

  12. Dual Targeting of a Processing Peptidase into Both Endosymbiotic Organelles Mediated by a Transport Signal of Unusual Architecture

    Institute of Scientific and Technical Information of China (English)

    Bianca Baudisch; Ralf Bernd Kl(o)sgen

    2012-01-01

    As a result of the endosymbiotic gene transfer,the majority of proteins of mitochondria and chloroplasts are encoded in the nucleus and synthesized in the cytosol as precursor proteins carrying N-terminal transport signals for the 're-import' into the respective target organelle.Most of these transport signals are monospecific,although some of them have dual targeting properties,that is,they are recognized both by mitochondria and by chloroplasts as target organelles.We have identified alpha-MPP2,one of the two isoforms of the substrate binding subunit of mitochondrial processing peptidase ofArabidopsis thaliana,as a novel member of this class of nuclear-encoded organelle proteins.As demonstrated by in organello transport experiments with isolated organelles and by in vivo localization studies employing fluorescent chimeric reporter proteins,the N-terminal region of the alpha-MPP2 precursor comprises transport signals for the import into mitochondria as well as into chloroplasts.Both signals are found within the N-terminal 79 residues of the precursor protein,where they occupy partly separated and partly overlapping regions.Deletion mapping combined with in organello and in vivo protein transport studies demonstrate an unusual architecture of this transport signal,suggesting a composition of three functionally separated domains.

  13. Use of dipeptidyl peptidase-4 inhibitors for the treatment of patients with type 2 diabetes mellitus and chronic kidney disease.

    Science.gov (United States)

    Mikhail, Nasser

    2012-07-01

    Choices of antidiabetic agents for patients with type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD) are limited. Available data suggest that the use of dipeptidyl peptidase-4 (DPP-4) inhibitors may be safe in patients at various stages of renal insufficiency. However, except for linagliptin, dosage adjustment is necessary. The efficacy of DPP-4 inhibitors in patients with renal insufficiency is generally similar to that of the general population with T2DM, with reductions in mean glycated hemoglobin (HbA(1c)) levels of 0.7% to 1.0% compared with baseline, and 0.4% to 0.7% compared with placebo. The frequency of moderate hypoglycemia is 21% to 80% higher with DPP-4 inhibitors compared with placebo, but the frequency of severe hypoglycemia is similar to that with placebo. The use of DPP-4 inhibitors in patients with renal insufficiency is associated with a slight weight loss of pioglitazone. They can also be used as add-on therapy to glipizide and/or pioglitazone in patients with HbA(1c) levels < 9%, but studies are needed to evaluate these combinations in patients with renal insufficiency. Long-term and large-scale clinical trials are underway to better determine the safety and efficacy of DPP-4 inhibitors in patients with T2DM with and without CKD.

  14. Structure of human aspartyl aminopeptidase complexed with substrate analogue: insight into catalytic mechanism, substrate specificity and M18 peptidase family

    Directory of Open Access Journals (Sweden)

    Chaikuad Apirat

    2012-06-01

    Full Text Available Abstract Backround Aspartyl aminopeptidase (DNPEP, with specificity towards an acidic amino acid at the N-terminus, is the only mammalian member among the poorly understood M18 peptidases. DNPEP has implicated roles in protein and peptide metabolism, as well as the renin-angiotensin system in blood pressure regulation. Despite previous enzyme and substrate characterization, structural details of DNPEP regarding ligand recognition and catalytic mechanism remain to be delineated. Results The crystal structure of human DNPEP complexed with zinc and a substrate analogue aspartate-β-hydroxamate reveals a dodecameric machinery built by domain-swapped dimers, in agreement with electron microscopy data. A structural comparison with bacterial homologues identifies unifying catalytic features among the poorly understood M18 enzymes. The bound ligands in the active site also reveal the coordination mode of the binuclear zinc centre and a substrate specificity pocket for acidic amino acids. Conclusions The DNPEP structure provides a molecular framework to understand its catalysis that is mediated by active site loop swapping, a mechanism likely adopted in other M18 and M42 metallopeptidases that form dodecameric complexes as a self-compartmentalization strategy. Small differences in the substrate binding pocket such as shape and positive charges, the latter conferred by a basic lysine residue, further provide the key to distinguishing substrate preference. Together, the structural knowledge will aid in the development of enzyme-/family-specific aminopeptidase inhibitors.

  15. Isolation of dipeptidyl peptidase IV (DP 4) isoforms from porcine kidney by preparative isoelectric focusing to improve crystallization.

    Science.gov (United States)

    Wagner, Leona; Wermann, Michael; Rosche, Fred; Rahfeld, Jens-Ulrich; Hoffmann, Torsten; Demuth, Hans-Ulrich

    2011-07-01

    Abstract In the present studies we resolved the post-translational microheterogeneity of purified porcine dipeptidyl peptidase IV (DP 4) from kidney cortex. Applying SDS-homogeneous DP 4 onto an analytical agarose isoelectric focusing (IEF) gel, pH 4-6, activity staining resulted in at least 17 isoforms between pH 4.8-6.0. These could be separated into fractions with only two to six isoforms by means of preparative liquid-phase IEF, using a Rotofor cell. Starting off with three parallel Rotofor runs under the same conditions at pH 5-6, the fractions were pooled according to the specific activity of DP 4, pH and analytical IEF profile, and further refractionated without any additional ampholytes. Since excessive dilution of ampholytes and proteins was kept to the minimum, a second refractionation step could be introduced, resulting in pH gradients between 0.022 and 0.028 pH increments per fraction. By performing two consecutive refractionation steps, the high resolution necessary for the separation of DP 4 isoforms could be achieved. This represents an alternative method if isolation of isoforms with similar pI's results in precipitation and denaturation in presence of a narrow pH range. Furthermore, it demonstrates that preparative IEF is a powerful tool to resolve post-translational microheterogeneity of a purified protein required for crystallization processing.

  16. Development and validation of a simple cell-based fluorescence assay for dipeptidyl peptidase 1 (DPP1) activity.

    Science.gov (United States)

    Thong, Bob; Pilling, James; Ainscow, Edward; Beri, Raj; Unitt, John

    2011-01-01

    Dipeptidyl peptidase 1 (DPP1) (EC 3.4.14.1; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.

  17. Dipeptidyl peptidase-4 inhibitor sitagliptin improves pancreatic β-cell function in hypertensive diabetic patients treated with angiotensin receptor blockers.

    Science.gov (United States)

    Fukui, Kensuke; Kawahito, Hiroyuki; Wakana, Noriyuki; Kikai, Masakazu; Terada, Kensuke; Yamamoto, Keita; Irie, Daisuke; Kato, Taku; Miyagawa, Sonoko; Yamada, Hiroyuki

    2015-12-01

    Dipeptidyl peptidase (DPP)-4 inhibitors, a novel oral anti-diabetic agents, exert a protective effect on pancreatic β-cell function in patients with type 2 diabetic mellitus (T2DM). However, their beneficial effect in hypertensive T2DM patients treated with angiotensin receptor blockers (ARBs) has not been investigated. In this open-label multicenter randomized study, a total of 55 hypertensive T2DM patients treated with ARBs were randomly assigned to receive the DPP-4 inhibitor sitagliptin or sulfonylurea (SU). After 24 weeks of treatment, a significant reduction in fasting blood glucose was only observed in the sitagliptin group, while HbA1c was significantly reduced in both groups. Homeostasis model assessment of insulin resistance was not significantly improved in either group. Indicators of pancreatic β-cell function, including proinsulin to insulin ratio and homeostasis model assessment of β-cell function, were significantly improved in the sitagliptin group, but not in the SU group. The beneficial effects of sitagliptin were observed in hypoglycemic drug naïve patients, but not in patients who had received SU monotherapy prior to the study. Treatment with the DPP-4 inhibitor sitagliptin might exert beneficial effects on pancreatic β-cell function in ARB-treated T2DM patients and its efficacy might be more pronounced in hypoglycemic drug naïve patients. © The Author(s) 2015.

  18. Dipeptidyl peptidase 4 inhibition may facilitate healing of chronic foot ulcers in patients with type 2 diabetes.

    Science.gov (United States)

    Marfella, Raffaele; Sasso, Ferdinando Carlo; Rizzo, Maria Rosaria; Paolisso, Pasquale; Barbieri, Michelangela; Padovano, Vincenzo; Carbonara, Ornella; Gualdiero, Pasquale; Petronella, Pasquale; Ferraraccio, Franca; Petrella, Antonello; Canonico, Raffaele; Campitiello, Ferdinando; Della Corte, Angela; Paolisso, Giuseppe; Canonico, Silvestro

    2012-01-01

    The pathophysiology of chronic diabetic ulcers is complex and still incompletely understood, both micro- and macroangiopathy strongly contribute to the development and delayed healing of diabetic wounds, through an impaired tissue feeding and response to ischemia. With adequate treatment, some ulcers may last only weeks; however, many ulcers are difficult to treat and may last months, in certain cases years; 19-35% of ulcers are reported as nonhealing. As no efficient therapy is available, it is a high priority to develop new strategies for treatment of this devastating complication. Because experimental and pathological studies suggest that incretin hormone glucagon-like peptide-1 may improves VEGF generation and promote the upregulation of HIF-1α through a reduction of oxidative stress, the study evaluated the effect of the augmentation of GLP-1, by inhibitors of the dipeptidyl peptidase-4, such as vildagliptin, on angiogenesis process and wound healing in diabetic chronic ulcers. Although elucidation of the pathophysiologic importance of these aspects awaits further confirmations, the present study evidences an additional aspect of how DPP-4 inhibition might contribute to improved ulcer outcome.

  19. Inhibitory effect of linalool-rich essential oil from Lippia alba on the peptidase and keratinase activities of dermatophytes.

    Science.gov (United States)

    Costa, Danielle Cristina Machado; Vermelho, Alane Beatriz; Almeida, Catia Amancio; de Souza Dias, Edilma Paraguai; Cedrola, Sabrina Martins Lage; Arrigoni-Blank, Maria de Fátima; Blank, Arie Fitzgerald; Alviano, Celuta Sales; Alviano, Daniela Sales

    2014-02-01

    Abstract Lippia alba (Miller) N.E. Brown is an aromatic plant known locally as "Erva-cidreira-do-campo" that has great importance in Brazilian folk medicine. The aim of our study was to evaluate the antidermatophytic potential of linalool-rich essential oil (EO) from L. alba and analyze the ability of this EO to inhibit peptidase and keratinase activities, which are important virulence factors in dermatophytes. The minimum inhibitory concentrations (MICs) of L. alba EO were 39, 156 and 312 µg/mL against Trichophyton rubrum, Epidermophyton floccosum and Microsporum gypseum, respectively. To evaluate the influence of L. alba EO on the proteolytic and keratinolytic activities of these dermatophytes, specific inhibitory assays were performed. The results indicated that linalool-rich EO from L. alba inhibited the activity of proteases and keratinases secreted from dermatophytes, and this inhibition could be a possible mechanism of action against dermatophytes. Due to the effective antidermatophytic activity of L. alba EO, further experiments should be performed to explore the potential of this linalool-rich EO as an alternative antifungal therapy.

  20. RNA Regulation by Estrogen

    Science.gov (United States)

    2011-08-01

    sterile alpha motif domain containing 4A 11.31 216923_at CDKL5 cyclin-dependent kinase-like 5 11.31 211533_at pdgfra platelet-derived growth factor...Prnd prion protein 2 (dublet) 4.29 209854_s_at KLK2 kallikrein-related peptidase 2 4.29 210964_s_at gyg2 glycogenin 2 4.29 207070_at Rgr retinal G

  1. Dental Enamel Development: Proteinases and Their Enamel Matrix Substrates

    OpenAIRE

    Bartlett, John D.

    2013-01-01

    This review focuses on recent discoveries and delves in detail about what is known about each of the proteins (amelogenin, ameloblastin, and enamelin) and proteinases (matrix metalloproteinase-20 and kallikrein-related peptidase-4) that are secreted into the enamel matrix. After an overview of enamel development, this review focuses on these enamel proteins by describing their nomenclature, tissue expression, functions, proteinase activation, and proteinase substrate specificity. These protei...

  2. Distinct effects of dipeptidyl peptidase-4 inhibitor and glucagon-like peptide-1 receptor agonist on islet morphology and function.

    Science.gov (United States)

    Morita, Asuka; Mukai, Eri; Hiratsuka, Ayano; Takatani, Tomozumi; Iwanaga, Toshihiko; Lee, Eun Young; Miki, Takashi

    2016-03-01

    Although the two anti-diabetic drugs, dipeptidyl peptidase-4 inhibitors (DPP4is) and glucagon-like peptide-1 (GLP-1) receptor agonists (GLP1RAs), have distinct effects on the dynamics of circulating incretins, little is known of the difference in their consequences on morphology and function of pancreatic islets. We examined these in a mouse model of β cell injury/regeneration. The model mice were generated so as to express diphtheria toxin (DT) receptor and a fluorescent protein (Tomato) specifically in β cells. The mice were treated with a DPP4i (MK-0626) and a GLP1RA (liraglutide), singly or doubly, and the morphology and function of the islets were compared. Prior administration of MK-0626 and/or liraglutide similarly protected β cells from DT-induced cell death, indicating that enhanced GLP-1 signaling can account for the cytoprotection. However, 2-week intervention of MK-0626 and/or liraglutide in DT-injected mice resulted in different islet morphology and function: β cell proliferation and glucose-stimulated insulin secretion (GSIS) were increased by MK-0626 but not by liraglutide; α cell mass was decreased by liraglutide but not by MK-0626. Although liraglutide administration nullified MK-0626-induced β cell proliferation, their co-administration resulted in increased GSIS, decreased α cell mass, and improved glucose tolerance. The pro-proliferative effect of MK-0626 was lost by co-administration of the GLP-1 receptor antagonist exendin-(9-39), indicating that GLP-1 signaling is required for this effect. Comparison of the effects of DPP4is and/or GLP1RAs treatment in a single mouse model shows that the two anti-diabetic drugs have distinct consequences on islet morphology and function.

  3. Homology models of dipeptidyl peptidases 8 and 9 with a focus on loop predictions near the active site.

    Science.gov (United States)

    Rummey, Christian; Metz, Günther

    2007-01-01

    Dipeptidyl peptidase 4 (DP4) inhibitors are currently under intensive investigation in late-stage clinical trials as a treatment for type II diabetes. Lack of selectivity toward the related enzymes DP8 and DP9 has recently emerged as a possible source of drug-induced toxicity. Unlike DP4, X-ray structures of DP8 and DP9 are not yet available. As an aid to understanding the structural basis for selectivity, the authors have constructed homology models of DP8 and DP9 based on the X-ray coordinates of DP4. Accurate sequence alignment reveals common structural features indicative for a well-preserved overall fold comprising two domains, namely, a hydrolase domain and a so-called beta-propeller, which together form the active site deeply buried within the protein. The conformation of two loops inside this deep cavity is particularly relevant for the active sites. The authors used a published protocol for loop prediction based on conformational sampling and energy analysis to generate plausible solutions for these two loops. The predictive power of the approach was successfully evaluated for the template protein DP4 and two additional known structures from the same protein family, namely, FAP and DPX. The authors also show that inclusion of the covalent ligand NVP-728 greatly enhances the refinement. Based on the established evaluation protocol, the corresponding loops of DP8 and DP9 were predicted and the resulting active sites were compared with DP4. In particular, the authors conclude that differences in the P2-pocket are relevant for the design of selective DP4 inhibitors. The loss of key interactions in DP8 and DP9 as predicted from their models is consistent with the selectivity profile of the DP4 clinical candidate MK-431.

  4. Structure and activity of Streptococcus pyogenes SipA: a signal peptidase-like protein essential for pilus polymerisation.

    Science.gov (United States)

    Young, Paul G; Proft, Thomas; Harris, Paul W R; Brimble, Margaret A; Baker, Edward N

    2014-01-01

    The pili expressed on the surface of the human pathogen Streptococcus pyogenes play an important role in host cell attachment, colonisation and pathogenesis. These pili are built from two or three components, an adhesin subunit at the tip, a major pilin that forms a polymeric shaft, and a basal pilin that is attached to the cell wall. Assembly is carried out by specific sortase (cysteine transpeptidase) enzyme. These components are encoded in a small gene cluster within the S. pyogenes genome, often together with another protein, SipA, whose function is unknown. We show through functional assays, carried out by expressing the S. pyogenes pilus components in Lactococcus lactis, SipA from the clinically important M1T1 strain is essential for pilus assembly, and that SipA function is likely to be conserved in all S. pyogenes. From the crystal structure of SipA we confirm that SipA belongs to the family of bacterial signal peptidases (SPases), which process the signal-peptides of secreted proteins. In contrast to a previous arm-swapped SipA dimer, this present structure shows that its principal domain closely resembles the catalytic domain of SPases and has a very similar peptide-binding cleft, but it lacks the catalytic Ser and Lys residues characteristic of SPases. In SipA these are replaced by Asp and Gly residues, which play no part in activity. We propose that SipA functions by binding a key component at the bacterial cell surface, in a conformation that facilitates pilus assembly.

  5. Di-peptidyl peptidase-4 inhibitor sitagliptin protects vascular function in metabolic syndrome: possible role of epigenetic regulation.

    Science.gov (United States)

    Cicek, Figen Amber; Amber, Cicek Figen; Tokcaer-Keskin, Zeynep; Zeynep, Tokcaer-Keskin; Ozcinar, Evren; Evren, Ozcinar; Bozkus, Yosuf; Yusuf, Bozkus; Akcali, Kamil Can; Can, Akcali Kamil; Turan, Belma; Belma, Turan

    2014-08-01

    Metabolic syndrome (MetS) is a complex medical disorder characterized by insulin resistance, hypertension, and high risk of coronary disease and stroke. Microvascular rarefaction and endothelial dysfunction have also been linked with MetS, and recent evidence from clinical studies supports the efficacy of incretin-based antidiabetic therapies for vascular protection in diabetes. Previous studies pointed out the importance of dipeptidyl peptidase-4 (DPP-4) inhibition in endothelial cells due to getting protection against metabolic pathologies. We therefore aimed to investigate the acute effects of a DPP-4 inhibitor, sitagliptin, on vascular function in rats with high-sucrose diet-induced MetS. In order to elucidate the mechanisms implicated in the effects of DPP-4 inhibition, we tested the involvement of NO pathway and epigenetic regulation in the MetS. Acute use of sitagliptin protects the vascular function in the rats with MetS in part due to NO pathway via restoring the depressed aortic relaxation responses mediated by receptors. Application of sitagliptin enhanced the depressed phosphorylation levels of both the endothelial NO synthase and the apoptotic status of protein kinase B, known as Akt, in endothelium-intact thoracic aorta from rats with MetS. One-hour application of sitagliptin on aortic rings from rats with MetS also induced remarkable histon posttranslational modifications such as increased expression of H3K27Me3, but not of H3K27Me2, resulting in an accumulation of the H3K27Me3. Our findings suggest that, in addition to its well-known hypoglycemic action, sitagliptin may also have beneficial effects on hyperglycemia-induced vascular changes in an endotheium-dependent manner. These present results with sitagliptin aside from the glycaemic control, may demonstrate its important role in the treatment of patients with MetS.

  6. Effect of zinc and calcium ions on the rat kidney membrane-bound form of dipeptidyl peptidase IV

    Indian Academy of Sciences (India)

    Hansel Gómez; Mae Chappé; Pedroa Valiente; Tirso Pons; Marí­a de Los Angeles Chávez; Jean-Louis Charli; Isel Pascual

    2013-09-01

    Dipeptidyl peptidase IV (DPP-IV) is an ectopeptidase with many roles, and a target of therapies for different pathologies. Zinc and calcium produce mixed inhibition of porcine DPP-IV activity. To investigate whether these results may be generalized to mammalian DPP-IV orthologues, we purified the intact membrane-bound form from rat kidney. Rat DPP-IV hydrolysed Gly-Pro--nitroanilide with an average Vmax of 0.86±0.01 mol min–1mL–1 and KM of 76±6 M. The enzyme was inhibited by the DPP-IV family inhibitor L-threo-Ile-thiazolidide (Ki=64.0±0.53 nM), competitively inhibited by bacitracin (Ki=0.16±0.01 mM) and bestatin (Ki=0.23±0.02 mM), and irreversibly inhibited by TLCK (IC50 value of 1.20±0.11 mM). The enzyme was also inhibited by divalent ions like Zn2+ and Ca2+, for which a mixed inhibition mechanism was observed (Ki values of the competitive component: 0.15±0.01 mM and 50.0±1.05 mM, respectively). According to bioinformatic tools, Ca2+ ions preferentially bound to the -propeller domain of the rat and human enzymes, while Zn2+ ions to the - hydrolase domain; the binding sites were essentially the same that were previously reported for the porcine DPP-IV. These data suggest that the cationic susceptibility of mammalian DPP-IV orthologues involves conserved mechanisms.

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

    Science.gov (United States)

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

    2014-07-02

    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.

  8. Structure activity relationship modelling of milk protein-derived peptides with dipeptidyl peptidase IV (DPP-IV) inhibitory activity.

    Science.gov (United States)

    Nongonierma, Alice B; FitzGerald, Richard J

    2016-05-01

    Quantitative structure activity type models were developed in an attempt to predict the key features of peptide sequences having dipeptidyl peptidase IV (DPP-IV) inhibitory activity. The models were then employed to help predict the potential of peptides, which are currently reported in the literature to be present in the intestinal tract of humans following milk/dairy product ingestion, to act as inhibitors of DPP-IV. Two models (z- and v-scale) for short (2-5 amino acid residues) bovine milk peptides, behaving as competitive inhibitors of DPP-IV, were developed. The z- and the v-scale models (p<0.05, R(2) of 0.829 and 0.815, respectively) were then applied to 56 milk protein-derived peptides previously reported in the literature to be found in the intestinal tract of humans which possessed a structural feature of DPP-IV inhibitory peptides (P at the N2 position). Ten of these peptides were synthetized and tested for their in vitro DPP-IV inhibitory properties. There was no agreement between the predicted and experimentally determined DPP-IV half maximal inhibitory concentrations (IC50) for the competitive peptide inhibitors. However, the ranking for DPP-IV inhibitory potency of the competitive peptide inhibitors was conserved. Furthermore, potent in vitro DPP-IV inhibitory activity was observed with two peptides, LPVPQ (IC50=43.8±8.8μM) and IPM (IC50=69.5±8.7μM). Peptides present within the gastrointestinal tract of human may have promise for the development of natural DPP-IV inhibitors for the management of serum glucose.

  9. Clinical and economical consequences of the combination of metformin with dipeptidyl peptidase inhibitors in type 2 diabetes patients.

    Science.gov (United States)

    Sicras Mainar, A; Roldán Suárez, C; Font Ramos, B; Navarro Artieda, R; Ibáñez Nolla, J

    2013-11-01

    There are different second line glucose lowering drugs whose efficacy, safety and economic profile have not been established in our setting. We have analyzed the clinical (diabetic treatment adherence, metabolic control, hypoglycemia and macrovascular complications) and economic (resource use and costs) consequences of the combination of metformin with dipeptidyl peptidase inhibitors (DPPIV) in patients with type 2 diabetes. We conducted a multicenter, observational and retrospective study. Patients ≥30 years treated with metformin who initiated a second antidiabetic treatment during 2008-2009 were enrolled in the study. Two groups of patients were established: a) metformin with DPPIV and metformin with other diabetic drugs. The main measurements were comorbidity, compliance/persistence, metabolic control (glycosylated hemoglobin <7%), complications (hypoglycemia, macrovascular) and total costs. Patients were followed-up for 2 years. A total of 2,067 patients were enrolled (mean age: 66.6 years, 53.1% male). Of these, 519 patients (25.1%) were analyzed in the metformin+DPPIV group and 1,548 patients (74.9%) in the group metformin+other antidiabetic drug. The DPPIV group patients showed better compliance (70.3 vs. 59.6%), persistence (63.4 vs. 51.0%) and metabolic control (64.3 vs. 59.6%), respectively (P<.001) compared to the other group. They also showed a lower proportion of hypoglycemia (13.9 vs. 44.3%), cardiovascular events (3.7 vs. 7.6%) and total costs (2,347 vs. € 2,682), P<.05. Despite the limitations of the study, patients treated with metformin associated to DPPIV were more likely to show increased adherence, metabolic control and lower rates of hypoglycemia than those treated with metformin associated to other antidiabetics. Copyright © 2013 Elsevier España, S.L. All rights reserved.

  10. Mitochondrial localization of the threonine peptidase PfHslV, a ClpQ ortholog in Plasmodium falciparum.

    Science.gov (United States)

    Tschan, Serena; Kreidenweiss, Andrea; Stierhof, York-Dieter; Sessler, Nicole; Fendel, Rolf; Mordmüller, Benjamin

    2010-11-01

    Plasmodium falciparum belongs to a group of eukaryotes expressing an ortholog of the prokaryotic T1-threonine peptidase, heat shock locus V (HslV). Bacterial HslV is a particularly well studied protease, due to its structural and biochemical similarity to the eukaryotic proteasome. Plasmodium falciparum HslV (PfHslV) is expressed in schizonts and merozoites of the asexual blood stage. Strong sequence conservation between plasmodial species, absence of HslV homologs in the human genome, and availability of specific inhibitors led us to explore its function and potential use as a drug target. In a first step, we investigated localization of PfHslV, using a bioinformatics approach and a transgenic P. falciparum line expressing a PfHslV-enhanced yellow fluorescent protein (EYFP) fusion protein from the endogenous pfhslV locus. PfHslV-EYFP was found in the mitochondrial matrix under fluorescence and immunoelectron microscopy. Endogenous, non-modified PfHslV was present in purified mitochondria and interference with mitochondrial membrane potential by drug treatment led to impairment of PfHslV processing. Import of heterologous EYFP into the plasmodial mitochondrion is mediated by the N-terminal 37 amino acids of PfHslV. PfHslV's targeting sequence is also functional in human cells, demonstrating strong conservation of mitochondrial targeting in eukaryotes. In conclusion, our data shows that PfHslV is located to the plasmodial mitochondrion and presumably has vital function within this organelle which makes it an attractive target for interventions.

  11. Cost-effectiveness of dipeptidyl peptidase-4 inhibitor monotherapy in elderly type 2 diabetes patients in Thailand

    Science.gov (United States)

    Permsuwan, Unchalee; Dilokthornsakul, Piyameth; Saokaew, Surasak; Thavorn, Kednapa; Chaiyakunapruk, Nathorn

    2016-01-01

    Background The management of type 2 diabetes mellitus (T2DM) in elderly population poses many challenges. Dipeptidyl peptidase-4 (DPP-4) inhibitors show particular promise due to excellent tolerability profiles, low risk of hypoglycemia, and little effect on body weight. This study evaluated, from the health care system’s perspective, the long-term cost-effectiveness of DPP-4 inhibitor monotherapy vs metformin and sulfonylurea (SFU) monotherapy in Thai elderly T2DM patients. Methods The clinical efficacy was estimated from a systematic review and meta-analysis. Baseline cohort characteristics and cost parameters were obtained from published studies and hospital databases in Thailand. A validated IMS CORE Diabetes Model version 8.5 was used to project clinical and economic outcomes over a lifetime horizon using a 3% annual discount rate. Costs were expressed in 2014 Thai Baht (THB) (US dollar value). Incremental cost-effectiveness ratios were calculated. Base-case assumptions were assessed through several sensitivity analyses. Results For treating elderly T2DM patients, DPP-4 inhibitors were more expensive and less effective, ie, a dominated strategy, than the metformin monotherapy. Compared with SFU, treatment with DPP-4 inhibitors gained 0.031 more quality-adjusted life years (QALYs) at a total cost incurred over THB113,701 or US$3,449.67, resulting in an incremental cost-effectiveness ratio of THB3.63 million or US$110,133.50 per QALY. At the acceptable Thai ceiling threshold of THB160,000/QALY (US$4,854.37/QALY), DPP-4 inhibitors were not a cost-effective treatment. Conclusion DPP-4 inhibitor monotherapy is not a cost-effective treatment for elderly T2DM patients compared with metformin monotherapy and SFU monotherapy, given current resource constraints in Thailand. PMID:27703387

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

    Directory of Open Access Journals (Sweden)

    Pryor Kellyann

    2009-04-01

    Full Text Available Abstract 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.

  13. Dipeptidyl peptidase-4 inhibitor decreases abdominal aortic aneurysm formation through GLP-1-dependent monocytic activity in mice.

    Directory of Open Access Journals (Sweden)

    Hsin Ying Lu

    Full Text Available Abdominal aortic aneurysm (AAA is a life-threatening situation affecting almost 10% of elders. There has been no effective medication for AAA other than surgical intervention. Dipeptidyl peptidase-4 (DPP-4 inhibitors have been shown to have a protective effect on cardiovascular disease. Whether DPP-4 inhibitors may be beneficial in the treatment of AAA is unclear. We investigated the effects of DPP-4 inhibitor sitagliptin on the angiotensin II (Ang II-infused AAA formation in apoE-deficient (apoE-/- mice. Mice with induced AAA were treated with placebo or 2.5, 5 or 10 mg/kg/day sitagliptin. Ang II-infused apoE-/- mice exhibited a 55.6% incidence of AAA formation, but treatment with sitagliptin decreased AAA formation. Specifically, administered sitagliptin in Ang II-infused mice exhibited decreased expansion of the suprarenal aorta, reduced elastin lamina degradation of the aorta, and diminished vascular inflammation by macrophage infiltration. Treatment with sitagliptin decreased gelatinolytic activity and apoptotic cells in aorta tissues. Sitaglipitn, additionally, was associated with increased levels of plasma active glucagon-like peptide-1 (GLP-1. In vitro studies, GLP-1 decreased reactive oxygen species (ROS production, cell migration, and MMP-2 as well as MMP-9 activity in Ang II-stimulated monocytic cells. The results conclude that oral administration of sitagliptin can prevent abdominal aortic aneurysm formation in Ang II-infused apoE-/-mice, at least in part, by increasing of GLP-1 activity, decreasing MMP-2 and MMP-9 production from macrophage infiltration. The results indicate that sitagliptin may have therapeutic potential in preventing the development of AAA.

  14. Serum activities of adenosine deaminase, dipeptidyl peptidase IV and prolyl endopeptidase in patients with fibromyalgia: diagnostic implications.

    Science.gov (United States)

    Čulić, Ognjen; Cordero, Mario D; Žanić-Grubišić, Tihana; Somborac-Bačura, Anita; Pučar, Lara Batičić; Detel, Dijana; Varljen, Jadranka; Barišić, Karmela

    2016-10-01

    Fibromyalgia (FM) is a chronic pain syndrome with number of symptoms that present challenge in terms of diagnosis and treatment. Patients with FM show abnormal profile of purines in plasma. In this work, we measured serum activities of enzymes involved in purine metabolism, namely total adenosine deaminase (ADE) and its isoforms (ADE1 and ADE2), ecto-ATPase, and 5'-nucleotidase (5'-NT). We also measured activity of dipeptidyl peptidase IV (DPPIV) and prolyl endopeptidase (PEP). Spectrophotometric and fluorometric methods were used for enzyme activity determinations. Enzyme activities were measured in sera of 24 patients with FM that were not undergoing pharmacological treatment during the study. Control group comprised 32 healthy control subjects. Significantly higher activities of total ADE (P = 0.025) and ADE2 (P = 0.011) were observed in FM patients, while no significant differences in ADE1, ecto-ATPase, and 5'-NT activities (P > 0.05) were found when compared to healthy controls. Moreover, increase in the activity of DPPIV (P = 0.015) and lower activity of PEP (P = 0.011) were also found in the FM group. ROC analysis pointed to different diagnostic sensitivities/specificities for individual enzyme activities measured as follows: ADE (50.0/87.5), ADE2 (41.7/90.6), DPPIV (62.5/71.9), and PEP (83.3/62.5). ADE2 and PEP were shown to be independent predictors of FM, while combination of the two gives AUC of 0.786 (95 % confidence interval of 0.656-0.885, P < 0.05). Our results are showing that serum activities of ADE2 and PEP could be useful as biomarkers for FM diagnosis. However, relatively low diagnostic sensitivity of ADE2 and specificity of PEP must be taken into account.

  15. Absorption, metabolism, and excretion of [14C]vildagliptin, a novel dipeptidyl peptidase 4 inhibitor, in humans.

    Science.gov (United States)

    He, Handan; Tran, Phi; Yin, Hequn; Smith, Harold; Batard, Yannick; Wang, Lai; Einolf, Heidi; Gu, Helen; Mangold, James B; Fischer, Volker; Howard, Dan

    2009-03-01

    The absorption, metabolism, and excretion of (1-[[3-hydroxy-1-adamantyl) amino] acetyl]-2-cyano-(S)-pyrrolidine (vildagliptin), an orally active and highly selective dipeptidyl peptidase 4 inhibitor developed for the treatment of type 2 diabetes, were evaluated in four healthy male subjects after a single p.o. 100-mg dose of [(14)C]vildagliptin. Serial blood and complete urine and feces were collected for 168 h postdose. Vildagliptin was rapidly absorbed, and peak plasma concentrations were attained at 1.1 h postdose. The fraction of drug absorbed was calculated to be at least 85.4%. Unchanged drug and a carboxylic acid metabolite (M20.7) were the major circulating components in plasma, accounting for 25.7% (vildagliptin) and 55% (M20.7) of total plasma radioactivity area under the curve. The terminal half-life of vildagliptin was 2.8 h. Complete recovery of the dose was achieved within 7 days, with 85.4% recovered in urine (22.6% unchanged drug) and the remainder in feces (4.54% unchanged drug). Vildagliptin was extensively metabolized via at least four pathways before excretion, with the major metabolite M20.7 resulting from cyano group hydrolysis, which is not mediated by cytochrome P450 (P450) enzymes. Minor metabolites resulted from amide bond hydrolysis (M15.3), glucuronidation (M20.2), or oxidation on the pyrrolidine moiety of vildagliptin (M20.9 and M21.6). The diverse metabolic pathways combined with a lack of significant P450 metabolism (1.6% of the dose) make vildagliptin less susceptible to potential pharmacokinetic interactions with comedications of P450 inhibitors/inducers. Furthermore, as vildagliptin is not a P450 inhibitor, it is unlikely that vildagliptin would affect the metabolic clearance of comedications metabolized by P450 enzymes.

  16. A molecular connection of Pterocarpus marsupium, Eugenia jambolana and Gymnema sylvestre with dipeptidyl peptidase-4 in the treatment of diabetes.

    Science.gov (United States)

    Kosaraju, Jayasankar; Dubala, Anil; Chinni, Santhivardhan; Khatwal, Rizwan Basha; Satish Kumar, M N; Basavan, Duraiswamy

    2014-02-01

    Pterocarpus marsupium (PM) (Leguminosae), Eugenia jambolana (EJ) (Myrtaceae) and Gymnema sylvestre (GS) (Asclepiadaceae) are the most important medicinal plants in the Indian system of traditional medicine for the treatment of hyperglycemia. Dipeptidyl peptidase-4 (DPP-4) inhibitors are the emerging class of anti-diabetic agents. However, only few compounds are commercially available. Therefore, in the present study we tried to explore the naturally occurring PM, EJ and GS semi-standardized extracts for their potential DPP-4 inhibition in vitro and in vivo. DPP-4 inhibition was evaluated by in vitro inhibitory assay, and enzyme kinetics were calculated using one-phase exponential decay equation. Glucose load (2 g/kg) was administered to control and diabetic rats 30 min following extract administration (100, 200 and 400 mg/kg) orally once, and blood samples were withdrawn at 0, 0.5, 1, 1.5, 2 and 3 h to measure plasma active glucagon-like peptide-1 (GLP-1) levels. PM and EJ inhibit DPP-4 potently with IC50 values of 273.73 ± 2.96 and 278.94 ± 6.73 µg/mL, respectively, compared to GS (773.22 ± 9.21 µg/mL). PM, EJ and GS exhibit long duration of action with enzyme inhibitory half-lives of 462.3, 317.2 and 153.8 min, respectively. Extracts significantly increase GLP-1 levels compared to negative control groups and peak GLP-1 level was observed at 2 h for PM and EJ, whereas for GS it was at 1.5 h Taken together, results suggest the extracts may have potent DPP-4 inhibitory action, and their hypoglycemic action attributed through an increase in plasma active GLP-1 levels.

  17. In vitro screening for protein tyrosine phosphatase 1B and dipeptidyl peptidase IV inhibitors from selected Nigerian medicinal plants.

    Science.gov (United States)

    Saidu, Yusuf; Muhammad, Suleiman Alhaji; Abbas, Abdullahi Yahaya; Onu, Andrew; Tsado, Ibrahim Mohammed; Muhammad, Luba

    2017-01-01

    Protein tyrosine phosphatase 1B (PTP 1B) and dipeptidyl peptidase IV (DPP IV) have been identified as one of the drug targets for the treatment of Type-2 diabetes. This study was designed to screen for PTP 1B and DPP-IV inhibitors from some Nigerian medicinal plants. PTP 1B and DPP-IV drug discovery kits from Enzo Life Sciences were used to investigate in vitro inhibitory effect of crude methanolic extract of 10 plants; Mangifera indica, Moringa oleifera, Acacia nilotica, Arachis hypogaea, Senna nigricans, Azadirachta indica, Calotropis procera, Leptadenia hastata, Ziziphus mauritiana, and Solanum incanum. The results indicated PTP IB inhibition by S. nigricans (68.2 ± 2.29%), A. indica (67.4 ± 2.80%), A. hypogaea (57.2 ± 2.50%), A. nilotica (55.1 ± 2.19%), and M. oleifera (41.2 ± 1.87%) were significantly (P 0.05) different from that of sumarin. The DPP-IV inhibition by S. incanum (68.1 ± 2.71%) was significantly higher as compared with a known inhibitor, P32/98. S. nigrican (57.0±1.91%), Z. mauritiana (56.6±2.01%), A. hypogaea (51.0±1.30%), M. indica (44.6 ± 2.40%), C. procera (36.2 ± 2.00%), A. nilotica (35.4 ± 2.10%), and A. indica (33.6 ± 1.50%) show significantly (P < 0.05) lower inhibitions toward DPP-IV. The work demonstrated that these plant materials could serve as sources of lead compounds in the development of anti-diabetic agent(s) targeting PTP 1B and/or DPP-IV.

  18. Bullous Pemphigoid Associated with the Dipeptidyl Peptidase-4 Inhibitor Sitagliptin in a Patient with Liver Cirrhosis Complicated with Rapidly Progressive Hepatocellular Carcinoma.

    Science.gov (United States)

    Harada, Masaru; Yoneda, Akitoshi; Haruyama, Sanehito; Yabuki, Kei; Honma, Yuichi; Hiura, Masaaki; Shibata, Michihiko; Matsuoka, Hidehiko; Uchiwa, Yasuhiro

    2017-09-15

    A 78-year-old man presented with cutaneous blisters of the limbs and abdominal distension. He had been treated for various diseases, including liver cirrhosis. He had begun receiving sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, for diabetes mellitus three years before the hospitalization. A skin biopsy demonstrated bullous pemphigoid. Ultrasonography (US) revealed multiple liver tumors, although he had been receiving regular US studies. We stopped sitagliptin and started insulin and corticosteroids. However, his renal dysfunction progressed, and he died 14 days after the hospitalization. We should therefore be careful of various complications, including bullous pemphigoid and progression of tumors, when using DPP-4 inhibitors.

  19. Muscle cell migrations of C. elegans are mediated by the alpha-integrin INA-1, Eph receptor VAB-1, and a novel peptidase homologue MNP-1.

    Science.gov (United States)

    Tucker, Morgan; Han, Min

    2008-06-15

    Cell migration is a fundamental process occurring during embryonic development and tissue morphogenesis. In the nematode Caenorhabditis elegans, morphogenesis of the body-wall musculature involves short-range migrations of 81 embryonic muscle cells from the lateral surface of the embryo towards the dorsal and ventral midlines. This study shows that mutations in ina-1 (alpha-integrin), as well as vab-1 (Eph receptor), and vab-2 (ephrin), display defects in embryonic muscle cell migration. Furthermore, an RNAi-based enhancer screen in an ina-1 weak loss-of-function background identified mnp-1 (matrix non-peptidase homologue-1) as a previously uncharacterized gene required for promoting proper migration of the embryonic muscle cells. mnp-1 encodes a membrane associated metalloproteinase homologue that is predicted to be catalytically inactive. Our data suggest that MNP-1 is expressed in migrating muscle cells and localizes to the plasma membrane with the non-peptidase domain exposed to the extra-cellular environment. Double-mutant analysis between mnp-1(RNAi), ina-1, and vab-1 mutations; as well as tissue specific rescue experiments; indicated that each of these gene products function predominantly independent of each other and from different cell types to affect muscle cell migration. Together these results suggest complex interactions between the adjacent epidermal, neuronal, and muscle cells are required to promote proper muscle cell migration during embryogenesis.

  20. Proteolytic cleavage by the IMP complex or Oct1 peptidase controls the localization of the yeast peroxiredoxin Prx1 to distinct mitochondrial compartments.

    Science.gov (United States)

    Gomes, Fernando; Palma, Flavio Romero; Barros, Mario H; Tsuchida, Eduardo T; Turano, Helena G; Alegria, Thiago G P; Demasi, Marilene; Netto, Luis E S

    2017-08-18

    Yeast Prx1 is a mitochondrial 1-Cys peroxiredoxin that catalyzes the reduction of endogenously generated H2O2 Prx1 is synthesized on cytosolic ribosomes as a preprotein with a cleavable N-terminal presequence that is the mitochondrial targeting signal, but the mechanisms underlying Prx1 distribution to distinct mitochondrial subcompartments are unknown. Here, we provide direct evidence of the following dual mitochondrial localization of Prx1: a soluble form in the intermembrane space and a form in the matrix weakly associated with the inner mitochondrial membrane. We show that Prx1 sorting into the intermembrane space likely involves the release of the protein precursor within the lipid bilayer of the inner membrane, followed by cleavage by the inner membrane peptidase (IMP). We also found that during its import into the matrix compartment, Prx1 is sequentially cleaved by mitochondrial processing peptidase (MPP) and then by octapeptidyl aminopeptidase 1 (Oct1). Oct1 cleaved eight amino acid residues from the N-terminal region of Prx1 inside the matrix, without interfering with its peroxidase activity in vitro Remarkably, the processing of Prx proteins by Oct1 appears to be an evolutionarily conserved process since yeast Oct1 could cleave the human mitochondrial peroxiredoxin Prx3 when expressed in Saccharomyces cerevisiae Altogether, the processing of peroxiredoxins by Imp2 or Oct1 likely represents systems that control the localization of Prxs into distinct compartments and thereby contribute to various mitochondrial redox processes.. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  1. ER type I signal peptidase subunit (LmSPC1) is essential for the survival of Locusta migratoria manilensis and affects moulting, feeding, reproduction and embryonic development.

    Science.gov (United States)

    Zhang, W; Xia, Y

    2014-06-01

    The endoplasmic reticulum type I signal peptidase complex (ER SPC) is a conserved enzyme that cleaves the signal peptides of secretory or membrane preproteins. The deletion of this enzyme leads to the accumulation of uncleaved proteins in biomembranes and cell death. However, the physiological functions of ER SPC in insects are not fully understood. Here, a catalytic subunit gene of ER SPC, LmSPC1, was cloned from Locusta migratoria manilensis and its physiological functions were analysed by RNA interference (RNAi). The LmSPC1 open reading frame encoded a protein of 178 amino acids with all five conserved regions of signal peptidases. RNAi-mediated knockdown of LmSPC1 resulted in high mortality. Sixty-nine per cent of dead nymphs died of abnormal moulting, corresponding to decreased activity of moulting fluid protease. Moreover, insects in the RNAi group experienced a decline in food intake, and a decrease in the secretion of total protein and digestive enzymes from midgut tissues to the midgut lumen. Furthermore, the females produced fewer eggs and eggs with disrupted embryogenesis. These results indicate that LmSPC1 is required for the secretion of secretory proteins, affects physiological functions, including moulting, feeding, reproduction and embryonic development, and is essential for survival. Therefore, LmSPC1 may be a potential target for locust control.

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

    Science.gov (United States)

    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.

  3. The dipeptidyl peptidase-4 inhibitor vildagliptin has the capacity to repair β-cell dysfunction and insulin resistance.

    Science.gov (United States)

    Horie, A; Tokuyama, Y; Ishizuka, T; Suzuki, Y; Marumo, K; Oshikiri, K; Ide, K; Sunaga, M; Kanatsuka, A

    2014-10-01

    The aim of the present study was to determine whether the dipeptidyl peptidase (DPP)-4 inhibitor could repair pancreatic β-cell dysfunction and insulin resistance. Ten subjects with type 2 diabetes who had never received DPP-4 inhibitor treatment were enrolled in the study. Just before and 3 months after twice-daily administration of vildagliptin (50 mg tablets), insulin secretion and insulin sensitivity were estimated using 2-compartment model analysis of C-peptide kinetics and insulin-modified minimal model parameters, respectively. The first-phase insulin secretion (CS1) was determined as the sum of the C-peptide secretion rate (CSR) from 0 to 5 min (normal range 6.8-18.5 ng/ml/min). The whole-body insulin sensitivity index (SI) was calculated using a minimal model software program (normal range 2.6-7.6×10(-4)/min/μU/ml). After vildagliptin treatment, reductions in mean (± SE) HbA1c were noted (43.28±1.53 vs. 40.98±1.77 mmol/mol; p=0.019). Vildagliptin treatment increased the area under the curve for the C peptide reactivity (CPR) (AUCCPR; 26.66±5.15 vs. 33.02±6.12 ng/ml · 20 min; p=0.003) and CS1 (0.80±0.20 vs. 1.35±0.38 ng/ml/min; p=0.037) in response to an intravenous glucose load. -Vildagliptin treatment significantly increased SI (0.46±0.27 vs. 1.21±0.48×10(-4)/min/μU/ml; p=0.037). The long-term administration of vildagliptin improved CS1 and Si suggesting that this drug has the capacity to repair impairments in pancreatic β-cell function and insulin resistance in type 2 diabetes.

  4. Cost-effectiveness of dipeptidyl peptidase-4 inhibitor monotherapy in elderly type 2 diabetes patients in Thailand

    Directory of Open Access Journals (Sweden)

    Permsuwan U

    2016-09-01

    Full Text Available Unchalee Permsuwan,1 Piyameth Dilokthornsakul,2 Surasak Saokaew,2–4 Kednapa Thavorn,5–7 Nathorn Chaiyakunapruk2,4,8,9 1Faculty of Pharmacy Chiang Mai University, Chiang Mai, 2Center of Pharmaceutical Outcomes Research, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, 3Center of Health Outcomes Research and Therapeutic Safety, School of Pharmaceutical Sciences, University of Phayao, Phayao, Thailand; 4School of Pharmacy, Monash University Malaysia, Sunway, Malaysia; 5Ottawa Hospital Research Institute, The Ottawa Hospital, 6School of Epidemiology, Public Health and Preventive Medicine, Faculty of Medicine, University of Ottawa, Ottawa, 7Institute for Clinical and Evaluative Sciences, Toronto, ON, Canada; 8School of Population Health, The University of Queensland, Brisbane, QLD, Australia; 9School of Pharmacy, University of Wisconsin–Madison, Madison, WI, USA Background: The management of type 2 diabetes mellitus (T2DM in elderly population poses many challenges. Dipeptidyl peptidase-4 (DPP-4 inhibitors show particular promise due to excellent tolerability profiles, low risk of hypoglycemia, and little effect on body weight. This study evaluated, from the health care system’s perspective, the long-term cost-effectiveness of DPP-4 inhibitor monotherapy vs metformin and sulfonylurea (SFU monotherapy in Thai elderly T2DM patients. Methods: The clinical efficacy was estimated from a systematic review and meta-analysis. Baseline cohort characteristics and cost parameters were obtained from published studies and hospital databases in Thailand. A validated IMS CORE Diabetes Model version 8.5 was used to project clinical and economic outcomes over a lifetime horizon using a 3% annual discount rate. Costs were expressed in 2014 Thai Baht (THB (US dollar value. Incremental cost-effectiveness ratios were calculated. Base-case assumptions were assessed through several sensitivity analyses. Results: For treating elderly T2DM

  5. Regulation of chemokine activity – a focus on the role of dipeptidyl peptidase IV/CD26

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    Mieke Metzemaekers

    2016-11-01

    Full Text Available Chemokines are small, chemotactic proteins that play a crucial role in leukocyte migration and are therefore essential for proper functioning of the immune system. Chemokines exert their chemotactic effect by activation of chemokine receptors, which are G protein-coupled receptors (GPCRs, and interaction with glycosaminoglycans (GAGs. Furthermore, the exact chemokine function is modulated at the level of posttranslational modifications. Among the different types of posttranslational modifications that were found to occur in vitro and in vivo, i.e. proteolysis, citrullination, glycosylation and nitration, NH2-terminal proteolysis of chemokines has been described most intensively. Since the NH2-terminal chemokine domain mediates receptor interaction, NH2-terminal modification by limited proteolysis or amino acid side chain modification can drastically affect their biological activity. An enzyme that has been shown to provoke NH2-terminal proteolysis of various chemokines is dipeptidyl peptidase IV or CD26. This multifunctional protein is a serine protease that preferably cleaves dipeptides from the NH2-terminal region of peptides and proteins with a proline or alanine residue in the penultimate position. Various chemokines possess such a proline or alanine residue and CD26-truncated forms of these chemokines have been identified in cell culture supernatant as well as in body fluids. The effects of CD26-mediated proteolysis in the context of chemokines turned out to be highly complex. Depending on the chemokine ligand, loss of these two NH2-terminal amino acids can result in either an increased or a decreased biological activity, enhanced receptor specificity, inactivation of the chemokine ligand or generation of receptor antagonists. Since chemokines direct leukocyte migration in homeostatic as well as pathophysiologic conditions, CD26-mediated proteolytic processing of these chemotactic proteins may have significant consequences for appropriate

  6. Treatment progression in sulfonylurea and dipeptidyl peptidase-4 inhibitor cohorts of type 2 diabetes patients on metformin

    Science.gov (United States)

    Peng, Xiaomei; Jiang, Dingfeng; Liu, Dongju; Varnado, Oralee J; Bae, Jay P

    2016-01-01

    Background Metformin is an oral antidiabetic drug (OAD) widely used as first-line therapy in type 2 diabetes (T2D) treatments. Numerous treatment pathways after metformin failure exist. It is important to understand how treatment choices influence subsequent therapy progressions. This retrospective study compares adherence to, persistence with, and treatment progression in sulfonylurea (SU) and dipeptidyl peptidase-4 (DPP-4) inhibitor patient cohorts with T2D on metformin. Methods Using health insurance claims data, matched patient cohorts were created and OAD use was compared in patients with T2D initiating SU or DPP-4 inhibitors (index drugs) since January 1, 2010, to December 31, 2010, with background metformin therapy. Propensity score matching adjusted for possible selection bias. Persistence was measured via Cox regression as days to a ≥60-day gap in index drug possession; adherence was defined as proportion of days covered (PDC) ≥80%. Evolving treatment patterns were traced at 6-month intervals for 24 months following index drug discontinuation. Results From among 19,621 and 7,484 patients in the SU and DPP-4 inhibitor cohorts, respectively, 6,758 patient pairs were matched. Persistence at 12 months in the SU cohort was 48.0% compared to 52.5% for the DPP-4 inhibitor cohort. PDC adherence (mean [SD]) during the 12-month follow-up period was 63.3 (29.7) for the SU cohort and 65.5 (28.7) for the DPP-4 inhibitor cohort. PDC ≥80% was 40.5% and 43.4% in the SU and DPP-4 inhibitor cohorts, respectively. A higher percentage of patients in the SU cohort remained untreated. Following index drug discontinuation, monotherapy was more common in the SU cohort, while use of two or three OADs was more common in the DPP-4 inhibitor cohort. Insulin therapy initiation was higher in the SU cohort. Conclusion Slightly better adherence and persistence were seen in the DPP-4 inhibitor cohort. Adherence and persistence remain a challenge to many patients; understanding

  7. Pharmacokinetic and pharmacodynamic interactions between metformin and a novel dipeptidyl peptidase-4 inhibitor, evogliptin, in healthy subjects

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    Rhee SJ

    2016-08-01

    Full Text Available Su-jin Rhee,1,* YoonJung Choi,1,* SeungHwan Lee,1,2 Jaeseong Oh,1 Sung-Jin Kim,3 Seo Hyun Yoon,1 Joo-Youn Cho,1 Kyung-Sang Yu1 1Department of Clinical Pharmacology and Therapeutics, Seoul National University Hospital, Seoul National University College of Medicine, Seoul, Republic of Korea; 2Clinical Trials Center, Seoul National University Hospital, Seoul, Republic of Korea; 3Department of Clinical Development, Dong-A ST Co., Ltd., Seoul, Republic of Korea *These authors contributed equally to this work Abstract: Evogliptin is a newly developed dipeptidyl peptidase-4 (DPP-4 inhibitor, which is expected to be combined with metformin for treating type 2 diabetes mellitus. We investigated the potential pharmacokinetic and pharmacodynamic interactions between evogliptin and metformin. A randomized, open-label, multiple-dose, six-sequence, three-period crossover study was conducted in 36 healthy male subjects. All subjects received three treatments, separated by 7-day washout intervals: evogliptin, 5 mg od for 7 days (EVO; metformin IR, 1,000 mg bid for 7 days (MET; and the combination of EVO and MET (EVO + MET. After the last dose in a period, serial blood samples were collected for 24 hours for pharmacokinetic assessments. During steady state, serial blood samples were collected for 2 hours after an oral glucose tolerance test, and DPP-4, active glucagon-like peptide-1, glucose, glucagon, insulin, and C-peptide were measured to assess pharmacodynamic properties. EVO + MET and EVO showed similar steady state maximum concentration and area under the concentration–time curve at steady state values for evogliptin; the geometric mean ratios (90% confidence interval were 1.06 (1.01–1.12 and 1.02 (0.99–1.06, respectively. EVO + MET slightly reduced steady state maximum concentration and area under the concentration–time curve at steady state values for metformin compared to MET, with geometric mean ratios (90% confidence interval of 0.84 (0.79

  8. Signal peptide peptidase (SPP dimer formation as assessed by fluorescence lifetime imaging microscopy (FLIM in intact cells

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    Nyborg Andrew C

    2006-11-01

    Full Text Available Abstract Background Signal peptide peptidase (SPP is an intramembrane cleaving protease identified by its cleavage of several type II membrane signal peptides. Conservation of intramembrane active site residues demonstrates that SPP, SPP family members, and presenilins (PSs make up a family of intramembrane cleaving proteases. Because SPP appears to function without additional protein cofactors, the study of SPP may provide structural insights into the mechanism of intramembrane proteolysis by this biomedically important family of proteins. Previous studies have shown that SPP isolated from cells appears to be a homodimer, but some evidence exists that in vitro SPP may be active as a monomer. We have conducted additional experiments to determine if SPP exists as a monomer or dimer in vivo. Results Fluorescence lifetime imaging microscopy (FLIM can be is used to determine intra- or intermolecular interactions by fluorescently labeling epitopes on one or two different molecules. If the donor and acceptor fluorophores are less than 10 nm apart, the donor fluorophore lifetime shortens proportionally to the distance between the fluorophores. In this study, we used two types of fluorescence energy transfer (FRET pairs; cyan fluorescent protein (CFP with yellow fluorescent protein (YFP or Alexa 488 with Cy3 to differentially label the NH2- or COOH-termini of SPP molecules. A cell based SPP activity assay was used to show that all tagged SPP proteins are proteolytically active. Using FLIM we were able to show that the donor fluorophore lifetime of the CFP tagged SPP construct in living cells significantly decreases when either a NH2- or COOH-terminally YFP tagged SPP construct is co-transfected, indicating close proximity between two different SPP molecules. These data were then confirmed in cell lines stably co-expressing V5- and FLAG-tagged SPP constructs. Conclusion Our FLIM data strongly suggest dimer formation between two separate SPP proteins

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

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    Zhu JY

    2014-02-01

    Full Text Available Jianyong Zhu,1,* XiaoDong Guo,2,* Baoan Qiu,1 Zhiyan Li,2 Nianxin Xia,1 Yingxiang Yang,1 Peng Liu1 1Department of Hepatobiliary Surgery, Navy General Hospital, PLA, Beijing, People's Republic of China; 2302 Hospital of PLA, Beijing, People's Republic of China *These authors contributed equally to this work 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

  10. Treatment progression in sulfonylurea and dipeptidyl peptidase-4-inhibitor cohorts of type 2 diabetes patients on metformin

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    Peng X

    2016-08-01

    Full Text Available Xiaomei Peng, Dingfeng Jiang, Dongju Liu, Oralee J Varnado, Jay P Bae Eli Lilly and Company, Global Patient Outcomes and Real World Evidence, Indianapolis, IN, USA Background: Metformin is an oral antidiabetic drug (OAD widely used as first-line therapy in type 2 diabetes (T2D treatments. Numerous treatment pathways after metformin failure exist. It is important to understand how treatment choices influence subsequent therapy progressions. This retrospective study compares adherence to, persistence with, and treatment progression in sulfonylurea (SU and dipeptidyl peptidase-4 (DPP-4 inhibitor patient cohorts with T2D on metformin. Methods: Using health insurance claims data, matched patient cohorts were created and OAD use was compared in patients with T2D initiating SU or DPP-4 inhibitors (index drugs since January 1, 2010, to December 31, 2010, with background metformin therapy. Propensity score matching adjusted for possible selection bias. Persistence was measured via Cox regression as days to a ≥60-day gap in index drug possession; adherence was defined as proportion of days covered (PDC ≥80%. Evolving treatment patterns were traced at 6-month intervals for 24 months following index drug discontinuation. Results: From among 19,621 and 7,484 patients in the SU and DPP-4 inhibitor cohorts, respectively, 6,758 patient pairs were matched. Persistence at 12 months in the SU cohort was 48.0% compared to 52.5% for the DPP-4 inhibitor cohort. PDC adherence (mean [SD] during the 12-month follow-up period was 63.3 (29.7 for the SU cohort and 65.5 (28.7 for the DPP-4 inhibitor cohort. PDC ≥80% was 40.5% and 43.4% in the SU and DPP-4 inhibitor cohorts, respectively. A higher percentage of patients in the SU cohort remained untreated. Following index drug discontinuation, monotherapy was more common in the SU cohort, while use of two or three OADs was more common in the DPP-4 inhibitor cohort. Insulin therapy initiation was higher in the SU

  11. Generation and confirmation of NAAG peptidase gene knockout model%NAAG肽酶基因剔除小鼠模型的建立及鉴定

    Institute of Scientific and Technical Information of China (English)

    崔振文; 张明坤; 钟春龙; 吴增宝; 蔡蕾; 匡颖; 王铸钢; 江基尧; 罗其中

    2012-01-01

    目的 建立N-乙酰天冬氨酰谷氨酸(NAAG)肽酶基因剔除小鼠模型,为在体研究NAAG肽酶基因的生物学功能并揭示其在脑损伤后继发性脑损害进程中的所起的作用创造条件.方法 根据小鼠NAAG肽酶基因组的序列,设计基因剔除策略,构建基因剔除载体NAAG-KO-pBR322,以电穿孔方法将基因剔除载体导入胚胎干细胞(ES),应用G418和更昔洛韦进行正负筛选,获得双抗性克隆,聚合酶链式反应(PCR)鉴定并测序获得正确同源重组的ES细胞克隆.结果 同源重组的ES细胞注入小鼠囊胚后获得11只嵌合率>50%嵌合体雄性小鼠,嵌合体小鼠与C57 BL/6J雌鼠交配后获得11只杂合子小鼠,其中雄性7只,雌性4只.在雌、雄杂合子小鼠交配的后代中获得7只纯合子小鼠,PCR鉴定其基因型,逆转录PCR (RT-PCR)提示该基因鼠未表达NAAG肽酶.结论 我们成功建立了NAAG肽酶基因剔除小鼠模型,其中纯合子小鼠未出现胚胎致死现象;初步的表型观察未发现NAAG肽酶基因剔除小鼠出现异常改变.%Objective To establish N-acetylaspartylglutamate (NAAG) peptidase gene knockout mouse model and to create the condition for farther in vivo study of its biological function and its role in the secondary brain damage after brain injury. Methods According to the NAAG peptidase genomic DNA sequence ,the strategy of gene targeting was established, and the gene knockout vector (NAAG-KO-pBR322) was constructed. Electroporation of embryonic stem (ES) cells with the gene knockout vector and screening of both G418 and Ganciclovir resistant clones were performed. The homologous recombined ES ceD clones were identified by polymerase chain reaction (PCR). Results After transplantation of homologous recombined ES cells into blastocysts through microinjection, there were 11 male chimeras bom with embedment rate >50%. The male chimeras then were bred with C57BL/6J female mice and 7(♂) and 4 (♀) offsprings with

  12. 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.

    Science.gov (United States)

    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.

  13. Effect of single oral doses of sitagliptin, a dipeptidyl peptidase-4 inhibitor, on incretin and plasma glucose levels after an oral glucose tolerance test in patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Herman, Gary A; Bergman, Arthur; Stevens, Catherine;

    2006-01-01

    CONTEXT: In response to a meal, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) are released and modulate glycemic control. Normally these incretins are rapidly degraded by dipeptidyl peptidase-4 (DPP-4). DPP-4 inhibitors are a novel class of oral antihyperglyce...

  14. Proteolytic degradation of neuropeptide Y (NPY) from head to toe: Identification of novel NPY-cleaving peptidases and potential drug interactions in CNS and Periphery.

    Science.gov (United States)

    Wagner, Leona; Wolf, Raik; Zeitschel, Ulrike; Rossner, Steffen; Petersén, Åsa; Leavitt, Blair R; Kästner, Florian; Rothermundt, Matthias; Gärtner, Ulf-Torsten; Gündel, Daniel; Schlenzig, Dagmar; Frerker, Nadine; Schade, Jutta; Manhart, Susanne; Rahfeld, Jens-Ulrich; Demuth, Hans-Ulrich; von Hörsten, Stephan

    2015-12-01

    The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes, aminopeptidases P, secreted meprin-A (Mep-A), and cathepsin D (CTSD) rapidly hydrolyze NPY, depending on the cell type and tissue under study. Novel degradation of NPY by cathepsins B, D, L, G, S, and tissue kallikrein could also be identified. The expression of DP4, CTSD, and Mep-A at the median eminence indicates that the bioactivity of NPY is regulated by peptidases at the interphase between the periphery and the CNS. Detailed ex vivo studies on human sera and CSF samples recognized CTSD as the major NPY-cleaving enzyme in the CSF, whereas an additional C-terminal truncation by angiotensin-converting enzyme could be detected in serum. The latter finding hints to potential drug interaction between antidiabetic DP4 inhibitors and anti-hypertensive angiotensin-converting enzyme inhibitors, while it ablates suspected hypertensive side effects of only antidiabetic DP4-inhibitors application. The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes

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

    Science.gov (United States)

    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 yam dioscorin might be beneficial in glycemic control in normal mice and need further investigations in diabetic animal models.

  16. The yrpAB operon of Yersinia ruckeri encoding two putative U32 peptidases is involved in virulence and induced under microaerobic conditions.

    Science.gov (United States)

    Navais, Roberto; Méndez, Jessica; Pérez-Pascual, David; Cascales, Desirée; Guijarro, José A

    2014-07-01

    In an attempt to dissect the virulence mechanisms of Yersinia ruckeri two adjacent genes, yrpA and yrpB, encoding putative peptidases belonging to the U32 family, were analyzed. Similar genes, with the same genetic organization were identified in genomic analysis of human-pathogenic yersiniae. RT-PCR studies indicated that these genes form an operon in Y. ruckeri. Transcriptional studies using an yrpB::lacZY fusion showed high levels of expression of these genes in the presence of peptone in the culture medium, as well as under oxygen-limited conditions. These two factors had a synergic effect on gene induction when both were present simultaneously during bacterial incubation, which indicates the important role that environmental conditions in the fish gut can play in the regulation of specific genes. LD 50 experiments using an yrpA insertional mutant strain demonstrated the participation of this gene in the virulence of Y. ruckeri.

  17. Identification and in silico characterization of two novel genes encoding peptidases S8 found by functional screening in a metagenomic library of Yucatán underground water.

    Science.gov (United States)

    Apolinar-Hernández, Max M; Peña-Ramírez, Yuri J; Pérez-Rueda, Ernesto; Canto-Canché, Blondy B; De Los Santos-Briones, César; O'Connor-Sánchez, Aileen

    2016-11-15

    Metagenomics is a culture-independent technology that allows access to novel and potentially useful genetic resources from a wide range of unknown microorganisms. In this study, a fosmid metagenomic library of tropical underground water was constructed, and clones were functionally screened for extracellular proteolytic activity. One of the positive clones, containing a 41,614-bp insert, had two genes with 60% and 68% identity respectively with a peptidase S8 of Chitinimonas koreensis. When these genes were individually sub-cloned, in both cases their sub-clones showed proteolytic phenotype, confirming that they both encode functional proteases. These genes -named PrAY5 and PrAY6- are next to each other. They are similar in size (1845bp and 1824bp respectively) and share 66.5% identity. An extensive in silico characterization showed that their ORFs encode complex zymogens having a signal peptide at their 5' end, followed by a pro-peptide, a catalytic region, and a PPC domain at their 3' end. Their translated sequences were classified as peptidases S8A by sequence comparisons against the non-redundant database and corroborated by Pfam and MEROPS. Phylogenetic analysis of the catalytic region showed that they encode novel proteases that clustered with the sub-family S8_13, which according to the CDD database at NCBI, is an uncharacterized subfamily. They clustered in a clade different from the other three proteases S8 found so far by functional metagenomics, and also different from proteases S8 found in sequenced environmental samples, thereby expanding the range of potentially useful proteases that have been identified by metagenomics. I-TASSER modeling corroborated that they may be subtilases, thus possibly they participate in the hydrolysis of proteins with broad specificity for peptide bonds, and have a preference for a large uncharged residue in P1.

  18. Inhibition of serine-peptidase activity enhances the generation of a survivin-derived HLA-A2-presented CTL epitope in colon-carcinoma cells.

    Science.gov (United States)

    Preta, G; Marescotti, D; Fortini, C; Carcoforo, P; Castelli, C; Masucci, M; Gavioli, R

    2008-12-01

    Cytotoxic T lymphocytes eliminate tumor cells expressing antigenic peptides in the context of MHC-I molecules. Peptides are generated during protein degradation by the proteasome and resulting products, surviving cytosolic amino-peptidases activity, may be presented by MHC-I molecules. The MHC-I processing pathway is altered in a large number of malignancies and modulation of antigen generation is one strategy employed by cells to evade immune control. In this study we analyzed the generation and presentation of a survivin-derived CTL epitope in HLA-A2-positive colon-carcinoma cells. Although all cell lines expressed the anti-apoptotic protein survivin, some tumors were poorly recognized by ELTLGEFLKL (ELT)-specific CTL cultures. The expression of MHC-I or TAP molecules was similar in all cell lines suggesting that tumors not recognized by CTLs may present defects in the generation of the ELT-epitope which could be due either to lack of generation or to subsequent degradation of the epitope. The cells were analyzed for the expression and the activity of extra-proteasomal peptidases. A significant overexpression and higher activity of TPPII was observed in colon-carcinoma cells which are not killed by ELT-specific CTLs, suggesting a possible role of TPPII in the degradation of the ELT-epitope. To confirm the role of TPPII in the degradation of the ELT-peptide, we showed that treatment of colon-carcinoma cells with a TPPII inhibitor resulted in a dose-dependent increased sensitivity to ELT-specific CTLs. These results suggest that TPPII is involved in degradation of the ELT-peptide, and its overexpression may contribute to the immune escape of colon-carcinoma cells.

  19. Characterization of a Recombinant Cathepsin B-Like Cysteine Peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae): A Putative Target for Control of Citrus Huanglongbing.

    Science.gov (United States)

    Ferrara, Taíse Fernanda da Silva; Schneider, Vanessa Karine; Kishi, Luciano Takeshi; Carmona, Adriana Karaoglanovic; Alves, Marcio Fernando Madureira; Belasque-Júnior, Jose; Rosa, José César; Hunter, Wayne Brian; Henrique-Silva, Flávio; Soares-Costa, Andrea

    2015-01-01

    Huanglonbing (HLB) is one of the most destructive disease affecting citrus plants. The causal agent is associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) and the psyllid Diaphorina citri, vector of disease, that transmits the bacterium associated with HLB. The control of disease can be achieved by suppressing either the bacterium or the vector. Among the control strategies for HLB disease, one of the widely used consists in controlling the enzymes of the disease vector, Diaphorina citri. The insect Diaphorina citri belongs to the order Hemiptera, which frequently have cysteine peptidases in the gut. The importance of this class of enzymes led us to search for enzymes in the D. citri transcriptome for the establishment of alternatives strategies for HLB control. In this study, we reported the identification and characterization of a cathepsin B-like cysteine peptidase from D. citri (DCcathB). DCcathB was recombinantly expressed in Pichia pastoris, presenting a molecular mass of approximately 50 kDa. The enzyme hydrolyzed the fluorogenic substrate Z-F-R-AMC (Km = 23.5 μM) and the selective substrate for cathepsin B, Z-R-R-AMC (Km = 6.13 μM). The recombinant enzyme was inhibited by the cysteine protease inhibitors E64 (IC50 = 0.014 μM) and CaneCPI-4 (Ki = 0.05 nM) and by the selective cathepsin B inhibitor CA-074 (IC50 = 0.095 nM). RT-qPCR analysis revealed that the expression of the DCcathB in nymph and adult was approximately 9-fold greater than in egg. Moreover, the expression of this enzyme in the gut was 175-fold and 3333-fold higher than in the remaining tissues and in the head, respectively, suggesting that DCcathB can be a target for HLB control.

  20. Characterization of a Recombinant Cathepsin B-Like Cysteine Peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae: A Putative Target for Control of Citrus Huanglongbing.

    Directory of Open Access Journals (Sweden)

    Taíse Fernanda da Silva Ferrara

    Full Text Available Huanglonbing (HLB is one of the most destructive disease affecting citrus plants. The causal agent is associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas and the psyllid Diaphorina citri, vector of disease, that transmits the bacterium associated with HLB. The control of disease can be achieved by suppressing either the bacterium or the vector. Among the control strategies for HLB disease, one of the widely used consists in controlling the enzymes of the disease vector, Diaphorina citri. The insect Diaphorina citri belongs to the order Hemiptera, which frequently have cysteine peptidases in the gut. The importance of this class of enzymes led us to search for enzymes in the D. citri transcriptome for the establishment of alternatives strategies for HLB control. In this study, we reported the identification and characterization of a cathepsin B-like cysteine peptidase from D. citri (DCcathB. DCcathB was recombinantly expressed in Pichia pastoris, presenting a molecular mass of approximately 50 kDa. The enzyme hydrolyzed the fluorogenic substrate Z-F-R-AMC (Km = 23.5 μM and the selective substrate for cathepsin B, Z-R-R-AMC (Km = 6.13 μM. The recombinant enzyme was inhibited by the cysteine protease inhibitors E64 (IC50 = 0.014 μM and CaneCPI-4 (Ki = 0.05 nM and by the selective cathepsin B inhibitor CA-074 (IC50 = 0.095 nM. RT-qPCR analysis revealed that the expression of the DCcathB in nymph and adult was approximately 9-fold greater than in egg. Moreover, the expression of this enzyme in the gut was 175-fold and 3333-fold higher than in the remaining tissues and in the head, respectively, suggesting that DCcathB can be a target for HLB control.

  1. COMPLEXES OF BISCITRATOGERMANATES AND BISCITRATOSTANATES WITH METALS ARE MODIFIERS OF ACTIVITY OF Bacillus thuringiensis var. іsraelensis PEPTIDASES AND α-Penicillium canescens, Cladosporium cladosporioides AND Aspergillus niger GALACTOIDASES

    Directory of Open Access Journals (Sweden)

    L. D. Varbanets

    2016-06-01

    Full Text Available The aim of the research was to study the effect of a number of coordination compounds of stanum and germanium (compounds 1‒8 as modifiers of activity of peptidases and α-galactosidases. The coordination compounds with the same type structure of were investigated as enzymes effectors. Two types of complexes: 1 [M(H2O6][Ge(НCitr2]4H2O (M = Mg(1, Mn(2, Co(3, Ni(4, Zn(5, containing biscitrate-stanate anion ([Ge(НCitr2]2-, and 2 [M(H2O6][Sn(НCitr2]4H2O (M = Mg(6, Co(7, Ni(8, containing biscitratostanate anion and various hexaaquacations ([M(H2O6]2+, М= Mg, Mn, Co, Ni, Zn were studied. It is shown that the compound 6 which is a biscitratostanate complex containing magnesium ions as metal, can be used for stimulation on 20‒25% of collagenase activity of B. thuringiensis var. israelensis IMV B-7465 peptidase 1 and peptidase 2. Compound 1 (biscitrate-stanate complex containing magnesium ions as metal and 7 (biscitratostanate complex containing cobalt ions as metal and compound 6 in a concentration of 0.001% are able to increase elastolytic activity of peptidase 1 on 55‒58%. However compound 7 has shown the greatest activating effect. It increased the elastolytic activity of peptidase 2 on 100‒140% (for both tested concentrations. This indicates that the compound 7 can further be used as of peptidase 2 elastolytic activity effector. In the study of effect of the considered coordination compounds on the activity of α-galactosidase of Penicillium canescens, Cladosporium cladosporioides and Aspergillus niger was found that when using a number of complexes (1‒2 and 4‒8, there is a slight increase on 12‒20% of enzyme activity of P. canescens, and the maximum effect (~20%, concentration 0.01% was provided by complex 6.

  2. Changes in glucose-induced plasma active glucagon-like peptide-1 levels by co-administration of sodium–glucose cotransporter inhibitors with dipeptidyl peptidase-4 inhibitors in rodents

    OpenAIRE

    Takahiro Oguma; Chiaki Kuriyama; Keiko Nakayama; Yasuaki Matsushita; Kumiko Hikida; Minoru Tsuda-Tsukimoto; Akira Saito; Kenji Arakawa; Kiichiro Ueta; Masabumi Minami; Masaharu Shiotani

    2016-01-01

    We investigated whether structurally different sodium–glucose cotransporter (SGLT) 2 inhibitors, when co-administered with dipeptidyl peptidase-4 (DPP4) inhibitors, could enhance glucagon-like peptide-1 (GLP-1) secretion during oral glucose tolerance tests (OGTTs) in rodents. Three different SGLT inhibitors—1-(β-d-Glucopyranosyl)-4-chloro-3-[5-(6-fluoro-2-pyridyl)-2-thienylmethyl]benzene (GTB), TA-1887, and canagliflozin—were examined to assess the effect of chemical structure. Oral treatment...

  3. Phenylalanine 664 of dipeptidyl peptidase (DPP) 7 and Phenylalanine 671 of DPP11 mediate preference for P2-position hydrophobic residues of a substrate.

    Science.gov (United States)

    Rouf, Shakh M A; Ohara-Nemoto, Yuko; Ono, Toshio; Shimoyama, Yu; Kimura, Shigenobu; Nemoto, Takayuki K

    2013-01-01

    Dipeptidyl peptidases (DPPs) are crucial for the energy metabolism in Porphyromonas gingivalis, a Gram-negative proteolytic and asaccharolytic anaerobic rod causing chronic periodontitis. Three DPPs, DPPIV specific for Pro, DPP7 for hydrophobic residues and DPP11 for Asp/Glu at the P1 position, are expressed in the bacterium. Like DPP7, DPP11 belongs to the S46 protease family, and they share 38.7% sequence identity. Although DPP11 is preferential for hydrophobic residues at the P2 position, it has been reported that DPP7 has no preference at the P2 position. In the present study, we defined the detailed P2 substrate preference of DPP7 and the amino acid residue responsible for the specificity. DPP7 most efficiently hydrolyzed Met-Leu-dipeptidyl-4-methylcoumaryl-7-amide (MCA) carrying hydrophobic residues at the P1 position with k cat/Km of 10.62 ± 2.51 μM(-1) s(-1), while it unexpectedly cleaved substrates with hydrophilic (Gln, Asn) or charged (Asp, Arg) residues. Examination with 21 dipeptidyl MCA demonstrated that DPP7-peptidase activity was dependent on hydrophobicity of the P2- as well as P1-position residue, thus it correlated best with the sum of the hydrophobicity index of P1- and P2-amino acid residues. Hydrophobicity of the P1 and P2 positions ensured efficient enzyme catalysis by increasing k cat and lowering Km values, respectively. Substitution of hydrophobic residues conserved in the S46 DPP7/DPP11 family to Ala revealed that Phe664 of DPP7 and Phe671 of DPP11 primarily afforded hydrophobic P2 preference. A modeling study suggested that Phe664 and Gly666 of DPP7 and Phe671 and Arg673 of DPP11 being associated with the P2- and P1-position residues, respectively, are located adjacent to the catalytic Ser648/Ser655. The present results expand the substrate repertoire of DPP7, which ensures efficient degradation of oligopeptides in asaccharolytic bacteria.

  4. Glycosaminoglycans Regulate CXCR3 Ligands at Distinct Levels: Protection against Processing by Dipeptidyl Peptidase IV/CD26 and Interference with Receptor Signaling

    Directory of Open Access Journals (Sweden)

    Mieke Metzemaekers

    2017-07-01

    Full Text Available CXC chemokine ligand (CXCL9, CXCL10 and CXCL11 direct chemotaxis of mainly T cells and NK cells through activation of their common CXC chemokine receptor (CXCR3. They are inactivated upon NH2-terminal cleavage by dipeptidyl peptidase IV/CD26. In the present study, we found that different glycosaminoglycans (GAGs protect the CXCR3 ligands against proteolytic processing by CD26 without directly affecting the enzymatic activity of CD26. In addition, GAGs were shown to interfere with chemokine-induced CXCR3 signaling. The observation that heparan sulfate did not, and heparin only moderately, altered CXCL10-induced T cell chemotaxis in vitro may be explained by a combination of protection against proteolytic inactivation and altered receptor interaction as observed in calcium assays. No effect of CD26 inhibition was found on CXCL10-induced chemotaxis in vitro. However, treatment of mice with the CD26 inhibitor sitagliptin resulted in an enhanced CXCL10-induced lymphocyte influx into the joint. This study reveals a dual role for GAGs in modulating the biological activity of CXCR3 ligands. GAGs protect the chemokines from proteolytic cleavage but also directly interfere with chemokine–CXCR3 signaling. These data support the hypothesis that both GAGs and CD26 affect the in vivo chemokine function.

  5. Sitagliptin, a dipeptidyl peptidase-4 inhibitor, increases the number of circulating CD34⁺CXCR4⁺ cells in patients with type 2 diabetes.

    Science.gov (United States)

    Aso, Yoshimasa; Jojima, T; Iijima, T; Suzuki, K; Terasawa, T; Fukushima, M; Momobayashi, A; Hara, K; Takebayashi, K; Kasai, K; Inukai, T

    2015-12-01

    We investigated the effects of sitagliptin, a dipeptidyl peptidase (DPP)-4 inhibitor, on the number of circulating CD34(+)CXCR4(+)cells, a candidate for endothelial progenitor cells (EPCs), plasma levels of stromal cell-derived factor (SDF)-1α, a ligand for CXCR4 receptor and a substrate for DPP-4, and plasma levels of interferon-inducible protein (IP)-10, for a substrate for DPP-4, in patients with type 2 diabetes. We studied 30 consecutive patients with type 2 diabetes who had poor glycemic control despite treatment with metformin and/or sulfonylurea. Thirty diabetic patients were randomized in a 2:1 ratio into a sitagliptin (50 mg/day) treatment group or an active placebo group (glimepiride 1 mg/day) for 12 weeks. Both groups showed similar improvements in glycemic control. The number of circulating CD34(+)CXCR4(+) cells was increased from 30.5 (20.0, 47.0)/10(6) cells at baseline to 55.5 (31.5, 80.5)/10(6) cells at 12 weeks of treatment with 50 mg/day sitagliptin (P = 0.0014), while showing no significant changes in patients treated with glimepiride. Plasma levels of SDF-1α and IP-10, both physiological substrates of endogenous DPP-4 and chemokines, were significantly decreased at 12 weeks of sitagliptin treatment. In conclusion, treatment with sitagliptin increased the number of circulating CD34(+)CXCR4(+) cells by approximately 2-fold in patients with type 2 diabetes.

  6. Preparation of Optically Active Alkoxy-serines from Amino-amide Racemate Catalyzed by Escherichia coil Cells with Peptidase B Activity

    Institute of Scientific and Technical Information of China (English)

    WANG Zhi-yuan; LIU Jun-zhong; XU Li-sheng; ZHANG Hong-juan; LIU Qian; JIAO Qing-cai

    2013-01-01

    Alkoxy-L-serines are useful for peptide syntheses.The demand for alkoxy-L-serines in the pharmaceutical industries continues to increase because of their multiple physiological effects.In this research,an improved method for alkoxy-L-serines synthesis is reported.A series of substrates,DL-β-alkoxy-α-amino propionamides,was used for the synthesis of alkoxy-serines catalyzed by Escherichia coli cells with peptidase B(PepB) activity.The results show that PepB has a high resolution activity with DL-β-alkoxy-α-amino propionamides as substrate.Reaction conditions were optimized,i.e.,DL-β-methoxy-α-amino propionamide as substrate at pH=9.0,40 ℃ and 14 h,and the optimal reaction concentration is 400 mmol/L.The results also show that divalent metal cations exhibit different effects on the PepB activity,for example,Zn2+ and Cu2+ can obviously inhibit the activity of PepB,whereas Co2+,Ca2+,Mn2+ and Mg2+ at low concentrations can activate PepB.This research provides access to enantiomerically enriched and valuable alkoxy-L-serines from a simple amino-amide racemate.

  7. Peptide array on cellulose support--a screening tool to identify peptides with dipeptidyl-peptidase IV inhibitory activity within the sequence of α-lactalbumin.

    Science.gov (United States)

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

    2014-11-13

    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 (αK(i) = 76 µM), 105LAHKALCSEK114 (K(i) = 217 µM) and 110LCSEKLDQWL119 (K(i) = 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.

  8. Discovery of a new biomarker for the mucopolysaccharidoses (MPS), dipeptidyl peptidase IV (DPP-IV; CD26), by SELDI-TOF mass spectrometry.

    Science.gov (United States)

    Beesley, Clare E; Young, Elisabeth P; Finnegan, Niamh; Jackson, Marie; Mills, Kevin; Vellodi, Ashok; Cleary, Maureen; Winchester, Bryan G

    2009-04-01

    Surface enhanced laser desorption/ionisation time of flight (SELDI-TOF) mass spectrometry has been used to search for new protein biomarkers in the plasma of patients with mucopolysacharidoses (MPS). Differences in the levels of some plasma proteins, particularly the apolipoprotein ApoCI, were observed between MPS patients and normal controls, using the different chromatographic surfaces (ProteinChips). ApoCI was identified by both its mass and by immunological techniques. In plasma, it exists in two forms, ApoCI and a truncated form which lacks two N-terminal amino acids, ApoCI'. In controls, the ratio of ApoCI':ApoCI observed using the cation-exchange surface (CM10) was approximately 1:2 whereas in most MPS patients it varied from 1:1 to 1:0.8. The ratio of ApoCI':ApoCI in plasma is determined by the activity of dipeptidyl peptidase IV, DPP-IV (also known as the leucocyte antigen CD26), which was found to be elevated up to 3-fold in MPS patients. The DPP-IV activity decreased in MPS I patients undergoing enzyme replacement therapy, indicating that it could be a useful biomarker for monitoring the efficacy of treatment in MPS disease. As DPP-IV has an important regulatory role in metabolism, it is possible that its elevation could cause some of the secondary pathology in MPS, and inhibition of DPP-IV might have a role in MPS therapy.

  9. Prevalence of the serpin peptidase inhibitor (alpha-1-antitrypsin PI*S and PI*Z alleles in Brazilian children with liver disease

    Directory of Open Access Journals (Sweden)

    Guilherme Baldo

    2008-01-01

    Full Text Available Serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin, member 1 (SERPINA1 deficiency is one of the main genetic causes related to liver disease in children. In SERPINA1 deficiency the most frequent SERPINA1 alleles found are the PI*S and PI*Z alleles. We used the polymerase chain reaction and the amplification created restriction site (ACRS technique to investigate the prevalence of the PI*S and PI*Z alleles in a group of Brazilian children (n = 200 with liver disease and established the general frequency of the PI*S allele in our population. We found a significant association of the PI*Z allele and liver disease, but no such relationship was found for the PI*S allele. Our results show that SERPINA1 deficiency due to the PI*Z allele, even when heterozygous, is a frequent cause of liver disease in our group of Brazilian children but that the PI*S allele does not confer an increased risk of hepatic disorders in our group of Brazilian children.

  10. Effect of pre-treatment on the generation of dipeptidyl peptidase-IV- and prolyl endopeptidase-inhibitory hydrolysates from bovine lung

    Directory of Open Access Journals (Sweden)

    Lafarga T.

    2017-05-01

    Full Text Available The aim of this work was to study the effect of two different pre-treatments, high temperature (100 °C, 5 min and high pressure (600 MPa, 3 min, on the potential of the enzymes papain, collagenase and Alcalase® to generate bioactive hydrolysates containing dipeptidyl peptidase-IV- (DPP-IV; EC 3.4.14.5 and prolyl endopeptidase- (PEP; EC 3.4.21.26 inhibitory peptides from bovine lung. Both pre-treatments resulted in an increase in the degree of hydrolysis over a 24 h period (P < 0.001 and significantly increased the DPP-IV- and PEP-inhibitory activities of the generated hydrolysates (P < 0.001. Generated hydrolysates included an Alcalase hydrolysate of pressure-treated bovine lung, which was the most active, and showed DPP-IV and PEP half-maximal inhibitory concentration (IC50 values of 1.43 ± 0.06 and 3.62 ± 0.07 mg/ mL, respectively. The major peptides contained in this hydrolysate were determined by liquid chromatography-tandem mass spectrometry, and results demonstrated that bovine lung is a good substrate for the release of bioactive peptides when proper pre-treatment and enzymatic treatment are applied.

  11. Stromal cell-derived factor-1 is upregulated by dipeptidyl peptidase-4 inhibition and has protective roles in progressive diabetic nephropathy.

    Science.gov (United States)

    Takashima, Satoru; Fujita, Hiroki; Fujishima, Hiromi; Shimizu, Tatsunori; Sato, Takehiro; Morii, Tsukasa; Tsukiyama, Katsushi; Narita, Takuma; Takahashi, Takamune; Drucker, Daniel J; Seino, Yutaka; Yamada, Yuichiro

    2016-10-01

    The role of stromal cell-derived factor-1 (SDF-1) in the pathogenesis of diabetic nephropathy and its modification by dipeptidyl peptidase-4 (DPP-4) inhibition are uncertain. Therefore, we studied this independent of glucagon-like peptide-1 receptor (GLP-1R) signaling using two Akita diabetic mouse models, the diabetic-resistant C57BL/6-Akita and diabetic-prone KK/Ta-Akita. Increased SDF-1 expression was found in glomerular podocytes and distal nephrons in the diabetic-prone mice, but not in kidneys from diabetic-resistant mice. The DPP-4 inhibitor linagliptin, but not the GLP-1R agonist liraglutide, further augmented renal SDF-1 expression in both Glp1r(+/+) and Glp1r(-/-) diabetic-prone mice. Along with upregulation of renal SDF-1 expression, the progression of albuminuria, glomerulosclerosis, periglomerular fibrosis, podocyte loss, and renal oxidative stress was suppressed in linagliptin-treated Glp1r(+/+) diabetic-prone mice. Linagliptin treatment increased urinary sodium excretion and attenuated the increase in glomerular filtration rate which reflects glomerular hypertension and hyperfiltration. In contrast, selective SDF-1 receptor blockade with AMD3100 reduced urinary sodium excretion and aggravated glomerular hypertension in the Glp1r(+/+) diabetic-prone mice. Thus, DPP-4 inhibition, independent of GLP-1R signaling, contributes to protection of the diabetic kidney through SDF-1-dependent antioxidative and antifibrotic effects and amelioration of adverse renal hemodynamics.

  12. Cell-free production of integral membrane aspartic acid proteases reveals zinc-dependent methyltransferase activity of the Pseudomonas aeruginosa prepilin peptidase PilD

    Science.gov (United States)

    Aly, Khaled A; Beebe, Emily T; Chan, Chi H; Goren, Michael A; Sepúlveda, Carolina; Makino, Shin-ichi; Fox, Brian G; Forest, Katrina T

    2013-01-01

    Integral membrane aspartic acid proteases are receiving growing recognition for their fundamental roles in cellular physiology of eukaryotes and prokaryotes, and may be medically important pharmaceutical targets. The Gram-negative Pseudomonas aeruginosa PilD and the archaeal Methanococcus voltae FlaK were synthesized in the presence of unilamellar liposomes in a cell-free translation system. Cosynthesis of PilD with its full-length substrate, PilA, or of FlaK with its full-length substrate, FlaB2, led to complete cleavage of the substrate signal peptides. Scaled-up synthesis of PilD, followed by solubilization in dodecyl-β-d-maltoside and chromatography, led to a pure enzyme that retained both of its known biochemical activities: cleavage of the PilA signal peptide and S-adenosyl methionine-dependent methylation of the mature pilin. X-ray fluorescence scans show for the first time that PilD is a zinc-binding protein. Zinc is required for the N-terminal methylation of the mature pilin, but not for signal peptide cleavage. Taken together, our work identifies the P. aeruginosa prepilin peptidase PilD as a zinc-dependent N-methyltransferase and provides a new platform for large-scale synthesis of PilD and other integral membrane proteases important for basic microbial physiology and virulence. PMID:23255525

  13. Determination of a dipeptidyl peptidase IV agonist, β-aminoacyl containing thiazolidine derivatives (KR-66223) in rat plasma by liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Kim, Min-Sun; Park, Jong-Shik; Jang, Su-Min; Lee, Byung Hoi; Ahn, Sung-Hoon; Ahn, Jin Hee; Yoo, Sung Eun; Song, Im-Sook; Silinski, Peter; Schneider, Stephen Edward; Bae, Myung Ae

    2011-07-15

    A sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for a novel dipeptidyl peptidase IV agonist (DDP-IV) agonist, KR-66223, in rat plasma. It involves liquid-liquid extraction (LLE) followed by HPLC separation and electrospray ionization tandem mass spectrometry. KR-66223 and imipramine (IS) was separated on Gemini-NX C18 column with mixture of acetonitrile-ammonium formate (10mM) (90:10, v/v) as mobile phase. The ion transitions monitored were m/z 553.2→206.2 for KR-66223, m/z 281.3→86.1 for imipramine in multiple reaction monitoring (MRM) mode. The linear ranges of the assay were 0.003-10μg/ml with a correlation coefficient (R(2)) greater than 0.99 and the lower limit of quantification was 3ng/ml. The average recovery was 78.9% and 87.1% from rat plasma for KR-66223 and imipramine, respectively. The coefficients of variation of intra- and inter-assay were 3.9-14.4% and the relative error was 0.8-11.5%. The method was validated and successfully applied to the pharmacokinetic study of KR-66223 in rat.

  14. Different molecular mechanisms involved in spontaneous and oxidative stress-induced mitochondrial fragmentation in tripeptidyl peptidase-1 (TPP-1)-deficient fibroblasts

    Science.gov (United States)

    Van Beersel, Guillaume; Tihon, Eliane; Demine, Stéphane; Hamer, Isabelle; Jadot, Michel; Arnould, Thierry

    2012-01-01

    NCLs (neuronal ceroid lipofuscinoses) form a group of eight inherited autosomal recessive diseases characterized by the intralysosomal accumulation of autofluorescent pigments, called ceroids. Recent data suggest that the pathogenesis of NCL is associated with the appearance of fragmented mitochondria with altered functions. However, even if an impairement in the autophagic pathway has often been evoked, the molecular mechanisms leading to mitochondrial fragmentation in response to a lysosomal dysfunction are still poorly understood. In this study, we show that fibroblasts that are deficient for the TPP-1 (tripeptidyl peptidase-1), a lysosomal hydrolase encoded by the gene mutated in the LINCL (late infantile NCL, CLN2 form) also exhibit a fragmented mitochondrial network. This morphological alteration is accompanied by an increase in the expression of the protein BNIP3 (Bcl2/adenovirus E1B 19 kDa interacting protein 3) as well as a decrease in the abundance of mitofusins 1 and 2, two proteins involved in mitochondrial fusion. Using RNAi (RNA interference) and quantitative analysis of the mitochondrial morphology, we show that the inhibition of BNIP3 expression does not result in an increase in the reticulation of the mitochondrial population in LINCL cells. However, this protein seems to play a key role in cell response to mitochondrial oxidative stress as it sensitizes mitochondria to antimycin A-induced fragmentation. To our knowledge, our results bring the first evidence of a mechanism that links TPP-1 deficiency and oxidative stress-induced changes in mitochondrial morphology. PMID:23249249

  15. Effects of dipeptidyl peptidase-4 inhibitors on beta-cell function and insulin resistance in type 2 diabetes: meta-analysis of randomized controlled trials

    Science.gov (United States)

    Lyu, Xiafei; Zhu, Xiaolin; Zhao, Bin; Du, Liang; Chen, Dawei; Wang, Chun; Liu, Guanjian; Ran, Xingwu

    2017-01-01

    Dipeptidyl peptidase-4 (DPP-4) inhibitors are a novel family of glucose-lowering agents. Accumulating evidence suggests that DPP-4 inhibitors preserve pancreatic beta-cell function, but results in previous studies have been inconsistent. We assessed the effects of DPP-4 inhibitors on the homoeostasis model assessment beta-cell function (HOMA-B) or insulin resistance (HOMA-IR) index in patients with type 2 diabetes through a systematic review and meta-analysis of randomized controlled trials (RCTs). Relevant articles were identified from PubMed, Embase, and Cochrane Library databases up to December 27, 2016. We calculated weighted mean differences (WMDs) and 95% confidence intervals (CIs) in each included trial and pooled the data using a random-effects model. Fifty-two trials were included in the present analysis. Compared with placebo control, DPP-4 inhibitors as monotherapy significantly improved HOMA-B (WMD 9.15; 95% CI 7.48, 10.81). Similarly, DPP-4 inhibitors as add-on therapy in combination with other drugs showed significant improvement in HOMA-B (WMD 9.04; 95% CI 5.72, 12.37). However, we found no significant improvement in HOMA-IR following treatment with DPP-4 inhibitors as mono-therapy or as add-on therapy. In conclusion, DPP-4 inhibitors as monotherapy or as add-on therapy significantly improved beta-cell function but had no significant effect on insulin resistance in type 2 diabetes. PMID:28322294

  16. An update on the clinical pharmacology of the dipeptidyl peptidase 4 inhibitor alogliptin used for the treatment of type 2 diabetes mellitus.

    Science.gov (United States)

    Chen, Xiao-Wu; He, Zhi-Xu; Zhou, Zhi-Wei; Yang, Tianxin; Zhang, Xueji; Yang, Yin-Xue; Duan, Wei; Zhou, Shu-Feng

    2015-12-01

    Alogliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor that is a class of relatively new oral hypoglycaemic drugs used in patients with type 2 diabetes (T2DM), can be used as monotherapy or in combination with other anti-diabetic agents, including metformin, pioglitazone, sulfonylureas and insulin with a considerable therapeutic effect. Alogliptin exhibits favorable pharmacokinetic and pharmacodynamic profiles in humans. Alogliptin is mainly metabolized by cytochrome P450 (CYP2D6) and CYP3A4. Dose reduction is recommended for patients with moderate or worse renal impairment. Side effects of alogliptin include nasopharyngitis, upper-respiratory tract infections and headache. Hypoglycaemia is seen in about 1.5% of the T2DM patients. Rare but severe adverse reactions such as acute pancreatitis, serious hypersensitivity including anaphylaxis, angioedema and severe cutaneous reactions such as Stevens-Johnson syndrome have been reported from post-marketing monitoring. Pharmacokinetic interactions have not been observed between alogliptin and other drugs including glyburide, metformin, pioglitazone, insulin and warfarin. The present review aimed to update the clinical information on pharmacodynamics, pharmacokinetics, adverse effects and drug interactions, and to discuss the future directions of alogliptin.

  17. Effects of dipeptidyl peptidase-4 inhibitors on blood pressure in patients with type 2 diabetes: A systematic review and meta-analysis.

    Science.gov (United States)

    Zhang, Xiaodan; Zhao, Qingyu

    2016-02-01

    This review was undertaken to assess the effects of dipeptidyl peptidase-4 (DPP-4) inhibitors on blood pressure (BP) in patients with type 2 diabetes (T2DM). We searched three main databases (PubMed, Web of Science, and Scopus) for relevant articles. Randomized controlled trials which reported BP changes from baseline to study endpoint in patients with T2DM receiving treatment of DPP-4 inhibitors were included for analysis. Random effects models were used to measure the mean differences with 95% confidence intervals (CIs). Fifteen trials involving 5636 participants were identified. When compared with placebo or nontreatment, DPP-4 inhibitors achieved greater reductions for both SBP (mean difference, -3.04  mmHg; 95% CI, -4.37 to -1.72; P pioglitazone, sulphonylureas, metformin, and α-glucosidase inhibitors. DPP-4 inhibitors may exert modest BP-lowering effects compared with placebo or nontreatment for patients with T2DM, but no significant BP improvement was seen with this drug class when compared with other antidiabetic medications.

  18. Susceptibility of Phytomonas serpens to calpain inhibitors in vitro: interference on the proliferation, ultrastructure, cysteine peptidase expression and interaction with the invertebrate host

    Science.gov (United States)

    de Oliveira, Simone Santiago Carvalho; Gonçalves, Diego de Souza; Garcia-Gomes, Aline dos Santos; Gonçalves, Inês Correa; Seabra, Sergio Henrique; Menna-Barreto, Rubem Figueiredo; Lopes, Angela Hampshire de Carvalho Santos; D’Avila-Levy, Claudia Masini; dos Santos, André Luis Souza; Branquinha, Marta Helena

    2016-01-01

    A pleiotropic response to the calpain inhibitor MDL28170 was detected in the tomato parasite Phytomonas serpens. Ultrastructural studies revealed that MDL28170 caused mitochondrial swelling, shortening of flagellum and disruption of trans Golgi network. This effect was correlated to the inhibition in processing of cruzipain-like molecules, which presented an increase in expression paralleled by decreased proteolytic activity. Concomitantly, a calcium-dependent cysteine peptidase was detected in the parasite extract, the activity of which was repressed by pre-incubation of parasites with MDL28170. Flow cytometry and Western blotting analyses revealed the differential expression of calpain-like proteins (CALPs) in response to the pre-incubation of parasites with the MDL28170, and confocal fluorescence microscopy confirmed their surface location. The interaction of promastigotes with explanted salivary glands of the insect Oncopeltus fasciatus was reduced when parasites were pre-treated with MDL28170, which was correlated to reduced levels of surface cruzipain-like and gp63-like molecules. Treatment of parasites with anti-Drosophila melanogaster (Dm) calpain antibody also decreased the adhesion process. Additionally, parasites recovered from the interaction process presented higher levels of surface cruzipain-like and gp63-like molecules, with similar levels of CALPs cross-reactive to anti-Dm-calpain antibody. The results confirm the importance of exploring the use of calpain inhibitors in studying parasites’ physiology. PMID:27925020

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

    Science.gov (United States)

    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.

  20. RNA sequencing identifies upregulated kyphoscoliosis peptidase and phosphatidic acid signaling pathways in muscle hypertrophy generated by transgenic expression of myostatin propeptide.

    Science.gov (United States)

    Miao, Yuanxin; Yang, Jinzeng; Xu, Zhong; Jing, Lu; Zhao, Shuhong; Li, Xinyun

    2015-04-09

    Myostatin (MSTN), a member of the transforming growth factor-β superfamily, plays a crucial negative role in muscle growth. MSTN mutations or inhibitions can dramatically increase muscle mass in most mammal species. Previously, we generated a transgenic mouse model of muscle hypertrophy via the transgenic expression of the MSTN N-terminal propeptide cDNA under the control of the skeletal muscle-specific MLC1 promoter. Here, we compare the mRNA profiles between transgenic mice and wild-type littermate controls with a high-throughput RNA sequencing method. The results show that 132 genes were significantly differentially expressed between transgenic mice and wild-type control mice; 97 of these genes were up-regulated, and 35 genes were down-regulated in the skeletal muscle. Several genes that had not been reported to be involved in muscle hypertrophy were identified, including up-regulated myosin binding protein H (mybph), and zinc metallopeptidase STE24 (Zmpste24). In addition, kyphoscoliosis peptidase (Ky), which plays a vital role in muscle growth, was also up-regulated in the transgenic mice. Interestingly, a pathway analysis based on grouping the differentially expressed genes uncovered that cardiomyopathy-related pathways and phosphatidic acid (PA) pathways (Dgki, Dgkz, Plcd4) were up-regulated. Increased PA signaling may increase mTOR signaling, resulting in skeletal muscle growth. The findings of the RNA sequencing analysis help to understand the molecular mechanisms of muscle hypertrophy caused by MSTN inhibition.

  1. The dipeptidyl peptidase-4 inhibitor teneligliptin improved endothelial dysfunction and insulin resistance in the SHR/NDmcr-cp rat model of metabolic syndrome.

    Science.gov (United States)

    Nakagami, Hironori; Pang, Zhengda; Shimosato, Takashi; Moritani, Toshinori; Kurinami, Hitomi; Koriyama, Hiroshi; Tenma, Akiko; Shimamura, Munehisa; Morishita, Ryuichi

    2014-07-01

    Diabetes mellitus, hypertension and metabolic syndrome are major risk factors for the occurrence of cardiovascular events. In this study, we used spontaneous hypertensive rat (SHR)/NDmcr-cp (cp/cp) (SHRcp) rats as a model for metabolic syndrome to examine the effects of dipeptidyl peptidase (DPP)-4 inhibition on hypertension, glucose metabolism and endothelial dysfunction. First, we confirmed that SHRcp rats showed very severe obesity, hypertension and endothelial dysfunction phenotypes from 14 to 54 weeks of age. Next, we examined whether the DPP-4 inhibitor teneligliptin (10 mg kg(-1) per day per os for 12 weeks) could modify any of these phenotypes. Treatment with teneligliptin significantly improved hyperglycemia and insulin resistance, as evidenced by an oral glucose tolerance test and homeostasis model assessment for insulin resistance, respectively. Teneligliptin showed no effects on systolic blood pressure or heart rate. In regard to endothelial function, the vasodilator response to acetylcholine was significantly impaired in SHRcp rats when compared with WKY rats. Long-term treatment with teneligliptin significantly attenuated endothelial dysfunction through the upregulation of endothelium-derived nitric oxide synthase mRNA. These results demonstrate that long-term treatment with teneligliptin significantly improved endothelial dysfunction and glucose metabolism in a rat model of metabolic syndrome, suggesting that teneligliptin treatment might be beneficial for patients with hypertension and/or diabetes.

  2. RNA Sequencing Identifies Upregulated Kyphoscoliosis Peptidase and Phosphatidic Acid Signaling Pathways in Muscle Hypertrophy Generated by Transgenic Expression of Myostatin Propeptide

    Directory of Open Access Journals (Sweden)

    Yuanxin Miao

    2015-04-01

    Full Text Available Myostatin (MSTN, a member of the transforming growth factor-β superfamily, plays a crucial negative role in muscle growth. MSTN mutations or inhibitions can dramatically increase muscle mass in most mammal species. Previously, we generated a transgenic mouse model of muscle hypertrophy via the transgenic expression of the MSTN N-terminal propeptide cDNA under the control of the skeletal muscle-specific MLC1 promoter. Here, we compare the mRNA profiles between transgenic mice and wild-type littermate controls with a high-throughput RNA sequencing method. The results show that 132 genes were significantly differentially expressed between transgenic mice and wild-type control mice; 97 of these genes were up-regulated, and 35 genes were down-regulated in the skeletal muscle. Several genes that had not been reported to be involved in muscle hypertrophy were identified, including up-regulated myosin binding protein H (mybph, and zinc metallopeptidase STE24 (Zmpste24. In addition, kyphoscoliosis peptidase (Ky, which plays a vital role in muscle growth, was also up-regulated in the transgenic mice. Interestingly, a pathway analysis based on grouping the differentially expressed genes uncovered that cardiomyopathy-related pathways and phosphatidic acid (PA pathways (Dgki, Dgkz, Plcd4 were up-regulated. Increased PA signaling may increase mTOR signaling, resulting in skeletal muscle growth. The findings of the RNA sequencing analysis help to understand the molecular mechanisms of muscle hypertrophy caused by MSTN inhibition.

  3. Influence of candidate polymorphisms on the dipeptidyl peptidase IV and μ-opioid receptor genes expression in aspect of the β-casomorphin-7 modulation functions in autism.

    Science.gov (United States)

    Cieślińska, Anna; Sienkiewicz-Szłapka, Edyta; Wasilewska, Jolanta; Fiedorowicz, Ewa; Chwała, Barbara; Moszyńska-Dumara, Małgorzata; Cieśliński, Tomasz; Bukało, Marta; Kostyra, Elżbieta

    2015-03-01

    Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder with population prevalence of approximately 60-70 per 10,000. Data shows that both opioid system function enhancement and opiate administration can result in autistic-like symptoms. Cow milk opioid peptides, including β-casomorphin-7 (BCM7, Tyr-Pro-Phe-Pro-Gly-Pro-Ile), affect the μ-opioid receptor (MOR) and are subjected to degradation resulting from the proline dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) enzyme activity. The presence of MOR and DPPIV activity are crucial factors determining biological activity of BCM7 in the human body. Our study examined the effect of β-casomorphin-7 on the MOR and DPPIV genes expression according to specific point mutations in these genes. In addition, we investigated frequency of A118G SNP in the MOR gene and rs7608798 of the DPPIV (A/G) gene in healthy and autistic children. Our research indicated correlation in DPPIV gene expression under the influence of BCM7 and hydrolyzed milk between healthy and ASD-affected children with genotype GG (Pautism symptoms.

  4. Postprandial incretin and islet hormone responses and dipeptidyl-peptidase 4 enzymatic activity in patients with maturity onset diabetes of the young

    DEFF Research Database (Denmark)

    Østoft, Signe Harring; Bagger, Jonatan Ising; Hansen, Torben;

    2015-01-01

    )), and dipeptidyl-peptidase 4 (DPP-4) enzymatic activity in patients with glucokinase (GCK)-diabetes (MODY2), hepatocyte nuclear factor 1α (HNF1A)-diabetes (MODY3), and in matched healthy individuals (CTRLs). Subjects and methods: Ten patients with GCK-diabetes (age: 43±5 years; BMI: 24±2 kg/m2; FPG: 7.1±0.3 mmol...... with a liquid test meal. Results: All groups exhibited similar baseline values of glucagon (7±1 (GCK-diabetes), 6±1 pmol/l (HNF1A-diabetes), 8±2 (CTRLs), P=0.787), but patients with HNF1A-diabetes exhibited postprandial hyperglucagonaemia (area under curve (AUC): 838±108 min×pmol/l) compared to CTRLs (182......±176 min×pmol/l, P=0.005) and tended to have a greater response than patients with GCK-diabetes (410±154 min×pmol/l, P=0.063). Similar peak concentrations and AUCs for plasma GIP and plasma GLP-1 were observed across the groups. Increased fasting DPP-4-activity was seen in patients with HNF1A-diabetes (17...

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

    Science.gov (United States)

    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.

  6. DA-1229, a dipeptidyl peptidase IV inhibitor, protects against renal injury by preventing podocyte damage in an animal model of progressive renal injury.

    Science.gov (United States)

    Eun Lee, Jee; Kim, Jung Eun; Lee, Mi Hwa; Song, Hye Kyoung; Ghee, Jung Yeon; Kang, Young Sun; Min, Hye Sook; Kim, Hyun Wook; Cha, Jin Joo; Han, Jee Young; Han, Sang Youb; Cha, Dae Ryong

    2016-05-01

    Although dipeptidyl peptidase IV (DPPIV) inhibitors are known to have renoprotective effects, the mechanism underlying these effects has remained elusive. Here we investigated the effects of DA-1229, a novel DPPIV inhibitor, in two animal models of renal injury including db/db mice and the adriamycin nephropathy rodent model of chronic renal disease characterized by podocyte injury. For both models, DA-1229 was administered at 300 mg/kg/day. DPPIV activity in the kidney was significantly higher in diabetic mice compared with their nondiabetic controls. Although DA-1229 did not affect glycemic control or insulin resistance, DA-1229 did improve lipid profiles, albuminuria and renal fibrosis. Moreover, DA-1229 treatment resulted in decreased urinary excretion of nephrin, decreased circulating and kidney DPPIV activity, and decreased macrophage infiltration in the kidney. In adriamycin-treated mice, DPPIV activity in the kidney and urinary nephrin loss were both increased, whereas glucagon-like peptide-1 concentrations were unchanged. Moreover, DA-1229 treatment significantly improved proteinuria, renal fibrosis and inflammation associated with decreased urinary nephrin loss, and kidney DPP4 activity. In cultured podocytes, DA-1229 restored the high glucose/angiotensin II-induced increase of DPPIV activity and preserved the nephrin levels in podocytes. These findings suggest that activation of DPPIV in the kidney has a role in the progression of renal disease, and that DA-1229 may exert its renoprotective effects by preventing podocyte injury.

  7. Dipeptidyl peptidase IV (CD26 activity in the hematopoietic system: differences between the membrane-anchored and the released enzyme activity

    Directory of Open Access Journals (Sweden)

    D.A. Pereira

    2003-05-01

    Full Text Available Dipeptidyl peptidase IV (DPP-IV; CD26 (EC 3.4.14.5 is a membrane-anchored ectoenzyme with N-terminal exopeptidase activity that preferentially cleaves X-Pro-dipeptides. It can also be spontaneously released to act in the extracellular environment or associated with the extracellular matrix. Many hematopoietic cytokines and chemokines contain DPP-IV-susceptible N-terminal sequences. We monitored DPP-IV expression and activity in murine bone marrow and liver stroma cells which sustain hematopoiesis, myeloid precursors, skin fibroblasts, and myoblasts. RT-PCR analysis showed that all these cells produced mRNA for DPP-IV. Partially purified protein reacted with a commercial antibody to CD26. The K M values for Gly-Pro-p-nitroanilide ranged from 0.43 to 0.98 mM for the membrane-associated enzyme of connective tissue stromas, and from 6.76 to 8.86 mM for the enzyme released from the membrane, corresponding to a ten-fold difference, but only a two-fold difference in K M was found in myoblasts. K M of the released soluble enzyme decreased in the presence of glycosaminoglycans, nonsulfated polysaccharide polymers (0.8-10 µg/ml or simple sugars (320-350 µg/ml. Purified membrane lipid rafts contained nearly 3/4 of the total cell enzyme activity, whose K M was three-fold decreased as compared to the total cell membrane pool, indicating that, in the hematopoietic environment, DPP-IV activity is essentially located in the lipid rafts. This is compatible with membrane-associated events and direct cell-cell interactions, whilst the long-range activity depending upon soluble enzyme is less probable in view of the low affinity of this form.

  8. A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

    Science.gov (United States)

    Rendón-Ramírez, Adela; Shukla, Manish; Oda, Masataka; Chakraborty, Sandeep; Minda, Renu; Dandekar, Abhaya M; Ásgeirsson, Bjarni; Goñi, Félix M; Rao, Basuthkar J

    2013-01-01

    Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

  9. The effects of a TGR5 agonist and a dipeptidyl peptidase IV inhibitor on dextran sulfate sodium-induced colitis in mice.

    Science.gov (United States)

    Sakanaka, Taisuke; Inoue, Takuya; Yorifuji, Naoki; Iguchi, Munetaka; Fujiwara, Kaori; Narabayashi, Ken; Kakimoto, Kazuki; Nouda, Sadaharu; Okada, Toshihiko; Kuramoto, Takanori; Ishida, Kumi; Abe, Yosuke; Takeuchi, Toshihisa; Umegaki, Eiji; Akiba, Yasutada; Kaunitz, Jonathan D; Higuchi, Kazuhide

    2015-03-01

    Luminal nutrients stimulate enteroendocrine L cells to release gut hormones, including intestinotrophic glucagon-like peptide-2 (GLP-2). Because L cells express the bile acid receptor TGR5 and dipeptidyl peptidase-IV (DPPIV) rapidly degrades GLPs, we hypothesized that luminal TGR5 activation may attenuate intestinal injury via GLP-2 release, which is enhanced by DPPIV inhibition. Intestinal injury was induced in mice by administration of dextran sulfate sodium (DSS) in drinking water (free access to water containing 5% DSS for 7 days). The selective TGR5 agonist betulinic acid (BTA) and the DPPIV inhibitor sitagliptin phosphate monohydrate (STG) were administered orally for 7 days. Male C57BL/6 mice (6-7 weeks old) were divided into five groups: normal control group, disease control group, BTA low group (drinking water containing 15 mg/L BTA), BTA high group (50 mg/L BTA), and BTA high + STG (3 mg/kg, i.g.) group. The selective TGR5 agonist BTA dose-dependently suppressed disease activity index and mRNA expression of the pro-inflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor-α in the colon. Nevertheless, STG administration had little additive effect on BTA-induced protection. Fibroblast activation protein mRNA expression, but not expression of other DPP family members, was increased in the colon of DSS-treated mice with increased mucosal DPPIV. Co-administration of the selective GLP-2 antagonist GLP-2 (3-33) reversed the effect of BTA. The selective TGR5 agonist BTA ameliorated DSS-induced colitis in mice via the GLP-2 pathway with no effect of DPPIV inhibition, suggesting that other DPP enzymatic activity is involved in GLP-2 degradation. © 2015 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

  10. Peptidases from Latex of Carica candamarcensis Upregulate COX-2 and IL-1 mRNA Transcripts against Salmonella enterica ser. Typhimurium-Mediated Inflammation

    Directory of Open Access Journals (Sweden)

    Maria Taciana Ralph

    2014-01-01

    Full Text Available The immunomodulatory properties of a mixture of cysteine peptidases (P1G10 obtained from the fruit lattice of Carica candamarcensis were investigated. P1G10 was obtained from fresh latex samples by chromatography in a Sephadex column and initially administered to Swiss mice (n=5; 1 or 10 mg/kg via i.p. After 30 min, the mice were injected with carrageenan (0.5 mg/mouse or heat-killed S. Typhimurium (107 CFU/mL; 100°C/30 min into the peritoneal cavity. Afterwards, two animal groups were i.p. administered with P1G10 (n=6; 1, 5, or 10 mg/Kg or PBS 24 hours prior to challenge with live S. Typhimurium (107 CFU/mL. P1G10 stimulated the proliferation of circulating neutrophils and lymphocytes, 6 h after injection of carrageenan or heat-killed bacteria, respectively. Furthermore, survival after infection was dose-dependent and reached 60% of the animal group. On the other hand, control mice died 1–3 days after infection. The examination of mRNA transcripts in liver cells 24 h after infection confirmed fold variation increases of 5.8 and 4.8 times on average for IL-1 and COX-2, respectively, in P1G10 pretreated mice but not for TNF-α, IL-10, γ-IFN and iNOS, for which the results were comparable to untreated animals. These data are discussed in light of previous reports.

  11. Peptidases from latex of Carica candamarcensis upregulate COX-2 and IL-1 mRNA transcripts against Salmonella enterica ser. Typhimurium-mediated inflammation.

    Science.gov (United States)

    Ralph, Maria Taciana; Silva, Ayrles Fernanda Brandão; da Silva, Dayane Laíse; do Nascimento, Danielle Cristina Oliveira; da Silva, Diogo Manoel Farias; Gomes-Filho, Manoel A; Souza, Paulo Roberto Eleutério; Evêncio-Neto, Joaquim; Ramos, Márcio Viana; Salas, Carlos Edmundo; Lima-Filho, José Vitor

    2014-01-01

    The immunomodulatory properties of a mixture of cysteine peptidases (P1G10) obtained from the fruit lattice of Carica candamarcensis were investigated. P1G10 was obtained from fresh latex samples by chromatography in a Sephadex column and initially administered to Swiss mice (n = 5; 1 or 10 mg/kg) via i.p. After 30 min, the mice were injected with carrageenan (0.5 mg/mouse) or heat-killed S. Typhimurium (10(7) CFU/mL; 100°C/30 min) into the peritoneal cavity. Afterwards, two animal groups were i.p. administered with P1G10 (n = 6; 1, 5, or 10 mg/Kg) or PBS 24 hours prior to challenge with live S. Typhimurium (10(7) CFU/mL). P1G10 stimulated the proliferation of circulating neutrophils and lymphocytes, 6 h after injection of carrageenan or heat-killed bacteria, respectively. Furthermore, survival after infection was dose-dependent and reached 60% of the animal group. On the other hand, control mice died 1-3 days after infection. The examination of mRNA transcripts in liver cells 24 h after infection confirmed fold variation increases of 5.8 and 4.8 times on average for IL-1 and COX-2, respectively, in P1G10 pretreated mice but not for TNF-α, IL-10, γ-IFN and iNOS, for which the results were comparable to untreated animals. These data are discussed in light of previous reports.

  12. Combination Therapy with a Sodium-Glucose Cotransporter 2 Inhibitor and a Dipeptidyl Peptidase-4 Inhibitor Additively Suppresses Macrophage Foam Cell Formation and Atherosclerosis in Diabetic Mice

    Science.gov (United States)

    Hiromura, Munenori; Mori, Yusaku; Kohashi, Kyoko; Kushima, Hideki; Ohara, Makoto; Watanabe, Takuya; Andersson, Olov

    2017-01-01

    Dipeptidyl peptidase-4 inhibitors (DPP-4is), in addition to their antihyperglycemic roles, have antiatherosclerotic effects. We reported that sodium-glucose cotransporter 2 inhibitors (SGLT2is) suppress atherosclerosis in a glucose-dependent manner in diabetic mice. Here, we investigated the effects of combination therapy with SGLT2i and DPP-4i on atherosclerosis in diabetic mice. SGLT2i (ipragliflozin, 1.0 mg/kg/day) and DPP-4i (alogliptin, 8.0 mg/kg/day), either alone or in combination, were administered to db/db mice or streptozotocin-induced diabetic apolipoprotein E-null (Apoe−/−) mice. Ipragliflozin and alogliptin monotherapies improved glucose intolerance; however, combination therapy did not show further improvement. The foam cell formation of peritoneal macrophages was suppressed by both the ipragliflozin and alogliptin monotherapies and was further enhanced by combination therapy. Although foam cell formation was closely associated with HbA1c levels in all groups, DPP-4i alone or the combination group showed further suppression of foam cell formation compared with the control or SGLT2i group at corresponding HbA1c levels. Both ipragliflozin and alogliptin monotherapies decreased scavenger receptors and increased cholesterol efflux regulatory genes in peritoneal macrophages, and combination therapy showed additive changes. In diabetic Apoe−/− mice, combination therapy showed the greatest suppression of plaque volume in the aortic root. In conclusion, combination therapy with SGLT2i and DPP4i synergistically suppresses macrophage foam cell formation and atherosclerosis in diabetic mice.

  13. Ubiquitin-specific peptidase 22 inhibits colon cancer cell invasion by suppressing the signal transducer and activator of transcription 3/matrix metalloproteinase 9 pathway.

    Science.gov (United States)

    Ao, Ning; Liu, Yanyan; Bian, Xiaocui; Feng, Hailiang; Liu, Yuqin

    2015-08-01

    Colon cancer is associated with increased cell migration and invasion. In the present study, the role of ubiquitin-specific peptidase 22 (USP22) in signal transducer and activator of transcription 3 (STAT3)-mediated colon cancer cell invasion was investigated. The messenger RNA levels of STAT3 target genes were measured by reverse transcription-quantitative polymerase chain reaction, following USP22 knockdown by RNA interference in SW480 colon cancer cells. The matrix metalloproteinase 9 (MMP9) proteolytic activity and invasion potential of SW480 cells were measured by zymography and Transwell assay, respectively, following combined USP22 and STAT3 short interfering (si)RNA treatment or STAT3 siRNA treatment alone. Similarly, a cell counting kit-8 assay was used to detect the proliferation potential of SW480 cells. The protein expression levels of USP22, STAT3 and MMP9 were detected by immunohistochemistry in colon cancer tissue microarrays (TMAs) and the correlation between USP22, STAT3 and MMP9 was analyzed. USP22/STAT3 co-depletion partly rescued the MMP9 proteolytic activity and invasion of SW480 cells, compared with that of STAT3 depletion alone. However, the proliferation of USP22/STAT3si-SW480 cells was decreased compared with that of STAT3si-SW480 cells. USP22 expression was positively correlated with STAT3 and MMP9 expression in colon cancer TMAs. In conclusion, USP22 attenuated the invasion capacity of colon cancer cells by inhibiting the STAT3/MMP9 signaling pathway.

  14. Teneligliptin, a dipeptidyl peptidase-4 inhibitor, attenuated pro-inflammatory phenotype of perivascular adipose tissue and inhibited atherogenesis in normoglycemic apolipoprotein-E-deficient mice.

    Science.gov (United States)

    Salim, Hotimah Masdan; Fukuda, Daiju; Higashikuni, Yasutomi; Tanaka, Kimie; Hirata, Yoichiro; Yagi, Shusuke; Soeki, Takeshi; Shimabukuro, Michio; Sata, Masataka

    2017-09-01

    Dipeptidyl peptidase-4 (DPP-4) inhibitors have various cellular effects that are associated with vascular protection. Here, we examined whether teneligliptin alters the pro-inflammatory phenotype of perivascular adipose tissue (PVAT) and inhibits atherogenesis. Teneligliptin (60mg/kg/day) was administered orally to apolipoprotein-E-deficient (ApoE(-/-)) mice for 20weeks. Teneligliptin significantly inhibited the development of atherosclerosis in the aortic arch compared with vehicle (P<0.05), without alteration of blood glucose level or blood pressure. Histological analyses demonstrated that teneligliptin decreased lipid deposition and MCP-1 expression (P<0.05, respectively), and tended to decrease macrophage accumulation in atherosclerotic plaques. The results of quantitative RT-PCR analysis demonstrated that teneligliptin reduced the expression of inflammatory molecules such as TNF-α and MCP-1 in the abdominal aorta. Furthermore, teneligliptin reduced the expression of a macrophage marker and Nox-4, a major NADPH oxidase subunit in adipocytes, in PVAT around the aortic arch. Administration of teneligliptin for 8weeks ameliorated endothelium-dependent vasodilation and reduced oxidative stress as determined by urinary 8-OHdG excretion (P<0.05) compared with vehicle. In vitro experiments demonstrated that exendin-4 (Ex-4), a GLP-1 analog, decreased the expression of inflammatory molecules in RAW264.7 cells. Also, Ex-4 decreased Nox4 expression in 3T3-L1 adipocytes. Teneligliptin inhibited atherogenesis with attenuation of the inflammatory phenotype in PVAT. A GLP-1 analog suppressed pro-inflammatory activation of macrophages and adipocytes. Suppression of the pro-inflammatory phenotype of PVAT might contribute, at least partially, to the cardioprotective effects of teneligliptin. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. The Maize Viviparous8 locus, encoding a putative ALTERED MERISTEM PROGRAM1-like peptidase, regulates abscisic acid accumulation and coordinates embryo and endosperm development.

    Science.gov (United States)

    Suzuki, Masaharu; Latshaw, Susan; Sato, Yutaka; Settles, A Mark; Koch, Karen E; Hannah, L Curtis; Kojima, Mikiko; Sakakibara, Hitoshi; McCarty, Donald R

    2008-03-01

    We describe a mutant of Zea mays isolated from a W22 inbred transposon population, widow's peak mutant1 (wpk1), with an altered pattern of anthocyanin synthesis and aleurone cell differentiation in endosperm. In addition, a failure of the developing mutant embryo to form leaf initials is associated with decreased expression of a subset of meristem regulatory genes that includes Abphyl1 and Td1. We show that the viviparous8 (vp8) mutant has a similar pleiotropic phenotype in the W22 inbred background in contrast to the viviparous embryo phenotype exhibited in the standard genetic background, and we confirmed that wpk1 is allelic to vp8. Further genetic analysis revealed that the standard vp8 stock contains an unlinked, partially dominant suppressor of the vp8 mutation that is not present in W22. Consistent with the early-onset viviparous phenotype of vp8, expression of several embryonic regulators, including LEC1/B3 domain transcription factors, was reduced in the mutant embryo. Moreover, reduced abscisic acid (ABA) content of vp8/wpk1 embryos was correlated with altered regulation of ABA biosynthesis, as well as ABA catabolic pathways. The ABA biosynthetic gene Vp14 was down-regulated in the nonsuppressed background, whereas the ZmABA8'oxA1a ABA 8'-hydroxylase gene was strongly up-regulated in both genetic backgrounds. Molecular analysis revealed that Vp8 encodes a putative peptidase closely related to Arabidopsis thaliana ALTERED MERISTEM PROGRAM1. Because the Vp8 regulates meristem development as well as seed maturation processes, including ABA accumulation, we propose that VP8 is required for synthesis of an unidentified signal that integrates meristem and embryo formation in seeds.

  16. The Maize Viviparous8 Locus, Encoding a Putative ALTERED MERISTEM PROGRAM1-Like Peptidase, Regulates Abscisic Acid Accumulation and Coordinates Embryo and Endosperm Development1[W

    Science.gov (United States)

    Suzuki, Masaharu; Latshaw, Susan; Sato, Yutaka; Settles, A. Mark; Koch, Karen E.; Hannah, L. Curtis; Kojima, Mikiko; Sakakibara, Hitoshi; McCarty, Donald R.

    2008-01-01

    We describe a mutant of Zea mays isolated from a W22 inbred transposon population, widow's peak mutant1 (wpk1), with an altered pattern of anthocyanin synthesis and aleurone cell differentiation in endosperm. In addition, a failure of the developing mutant embryo to form leaf initials is associated with decreased expression of a subset of meristem regulatory genes that includes Abphyl1 and Td1. We show that the viviparous8 (vp8) mutant has a similar pleiotropic phenotype in the W22 inbred background in contrast to the viviparous embryo phenotype exhibited in the standard genetic background, and we confirmed that wpk1 is allelic to vp8. Further genetic analysis revealed that the standard vp8 stock contains an unlinked, partially dominant suppressor of the vp8 mutation that is not present in W22. Consistent with the early-onset viviparous phenotype of vp8, expression of several embryonic regulators, including LEC1/B3 domain transcription factors, was reduced in the mutant embryo. Moreover, reduced abscisic acid (ABA) content of vp8/wpk1 embryos was correlated with altered regulation of ABA biosynthesis, as well as ABA catabolic pathways. The ABA biosynthetic gene Vp14 was down-regulated in the nonsuppressed background, whereas the ZmABA8′oxA1a ABA 8′-hydroxylase gene was strongly up-regulated in both genetic backgrounds. Molecular analysis revealed that Vp8 encodes a putative peptidase closely related to Arabidopsis thaliana ALTERED MERISTEM PROGRAM1. Because the Vp8 regulates meristem development as well as seed maturation processes, including ABA accumulation, we propose that VP8 is required for synthesis of an unidentified signal that integrates meristem and embryo formation in seeds. PMID:18203869

  17. Quality measure and weight loss assessment in patients with type 2 diabetes mellitus treated with canagliflozin or dipeptidyl peptidase-4 inhibitors.

    Science.gov (United States)

    Wysham, Carol H; Lefebvre, Patrick; Pilon, Dominic; Ingham, Mike; Lafeuille, Marie-Hélène; Emond, Bruno; Kamstra, Rhiannon; Chow, Wing; Pfeifer, Michael; Duh, Mei Sheng

    2017-06-08

    Achieving control of glycated hemoglobin (HbA1c), blood pressure (BP), and body weight (BW) remains a challenge for most patients with type 2 diabetes mellitus (T2DM). In clinical trials, canagliflozin (CANA), an inhibitor of sodium-glucose co-transporter 2, has shown significant improvement compared to some dipeptidyl peptidase-4 (DPP-4) inhibitors in the achievement of such quality measures. This study used recent electronic medical records (EMR) data to assess quality measure achievement of HbA1C, BP, and BW loss in patients treated with CANA versus DPP-4 inhibitors. Adult patients with ≥1 T2DM diagnosis and ≥12 months of clinical activity (baseline) before first CANA or DPP-4 prescription (index) were identified in the QuintilesIMS Health Real-World Data EMRs-US database (03/29/2012-10/30/2015). Patients were observed from the index to last encounter. Inverse probability of treatment weighting (IPTW) was used to adjust for observed baseline confounders between groups. Kaplan-Meier (KM) rates and Cox proportional hazard models were used to compare achievement of HbA1c CANA and 17,679 DPP-4 patients were selected. IPTW resulted in balanced baseline demographic, comorbidity, and disease characteristics (CANA: N = 13,793, mean age: 59.0 years; DPP-4: N = 14,588, mean age: 58.9 years). Up until 24 months post-index, CANA patients were more likely to reach an HbA1c CANA patients were more likely to reach a systolic BP CANA approval (March 2013) suggests that patients initiated on CANA were more likely to reach HbA1c, systolic BP, and weight loss objectives specified by general diabetes care guidelines than patients initiated on DPP-4 inhibitors.

  18. Signal peptide peptidase-mediated nuclear localization of heme oxygenase-1 promotes cancer cell proliferation and invasion independent of its enzymatic activity.

    Science.gov (United States)

    Hsu, F-F; Yeh, C-T; Sun, Y-J; Chiang, M-T; Lan, W-M; Li, F-A; Lee, W-H; Chau, L-Y

    2015-04-30

    Heme oxygenase-1 (HO-1) is a heme-degrading enzyme anchored in the endoplasmic reticulum by a carboxyl-terminal transmembrane segment (TMS). HO-1 is highly expressed in various cancers and its nuclear localization is associated with the progression of some cancers. Nevertheless, the mechanism underlying HO-1 nuclear translocation and its pathological significance remain elusive. Here we show that the signal peptide peptidase (SPP) catalyzes the intramembrane cleavage of HO-1. Coexpression of HO-1 with wild-type SPP, but not a dominant-negative SPP, promoted the nuclear localization of HO-1 in cells. Mass spectrometry analysis of cytosolic HO-1 isolated from HeLa cells overexpressing HO-1 and SPP revealed two adjacent intramembrane cleavage sites located after S275 and F276 within the TMS. Mutations of S275F276 to A275L276 significantly hindered SPP-mediated HO-1 cleavage and nuclear localization. Nuclear HO-1 was detected in A549 and DU145 cancer cell lines expressing high levels of endogenous HO-1 and SPP. SPP knockdown or inhibition significantly reduced nuclear HO-1 localization in A549 and DU145 cells. The positive nuclear HO-1 stain was also evident in lung cancer tissues expressing high levels of HO-1 and SPP. Overexpression of a truncated HO-1 (t-HO-1) lacking the TMS in HeLa and H1299 cells promoted cell proliferation and migration/invasion. The effect of t-HO-1 was not affected by a mutation in the catalytic site. However, blockade of t-HO-1 nuclear localization abolished t-HO-1-mediated effect. The tumorigenic effect of t-HO-1 was also demonstrated in the mouse model. These findings disclose that SPP-mediated intramembrane cleavage of HO-1 promotes HO-1 nuclear localization and cancer progression independent of HO-1 enzymatic activity.

  19. Incretin therapies: highlighting common features and differences in the modes of action of glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors.

    Science.gov (United States)

    Nauck, M

    2016-03-01

    Over the last few years, incretin-based therapies have emerged as important agents in the treatment of type 2 diabetes (T2D). These agents exert their effect via the incretin system, specifically targeting the receptor for the incretin hormone glucagon-like peptide 1 (GLP-1), which is partly responsible for augmenting glucose-dependent insulin secretion in response to nutrient intake (the 'incretin effect'). In patients with T2D, pharmacological doses/concentrations of GLP-1 can compensate for the inability of diabetic β cells to respond to the main incretin hormone glucose-dependent insulinotropic polypeptide, and this is therefore a suitable parent compound for incretin-based glucose-lowering medications. Two classes of incretin-based therapies are available: GLP-1 receptor agonists (GLP-1RAs) and dipeptidyl peptidase-4 (DPP-4) inhibitors. GLP-1RAs promote GLP-1 receptor (GLP-1R) signalling by providing GLP-1R stimulation through 'incretin mimetics' circulating at pharmacological concentrations, whereas DPP-4 inhibitors prevent the degradation of endogenously released GLP-1. Both agents produce reductions in plasma glucose and, as a result of their glucose-dependent mode of action, this is associated with low rates of hypoglycaemia; however, there are distinct modes of action resulting in differing efficacy and tolerability profiles. Furthermore, as their actions are not restricted to stimulating insulin secretion, these agents have also been associated with additional non-glycaemic benefits such as weight loss, improvements in β-cell function and cardiovascular risk markers. These attributes have made incretin therapies attractive treatments for the management of T2D and have presented physicians with an opportunity to tailor treatment plans. This review endeavours to outline the commonalities and differences among incretin-based therapies and to provide guidance regarding agents most suitable for treating T2D in individual patients.

  20. MIP1, a new yeast gene homologous to the rat mitochondrial intermediate peptidase gene, is required for oxidative metabolism in Saccharomyces cerevisiae.

    Science.gov (United States)

    Isaya, G; Miklos, D; Rollins, R A

    1994-08-01

    Cleavage of amino-terminal octapeptides, F/L/IXXS/T/GXXXX, by mitochondrial intermediate peptidase (MIP) is typical of many mitochondrial precursor proteins imported to the matrix and the inner membrane. We previously described the molecular characterization of rat liver MIP (RMIP) and indicated a putative homolog in the sequence predicted from gene YCL57w of yeast chromosome III. A new yeast gene, MIP1, has now been isolated by screening a Saccharomyces cerevisiae genomic library with an RMIP cDNA probe. MIP1 predicts a protein of 772 amino acids (YMIP), which is 54% similar and 31% identical to RMIP and includes a putative 37-residue mitochondrial leader peptide. RMIP and YMIP contain the sequence LFHEMGHAM HSMLGRT, which includes a zinc-binding motif, HEXXH, while the predicted YCL57w protein contains a comparable sequence with a lower degree of homology. No obvious biochemical phenotype was observed in a chromosomally disrupted ycl57w mutant. In contrast, a mip1 mutant was unable to grow on nonfermentable substrates, while a mip1 ycl57w double disruption did not result in a more severe phenotype. The mip1 mutant exhibited defects of complexes III and IV of the respiratory chain, caused by failure to carry out the second MIP-catalyzed cleavage of the nuclear-encoded precursors for cytochrome oxidase subunit IV (CoxIV) and the iron-sulfur protein (Fe-S) of the bc1 complex to mature proteins. In vivo, intermediate-size CoxIV was accumulated in the mitochondrial matrix, while intermediate-size Fe-S was targeted to the inner membrane. Moreover, mip1 mitochondrial fractions failed to carry out maturation of the human ornithine transcarbamylase intermediate (iOTC), specifically cleaved by RMIP. A CEN plasmid-encoded YMIP protein restored normal MIP activity along with respiratory competence. Thus, YMIP is a functional homolog of RMIP and represents a new component of the yeast mitochondrial import machinery.

  1. In vivo dual-delivery of glucagon like peptide-1 (GLP-1) and dipeptidyl peptidase-4 (DPP4) inhibitor through composites prepared by microfluidics for diabetes therapy

    Science.gov (United States)

    Araújo, F.; Shrestha, N.; Gomes, M. J.; Herranz-Blanco, B.; Liu, D.; Hirvonen, J. J.; Granja, P. L.; Santos, H. A.; Sarmento, B.

    2016-05-01

    Oral delivery of proteins is still a challenge in the pharmaceutical field. Nanoparticles are among the most promising carrier systems for the oral delivery of proteins by increasing their oral bioavailability. However, most of the existent data regarding nanosystems for oral protein delivery is from in vitro studies, lacking in vivo experiments to evaluate the efficacy of these systems. Herein, a multifunctional composite system, tailored by droplet microfluidics, was used for dual delivery of glucagon like peptide-1 (GLP-1) and dipeptidyl peptidase-4 inhibitor (iDPP4) in vivo. Oral delivery of GLP-1 with nano- or micro-systems has been studied before, but the simultaneous nanodelivery of GLP-1 with iDPP4 is a novel strategy presented here. The type 2 diabetes mellitus (T2DM) rat model, induced through the combined administration of streptozotocin and nicotinamide, a non-obese model of T2DM, was used. The combination of both drugs resulted in an increase in the hypoglycemic effects in a sustained, but prolonged manner, where the iDPP4 improved the therapeutic efficacy of GLP-1. Four hours after the oral administration of the system, blood glucose levels were decreased by 44%, and were constant for another 4 h, representing half of the glucose area under the curve when compared to the control. An enhancement of the plasmatic insulin levels was also observed 6 h after the oral administration of the dual-drug composite system and, although no statistically significant differences existed, the amount of pancreatic insulin was also higher. These are promising results for the oral delivery of GLP-1 to be pursued further in a chronic diabetic model study.

  2. Sitagliptin, a dipeptidyl peptidase-4 inhibitor, improves recognition memory, oxidative stress and hippocampal neurogenesis and upregulates key genes involved in cognitive decline.

    Science.gov (United States)

    Gault, V A; Lennox, R; Flatt, P R

    2015-04-01

    To examine whether prolonged dipeptidyl peptidase-4 (DPP-4) inhibition can reverse learning and memory impairment in high-fat-fed mice. High-fat-fed mice received oral sitagliptin (50 mg/kg body weight) once daily or saline vehicle over 21 days. An additional group of mice on standard chow received saline vehicle. Energy intake, body weight, glucose and insulin concentrations were measured at regular intervals. Glucose tolerance, insulin sensitivity, novel object recognition, DPP-4 activity, hormone analysis, hippocampal gene expression and histology were performed. Sitagliptin decreased circulating DPP-4 activity and improved glucose tolerance, glucose-stimulated insulin secretion and insulin sensitivity, and reduced plasma triglycerides and cholesterol levels. DPP-4 inhibition improved recognition memory (1.2-fold increase) without affecting hypermoteric activity or anxiety levels. Improvement in memory and learning was linked to reduced immunostaining for 8-oxoguanine and increased doublecortin staining in the hippocampus, which were indicative of reduced brain oxidative stress and increased hippocampal neurogenesis, respectively. These effects were associated with significant upregulation of hippocampal gene expression of glucagon-like peptide-1 (GLP-1) receptor, glucose-dependent insulinotropic polypeptide receptor, synaptophysin, sirtuin 1, glycogen synthase kinase 3β, superdioxide mutase 2, nuclear factor (erythroid-derived 2)-like 2 and vascular endothelial growth factor. Total plasma and brain GLP-1 concentrations were significantly increased after sitagliptin therapy, whereas DPP-4 activity in brain tissue was not altered. These studies show that sitagliptin can reverse memory impairment in high-fat-fed mice and is also associated with improved insulin sensitivity, enhanced hippocampal neurogenesis and reduced oxidative stress. DPP-4 inhibitors may therefore exhibit dual benefits by improving metabolic control and reducing the decline in cognitive

  3. Increased plasma dipeptidyl peptidase-4 activities are associated with high prevalence of subclinical atherosclerosis in Chinese patients with newly diagnosed type 2 diabetes: a cross-sectional study.

    Science.gov (United States)

    Zheng, T P; Liu, Y H; Yang, L X; Qin, S H; Liu, H B

    2015-10-01

    Hyperglycemia, insulin resistance, dislipidemia, oxidative stress and inflammation are well-documented risk factors for subclinical atherosclerosis. Dipeptidyl peptidase-4(DPP4) is a newly identified adipokine related to these risk factors. Hence, we aimed to investigate the association between plasma DPP4 activities and subclinical atherosclerosis in type 2 diabetes. A total of 985 newly diagnosed type 2 diabetic subjects were studied. Plasma DPP4 activity, mannose 6-phosphate receptor (M6P-R), oxidative stress parameters, inflammatory markers and common carotid artery Intima-Media Thickness (c-IMT) were measured in all participants. Participants in the highest quartile of DPP4 activity had higher HbA1c, homeostatic model assessment of insulin resistance(HOMA-IR), triglyceride, low-density lipoprotein cholesterol(LDL-C), oxidized LDL, nitrotyrosine, 8-iso-PGF2a, interleukin-6 (IL-6), high-sensitivity C-reactive protein (hs-CRP), M6P-R, c-IMT compared with participants in the lowest quartile (all P dislipidemia, oxidative stress and inflammation were higher with increasing DPP4 quartiles (P < 0.001 for trend). In the highest DPP4 quartile, subclinical atherosclerosis risk was significantly higher (OR 4.97; 95% CI 3.03-8.17) than in the lowest quartile. This association remained strong (2.17; 1.21-3.89) after further controlling for HbA1c, HOMA-IR, triglyceride, oxidized LDL, nitrotyrosine, and IL-6. This study shows that increased DPP4 activities are positively and independently associated with subclinical atherosclerosis in type 2 diabetes. Our findings suggest of potential role of DPP4 in the pathogenesis of subclinical atherosclerosis and in the prevention and management of this disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  4. Chronic Psychological Stress Accelerates Vascular Senescence and Impairs Ischemia-Induced Neovascularization: The Role of Dipeptidyl Peptidase-4/Glucagon-Like Peptide-1-Adiponectin Axis.

    Science.gov (United States)

    Piao, Limei; Zhao, Guangxian; Zhu, Enbo; Inoue, Aiko; Shibata, Rei; Lei, Yanna; Hu, Lina; Yu, Chenglin; Yang, Guang; Wu, Hongxian; Xu, Wenhu; Okumura, Kenji; Ouchi, Noriyuki; Murohara, Toyoaki; Kuzuya, Masafumi; Cheng, Xian Wu

    2017-09-28

    Exposure to psychosocial stress is a risk factor for cardiovascular disease, including vascular aging and regeneration. Given that dipeptidyl peptidase-4 (DPP4) regulates several intracellular signaling pathways associated with the glucagon-like peptide-1 (GLP-1) metabolism, we investigated the role of DPP4/GLP-1 axis in vascular senescence and ischemia-induced neovascularization in mice under chronic stress, with a special focus on adiponectin -mediated peroxisome proliferator activated receptor-γ/its co-activator 1α (PGC-1α) activation. Seven-week-old mice subjected to restraint stress for 4 weeks underwent ischemic surgery and were kept under immobilization stress conditions. Mice that underwent ischemic surgery alone served as controls. We demonstrated that stress impaired the recovery of the ischemic/normal blood-flow ratio throughout the follow-up period and capillary formation. On postoperative day 4, stressed mice showed the following: increased levels of plasma and ischemic muscle DPP4 and decreased levels of GLP-1 and adiponectin in plasma and phospho-AMP-activated protein kinase α (p-AMPKα), vascular endothelial growth factor, peroxisome proliferator activated receptor-γ, PGC-1α, and Sirt1 proteins and insulin receptor 1 and glucose transporter 4 genes in the ischemic tissues, vessels, and/or adipose tissues and numbers of circulating endothelial CD31(+)/c-Kit(+) progenitor cells. Chronic stress accelerated aortic senescence and impaired aortic endothelial sprouting. DPP4 inhibition and GLP-1 receptor activation improved these changes; these benefits were abrogated by adiponectin blocking and genetic depletion. These results indicate that the DPP4/GLP-1-adiponectin axis is a novel therapeutic target for the treatment of vascular aging and cardiovascular disease under chronic stress conditions. © 2017 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

  5. Reversal of new-onset diabetes through modulating inflammation and stimulating beta-cell replication in nonobese diabetic mice by a dipeptidyl peptidase IV inhibitor.

    Science.gov (United States)

    Tian, Lei; Gao, Jie; Hao, Jianqiang; Zhang, Yu; Yi, Huimin; O'Brien, Timothy D; Sorenson, Robert; Luo, Jian; Guo, Zhiguang

    2010-07-01

    Inhibition of dipeptidyl peptidase IV (DPP-IV) activity by NVP-DPP728, a DPP-IV inhibitor, improves the therapeutic efficacy of glucagon-like peptide-1 (GLP-1). CD26 is a membrane-associated glycoprotein with DPP-IV activity and is expressed on lymphocytes. We investigated the effect of NVP-DPP728 on reversing new-onset diabetes in nonobese diabetic (NOD) mice and modulating the inflammatory response and stimulating beta-cell regeneration. New-onset diabetic NOD mice were treated with NVP-DPP728 for 2, 4, and 6 wk. Blood glucose level was monitored. Regulatory T cells in thymus and secondary lymph nodes, TGF-beta1 and GLP-1 in plasma, and the insulin content in the pancreas were measured. Immunostaining for insulin and bromodeoxyuridine (BrdU) were performed. The correlation of beta-cell replication with inflammation was determined. In NVP-DPP728-treated NOD mice, diabetes could be reversed in 57, 74, and 73% of mice after 2, 4, and 6 wk treatment, respectively. Insulitis was reduced and the percentage of CD4(+)CD25(+)FoxP3(+) regulatory T cells was increased in treated NOD mice with remission. Plasma TGF-beta1 and GLP-1, the insulin content, and both insulin(+) and BrdU(+) beta-cells in pancreas were also significantly increased. No significant correlations were found between numbers of both insulin(+) and BrdU(+) beta-cells in islets and beta-cell area or islets with different insulitis score in NOD mice with remission of diabetes. In conclusion, NVP-DPP728 treatment can reverse new-onset diabetes in NOD mice by reducing insulitis, increasing CD4(+)CD25(+)FoxP3(+) regulatory T cells, and stimulating beta-cell replication. beta-Cell replication is not associated with the degree of inflammation in NVP-DPP728-treated NOD mice.

  6. Sodium-glucose co-transporter-2 inhibitors and dipeptidyl peptidase-4 inhibitors combination therapy in type 2 diabetes: A systematic review of current evidence

    Directory of Open Access Journals (Sweden)

    Awadhesh Kumar Singh

    2016-01-01

    Full Text Available As type 2 diabetes mellitus (T2DM is a chronic and progressive disease with multiple pathophysiologic defects, no single anti-diabetic agent can tackle all these multi-factorial pathways. Consequently, multiple agents working through the different mechanisms will be required for the optimal glycemic control. Moreover, the combination therapies of different anti-diabetic agents may complement their actions and possibly act synergistic. Furthermore, these combinations could possess the additional properties to counter their undesired physiological compensatory response. Sodium-glucose co-transporter-2 inhibitors (SGLT-2I are newly emerging class of drugs, with a great potential to reduce glucose effectively with an additional quality of lowering cardiovascular events as demonstrated very recently by one of the agents of this class. However, increase in endogenous glucose production (EGP from the liver, either due to the increase in glucagon or compensatory response to glucosuria can offset the glucose-lowering potential of SGLT-2I. Interestingly, another class of drugs such as dipeptidyl peptidase-4 inhibitors (DPP-4I effectively decrease glucagon and reduce EGP. In light of these findings, combination therapies with SGLT-2I and DPP-4I are particularly appealing and are expected to produce a synergistic effect. Preclinical studies of combination therapies with DPP-4I and SGLT-2I have already demonstrated a significant lowering of hemoglobin A1c potential and human studies also find no drug-drug interaction between these agents. This article aims to systematically review the efficacy and safety of combination therapy of SGLT-2I and DPP-4I in T2DM.

  7. Crosstalk between advanced glycation end products (AGEs)-receptor RAGE axis and dipeptidyl peptidase-4-incretin system in diabetic vascular complications.

    Science.gov (United States)

    Yamagishi, Sho-ichi; Fukami, Kei; Matsui, Takanori

    2015-01-13

    Advanced glycation end products (AGEs) consist of heterogenous group of macroprotein derivatives, which are formed by non-enzymatic reaction between reducing sugars and amino groups of proteins, lipids and nucleic acids, and whose process has progressed at an accelerated rate under diabetes. Non-enzymatic glycation and cross-linking of protein alter its structural integrity and function, contributing to the aging of macromolecules. Furthermore, engagement of receptor for AGEs (RAGE) with AGEs elicits oxidative stress generation and subsequently evokes proliferative, inflammatory, and fibrotic reactions in a variety of cells. Indeed, accumulating evidence has suggested the active involvement of accumulation of AGEs in diabetes-associated disorders such as diabetic microangiopathy, atherosclerotic cardiovascular diseases, Alzheimer's disease and osteoporosis. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are incretins, gut hormones secreted from the intestine in response to food intake, both of which augment glucose-induced insulin release, suppress glucagon secretion, and slow gastric emptying. Since GLP-1 and GIP are rapidly degraded and inactivated by dipeptidyl peptidase-4 (DPP-4), inhibition of DPP-4 and/or DPP-4-resistant GLP-1 analogues have been proposed as a potential target for the treatment of diabetes. Recently, DPP-4 has been shown to cleave multiple peptides, and blockade of DPP-4 could exert diverse biological actions in GLP-1- or GIP-independent manner. This article summarizes the crosstalk between AGEs-RAGE axis and DPP-4-incretin system in the development and progression of diabetes-associated disorders and its therapeutic intervention, especially focusing on diabetic vascular complications.

  8. A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

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    Adela Rendón-Ramírez

    Full Text Available Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP. CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP and a scaffold recognizing a β-lactam (imipenem in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV, we sought to query other OMV proteins, like phospholipase C (PLC, using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM. Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

  9. Purification and characterization of tenerplasminin-1, a serine peptidase inhibitor with antiplasmin activity from the coral snake (Micrurus tener tener) venom.

    Science.gov (United States)

    Vivas, Jeilyn; Ibarra, Carlos; Salazar, Ana M; Neves-Ferreira, Ana G C; Sánchez, Elda E; Perales, Jonás; Rodríguez-Acosta, Alexis; Guerrero, Belsy

    2016-01-01

    A plasmin inhibitor, named tenerplasminin-1 (TP1), was isolated from Micrurus tener tener (Mtt) venom. It showed a molecular mass of 6542Da, similarly to Kunitz-type serine peptidase inhibitors. The amidolytic activity of plasmin (0.5nM) on synthetic substrate S-2251 was inhibited by 91% following the incubation with TP1 (1nM). Aprotinin (2nM) used as the positive control of inhibition, reduced the plasmin amidolytic activity by 71%. Plasmin fibrinolytic activity (0.05nM) was inhibited by 67% following incubation with TP1 (0.1nM). The degradation of fibrinogen chains induced by plasmin, trypsin or elastase was inhibited by TP1 at a 1:2, 1:4 and 1:20 enzyme:inhibitor ratio, respectively. On the other hand, the proteolytic activity of crude Mtt venom on fibrinogen chains, previously attributed to metallopeptidases, was not abolished by TP1. The tPA-clot lysis assay showed that TP1 (0.2nM) acts like aprotinin (0.4nM) inducing a delay in lysis time and lysis rate which may be associated with the inhibition of plasmin generated from the endogenous plasminogen activation. TP1 is the first serine protease plasmin-like inhibitor isolated from Mtt snake venom which has been characterized in relation to its mechanism of action, formation of a plasmin:TP1 complex and therapeutic potential as anti-fibrinolytic agent, a biological characteristic of great interest in the field of biomedical research. They could be used to regulate the fibrinolytic system in pathologies such as metastatic cancer, parasitic infections, hemophilia and other hemorrhagic syndromes, in which an intense fibrinolytic activity is observed.

  10. Dipeptidyl peptidase-4 inhibitor ameliorates early renal injury through its anti-inflammatory action in a rat model of type 1 diabetes

    Energy Technology Data Exchange (ETDEWEB)

    Kodera, Ryo, E-mail: kodera@cc.okayama-u.ac.jp [Center for Innovative Clinical Medicine, Okayama University Hospital, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan); Shikata, Kenichi [Center for Innovative Clinical Medicine, Okayama University Hospital, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan); Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan); Takatsuka, Tetsuharu; Oda, Kaori; Miyamoto, Satoshi; Kajitani, Nobuo; Hirota, Daisho; Ono, Tetsuichiro [Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan); Usui, Hitomi Kataoka [Department of Primary Care and Medical Education, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan); Makino, Hirofumi [Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan)

    2014-01-17

    Highlights: •DPP-4 inhibitor decreased urinary albumin excretion in a rat of type 1 diabetes. •DPP-4 inhibitor ameliorated histlogical changes of diabetic nephropathy. •DPP-4 inhibitor has reno-protective effects through anti-inflammatory action. •DPP-4 inhibitor is beneficial on diabetic nephropathy besides lowering blood glucose. -- Abstract: Introduction: Dipeptidyl peptidase-4 (DPP-4) inhibitors are incretin-based drugs in patients with type 2 diabetes. In our previous study, we showed that glucagon-like peptide-1 (GLP-1) receptor agonist has reno-protective effects through anti-inflammatory action. The mechanism of action of DPP-4 inhibitor is different from that of GLP-1 receptor agonists. It is not obvious whether DPP-4 inhibitor prevents the exacerbation of diabetic nephropathy through anti-inflammatory effects besides lowering blood glucose or not. The purpose of this study is to clarify the reno-protective effects of DPP-4 inhibitor through anti-inflammatory actions in the early diabetic nephropathy. Materials and methods: Five-week-old male Sprague–Dawley (SD) rats were divided into three groups; non-diabetes, diabetes and diabetes treated with DPP-4 inhibitor (PKF275-055; 3 mg/kg/day). PKF275-055 was administered orally for 8 weeks. Results: PKF275-055 increased the serum active GLP-1 concentration and the production of urinary cyclic AMP. PKF275-055 decreased urinary albumin excretion and ameliorated histological change of diabetic nephropathy. Macrophage infiltration was inhibited, and inflammatory molecules were down-regulated by PKF275-055 in the glomeruli. In addition, nuclear factor-κB (NF-κB) activity was suppressed in the kidney. Conclusions: These results indicate that DPP-4 inhibitor, PKF275-055, have reno-protective effects through anti-inflammatory action in the early stage of diabetic nephropathy. The endogenous biological active GLP-1 might be beneficial on diabetic nephropathy besides lowering blood glucose.

  11. 代谢综合征孕妇血清丙氨酸氨基肽酶、亮氨酸氨基肽酶表达和临床意义%Clinical significance and expression of alanine amino peptidase and leucine amino peptidase in the serum of pregnant women with metabolic syndrome

    Institute of Scientific and Technical Information of China (English)

    蔡仲仁

    2011-01-01

    目的:探讨代谢综合征孕妇血清丙氨酸氨基肽酶(AAP)、亮氨酸氨基肽酶(LAP)水平的表达,分析其临床意义.方法:选取我院2009年1月~2010年12月收治的42例代谢综合征孕妇作为研究组,入院后检测血清AAP、LAP的表达.另外选择同期我院的40例正常孕妇作为对照组,入院后检测血清AAP、LAP的表达.所有孕妇均为妊娠晚期.结果:研究组血清AAP[(64.21±9.46)U/L]、LAP[(53.53±7.67)U/L]均高于对照组血清AAP[(44.64±8.36)U/L]、LAP[(36.75±7.86)U/L],两组比较,差异有统计学意义(P<0.05).结论:代谢综合征孕妇血清AAP、LAP明显增高,可能是机体对代谢综合征的一种保护措施.%Objective: To discuss the expression of aianine ammo peptidase (APP) and leucine amino peptidase (LAP) in the serum of pregnant women with metabolic syndrome, and analyze the clinical significance. Methods: 42 pregnant women with metabolic syndrome were collected from January 2009 to December 2010 in our hospital, after admission the serum AAP, LAP expression were detected. Another 40 patients with normal pregnant women were chose as control group, as the same time after admission the serum AAP, LAP expression were delected. All pregnant women were late trimester of pregnancy. Results: After the detection, the semm AAP [(64.21 ±9.46)U/L], LAP [(53.53-7.67)LVL] in the research group were higher than those in the control group serum AAP [(44.64 ±8.36)U/Lj, LAP[(36.75±7.86)U/L], there were significant differences in the two groups (P<0.05). Conclusion: Serum AAP, LAP of pregnant women wiih metabolic syndrome is significantly higher, which may be a protection measure for the body of the metabolic syndrome.

  12. The first structure in a family of peptidase inhibitors reveals an unusual Ig-like fold [v2; ref status: indexed, http://f1000r.es/1nx

    Directory of Open Access Journals (Sweden)

    Daniel J Rigden

    2013-08-01

    Full Text Available We report the crystal structure solution of the Intracellular Protease Inhibitor (IPI protein from Bacillus subtilis, which has been reported to be an inhibitor of the intracellular subtilisin Isp1 from the same organism. The structure of IPI is a variant of the all-beta, immunoglobulin (Ig fold. It is possible that IPI is important for protein-protein interactions, of which inhibition of Isp1 is one. The intracellular nature of ISP is questioned, because an alternative ATG codon in the ipi gene would produce a protein with an N-terminal extension containing a signal peptide. It is possible that alternative initiation exists, producing either an intracellular inhibitor or a secreted form that may be associated with the cell surface.  Homologues of the IPI protein from other species are multi-domain proteins, containing signal peptides and domains also associated with the bacterial cell-surface. The cysteine peptidase inhibitors chagasin and amoebiasin also have Ig-like folds, but their topology differs significantly from that of IPI, and they share no recent common ancestor. A model of IPI docked to Isp1 shows similarities to other subtilisin:inhibitor complexes, particularly where the inhibitor interacts with the peptidase active site.

  13. Real-world evaluation of glycemic control among patients with type 2 diabetes mellitus treated with canagliflozin versus dipeptidyl peptidase-4 inhibitors.

    Science.gov (United States)

    Thayer, Sarah; Chow, Wing; Korrer, Stephanie; Aguilar, Richard

    2016-06-01

    Objective To evaluate glycemic control among patients with type 2 diabetes mellitus (T2DM) treated with canagliflozin (CANA) vs. dipeptidyl peptidase-4 (DPP-4) inhibitors. Methods Using integrated claims and lab data from a US health plan of commercial and Medicare Advantage enrollees, this matched-control cohort study assessed adult T2DM patients receiving treatment with CANA or DPP-4 inhibitors (1 April 2013-31 December 2013). Cohorts were chosen hierarchically; the first pharmacy claim for CANA was identified as the index date; then the first pharmacy claim for a DPP-4 inhibitor was identified and index date set. Eligible patients had 6 months of continuous health plan enrollment before the index date (baseline) and 9 months after (follow-up) and no evidence of index drug in baseline. Patients were matched 1:1 using propensity score matching. Changes in glycated hemoglobin (HbA1c) and percentages of patients with HbA1c <8% and <7% during the follow-up were evaluated. Results The matched CANA and DPP-4 inhibitor cohorts (53.2% treated with sitagliptin) included 2766 patients each (mean age: 55.7 years). Among patients with baseline and follow-up HbA1c results, mean baseline HbA1c values were similar, 8.62% and 8.57% (p = 0.615) for the CANA (n = 729) and DPP-4 inhibitor (n = 710) cohorts, respectively. Change in HbA1c was greater among patients in the CANA cohort than for those in the DPP-4 inhibitor cohort (-0.92% vs. -0.63%, p < 0.001), and also among the subset of patients with baseline HbA1c ≥7% (-1.07% [n = 624] vs. -0.79% [n = 603], p = 0.004). During follow-up, greater percentages of the CANA cohort relative to the DPP-4 inhibitor cohort achieved HbA1c of <8% (66.0% vs. 58.6%, p = 0.004) and <7% (35.4% vs. 29.9%, p = 0.022). Limitations This study was observational and residual confounding remains a possibility. Conclusions In this real-world study of patients with T2DM, CANA use was associated with greater HbA1c

  14. Efficacy and safety of dipeptidyl peptidase-4 inhibitors in type 2 diabetes mellitus patients with moderate to severe renal impairment: a systematic review and meta-analysis.

    Directory of Open Access Journals (Sweden)

    Dongsheng Cheng

    Full Text Available To perform a systematic review and meta-analysis regarding the efficacy and safety of dipeptidyl peptidase-4 (DDP-4 inhibitors ("gliptins" for the treatment of type 2 diabetes mellitus (T2DM patients with moderate to severe renal impairment.All available randomized-controlled trials (RCTs that assessed the efficacy and safety of DDP-4 inhibitors compared with placebo, no treatment, or active drugs were identified using PubMed, EMBASE, Cochrane CENTRAL, conference abstracts, clinical trials.gov, pharmaceutical company websites, the FDA, and the EMA (up to June 2014. Two independent reviewers extracted the data, and a random-effects model was applied to estimate summary effects.Thirteen reports of ten studies with a total of 1,915 participants were included in the final analysis. Compared with placebo or no treatment, DPP-4 inhibitors reduced HbA1c significantly (-0.52%, 95%CI -0.64 to -0.39 and had no increased risk of hypoglycemia (RR 1.10, 95%CI 0.92 to 1.32 or weight gain. In contrast to glipizide monotherapy, DPP-4 inhibitors showed no difference in HbA1c lowering effect (-0.08%, 95% CI -0.40 to 0.25 but had a lower incidence of hypoglycemia (RR 0.40, 95%CI 0.23 to 0.69. Furthermore, DPP-4 inhibitors were well-tolerated, without any additional mortality and adverse events. However, the quality of evidence was mostly as low, as assessed using the GRADE system for each outcome.DPP-4 inhibitors are effective at lowering HbA1c in T2DM patients with moderate to severe renal impairment. DPP-4 inhibitors also have a potential advantage in lowering the risk of adverse events. Regarding the low quality of the evidence according to GRADE, additional well-designed randomized trials that focus on the safety and efficacy of DPP-4 inhibitors in various CKD stages are needed urgently.

  15. Dipeptidyl peptidase-IV inhibitors are efficient adjunct therapy in HNF1A maturity-onset diabetes of the young patients--report of two cases.

    Science.gov (United States)

    Katra, Barbara; Klupa, Tomasz; Skupien, Jan; Szopa, Magdalena; Nowak, Natalia; Borowiec, Maciej; Kozek, Elzbieta; Malecki, Maciej T

    2010-04-01

    In HNF1A maturity-onset diabetes of the young (MODY), sulfonylurea (SU) is the first-line treatment. Over time, such therapy fails, and additional treatment is required. Dipeptidyl peptidase IV (DPP-IV) inhibitors are new agents that lower blood glucose by prolonging the activity of circulating incretins. We applied DPP-IV inhibitors in two HNF1A MODY patients whose earlier therapeutic regimen included SU. Case 1, a 39-year-old woman, a carrier of the ArgR171X HNF1A mutation, with a 7-year history of diabetes was on 160 mg of gliclazide and 2,000 mg of metformin. Her initial hemoglobin A1c (HbA1c) level was 7.2%, while the mean glucose level on the CGMS((R)) (Medtronic, Northridge, CA) record was 162 mg/dL. Sitagliptine, in a dose of 100 mg/day, was added to the previous treatment. Case 2, a 62-year-old woman, a carrier of the IVS7nt-6G>A mutation, with a 41-year history of diabetes was treated with 240 mg/day gliclazide and 6 IU of insulin/day. Her initial HbA1c was 8.8%, and average glycemia reached 172 mg/dL. In her case, we started the combined therapy with 50 mg of vildagliptine twice daily. Patients were reexamined after 3 months, and HbA1c fell to 6.3% in both subjects. Similarly, significant improvement in glycemic control on CGMS was observed as the average glycemia decreased to 114 mg/dL and 134 mg/dL in Case 1 and Case 2, respectively. No episodes of hypoglycemia or other side effects were recorded. As intravenous glucose tolerance tests (IVGTTs) were performed before and after DPP-IV implementation, we were able to assess their impact on insulin secretion under fasting conditions. We saw a substantial rise in insulin level increment during IVGTT (by 9.8 and13.4 mIU/L in Case 1 and Case 2, respectively). DPP-IV inhibitors may be an effective tool of combined therapy in HNF1A MODY, and they seem to improve beta-cell function under fasting conditions.

  16. Treatment of both native and deamidated gluten peptides with an endo-peptidase from Aspergillus niger prevents stimulation of gut-derived gluten-reactive T cells from either children or adults with celiac disease

    DEFF Research Database (Denmark)

    Toft-Hansen, Henrik; Rasmussen, Karina Søndergård; Nielsen, Anne Staal

    2014-01-01

    Celiac disease (CD) is characterized by an inappropriate immunological reaction against gluten driven by gluten-specific CD4+ T cells. We screened 25 proteases and tested 10 for their potential to degrade gluten in vitro. Five proteases were further tested for their ability to prevent...... the proliferative response by a gluten-specific CD4+ T cell clone and seven gluten-reactive T cell lines to protease-digested gluten peptides. A proline-specific endo-peptidase from Aspergillus niger (AnP2), was particularly efficient at diminishing proliferation after stimulation with cleaved antigen, and could...... completely block the response against both native and deamidated gluten peptides. We found that AnP2 was efficient down to a 1:64 protease:substrate ratio (w:w). When AnP2 was tested in assays using seven gluten-reactive T cell lines from individual CD patients (three adults and four children), the response...

  17. A locus on chromosome 20 encompassing genes that are highly expressed in the epididymis

    Institute of Scientific and Technical Information of China (English)

    (A)ke Lundwall

    2007-01-01

    During liquefaction of the ejaculate, the semen coagulum proteins semenogelin Ⅰ (SEMG1) and semenogelin Ⅱ (SEMG2) are degraded to low molecular mass fragments by kallikrein-related peptidase 3 (KLK3), also known as prostate-specific antigen. Semenogelin molecules initiate their own destruction by chelating Zn2+ that normally would completely inhibit the proteolytic activity of KLK3. In a similar way, semenogelins might regulate the activity of kallikrein-related peptidases in the epididymis, something that might be of importance for the maturation of spermatozoa or generation of anti-bacterial peptides. Studies on the evolution of semen coagulum proteins have revealed that most of them carry an exon that displays a rapid and unusual evolution. As a consequence, homologous proteins in rodents and primates show almost no conservation in primary structure. Further studies on their evolution suggest that the progenitor of the semen coagulum proteins probably was a protease inhibitor that might have displayed antimicrobial activity. The semenogelin locus on chromosome 20 contains at least 17 homologous genes encoding probable protease inhibitors with homology to semen coagulum proteins. All of these are highly expressed in the epididymis where they, similar to the semenogelins, could affect the maturation of spermatozoa or display antibacterial properties.

  18. Relationship between Plasma Serine Peptidase Inhibitor and Calcium of Dairy Cows during Transition Period%丝氨酸蛋白抑制剂与围产期奶牛血钙的关系

    Institute of Scientific and Technical Information of China (English)

    舒适; 夏成; 徐闯; 张洪友; 吴凌; 郑家三; 杨龙蛟

    2012-01-01

    This study investigated relationship between plasma serine peptidase inhibitor (SERPIN) and calcium of dairy cows in transition period. At calving, days 14-7 before calving, and days after calving the dairy cows were chosen and divided into hypocaleaemia group { 〈 2.20 mmol/L, T} and normal group (2.20-3.5 mmol/L, C}, 3 each group. Plasma SERPIN abundance were measured using western blotting. Results showed that abundance of plasma SERPIN was upregulated in plasma of dairy cows with hypocal- caemia. Conclusion obtained that we first confirm that there is a negative association of SERPIN and calci- um in plasma, which implies that calcium may regulate blood coagulation and immune function of dairy cows by SERPIN pathway.%本研究旨在了解围产期低血钙奶牛与正常奶牛的血浆Serine peptidase inhibitor(SERPIN)与血钙之间的关系。在分娩当天、产前14~7d和产后7~14d根据血钙浓度将实验牛分为正常对照组(2.20~3.5mmol/L,C)和低血钙组(〈2.20mmol/L,T),每组3头。利用Westernblotting检测试验牛血浆SERPIN丰度。结果显示:SPI蛋白在奶牛发生低血钙时,表达上调。结论:本试验首次证明了血浆SERPIN与血钙浓度呈负关联,血钙可能通过SERPIN途径调节机体的凝血过程和免疫功能。

  19. (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

    Energy Technology Data Exchange (ETDEWEB)

    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. (Merck)

    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.

  20. Neuroprotective effects of NAAG peptidase inhibition on the treatment of traumatic brain injury%脑损伤后N-乙酰天冬氨酰谷氨酸肽酶抑制的神经保护

    Institute of Scientific and Technical Information of China (English)

    钟春龙; 江基尧; 罗其中; 赵学仁; Bruce G.Lyeth

    2009-01-01

    Objective To explore the neuroprotective effects of NAAG peptidase inhibition on the treatment of traumatic brain injury.Method A total of 20 male Sprague-Dawley rats were divided into 4 groups,then sustained to moderate lateral fluid percussion TBI.A novel NAAG peptidase inhibitor,ZJ-43,was used in4 different doses (0,50,100,or 150 mg/kg).Each dose was repeatedly administered i.p.(n=5/group) by multiple injections at 3 times (0 h,8 h,16 h) after TBI.Rats were euthanized at 24 hrs after TBI and brains were processed and stained according to the Fluoro-Jade B Histofluorescence and GFAP Immunohistochemistry protocols,lpsilateral neuronal degeneration and bilateral astrocyte loss in the CA2/3 regions of the hippocampus were quantified using stereological techniques.Results 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,50 mg/kg of ZJ-43 also significantly reduced the ipsilateral astrocyte loss (P<0.05 ).Conclusions Blockade of NAAG peptidases may augment an endogenous neuroprotective mechanism in the brain and limit glutamate excitotoxicity.The strategy may represent a new therapeutic approach to secondary brain damage after traumatic brain injury.%目的 探索颅脑损伤后N-乙酰天冬氨酰谷氨酸(NAAG)肽酶抑制的神经保护效应.方法 将20只SD大鼠随机分成4组后制作中度液压颅脑损伤模型.伤后3个时间点(即时,8 h,16 h)分别经腹腔注射新型NAAG肽酶抑制剂ZJ-43(各组剂量:0,50,100,150 mg/kg).伤后24 h处死动物取脑组织行Fluoro-Jade B组织荧光染色以及GFAP免疫组化染色.通过立体细胞计数着重观察大鼠海马CA2/3区退变神经元及正常胶质细胞的数量变化.结果 ZJ-43能显著减少颅脑损伤后的神经元退变(P<0.01),其中小剂量处理组(50 mg/kg)保护作用最佳,还能同时显著减少儋颅脑损伤后的胶质细胞丢失(P<0.05).

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

    Science.gov (United States)

    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.

  2. Changes in glucose-induced plasma active glucagon-like peptide-1 levels by co-administration of sodium–glucose cotransporter inhibitors with dipeptidyl peptidase-4 inhibitors in rodents

    Directory of Open Access Journals (Sweden)

    Takahiro Oguma

    2016-12-01

    Full Text Available We investigated whether structurally different sodium–glucose cotransporter (SGLT 2 inhibitors, when co-administered with dipeptidyl peptidase-4 (DPP4 inhibitors, could enhance glucagon-like peptide-1 (GLP-1 secretion during oral glucose tolerance tests (OGTTs in rodents. Three different SGLT inhibitors—1-(β-d-Glucopyranosyl-4-chloro-3-[5-(6-fluoro-2-pyridyl-2-thienylmethyl]benzene (GTB, TA-1887, and canagliflozin—were examined to assess the effect of chemical structure. Oral treatment with GTB plus a DPP4 inhibitor enhanced glucose-induced plasma active GLP-1 (aGLP-1 elevation and suppressed glucose excursions in both normal and diabetic rodents. In DPP4-deficient rats, GTB enhanced glucose-induced aGLP-1 elevation without affecting the basal level, whereas metformin, previously reported to enhance GLP-1 secretion, increased both the basal level and glucose-induced elevation. Oral treatment with canagliflozin and TA-1887 also enhanced glucose-induced aGLP-1 elevation when co-administered with either teneligliptin or sitagliptin. These data suggest that structurally different SGLT2 inhibitors enhance plasma aGLP-1 elevation and suppress glucose excursions during OGTT when co-administered with DPP4 inhibitors, regardless of the difference in chemical structure. Combination treatment with DPP4 inhibitors and SGLT2 inhibitors having moderate SGLT1 inhibitory activity may be a promising therapeutic option for improving glycemic control in patients with type 2 diabetes mellitus.

  3. Is the Inhibition of Dipeptidyl Peptidase-4 (DDP-4) Enzyme Route Dependent and/or Driven by High Peak Concentration?- Seeking Answers with ZYDPLA1, a Novel Long Acting DPP-4 Inhibitor, in a Rodent Model.

    Science.gov (United States)

    Patel, Harilal; Joharapurkar, Amit A; Bahekar, Rajesh; Patel, Prakash; Kshirsagar, Samadhan G; Modi, Nirav; Ghoghari, Ashok; Patel, Vishal J; Jain, Mukul R; Srinivas, Nuggehally R; Patel, Pankaj R; Desai, Ranjit C

    2017-04-01

    ZYDPLA1 is a long acting enzyme dipeptidyl peptidase-4 (DPP-4) inhibitor. The comparative effect of DPP-4 inhibition after intravenous (IV) and oral administration of ZYDPLA1 in a rat model was evaluated to answer the question of route dependency and/or the need of high plasma levels of ZYDPLA1. The study was conducted using parallel design in male Wistar rats for IV/oral route (n=9 and 6, for IV and oral respectively). A single 30 mg/kg dose of ZYDPLA1 was administered. Plasma samples were analysed for ZYDPLA1 concentration and DPP-4 inhibition. Pharmacokinetic analysis was carried out to assess peak concentration, area under the concentration-time curve, total body clearance, elimination half-life, and mean residence time. The PK/PD correlation was performed using standard sigmoidal Emax modelling to derive; maximum effect (Emax) and concentration to exert 50% Emax effect (EC50). ZYDPLA1 showed rapid absorption, high volume of distribution, low clearance, and complete oral bioavailability. The Emax derived after both routes and corresponding PK/PD profile showed comparable DDP-4 inhibition. The EC50 for IV (0.021 µg/mL) was comparable to the oral route (0.019 µg/mL). ZYDPLA1 showed full DPP-4 inhibition without regard to the route of administration. Higher systemic peak levels showed no bearing on the DDP-4 inhibition. © Georg Thieme Verlag KG Stuttgart · New York.

  4. Combined treatment with dipeptidyl peptidase 4 (DPP4) inhibitor sitagliptin and elemental diets reduced indomethacin-induced intestinal injury in rats via the increase of mucosal glucagon-like peptide-2 concentration.

    Science.gov (United States)

    Fujiwara, Kaori; Inoue, Takuya; Yorifuji, Naoki; Iguchi, Munetaka; Sakanaka, Taisuke; Narabayashi, Ken; Kakimoto, Kazuki; Nouda, Sadaharu; Okada, Toshihiko; Ishida, Kumi; Abe, Yosuke; Masuda, Daisuke; Takeuchi, Toshihisa; Fukunishi, Shinya; Umegaki, Eiji; Akiba, Yasutada; Kaunitz, Jonathan D; Higuchi, Kazuhide

    2015-03-01

    The gut incretin glucagon-like peptide-1 (GLP-1) and the intestinotropic hormone GLP-2 are released from enteroendocrine L cells in response to ingested nutrients. Treatment with an exogenous GLP-2 analogue increases intestinal villous mass and prevents intestinal injury. Since GLP-2 is rapidly degraded by dipeptidyl peptidase 4 (DPP4), DPP4 inhibition may be an effective treatment for intestinal ulcers. We measured mRNA expression and DPP enzymatic activity in intestinal segments. Mucosal DPP activity and GLP concentrations were measured after administration of the DPP4 inhibitor sitagliptin (STG). Small intestinal ulcers were induced by indomethacin (IM) injection. STG was given before IM treatment, or orally administered after IM treatment with or without an elemental diet (ED). DPP4 mRNA expression and enzymatic activity were high in the jejunum and ileum. STG dose-dependently suppressed ileal mucosal enzyme activity. Treatment with STG prior to IM reduced small intestinal ulcer scores. Combined treatment with STG and ED accelerated intestinal ulcer healing, accompanied by increased mucosal GLP-2 concentrations. The reduction of ulcers by ED and STG was reversed by co-administration of the GLP-2 receptor antagonist. DPP4 inhibition combined with luminal nutrients, which up-regulate mucosal concentrations of GLP-2, may be an effective therapy for the treatment of small intestinal ulcers.

  5. Treatment of both native and deamidated gluten peptides with an endo-peptidase from Aspergillus niger prevents stimulation of gut-derived gluten-reactive T cells from either children or adults with celiac disease.

    Science.gov (United States)

    Toft-Hansen, Henrik; Rasmussen, Karina S; Staal, Anne; Roggen, Erwin L; Sollid, Ludvig M; Lillevang, Søren T; Barington, Torben; Husby, Steffen

    2014-08-01

    Celiac disease (CD) is characterized by an inappropriate immunological reaction against gluten driven by gluten-specific CD4+ T cells. We screened 25 proteases and tested 10 for their potential to degrade gluten in vitro. Five proteases were further tested for their ability to prevent the proliferative response by a gluten-specific CD4+ T cell clone and seven gluten-reactive T cell lines to protease-digested gluten peptides. A proline-specific endo-peptidase from Aspergillus niger (AnP2) was particularly efficient at diminishing proliferation after stimulation with cleaved antigen, and could completely block the response against both native and deamidated gluten peptides. We found that AnP2 was efficient down to a 1:64 protease:substrate ratio (w:w). When AnP2 was tested in assays using seven gluten-reactive T cell lines from individual CD patients (three adults and four children), the response to gluten was diminished in all cases. Our study indicates a therapeutic benefit of AnP2 to CD patients.

  6. 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).

    Science.gov (United States)

    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.

  7. 二肽基肽酶IV抑制剂在2型糖尿病中治疗的研究进展%Review of Dipeptidyl Peptidase IV Inhibitors in the Treatment of Type 2 Diabetes Mellitus

    Institute of Scientific and Technical Information of China (English)

    黎健南; 黄汉伟

    2015-01-01

    Dipeptide peptidase IV (DPP - 4) inhibitors as a novel oral hypoglycemic agents , glucose-dependent mechanism of action based on incretin , can reduce the overall three key indicators (fasting blood glucose, postprandial blood glucose and glycosylated hemoglobin) , while reducing the risk of hypoglycemia and weight gain , with the protection of islet function , may delay disease progression , good safety and other therapeutic advantage .In this article the pharmacodynamic , pharmacokinetic characteristics and security of DPP 4 inhibitors were reviewed.%二肽基肽酶IV(DPP-4)抑制剂作为一种新型口服降糖药,其作用机理基于葡萄糖依赖性肠促胰素,能全面降低空腹血糖、餐后血糖和糖化血红蛋白(HbAlc),同时减少低血糖发生和体重增加的风险,具有保护胰岛β细胞、可能延缓疾病进程、安全性良好等治疗优势。本文对DPP-4抑制剂的药效学、药代动力学特点及安全性做一综述。

  8. Changes in glucose-induced plasma active glucagon-like peptide-1 levels by co-administration of sodium-glucose cotransporter inhibitors with dipeptidyl peptidase-4 inhibitors in rodents.

    Science.gov (United States)

    Oguma, Takahiro; Kuriyama, Chiaki; Nakayama, Keiko; Matsushita, Yasuaki; Hikida, Kumiko; Tsuda-Tsukimoto, Minoru; Saito, Akira; Arakawa, Kenji; Ueta, Kiichiro; Minami, Masabumi; Shiotani, Masaharu

    2016-12-01

    We investigated whether structurally different sodium-glucose cotransporter (SGLT) 2 inhibitors, when co-administered with dipeptidyl peptidase-4 (DPP4) inhibitors, could enhance glucagon-like peptide-1 (GLP-1) secretion during oral glucose tolerance tests (OGTTs) in rodents. Three different SGLT inhibitors-1-(β-d-Glucopyranosyl)-4-chloro-3-[5-(6-fluoro-2-pyridyl)-2-thienylmethyl]benzene (GTB), TA-1887, and canagliflozin-were examined to assess the effect of chemical structure. Oral treatment with GTB plus a DPP4 inhibitor enhanced glucose-induced plasma active GLP-1 (aGLP-1) elevation and suppressed glucose excursions in both normal and diabetic rodents. In DPP4-deficient rats, GTB enhanced glucose-induced aGLP-1 elevation without affecting the basal level, whereas metformin, previously reported to enhance GLP-1 secretion, increased both the basal level and glucose-induced elevation. Oral treatment with canagliflozin and TA-1887 also enhanced glucose-induced aGLP-1 elevation when co-administered with either teneligliptin or sitagliptin. These data suggest that structurally different SGLT2 inhibitors enhance plasma aGLP-1 elevation and suppress glucose excursions during OGTT when co-administered with DPP4 inhibitors, regardless of the difference in chemical structure. Combination treatment with DPP4 inhibitors and SGLT2 inhibitors having moderate SGLT1 inhibitory activity may be a promising therapeutic option for improving glycemic control in patients with type 2 diabetes mellitus. Copyright © 2016 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  9. The Role of DmCatD, a Cathepsin D-Like Peptidase, and Acid Phosphatase in the Process of Follicular Atresia in Dipetalogaster maxima (Hemiptera: Reduviidae), a Vector of Chagas' Disease

    Science.gov (United States)

    Leyria, Jimena; Fruttero, Leonardo L.; Nazar, Magalí; Canavoso, Lilián E.

    2015-01-01

    In this work, we have investigated the involvement of DmCatD, a cathepsin D-like peptidase, and acid phosphatase in the process of follicular atresia of Dipetalogaster maxima, a hematophagous insect vector of Chagas’ disease. For the studies, fat bodies, ovaries and hemolymph were sampled from anautogenous females at representative days of the reproductive cycle: pre-vitellogenesis, vitellogenesis as well as early and late atresia. Real time PCR (qPCR) and western blot assays showed that DmCatD was expressed in fat bodies and ovaries at all reproductive stages, being the expression of its active form significantly higher at the atretic stages. In hemolymph samples, only the immunoreactive band compatible with pro-DmCatD was observed by western blot. Acid phosphatase activity in ovarian tissues significantly increased during follicular atresia in comparison to pre-vitellogenesis and vitellogenesis. A further enzyme characterization with inhibitors showed that the high levels of acid phosphatase activity in atretic ovaries corresponded mainly to a tyrosine phosphatase. Immunofluorescence assays demonstrated that DmCatD and tyrosine phosphatase were associated with yolk bodies in vitellogenic follicles, while in atretic stages they displayed a different cellular distribution. DmCatD and tyrosine phosphatase partially co-localized with vitellin. Moreover, their interaction was supported by FRET analysis. In vitro assays using homogenates of atretic ovaries as the enzyme source and enzyme inhibitors demonstrated that DmCatD, together with a tyrosine phosphatase, were necessary to promote the degradation of vitellin. Taken together, the results strongly suggested that both acid hydrolases play a central role in early vitellin proteolysis during the process of follicular atresia. PMID:26091289

  10. Gemfibrozil and fenofibrate, Food and Drug Administration-approved lipid-lowering drugs, up-regulate tripeptidyl-peptidase 1 in brain cells via peroxisome proliferator-activated receptor α: implications for late infantile Batten disease therapy.

    Science.gov (United States)

    Ghosh, Arunava; Corbett, Grant T; Gonzalez, Frank J; Pahan, Kalipada

    2012-11-09

    The classical late infantile neuronal ceroid lipofuscinosis (LINCLs) is an autosomal recessive disease, where the defective gene is Cln2, encoding tripeptidyl-peptidase I (TPP1). At the molecular level, LINCL is caused by accumulation of autofluorescent storage materials in neurons and other cell types. Currently, there is no established treatment for this fatal disease. This study reveals a novel use of gemfibrozil and fenofibrate, Food and Drug Administration-approved lipid-lowering drugs, in up-regulating TPP1 in brain cells. Both gemfibrozil and fenofibrate up-regulated mRNA, protein, and enzymatic activity of TPP1 in primary mouse neurons and astrocytes as well as human astrocytes and neuronal cells. Because gemfibrozil and fenofibrate are known to activate peroxisome proliferator-activated receptor-α (PPARα), the role of PPARα in gemfibrozil- and fenofibrate-mediated up-regulation of TPP1 was investigated revealing that both drugs up-regulated TPP1 mRNA, protein, and enzymatic activity both in vitro and in vivo in wild type (WT) and PPARβ(-/-), but not PPARα(-/-), mice. In an attempt to delineate the mechanism of TPP1 up-regulation, it was found that the effects of the fibrate drugs were abrogated in the absence of retinoid X receptor-α (RXRα), a molecule known to form a heterodimer with PPARα. Accordingly, all-trans-retinoic acid, alone or together with gemfibrozil, up-regulated TPP1. Co-immunoprecipitation and ChIP studies revealed the formation of a PPARα/RXRα heterodimer and binding of the heterodimer to an RXR-binding site on the Cln2 promoter. Together, this study demonstrates a unique mechanism for the up-regulation of TPP1 by fibrate drugs via PPARα/RXRα pathway.

  11. Correlation between ubiquitin specific peptidase 7 expression and male infertility%泛素特异性蛋白酶7的表达与男性不育的相关性

    Institute of Scientific and Technical Information of China (English)

    王美姣; 于廷和; 胡丽娜; 程志; 李敏; 郭倩文

    2016-01-01

    目的 探讨弱精子症(asthenozoospermia)和少弱精子症(oligoasthenozoospermia)患者精子中泛素特异性蛋白酶7(ubiquitin specific peptidase 7,USP7)的表达及其与男性不育的相关性.方法 随机选取就诊于重庆医科大学附属第二医院妇产科生殖医学中心的不育症患者120例,根据2010年世界卫生组织标准进行精液参数常规分析,包括精子浓度和运动能力,分为弱精子症组(A组)37例、少弱精子症组(OA组)26例和正常精子(normozoospcrmia)组(N组)57例.采用免疫荧光染色和蛋白质印迹法检测USP7在人精子中的定位与表达变化,并进行相关性分析.结果 USP7定位于精子尾部中段和主段,与N组(1.63±0.76)相比,在A组(0.86±0.53)或OA组(0.62±0.43)精子中USP7蛋白的表达下降(P<0.01).经相关分析,USP7蛋白水平与精子浓度(r=0.455 0,P=0.005 3)、前向运动精子百分率(r=0.431 2,P=0.008 7)和总活动率(r=0.443 8,P=0.006 7)呈正相关.结论 USP7表达下降与人精子活力和浓度降低有关,其可作为评估男性生育力的指标之一.

  12. 二肽基肽酶-4抑制剂对成人隐匿性自身免疫糖尿病早期患者的治疗价值及药学评估与监护%Therapeutic value, pharmaceutical assessment and monitoring of dipeptidyl peptidase-4 inhibitor in early latent autoimmune diabetes adult patients

    Institute of Scientific and Technical Information of China (English)

    陈红利; 邓昂

    2016-01-01

    目的:探讨二肽基肽酶-4(dipeptidyl peptidase-4, DPP-4)抑制剂对于LADA早期患者的治疗价值及药学评估与监护。方法:对1例LADA早期患者的病例进行分析,该患者糖尿病诊断8个月,此次为调整血糖,优化用药方案入院。结果:经过药学评估,分析该患者使用DPP-4抑制剂的风险与获益,该患者无DPP-4抑制剂使用的禁忌,给予磷酸西格列片100 mg每日1次经口服治疗后,患者血糖平稳达标出院。结论:对于早期LADA患者用药前进行药学评估及用药教育,用药后加强对患者的药学监护,可以提高患者用药依从性,降低用药风险。%Objective:To explore the value of Dipeptidyl peptidase-4 inhibitor in early-stage LADA patients as well as its pharmaceutical assessment and monitoring.Methods: As it is showed in one case, the early-stage LADA patient was diagnosed with diabetes for eight months. We chose to optimize the medication scheme to help regulate blood sugar. By analyzing risks and proifts of the usage of dipeptidyl peptidase-4 inhibitor, the patient didn’t have any potential contraindications. We gave prescription and advice as follows: Oral take, 100 mg Sitagliptin Phosphate Tablets each time, once a day. The patient was discharged from hospital maintaining stable blood sugar level.Results and Conclusion: Before the usage of medication on early-stage LADA patients, we must take pharmaceutical assessment and medical education into consideration. After the usage of medication on early-stage LADA patients, we should pay attention to the pharmaceutical supervision of patients in order to raise their awareness and lower the risk of the usage of medication.

  13. Focal inhibitory interneuron loss and principal cell hyperexcitability in the rat hippocampus after microinjection of a neurotoxic conjugate of saporin and a peptidase-resistant analog of Substance P.

    Science.gov (United States)

    Martin, J L; Sloviter, R S

    2001-07-23

    Episodes of prolonged seizures or head trauma produce chronic hippocampal network hyperexcitability hypothesized to result primarily from inhibitory interneuron loss or dysfunction. The possibly causal role of inhibitory neuron failure in the development of epileptiform pathophysiology remains unclear because global neurologic injuries produce such a multitude of effects. The recent finding that Substance P receptors (SPRs) are expressed exclusively in the rat hippocampus by inhibitory interneurons provided the rationale for attempting to ablate interneurons selectively by using neurotoxic conjugates of SPR ligands and the ribosome inactivating protein saporin that specifically target Substance P receptor-expressing cells. Whereas intrahippocampal microinjection of a conjugate of native SP and saporin produced significant nonspecific damage at concentrations needed to produce even limited selective loss of SPR-positive cells, a conjugate of saporin and the more potent and peptidase-resistant SP analog [Sar(9), Met(O(2))(11)] Substance P (SSP-saporin) caused negligible nonspecific damage at the injection site, and a virtually complete loss of SPR-like immunoreactivity (LI) up to 1 mm from the injection site. Within the SPR depletion zone, immunoreactivities for most GABA-, parvalbumin-, somatostatin-, and cholecystokinin-immunoreactive cells and fibers were eliminated. The few interneurons detectable within the affected zone were devoid of SPR-LI. The apparent loss of interneurons was selective in that calbindin- and glutamate receptor subunit 2 (GluR2) -positive principal cells survived within the affected zone, as did myelinated fibers and the extrinsic calretinin- and tyrosine hydroxylase--immunoreactive terminals of subcortical afferents. An apparent lack of reactive synaptic reorganization in response to interneuron loss was indicated by zinc transporter-3 (ZnT3)-- and beta-synuclein--LI, as well as by Timm staining, all of which revealed relatively normal

  14. Dipeptidyl-peptidase (DPP)-4 inhibitors and glucagon-like peptide (GLP)-1 analogues for prevention or delay of type 2 diabetes mellitus and its associated complications in people at increased risk for the development of type 2 diabetes mellitus.

    Science.gov (United States)

    Hemmingsen, Bianca; Sonne, David P; Metzendorf, Maria-Inti; Richter, Bernd

    2017-05-10

    The projected rise in the incidence of type 2 diabetes mellitus (T2DM) could develop into a substantial health problem worldwide. Whether dipeptidyl-peptidase (DPP)-4 inhibitors or glucagon-like peptide (GLP)-1 analogues are able to prevent or delay T2DM and its associated complications in people at risk for the development of T2DM is unknown. To assess the effects of DPP-4 inhibitors and GLP-1 analogues on the prevention or delay of T2DM and its associated complications in people with impaired glucose tolerance, impaired fasting blood glucose, moderately elevated glycosylated haemoglobin A1c (HbA1c) or any combination of these. We searched the Cochrane Central Register of Controlled Trials; MEDLINE; PubMed; Embase; ClinicalTrials.gov; the World Health Organization (WHO) International Clinical Trials Registry Platform; and the reference lists of systematic reviews, articles and health technology assessment reports. We asked investigators of the included trials for information about additional trials. The date of the last search of all databases was January 2017. We included randomised controlled trials (RCTs) with a duration of 12 weeks or more comparing DPP-4 inhibitors and GLP-1 analogues with any pharmacological glucose-lowering intervention, behaviour-changing intervention, placebo or no intervention in people with impaired fasting glucose, impaired glucose tolerance, moderately elevated HbA1c or combinations of these. Two review authors read all abstracts and full-text articles and records, assessed quality and extracted outcome data independently. One review author extracted data which were checked by a second review author. We resolved discrepancies by consensus or the involvement of a third review author. For meta-analyses, we planned to use a random-effects model with investigation of risk ratios (RRs) for dichotomous outcomes and mean differences (MDs) for continuous outcomes, using 95% confidence intervals (CIs) for effect estimates. We assessed the

  15. Cardioprotective effects of glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors%GLP-1受体激动剂和DPP-4抑制剂对心血管系统的保护作用

    Institute of Scientific and Technical Information of China (English)

    张雪松; 秦明照

    2013-01-01

      胰高糖素样肽1(GLP-1)受体激动剂和二肽基肽酶-4( DPP-4)抑制剂是近年上市的治疗2型糖尿病的新型药物。GLP-1主要通过抑制心肌细胞凋亡、改善内皮细胞功能、减轻体重、降低血糖、降低血压、改善心肌收缩力、舒张血管等直接或间接发挥其心脏保护作用,GLP-1受体激动剂和DPP-4抑制剂对体重、血压和血脂等心血管危险因素有改善作用。GLP-1受体激动剂和DPP-4抑制剂作用于新靶点,具有其独特的优点,将会是一类抗糖尿病新药。%Glucagon-like Peptide-1 (GLP-1) receptor agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors are new kinds of drugs to treat type 2 diabetes mel itus. It was demonstrated that GLP-1 has an obvious cardioprotective effect mainly achieved by relaxing vascular vessels, improving endothelial function, improving endothelial function and promoting the functional recovery of pathological ventricle, which can directly or indirectly protect the heart. GLP-1 receptor agonists and DPP4 inhibitors also showed important effects on the body weight, blood pressure, lipids, and other cardiovascular risk factors. GLP-1 receptor agonists and DPP4 inhibitors have more advantages than other anti-diabetes drugs because they act on a new target. They wil be a useful new class of anti-diabetes drugs.

  16. Comparison of glucagon-like peptide 1 receptor agonists and dipeptidyl peptidase 4 inhibitors in the treatment of type 2 diabetes mettilus%GLP-1受体激动剂与二肽基肽酶4抑制剂治疗2型糖尿病的比较

    Institute of Scientific and Technical Information of China (English)

    骆天红

    2012-01-01

    随着对肠促胰素在维持葡萄糖稳定作用认识的日益增多,促进了针对2型糖尿病患者肠促胰素活性缺乏治疗药物的研发.根据肠促胰素治疗药物不同的作用机制,可分为以下2类:(1)胰升糖素样肽1(GLP-1)受体激动剂,包括利拉鲁肽(liraglutide)、艾塞那肽每日2次制剂和艾塞那肽每周1次制剂;(2)二肽基肽酶4(DPP-4)抑制剂,包括西格列汀(sitagliptin)、利拉利汀(linagliptin)、沙格列汀(saxagliptin)和维格列汀(vildagliptin),DPP-4抑制剂可限制内源性GLP-1的降解.这2类药物具有某些共性,如葡萄糖依赖性刺激胰岛素分泌,低血糖发生率低.然而这2类药物在疗效方面的表现却有所不同.本文综述了这2类基于肠促胰素治疗药物的药代动力学及其临床方面的疗效和安全性,阐明了此类药物在2型糖尿病治疗中的地位.%Increased understanding of the role of incretin hormones in maintaining glucose homeostasis has enabled the development of pharmacotherapies that target deficient incretin activity in type 2 diabetes mellitus.lncretin-based therapies could be classified into 2 categories with different mechanisms of action:glucagon-like peptide 1 ( GLP-1 ) receptor agonists ( e.g.liraglutide,exenatide twice daily,and exenatide once weekly),and the dipeptidyl peptidase 4 ( DPP-4 ) inhibitors ( e.g.sitagliptin,linagliptin,saxagliptin,and vildagliptin),which inhibit the breakdown of endogenous GLP-1.Both categories share some specific characteristics,such as glucose-dependent insulin stimulation and low incidence of hypoglycemia.But there are also different profiles in efficacy between these 2 classes.This article reviews the pharmacokinetics and clinical aspects of efficacy and safety of these 2 classes of incretin-based therapies and elucidates their roles in treating type 2 diabetes mellitus.

  17. Cysteine peptidases and their inhibitors in breast and genital cancer.

    Directory of Open Access Journals (Sweden)

    Magdalena Milan

    2010-11-01

    Full Text Available Cysteine proteinases and their inhibitors probably play the main role in carcinogenesis and metastasis. The metastasis process need external proteolytic activities that pass several barriers which are membranous structures of the connective tissue which includes, the basement membrane of blood vessels. Activities of the proteinases are regulated by endogenous inhibitors and activators. The imbalance between cysteine proteinases and cystatins seems to be associated with an increase in metastatic potential in some tumors. It has also been reported that proteinase inhibitors, specific antibodies for these enzymes and inhibition of the urokinase receptor may prevent cancer cell invasion. Some proteinase inhibitor could serve as agents for cancer treatment.

  18. 新生儿巨细胞病毒感染的胞外体二肽基肽酶Ⅳ及血清中白细胞介素-6,-8水平的研究%Levels of Dipeptidyl Peptidase Ⅳ and Interleukin-6,-8 in Neonates of Cytomegalovirus Infection

    Institute of Scientific and Technical Information of China (English)

    李晓莺; 卢宪梅; 阎贝贝; 胡晓燕

    2012-01-01

    Objective To study the levels of dipeptidyl peptidase Ⅳ (DPPⅣ)of exosome(EXO) and interleukin(IL)-6,-8 in serum of neonates with cytomegalovirus(CMV) infection,to investigate the influence of CMV on neonates' immune function,and the roles of DPPⅣ,and IL-6,-8 in serum which played in the process of CMV infection.Methods From January 2008 to December 2010,a total of 53 neonates with CMV infection were collected as CMV group,meanwhile the other 52 non-CMV infection neonates were recruited as control group.By ELASA method to detect the levels of EXO-DPPⅣ and IL-6,-8 in serum,then analysis the differences between two groups.Informed consent was obtained from the parents of each participating neonate.There had no significant difference between two groups among gender,age,gestational age etc.(P>0.05).Results The levels of EXO-DPPⅣ,and IL-6,-8 in serum in CMV group were much higher than those in control group,and had significance difference (P<0.05).Conclusions CMV can break normal immune balance,leading abnormality of enzymes and cytokine.There is close correlation between inflammatory reaction,DPPⅣ,and IL-6,-8.%目的 探讨巨细胞病毒(CMV)感染对新生儿免疫功能的影响及胞外体(EXO)二肽基肽酶Ⅳ(DPPⅣ,CD26)及血清白细胞介素(IL)-6,-8水平在CMV感染发生发展过程中的作用.方法 选择2008年1月至2010年12月山东大学齐鲁医院儿科收治的53例CMV感染新生儿为研究对象,纳入CMV组,选择同期入院的42例非CMV感染新生儿(CMV IgM呈阴性,血、尿中CMV-DNA< 103拷贝/mL)纳入对照组.采用ELASA法检测EXO-DPPⅣ活性及血清IL-6,-8水平,并进行相关统计学分析(本研究遵循的程序符合山东大学齐鲁医院人体试验委员会制定的伦理学标准,得到该委员会批准,分组征得受试对象监护人的知情同意,并与其签署临床研究知情同意书).两组新生儿的性别、年龄及胎龄等比较,差异无统计学意义(P>0.05).结果 CMV组

  19. Efficacy Of Dipeptidyl Peptidase_4 Inhibitor Saxagliptin Combind With Insulin Therpy In Patients With Type 2 Diabetes%为应用胰岛素控制血糖不佳的2型糖尿病患者加用沙格列汀的疗效观察

    Institute of Scientific and Technical Information of China (English)

    朱凤; 宋晓燕

    2014-01-01

    The efficacy and safety of the dipeptidyl peptidase_4 inhibitor saxagliptin added to ongoing insulin therapy, were assessed in patients with type 2 diabetes who had inadequate glycemic control with insulin alone.Methods:The85 patients with type 2 diabetes were randomly devided into two groups,therpy group (42 cases)were given saxagliptin and insulin,and control group (43 cases) were given insulin only for 12 weeks.The changes of FPG、proinsulin-to-insulin ratio were observed in two groups(p<0.05).Result: Compared with pretherapy,the levels of FBG,2HPG,HbA1c in twe groups were obviously descendent(p<0.05),after 12 weeks,the insulin dosage of control group increased 28 U ,but that of therpy group did not changed greatly;26 patients in therpy group achieved the ideal and the success rate was 62%,21 patients in control group achieved the ideal and the success rate was 49%.Conclusion: Saxagliptin 5mg once-daily added to ongoing insulin therapy was efficacious in patients with type 2 diabetes.%观察为应用胰岛素控制血糖不佳的2型糖尿病患者加用沙格列汀进行治疗的疗效与安全性。方法:将我院收治的85例应用胰岛素进行治疗但血糖控制不佳的2型糖尿病患者随机分为对照组(43例)和治疗组(42例),为对照组患者增加胰岛素的用量进行治疗,为治疗组患者在使用胰岛素的基础上加用5mg的沙格列汀(口服)进行治疗,在用药12周后观察其临床疗效及发生不不良反应的情况。结果:两组患者治疗12周后的血糖均较治疗前有所下降,差异显著(p<0.05),有统计学意义。对照组患者在治疗12周后胰岛素的用量增加28U,治疗组患者在治疗12周后胰岛素的用量无明显改变。在治疗组患者中,有26例患者的血糖达标,达标率为62%。在对照组患者中,有21例患者的血糖达标,达标率为49%。结论:联合应用胰岛素和沙格列汀为2型糖尿病患者进行

  20. Mastering the canonical loop of serine protease inhibitors: enhancing potency by optimising the internal hydrogen bond network.

    Directory of Open Access Journals (Sweden)

    Joakim E Swedberg

    Full Text Available BACKGROUND: Canonical serine protease inhibitors commonly bind to their targets through a rigid loop stabilised by an internal hydrogen bond network and disulfide bond(s. The smallest of these is sunflower trypsin inhibitor (SFTI-1, a potent and broad-range protease inhibitor. Recently, we re-engineered the contact β-sheet of SFTI-1 to produce a selective inhibitor of kallikrein-related peptidase 4 (KLK4, a protease associated with prostate cancer progression. However, modifications in the binding loop to achieve specificity may compromise structural rigidity and prevent re-engineered inhibitors from reaching optimal binding affinity. METHODOLOGY/PRINCIPAL FINDINGS: In this study, the effect of amino acid substitutions on the internal hydrogen bonding network of SFTI were investigated using an in silico screen of inhibitor variants in complex with KLK4 or trypsin. Substitutions favouring internal hydrogen bond formation directly correlated with increased potency of inhibition in vitro. This produced a second generation inhibitor (SFTI-FCQR Asn(14 which displayed both a 125-fold increased capacity to inhibit KLK4 (K(i = 0.0386±0.0060 nM and enhanced selectivity over off-target serine proteases. Further, SFTI-FCQR Asn(14 was stable in cell culture and bioavailable in mice when administered by intraperitoneal perfusion. CONCLUSION/SIGNIFICANCE: These findings highlight the importance of conserving structural rigidity of the binding loop in addition to optimising protease/inhibitor contacts when re-engineering canonical serine protease inhibitors.

  1. Relationships between protein and mineral during enamel development in normal and genetically altered mice

    Science.gov (United States)

    Smith, Charles E.; Hu, Yuanyuan; Richardson, Amelia S.; Bartlett, John D.; Hu, Jan C-C.; Simmer, James P.

    2012-01-01

    The purpose of this study was to quantify and compare the amounts of volatiles (mostly protein) and mineral present in developing incisor enamel in normal mice and in those genetically engineered for absence of intact enamelin, ameloblastin, matrix metalloproteinase 20 (MMP20) or kallikrein-related peptidase 4 (KLK4). Data indicated that all mice showed peaks in the gross weight of volatiles and a similar weight of mineral at locations on incisors normally associated with early maturation. Thereafter, the content of volatiles on normal incisors declined rapidly by as much as 62%, but not by 100%, over 2 mm, accompanied by increases of ~threefold in mineral weights. Enamelin heterozygous mice (lower incisors) showed a decrease in volatile content across the maturation stage, yet mineral failed to increase significantly. Mmp20 null mice showed no significant loss of volatiles from maturing enamel, yet the amount of mineral increased. Klk4 null mice showed normal mineral acquisition up to early maturation, but the input of new volatiles in mid to late maturation caused the final mineralization to slow below normal levels. These results suggest that it is not only the amount of protein but also the nature or type of protein or fragments present in the local crystallite environment that affects their volumetric expansion as they mature. PMID:22243238

  2. Circadian rhythms regulate amelogenesis.

    Science.gov (United States)

    Zheng, Li; Seon, Yoon Ji; Mourão, Marcio A; Schnell, Santiago; Kim, Doohak; Harada, Hidemitsu; Papagerakis, Silvana; Papagerakis, Petros

    2013-07-01

    Ameloblasts, the cells responsible for making enamel, modify their morphological features in response to specialized functions necessary for synchronized ameloblast differentiation and enamel formation. Secretory and maturation ameloblasts are characterized by the expression of stage-specific genes which follows strictly controlled repetitive patterns. Circadian rhythms are recognized as key regulators of the development and diseases of many tissues including bone. Our aim was to gain novel insights on the role of clock genes in enamel formation and to explore the potential links between circadian rhythms and amelogenesis. Our data shows definitive evidence that the main clock genes (Bmal1, Clock, Per1 and Per2) oscillate in ameloblasts at regular circadian (24 h) intervals both at RNA and protein levels. This study also reveals that the two markers of ameloblast differentiation i.e. amelogenin (Amelx; a marker of secretory stage ameloblasts) and kallikrein-related peptidase 4 (Klk4, a marker of maturation stage ameloblasts) are downstream targets of clock genes. Both, Amelx and Klk4 show 24h oscillatory expression patterns and their expression levels are up-regulated after Bmal1 over-expression in HAT-7 ameloblast cells. Taken together, these data suggest that both the secretory and the maturation stages of amelogenesis might be under circadian control. Changes in clock gene expression patterns might result in significant alterations of enamel apposition and mineralization.

  3. Target gene analyses of 39 amelogenesis imperfecta kindreds.

    Science.gov (United States)

    Chan, Hui-Chen; Estrella, Ninna M R P; Milkovich, Rachel N; Kim, Jung-Wook; Simmer, James P; Hu, Jan C-C

    2011-12-01

    Previously, mutational analyses identified six disease-causing mutations in 24 amelogenesis imperfecta (AI) kindreds. We have since expanded the number of AI kindreds to 39, and performed mutation analyses covering the coding exons and adjoining intron sequences for the six proven AI candidate genes [amelogenin (AMELX), enamelin (ENAM), family with sequence similarity 83, member H (FAM83H), WD repeat containing domain 72 (WDR72), enamelysin (MMP20), and kallikrein-related peptidase 4 (KLK4)] and for ameloblastin (AMBN) (a suspected candidate gene). All four of the X-linked AI families (100%) had disease-causing mutations in AMELX, suggesting that AMELX is the only gene involved in the aetiology of X-linked AI. Eighteen families showed an autosomal-dominant pattern of inheritance. Disease-causing mutations were identified in 12 (67%): eight in FAM83H, and four in ENAM. No FAM83H coding-region or splice-junction mutations were identified in three probands with autosomal-dominant hypocalcification AI (ADHCAI), suggesting that a second gene may contribute to the aetiology of ADHCAI. Six families showed an autosomal-recessive pattern of inheritance, and disease-causing mutations were identified in three (50%): two in MMP20, and one in WDR72. No disease-causing mutations were found in 11 families with only one affected member. We conclude that mutation analyses of the current candidate genes for AI have about a 50% chance of identifying the disease-causing mutation in a given kindred.

  4. Identification of IGFBP-3 fragments generated by KLK2 and prevention of fragmentation by KLK2-inhibiting peptides.

    Science.gov (United States)

    Hekim, Can; Riipi, Tero; Weisell, Janne; Närvänen, Ale; Koistinen, Riitta; Stenman, Ulf-Håkan; Koistinen, Hannu

    2010-04-01

    Kallikrein-related peptidase 2 (KLK2) degrades insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) in vitro. IGFBP-3 forms complexes with IGFs, preventing them from binding to their receptors and stimulating cell proliferation and survival. IGF-independent actions have also been described for IGFBP-3. The degradation of IGFBP-3 by KLK2 or other proteases in the prostate may promote the growth of prostate cancer. We studied IGFBP-3 degradation by immunoblotting and two specific immunoassays, one recognizing only native non-fragmented IGFBP-3 and the other one recognizing both intact and proteolytically cleaved IGFBP-3. Peptides were used to inhibit the enzyme activity of KLK2 and cleavage sites in IGFBP-3 were identified by mass spectrometry. KLK2 proteolyzed IGFBP-3 into several small fragments, mostly after Arg residues, in keeping with the trypsin-like activity of KLK2. The fragmentation could be inhibited by KLK2-inhibiting peptides in a dose-dependent fashion. As degradation of IGFBP-3 could lead to a more aggressive cancer phenotype, inhibition of KLK2 activity might be useful for treatment of prostate cancer and other diseases associated with increased KLK2 activity.

  5. Dental Enamel Development: Proteinases and Their Enamel Matrix Substrates

    Science.gov (United States)

    Bartlett, John D.

    2013-01-01

    This review focuses on recent discoveries and delves in detail about what is known about each of the proteins (amelogenin, ameloblastin, and enamelin) and proteinases (matrix metalloproteinase-20 and kallikrein-related peptidase-4) that are secreted into the enamel matrix. After an overview of enamel development, this review focuses on these enamel proteins by describing their nomenclature, tissue expression, functions, proteinase activation, and proteinase substrate specificity. These proteins and their respective null mice and human mutations are also evaluated to shed light on the mechanisms that cause nonsyndromic enamel malformations termed amelogenesis imperfecta. Pertinent controversies are addressed. For example, do any of these proteins have a critical function in addition to their role in enamel development? Does amelogenin initiate crystallite growth, does it inhibit crystallite growth in width and thickness, or does it do neither? Detailed examination of the null mouse literature provides unmistakable clues and/or answers to these questions, and this data is thoroughly analyzed. Striking conclusions from this analysis reveal that widely held paradigms of enamel formation are inadequate. The final section of this review weaves the recent data into a plausible new mechanism by which these enamel matrix proteins support and promote enamel development. PMID:24159389

  6. Proteolytic activity of prostate-specific antigen (PSA towards protein substrates and effect of peptides stimulating PSA activity.

    Directory of Open Access Journals (Sweden)

    Johanna M Mattsson

    Full Text Available Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3 exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

  7. Lanthanide chelate complementation and hydrolysis enhanced luminescent chelate in real-time reverse transcription polymerase chain reaction assays for KLK3 transcripts.

    Science.gov (United States)

    Alinezhad, Saeid; Väänänen, Riina-Minna; Lehmusvuori, Ari; Karhunen, Ulla; Soukka, Tero; Kähkönen, Esa; Taimen, Pekka; Alanen, Kalle; Pettersson, Kim

    2014-01-01

    The requirement for high-performance reporter probes in real-time detection of polymerase chain reaction (PCR) has led to the use of time-resolved fluorometry of lanthanide chelates. The aim of this study was to investigate the applicability of the principle of lanthanide chelate complementation (LCC) in comparison with a method based on hydrolysis enhancement and quenching of intact probes. A real-time reverse transcription (RT) PCR assay for kallikrein-related peptidase 3 (KLK3, model analyte) was developed by using the LCC detection method. Both detection methods were tested with a standard series of purified PCR products, 20 prostatic tissues, 20 healthy and prostate cancer patient blood samples, and female blood samples spiked with LNCaP cells. The same limit of detection was obtained with both methods, and two cycles earlier detection with the LCC method was observed. KLK3 messenger RNA (mRNA) was detected in all tissue samples and in 1 of 20 blood samples identically with both methods. The background was 30 times lower, and the signal-to-background (S/B) ratio was 3 times higher, when compared with the reference method. Use of the new reporter method provided similar sensitivity and specificity as the reference method. The lower background, the improved S/B ratio, and the possibility of melting curve analysis and single nucleotide polymorphism (SNP) detection could be advantages for this new reporter probe.

  8. Investigation of serum dipeptidyl peptidase-4, body fat content and distribution in type 2 diabetes%血清二肽基肽酶4与2型糖尿病患者体脂含量及分布的关系探讨

    Institute of Scientific and Technical Information of China (English)

    蔡芸莹; 苏恒; 欧杨; 李清竹; 杨凡; 思永婷; 张云; 薛元明

    2014-01-01

    目的 探讨2型糖尿病(T2DM)患者血清二肽基肽酶4(DPP-Ⅳ)水平与体脂含量及分布的关系.方法 选取2013年7月至11月入住云南省第一人民医院内分泌科的T2DM患者共40例为研究对象,测量其身高、体重,计算体质指数(BMI);电化学发光法测定总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C);酶联免疫法测定DPP-Ⅳ水平;双能X线骨密度分析仪(DEXA)测定体脂及瘦肉组织含量.将所有患者分为:体重正常组(NC组)14例(BMI 18.5~24.0 kg/m2),超重/肥胖组(OB组)26例(BMI≥24.0 kg/m2).组间比较采用t检验,相关性分析采用Spearman法.结果 与NC组比较,OB组血清DPP-Ⅳ水平显著升高[(765±72)比(706±91) μg/L,t=2.2480,P<0.05].血清DPP-Ⅳ水平与全身脂肪含量(FAT)之间呈正相关(r=0.498,P<0.05).血清DPP-Ⅳ水平与T2DM患者躯干脂肪含量、上肢脂肪含量及下肢脂肪含量呈正相关(r=0.567、0.391、0.344,均P<0.05),与全身及各部位瘦肉组织含量无相关关系.结论 血清DPP-Ⅳ水平可能与T2DM患者体脂含量及分布相关.%Objective To investigate the relationship between serum dipeptidyl peptidase 4 (DPP-Ⅳ) and body fat content and distribution in type 2 diabetes (T2DM).Methods A total of 40 T2DM inpatients treated in Department of Endocrinology of the First People's Hospital of Yunnan Province from July 2013 to November 2013 were enrolled.The height,weight,body mass index (BMI) were measured; total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol (LDL-C) were detected by electrochemiluminescence assay and serum DPP-Ⅳ were detected by enzyme linked immunosorbent assay (Ray Biotech).Body fat content and lean mass was measured by dual energy X-ray (DEXA).Based on BMI,the subjects were divided into 2 groups:the normal weight group (BMI 18.5-24.0 kg/m2,NC group,n=14) and the over weight/obese group (BMI≥24.0 kg/m2,OB group,n=26).Two-tailed independent samples t test was

  9. 尿胞外体亮氨酸氨基肽酶及二肽基肽酶在糖尿病肾病中的变化%Changes of urinary exosomal leucine aminopeptidase and dipeptidyl peptidase 4 in diabetic nephropathy

    Institute of Scientific and Technical Information of China (English)

    孙爱丽; 胡晓燕; 关广聚; 邓浩萍; 刘元涛; 陈诗鸿; 刘军莉; 邓景惕

    2011-01-01

    AIM: To investigate the changes of urinary exosomal enzymes and the correlation with diabetic nephropathy.METHODS: Thirty - four healthy volunteers and 127 patients of type 2 diabetes mellitus ( T2DM ) were included in the study.The healthy volunteers served as control.The patients with T2DM were divided into 3 groups based on their 24 h urinary albumin/creatinine ratio ( UACR ): 50 patients with microalbuminuria in early DN group ( DN1 ), 34 patients with macroalbuminuria in overt DN group ( DN2 ) and 43 patients without albuminuria in DM group.The levels of urine exosomal leucine aminopeptidase( exosome - LAP ) and exosomal dipeptidyl peptidase 4( exosome - DPP4 ) were determined by enzyme - linked immunosorbent assay ( ELISA ).The following methods were used to determine the biochemical parameters: liquid chromatography for glycated hemoglobin ( HbA1c ), chemical modification method for cholesterol ( CH ), Jaffe - kinetic assay for creatinine ( CR ) and urease - GLDH method for blood urea nitrogen ( BUN ).Multiple stepwise linear regressions were used to analyze the relationship of exosome - LAP or exosome - DPP4 with HbA1c, CH, UACR, CR and BUN.RESULTS: The levels of exosome - LAP and exosome - DPP4 in DM, DN1 and DN2 groups were significantly higher than those in control group ( P <0.01 ).The exosome - LAP in DN2 group was significantly higher than that in DM group.Correlation analysis showed that the levels of urinary exosome - LAP and exosome - DPP4 were positively correlated with HbA1c, CH, UACR, CR and BUN.Multiple stepwise linear regression analysis showed that CH and UACR were independent determinants for exosome - LAP ( P <0.01 ), and UACR and HbAlc were independent determinants for exosome - DPP4 ( P <0.01 ).CONCLUSION: Urine exosome - LAP and exosome - DPP4 are correlated with the severity of diabetic nephropathy.These parameters may serve as clinical markers for the diagnosis and prognosis evaluation of diabetic nephropathy.%目的:探讨 2

  10. Genetic association of the KLK4 locus with risk of prostate cancer.

    Directory of Open Access Journals (Sweden)

    Felicity Lose

    Full Text Available The Kallikrein-related peptidase, KLK4, has been shown to be significantly overexpressed in prostate tumours in numerous studies and is suggested to be a potential biomarker for prostate cancer. KLK4 may also play a role in prostate cancer progression through its involvement in epithelial-mesenchymal transition, a more aggressive phenotype, and metastases to bone. It is well known that genetic variation has the potential to affect gene expression and/or various protein characteristics and hence we sought to investigate the possible role of single nucleotide polymorphisms (SNPs in the KLK4 gene in prostate cancer. Assessment of 61 SNPs in the KLK4 locus (± 10 kb in approximately 1300 prostate cancer cases and 1300 male controls for associations with prostate cancer risk and/or prostate tumour aggressiveness (Gleason score <7 versus ≥ 7 revealed 7 SNPs to be associated with a decreased risk of prostate cancer at the P(trend<0.05 significance level. Three of these SNPs, rs268923, rs56112930 and the HapMap tagSNP rs7248321, are located several kb upstream of KLK4; rs1654551 encodes a non-synonymous serine to alanine substitution at position 22 of the long isoform of the KLK4 protein, and the remaining 3 risk-associated SNPs, rs1701927, rs1090649 and rs806019, are located downstream of KLK4 and are in high linkage disequilibrium with each other (r(2 ≥ 0.98. Our findings provide suggestive evidence of a role for genetic variation in the KLK4 locus in prostate cancer predisposition.

  11. Copy number variants in the kallikrein gene cluster.

    Directory of Open Access Journals (Sweden)

    Pernilla Lindahl

    Full Text Available The kallikrein gene family (KLK1-KLK15 is the largest contiguous group of protease genes within the human genome and is associated with both risk and outcome of cancer and other diseases. We searched for copy number variants in all KLK genes using quantitative PCR analysis and analysis of inheritance patterns of single nucleotide polymorphisms. Two deletions were identified: one 2235-bp deletion in KLK9 present in 1.2% of alleles, and one 3394-bp deletion in KLK15 present in 4.0% of alleles. Each deletion eliminated one complete exon and created out-of-frame coding that eliminated the catalytic triad of the resulting truncated gene product, which therefore likely is a non-functional protein. Deletion breakpoints identified by DNA sequencing located the KLK9 deletion breakpoint to a long interspersed element (LINE repeated sequence, while the deletion in KLK15 is located in a single copy sequence. To search for an association between each deletion and risk of prostate cancer (PC, we analyzed a cohort of 667 biopsied men (266 PC cases and 401 men with no evidence of PC at biopsy using short deletion-specific PCR assays. There was no association between evidence of PC in this cohort and the presence of either gene deletion. Haplotyping revealed a single origin of each deletion, with most recent common ancestor estimates of 3000-8000 and 6000-14 000 years for the deletions in KLK9 and KLK15, respectively. The presence of the deletions on the same haplotypes in 1000 Genomes data of both European and African populations indicate an early origin of both deletions. The old age in combination with homozygous presence of loss-of-function variants suggests that some kallikrein-related peptidases have non-essential functions.

  12. Bcl11b regulates enamel matrix protein expression and dental epithelial cell differentiation during rat tooth development.

    Science.gov (United States)

    Li, Ziyue; Chen, Guoqing; Yang, Yaling; Guo, Weihua; Tian, Weidong

    2017-01-01

    Amelogenesis, beginning with thickened epithelial aggregation and ending with highly mineralized enamel formation, is a process mediated by a complex signaling network that involves several molecules, including growth and transcription factors. During early tooth development, the transcription factor B‑cell CLL/lymphoma 11B (Bcl11b) participates in dental epithelial cell proliferation and differentiation. However, whether it affects the postnatal regulation of enamel matrix protein expression and ameloblast differentiation remains unclear. To clarify the role of Bcl11b in enamel development, the present study initially detected the protein expression levels of Bcl11b during tooth development using immunohistochemistry, from the embryonic lamina stage to the postnatal period, and demonstrated that Bcl11b is predominantly restricted to cervical loop epithelial cells at the cap and bell stages, whereas expression is reduced in ameloblasts. Notably, the expression pattern of Bcl11b during tooth development differed between rats and mice. Knockdown of Bcl11b by specific small interfering RNA attenuated the expression of enamel‑associated genes, including amelogenin, X‑linked (Amelx), ameloblastin (Ambn), enamelin (Enam), kallikrein related peptidase 4 (Klk4), matrix metallopeptidase 20 and Msh homeobox 2 (Msx2). Chromatin immunoprecipitation assay verified that Msx2 was a transcriptional target of Bcl11b. However, overexpression of Msx2 resulted in downregulation of enamel‑associated genes, including Ambn, Amelx, Enam and Klk4. The present study suggested that Bcl11b serves a potentially important role in the regulation of ameloblast differentiation and enamel matrix protein expression. In addition, a complex feedback regulatory network may exist between Bcl11b and Msx2.

  13. Up-regulation and clinical significance of serine protease kallikrein 6 in colon cancer.

    Science.gov (United States)

    Kim, Jong-Tae; Song, Eun Young; Chung, Kyung-Sook; Kang, Min Ah; Kim, Jae Wha; Kim, Sang Jick; Yeom, Young Il; Kim, Joo Heon; Kim, Kyo Hyun; Lee, Hee Gu

    2011-06-15

    Kallikrein-related peptidase 6 (KLK6) encodes a trypsin-like serine protease that is up-regulated in several cancers, although the putative functions of KLK6 in cancer have not been elucidated. In the current study, overexpression of KLK6 was identified in colon cancer, and the possibility that KLK6 may be a suitable candidate as a tumor marker was examined. Messenger RNA (mRNA) transcript levels and protein up-regulation of KLK6 in colon cancer tissues was examined using reverse transcriptase-polymerase chain reaction, immunohistochemistry, and clinicopathologic analyses. Cell proliferation, invasiveness, and antiapoptotic activity were determined in colon cancer cells that were transfected with small-interfering RNA (siRNA) of KLK6. KLK6 mRNA was up-regulated significantly in tumor tissues compared with nontumor regions. KLK6 protein was strongly expressed in adenocarcinomas but was not expressed in normal mucosa or in premalignant dysplastic lesions. Sera from patients with colon cancer revealed an increase in KLK6 secretion (0.25 μg/mL; P = .031) compared with noncancer cells (0.19 μg/mL). Clinicopathologic and immunohistochemical studies of 143 patients with colon cancer revealed a significant correlation between KLK6 expression and Dukes disease stage (P = .005). High KLK6 expression was associated significantly with shorter overall (P = .001) and recurrence-free survival (P = .001). The rates of proliferation and invasiveness were decreased by 50% in cells that were transfected with KLK6 siRNA. The overexpression of KLK6 led to decreased activity of the E-cadherin promoter. KLK6 was up-regulated significantly in tissues and sera from patients with colon cancer and was associated closely with a poor prognosis, suggesting that KLK6 may be used as a potential biomarker and a therapeutic target for colon cancer. Copyright © 2010 American Cancer Society.

  14. Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Rama Garimella

    2017-03-01

    Full Text Available Osteosarcoma (OS is an aggressive malignancy of bone affecting children, adolescents and young adults. Understanding vitamin D metabolism and vitamin D regulated genes in OS is an important aspect of vitamin D/cancer paradigm, and in evaluating vitamin D as adjuvant therapy for human OS. Vitamin D treatment of 143B OS cells induced significant and novel changes in the expression of genes that regulate: (a inflammation and immunity; (b formation of reactive oxygen species, metabolism of cyclic nucleotides, sterols, vitamins and mineral (calcium, quantity of gap junctions and skeletogenesis; (c bone mineral density; and (d cell viability of skeletal cells, aggregation of bone cancer cells and exocytosis of secretory vesicles. Ingenuity pathway analysis revealed significant reduction in Runx2 target genes such as fibroblast growth factor -1, -12 (FGF1 and FGF12, bone morphogenetic factor-1 (BMP1, SWI/SNF related, matrix associated actin dependent regulator of chromatin subfamily a, member 4 (SMARCA4, Matrix extracellular phosphoglycoprotein (MEPE, Integrin, β4 (ITGBP4, Matrix Metalloproteinase -1, -28 (MMP1 and MMP28, and signal transducer and activator of transcription-4 (STAT4 in vitamin D treated 143B OS cells. These genes interact with the inflammation, oxidative stress and membrane vesicle biogenesis gene networks. Vitamin D not only inhibited the expression of Runx2 target genes MMP1, MMP28 and kallikrein related peptidase-7 (KLK7, but also migration and invasion of 143B OS cells. Vitamin D regulated Runx2 target genes or their products represent potential therapeutic targets and laboratory biomarkers for applications in translational oncology.

  15. Blood biomarker levels to aid discovery of cancer-related single-nucleotide polymorphisms: kallikreins and prostate cancer.

    Science.gov (United States)

    Klein, Robert J; Halldén, Christer; Cronin, Angel M; Ploner, Alexander; Wiklund, Fredrik; Bjartell, Anders S; Stattin, Pär; Xu, Jianfeng; Scardino, Peter T; Offit, Kenneth; Vickers, Andrew J; Grönberg, Henrik; Lilja, Hans

    2010-05-01

    Polymorphisms associated with prostate cancer include those in three genes encoding major secretory products of the prostate: KLK2 (encoding kallikrein-related peptidase 2; hK2), KLK3 (encoding prostate-specific antigen; PSA), and MSMB (encoding beta-microseminoprotein). PSA and hK2, members of the kallikrein family, are elevated in sera of men with prostate cancer. In a comprehensive analysis that included sequencing of all coding, flanking, and 2 kb of putative promoter regions of all 15 kallikrein (KLK) genes spanning approximately 280 kb on chromosome 19q, we identified novel single-nucleotide polymorphisms (SNP) and genotyped 104 SNPs in 1,419 cancer cases and 736 controls in Cancer Prostate in Sweden 1, with independent replication in 1,267 cases and 901 controls in Cancer Prostate in Sweden 2. This verified prior associations of SNPs in KLK2 and in MSMB (but not in KLK3) with prostate cancer. Twelve SNPs in KLK2 and KLK3 were associated with levels of PSA forms or hK2 in plasma of control subjects. Based on our comprehensive approach, this is likely to represent all common KLK variants associated with these phenotypes. A T allele at rs198977 in KLK2 was associated with increased cancer risk and a striking decrease of hK2 levels in blood. We also found a strong interaction between rs198977 genotype and hK2 levels in blood in predicting cancer risk. Based on this strong association, we developed a model for predicting prostate cancer risk from standard biomarkers, rs198977 genotype, and rs198977 x hK2 interaction; this model had greater accuracy than did biomarkers alone (area under the receiver operating characteristic curve, 0.874 versus 0.866), providing proof in principle to clinical application for our findings.

  16. Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells.

    Science.gov (United States)

    Garimella, Rama; Tadikonda, Priyanka; Tawfik, Ossama; Gunewardena, Sumedha; Rowe, Peter; Van Veldhuizen, Peter

    2017-03-16

    Osteosarcoma (OS) is an aggressive malignancy of bone affecting children, adolescents and young adults. Understanding vitamin D metabolism and vitamin D regulated genes in OS is an important aspect of vitamin D/cancer paradigm, and in evaluating vitamin D as adjuvant therapy for human OS. Vitamin D treatment of 143B OS cells induced significant and novel changes in the expression of genes that regulate: (a) inflammation and immunity; (b) formation of reactive oxygen species, metabolism of cyclic nucleotides, sterols, vitamins and mineral (calcium), quantity of gap junctions and skeletogenesis; (c) bone mineral density; and (d) cell viability of skeletal cells, aggregation of bone cancer cells and exocytosis of secretory vesicles. Ingenuity pathway analysis revealed significant reduction in Runx2 target genes such as fibroblast growth factor -1, -12 (FGF1 and FGF12), bone morphogenetic factor-1 (BMP1), SWI/SNF related, matrix associated actin dependent regulator of chromatin subfamily a, member 4 (SMARCA4), Matrix extracellular phosphoglycoprotein (MEPE), Integrin, β4 (ITGBP4), Matrix Metalloproteinase -1, -28 (MMP1 and MMP28), and signal transducer and activator of transcription-4 (STAT4) in vitamin D treated 143B OS cells. These genes interact with the inflammation, oxidative stress and membrane vesicle biogenesis gene networks. Vitamin D not only inhibited the expression of Runx2 target genes MMP1, MMP28 and kallikrein related peptidase-7 (KLK7), but also migration and invasion of 143B OS cells. Vitamin D regulated Runx2 target genes or their products represent potential therapeutic targets and laboratory biomarkers for applications in translational oncology.

  17. Diversity of neuropsin (KLK8)-dependent synaptic associativity in the hippocampal pyramidal neuron.

    Science.gov (United States)

    Ishikawa, Yasuyuki; Tamura, Hideki; Shiosaka, Sadao

    2011-07-15

    Hippocampal early (E-) long-term potentiation (LTP) and long-term depression (LTD) elicited by a weak stimulus normally fades within 90 min. Late (L-) LTP and LTD elicited by strong stimuli continue for >180 min and require new protein synthesis to persist. If a strong tetanus is applied once to synaptic inputs, even a weak tetanus applied to another synaptic input can evoke persistent LTP. A synaptic tag is hypothesized to enable the capture of newly synthesized synaptic molecules. This process, referred to as synaptic tagging, is found between not only the same processes (i.e. E- and L-LTP; E- and L-LTD) but also between different processes (i.e. E-LTP and L-LTD; E-LTD and L-LTP) induced at two independent synaptic inputs (cross-tagging). However, the mechanisms of synaptic tag setting remain unclear. In our previous study, we found that synaptic associativity in the hippocampal Schaffer collateral pathway depended on neuropsin (kallikrein-related peptidase 8 or KLK8), a plasticity-related extracellular protease. In the present study, we investigated how neuropsin participates in synaptic tagging and cross-tagging. We report that neuropsin is involved in synaptic tagging during LTP at basal and apical dendritic inputs. Moreover, neuropsin is involved in synaptic tagging and cross-tagging during LTP at apical dendritic inputs via integrin β1 and calcium/calmodulin-dependent protein kinase II signalling. Thus, neuropsin is a candidate molecule for the LTP-specific tag setting and regulates the transformation of E- to L-LTP during both synaptic tagging and cross-tagging.

  18. Protein-lipid interactions in assembly and function of Leader Peptidase

    NARCIS (Netherlands)

    Klompenburg, W. van

    1997-01-01

    Cells are the entities of life and they at least consist of one aqueous compartment separated from the environment by a membrane. Lipids and proteins are important constituents of membranes and the interactions between these components are the subject of this thesis. The studies were performed usi

  19. Protein secretion and possible roles for multiple signal peptidases for precursor processing in Bacilli

    NARCIS (Netherlands)

    Bron, S; Bolhuis, A; Tjalsma, H; Holsappel, S; Venema, G; van Dijl, J.M

    1998-01-01

    Bacillus subtilis is one of the best known Gram-positive bacteria at both the genetic and physiological level. The entire sequence of its chromosome is known and efficient tools for the genetic modification of this bacterium are available. Moreover, B. subtilis and related Bacillus species are

  20. Tyrosine nitration moderates the peptidase activity of human methionyl aminopeptidase 2.

    Science.gov (United States)

    Ng, John Y; Chiu, Joyce; Hogg, Philip J; Wong, Jason W H

    2013-10-11

    Methionyl aminopeptidase 2 (MetAP2) plays an important role in the regulation of angiogenesis. This study examined whether nitration of MetAP2 alters its enzymatic activity in vitro. The activity of unmodified, nitrated and oxidised MetAP2 was assessed and it was found that nitration significantly reduced its ability to cleave a chromogenic substrate. Mass spectrometry analysis identified Tyr336 as a nitrated residue in MetAP2. Structural and evolutionary analysis indicate that this is an important residue for MetAP2 activity. Combined, the results show that the activity of MetAP2 is reduced by nitration and raise the possibility that nitration of MetAP2 is a mechanism contributing to endothelial dysfunction. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Computational Analysis of Gynura bicolor Bioactive Compounds as Dipeptidyl Peptidase-IV Inhibitor

    National Research Council Canada - National Science Library

    Lina Rozano; Muhammad Redha Abdullah Zawawi; Muhamad Aizuddin Ahmad; Indu Bala Jaganath

    2017-01-01

    .... However, an understanding of the inhibitory actions of G. bicolor bioactive compounds on DPPIV is still lacking and this may provide crucial information for the development of more potent and natural sources of DPPIV inhibitors. Evaluation of G...

  2. Metabolism of glucagon-like peptide-2 in pigs: role of dipeptidyl peptidase IV

    DEFF Research Database (Denmark)

    Hansen, Lene; Hare, Kristine J; Hartmann, Bolette;

    2006-01-01

    -33). During infusion of GLP-2 alone, 30.9+/-1.7% of the infused peptide was degraded to GLP-2 (3-33). After valine-pyrrolidide, there was no significant formation of the metabolite. Significant extraction of intact GLP-2 was observed across the kidneys, the extremities (represented by a leg......), and the splanchnic bed, resulting in a metabolic clearance rate (MCR) of 6.80+/-0.47 ml/kg/min and a plasma half-life of 6.8+/-0.8 min. Hepatic extraction was not detected. Valine-pyrrolidide addition did not affect extraction ratios significantly, but decreased (p=0.003) MCR to 4.18+/-0.27 ml/kg/min and increased...

  3. The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells.

    Directory of Open Access Journals (Sweden)

    Hanjun Guan

    Full Text Available Amyloid formation and mitochondrial dysfunction are characteristics of type 2 diabetes. The major peptide constituent of the amyloid deposits in type 2 diabetes is islet amyloid polypeptide (IAPP. In this study, we found that pitrilysin, a zinc metallopeptidase of the inverzincin family, degrades monomeric, but not oligomeric, islet amyloid polypeptide in vitro. In insulinoma cells when pitrilysin expression was decreased to 5% of normal levels, there was a 60% increase in islet amyloid polypeptide-induced apoptosis. In contrast, overexpression of pitrilysin protects insulinoma cells from human islet amyloid polypeptide-induced apoptosis. Since pitrilysin is a mitochondrial protein, we used immunofluorescence staining of pancreases from human IAPP transgenic mice and Western blot analysis of IAPP in isolated mitochondria from insulinoma cells to provide evidence for a putative intramitochondrial pool of IAPP. These results suggest that pitrilysin regulates islet amyloid polypeptide in beta cells and suggest the presence of an intramitochondrial pool of islet amyloid polypeptide involved in beta-cell apoptosis.

  4. Dipeptidyl peptidase-4 inhibitors for the treatment of type 2 diabetes

    DEFF Research Database (Denmark)

    Deacon, Carolyn F; Holst, Jens Juul

    2013-01-01

    of the different DPP-4 inhibitors, all are small orally active compounds with broadly similar HbA1c-lowering efficacy. They improve glycaemic control in T2D, without increasing the risk of hypoglycaemia or causing weight gain. They can be used as monotherapy or in combination with other anti-diabetic therapies...

  5. Isolation and characterization of hieronymain II, another peptidase isolated from fruits of Bromelia hieronymi Mez (Bromeliaceae).

    Science.gov (United States)

    Bruno, Mariela A; Trejo, Sebastián A; Avilés, Xavier F; Caffini, Néstor O; López, Laura M I

    2006-04-01

    From unripe fruits of Bromelia hieronymi Mez (Bromeliaceae), a partially purified protease preparation was obtained by acetone fractionation of the crude extract. Purification was achieved by anionic exchange chromatography (FPLC) on Q-Sepharose HP followed by cationic exchange chromatography (SP-Sepharose HP). Homogeneity of the new enzyme, named hieronymain II, was confirmed by SDS-PAGE and mass spectroscopy (MALDI-TOF-TOF). The molecular mass of was 23,411 Da, and maximum proteolytic activity (more than 90% of maximum activity) was achieved at pH 7.5-9.0 on casein and at pH 7.30-8.3 on Z-Phe-Arg-p-nitroanilide. The enzyme was completely inhibited by E-64 and iodoacetic acid and activated by the addition of cysteine. The N-terminal sequence of hieronymain II (AVPQSIDWRVYGAV) was compared with those of 12 plant cysteine proteases which showed more than 70% of identity. Kinetic enzymatic assays were made on Z-Phe-Arg-p-nitroanilide (Km = 0.72mM, kcat = 1.82 seg(-1) , kcat/ Km = 2.54seg(-1) mM(-l)). No detectable activity could be found on PFLNA or Z-Arg-Arg-p-nitroanilide.

  6. Archaeal type IV prepilin-like signal peptidases : substrates and biochemistry

    NARCIS (Netherlands)

    Szabo, Zalan

    2006-01-01

    De wereld van de micro-organismen is oneindig en fascinerend. Voor ons is deze wereld echter nauwelijks waar te nemen. Micro-organismen zijn met het blote oog niet te zien, maar ze laten op vele manieren hun sporen achter. Denk aan de manier waarop yoghurt en kaas wordt gemaakt, of de heerlijke bosl

  7. Minimal motif peptide structure of metzincin clan zinc peptidases in micelles.

    Science.gov (United States)

    Onoda, Akira; Suzuki, Takako; Ishizuka, Hiroaki; Sugiyama, Rumiko; Ariyasu, Shinya; Yamamura, Takeshi

    2009-12-01

    It is well known that the functions of metalloproteins generally originate from their metal-binding motifs. However, the intrinsic nature of individual motifs remains unknown, particularly the details about metal-binding effects on the folding of motifs; the converse is also unknown, although there is no doubt that the motif is the core of the reactivity for each metalloprotein. In this study, we focused our attention on the zinc-binding motif of the metzincin clan family, HEXXHXXGXXH; this family contains the general zinc-binding sequence His-Glu-Xaa-Xaa-His (HEXXH) and the extended GXXH region. We adopted the motif sequence of stromelysin-1 and investigated the folding properties of the Trp-labeled peptides WAHEIAHSLGLFHA (STR-W1), AWHEIAHSLGLFHA (STR-W2), AHEIAHSLGWFHA (STR-W11), and AHEIAHSLGLFHWA (STR-W14) in the presence and absence of zinc ions in hydrophobic micellar environments by circular dichroism (CD) measurements. We accessed successful incorporation of these zinc peptides into micelles using quenching of Trp fluorescence. Results of CD studies indicated that two of the Trp-incorporated peptides, STR-W1 and STR-W14, exhibited helical folding in the hydrophobic region of cetyltrimethylammonium chloride micelle. The NMR structural analysis of the apo STR-W14 revealed that the conformation in the C-terminus GXXH region significantly differred between the apo state in the micelle and the reported Zn-bound state of stromelysin-1 in crystal structures. The structural analyses of the qualitative Zn-binding properties of this motif peptide provide an interesting Zn-binding mechanism: the minimum consensus motif in the metzincin clan, a basic zinc-binding motif with an extended GXXH region, has the potential to serve as a preorganized Zn binding scaffold in a hydrophobic environment.

  8. The Alzheimer's Amyloid-Degrading Peptidase, Neprilysin: Can We Control It?

    Directory of Open Access Journals (Sweden)

    N. N. Nalivaeva

    2012-01-01

    Full Text Available The amyloid cascade hypothesis of Alzheimer's disease (AD postulates that accumulation in the brain of amyloid β-peptide (Aβ is the primary trigger for neuronal loss specific to this pathology. In healthy brain, Aβ levels are regulated by a dynamic equilibrium between Aβ release from the amyloid precursor protein (APP and its removal by perivascular drainage or by amyloid-degrading enzymes (ADEs. During the last decade, the ADE family was fast growing, and currently it embraces more than 20 members. There are solid data supporting involvement of each of them in Aβ clearance but a zinc metallopeptidase neprilysin (NEP is considered as a major ADE. NEP plays an important role in brain function due to its role in terminating neuropeptide signalling and its decrease during ageing or after such pathologies as hypoxia or ischemia contribute significantly to the development of AD pathology. The recently discovered mechanism of epigenetic regulation of NEP by the APP intracellular domain (AICD opens new avenues for its therapeutic manipulation and raises hope for developing preventive strategies in AD. However, consideration needs to be given to the diverse physiological roles of NEP. This paper critically evaluates general biochemical and physiological functions of NEP and their therapeutic relevance.

  9. Archaeal type IV prepilin-like signal peptidases : substrates and biochemistry

    NARCIS (Netherlands)

    Szabo, Zalan

    2006-01-01

    De wereld van de micro-organismen is oneindig en fascinerend. Voor ons is deze wereld echter nauwelijks waar te nemen. Micro-organismen zijn met het blote oog niet te zien, maar ze laten op vele manieren hun sporen achter. Denk aan de manier waarop yoghurt en kaas wordt gemaakt, of de heerlijke

  10. Structure-function relationships in type I signal peptidases of bacilli

    NARCIS (Netherlands)

    Roosmalen, Maarten Leonardus van

    2001-01-01

    Every living cell, either being part of a complex cellular netwerk or just a unicellular organism, produces protiens inside the cell that are destined for extracellular locations. Consequently, these protiens have to be transported across one or more membrane(s) that subdived, or surround the cell.

  11. Saxagliptin: a new dipeptidyl peptidase-4 inhibitor for the treatment of type 2 diabetes

    DEFF Research Database (Denmark)

    Deacon, Carolyn F; Holst, Jens J

    2009-01-01

    in vivo to form an active metabolite, and both parent drug and metabolite are excreted primarily via the kidneys. Saxagliptin reduces the degradation of the incretin hormone glucagon-like peptide-1, thereby enhancing its actions, and is associated with improved beta-cell function and suppression...... of glucagon secretion. Clinical trials of up to 24 weeks duration have shown that saxagliptin improves glycemic control in monotherapy and provides additional efficacy when used in combination with other oral antidiabetic agents (metformin, sulfonylurea, thiazolidinedione). Both fasting and postprandial...

  12. Bacterial calpains and the evolution of the calpain (C2) family of peptidases.

    Science.gov (United States)

    Rawlings, Neil D

    2015-11-02

    Homologues of calpain, often thought to be an essential, cytoplasmic, calcium-dependent cysteine endopeptidase found exclusively in eukaryotes, have been found in bacterial proteomes. The homologues lack calcium-binding sites, have differing domain architectures, and can be secreted or membrane-associated. Homologues are rare and occur in a minority of bacterial phyla and often in a minority of species in a genus. However, the differences in domain architecture argue against a recent, horizontal gene transfer from a eukaryote. From analysis of a phylogenetic tree and absence of homologues in archaea, calpains in eukaryotes may be derived from genes horizontally transferred from a bacterium.

  13. Structure of the archaeal pab87 peptidase reveals a novel self-compartmentalizing protease family.

    Directory of Open Access Journals (Sweden)

    Vanessa Delfosse

    Full Text Available Self-compartmentalizing proteases orchestrate protein turnover through an original architecture characterized by a central catalytic chamber. Here we report the first structure of an archaeal member of a new self-compartmentalizing protease family forming a cubic-shaped octamer with D(4 symmetry and referred to as CubicO. We solved the structure of the Pyrococcus abyssi Pab87 protein at 2.2 A resolution using the anomalous signal of the high-phasing-power lanthanide derivative Lu-HPDO3A. A 20 A wide channel runs through this supramolecular assembly of 0.4 MDa, giving access to a 60 A wide central chamber holding the eight active sites. Surprisingly, activity assays revealed that Pab87 degrades specifically d-amino acid containing peptides, which have never been observed in archaea. Genomic context of the Pab87 gene showed that it is surrounded by genes involved in the amino acid/peptide transport or metabolism. We propose that CubicO proteases are involved in the processing of d-peptides from environmental origins.

  14. Extracellular methionine amino peptidase (MAP production by Streptomyces gedanensis in solid-state fermentation

    Directory of Open Access Journals (Sweden)

    Raji Rahulan

    2014-04-01

    Full Text Available A bioprocess was developed for extracellular MAP production from Streptomyces gedanensis by solid-state fermentation. Response surface methodology of Box Behken Design was performed to evaluate the interaction effects of most significant variables {inoculum size, (NH42SO4 concentration, MgSO4.7H2O and tryptone on MAP production after the single parameter optimization and it resulted a maximum MAP production of 55.26 IU/g PUF after 120 h of fermentation. The concentrated crude MAP displayed a pH and temperature optimum of 8.5 and 50°C. By analyzing the thermal stability, the MAP was found to be stable in a temperature range of 50 to 55°C but lost about 50% of its activity at 65°C after 30 min. This is a first report of this kind of study for MAP.

  15. MMP20, KLK4, and MMP20/KLK4 double null mice define roles for matrix proteases during dental enamel formation.

    Science.gov (United States)

    Hu, Yuanyuan; Smith, Charles E; Richardson, Amelia S; Bartlett, John D; Hu, Jan C C; Simmer, James P

    2016-03-01

    Matrix metalloproteinase 20 (MMP20) and kallikrein-related peptidase 4 (KLK4) are secreted proteinases that are essential for proper dental enamel formation. We characterized and compared enamel formed in wild-type, Mmp20 (-/-), Klk4 (-/-), Mmp20 (+/-) Klk4 (+/-), and Mmp20 (-/-) Klk4 (-/-) mice using dissecting and light microscopy, backscattered scanning electron microscopy (bSEM), SEM, microcomputed tomography (μCT), and energy-dispersive X-ray analysis (EDX). Following eruption, fractures were observed on Mmp20 (-/-), Klk4 (-/-), Mmp20 (+/-) Klk4 (+/-), and Mmp20 (-/-) Klk4 (-/-) molars. Failure of the enamel in the Mmp20 (+/-) Klk4 (+/-) molars was unexpected and suggested that digenic effects could contribute to the etiology of amelogenesis imperfecta in humans. Micro-CT analyses of hemimandibles demonstrated significantly reduced high-density enamel volume in the Mmp20 (-/-) and Klk4 (-/-) mice relative to the wild-type, which was further reduced in Mmp20 (-/-) Klk4 (-/-) mice. bSEM images of 7-week Mmp20 (-/-) and Mmp20 (-/-) Klk4 (-/-) mandibular incisors showed rough, pitted enamel surfaces with numerous indentations and protruding nodules. The Mmp20 (+/-) and Mmp20 (+/-) Klk4 (+/-) incisors showed prominent, evenly spaced, horizontal ridges that were more distinct in Mmp20 (+/-) Klk4 (+/-) incisors relative to Mmp20 (+/-) incisors due to the darkening of the valleys between the ridges. In cross sections, the Mmp20 (-/-) and Mmp20 (-/-) Klk4 (-/-) exhibited three distinct layers. The outer layer exhibited a disturbed elemental composition and an irregular enamel surface covered with nodules. The Mmp20 null enamel was apparently unable to withstand the sheer forces associated with eruption and separated from dentin during development. Cells invaded the cracks and interposed between the dentin and enamel layers. MMP20 and KLK4 serve overlapping and complementary functions to harden enamel by removing protein, but MMP20 potentially serves multiple

  16. Evolutionary conservation of zinc finger transcription factor binding sites in promoters of genes co-expressed with WT1 in prostate cancer

    Directory of Open Access Journals (Sweden)

    Brett Adina

    2008-07-01

    Full Text Available Abstract Background Gene expression analyses have led to a better understanding of growth control of prostate cancer cells. We and others have identified the presence of several zinc finger transcription factors in the neoplastic prostate, suggesting a potential role for these genes in the regulation of the prostate cancer transcriptome. One of the transcription factors (TFs identified in the prostate cancer epithelial cells was the Wilms tumor gene (WT1. To rapidly identify coordinately expressed prostate cancer growth control genes that may be regulated by WT1, we used an in silico approach. Results Evolutionary conserved transcription factor binding sites (TFBS recognized by WT1, EGR1, SP1, SP2, AP2 and GATA1 were identified in the promoters of 24 differentially expressed prostate cancer genes from eight mammalian species. To test the relationship between sequence conservation and function, chromatin of LNCaP prostate cancer and kidney 293 cells were tested for TF binding using chromatin immunoprecipitation (ChIP. Multiple putative TFBS in gene promoters of placental mammals were found to be shared with those in human gene promoters and some were conserved between genomes that diverged about 170 million years ago (i.e., primates and marsupials, therefore implicating these sites as candidate binding sites. Among those genes coordinately expressed with WT1 was the kallikrein-related peptidase 3 (KLK3 gene commonly known as the prostate specific antigen (PSA gene. This analysis located several potential WT1 TFBS in the PSA gene promoter and led to the rapid identification of a novel putative binding site confirmed in vivo by ChIP. Conversely for two prostate growth control genes, androgen receptor (AR and vascular endothelial growth factor (VEGF, known to be transcriptionally regulated by WT1, regulatory sequence conservation was observed and TF binding in vivo was confirmed by ChIP. Conclusion Overall, this targeted approach rapidly identified

  17. A Fourth KLK4 Mutation Is Associated with Enamel Hypomineralisation and Structural Abnormalities

    Science.gov (United States)

    Smith, Claire E. L.; Kirkham, Jennifer; Day, Peter F.; Soldani, Francesca; McDerra, Esther J.; Poulter, James A.; Inglehearn, Christopher F.; Mighell, Alan J.; Brookes, Steven J.

    2017-01-01

    “Amelogenesis imperfecta” (AI) describes a group of genetic conditions that result in defects in tooth enamel formation. Mutations in many genes are known to cause AI, including the gene encoding the serine protease, kallikrein related peptidase 4 (KLK4), expressed during the maturation stage of amelogenesis. In this study we report the fourth KLK4 mutation to be identified in autosomal recessively-inherited hypomaturation type AI, c.632delT, p.(L211Rfs*37) (NM_004917.4, NP_004908.4). This homozygous variant was identified in five Pakistani AI families and is predicted to result in a transcript with a premature stop codon that escapes nonsense mediated decay. However, the protein may misfold, as three of six disulphide bonds would be disrupted, and may be degraded or non-functional as a result. Primary teeth were obtained from one affected individual. The enamel phenotype was characterized using high-resolution computerized X-ray tomography (CT), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and microhardness testing (MH). Enamel from the affected individual (referred to as KLK4 enamel) was hypomineralised in comparison with matched control enamel. Furthermore, KLK4 inner enamel was hypomineralised compared with KLK4 outer enamel. SEM showed a clear structural demarcation between KLK4 inner and outer enamel, although enamel structure was similar to control tissue overall. EDX showed that KLK4 inner enamel contained less calcium and phosphorus and more nitrogen than control inner enamel and KLK4 outer enamel. MH testing showed that KLK4 inner enamel was significantly softer than KLK4 outer enamel (p enamel was not significantly different to that of control outer enamel. Overall, these findings suggest that the KLK4 c.632delT mutation may be a common cause of autosomal recessive AI in the Pakistani population. The phenotype data obtained mirror findings in the Klk4−/− mouse and suggest that KLK4 is required for the hardening

  18. A Fourth KLK4 Mutation Is Associated with Enamel Hypomineralisation and Structural Abnormalities

    Directory of Open Access Journals (Sweden)

    Claire E. L. Smith

    2017-05-01

    Full Text Available “Amelogenesis imperfecta” (AI describes a group of genetic conditions that result in defects in tooth enamel formation. Mutations in many genes are known to cause AI, including the gene encoding the serine protease, kallikrein related peptidase 4 (KLK4, expressed during the maturation stage of amelogenesis. In this study we report the fourth KLK4 mutation to be identified in autosomal recessively-inherited hypomaturation type AI, c.632delT, p.(L211Rfs*37 (NM_004917.4, NP_004908.4. This homozygous variant was identified in five Pakistani AI families and is predicted to result in a transcript with a premature stop codon that escapes nonsense mediated decay. However, the protein may misfold, as three of six disulphide bonds would be disrupted, and may be degraded or non-functional as a result. Primary teeth were obtained from one affected individual. The enamel phenotype was characterized using high-resolution computerized X-ray tomography (CT, scanning electron microscopy (SEM, energy dispersive X-ray spectroscopy (EDX, and microhardness testing (MH. Enamel from the affected individual (referred to as KLK4 enamel was hypomineralised in comparison with matched control enamel. Furthermore, KLK4 inner enamel was hypomineralised compared with KLK4 outer enamel. SEM showed a clear structural demarcation between KLK4 inner and outer enamel, although enamel structure was similar to control tissue overall. EDX showed that KLK4 inner enamel contained less calcium and phosphorus and more nitrogen than control inner enamel and KLK4 outer enamel. MH testing showed that KLK4 inner enamel was significantly softer than KLK4 outer enamel (p < 0.001. However, the hardness of control inner enamel was not significantly different to that of control outer enamel. Overall, these findings suggest that the KLK4 c.632delT mutation may be a common cause of autosomal recessive AI in the Pakistani population. The phenotype data obtained mirror findings in the Klk4

  19. The role of lipoprotein processing by signal peptidase II in the Gram-positive eubacterium Bacillus subtilis : Signal peptidase II is required for the efficient secretion of alpha-amylase, a non-lipoprotein

    NARCIS (Netherlands)

    Tjalsma, H; Kontinen, VP; Pragai, Z; Meima, R; Venema, G; Bron, S; Sarvas, M; van Dijl, JM

    1999-01-01

    Computer-assisted analyses indicate that Bacillus subtilis contains approximately 300 genes for exported proteins with an amino-terminal signal peptide. About 114 of these are lipoproteins, which are retained in the cytoplasmic membrane. We have investigated the importance of lipoprotein processing

  20. Identification of novel human dipeptidyl peptidase-IV inhibitors of natural origin (part I: virtual screening and activity assays.

    Directory of Open Access Journals (Sweden)

    Laura Guasch

    Full Text Available BACKGROUND: There has been great interest in determining whether natural products show biological activity toward protein targets of pharmacological relevance. One target of particular interest is DPP-IV whose most important substrates are incretins that, among other beneficial effects, stimulates insulin biosynthesis and secretion. Incretins have very short half-lives because of their rapid degradation by DPP-IV and, therefore, inhibiting this enzyme improves glucose homeostasis. As a result, DPP-IV inhibitors are of considerable interest to the pharmaceutical industry. The main goals of this study were (a to develop a virtual screening process to identify potential DPP-IV inhibitors of natural origin; (b to evaluate the reliability of our virtual-screening protocol by experimentally testing the in vitro activity of selected natural-product hits; and (c to use the most active hit for predicting derivatives with higher binding affinities for the DPP-IV binding site. METHODOLOGY/PRINCIPAL FINDINGS: We predicted that 446 out of the 89,165 molecules present in the natural products subset of the ZINC database would inhibit DPP-IV with good ADMET properties. Notably, when these 446 molecules were merged with 2,342 known DPP-IV inhibitors and the resulting set was classified into 50 clusters according to chemical similarity, there were 12 clusters that contained only natural products for which no DPP-IV inhibitory activity has been previously reported. Nine molecules from 7 of these 12 clusters were then selected for in vitro activity testing and 7 out of the 9 molecules were shown to inhibit DPP-IV (where the remaining two molecules could not be solubilized, preventing the evaluation of their DPP-IV inhibitory activity. Then, the hit with the highest activity was used as a lead compound in the prediction of more potent derivatives. CONCLUSIONS/SIGNIFICANCE: We have demonstrated that our virtual-screening protocol was successful in identifying novel lead compounds for developing more potent DPP-IV inhibitors.

  1. Two galactose-α-1,3-galactose carrying peptidases from pork kidney mediate anaphylactogenic responses in delayed meat allergy

    DEFF Research Database (Denmark)

    Hilger, Christiane; Fischer, Jörg; Swiontek, Kyra;

    2016-01-01

    BACKGROUND: Serum IgE-antibodies directed at galactose-α-1,3-galactose (α-Gal) are associated with a novel form of delayed anaphylaxis occuring upon consumption of red meat or innards. Pork kidney is known as the most potent trigger of this syndrome, but the culprit allergens have not yet been....... Proteins were identified by peptide mass fingerprinting. Isolated proteins were assayed in ELISA and ELISA inhibition, basophil activation and skin prick test. RESULTS: Several IgE-binding proteins of high molecular weight (100 - >200 kDa) were detected in pork kidney extracts by immunoblot using patient...... resistent to heat denaturation. Pork kidney extract, isolated ACE I and AP-N were able to activate patient basophils and elicit positive responses in skin prick tests. CONCLUSION: Two cell-membrane proteins carrying α-Gal epitopes were identified in pork kidney. For the first time, isolated meat proteins...

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

    OpenAIRE

    Suman RK; Mohanty IR; Maheshwari U; Borde MK; Deshmukh YA

    2016-01-01

    Rajesh Kumar Suman,1 Ipseeta Ray Mohanty,1 Ujwala Maheshwari,2 Manjusha K Borde,1 YA Deshmukh1 1Department of Pharmacology, 2Department of Pathology, MGM Medical College, Kamothe, Navi Mumbai, India 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 ...

  3. Association of variants in the carnosine peptidase 1 gene (CNDP1) with diabetic nephropathy in American Indians.

    Science.gov (United States)

    Chakkera, Harini A; Hanson, Robert L; Kobes, Sayuko; Millis, Meredith P; Nelson, Robert G; Knowler, William C; Distefano, Johanna K

    2011-06-01

    CNDP1 is located on 18q22.3, where linkage with diabetic nephropathy has been observed in several populations, including Pima Indians. However, evidence for association between CNDP1 alleles and diabetic nephropathy is equivocal and population-dependent. This study investigated CNDP1 as a candidate for diabetic kidney disease in Pima Indians. Nineteen tag single nucleotide polymorphisms spanning the CNDP1 locus were selected using genotype data from Chinese individuals in the HapMap resource along with 2 variants previously associated with diabetic nephropathy. All variants were genotyped in 3 different samples including a diabetic end-stage renal disease (ESRD) case-control study, a family-based study of diabetic individuals who participated in the linkage study for nephropathy, and a cohort of diabetic individuals in whom longitudinal measures of glomerular filtration rates (GFR) were performed. There was no statistically significant evidence for association with diabetic ESRD. However, nominal evidence for association was found in the family study, where markers rs12957330 (Odds ratio [OR]=0.29 per copy of G allele; p=0.04) and rs17817077 (OR=0.46 per copy of G allele; p=0.05) were associated with diabetic nephropathy. In addition, markers rs12964454, rs7244647, and rs7229005 were associated with changes in GFR (-8.5ml/min per copy of the G allele; p=0.04; 18.8ml/min per copy of the C allele; p=0.03; and -13.4ml/min per copy of the C allele; p=0.001, respectively). These findings provide nominal evidence supporting a role between CNDP1 variants and diabetic kidney disease.

  4. Monitoring of the effects of transfection with baculovirus on Sf9 cell line and expression of human dipeptidyl peptidase IV.

    Science.gov (United States)

    Ustün-Aytekin, Ozlem; Gürhan, Ismet Deliloğlu; Ohura, Kayoko; Imai, Teruko; Ongen, Gaye

    2014-01-01

    Human dipeptidylpeptidase IV (hDPPIV) is an enzyme that is in hydrolase class and has various roles in different parts of human body. Its deficiency may cause some disorders in the gastrointestinal, neurologic, endocrinological and immunological systems of humans. In the present study, hDPPIV enzyme was expressed on Spodoptera frugiperda (Sf9) cell lines as a host cell, and the expression of hDPPIV was obtained by a baculoviral expression system. The enzyme production, optimum multiplicity of infection, optimum transfection time, infected and uninfected cell size and cell behavior during transfection were also determined. For maximum hDPPIV (269 mU mL(-1)) enzyme, optimum multiplicity of infection (MOI) and time were 0.1 and 72 h, respectively. The size of infected cells increased significantly (P Sf9 cell line was applicable to the large scale for hDPPIV expression by using optimized parameters (infection time and MOI) because of its high productivity (4.03 mU m L(-1) h(-1)).

  5. Conversion of des-tyrosine-y-endorphin by brain synaptic membrane associated peptidases: identification of generated peptide fragments

    NARCIS (Netherlands)

    Burbach, J.P.H.; Schotman, P.; Verhoef, J.; Kloet, E.R. de; Wied, D. de

    1980-01-01

    Des-tyrosine-γ-endorphin, a β-endorphin fragment with neuroleptic-like properties, was digested with a cSPM fraction of rat brain. A profile of metabolites and a time course of conversion were obtained by HPLC analysis of the digests. Quantitative amino acid analysis and a second HPLC fractionation

  6. Dipeptidyl peptidase IV inhibition enhances the intestinotrophic effect of glucagon-like peptide-2 in rats and mice

    DEFF Research Database (Denmark)

    Hartmann, B; Thulesen, J; Kissow, Hannelouise;

    2000-01-01

    s.c. injection of GLP-2 with or without the specific DPP-IV inhibitor, valine-pyrrolidide (VP). Rats were injected s.c. with 40 microg GLP-2 or 40 microg GLP-2+15 mg VP. Plasma was collected at different time points and analyzed, by RIA, for intact GLP-2. Rats were treated for 14 days with: saline...

  7. Dipeptidyl Peptidase IV Inhibitor Improves Insulin Resistance and Steatosis in a Refractory Nonalcoholic Fatty Liver Disease Patient: A Case Report

    Directory of Open Access Journals (Sweden)

    Minoru Itou

    2012-08-01

    Full Text Available A 67-year-old Asian woman was referred to Kurume University Hospital due to abnormal liver function tests. She was diagnosed with nonalcoholic fatty liver disease (NAFLD. NAFLD was treated by diet therapy with medication of metformin and pioglitazone; however, NAFLD did not improve. Subsequently, the patient was administered sitagliptin. Although her energy intake and physical activity did not change, her hemoglobin A1c level was decreased from 7.8 to 6.4% 3 months after treatment. Moreover, her serum insulin level and homeostasis model assessment-insulin resistance value were also improved, as was the severity of hepatic steatosis. These findings indicate that sitagliptin may improve insulin resistance and steatosis in patients with refractory NAFLD.

  8. Intestinal physiology and peptidase activity in male pigs are modulated by consumption of corn culture extracts containing fumonisins

    National Research Council Canada - National Science Library

    Lessard, Martin; Boudry, Gaëlle; Sève, Bernard; Oswald, Isabelle P; Lallès, Jean-Paul

    2009-01-01

    .... Therefore, we tested the hypothesis that repeated intake of a corn culture extract rich in fumonisins, mainly in FB1, alters indices of intestinal absorptive and secretory physiology and barrier function in vivo...

  9. Cost-effectiveness of dipeptidyl peptidase-4 inhibitor monotherapy in elderly type 2 diabetes patients in Thailand

    OpenAIRE

    Permsuwan U; Dilokthornsakul P; Saokaew S; Thavorn K; Chaiyakunapruk N

    2016-01-01

    Unchalee Permsuwan,1 Piyameth Dilokthornsakul,2 Surasak Saokaew,2–4 Kednapa Thavorn,5–7 Nathorn Chaiyakunapruk2,4,8,9 1Faculty of Pharmacy Chiang Mai University, Chiang Mai, 2Center of Pharmaceutical Outcomes Research, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, 3Center of Health Outcomes Research and Therapeutic Safety, School of Pharmaceutical Sciences, University of Phayao, Phayao, Thailand; 4School of Pharmacy, Monash University Malaysia, Sunway, Mala...

  10. Cost-effectiveness of dipeptidyl peptidase-4 inhibitor monotherapy in elderly type 2 diabetes patients in Thailand

    OpenAIRE

    Permsuwan, Unchalee; Dilokthornsakul, Piyameth; Saokaew, Surasak; Thavorn, Kednapa; Chaiyakunapruk, Nathorn

    2016-01-01

    Unchalee Permsuwan,1 Piyameth Dilokthornsakul,2 Surasak Saokaew,2–4 Kednapa Thavorn,5–7 Nathorn Chaiyakunapruk2,4,8,9 1Faculty of Pharmacy Chiang Mai University, Chiang Mai, 2Center of Pharmaceutical Outcomes Research, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, 3Center of Health Outcomes Research and Therapeutic Safety, School of Pharmaceutical Sciences, University of Phayao, Phayao, Thailand; 4School of Pharmacy, Monash University Malaysia, Sunw...

  11. Preliminary functional characterization, cloning and primary sequence of Fastuosain, a cysteine peptidase isolated from fruits of Bromelia fastuosa.

    Science.gov (United States)

    Cabral, Hamilton; Leopoldino, Andréia M; Tajara, Eloiza H; Greene, Lewis J; Faça, Vitor M; Mateus, Rogério P; Ceron, Carlos R; de Souza Judice, Wagner A; Julianod, Luiz; Bonilla-Rodriguez, Gustavo O

    2006-01-01

    The present work reports the characterization of Fastuosain, a novel cysteine protease of 25kDa, purified from the unripe fruits of Bromelia fastuosa, a wild South American Bromeliaceae. Proteolytic activity, measured using casein and synthetic substrates, was dependent on the presence of thiol reagents, having maximum activity at pH 7.0. The present work reports cDNA cloning of Fastuosain; cDNA was amplified by PCR using specific primers. The product was 1096pb long. Mature fastuosain has 217 residues, and with the proregion has a total length of 324 residues. Its primary sequence showed high homology with ananain(74%), stem bromelain (66%) and papain (44%).

  12. Dipeptidyl peptidase-4 (DPP-4) inhibitors are favourable to glucagon-like peptide-1 (GLP-1) agonists

    DEFF Research Database (Denmark)

    Madsbad, Sten

    2012-01-01

    Incretin-based therapies, which include the GLP-1 receptor agonists and DPP-4 inhibitors, use the antidiabetic properties of potentiating the GLP-1 receptor signalling via the regulation of insulin and glucagon secretion, inhibition of gastric emptying and suppression of appetite. Most physicians...... a DPP-4 inhibitor in obese type 2 diabetic patients, who want to lose weight. Furthermore, the GLP-1 receptor agonists cover the whole spectrum of treatment from time of diagnosis with lifestyle treatment to combination treatment with basal insulin....... are also more effective in weight and systolic blood pressure control than DPP-4 inhibitors. The side effects of the GLP-1 receptor agonists are primarily nausea and vomiting, which is less pronounced with the long acting agonists and often transient. A GLP-1 receptor agonist can be recommended before...

  13. Effect of a dipeptidyl peptidase-IV inhibitor, des-fluoro-sitagliptin, on neointimal formation after balloon injury in rats.

    Directory of Open Access Journals (Sweden)

    Soo Lim

    Full Text Available 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.

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

    Science.gov (United States)

    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...

  15. Exploring the bioprospecting and biotechnological potential of white-rot and anaerobic Neocallimastigomycota fungi: peptidases, esterases, and lignocellulolytic enzymes.

    Science.gov (United States)

    da Silva, Ronivaldo Rodrigues; Pedezzi, Rafael; Souto, Tatiane Beltramini

    2017-04-01

    Fungi constitute an invaluable natural resource for scientific research, owing to their diversity; they offer a promising alternative for bioprospecting, thus contributing to biotechnological advances. For a long time, extensive information has been exploited and fungal products have been tested as a source of natural compounds. In this context, enzyme production remains a field of interest, since it offers an efficient alternative to the hazardous processes of chemical transformations. Owing to their vast biodiversity and peculiar biochemical characteristics, two fungal categories, white-rot and anaerobic Neocallimastigomycota, have gathered considerable attention for biotechnological applications. These fungi are known for their ability to depolymerize complex molecular structures and are used in degradation of lignocellulosic biomass, improvement of animal feed digestibility, biogas and bioethanol production, and various other applications. However, there are only limited reports that describe proteolytic enzymes and esterases in these fungi and their synergistic action with lignocellulolytic enzymes on degradation of complex polymers. Thus, in this minireview, we focus on the importance of these organisms in enzyme technology, their bioprospecting, possibility of integration of their enzyme repertoire, and their prospects for future biotechnological innovation.

  16. In vitro and in vivo anti-leukemic activity of the peptidase-potentiated alkylator melflufen in acute myeloid leukemia

    Science.gov (United States)

    Strese, Sara; Bashir Saadia, Hassan; Velander, Ebba; Haglund, Caroline; Höglund, Martin; Larsson, Rolf; Gullbo, Joachim

    2017-01-01

    The novel aminopeptidase potentiated alkylating agent melflufen, was evaluated for activity in acute myeloid leukemia in a range of in vitro models, as well as in a patient derived xenograft study. All tested AML cell lines were highly sensitive to melflufen while melphalan was considerably less potent. In the HL-60 cell line model, synergy was observed for the combination of melflufen and cytarabine, an interaction that appeared sequence dependent with increased synergy when melflufen was added before cytarabine. Also, in primary cultures of AML cells from patients melflufen was highly active, while normal PBMC cultures appeared less sensitive, indicating a 7-fold in vitro therapeutic index. Melphalan, on the other hand, was only 2-fold more potent in the AML patient samples compared with PBMCs. Melflufen was equally active against non-malignant, immature CD34+ progenitor cells and a more differentiated CD34+ derived cell population (GM14), whereas the stem cell like cells were less sensitive to melphalan. Finally, melflufen treatment showed significant anti-leukemia activity and increased survival in a patient derived xenograft of AML in mice. In conclusion, melflufen demonstrates high and significant preclinical activity in AML and further clinical evaluation seem warranted in this disease. PMID:27974676

  17. Human lysozyme peptidase resistance is perturbed by the anionic glycolipid biosurfactant rhamnolipid produced by the opportunistic pathogen Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Andersen, Kell K; Vad, Brian Stougaard; Scavenius, Carsten;

    2016-01-01

    Infection by the opportunistic pathogen Pseudomonas aeruginosa (PA) is accompanied by the secretion of virulence factors such as the secondary metabolite rhamnolipid (RL) as well as an array of bacterial enzymes, including the protease elastase. The human immune system tries to counter this via...... defensive proteins such as human lysozyme (HLZ). HLZ targets the bacterial cell wall but may also have other antimicrobial activities. The enzyme contains four disulfide bonds and shows high thermodynamic stability and resistance to proteolytic attack. Here we show that RL promotes HLZ degradation...... by several unrelated proteases, including the PA elastase and human proteases. This occurs although RL does not by itself denature HLZ. Nevertheless, RL binds in a sufficiently high stoichiometry (8 RL:1 HLZ) to neutralize the highly cationic surface of HLZ. The initial cleavage sites agree well...

  18. SwissProt search result: AK100736 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100736 J023118B01 (P22411) Dipeptidyl peptidase 4 (EC 3.4.14.5) (Dipeptidyl pepti...dase IV) (DPP IV) (T-cell activation antigen CD26) [Contains: Dipeptidyl peptidase 4 membrane form (Dipeptidyl pep...tidase IV membrane form); Dipeptidyl peptidase 4 soluble form (Dipep DPP4_PIG 6e-45 ...

  19. SwissProt search result: AK060335 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060335 001-008-C02 (P22411) Dipeptidyl peptidase 4 (EC 3.4.14.5) (Dipeptidyl pept...idase IV) (DPP IV) (T-cell activation antigen CD26) [Contains: Dipeptidyl peptidase 4 membrane form (Dipeptidyl pep...tidase IV membrane form); Dipeptidyl peptidase 4 soluble form (Dipep DPP4_PIG 6e-31 ...

  20. SwissProt search result: AK110189 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110189 002-162-A04 (P22411) Dipeptidyl peptidase 4 (EC 3.4.14.5) (Dipeptidyl pept...idase IV) (DPP IV) (T-cell activation antigen CD26) [Contains: Dipeptidyl peptidase 4 membrane form (Dipeptidyl pep...tidase IV membrane form); Dipeptidyl peptidase 4 soluble form (Dipep DPP4_PIG 4e-76 ...

  1. SwissProt search result: AK061101 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK061101 006-207-A12 (P07647) Submandibular glandular kallikrein-9 precursor (EC 3....4.21.35) (Tissue kallikrein) (S3 kallikrein) (Submandibular enzymatic vasoconstrictor) (SEV) (KLK-S3) [Contains: Submandibular glandu...lar kallikrein-9 light chain; Submandibular glandu KLK9_RAT 2e-18 ...

  2. SwissProt search result: AK112005 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112005 006-202-F10 (P07647) Submandibular glandular kallikrein-9 precursor (EC 3....4.21.35) (Tissue kallikrein) (S3 kallikrein) (Submandibular enzymatic vasoconstrictor) (SEV) (KLK-S3) [Contains: Submandibular glandu...lar kallikrein-9 light chain; Submandibular glandu KLK9_RAT 4e-11 ...

  3. SwissProt search result: AK109059 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK109059 002-154-F02 (P07647) Submandibular glandular kallikrein-9 precursor (EC 3....4.21.35) (Tissue kallikrein) (S3 kallikrein) (Submandibular enzymatic vasoconstrictor) (SEV) (KLK-S3) [Contains: Submandibular glandu...lar kallikrein-9 light chain; Submandibular glandu KLK9_RAT 2e-17 ...

  4. SwissProt search result: AK112119 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK112119 006-303-H09 (P07647) Submandibular glandular kallikrein-9 precursor (EC 3....4.21.35) (Tissue kallikrein) (S3 kallikrein) (Submandibular enzymatic vasoconstrictor) (SEV) (KLK-S3) [Contains: Submandibular glandu...lar kallikrein-9 light chain; Submandibular glandu KLK9_RAT 3e-11 ...

  5. SwissProt search result: AK110189 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK110189 002-162-A04 (Q86TI2) Dipeptidyl peptidase 9 (EC 3.4.14.5) (Dipeptidyl peptidase IX) (DP9) (Dipep...tidyl peptidase-like protein 9) (DPLP9) (Dipeptidyl peptidase IV-related protein 2) (DPRP-2) DPP9_HUMAN 9e-27 ...

  6. SwissProt search result: AK100736 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK100736 J023118B01 (Q86TI2) Dipeptidyl peptidase 9 (EC 3.4.14.5) (Dipeptidyl peptidase IX) (DP9) (Dipep...tidyl peptidase-like protein 9) (DPLP9) (Dipeptidyl peptidase IV-related protein 2) (DPRP-2) DPP9_HUMAN 2e-78 ...

  7. SwissProt search result: AK060335 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK060335 001-008-C02 (Q86TI2) Dipeptidyl peptidase 9 (EC 3.4.14.5) (Dipeptidyl peptidase IX) (DP9) (Dipep...tidyl peptidase-like protein 9) (DPLP9) (Dipeptidyl peptidase IV-related protein 2) (DPRP-2) DPP9_HUMAN 1e-63 ...

  8. The metabolite generated by dipeptidyl-peptidase 4 metabolism of glucagon-like peptide-1 has no influence on plasma glucose levels in patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Zander, M; Madsbad, S; Deacon, C F

    2006-01-01

    /INTERPRETATION: At approximately similar concentrations of intact GLP-1 (IV, IB, HSC), but with widely ranging metabolite concentrations, the effect on plasma glucose levels was equal, indicating that the presence of the metabolite does not antagonise the glucose-lowering effect of GLP-1....

  9. Effect of vitamin E and human placenta cysteine peptidase inhibitor on expression of cathepsins B and L in implanted hepatoma Morris 5123 tumor model in Wistar rats

    Institute of Scientific and Technical Information of China (English)

    Tadeusz Sebzda; Piotr Hanczyc; Yousif Saleh; Bernice F Akinpelumi; Maciej Siewinski; Jerzy Rudnicki

    2005-01-01

    AIM: To examine the effectiveness of human placental inhibitors, by injecting vitamin E to rats with transplanted Norris-5123 hepatoma, on the expression of cathepsins B and L in tumor, liver, lung and blood sera after transplantation of Norris 5123 hepatoma.METHODS: Animals were divided into 10 groups receiving three different concentrations of vitamin E and inhibitors along or in combination and compared with negative control (healthy rats) and positive control (tumor rats). Effectiveness of treatment was evaluated with regard to survival time,tumor response and determination of the activities of proteolytic enzymes and their inhibitors using flurogenic substrates.RESULTS: Cathepsins B and L activities were elevated by 16-fold in comparison with negative control tissues, and their endogenous inhibitor activity decreased by 1.2-fold before treatment. In several cases, tumors completely disappeared following vitamin E plus human placental cyteine protease inhibitor (CPI) compared with controls.The number of complete tumor responses was higher when 20 m/kg vitamin E plus 400 μg of CPI was used, i.e.7/10 rats survived more than two mo. Cathepsins B and L were expressed significantly in tumor, liver, lung tissues and sera in parallel to the increasing of the endogenous inhibitor activity compared with the controls after treatment (P<0.0001).CONCLUSION: The data indicate formation of metastasis significantly reduced in treated rats, which might provide a therapeutic basis for anti-cancer therapy.

  10. Vildagliptin, a dipeptidyl peptidase-IV inhibitor, improves model-assessed beta-cell function in patients with type 2 diabetes

    DEFF Research Database (Denmark)

    Mari, A; Sallas, W M; He, Y L;

    2005-01-01

    .8 +/- 1.6 pmol/liter; P P function in diabetic patients by increasing the insulin secretory tone....... in diabetic patients, suggesting that more sophisticated measures are necessary to ascertain the influence of vildagliptin on beta-cell function. METHODS: This study examined the effects of 28-d treatment with vildagliptin (100 mg, twice daily; n = 9) vs. placebo (n = 11) on beta-cell function in diabetic...... patients using a mathematical model that describes the insulin secretory rate as a function of glucose levels (beta-cell dose response), the change in glucose with time (derivative component), and a potentiation factor, which is a function of time and may reflect the actions of nonglucose secretagogues...

  11. Effects of the dipeptidyl peptidase-IV inhibitor vildagliptin on incretin hormones, islet function, and postprandial glycemia in subjects with impaired glucose tolerance

    DEFF Research Database (Denmark)

    Rosenstock, Julio; Foley, James E; Rendell, Marc;

    2008-01-01

    OBJECTIVE: This study was conducted to determine the effects of vildagliptin on incretin hormone levels, islet function, and postprandial glucose control in subjects with impaired glucose tolerance (IGT). RESEARCH DESIGN AND METHODS: A 12-week, double-blind, randomized, parallel-group study compa...

  12. Dipeptidyl Peptidase-4 Inhibitor Use Is Not Associated With Acute Pancreatitis in High-Risk Type 2 Diabetic Patients: A Nationwide Cohort Study.

    Science.gov (United States)

    Chang, Chia-Hsuin; Lin, Jou-Wei; Chen, Shu-Ting; Lai, Mei-Shu; Chuang, Lee-Ming; Chang, Yi-Cheng

    2016-02-01

    To analyze the association between use of DPP-4 inhibitors and acute pancreatitis in high-risk type 2 diabetic patients. A retrospective nationwide cohort study was conducted using the Taiwan National Health Insurance claim database. The risk associated with sitagliptin was compared to that with acarbose, a second-line antidiabetic drug prescribed for patients with similar diabetes severity and with a known neutral effect on pancreatitis. Between January 1, 2009 and December 31, 2010, a total of 8526 sitagliptin initiators and 8055 acarbose initiators who had hypertriglyceridemia or prior hospitalization history for acute pancreatitis were analyzed for the risk of hospitalization due to acute pancreatitis stratified for baseline propensity score. In the crude analysis, sitagliptin was associated with a decreased risk of acute pancreatitis (hazard ratio [HR] 0.74; 95% confidence interval [CI]: 0.62-0.88) compared to acarbose in diabetic patients with prior history of hospitalization for pancreatitis or hypertriglyceridemia. The association was abolished after stratification for propensity score quintiles (adjusted HR 0.95; 95% CI: 0.79-1.16). Similar results were found separately in both patients' histories of prior hospitalization of acute pancreatitis (adjusted HR 0.97; 95% CI: 0.76-1.24) and those with hypertriglyceridemia (adjusted HR 0.86; 95% CI: 0.65-1.13). No significant association was found for different durations or accumulative doses of sitagliptin. In the stratified analysis, no significant effect modification was found in relation to patients' characteristics. Use of sitagliptin was not associated with an increased risk of acute pancreatitis in high-risk diabetic patients with hypertriglyceridemia or with history of acute pancreatitis.

  13. Characterization of dedifferentiating human mature adipocytes from the visceral and subcutaneous fat compartments: fibroblast-activation protein alpha and dipeptidyl peptidase 4 as major components of matrix remodeling.

    Directory of Open Access Journals (Sweden)

    Julie Lessard

    Full Text Available Mature adipocytes can reverse their phenotype to become fibroblast-like cells. This is achieved by ceiling culture and the resulting cells, called dedifferentiated fat (DFAT cells, are multipotent. Beyond the potential value of these cells for regenerative medicine, the dedifferentiation process itself raises many questions about cellular plasticity and the pathways implicated in cell behavior. This work has been performed with the objective of obtaining new information on adipocyte dedifferentiation, especially pertaining to new targets that may be involved in cellular fate changes. To do so, omental and subcutaneous mature adipocytes sampled from severely obese subjects have been dedifferentiated by ceiling culture. An experimental design with various time points along the dedifferentiation process has been utilized to better understand this process. Cell size, gene and protein expression as well as cytokine secretion were investigated. Il-6, IL-8, SerpinE1 and VEGF secretion were increased during dedifferentiation, whereas MIF-1 secretion was transiently increased. A marked decrease in expression of mature adipocyte transcripts (PPARγ2, C/EBPα, LPL and Adiponectin was detected early in the process. In addition, some matrix remodeling transcripts (FAP, DPP4, MMP1 and TGFβ1 were rapidly and strongly up-regulated. FAP and DPP4 proteins were simultaneously induced in dedifferentiating mature adipocytes supporting a potential role for these enzymes in adipose tissue remodeling and cell plasticity.

  14. THE ENDOGENOUS BACILLUS-SUBTILIS (NATTO) PLASMIDS PTA1015 AND PTA1040 CONTAIN SIGNAL PEPTIDASE-ENCODING GENES - IDENTIFICATION OF A NEW STRUCTURAL MODULE ON CRYPTIC PLASMIDS

    NARCIS (Netherlands)

    MEIJER, WJJ; DEJONG, A; BEA, G; WISMAN, A; TJALSMA, H; VENEMA, G; BRON, S; MAARTEN, J; VANDIJL, JM

    Various strains of Bacillus subtilis (natto) contain small cryptic plasmids that replicate via the rolling-circle mechanism. Like plasmids from other Gram-positive bacteria, these plasmids are composed of several distinct structural modules. A new structural module was identified on the B. subtilis

  15. The Caenorhabditis elegans matrix non-peptidase MNP-1 is required for neuronal cell migration and interacts with the Ror receptor tyrosine kinase CAM-1.

    Science.gov (United States)

    Craft, Teresa R; Forrester, Wayne C

    2017-04-01

    Directed cell migration is critical for metazoan development. During Caenorhabditis elegans development many neuronal, muscle and other cell types migrate. Multiple classes of proteins have been implicated in cell migration including secreted guidance cues, receptors for guidance cues and intracellular proteins that respond to cues to polarize cells and produce the forces that move them. In addition, cell surface and secreted proteases have been identified that may clear the migratory route and process guidance cues. We report here that mnp-1 is required for neuronal cell and growth cone migrations. MNP-1 is expressed by migrating cells and functions cell autonomously for cell migrations. We also find a genetic interaction between mnp-1 and cam-1, which encodes a Ror receptor tyrosine kinase required for some of the same cell migrations.

  16. Characterization of VanYn, a novel D,D-peptidase/D,D-carboxypeptidase involved in glycopeptide antibiotic resistance in Nonomuraea sp. ATCC 39727.

    Science.gov (United States)

    Binda, Elisa; Marcone, Giorgia L; Pollegioni, Loredano; Marinelli, Flavia

    2012-09-01

    VanY(n) is a novel protein involved in the mechanism of self-resistance in Nonomuraea sp. ATCC 39727, which produces the glycopeptide antibiotic A40926, the precursor of the second-generation dalbavancin, which is in phase III of clinical development. VanY(n) (196 residues) is encoded by the dbv7 gene within the dbv biosynthetic cluster devoted to A40926 production. C-terminal His6-tagged VanY(n) was successfully expressed as a soluble and active protein in Escherichia coli. The analysis of the sequence suggests the presence of a hydrophobic transmembrane portion and two conserved sequences (SxHxxGxAxD and ExxH) in the extracytoplasmic domain that are potentially involved in coordination of Zn(2+) and catalytic activity. The presence of these conserved sequences indicates a similar mechanism of action and substrate binding in VanY(n) as in VanY, VanX and VanXY Zn(2+)-dependent D,D-carboxypeptidases and D-Ala-D-Ala dipeptidases acting on peptidoglycan maturation and involved in glycopeptide resistance in pathogens. On substrates mimicking peptidoglycan precursors, VanY(n) shows D,D-carboxypeptidase and D,D-dipeptidase activity, but lacks D,D-carboxyesterase ability on D-Ala-D-Lac-terminating peptides. VanY(n) belongs to the metallo-D,D-carboxypeptidase family, but it is inhibited by β-lactams. Its characterization provides new insights into the evolution and transfer of resistance determinants from environmental glycopeptide-producing actinomycetes (such as Nonomuraea sp.) to glycopeptide-resistant pathogens (enterococci and staphylococci). It may also contribute to an early warning system for emerging resistance mechanisms following the introduction into clinics of a second-generation glycopeptide such as dalbavancin.

  17. Up-regulated dipeptidyl-peptidase IV (CD26) on monocytes was unaffected by effective DMARD treatment in early steroid and DMARD-naive rheumatoid arthritis

    DEFF Research Database (Denmark)

    Ellingsen, Torkell Juulsgaad; Hansen, I; Thorsen, J

    2012-01-01

    To study the CD26 density on monocytes and CD4+ T-lymphocytes in steroid and DMARD-naïve, early rheumatoid arthritis (RA) patients and to analyse for correlations with disease activity, including long-term radiographic progression.......To study the CD26 density on monocytes and CD4+ T-lymphocytes in steroid and DMARD-naïve, early rheumatoid arthritis (RA) patients and to analyse for correlations with disease activity, including long-term radiographic progression....

  18. Characterization of a recombinant Cathepsin B-Like cysteine peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae): A putative target control of citrus huanglongbing

    Science.gov (United States)

    Huanglongbing (HLB) is one of the most destructive disease affecting citrus plants. The causal agent is associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) spread by the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae). Among the control strategies for H...

  19. RNA Sequencing Identifies Upregulated Kyphoscoliosis Peptidase and Phosphatidic Acid Signaling Pathways in Muscle Hypertrophy Generated by Transgenic Expression of Myostatin Propeptide

    OpenAIRE

    Yuanxin Miao; Jinzeng Yang; Zhong Xu; Lu Jing; Shuhong Zhao; Xinyun Li

    2015-01-01

    Myostatin (MSTN), a member of the transforming growth factor-β superfamily, plays a crucial negative role in muscle growth. MSTN mutations or inhibitions can dramatically increase muscle mass in most mammal species. Previously, we generated a transgenic mouse model of muscle hypertrophy via the transgenic expression of the MSTN N-terminal propeptide cDNA under the control of the skeletal muscle-specific MLC1 promoter. Here, we compare the mRNA profiles between transgenic mice and wild-type li...

  20. Sensory nerve desensitization by resiniferatoxin improves glucose tolerance and increases insulin secretion in Zucker Diabetic Fatty rats and is associated with reduced plasma activity of dipeptidyl peptidase IV

    DEFF Research Database (Denmark)

    Gram, Dorte X; Hansen, Anker J; Deacon, Carolyn F

    2005-01-01

    Sensory nerve desensitization by capsaicin has been shown to improve the diabetic condition in Zucker Diabetic Fatty rats. However, administration of capsaicin to adult rats is associated with an increased mortality. Therefore, in this experiment, we examined the influence of resiniferatoxin...

  1. Plasma dipeptidyl peptidase-IV activity in patients with type-2 diabetes mellitus correlates positively with HbAlc levels, but is not acutely affected by food intake

    DEFF Research Database (Denmark)

    Ryskjaer, Jakob; Deacon, Carolyn F.; Carr, Richard D;

    2006-01-01

    a standard meal test (566 kcal) was estimated. RESULTS: Mean fasting plasma DPP-IV activity (expressed as degradation of GLP-1) was significantly higher in this patient group compared with the control subjects (67.5 +/- 1.9 vs 56.8 +/- 2.2 fmol GLP-1/h (mean +/- s.e.m.); P=0.001). In the type-2 diabetic...... patients and control subjects. DESIGN: The study included two protocols. Protocol one involved 40 fasting type-2 diabetic patients (28 men); age 61 +/- 1.4 (mean +/- s.e.m.) years; body mass index (BMI) 31 +/- 0.6 kg/m(2); HbAlc 7.2 +/- 0.2%; and 20 matched control subjects (14 men) were studied. Protocol...

  2. The Stable Interaction Between Signal Peptidase LepB of Escherichia coli and Nuclease Bacteriocins Promotes Toxin Entry into the Cytoplasm.

    Science.gov (United States)

    Mora, Liliana; Moncoq, Karine; England, Patrick; Oberto, Jacques; de Zamaroczy, Miklos

    2015-12-25

    LepB is a key membrane component of the cellular secretion machinery, which releases secreted proteins into the periplasm by cleaving the inner membrane-bound leader. We showed that LepB is also an essential component of the machinery hijacked by the tRNase colicin D for its import. Here we demonstrate that this non-catalytic activity of LepB is to promote the association of the central domain of colicin D with the inner membrane before the FtsH-dependent proteolytic processing and translocation of the toxic tRNase domain into the cytoplasm. The novel structural role of LepB results in a stable interaction with colicin D, with a stoichiometry of 1:1 and a nanomolar Kd determined in vitro. LepB provides a chaperone-like function for the penetration of several nuclease-type bacteriocins into target cells. The colicin-LepB interaction is shown to require only a short peptide sequence within the central domain of these bacteriocins and to involve residues present in the short C-terminal Box E of LepB. Genomic screening identified the conserved LepB binding motif in colicin-like ORFs from 13 additional bacterial species. These findings establish a new paradigm for the functional adaptability of an essential inner-membrane enzyme.

  3. RNA Sequencing Identifies Upregulated Kyphoscoliosis Peptidase and Phosphatidic Acid Signaling Pathways in Muscle Hypertrophy Generated by Transgenic Expression of Myostatin Propeptide

    OpenAIRE

    Yuanxin Miao; Jinzeng Yang; Zhong Xu; Lu Jing; Shuhong Zhao; Xinyun Li

    2015-01-01

    Myostatin (MSTN), a member of the transforming growth factor-β superfamily, plays a crucial negative role in muscle growth. MSTN mutations or inhibitions can dramatically increase muscle mass in most mammal species. Previously, we generated a transgenic mouse model of muscle hypertrophy via the transgenic expression of the MSTN N-terminal propeptide cDNA under the control of the skeletal muscle-specific MLC1 promoter. Here, we compare the mRNA profiles between transgenic mice and wild-type li...

  4. Characterization of dedifferentiating human mature adipocytes from the visceral and subcutaneous fat compartments: fibroblast-activation protein alpha and dipeptidyl peptidase 4 as major components of matrix remodeling.

    Science.gov (United States)

    Lessard, Julie; Pelletier, Mélissa; Biertho, Laurent; Biron, Simon; Marceau, Simon; Hould, Frédéric-Simon; Lebel, Stéfane; Moustarah, Fady; Lescelleur, Odette; Marceau, Picard; Tchernof, André

    2015-01-01

    Mature adipocytes can reverse their phenotype to become fibroblast-like cells. This is achieved by ceiling culture and the resulting cells, called dedifferentiated fat (DFAT) cells, are multipotent. Beyond the potential value of these cells for regenerative medicine, the dedifferentiation process itself raises many questions about cellular plasticity and the pathways implicated in cell behavior. This work has been performed with the objective of obtaining new information on adipocyte dedifferentiation, especially pertaining to new targets that may be involved in cellular fate changes. To do so, omental and subcutaneous mature adipocytes sampled from severely obese subjects have been dedifferentiated by ceiling culture. An experimental design with various time points along the dedifferentiation process has been utilized to better understand this process. Cell size, gene and protein expression as well as cytokine secretion were investigated. Il-6, IL-8, SerpinE1 and VEGF secretion were increased during dedifferentiation, whereas MIF-1 secretion was transiently increased. A marked decrease in expression of mature adipocyte transcripts (PPARγ2, C/EBPα, LPL and Adiponectin) was detected early in the process. In addition, some matrix remodeling transcripts (FAP, DPP4, MMP1 and TGFβ1) were rapidly and strongly up-regulated. FAP and DPP4 proteins were simultaneously induced in dedifferentiating mature adipocytes supporting a potential role for these enzymes in adipose tissue remodeling and cell plasticity.

  5. The impact of dipeptidyl peptidase 4 inhibition on incretin effect, glucose tolerance, and gastrointestinal-mediated glucose disposal in healthy subjects

    DEFF Research Database (Denmark)

    Rhee, Nicolai Alexander; Østoft, Signe Harring; Holst, Jens Juul

    2014-01-01

    /mol [5.3±0.1%]) were randomised to two paired study days comprising a 4h 50 g-OGTT with paracetamol (A) and an isoglycaemic i.v. glucose infusion (IIGI) (B), with (A1+B1) and without (A2+B2) preceding administration of the DPP-4 inhibitor sitagliptin. Results Isoglycaemia was obtained in all subjects...... gastric emptying (Tmax for plasma paracetamol: 86±9 vs 80±12 min, p=0.60) changed following DPP-4 inhibition. Conclusions These results suggest that acute increases in active incretin hormone levels do not affect glucose tolerance, GIGD, incretin effect, glucagon responses or gastric emptying in healthy...

  6. CD26/dipeptidyl peptidase IV (CD26/DPPIV is highly expressed in peripheral blood of HIV-1 exposed uninfected Female sex workers

    Directory of Open Access Journals (Sweden)

    Meyers Adrienne FA

    2010-11-01

    Full Text Available Abstract Background Design of effective vaccines against the human immunodeficiency virus (HIV-1 continues to present formidable challenges. However, individuals who are exposed HIV-1 but do not get infected may reveal correlates of protection that may inform on effective vaccine design. A preliminary gene expression analysis of HIV resistant female sex workers (HIV-R suggested a high expression CD26/DPPIV gene. Previous studies have indicated an anti-HIV effect of high CD26/DPPIV expressing cells in vitro. Similarly, high CD26/DPPIV protein levels in vivo have been shown to be a risk factor for type 2 diabetes. We carried out a study to confirm if the high CD26/DPPIV gene expression among the HIV-R were concordant with high blood protein levels and its correlation with clinical type 2 diabetes and other perturbations in the insulin signaling pathway. Results A quantitative CD26/DPPIV plasma analysis from 100 HIV-R, 100 HIV infected (HIV + and 100 HIV negative controls (HIV Neg showed a significantly elevated CD26/DPPIV concentration among the HIV-R group (mean 1315 ng/ml than the HIV Neg (910 ng/ml and HIV + (870 ng/ml, p Conclusion HIV resistant sex workers have a high expression of CD26/DPPIV in tandem with lowered immune activation markers. This may suggest a novel role for CD26/DPPIV in protection against HIV infection in vivo.

  7. Evidence for Cleavage of the Metalloprotease Vsm from Vibrio splendidus Strain JZ6 by an M20 Peptidase (PepT-like Protein) at Low Temperature

    OpenAIRE

    2016-01-01

    Metalloprotease Vsm is a major extracellular virulence factor of Vibrio splendidus. The toxicity of Vsm from V. splendidus strain JZ6 has been characterized, and production of this virulence factor proved to be temperature-regulated. The present study provides evidence that two forms (JZE1 and JZE2) of Vsm protein exist in extracellular products (ECPs) of strain JZ6, and a significant conversion of these two forms was detected by SDS-PAGE and immunoblotting analyses of samples obtained from c...

  8. Neutral endopeptidase 24.11 and dipeptidyl peptidase IV are both mediators of the degradation of glucagon-like peptide 1 in the anaesthetised pig

    DEFF Research Database (Denmark)

    Plamboeck, A; Holst, Jens Juul; Carr, R D;

    2005-01-01

    glucose were unaffected by candoxatril, but glucose tolerance was improved (DeltaAUC(min 27-87) 118+/-5 to 74+/-14 min.mmol.l(-1); glucose elimination rate [k] 6.6+/-0.5 to 8.6+/-0.5%; pvaline pyrrolidide (a DPP-IV inhibitor), changes in C-terminal GLP-1...... pharmacokinetics mirrored those seen when candoxatril alone was administered (t(1/2) 2.7+/-0.3 and 7.7+/-0.8 min; MCR 17.3+/-2.6 and 6.5+/-0.8 ml.kg(-1).min(-1) for valine pyrrolidide without and with candoxatril, respectively). However, intact GLP-1 pharmacokinetics were improved (t(1/2) 2.8+/-0.3 and 7...

  9. The efficacy and safety of the dipeptidyl peptidase-4 inhibitor saxagliptin in treatment-naïve patients with type 2 diabetes mellitus: a randomized controlled trial

    Directory of Open Access Journals (Sweden)

    Frederich Robert

    2012-07-01

    Full Text Available Abstract Background The aim of this study was to assess efficacy and safety of saxagliptin monotherapy for up to 76 weeks in patients with type 2 diabetes mellitus (T2DM and inadequate glycemic control, with main efficacy assessment at 24 weeks. Methods 365 treatment-naïve patients with T2DM (HbA1c 7.0%–10.0% were treated with saxagliptin 2.5 mg q.A.M., saxagliptin 2.5 mg q.A.M. with possible titration to saxagliptin 5 mg, saxagliptin 5 mg q.A.M., saxagliptin 5 mg q.P.M., or placebo. After week 24, patients in all groups were eligible for titration to saxagliptin 10 mg based on HbA1c ≥7%, and all unrescued placebo patients began blinded metformin 500 mg/day. Rescue with open-label metformin was available for patients with inadequate glycemic control. Results At week 24, placebo-subtracted mean HbA1c reduction from baseline (LOCF was significantly greater in the saxagliptin treatment groups vs placebo, and remained greater through week 76. Serious adverse events (AEs and discontinuations due to AEs were similar in saxagliptin and control groups; incidence of confirmed hypoglycemia was low across all treatment groups (saxagliptin-treated, 2 [0.7]; control, 1 [1.4]. Conclusions In treatment-naïve patients with T2DM, saxagliptin monotherapy demonstrated statistically significant improvement in HbA1c compared with placebo at 24 weeks and was generally well tolerated for up to 76 weeks. Trial registration ClinicalTrials.gov Identifier: NCT00316082

  10. Serum dipeptidyl peptidase-4 activity in insulin resistant patients with non-alcoholic fatty liver disease: a novel liver disease biomarker.

    Directory of Open Access Journals (Sweden)

    Gábor Firneisz

    Full Text Available BACKGROUND: In a cross-sectional study we studied the fasting serum DPP-4 enzymatic activity (sDPP-4 and the insulin resistance index (HOMA2-IR in gliptin naïve patients with type 2 diabetes and in non-alcoholic fatty liver disease (NAFLD and in healthy controls (CNTRL. METHODS AND FINDINGS: sDPP-4 was measured by kinetic assay in 39 NAFLD (F/M:19/20, mean age: 47.42 yrs and 82 type 2 diabetes (F/M:48/34, 62.8 yrs patients and 26 (F/M:14/12, 35.3 yrs controls. Definition of T2D group as patients with type 2 diabetes but without clinically obvious liver disease created non-overlapping study groups. Diagnosis of NAFLD was based on ultrasonography and the exclusion of other etiololgy. Patients in T2D and NAFLD groups were similarly obese. 75 g CH OGTT in 39 NAFLD patients: 24-NGT, 4-IGT or IFG ("prediabetes", 11-type 2 diabetes. HOMA2-IR: CNTRL: 1.44; T2D-group: 2.62 (p = 0.046 vs CNTRL, parametric tests; NAFLD(NGTonly: 3.23 (p = 0.0013 vs CNTRL; NAFLD(IFG/IGT/type 2 diabetes: 3.82 (p<0.001 vs CNTRL, p = 0.049 vs 2TD group. sDPP-4 activity was higher in NAFLD both with NGT (mean:33.08U/L and abnormal glucose metabolism (30.38U/L than in CNTRL (25.89U/L, p<0.001 and p = 0.013 or in T2D groups (23.97U/L, p<0.001 and p = 0.004. Correlations in NAFLD among sDPP-4 and ALT: r = 0.4637,p = 0.0038 and gammaGT: r = 0.4991,p = 0.0017 and HOMA2-IR: r = 0.5295,p = 0.0026 and among HOMA2-IR and ALT: r = 0.4340,p = 0.0147 and gammaGT: r = 0.4128,p = 0.0210. CONCLUSIONS: The fasting serum DPP-4 activity was not increased in T2D provided that patients with liver disease were intentionally excluded. The high serum DPP-4 activities in NAFLD were correlated with liver tests but not with the fasting plasma glucose or HbA1C supporting that the excess is of hepatic origin and it might contribute to the speedup of metabolic deterioration. The correlation among gammaGT, ALT and serum DPP-4 activity and also between serum DPP-4 activity and HOMA2-IR in NAFLD strongly suggests that serum DPP-4 activity should be considered as a novel liver disease biomarker.

  11. The