WorldWideScience

Sample records for metalloproteinase cleavage sites

  1. Degradation of tropoelastin by matrix metalloproteinases--cleavage site specificities and release of matrikines.

    Science.gov (United States)

    Heinz, Andrea; Jung, Michael C; Duca, Laurent; Sippl, Wolfgang; Taddese, Samuel; Ihling, Christian; Rusciani, Anthony; Jahreis, Günther; Weiss, Anthony S; Neubert, Reinhard H H; Schmelzer, Christian E H

    2010-04-01

    To provide a basis for the development of approaches to treat elastin-degrading diseases, the aim of this study was to investigate the degradation of the natural substrate tropoelastin by the elastinolytic matrix metalloproteinases MMP-7, MMP-9, and MMP-12 and to compare the cleavage site specificities of the enzymes using complementary MS techniques and molecular modeling. Furthermore, the ability of the three proteases to release bioactive peptides was studied. Tropoelastin was readily degraded by all three MMPs. Eighty-nine cleavage sites in tropoelastin were identified for MMP-12, whereas MMP-7 and MMP-9 were found to cleave at only 58 and 63 sites, respectively. Cleavages occurred predominantly in the N-terminal and C-terminal regions of tropoelastin. With respect to the cleavage site specificities, the study revealed that all three MMPs similarly tolerate hydrophobic and/or aliphatic amino acids, including Pro, Gly, Ile, and Val, at P(1)'. MMP-7 shows a strong preference for Leu at P(1)', which is also well accepted by MMP-9 and MMP-12. Of all three MMPs, MMP-12 best tolerates bulky charged and aromatic amino acids at P(1)'. All three MMPs showed a clear preference for Pro at P(3) that could be structurally explained by molecular modeling. Analysis of the generated peptides revealed that all three MMPs show a similar ability to release bioactive sequences, with MMP-12 producing the highest number of these peptides. Furthermore, the generated peptides YTTGKLPYGYGPGG, YGARPGVGVGGIP, and PGFGAVPGA, containing GxxPG motifs that have not yet been proven to be bioactive, were identified as new matrikines upon biological activity testing.

  2. Active site specificity profiling of the matrix metalloproteinase family: Proteomic identification of 4300 cleavage sites by nine MMPs explored with structural and synthetic peptide cleavage analyses.

    Science.gov (United States)

    Eckhard, Ulrich; Huesgen, Pitter F; Schilling, Oliver; Bellac, Caroline L; Butler, Georgina S; Cox, Jennifer H; Dufour, Antoine; Goebeler, Verena; Kappelhoff, Reinhild; Keller, Ulrich Auf dem; Klein, Theo; Lange, Philipp F; Marino, Giada; Morrison, Charlotte J; Prudova, Anna; Rodriguez, David; Starr, Amanda E; Wang, Yili; Overall, Christopher M

    2016-01-01

    Secreted and membrane tethered matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Sites (PICS) method to simultaneously profile both the prime and non-prime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6' in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1' (with a ~32-93% preference for leucine depending on the MMP), and basic and small residues in P2' and P3', respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptide with the universally preferred P1' leucine, an unexpected negative cooperativity emerged. This was not observed in previous studies, probably due to the paucity of approaches that profile both the prime and non-prime sides together, and the masking of subsite cooperativity effects by global heat maps and iceLogos. These caveats make it critical to check for these biologically highly important effects by fixing all 20 amino acids one-by-one in the respective subsites and thorough assessing of the inferred specificity logo changes. Indeed an analysis of bona fide MEROPS physiological substrate cleavage data revealed that of the 37 natural substrates with either a P3-Pro or a P1'-Leu only 5 shared both features, confirming the PICS data. Upon probing with several new quenched-fluorescent peptides, rationally designed on our specificity data, the negative cooperativity was explained by reduced non-prime side flexibility constraining accommodation of the rigidifying P3 proline with

  3. Mapping of matrix metalloproteinase cleavage sites on syndecan-1 and syndecan-4 ectodomains

    DEFF Research Database (Denmark)

    Manon-Jensen, Tina; Multhaupt, Hinke A B; Couchman, John R

    2013-01-01

    , which makes them key effectors in the pericellular microenvironment. Extracellular shedding of syndecans by tumour-associated matrix metalloproteinases (MMPs) may have an important role in tumour progression. Such ectodomain shedding generates soluble ectodomains that may function as paracrine......Syndecans are transmembrane heparan sulfate proteoglycans with roles in cell proliferation, differentiation, adhesion, and migration. They have been associated with multiple functions in tumour progression, through their ability to interact with a wide range of ligands as well as other receptors...

  4. Site specificity of DSP-PP cleavage by BMP1.

    Science.gov (United States)

    Yang, Robert T; Lim, Glendale L; Yee, Colin T; Fuller, Robert S; Ritchie, Helena H

    2014-08-01

    Bone morphogenic protein 1 (BMP1), a metalloproteinase, is known to cleave a wide variety of extracellular matrix proteins, suggesting that a consensus substrate cleavage amino acid sequence might exist. However, while such a consensus sequence has been proposed based on P4 to P4' (i.e. the four amino acids flanking either side of the BMP1 cleavage site; P4P3P2P1|P1'P2'P3'P4') sequence homologies between two BMP1 substrates, dentin matrix protein 1 and dentin sialoprotein phosphophoryn (DSP-PP) (i.e. xMQx|DDP), no direct testing has so far been attempted. Using an Sf9 cell expression system, we have been able to produce large amounts of uncleaved DSP-PP. Point mutations introduced into this recombinant DSP-PP were then tested for their effects on DSP-PP cleavage by either Sf9 endogenous tolloid-related protein 1 (TLR-1) or by its human homolog, BMP1. Here, we have measured DSP-PP cleavage efficiencies after modifications based on P4-P4' sequence comparisons with dentin matrix protein 1, as well as for prolysyl oxidase and chordin, two other BMP1 substrates. Our results demonstrate that any mutations within or outside of the DSP-PP P4 to P4' cleavage site can block, impair or accelerate DSP-PP cleavage, and suggest that its BMP1 cleavage site is highly conserved in order to regulate its cleavage efficiency, possibly with additional assistance from its conserved exosites. Thus, BMP1 cleavage cannot be based on a consensus substrate cleavage site.

  5. Trichomonas vaginalis metalloproteinase induces mTOR cleavage of SiHa cells.

    Science.gov (United States)

    Quan, Juan-Hua; Choi, In-Wook; Yang, Jung-Bo; Zhou, Wei; Cha, Guang-Ho; Zhou, Yu; Ryu, Jae-Sook; Lee, Young-Ha

    2014-12-01

    Trichomonas vaginalis secretes a number of proteases which are suspected to be the cause of pathogenesis; however, little is understood how they manipulate host cells. The mammalian target of rapamycin (mTOR) regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. We detected various types of metalloproteinases including GP63 protein from T. vaginalis trophozoites, and T. vaginalis GP63 metalloproteinase was confirmed by sequencing and western blot. When SiHa cells were stimulated with live T. vaginalis, T. vaginalis excretory-secretory products (ESP) or T. vaginalis lysate, live T. vaginalis and T. vaginalis ESP induced the mTOR cleavage in both time- and parasite load-dependent manner, but T. vaginalis lysate did not. Pretreatment of T. vaginalis with a metalloproteinase inhibitor, 1,10-phenanthroline, completely disappeared the mTOR cleavage in SiHa cells. Collectively, T. vaginalis metallopeptidase induces host cell mTOR cleavage, which may be related to survival of the parasite.

  6. Dataset of cocoa aspartic protease cleavage sites

    Directory of Open Access Journals (Sweden)

    Katharina Janek

    2016-09-01

    Full Text Available The data provide information in support of the research article, “The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors” (Janek et al., 2016 [1]. Three different protein substrates were partially digested with the aspartic protease isolated from cocoa beans and commercial pepsin, respectively. The obtained peptide fragments were analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS and identified using the MASCOT server. The N- and C-terminal ends of the peptide fragments were used to identify the corresponding in-vitro cleavage sites by comparison with the amino acid sequences of the substrate proteins. The same procedure was applied to identify the cleavage sites used by the cocoa aspartic protease during cocoa fermentation starting from the published amino acid sequences of oligopeptides isolated from fermented cocoa beans.

  7. Cleavage site analysis in picornaviral polyproteins

    DEFF Research Database (Denmark)

    Blom, Nikolaj; Hansen, Jan; Blaas, Dieter

    1996-01-01

    are indeed cleaved awaits experimental verification. Additionally, we report several errors detected in the protein databases. A computer server for prediction of cleavage sites by picornaviral proteinases is publicly available at the e-mail address NetPicoRNA@cbs.dtu.dk or via WWW at http://www.cbs.dtu.dk/services/NetPicoRNA...

  8. SVM-based prediction of caspase substrate cleavage sites

    Directory of Open Access Journals (Sweden)

    Ranganathan Shoba

    2006-12-01

    Full Text Available Abstract Background Caspases belong to a class of cysteine proteases which function as critical effectors in apoptosis and inflammation by cleaving substrates immediately after unique sites. Prediction of such cleavage sites will complement structural and functional studies on substrates cleavage as well as discovery of new substrates. Recently, different computational methods have been developed to predict the cleavage sites of caspase substrates with varying degrees of success. As the support vector machines (SVM algorithm has been shown to be useful in several biological classification problems, we have implemented an SVM-based method to investigate its applicability to this domain. Results A set of unique caspase substrates cleavage sites were obtained from literature and used for evaluating the SVM method. Datasets containing (i the tetrapeptide cleavage sites, (ii the tetrapeptide cleavage sites, augmented by two adjacent residues, P1' and P2' amino acids and (iii the tetrapeptide cleavage sites with ten additional upstream and downstream flanking sequences (where available were tested. The SVM method achieved an accuracy ranging from 81.25% to 97.92% on independent test sets. The SVM method successfully predicted the cleavage of a novel caspase substrate and its mutants. Conclusion This study presents an SVM approach for predicting caspase substrate cleavage sites based on the cleavage sites and the downstream and upstream flanking sequences. The method shows an improvement over existing methods and may be useful for predicting hitherto undiscovered cleavage sites.

  9. Site Specificity of Cleavage of DSP-PP by BMP1

    Science.gov (United States)

    Yang, Robert T.; Lim, Glendale L.; Yee, Colin T.; Fuller, Robert S.; Ritchie, Helena H.

    2015-01-01

    Bone morphogenic protein 1 (BMP1), a metalloproteinase, is known to cleave a wide variety of extracellular matrix proteins, suggesting that a consensus substrate cleavage amino acid sequence might exist. However, while such a consensus sequence has been proposed based on P4 to P4′ (i.e., the four amino acids flanking either side of the BMP1 cleavage site; P4P3P2P1|P1′P2′P3′P4′) sequence homologies between two BMP1 substrates, dentin matrix protein 1 and dentin sialoprotein phosphophoryn (DSP-PP) (i.e., xMQx | DDP), no direct testing has so far been attempted. Using an Sf9 cell expression system, we have been able to produce large amounts of uncleaved DSP-PP,. Point mutations introduced into this recombinant DSP-PP were then tested for their affects on DSP-PP cleavage by either Sf9 endogenous tolloid-related protein 1 (TLR-1) or by its human homolog, BMP1. Here we have measured DSP-PP cleavage efficiencies after modifications based on P4-P4′ sequence comparisons with dentin matrix protein 1, as well as for prolysyl oxidase and chordin, two other BMP1 substrates. Our results demonstrate that any mutations within or outside of the DSP-PP P4 to P4′ cleavage site can block, impair or accelerate DSP-PP cleavage, and suggest that its BMP1 cleavage site is highly conserved in order to regulate its cleavage efficiency, possibly with additional assistance from its conserved exosites. Thus, BMP1 cleavage cannot be based on a consensus substrate cleavage site. PMID:25158199

  10. Mapping of macrophage elastase cleavage sites in insoluble human skin elastin.

    Science.gov (United States)

    Taddese, Samuel; Weiss, Anthony S; Neubert, Reinhard H H; Schmelzer, Christian E H

    2008-06-01

    Macrophage elastase (MMP-12) is a member of the family of matrix metalloproteinases (MMPs) and is active against multiple extracellular protein substrates such as elastin. Its effect on elastin is central to emphysema in the lung and photoaging of skin. Its expression in the skin increases on photodamaged skin and upon aging. Detecting and characterizing peptides cleaved in elastin, therefore, helps to understand such degradative disease processes in the skin and is also needed to assist in the rational design of agents that specifically inhibit the degradation. In this study, cleavage sites of MMP-12 in human skin elastin were extensively investigated. The peptides formed as a result of cleavages by this enzyme in the human skin elastin were characterized using mass spectrometry. A total of 41 peptides ranging from 4 to 41 amino acids were identified and 36 cleavage sites were determined. Amino acids encoded by exons 5, 6, 26, 28-31 were particularly susceptible to cleavages by MMP-12 and none or very few cleavages were detected from domains encoded by the remaining exons. The amino acid preferences of the different subsites on the catalytic domain of MMP-12 were analyzed.

  11. Variable context Markov chains for HIV protease cleavage site prediction.

    Science.gov (United States)

    Oğul, Hasan

    2009-06-01

    Deciphering the knowledge of HIV protease specificity and developing computational tools for detecting its cleavage sites in protein polypeptide chain are very desirable for designing efficient and specific chemical inhibitors to prevent acquired immunodeficiency syndrome. In this study, we developed a generative model based on a generalization of variable order Markov chains (VOMC) for peptide sequences and adapted the model for prediction of their cleavability by certain proteases. The new method, called variable context Markov chains (VCMC), attempts to identify the context equivalence based on the evolutionary similarities between individual amino acids. It was applied for HIV-1 protease cleavage site prediction problem and shown to outperform existing methods in terms of prediction accuracy on a common dataset. In general, the method is a promising tool for prediction of cleavage sites of all proteases and encouraged to be used for any kind of peptide classification problem as well.

  12. PACdb: PolyA Cleavage Site and 3'-UTR Database.

    Science.gov (United States)

    Brockman, J Michael; Singh, Priyam; Liu, Donglin; Quinlan, Sean; Salisbury, Jesse; Graber, Joel H

    2005-09-15

    The PolyA Cleavage Site and 3'-UTR Database (PACdb) is a web-accessible database that catalogs putative 3'-processing sites and 3'-UTR sequences for multiple organisms. Sites have been identified primarily via expressed sequence tag-genome alignments, enabling delineation of both the specificities and heterogeneity of 3'-processing events. By web browser or CGI: PACdb: http://harlequin.jax.org/pacdb/; AtPACdb: http://harlequin.jax.org/atpacdb/. Available online at http://harlequin.jax.org/pacdb/supplemental.php.

  13. β-Dystroglycan cleavage by matrix metalloproteinase-2/-9 disturbs aquaporin-4 polarization and influences brain edema in acute cerebral ischemia.

    Science.gov (United States)

    Yan, W; Zhao, X; Chen, H; Zhong, D; Jin, J; Qin, Q; Zhang, H; Ma, S; Li, G

    2016-06-21

    Dystroglycan (DG) is widely expressed in various tissues, and throughout the cerebral microvasculature. It consists of two subunits, α-DG and β-DG, and the cleavage of the latter by matrix metalloproteinase (MMP)-2 and -9 underlies a number of physiological and pathological processes. However, the involvement of MMP-2/-9-mediated β-DG cleavage in cerebral ischemia remains uncertain. In astrocytes, DG is crucial for maintaining the polarization of aquaporin-4 (AQP4), which plays a role in the regulation of cytotoxic and vasogenic edema. The present study aimed to explore the effects of MMP-2/-9-mediated β-DG cleavage on AQP4 polarization and brain edema in acute cerebral ischemia. A model of cerebral ischemia was established via permanent middle cerebral artery occlusion (pMCAO) in male C57BL/6 mice. Western blotting, real-time polymerase chain reaction (PCR), immunohistochemical staining, immunofluorescent staining, electron microscopy, and light microscopy were used. Captopril was applied as a selective MMP-2/-9 inhibitor. Recombinant mouse MMP (rmMMP)-2 and -9 were used in an in vitro cleavage experiment. The present study demonstrated evidence of β-DG cleavage by MMP-2/-9 in pMCAO mouse brains; this cleavage was implicated in AQP4 redistribution and brain edema in cerebral ischemia. In addition, captopril exacerbated cytotoxic edema and ameliorated vasogenic edema at 24h after pMCAO, and alleviated brain edema and neurological deficit at 48h and 72h. In conclusion, this study provides novel insight into the effects of MMP-2/-9-mediated β-DG cleavage in acute cerebral ischemia. Such findings might facilitate the development of a therapeutic strategy for the optimization of MMP-2/-9 targeted treatment in cerebral ischemia.

  14. CD40 ligand induced cytotoxicity in carcinoma cells is enhanced by inhibition of metalloproteinase cleavage and delivery via a conditionally-replicating adenovirus

    Directory of Open Access Journals (Sweden)

    Young Lawrence S

    2010-03-01

    Full Text Available Abstract Background CD40 and its ligand (CD40L play a critical role in co-ordinating immune responses. CD40 is also expressed in lymphoid malignancies and a number of carcinomas. In carcinoma cells the physiological outcome of CD40 ligation depends on the level of receptor engagement with low levels promoting cell survival and high levels inducing cell death. The most profound induction of cell death in carcinoma cells is induced by membrane-bound rather than recombinant soluble CD40L, but like other TNF family ligands, it is cleaved from the membrane by matrix metalloproteinases. Results We have generated a replication-deficient adenovirus expressing a mutant CD40L that is resistant to metalloproteinase cleavage such that ligand expression is retained at the cell membrane. Here we show that the mutated, cleavage-resistant form of CD40L is a more potent inducer of apoptosis than wild-type ligand in CD40-positive carcinoma cell lines. Since transgene expression via replication-deficient adenovirus vectors in vivo is low, we have also engineered a conditionally replicating E1A-CR2 deleted adenovirus to express mutant CD40L, resulting in significant amplification of ligand expression and consequent enhancement of its therapeutic effect. Conclusions Combined with numerous studies demonstrating its immunotherapeutic potential, these data provide a strong rationale for the exploitation of the CD40-CD40L pathway for the treatment of solid tumours.

  15. Coronavirus 3CL(pro) proteinase cleavage sites: Possible relevance to SARS virus pathology

    DEFF Research Database (Denmark)

    Kiemer, Lars; Lund, Ole; Brunak, Søren

    2004-01-01

    such as the cystic fibrosis transmembrane conductance regulator ( CFTR), transcription factors CREB-RP and OCT-I, and components of the ubiquitin pathway. Conclusions: Our prediction method NetCorona predicts coronavirus cleavage sites with high specificity and several potential cleavage candidates were identified...

  16. Cleavage of model substrates by archaeal RNase P: role of protein cofactors in cleavage-site selection.

    Science.gov (United States)

    Sinapah, Sylvie; Wu, Shiying; Chen, Yu; Pettersson, B M Fredrik; Gopalan, Venkat; Kirsebom, Leif A

    2011-02-01

    RNase P is a catalytic ribonucleoprotein primarily involved in tRNA biogenesis. Archaeal RNase P comprises a catalytic RNase P RNA (RPR) and at least four protein cofactors (RPPs), which function as two binary complexes (POP5•RPP30 and RPP21• RPP29). Exploiting the ability to assemble a functional Pyrococcus furiosus (Pfu) RNase P in vitro, we examined the role of RPPs in influencing substrate recognition by the RPR. We first demonstrate that Pfu RPR, like its bacterial and eukaryal counterparts, cleaves model hairpin loop substrates albeit at rates 90- to 200-fold lower when compared with cleavage by bacterial RPR, highlighting the functionally comparable catalytic cores in bacterial and archaeal RPRs. By investigating cleavage-site selection exhibited by Pfu RPR (±RPPs) with various model substrates missing consensus-recognition elements, we determined substrate features whose recognition is facilitated by either POP5•RPP30 or RPP21•RPP29 (directly or indirectly via the RPR). Our results also revealed that Pfu RPR + RPP21•RPP29 displays substrate-recognition properties coinciding with those of the bacterial RPR-alone reaction rather than the Pfu RPR, and that this behaviour is attributable to structural differences in the substrate-specificity domains of bacterial and archaeal RPRs. Moreover, our data reveal a hierarchy in recognition elements that dictates cleavage-site selection by archaeal RNase P.

  17. Handling Metalloproteinases.

    Science.gov (United States)

    Fridrich, Sven; Karmilin, Konstantin; Stöcker, Walter

    2016-02-02

    Substrate cleavage by metalloproteinases involves nucleophilic attack on the scissile peptide bond by a water molecule that is polarized by a catalytic metal, usually a zinc ion, and a general base, usually the carboxyl group of a glutamic acid side chain. The zinc ion is most often complexed by imidazole nitrogens of histidine side chains. This arrangement suggests that the physiological pH optimum of most metalloproteinases is in the neutral range. In addition to their catalytic metal ion, many metalloproteinases contain additional transition metal or alkaline earth ions, which are structurally important or modulate the catalytic activity. As a consequence, these enzymes are generally sensitive to metal chelators. Moreover, the catalytic metal can be displaced by adventitious metal ions from buffers or biological fluids, which may fundamentally alter the catalytic function. Therefore, handling, purification, and assaying of metalloproteinases require specific precautions to warrant their stability. Copyright © 2016 John Wiley & Sons, Inc.

  18. Identification of BACE1 cleavage sites in human voltage-gated sodium channel beta 2 subunit

    Directory of Open Access Journals (Sweden)

    Kovacs Dora M

    2010-12-01

    Full Text Available Abstract Background The voltage-gated sodium channel β2 subunit (Navβ2 is a physiological substrate of BACE1 (β-site APP cleaving enzyme and γ-secretase, two proteolytic enzymes central to Alzheimer's disease pathogenesis. Previously, we have found that the processing of Navβ2 by BACE1 and γ-secretase regulates sodium channel metabolism in neuronal cells. In the current study we identified the BACE1 cleavage sites in human Navβ2. Results We found a major (147-148 L↓M, where ↓ indicates the cleavage site and a minor (144145 L↓Q BACE1 cleavage site in the extracellular domain of human Navβ2 using a cell-free BACE1 cleavage assay followed by mass spectrometry. Next, we introduced two different double mutations into the identified major BACE1 cleavage site in human Navβ2: 147LM/VI and 147LM/AA. Both mutations dramatically decreased the cleavage of human Navβ2 by endogenous BACE1 in cell-free BACE1 cleavage assays. Neither of the two mutations affected subcellular localization of Navβ2 as confirmed by confocal fluorescence microscopy and subcellular fractionation of cholesterol-rich domains. Finally, wildtype and mutated Navβ2 were expressed along BACE1 in B104 rat neuroblastoma cells. In spite of α-secretase still actively cleaving the mutant proteins, Navβ2 cleavage products decreased by ~50% in cells expressing Navβ2 (147LM/VI and ~75% in cells expressing Navβ2 (147LM/AA as compared to cells expressing wildtype Navβ2. Conclusion We identified a major (147-148 L↓M and a minor (144-145 L↓Q BACE1 cleavage site in human Navβ2. Our in vitro and cell-based results clearly show that the 147-148 L↓M is the major BACE1 cleavage site in human Navβ2. These findings expand our understanding of the role of BACE1 in voltage-gated sodium channel metabolism.

  19. The matrix metalloproteinase stromelysin-3 cleaves laminin receptor at two distinct sites between the transmembrane domain and laminin binding sequence within the extracellular domain

    Institute of Scientific and Technical Information of China (English)

    Tosikazu AMANO; Olivia KWAK; Liezhen FU; Anastasia MARSHAK; Yun-Bo SHI

    2005-01-01

    The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has long been implicated to play an important role in extracellular matrix (ECM) remodeling and cell fate determination during normal and pathological processes. However,like other MMPs, the molecular basis of ST3 function in vivo remains unclear due to the lack of information on its physiological substrates. Furthermore, ST3 has only weak activities toward all tested ECM proteins. Using thyroid hormone-dependent Xenopus laevis metamorphosis as a model, we demonstrated previously that ST3 is important for apoptosis and tissue morphogenesis during intestinal remodeling. Here, we used yeast two-hybrid screen with mRNAs from metamorphosing tadpoles to identify potential substrate of ST3 during development. We thus isolated the 37 kd laminin receptor precursor (LR). We showed that LR binds to ST3 in vitro and can be cleaved by ST3 at two sites,distinct from where other MMPs cleave. Through peptide sequencing, we determined that the two cleavage sites are in the extracellular domain between the transmembrane domain and laminin binding sequence. Furthermore, we demonstrated that these cleavage sites are conserved in human LR. These results together with high levels of human LR and ST3 expression in carcinomas suggest that LR is a likely in vivo substrate of ST3 and that its cleavage by ST3 may alter cell-extracellular matrix interaction, thus, playing a role in mediating the effects of ST3 on cell fate and behavior observed during development and pathogenesis.

  20. Genetic Predisposition To Acquire a Polybasic Cleavage Site for Highly Pathogenic Avian Influenza Virus Hemagglutinin

    Science.gov (United States)

    Nao, Naganori; Yamagishi, Junya; Miyamoto, Hiroko; Igarashi, Manabu; Manzoor, Rashid; Ohnuma, Aiko; Tsuda, Yoshimi; Furuyama, Wakako; Shigeno, Asako; Kajihara, Masahiro; Kishida, Noriko; Yoshida, Reiko

    2017-01-01

    ABSTRACT Highly pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Here we show that consecutive adenine residues and a stem-loop structure, which are frequently found in the viral RNA region encoding amino acids around the cleavage site of low-pathogenic H5 and H7 viruses isolated from waterfowl reservoirs, are important for nucleotide insertions into this RNA region. A reporter assay to detect nontemplated nucleotide insertions and deep-sequencing analysis of viral RNAs revealed that an increased number of adenine residues and enlarged stem-loop structure in the RNA region accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids. Interestingly, nucleotide insertions associated with the HA cleavage site motif were not observed principally in the viral RNA of other subtypes tested (H1, H2, H3, and H4). Our findings suggest that the RNA editing-like activity is the key mechanism for nucleotide insertions, providing a clue as to why the acquisition of the polybasic HA cleavage site is restricted to the particular HA subtypes. PMID:28196963

  1. Site-Specific Pyrolysis Induced Cleavage at Aspartic Acid Residue in Peptides and Proteins

    Science.gov (United States)

    Zhang, Shaofeng; Basile, Franco

    2011-01-01

    A simple and site-specific non-enzymatic method based on pyrolysis has been developed to cleave peptides and proteins. Pyrolytic cleavage was found to be specific and rapid as it induced a cleavage at the C-terminal side of aspartic acid in the temperature range of 220–250 °C in 10 seconds. Electrospray Ionization (ESI) mass spectrometry (MS) and tandem-MS (MS/MS) were used to characterize and identify pyrolysis cleavage products, confirming that sequence information is conserved after the pyrolysis process in both peptides and protein tested. This suggests that pyrolysis-induced cleavage at aspartyl residues can be used as a rapid protein digestion procedure for the generation of sequence specific protein biomarkers. PMID:17388620

  2. Coronavirus 3CLpro proteinase cleavage sites: Possible relevance to SARS virus pathology

    Directory of Open Access Journals (Sweden)

    Blom Nikolaj

    2004-06-01

    Full Text Available Abstract Background Despite the passing of more than a year since the first outbreak of Severe Acute Respiratory Syndrome (SARS, efficient counter-measures are still few and many believe that reappearance of SARS, or a similar disease caused by a coronavirus, is not unlikely. For other virus families like the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested using specific proteinase inhibitors supporting the belief that proteinases are indeed important during infection. Prompted by this, we set out to analyse and predict cleavage by the coronavirus main proteinase using computational methods. Results We retrieved sequence data on seven fully sequenced coronaviruses and identified the main 3CL proteinase cleavage sites in polyproteins using alignments. A neural network was trained to recognise the cleavage sites in the genomes obtaining a sensitivity of 87.0% and a specificity of 99.0%. Several proteins known to be cleaved by other viruses were submitted to prediction as well as proteins suspected relevant in coronavirus pathology. Cleavage sites were predicted in proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR, transcription factors CREB-RP and OCT-1, and components of the ubiquitin pathway. Conclusions Our prediction method NetCorona predicts coronavirus cleavage sites with high specificity and several potential cleavage candidates were identified which might be important to elucidate coronavirus pathology. Furthermore, the method might assist in design of proteinase inhibitors for treatment of SARS and possible future diseases caused by coronaviruses. It is made available for public use at our website: http://www.cbs.dtu.dk/services/NetCorona/.

  3. Cleavage of proBDNF to BDNF by a tolloid-like metalloproteinase is required for acquisition of in vitro eyeblink classical conditioning.

    Science.gov (United States)

    Keifer, Joyce; Sabirzhanov, Boris E; Zheng, Zhaoqing; Li, Wei; Clark, Timothy G

    2009-11-25

    The tolloid/bone morphogenetic protein-1 family of metalloproteinases have an important role in the regulation of embryonic pattern formation and tissue morphogenesis. Studies suggest that they participate in mechanisms of synaptic plasticity in adults, but very little is known about their function. Recently, we isolated a reptilian ortholog of the tolloid gene family designated turtle tolloid-like gene (tTll). Here, we examined the role of tTLL in an in vitro model of eyeblink classical conditioning using an isolated brainstem preparation to assess its role in synaptic plasticity during conditioning. Analysis by real-time reverse transcription-PCR shows that an extracellularly secreted form of tTLL, tTLLs, is transiently expressed in the early stages of conditioning during conditioned response acquisition, whereas a cytosolic form, tTLLc, is not. Short interfering RNA (siRNA)-directed gene knockdown and rescue of tTLL expression demonstrate that it is required for conditioning. Significantly, we show that tTLLs cleaves the precursor proBDNF into mature BDNF in cleavage assay studies, and application of recombinant tTLLs protein alone to preparations results in induction of mature BDNF expression. The mature form of BDNF is minimally expressed in preparations treated with anti-tTLL siRNA, and the synaptic incorporation of both GluR1- and GluR4-containing AMPA receptors is significantly reduced, resulting in suppression of conditioning. This is the first study to demonstrate that expression of an extracellularly secreted tolloid-like metalloproteinase is regulated in the early stages of classical conditioning and functions in the conversion of proBDNF to mature BDNF. The mature form of BDNF is required for synaptic delivery of AMPA receptors and acquisition of conditioned responses.

  4. PROSPER: an integrated feature-based tool for predicting protease substrate cleavage sites.

    Directory of Open Access Journals (Sweden)

    Jiangning Song

    Full Text Available The ability to catalytically cleave protein substrates after synthesis is fundamental for all forms of life. Accordingly, site-specific proteolysis is one of the most important post-translational modifications. The key to understanding the physiological role of a protease is to identify its natural substrate(s. Knowledge of the substrate specificity of a protease can dramatically improve our ability to predict its target protein substrates, but this information must be utilized in an effective manner in order to efficiently identify protein substrates by in silico approaches. To address this problem, we present PROSPER, an integrated feature-based server for in silico identification of protease substrates and their cleavage sites for twenty-four different proteases. PROSPER utilizes established specificity information for these proteases (derived from the MEROPS database with a machine learning approach to predict protease cleavage sites by using different, but complementary sequence and structure characteristics. Features used by PROSPER include local amino acid sequence profile, predicted secondary structure, solvent accessibility and predicted native disorder. Thus, for proteases with known amino acid specificity, PROSPER provides a convenient, pre-prepared tool for use in identifying protein substrates for the enzymes. Systematic prediction analysis for the twenty-four proteases thus far included in the database revealed that the features we have included in the tool strongly improve performance in terms of cleavage site prediction, as evidenced by their contribution to performance improvement in terms of identifying known cleavage sites in substrates for these enzymes. In comparison with two state-of-the-art prediction tools, PoPS and SitePrediction, PROSPER achieves greater accuracy and coverage. To our knowledge, PROSPER is the first comprehensive server capable of predicting cleavage sites of multiple proteases within a single substrate

  5. Matrix metalloproteinase production in regenerating axolotl spinal cord.

    Science.gov (United States)

    Chernoff, E A; O'Hara, C M; Bauerle, D; Bowling, M

    2000-01-01

    In urodele amphibian spinal cord regeneration, the ependymal cells lining the central canal remodel the lesion site to favor axonal regrowth. We profiled the production of matrix metalloproteinases by injury-reactive mesenchymal ependymal cells in vivo and in vitro and found that matrix metalloproteinases are involved in this remodeling process in the axolotl (Ambystoma mexicanum). The production of cell-associated matrix metalloproteinases in vivo was shown to be identical to that in our cultured ependymal cell model system. Activated and zymogen forms of matrix metalloproteinases were identified using zymography, chemical inhibitors of matrix metalloproteinases, and cleavage of propeptides by organomercurials. The principal cellular proteinases consisted of matrix metalloproteinase-2 (gelatinase A) and matrix metalloproteinase-1 (type I collagenase), which display characteristic shifts in molecular weight following proenzyme processing by organomercurials. In addition, ependymal cell conditioned medium contained secreted forms of the enzyme undetectable in situ. Matrix metalloproteinase-9 (gelatinase B) as well as matrix metalloproteinase-2 and matrix metalloproteinase-1 were secreted and casein substrate zymography showed the presence of a small amount of a very high molecular weight matrix metalloproteinase-3 (prostromelysin) secreted into the culture medium. Matrix metalloproteinases were still present at 4 weeks post-lesioning when the ependymal cells have just re-epithelialized, but decreased near the completion of regeneration (8 weeks post-lesioning). Zymography showed no detectable matrix metalloproteinases in unlesioned cord but the presence of tissue inhibitor of metalloproteinase-1 in intact cord was seen by Western blotting. This study shows that matrix metalloproteinases are associated with urodele spinal cord regeneration and validates the use of our ependymal cell tissue culture model system to evaluate ependymal cell behavior during spinal cord

  6. Site-specifically Hydrolytic Cleavage of Oxidized Insulin B Chain With Cu(II) Ion

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Electrospray mass spectrometry investigation shows that denatured oxidized insulin B chain can be selectively cleaved by simple Cu(II) ion and the site of cleavage is at Gly8-Ser9 bond which is second amide bond left from His 10 in the sequence of oxidized insulin B chain.

  7. A novel purification method for histidine-tagged proteins containing a thrombin cleavage site

    NARCIS (Netherlands)

    Hefti, M.H.; Vugt-Toorn, van der C.J.; Dixon, R.; Vervoort, J.J.M.

    2001-01-01

    A general procedure for the purification of histidine-tagged proteins has been developed using immobilized metal-ion affinity chromatography. This two-step purification method can be used for proteins containing a hexahistidine tag and a thrombin cleavage site, yielding high amounts of purified prot

  8. Prediction of Signal Peptide Cleavage Sites with Subsite-Coupled and Template Matching Fusion Algorithm.

    Science.gov (United States)

    Zhang, Shao-Wu; Zhang, Ting-He; Zhang, Jun-Nan; Huang, Yufei

    2014-03-01

    Fast and effective prediction of signal peptides (SP) and their cleavage sites is of great importance in computational biology. The approaches developed to predict signal peptide can be roughly divided into machine learning based, and sliding windows based. In order to further increase the prediction accuracy and coverage of organism for SP cleavage sites, we propose a novel method for predicting SP cleavage sites called Signal-CTF that utilizes machine learning and sliding windows, and is designed for N-termial secretory proteins in a large variety of organisms including human, animal, plant, virus, bacteria, fungi and archaea. Signal-CTF consists of three distinct elements: (1) a subsite-coupled and regularization function with a scaled window of fixed width that selects a set of candidates of possible secretion-cleavable segment for a query secretory protein; (2) a sum fusion system that integrates the outcomes from aligning the cleavage site template sequence with each of the aforementioned candidates in a scaled window of fixed width to determine the best candidate cleavage sites for the query secretory protein; (3) a voting system that identifies the ultimate signal peptide cleavage site among all possible results derived from using scaled windows of different width. When compared with Signal-3L and SignalP 4.0 predictors, the prediction accuracy of Signal-CTF is 4-12 %, 10-25 % higher than that of Signal-3L for human, animal and eukaryote, and SignalP 4.0 for eukaryota, Gram-positive bacteria and Gram-negative bacteria, respectively. Comparing with PRED-SIGNAL and SignalP 4.0 predictors on the 32 archaea secretory proteins of used in Bagos's paper, the prediction accuracy of Signal-CTF is 12.5 %, 25 % higher than that of PRED-SIGNAL and SignalP 4.0, respectively. The predicting results of several long signal peptides show that the Signal-CTF can better predict cleavage sites for long signal peptides than SignalP, Phobius, Philius, SPOCTOPUS, Signal

  9. Genetic Predisposition To Acquire a Polybasic Cleavage Site for Highly Pathogenic Avian Influenza Virus Hemagglutinin.

    Science.gov (United States)

    Nao, Naganori; Yamagishi, Junya; Miyamoto, Hiroko; Igarashi, Manabu; Manzoor, Rashid; Ohnuma, Aiko; Tsuda, Yoshimi; Furuyama, Wakako; Shigeno, Asako; Kajihara, Masahiro; Kishida, Noriko; Yoshida, Reiko; Takada, Ayato

    2017-02-14

    Highly pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Here we show that consecutive adenine residues and a stem-loop structure, which are frequently found in the viral RNA region encoding amino acids around the cleavage site of low-pathogenic H5 and H7 viruses isolated from waterfowl reservoirs, are important for nucleotide insertions into this RNA region. A reporter assay to detect nontemplated nucleotide insertions and deep-sequencing analysis of viral RNAs revealed that an increased number of adenine residues and enlarged stem-loop structure in the RNA region accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids. Interestingly, nucleotide insertions associated with the HA cleavage site motif were not observed principally in the viral RNA of other subtypes tested (H1, H2, H3, and H4). Our findings suggest that the RNA editing-like activity is the key mechanism for nucleotide insertions, providing a clue as to why the acquisition of the polybasic HA cleavage site is restricted to the particular HA subtypes.IMPORTANCE Influenza A viruses are divided into subtypes based on the antigenicity of the viral surface glycoproteins hemagglutinin (HA) and neuraminidase. Of the 16 HA subtypes (H1 to -16) maintained in waterfowl reservoirs of influenza A viruses, H5 and H7 viruses often become highly pathogenic through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been known since the 1980s, the genetic basis for nucleotide insertions has remained unclear. This study shows the potential role of the viral RNA secondary structure for

  10. Hemoglobin cleavage site-specificity of the Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3.

    Directory of Open Access Journals (Sweden)

    Shoba Subramanian

    Full Text Available The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P(1 - P(4 amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P(2 position. Second, with overlapping peptides spanning alpha and beta globin and proteolysis-dependent (18O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P(2 Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents.

  11. Caspase cleavage sites in the human proteome: CaspDB, a database of predicted substrates.

    Directory of Open Access Journals (Sweden)

    Sonu Kumar

    Full Text Available Caspases are enzymes belonging to a conserved family of cysteine-dependent aspartic-specific proteases that are involved in vital cellular processes and play a prominent role in apoptosis and inflammation. Determining all relevant protein substrates of caspases remains a challenging task. Over 1500 caspase substrates have been discovered in the human proteome according to published data and new substrates are discovered on a daily basis. To aid the discovery process we developed a caspase cleavage prediction method using the recently published curated MerCASBA database of experimentally determined caspase substrates and a Random Forest classification method. On both internal and external test sets, the ranking of predicted cleavage positions is superior to all previously developed prediction methods. The in silico predicted caspase cleavage positions in human proteins are available from a relational database: CaspDB. Our database provides information about potential cleavage sites in a verified set of all human proteins collected in Uniprot and their orthologs, allowing for tracing of cleavage motif conservation. It also provides information about the positions of disease-annotated single nucleotide polymorphisms, and posttranslational modifications that may modulate the caspase cleaving efficiency.

  12. Evaluation of fusion protein cleavage site sequences of Newcastle disease virus in genotype matched vaccines.

    Science.gov (United States)

    Kim, Shin-Hee; Chen, Zongyan; Yoshida, Asuka; Paldurai, Anandan; Xiao, Sa; Samal, Siba K

    2017-01-01

    Newcastle disease virus (NDV) causes a devastating poultry disease worldwide. Frequent outbreaks of NDV in chickens vaccinated with conventional live vaccines suggest a need to develop new vaccines that are genetically matched against circulating NDV strains, such as the genotype V virulent strains currently circulating in Mexico and Central America. In this study, a reverse genetics system was developed for the virulent NDV strain Mexico/01/10 strain and used to generate highly attenuated vaccine candidates by individually modifying the cleavage site sequence of fusion (F) protein. The cleavage site sequence of parental virus was individually changed to those of the avirulent NDV strain LaSota and other serotypes of avian paramyxoviruses (APMV serotype-2, -3, -4, -6, -7, -8, and -9). In general, these mutations affected cell-to-cell fusion activity in vitro and the efficiency of the F protein cleavage and made recombinant Mexico/01/10 (rMex) virus highly attenuated in chickens. When chickens were immunized with the rMex mutant viruses and challenged with the virulent parent virus, there was reduced challenge virus shedding compared to birds immunized with the heterologous vaccine strain LaSota. Among the vaccine candidates, rMex containing the cleavage site sequence of APMV-2 induced the highest neutralizing antibody titer and completely protected chickens from challenge virus shedding. These results show the role of the F protein cleavage site sequence of each APMV type in generating genotype V-matched vaccines and the efficacy of matched vaccine strains to provide better protection against NDV strains currently circulating in Mexico.

  13. A Python analytical pipeline to identify prohormone precursors and predict prohormone cleavage sites

    Directory of Open Access Journals (Sweden)

    Bruce Southey

    2008-12-01

    Full Text Available Neuropeptides and hormones are signaling molecules that support cell-cell communication in the central nervous system. Experimentally characterizing neuropeptides requires significant efforts because of the complex and variable processing of prohormone precursor proteins into neuropeptides and hormones. We demonstrate the power and flexibility of the Python language to develop components of an bioinformatic analytical pipeline to identify precursors from genomic data and to predict cleavage as these precursors are en route to the final bioactive peptides. We identified 75 precursors in the rhesus genome, predicted cleavage sites using support vector machines and compared the rhesus predictions to putative assignments based on homology to human sequences. The correct classification rate of cleavage using the support vector machines was over 97% for both human and rhesus data sets. The functionality of Python has been important to develop and maintain NeuroPred (http://neuroproteomics.scs.uiuc.edu/neuropred.html, a user-centered web application for the neuroscience community that provides cleavage site prediction from a wide range of models, precision and accuracy statistics, post-translational modifications, and the molecular mass of potential peptides. The combined results illustrate the suitability of the Python language to implement an all-inclusive bioinformatics approach to predict neuropeptides that encompasses a large number of interdependent steps, from scanning genomes for precursor genes to identification of potential bioactive neuropeptides.

  14. A python analytical pipeline to identify prohormone precursors and predict prohormone cleavage sites.

    Science.gov (United States)

    Southey, Bruce R; Sweedler, Jonathan V; Rodriguez-Zas, Sandra L

    2008-01-01

    Neuropeptides and hormones are signaling molecules that support cell-cell communication in the central nervous system. Experimentally characterizing neuropeptides requires significant efforts because of the complex and variable processing of prohormone precursor proteins into neuropeptides and hormones. We demonstrate the power and flexibility of the Python language to develop components of an bioinformatic analytical pipeline to identify precursors from genomic data and to predict cleavage as these precursors are en route to the final bioactive peptides. We identified 75 precursors in the rhesus genome, predicted cleavage sites using support vector machines and compared the rhesus predictions to putative assignments based on homology to human sequences. The correct classification rate of cleavage using the support vector machines was over 97% for both human and rhesus data sets. The functionality of Python has been important to develop and maintain NeuroPred (http://neuroproteomics.scs.uiuc.edu/neuropred.html), a user-centered web application for the neuroscience community that provides cleavage site prediction from a wide range of models, precision and accuracy statistics, post-translational modifications, and the molecular mass of potential peptides. The combined results illustrate the suitability of the Python language to implement an all-inclusive bioinformatics approach to predict neuropeptides that encompasses a large number of interdependent steps, from scanning genomes for precursor genes to identification of potential bioactive neuropeptides.

  15. Identification and characterization of a cleavage site in the proteolysis of orf virus 086 protein

    Directory of Open Access Journals (Sweden)

    Xiaoping eWang

    2016-04-01

    Full Text Available The ORF virus (ORFV is among the parapoxvirus genus of the poxviridae family, but little is known about the proteolytic pathways of ORFV encoding proteins. By contrast, the proteolysis mechanism of the vaccinia virus has been extensively explored. Vaccinia virus core protein P4a undergoes a proteolytic process that takes place at a conserved cleavage site Ala-Gly-X (where X is any amino acid and participates in virus assembly. Bioinformatics analysis revealed that an ORFV encoding protein, ORFV086, has a similar structure to the Vaccinia virus P4a core protein. In this study, we focus on the kinetic analysis and proteolysis mechanism of ORFV086. We found, via kinetic analysis, that ORFV086 is a late gene that starts to express at 8 hours post infection at mRNA level and 12 to 24 hours post infection at the protein level. The ORFV086 precursor and a 21kDa fragment can be observed in mature ORFV virions. The same bands were detected at only 3 hours post infection, suggesting that both the ORFV086 precursor and the 21kDa fragment are viral structural proteins. ORFV086 was cleaved from 12 to 24 hours post infection. The cleavage took place at different sites,resulting in seven bands with differing molecular weights. Sequence alignment revealed that five putative cleavage sites were predicted at C-terminal and internal regions of ORFV086. To investigate whether those cleavage sites are involved in proteolytic processing, full length and several deletion mutant ORFV086 recombinant proteins were expressed and probed. The GGS site that produced a 21kDa cleavage fragment was confirmed by identification of N/C-terminal FLAG epitope recombinant proteins, site-directed mutagenesis and Pulse-chase analysis. Interestingly, chase results demonstrated that, at late times, ORFV086 is partially cleaved. Taken together, we concluded that GGS is a cleavage site in ORFV086 and produces a 21kDa fragment post infection. Both ORFV086 precursor and the 21kDa fragment

  16. ADAM10 overexpression shifts lympho- and myelopoiesis by dysregulating site 2/site 3 cleavage products of Notch.

    Science.gov (United States)

    Gibb, David R; Saleem, Sheinei J; Kang, Dae-Joong; Subler, Mark A; Conrad, Daniel H

    2011-04-01

    Although the physiological consequences of Notch signaling in hematopoiesis have been extensively studied, the differential effects of individual notch cleavage products remain to be elucidated. Given that ADAM10 is a critical regulator of Notch and that its deletion is embryonically lethal, we generated mice that overexpress ADAM10 (ADAM10 transgenic [A10Tg]) at early stages of lympho- and myeloid development. Transgene expression resulted in abrogated B cell development, delayed T cell development in the thymus, and unexpected systemic expansion of CD11b(+)Gr-1(+) cells, also known as myeloid-derived suppressor cells. Mixed bone marrow reconstitution assays demonstrated that transgene expression altered hematopoiesis via a cell-intrinsic mechanism. Consistent with previously reported observations, we hypothesized that ADAM10 overexpression dysregulated Notch by uncoupling the highly regulated proteolysis of Notch receptors. This was confirmed using an in vitro model of hematopoiesis via culturing A10Tg hematopoietic Lineage(-)Sca-1(+)c-Kit(+) cells with OP-9 stromal cells in the presence or absence of Delta-like 1, a primary ligand for Notch. Blockade of the site 2 (S2) and site 3 (S3) cleavage of the Notch receptor demonstrated differential effects on hematopoiesis. OP9-DL1 cultures containing the ADAM10 inhibitor (S2 cleavage site) enhanced and rescued B cell development from wild-type and A10Tg Lineage(-)Sca-1(+)c-Kit(+) cells, respectively. In contrast, blockade of γ-secretase at the S3 cleavage site induced accumulation of the S2 product and consequently prevented B cell development and resulted in myeloid cell accumulation. Collectively, these findings indicate that the differential cleavage of Notch into S2 and S3 products regulated by ADAM10 is critical to hematopoietic cell-fate determination.

  17. Talin contains a C-terminal calpain2 cleavage site important in focal adhesion dynamics.

    Directory of Open Access Journals (Sweden)

    Neil Bate

    Full Text Available Talin is a large (∼2540 residues dimeric adaptor protein that associates with the integrin family of cell adhesion molecules in cell-extracellular matrix junctions (focal adhesions; FAs, where it both activates integrins and couples them to the actin cytoskeleton. Calpain2-mediated cleavage of talin between the head and rod domains has previously been shown to be important in FA turnover. Here we identify an additional calpain2-cleavage site that removes the dimerisation domain from the C-terminus of the talin rod, and show that an E2492G mutation inhibits calpain cleavage at this site in vitro, and increases the steady state levels of talin1 in vivo. Expression of a GFP-tagged talin1 E2492G mutant in CHO.K1 cells inhibited FA turnover and the persistence of cell protrusion just as effectively as a L432G mutation that inhibits calpain cleavage between the talin head and rod domains. Moreover, incorporation of both mutations into a single talin molecule had an additive effect clearly demonstrating that calpain cleavage at both the N- and C-terminal regions of talin contribute to the regulation of FA dynamics. However, the N-terminal site was more sensitive to calpain cleavage suggesting that lower levels of calpain are required to liberate the talin head and rod fragments than are needed to clip off the C-terminal dimerisation domain. The talin head and rod liberated by calpain2 cleavage have recently been shown to play roles in an integrin activation cycle important in FA turnover and in FAK-dependent cell cycle progression respectively. The half-life of the talin head is tightly regulated by ubiquitination and we suggest that removal of the C-terminal dimerisation domain from the talin rod may provide a mechanism both for terminating the signalling function of the talin rod and indeed for inactivating full-length talin thereby promoting FA turnover at the rear of the cell.

  18. Signal peptide discrimination and cleavage site identification using SVM and NN.

    Science.gov (United States)

    Kazemian, H B; Yusuf, S A; White, K

    2014-02-01

    About 15% of all proteins in a genome contain a signal peptide (SP) sequence, at the N-terminus, that targets the protein to intracellular secretory pathways. Once the protein is targeted correctly in the cell, the SP is cleaved, releasing the mature protein. Accurate prediction of the presence of these short amino-acid SP chains is crucial for modelling the topology of membrane proteins, since SP sequences can be confused with transmembrane domains due to similar composition of hydrophobic amino acids. This paper presents a cascaded Support Vector Machine (SVM)-Neural Network (NN) classification methodology for SP discrimination and cleavage site identification. The proposed method utilises a dual phase classification approach using SVM as a primary classifier to discriminate SP sequences from Non-SP. The methodology further employs NNs to predict the most suitable cleavage site candidates. In phase one, a SVM classification utilises hydrophobic propensities as a primary feature vector extraction using symmetric sliding window amino-acid sequence analysis for discrimination of SP and Non-SP. In phase two, a NN classification uses asymmetric sliding window sequence analysis for prediction of cleavage site identification. The proposed SVM-NN method was tested using Uni-Prot non-redundant datasets of eukaryotic and prokaryotic proteins with SP and Non-SP N-termini. Computer simulation results demonstrate an overall accuracy of 0.90 for SP and Non-SP discrimination based on Matthews Correlation Coefficient (MCC) tests using SVM. For SP cleavage site prediction, the overall accuracy is 91.5% based on cross-validation tests using the novel SVM-NN model.

  19. ChloroP, a neural network-based method for predicting chloroplast transitpeptides and their cleavage sites

    DEFF Research Database (Denmark)

    Emanuelsson, O.; Nielsen, Henrik; von Heijne, Gunnar

    1999-01-01

    We present a neural network based method (ChloroP) for identifying chloroplast transit peptides and their cleavage sites. Using cross-validation, 88% of the sequences in our homology reduced training set were correctly classified as transit peptides or nontransit peptides. This performance level...... is well above that of the publicly available chloroplast localization predictor PSORT. Cleavage sites are predicted using a scoring matrix derived by an automatic motif-finding algorithm. Approximately 60% of the known cleavage sites in our sequence collection were predicted to within +/-2 residues from...

  20. A 20 Residues Motif Delineates the Furin Cleavage Site and its Physical Properties May Influence Viral Fusion

    Directory of Open Access Journals (Sweden)

    Sun Tian

    2009-01-01

    Full Text Available Furin is a proprotein convertase that proteolytically cleaves protein precursors to yield functional proteins. Efficient cleavage depends on the presence of a specific sequence motif on the substrate. Currently, the cleavage site motif is described as a four amino acid pattern: R-X-[K/R]-R↓. However, not all furin cleavage recognition sites can be described by this pattern and not all R-X-[K/R]-R↓ sites are cleaved by furin. Since many furin substrates are involved in the pathogenesis of viral infection and human diseases, it is important to accurately characterize the furin cleavage site motif. In this study, the furin cleavage site motif was characterized using statistical analysis. The data were interpreted within the 3D crystal structure of the furin catalytic domain. The results indicate that the furin cleavage site motif is comprised of about 20 residues, P14–P6´. Specific physical properties such as volume, charge, and hydrophilicity are required at specific positions. The furin cleavage site motif is divided into two parts: 1 one core region (8 amino acids, positions P6–P2´ packed inside the furin binding pocket; 2 two polar regions (8 amino acids, positions P7–P14; and 4 amino acids, positions P3´–P6´ located outside the furin binding pocket. The physical properties of the core region contribute to the binding strength of the furin substrate, while the polar regions provide a solvent accessible environment and facilitate the accessibility of the core region to the furin binding pocket. This furin cleavage site motif also revealed a dynamic relationship linking the evolution of physical properties in region P1´–P6´ of viral fusion peptides, furin cleavage efficacy, and viral infectivity.

  1. Genetic insight of the H5N1 hemagglutinin cleavage site

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    The cleavability of the hemagglutinin (HA) plays a major role in virulence of avian influenza viruses. Detailed analyses of the cleavage sequences and their evolution would give insights into the high pathogenicity of the H5N1 virus. HA segments were visually identifiable in the cellular automata (CA) image, and a feature gene segment (FGS) was only found in H5N1 rather than any other subtype. This FGS is a 30-bp gene segment mainly consisting of 'A' and 'G'. When translated into amino acids the FGS converted into a sequence of mainly basic amino acids with positive charges. This feature amino acid segment (FAAS) was located in the cleavage site loop of HA which was potentially cleavable by various proteases. The 3D structure of H5N1 HA was reconstructed using homology modelling. It was found that the cleavage site loop was well exposed to potential proteases. The molecular surfaces were reconstructed to study how mutation and deletion of some amino acids in the FAAS affected the charge distribution. It was found that some mutations had severely changed the landscape of the charge distribution. Statistical analyses of FAAS were made with respect to when and where the H5N1 viruses were found. In 2005, there were less un-mutated FAAS than the other years according to temporal evolution, and more mutated FAAS appeared in China than other regions according to geographic distribution. These results are helpful for exploring the evolution of virus high pathogenicity.

  2. ADAM13 cleavage of cadherin-11 promotes CNC migration independently of the homophilic binding site.

    Science.gov (United States)

    Abbruzzese, Genevieve; Becker, Sarah F; Kashef, Jubin; Alfandari, Dominique

    2016-07-15

    The cranial neural crest (CNC) is a highly motile population of cells that is responsible for forming the face and jaw in all vertebrates and perturbing their migration can lead to craniofacial birth defects. Cell motility requires a dynamic modification of cell-cell and cell-matrix adhesion. In the CNC, cleavage of the cell adhesion molecule cadherin-11 by ADAM13 is essential for cell migration. This cleavage generates a shed extracellular fragment of cadherin-11 (EC1-3) that possesses pro-migratory activity via an unknown mechanism. Cadherin-11 plays an important role in modulating contact inhibition of locomotion (CIL) in the CNC to regulate directional cell migration. Here, we show that while the integral cadherin-11 requires the homophilic binding site to promote CNC migration in vivo, the EC1-3 fragment does not. In addition, we show that increased ADAM13 activity or expression of the EC1-3 fragment increases CNC invasiveness in vitro and blocks the repulsive CIL response in colliding cells. This activity requires the presence of an intact homophilic binding site on the EC1-3 suggesting that the cleavage fragment may function as a competitive inhibitor of cadherin-11 adhesion in CIL but not to promote cell migration in vivo.

  3. Positively Selected Sites at HCMV gB Furin Processing Region and Their Effects in Cleavage Efficiency.

    Science.gov (United States)

    Stangherlin, Lucas M; de Paula, Felipe N; Icimoto, Marcelo Y; Ruiz, Leonardo G P; Nogueira, Maurício L; Braz, Antônio S K; Juliano, Luiz; da Silva, Maria C C

    2017-01-01

    Human cytomegalovirus is a ubiquitous infectious agent that affects mainly immunosuppressed, fetuses, and newborns. The virus has several polymorphic regions, in particular in the envelope glycoproteins. The UL55 gene encodes the glycoprotein B that has a variable region, containing a furin cleavage site and according to the variability different genotypes are characterized. Here we investigated variability and existence of selective pressure on the UL55 variable region containing the furin cleavage site in 213 clinical sequences from patients worldwide. We showed the occurrence of positive selective pressure on gB codons 461 and 462, near the furin cleavage site. Cleavage analysis of synthesized peptides demonstrated that most mutations confer better cleavage by furin, suggesting that evolution is acting in order to increase the efficiency cleavage and supporting the hypothesis that gB processing is important in the host. We also demonstrated that peptides containing sequences, that characterize genotypes gB2 and 3, are differentially cleaved by furin. Our data demonstrate for the first time that variability in the cleavage site is related to degree of gB processing by furin.

  4. Positively Selected Sites at HCMV gB Furin Processing Region and Their Effects in Cleavage Efficiency

    Directory of Open Access Journals (Sweden)

    Lucas M. Stangherlin

    2017-05-01

    Full Text Available Human cytomegalovirus is a ubiquitous infectious agent that affects mainly immunosuppressed, fetuses, and newborns. The virus has several polymorphic regions, in particular in the envelope glycoproteins. The UL55 gene encodes the glycoprotein B that has a variable region, containing a furin cleavage site and according to the variability different genotypes are characterized. Here we investigated variability and existence of selective pressure on the UL55 variable region containing the furin cleavage site in 213 clinical sequences from patients worldwide. We showed the occurrence of positive selective pressure on gB codons 461 and 462, near the furin cleavage site. Cleavage analysis of synthesized peptides demonstrated that most mutations confer better cleavage by furin, suggesting that evolution is acting in order to increase the efficiency cleavage and supporting the hypothesis that gB processing is important in the host. We also demonstrated that peptides containing sequences, that characterize genotypes gB2 and 3, are differentially cleaved by furin. Our data demonstrate for the first time that variability in the cleavage site is related to degree of gB processing by furin.

  5. Mutation in spike protein cleavage site and pathogenesis of feline coronavirus.

    Science.gov (United States)

    Licitra, Beth N; Millet, Jean K; Regan, Andrew D; Hamilton, Brian S; Rinaldi, Vera D; Duhamel, Gerald E; Whittaker, Gary R

    2013-07-01

    Feline coronaviruses (FCoV) exist as 2 biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV causes subclinical infections; FIPV causes feline infectious peritonitis (FIP), a systemic and fatal disease. It is thought that mutations in FECV enable infection of macrophages, causing FIP. However, the molecular basis for this biotype switch is unknown. We examined a furin cleavage site in the region between receptor-binding (S1) and fusion (S2) domains of the spike of serotype 1 FCoV. FECV sequences were compared with FIPV sequences. All FECVs had a conserved furin cleavage motif. For FIPV, there was a correlation with the disease and >1 substitution in the S1/S2 motif. Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage. We document a functionally relevant S1/S2 mutation that arises when FIP develops in a cat. These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses.

  6. Analysis of the Proteolytic Processing of ABCA3: Identification of Cleavage Site and Involved Proteases.

    Directory of Open Access Journals (Sweden)

    Nicole Hofmann

    Full Text Available ABCA3 is a lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ABCA3 gene cause respiratory distress syndrome in new-borns and childhood interstitial lung disease. ABCA3 is N-terminally cleaved by an as yet unknown protease, a process believed to regulate ABCA3 activity.The exact site where ABCA3 is cleaved was localized using mass spectrometry (MS. Proteases involved in ABCA3 processing were identified using small molecule inhibitors and siRNA mediated gene knockdown. Results were verified by in vitro digestion of a synthetic peptide substrate mimicking ABCA3's cleavage region, followed by MS analysis.We found that cleavage of ABCA3 occurs after Lys174 which is located in the proteins' first luminal loop. Inhibition of cathepsin L and, to a lesser extent, cathepsin B resulted in attenuation of ABCA3 cleavage. Both enzymes showed activity against the ABCA3 peptide in vitro with cathepsin L being more active.We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L. Therefore, cathepsin L may represent a potential target to therapeutically influence ABCA3 activity in ABCA3-associated lung disease.

  7. The Prediction of Calpain Cleavage Sites with the mRMR and IFS Approaches

    Directory of Open Access Journals (Sweden)

    Wenyi Zhang

    2013-01-01

    Full Text Available Calpains are an important family of the Ca2+-dependent cysteine proteases which catalyze the limited proteolysis of many specific substrates. Calpains play crucial roles in basic physiological and pathological processes, and identification of the calpain cleavage sites may facilitate the understanding of the molecular mechanisms and biological function. But traditional experiment approaches to predict the sites are accurate, and are always labor-intensive and time-consuming. Thus, it is common to see that computational methods receive increasing attention due to their convenience and fast speed in recent years. In this study, we develop a new predictor based on the support vector machine (SVM with the maximum relevance minimum redundancy (mRMR method followed by incremental feature selection (IFS. And we concern the feature of physicochemical/biochemical properties, sequence conservation, residual disorder, secondary structure, and solvent accessibility to represent the calpain cleavage sites. Experimental results show that the performance of our predictor is better than several other state-of- the-art predictors, whose average prediction accuracy is 79.49%, sensitivity is 62.31%, and specificity is 88.12%. Since user-friendly and publicly accessible web servers represent the future direction for developing practically more useful predictors, here we have provided a web-server for the method presented in this paper.

  8. Defining a similarity threshold for a functional proteinsequence pattern: The signal peptide cleavage site

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Engelbrecht, Jacob; von Heijne, Gunnar

    1996-01-01

    prediction. We have developed a similar procedure based on pair-wise alignments for sequences with functional sites. We show how a correlation coefficient between sequence similarity and functional homology can be used to compare the efficiency of different similarity measures and choose a nonarbitrary...... threshold value for excluding redundant sequences. The impact of the choice of scoring matrix used in the alignments is examined. We demonstrate that the parameter determining the quality of the correlation is the relative entropy of the matrix, rather than the assumed(PAM or identity) substitution model....... Results are presented for the case of prediction of cleavage sites in signal peptides. By inspection of the false positives, several errors in the database were found. The procedure presented may be used as a general outline for finding a problem-specific similarity measure and threshold value...

  9. prpC-related signal transduction is influenced by copper, membrane integrity and the alpha cleavage site

    Institute of Scientific and Technical Information of China (English)

    Cathryn L Haigh; Victoria A Lewis; Laura J Vella; Colin L Masters; Andrew F Hill; Victoria A Lawson; Steven J Collins

    2009-01-01

    The copper-binding, membrane-anchored, cellular prion protein (PrPC) has two constitutive cleavage sites pro-ducing distinct N- and C-terminal fragments (N1/C1 and N2/C2). Using RKI3 cells expressing either human PrPC, mouse PrPC or mouse PrPC carrying the 3F4 epitope, this study explored the influence of the PrPC primary sequence on endoproteolytic cleavage and one putative PrPC function, MAP kinase signal transduction, in response to exoge-nous copper with or without a perturbed membrane environment. PrPC primary sequence, especially that around the N1/C1 cleavage site, appeared to influence basal levels of proteolysis at this location and extracellular signal-regulat-ed kinase 1/2 (ERK1/2) phosphorylation, with increased processing demonstrating an inverse relationship with basal ERK1/2 activation. Human PrPC showed increased N1/C1 cleavage in response to copper alone, accompanied by spe-cific p38 and JNK/SAPK phosphorylation. Combined exposure to copper plus the cholesterol-sequestering antibiotic filipin resulted in a mouse PrPC-specific substantial increase in signal protein phosphorylation, accompanied by an increase in N1/C1 cleavage. Mouse PrPC harboring the human N1/C1 cleavage site assumed more human-like profiles basally and in response to copper and altered membrane environments. Our results demonstrate that the PrPC pri-mary sequence around the N1/C1 cleavage site influences endoproteolytic processing at this location, which appears linked to MAP kinase signal transduction both basally and in response to copper. Further, the primary sequence ap-pears to confer a mutual dependence of N1/C1 cleavage and membrane integrity on the fidelity of prpC-related signal transduction in response to exogenous stimuli.

  10. DNA cleavage at the AP site via β-elimination mediated by the AP site-binding ligands.

    Science.gov (United States)

    Abe, Yukiko S; Sasaki, Shigeki

    2016-02-15

    DNA is continuously damaged by endogenous and exogenous factors such as oxidation and alkylation. In the base excision repair pathway, the damaged nucleobases are removed by DNA N-glycosylase to form the abasic sites (AP sites). The alkylating antitumor agent exhibits cytotoxicity through the formation of the AP site. Therefore blockage or modulation of the AP site repair pathway may enhance the antitumor efficacy of DNA alkylating agents. In this study, we have examined the effects of the nucleobase-polyamine conjugated ligands (G-, A-, C- and T-ligands) on the cleavage of the AP site. The G- and A-ligands cleaved DNA at the AP site by promoting β-elimination in a non-selective manner by the G-ligand, and in a selective manner for the opposing dT by the A-ligand. These results suggest that the nucleobase-polyamine conjugate ligands may have the potential for enhancement of the cytotoxicities of the AP site.

  11. The bacterial toxin RelE induces specific mRNA cleavage in the A site of the eukaryote ribosome

    Science.gov (United States)

    Andreev, Dmitri; Hauryliuk, Vasili; Terenin, Ilya; Dmitriev, Sergey; Ehrenberg, Måns; Shatsky, Ivan

    2008-01-01

    RelE/RelB is a well-characterized toxin–anti-toxin pair involved in nutritional stress responses in Bacteria and Archae. RelE lacks any eukaryote homolog, but we demonstrate here that it efficiently and specifically cleaves mRNA in the A site of the eukaryote ribosome. The cleavage mechanism is similar to that in bacteria, showing the feasibility of A-site cleavage of mRNA for regulatory purposes also in eukaryotes. RelE cleavage in the A-site codon of a stalled eukaryote ribosome is precise and easily monitored, making “RelE printing” a useful complement to toeprinting to determine the exact mRNA location on the eukaryote ribosome and to probe the occupancy of its A site. PMID:18083838

  12. CTAG-containing cleavage site profiling to delineate Salmonella into natural clusters.

    Directory of Open Access Journals (Sweden)

    Le Tang

    Full Text Available The bacterial genus Salmonella contains thousands of serotypes that infect humans or other hosts, causing mild gastroenteritis to potentially fatal systemic infections in humans. Pathogenically distinct Salmonella serotypes have been classified as individual species or as serological variants of merely one or two species, causing considerable confusion in both research and clinical settings. This situation reflects a long unanswered question regarding whether the Salmonella serotypes exist as discrete genetic clusters (natural species of organisms or as phenotypic (e.g. pathogenic variants of a single (or two natural species with a continuous spectrum of genetic divergence among them. Our recent work, based on genomic sequence divergence analysis, has demonstrated that genetic boundaries exist among Salmonella serotypes, circumscribing them into clear-cut genetic clusters of bacteria.To further test the genetic boundary concept for delineating Salmonella into clearly defined natural lineages (e.g., species, we sampled a small subset of conserved genomic DNA sequences, i.e., the endonuclease cleavage sites that contain the highly conserved CTAG sequence such as TCTAGA for XbaI. We found that the CTAG-containing cleavage sequence profiles could be used to resolve the genetic boundaries as reliably and efficiently as whole genome sequence comparisons but with enormously reduced requirements for time and resources.Profiling of CTAG sequence subsets reflects genetic boundaries among Salmonella lineages and can delineate these bacteria into discrete natural clusters.

  13. Polymorphisms at activated protein C cleavage sites of factor V: Are they important in the absence of factor V Leiden?

    Directory of Open Access Journals (Sweden)

    Ehsan Kheradmand

    2017-01-01

    Full Text Available Background: Activated protein C (APC inactivates factor V (FV by cleavage of its heavy chain at Arg306, Arg506, Arg679, and Lys994. Mutational changes, which abolish APC cleavage sites, may predispose thrombosis by altering the inactivation process of FV. FV Leiden (FVL (Arg506Glu has been demonstrated as a strong risk factor for thrombosis. In the current study, we have studied whether mutations in the cleavage sites of FV for APC, not due to FVL, would have a role in presenting APC resistance (APCR and initiation of a cerebral thrombotic event.Methods: A group of 22 patients with a history of cerebral venous thrombosis (CVT, who were not carriers of FVL enrolled in the study. The patients who had conditions associated with acquired APCR were excluded from the study. APCR test was performed on the remaining 16 patients, which showed APCR in 4 plasma samples. DNA sequencing was performed on four exons of FV of APCR patients, encoding Arg306, Arg506, Arg679, and Lys994.Results: Mutations were not found within nucleotides encoding the cleavage sites; neither was found within their close upstream and downstream sequences.Conclusion: Our results show that polymorphisms affecting cleavage sites of FV other than Arg506Glu it would be less likely to be the basis for APCR and its increased thrombosis susceptibility. In addition, it emphasizes on the importance of screening for APCR in the patients diagnosed with CVT.

  14. Effect of multifunctional protein YB-1 on the AP site cleavage by AP endonuclease 1 and tyrosyl phosphodiesterase 1

    Directory of Open Access Journals (Sweden)

    Ovchinnikov L. P.

    2012-07-01

    Full Text Available Apurinic/apyrimidinic sites (AP sites which represent one of the most abundantly generated DNA lesions in the cell are generally repaired by base excision repair (BER pathway. Multifunctional protein YB-1 is known to participate in cellular response to genotoxic stress and was shown to interact with several components of BER – DNA glycosylases NTH1, NEIL2, DNA polymerase and DNA ligase III. Therefore, it is of great interest to investigate the influence of YB-1 on one of the major BER enzymes, responsible for AP site cleavage, AP endonuclease APE1, and on tyrosyl phosphodiesterase Tdp1, participating in APE1 independent pathway of AP site repair. Aim. Effect of multifunctional protein YB-1 on the AP site cleavage by the activities of APE1 and Tdp1 was studied. Methods. Gel-mobility shift assays and enzyme activity tests. Results. YB-1 was shown to inhibit the cleavage of AP site located in single-stranded DNA by both APE1 and Tdp1. Stimulation of APE1 activity on protruding double-stranded DNA in the presence of YB-1 was observed, whereas no effect on Tdp1-mediated cleavage of AP site in double-stranded DNA was found. Conclusions. YB-1 can modulate the repair of AP sites in DNA by both positively stimulating APE1 during the classic BER of AP sites and avoiding a possible generation of doublestrand breaks, arising from the cleavage of single-stranded portion of DNA substrate already used by different DNA-processing pathway

  15. Triple helix-forming oligonucleotides conjugated to indolocarbazole poisons direct topoisomerase I-mediated DNA cleavage to a specific site.

    Science.gov (United States)

    Arimondo, P B; Bailly, C; Boutorine, A S; Moreau, P; Prudhomme, M; Sun, J S; Garestier, T; Hélène, C

    2001-01-01

    Topoisomerase I is an ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins as well as for indolocarbazole antibiotics such as rebeccamycin. To achieve a sequence-specific cleavage of DNA by topoisomerase I, a triple helix-forming oligonucleotide was covalently linked to indolocarbazole-type topoisomerase I poisons. The three indolocarbazole-oligonucleotide conjugates investigated were able to direct topoisomerase I cleavage at a specific site based upon sequence recognition by triplex formation. The efficacy of topoisomerase I-mediated DNA cleavage depends markedly on the intrinsic potency of the drug. We show that DNA cleavage depends also upon the length of the linker arm between the triplex-forming oligonucleotide and the drug. Based on a known structure of the DNA-topoisomerase I complex, a molecular model of the oligonucleotide conjugates bound to the DNA-topoisomerase I complex was elaborated to facilitate the design of a potent topoisomerase I inhibitor-oligonucleotide conjugate with an optimized linker between the two moieties. The resulting oligonucleotide-indolocarbazole conjugate at 10 nM induced cleavage at the triple helix site 2-fold more efficiently than 5 microM of free indolocarbazole, while the other drug-sensitive sites were not cleaved. The rational design of drug-oligonucleotide conjugates carrying a DNA topoisomerase poison may be exploited to improve the efficacy and selectivity of chemotherapeutic cancer treatments by targeting specific genes and reducing drug toxicity.

  16. Site-specific O-glycosylation by Polypeptide GalNAc-transferase T2 Co-regulates Beta1-adrenergic Receptor N-terminal Cleavage.

    Science.gov (United States)

    Goth, Christoffer K; Tuhkanen, Hanna E; Khan, Hamayun; Lackman, Jarkko J; Wang, Shengjun; Narimatsu, Yoshiki; Holst Hansen, Lasse; Overall, Christopher; Clausen, Henrik; Schjoldager, Katrine T; Petäjä-Repo, Ulla E

    2017-02-06

    The β1-adrenergic receptor (β1AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for β-adrenergic antagonists, such as beta-blockers, relatively little is still known about its regulation. We have previously shown that β1AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation, and moreover that the receptor is modified by O-glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O-glycosylates β1AR at five residues in the extracellular N-terminus, including the Ser49 residue at a location of the common Ser49Gly single-nucleotide polymorphism. Using in vitro O-glycosylation and proteolytic cleavage assays, a cell line deficient in O-glycosylation, GalNAc-T edited cell line model systems, and a GalNAc-T2 knockout rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of β1AR. Furthermore, we demonstrate that impaired O-glycosylation and enhanced proteolysis leads to attenuated receptor signaling, as the maximal response elicited by the βAR agonist isoproterenol and it potency in a cAMP accumulation assay was decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O-glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of β1AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases.

  17. A single molecule assay for measuring site-specific DNA cleavage.

    Science.gov (United States)

    Gambino, Stefano; Mousley, Briana; Cathcart, Lindsay; Winship, Janelle; Loparo, Joseph J; Price, Allen C

    2016-02-15

    Sequence-specific DNA cleavage is a key step in a number of genomic transactions. Here, we report a single-molecule technique that allows the simultaneous measurement of hundreds of DNAs, thereby collecting significant statistics in a single experiment. Microbeads are tethered with single DNA molecules in a microfluidic channel. After the DNA cleavage reaction is initiated, the time of cleavage of each DNA is recorded using video microscopy. We demonstrate the utility of our method by measuring the cleavage kinetics of NdeI, a type II restriction endonuclease.

  18. Isolation of recombinant phage antibodies targeting the hemagglutinin cleavage site of highly pathogenic avian influenza virus.

    Directory of Open Access Journals (Sweden)

    Jinhua Dong

    Full Text Available Highly pathogenic avian influenza (HPAI H5N1 viruses, which have emerged in poultry and other wildlife worldwide, contain a characteristic multi-basic cleavage site (CS in the hemagglutinin protein (HA. Because this arginine-rich CS is unique among influenza virus subtypes, antibodies against this site have the potential to specifically diagnose pathogenic H5N1. By immunizing mice with the CS peptide and screening a phage display library, we isolated four antibody Fab fragment clones that specifically bind the antigen peptide and several HPAI H5N1 HA proteins in different clades. The soluble Fab fragments expressed in Escherichia coli bound the CS peptide and the H5N1 HA protein with nanomolar affinity. In an immunofluorescence assay, these Fab fragments stained cells infected with HPAI H5N1 but not those infected with a less virulent strain. Lastly, all the Fab clones could detect the CS peptide and H5N1 HA protein by open sandwich ELISA. Thus, these recombinant Fab fragments will be useful novel reagents for the rapid and specific detection of HPAI H5N1 virus.

  19. Identification of off-target cleavage sites of zinc finger nucleases and TAL effector nucleases using predictive models.

    Science.gov (United States)

    Fine, Eli J; Cradick, Thomas J; Bao, Gang

    2014-01-01

    Using engineered nucleases, such as Zinc Finger Nucleases (ZFNs) or Transcription Activator-Like Effector Nucleases (TALENs), to make targeted genomic modifications has become a common technique to create new model organisms and custom cell lines, and has shown great promise for disease treatment. However, these nucleases have the potential for off-target cleavage that could confound interpretation of experimental results and be detrimental for therapeutic use. Here, we describe a method to test for nuclease cleavage at potential off-target sites predicted by bioinformatics models.

  20. Relaxase DNA Binding and Cleavage Are Two Distinguishable Steps in Conjugative DNA Processing That Involve Different Sequence Elements of the nic Site*

    Science.gov (United States)

    Lucas, María; González-Pérez, Blanca; Cabezas, Matilde; Moncalian, Gabriel; Rivas, Germán; de la Cruz, Fernando

    2010-01-01

    TrwC, the relaxase of plasmid R388, catalyzes a series of concerted DNA cleavage and strand transfer reactions on a specific site (nic) of its origin of transfer (oriT). nic contains the cleavage site and an adjacent inverted repeat (IR2). Mutation analysis in the nic region indicated that recognition of the IR2 proximal arm and the nucleotides located between IR2 and the cleavage site were essential for supercoiled DNA processing, as judged either by in vitro nic cleavage or by mobilization of a plasmid containing oriT. Formation of the IR2 cruciform and recognition of the distal IR2 arm and loop were not necessary for these reactions to take place. On the other hand, IR2 was not involved in TrwC single-stranded DNA processing in vitro. For single-stranded DNA nic cleavage, TrwC recognized a sequence embracing six nucleotides upstream of the cleavage site and two nucleotides downstream. This suggests that TrwC DNA binding and cleavage are two distinguishable steps in conjugative DNA processing and that different sequence elements are recognized by TrwC in each step. IR2-proximal arm recognition was crucial for the initial supercoiled DNA binding. Subsequent recognition of the adjacent single-stranded DNA binding site was required to position the cleavage site in the active center of the protein so that the nic cleavage reaction could take place. PMID:20061574

  1. Relaxase DNA binding and cleavage are two distinguishable steps in conjugative DNA processing that involve different sequence elements of the nic site.

    Science.gov (United States)

    Lucas, María; González-Pérez, Blanca; Cabezas, Matilde; Moncalian, Gabriel; Rivas, Germán; de la Cruz, Fernando

    2010-03-19

    TrwC, the relaxase of plasmid R388, catalyzes a series of concerted DNA cleavage and strand transfer reactions on a specific site (nic) of its origin of transfer (oriT). nic contains the cleavage site and an adjacent inverted repeat (IR(2)). Mutation analysis in the nic region indicated that recognition of the IR(2) proximal arm and the nucleotides located between IR(2) and the cleavage site were essential for supercoiled DNA processing, as judged either by in vitro nic cleavage or by mobilization of a plasmid containing oriT. Formation of the IR(2) cruciform and recognition of the distal IR(2) arm and loop were not necessary for these reactions to take place. On the other hand, IR(2) was not involved in TrwC single-stranded DNA processing in vitro. For single-stranded DNA nic cleavage, TrwC recognized a sequence embracing six nucleotides upstream of the cleavage site and two nucleotides downstream. This suggests that TrwC DNA binding and cleavage are two distinguishable steps in conjugative DNA processing and that different sequence elements are recognized by TrwC in each step. IR(2)-proximal arm recognition was crucial for the initial supercoiled DNA binding. Subsequent recognition of the adjacent single-stranded DNA binding site was required to position the cleavage site in the active center of the protein so that the nic cleavage reaction could take place.

  2. Feature Selection Combined with Neural Network Structure Optimization for HIV-1 Protease Cleavage Site Prediction

    Directory of Open Access Journals (Sweden)

    Hui Liu

    2015-01-01

    Full Text Available It is crucial to understand the specificity of HIV-1 protease for designing HIV-1 protease inhibitors. In this paper, a new feature selection method combined with neural network structure optimization is proposed to analyze the specificity of HIV-1 protease and find the important positions in an octapeptide that determined its cleavability. Two kinds of newly proposed features based on Amino Acid Index database plus traditional orthogonal encoding features are used in this paper, taking both physiochemical and sequence information into consideration. Results of feature selection prove that p2, p1, p1′, and p2′ are the most important positions. Two feature fusion methods are used in this paper: combination fusion and decision fusion aiming to get comprehensive feature representation and improve prediction performance. Decision fusion of subsets that getting after feature selection obtains excellent prediction performance, which proves feature selection combined with decision fusion is an effective and useful method for the task of HIV-1 protease cleavage site prediction. The results and analysis in this paper can provide useful instruction and help designing HIV-1 protease inhibitor in the future.

  3. Specificity of human rhinovirus 2A(pro) is determined by combined spatial properties of four cleavage site residues.

    Science.gov (United States)

    Neubauer, David; Aumayr, Martina; Gösler, Irene; Skern, Tim

    2013-07-01

    The 2A proteinase (2A(pro)) of human rhinoviruses cleaves the virally encoded polyprotein between the C terminus of VP1 and its own N terminus. Poor understanding of the 2A(pro) substrate specificity of this enzyme has hampered progress in developing inhibitors that may serve as antiviral agents. We show here that the 2A(pro) of human rhinovirus (HRV) 1A and 2 (rhinoviruses from genetic group A) cannot self-process at the HRV14 (a genetic group B rhinovirus) cleavage site. When the amino acids in the cleavage site of HRV2 2A(pro) (Ile-Ile-Thr-Thr-Ala*Gly-Pro-Ser-Asp) were singly or doubly replaced with the corresponding HRV14 residues (Asp-Ile-Lys-Ser-Tyr*Gly-Leu-Gly-Pro) at positions from P3 to P2', HRV1A and HRV2 2A(pro) cleavage took place at WT levels. However, when three or more positions of the HRV1A or 2 2A(pro) were substituted (e.g. at P2, P1 and P2'), cleavage in vitro was essentially eliminated. Introduction of the full HRV14 cleavage site into a full-length clone of the HRV1A and transfection of HeLa cells with a transcribed RNA did not give rise to viable virus. In contrast, revertant viruses bearing cysteine at the P1 position or proline at P2' were obtained when an RNA bearing the three inhibitory amino acids was transfected. Reversions in the enzyme affecting substrate specificity were not found in any of the in vivo experiments. Modelling of oligopeptide substrates onto the structure of HRV2 2A(pro) revealed no appreciable differences in residues of HRV2 and HRV14 in the respective substrate binding sites, suggesting that the overall shape of the substrate is important in determining binding efficiency.

  4. Action of matrix metalloproteinases at restricted sites in colon anastomosis repair

    DEFF Research Database (Denmark)

    Ågran, Magnus S.; Levin Andersen, Thomas; Mirastschijski, Ursula;

    2006-01-01

    BACKGROUND: Dehiscence of colon anastomosis is a common, serious and potentially life-threatening complication after colorectal operation. In experimental models, impaired biomechanic strength of colon anastomoses is preventable by general inhibitors of matrix metalloproteinases (MMPs) and associ......BACKGROUND: Dehiscence of colon anastomosis is a common, serious and potentially life-threatening complication after colorectal operation. In experimental models, impaired biomechanic strength of colon anastomoses is preventable by general inhibitors of matrix metalloproteinases (MMPs....... CONCLUSIONS: The unique finding of this study was that the specific tissue holding the sutures of a colon anastomosis lost the most collagen presumably through induction and activation of multiple MMPs that may explain the beneficial effects of treatment with non-selective MMP antagonists....

  5. A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites

    DEFF Research Database (Denmark)

    Nielsen, Henrik; Engelbrecht, Jacob; Brunak, Søren;

    1997-01-01

    We have developed a new method for the identication of signal peptides and their cleavage sites based on neural networks trained on separate sets of prokaryotic and eukaryotic sequences. The method performs signicantly better than previous prediction schemes, and can easily be applied to genome-w......-wide data sets. Discrimination between cleaved signal peptides and uncleaved N-terminal signal-anchor sequences is also possible, though with lower precision....

  6. Site of cleavage in infected cells and polypeptides of representative paramyxoviruses grown in cultured cells of the chorioallantoic membrane

    Energy Technology Data Exchange (ETDEWEB)

    Seto, J.T.; Garten, W.; Rott, R. (Giessen Univ. (Germany, F.R.))

    1981-01-01

    Cultured cells of the chorioallantoic membrane (CAM) fulfilled the need of using the same cell system that was permissive for representative paramyxoviruses to carry out studies on the biosynthesis of their glycoproteins in infected cells. The polypeptide composition of the respective paramyxoviruses (Newcastle disease virus (NDV), paramyxovirus Yucaipa (PMY), and Sendai virus), grown in eggs and CAM-cells, was essentially identical. In egg-grown PMY a large glycoprotein (LGP) was present but only in some CAM-grown preparations of virus labeled with (/sup 3/H)-glucosamine and rarely in (/sup 35/S)-methionine or (/sup 3/H)-amino acids (valine, leucine, and tyrosine) labeled viruses. The site of cleavage of precursor F/sub 0/ to Fsub(1,2) was not the same. In contrast to the cleavage of Sendai virus glycoprotein, cleavage was intracellular in NDV and PMY infected cells. Homologous antisera against the glycoproteins failed to inhibit cleavage of HN/sub 0/ or F/sub 0/ in cells infected with the representative paramyxoviruses.

  7. Acquisition of a novel eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site confers intracellular cleavage of an H7N7 influenza virus hemagglutinin

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, Brian S.; Sun, Xiangjie; Chung, Changik [Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca NY 14853 (United States); New York Center of Excellence for Influenza Research and Surveillance, University of Rochester Medical Center, Rochester NY 14627 (United States); Whittaker, Gary R., E-mail: grw7@cornell.edu [Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca NY 14853 (United States); New York Center of Excellence for Influenza Research and Surveillance, University of Rochester Medical Center, Rochester NY 14627 (United States)

    2012-12-05

    A critical feature of highly pathogenic avian influenza viruses (H5N1 and H7N7) is the efficient intracellular cleavage of the hemagglutinin (HA) protein. H7N7 viruses also exist in equine species, and a unique feature of the equine H7N7 HA is the presence of an eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site. Here, we show that three histidine residues within the unique insertion of the equine H7N7 HA are essential for intracellular cleavage. An asparagine residue within the insertion-derived glycosylation site was also found to be essential for intracellular cleavage. The presence of the histidine residues also appear to be involved in triggering fusion, since mutation of the histidine residues resulted in a destabilizing effect. Importantly, the addition of a tetrabasic site and the eleven amino acid insertion conferred efficient intracellular cleavage to the HA of an H7N3 low pathogenicity avian influenza virus. Our studies show that acquisition of the eleven amino acid insertion offers an alternative mechanism for intracellular cleavage of influenza HA.

  8. DNA cleavage site selection by Type III restriction enzymes provides evidence for head-on protein collisions following 1D bidirectional motion

    Science.gov (United States)

    Schwarz, Friedrich W.; van Aelst, Kara; Tóth, Júlia; Seidel, Ralf; Szczelkun, Mark D.

    2011-01-01

    DNA cleavage by the Type III Restriction–Modification enzymes requires communication in 1D between two distant indirectly-repeated recognitions sites, yet results in non-specific dsDNA cleavage close to only one of the two sites. To test a recently proposed ATP-triggered DNA sliding model, we addressed why one site is selected over another during cleavage. We examined the relative cleavage of a pair of identical sites on DNA substrates with different distances to a free or protein blocked end, and on a DNA substrate using different relative concentrations of protein. Under these conditions a bias can be induced in the cleavage of one site over the other. Monte-Carlo simulations based on the sliding model reproduce the experimentally observed behaviour. This suggests that cleavage site selection simply reflects the dynamics of the preceding stochastic enzyme events that are consistent with bidirectional motion in 1D and DNA cleavage following head-on protein collision. PMID:21724613

  9. Processing Map and Essential Cleavage Sites of the Nonstructural Polyprotein Encoded by ORF1 of the Feline Calicivirus Genome

    OpenAIRE

    Sosnovtsev, Stanislav V.; Garfield, Mark; Green, Kim Y.

    2002-01-01

    Feline calicivirus (FCV) nonstructural proteins are translated as part of a large polyprotein that undergoes autocatalytic processing by the virus-encoded 3C-like proteinase. In this study, we mapped three new cleavage sites (E46/A47, E331/D332, and E685/N686) recognized by the virus proteinase in the N-terminal part of the open reading frame 1 (ORF1) polyprotein to complete the processing map. Taken together with two sites we identified previously (E960/A961 and E1071/S1072), the FCV ORF1 po...

  10. DNA cleavage is independent of synapsis during Streptomyces phage phiBT1 integrase-mediated site-specific recombination.

    Science.gov (United States)

    Zhang, Lin; Wang, Lu; Wang, Jin; Ou, Xijun; Zhao, Guoping; Ding, Xiaoming

    2010-10-01

    Bacteriophage-encoded serine recombinases have great potential in genetic engineering but their catalytic mechanisms have not been adequately studied. Integration of ϕBT1 and ϕC31 via their attachment (att) sites is catalyzed by integrases of the large serine recombinase subtype. Both ϕBT1 and ϕC31 integrases were found to cleave single-substrate att sites without synaptic complex formation, and ϕBT1 integrase relaxed supercoiled DNA containing a single integration site. Systematic mutation of the central att site dinucleotide revealed that cleavage was independent of nucleotide sequence, but rejoining was crucially dependent upon complementarity of the cleavage products. Recombination between att sites containing dinucleotides with antiparallel complementarity led to antiparallel recombination. Integrase-substrate pre-incubation experiments revealed that the enzyme can form an attP-integrase tetramer complex that then captures naked attB DNA, and suggested that two alternative assembly pathways can lead to synaptic complex formation.

  11. LH-Induced Steroidogenesis in the Mouse Ovary, but Not Testis, Requires Matrix Metalloproteinase 2- and 9-Mediated Cleavage of Upregulated EGF Receptor Ligands.

    Science.gov (United States)

    Light, Allison; Hammes, Stephen R

    2015-09-01

    Oocyte maturation and cumulus cell expansion depend on luteinizing hormone (LH)-mediated upregulation of membrane-bound epidermal growth factor (EGF)-like ligands, including amphiregulin, epiregulin, and betacellulin. These ligands then transactivate the EGF receptor (EGFR) after release by matrix metalloproteinases (MMPs). However, direct measurement of released EGF-like ligands or MMPs from granulosa cells has not been formally evaluated, nor has direct identification of responsible MMPs. Here we address these issues by analyzing LH-induced steroidogenesis, which is also MMP and EGFR dependent, in freshly isolated mouse primary granulosa cells. We demonstrate a correlation between amphiregulin and epiregulin mRNA induction and steroid production in LH-treated granulosa cells as well as in ovaries of human chorionic gonadotropin-treated mice. In contrast, LH does not alter Mmp1, Mmp2, Mmp3, Mmp8, Mmp9, or Adam17 mRNA expression. We demonstrate that, in primary mouse granulosa cells, LH triggers release of soluble amphiregulin that correlates with steroid production, both of which are blocked by MMP2/9 inhibition, confirming that MMP2/9 likely regulates LH-induced amphiregulin release and downstream processes. Notably, LH does not alter secretion of MMP2/9 from primary granulosa cells, nor does it modulate MMP activity. These findings indicate that, in the ovary, LH dictates EGFR-mediated processes not by regulating MMPs, but instead by increasing EGF-like ligand availability. In contrast, LH stimulation of primary mouse Leydig cells does not induce EGF-like ligand expression or require MMP2/9 for steroidogenesis, confirming marked differences in LH receptor-induced processes in the testes. Our results suggest that MMP inhibition may be a means of attenuating excess ovarian steroid production in diseases like polycystic ovary syndrome.

  12. Identification of E-cadherin signature motifs functioning as cleavage sites for Helicobacter pylori HtrA

    Science.gov (United States)

    Schmidt, Thomas P.; Perna, Anna M.; Fugmann, Tim; Böhm, Manja; Jan Hiss; Haller, Sarah; Götz, Camilla; Tegtmeyer, Nicole; Hoy, Benjamin; Rau, Tilman T.; Neri, Dario; Backert, Steffen; Schneider, Gisbert; Wessler, Silja

    2016-03-01

    The cell adhesion protein and tumour suppressor E-cadherin exhibits important functions in the prevention of gastric cancer. As a class-I carcinogen, Helicobacter pylori (H. pylori) has developed a unique strategy to interfere with E-cadherin functions. In previous studies, we have demonstrated that H. pylori secretes the protease high temperature requirement A (HtrA) which cleaves off the E-cadherin ectodomain (NTF) on epithelial cells. This opens cell-to-cell junctions, allowing bacterial transmigration across the polarised epithelium. Here, we investigated the molecular mechanism of the HtrA-E-cadherin interaction and identified E-cadherin cleavage sites for HtrA. Mass-spectrometry-based proteomics and Edman degradation revealed three signature motifs containing the [VITA]-[VITA]-x-x-D-[DN] sequence pattern, which were preferentially cleaved by HtrA. Based on these sites, we developed a substrate-derived peptide inhibitor that selectively bound and inhibited HtrA, thereby blocking transmigration of H. pylori. The discovery of HtrA-targeted signature sites might further explain why we detected a stable 90 kDa NTF fragment during H. pylori infection, but also additional E-cadherin fragments ranging from 105 kDa to 48 kDa in in vitro cleavage experiments. In conclusion, HtrA targets E-cadherin signature sites that are accessible in in vitro reactions, but might be partially masked on epithelial cells through functional homophilic E-cadherin interactions.

  13. The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles

    Directory of Open Access Journals (Sweden)

    Nguyen Albert T

    2011-12-01

    Full Text Available Abstract Background Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1 maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1 is cleaved to p24 (CA and SP1. Results In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR. Extensive prior genetic evidence suggests that the MHR is critical for virus assembly. Conclusions This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.

  14. The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles

    Science.gov (United States)

    2011-01-01

    Background Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1) maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1) is cleaved to p24 (CA) and SP1. Results In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR). Extensive prior genetic evidence suggests that the MHR is critical for virus assembly. Conclusions This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors. PMID:22151792

  15. Cleavage sites in the polypeptide precursors of poliovirus protein P2-X

    Energy Technology Data Exchange (ETDEWEB)

    Selmer, B.L. (State Univ. of New York, Stony Brook); Hanecak, R.; Anderson, C.W.; Wimmer, E.

    1981-01-01

    Partial amino-terminal sequence analysis has been performed on the three major polypeptide products (P2-3b, P2-5b, and P2-X) from the central region (P2) of the poliovirus polyprotein, and this analysis precisely locates the amino termini of these products with respect to the nucleotide sequence of the poliovirus RNA genome. Like most of the products of the replicase region (P3), the amino termini of P2-5b and P2-X are generated by cleavage between glutamine and glycine residues. Thus, P2-5b and P2-X are probably both produced by the action of a singly (virus-encoded.) proteinase. The amino terminus of P2-3b, on the other hand, is produced by a cleavage between the carboxy-terminal tyrosine of VP1 and the glycine encoded by nucleotides 3381-3383. This result may suggest that more than one proteolytic activity is required for the complete processing of the poliovirus polyprotein.

  16. SVM-based prediction of propeptide cleavage sites in spider toxins identifies toxin innovation in an Australian tarantula.

    Directory of Open Access Journals (Sweden)

    Emily S W Wong

    Full Text Available Spider neurotoxins are commonly used as pharmacological tools and are a popular source of novel compounds with therapeutic and agrochemical potential. Since venom peptides are inherently toxic, the host spider must employ strategies to avoid adverse effects prior to venom use. It is partly for this reason that most spider toxins encode a protective proregion that upon enzymatic cleavage is excised from the mature peptide. In order to identify the mature toxin sequence directly from toxin transcripts, without resorting to protein sequencing, the propeptide cleavage site in the toxin precursor must be predicted bioinformatically. We evaluated different machine learning strategies (support vector machines, hidden Markov model and decision tree and developed an algorithm (SpiderP for prediction of propeptide cleavage sites in spider toxins. Our strategy uses a support vector machine (SVM framework that combines both local and global sequence information. Our method is superior or comparable to current tools for prediction of propeptide sequences in spider toxins. Evaluation of the SVM method on an independent test set of known toxin sequences yielded 96% sensitivity and 100% specificity. Furthermore, we sequenced five novel peptides (not used to train the final predictor from the venom of the Australian tarantula Selenotypus plumipes to test the accuracy of the predictor and found 80% sensitivity and 99.6% 8-mer specificity. Finally, we used the predictor together with homology information to predict and characterize seven groups of novel toxins from the deeply sequenced venom gland transcriptome of S. plumipes, which revealed structural complexity and innovations in the evolution of the toxins. The precursor prediction tool (SpiderP is freely available on ArachnoServer (http://www.arachnoserver.org/spiderP.html, a web portal to a comprehensive relational database of spider toxins. All training data, test data, and scripts used are available from

  17. The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair.

    Science.gov (United States)

    Jasin, Maria; Haber, James E

    2016-08-01

    DNA double-strand breaks (DSBs) are dangerous lesions that if not properly repaired can lead to genomic change or cell death. Organisms have developed several pathways and have many factors devoted to repairing DSBs, which broadly occurs by homologous recombination, which relies on an identical or homologous sequence to template repair, or nonhomologous end-joining. Much of our understanding of these repair mechanisms has come from the study of induced DNA cleavage by site-specific endonucleases. In addition to their biological role, these cellular pathways can be co-opted for gene editing to study gene function or for gene therapy or other applications. While the first gene editing experiments were done more than 20 years ago, the recent discovery of RNA-guided endonucleases has simplified approaches developed over the years to make gene editing an approach that is available to the entire biomedical research community. Here, we review DSB repair mechanisms and site-specific cleavage systems that have provided insight into these mechanisms and led to the current gene editing revolution.

  18. Identification of protein SUMOylation sites by mass spectrometry using combined microwave-assisted aspartic acid cleavage and tryptic digestion

    Science.gov (United States)

    Osula, Omoruyi; Swatkoski, Stephen; Cotter, Robert J.

    2012-01-01

    SUMO (Small-Ubiquitin-like MOdifier) is a post-translational modifier of protein substrates at lysine residues that conjugates to proteins in response to various changes in the cell. As a result of SUMO modification, marked changes in transcription regulation, DNA repair, subcellular localization, and mitosis, among other cellular processes, are known to occur. However, while the identification of ubiquitylation sites by mass spectrometry is aided in part by the presence of a small di-amino acid GlyGly “tag” that remains on lysine residues following tryptic digestion, SUMOylation poses a particular challenge as the absence of a basic residue near to the SUMO C-terminus results in a significant 27 or 32 amino acid sequence branch conjugated to the substrate peptide. MS/MS analyses of these branch peptides generally reveal abundant fragment ions resulting from cleavage of the SUMO tail, but which obscure those needed for characterizing the target peptide sequence. Other approaches for identifying SUMO substrates exist and include overexpression of the SUMO isoforms using an N-terminal histidine tag, as well as site-directed mutagenesis of the C-terminal end of the SUMO sequence. Here, we employ combined enzymatic/chemical approaches which serve to shorten the SUMO tag, and thus help to simplify SUMO spectra, making interpretation of mass spectra and location of the SUMOylation site easier. As described in this report, we demonstrate a method for identifying SUMOylation sites using three commercially available SUMO- modified isoforms, and by employing acid-only and acid/trypsin cleavage strategies. These approaches were carried out using MALDI-TOF and LC/MS instrumentation, along with CID and ETD fragmentation. PMID:22576878

  19. A minimalist chemical model of matrix metalloproteinases--can small peptides mimic the more rigid metal binding sites of proteins?

    Science.gov (United States)

    Árus, Dávid; Nagy, Nóra Veronika; Dancs, Ágnes; Jancsó, Attila; Berkecz, Róbert; Gajda, Tamás

    2013-09-01

    In order to mimic the active center of matrix metalloproteinases (MMPs), we synthesized a pentadecapeptide (Ac-KAHEFGHSLGLDHSK-NH2) corresponding to the catalytic zinc(II) binding site of human MMP-13. The multi-domain structural organization of MMPs fundamentally determines their metal binding affinity, catalytic activity and selectivity. Our potentiometric, UV-visible, CD, EPR, NMR, mass spectrometric and kinetic studies are aimed to explore the usefulness of such flexible peptides to mimic the more rigid metal binding sites of proteins, to examine the intrinsic metal binding properties of this naked sequence, as well as to contribute to the development of a minimalist, peptide-based chemical model of MMPs, including the catalytic properties. Since the multiimidazole environment is also characteristic for copper(II), and recently copper(II) containing variants of MMPs have been identified, we also studied the copper(II) complexes of the above peptide. Around pH 6-7 the peptide, similarly to MMPs, offers a {3Nim} coordination binding site for both zinc(II) and copper(II). In the case of copper(II), the formation of amide coordinated species at higher pH abolished the analogy with the copper(II) containing MMP variant. On the other hand, the zinc(II)-peptide system mimics some basic features of the MMP active sites: the main species around pH7 (ZnH2L) possesses a {3Nim,H2O} coordination environment, the deprotonation of the zinc-bound water takes place near the physiological pH, it forms relatively stable ternary complexes with hydroxamic acids, and the species ZnH2L(OH) and ZnH2L(OH)2 have notable hydrolytic activity between pH7 and 9.

  20. Two tandem RNase III cleavage sites determine betT mRNA stability in response to osmotic stress in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Minji Sim

    Full Text Available While identifying genes regulated by ribonuclease III (RNase III in Escherichia coli, we observed that steady-state levels of betT mRNA, which encodes a transporter mediating the influx of choline, are dependent on cellular concentrations of RNase III. In the present study, we also observed that steady-state levels of betT mRNA are dependent on RNase III activity upon exposure to osmotic stress, indicating the presence of cis-acting elements controlled by RNase III in betT mRNA. Primer extension analyses of betT mRNA revealed two tandem RNase III cleavage sites in its stem-loop region, which were biochemically confirmed via in vitro cleavage assays. Analyses of cleavage sites suggested the stochastic selection of cleavage sites by RNase III, and mutational analyses indicated that RNase III cleavage at either site individually is insufficient for efficient betT mRNA degradation. In addition, both the half-life and abundance of betT mRNA were significantly increased in association with decreased RNase III activity under hyper-osmotic stress conditions. Our findings demonstrate that betT mRNA stability is controlled by RNase III at the post-transcriptional level under conditions of osmotic stress.

  1. Secondary structural analysis of the mRNA regions encoding the hemagglutinin cleavage site basic amino acids of the avian influenza virus H5N1 subtype samples

    Institute of Scientific and Technical Information of China (English)

    ZHANG SuXia; WANG Xin; CHEN XueFeng; CAO Huai; ZHANG Wen; LIU CiQuan

    2008-01-01

    Here we report the codon bias and the mRNA secondary structural features of the hemagglutinin (HA) cleavage site basic amino acid regions of avian influenza virus H5N1 subtypes. We have developed a dynamic extended folding strategy to predict RNA secondary structure with RNAstructure 4.1 program in an iterative extension process. Statistical analysis of the sequences showed that the HA cleavage site basic amino acids favor the adenine-rich codons, and the corresponding mRNA fragments are mainly in the folding states of single-stranded loops. Our sequential and structural analyses showed that to prevent and control these highly pathogenic viruses, that is, to inhibit the gene expression of avian influenza virus H5N1 subtypes, we should consider the single-stranded loop regions of the HA cleavage site-coding sequences as the targets of RNA interference.

  2. Production and cleavage of Drosophila hsp70 transcripts extending beyond the polyadenylation site.

    OpenAIRE

    Berger, S L; Meselson, M

    1994-01-01

    Transcription downstream of the polyadenylation site was studied in the Drosophila hsp70 gene, whose high level of transcription in response to temperature elevation facilitates detection of rare and possibly short-lived transcripts. Transcription downstream of the polyadenylation site was detected both in cultured cells and in intact animals. Even shortly after temperature elevation the extended nonpolyadenylated RNAs were rare relative to mature message, and their level continued to increas...

  3. Determination of optimal sites of antisense oligonucleotide cleavage within TNFα mRNA

    Science.gov (United States)

    Lloyd, B. H.; Giles, R. V.; Spiller, D. G.; Grzybowski, J.; Tidd, D. M.; Sibson, D. R.

    2001-01-01

    Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFα mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity. PMID:11522838

  4. Positive selection pressure introduces secondary mutations at Gag cleavage sites in human immunodeficiency virus type 1 harboring major protease resistance mutations

    DEFF Research Database (Denmark)

    Banke, S.; Lillemark, M.R.; Gerstoft, J.;

    2009-01-01

    mutations). Additional sequences from 13 patients were included for longitudinal analysis. We assessed positive selection pressure on the gag/protease region using a test for the overall influence of positive selection and a total of five tests to identify positively selected single codons. We found...... that positive selection pressure was the driving evolutionary force for the gag region in all three patient groups. An increase in positive selection was observed in gag cleavage site regions p7/p1/p6 only after the acquisition of major PI mutations, suggesting that amino acids in gag cleavage sites under...

  5. Site controlled transgenic mice validating increased expression from human matrix metalloproteinase (MMP-1) promoter due to a naturally occurring SNP.

    Science.gov (United States)

    Coon, Charles I; Fiering, Steven; Gaudet, Justin; Wyatt, Colby A; Brinckerhoff, Constance E

    2009-09-01

    Matrix metalloproteinases (MMPs) comprise a family of more than 20 members, each with the ability to degrade components of the extracellular matrix. The interstitial collagenases have the unique capacity to degrade the stromal collagens, types I, II and III, the body's most abundant proteins. These collagenases include MMP-1, MMP-8, MMP-13 and MMP-14. MMP-1, with a very broad expression pattern, has major roles in mediating matrix destruction in many diseases. We have described a single nucleotide polymorphism (SNP) in the MMP-1 promoter that augments transcription. This SNP is the presence or absence of an extra guanine (G) at -1607 bp, which creates the sequence 5'-GGAA-3'(2G allele), and which is an ETS binding site. Compared to the 1G allele (5'-GAA-3'), the 2G SNP is associated with enhanced transcription of MMP-1 and increased enzymatic activity. Although murine systems are often used to model human diseases, mice have only distant homologues of human MMP-1. Therefore, we used a technique for the targeted insertion of a single copy of a gene at the HPRT locus to compare expression of the 1G and 2G alleles. We generated transgenic mice with -4372 bp of the human MMP-1 promoter containing either the 1G or 2G SNP in front of the lac Z (E.coli ss-galactosidase) gene. We measured the relative expression of the transgenes in vitro in embryonic stem (ES) cells and in fibroblasts derived from embryonic mice. Our data show modest constitutive expression of ss-galactosidase mRNA and protein from these alleles, with the 2G allele more transcriptionally active than the 1G allele. We conclude that these mice represent a model for integration of a single copy of the human MMP-1 promoter into the murine genome, and could be used to study MMP-1 gene expression in a murine system.

  6. Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities.

    Science.gov (United States)

    Fukuyo, Masaki; Nakano, Toshiaki; Zhang, Yingbiao; Furuta, Yoshikazu; Ishikawa, Ken; Watanabe-Matsui, Miki; Yano, Hirokazu; Hamakawa, Takeshi; Ide, Hiroshi; Kobayashi, Ichizo

    2015-03-11

    The restriction-modification systems use epigenetic modification to distinguish between self and nonself DNA. A modification enzyme transfers a methyl group to a base in a specific DNA sequence while its cognate restriction enzyme introduces breaks in DNA lacking this methyl group. So far, all the restriction enzymes hydrolyze phosphodiester bonds linking the monomer units of DNA. We recently reported that a restriction enzyme (R.PabI) of the PabI superfamily with half-pipe fold has DNA glycosylase activity that excises an adenine base in the recognition sequence (5'-GTAC). We now found a second activity in this enzyme: at the resulting apurinic/apyrimidinic (AP) (abasic) site (5'-GT#C, # = AP), its AP lyase activity generates an atypical strand break. Although the lyase activity is weak and lacks sequence specificity, its covalent DNA-R.PabI reaction intermediates can be trapped by NaBH4 reduction. The base excision is not coupled with the strand breakage and yet causes restriction because the restriction enzyme action can impair transformation ability of unmethylated DNA even in the absence of strand breaks in vitro. The base excision of R.PabI is inhibited by methylation of the target adenine base. These findings expand our understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes.

  7. Inhibition of Lassa virus glycoprotein cleavage and multicycle replication by site 1 protease-adapted alpha(1-antitrypsin variants.

    Directory of Open Access Journals (Sweden)

    Anna Maisa

    Full Text Available BACKGROUND: Proteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease (S1P is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and, hence, essential for infectivity and virus spread. Therefore, we tested in this study the concept of using S1P as a target to block efficient virus replication. METHODOLOGY/PRINCIPAL FINDING: We demonstrate that stable cell lines inducibly expressing S1P-adapted alpha(1-antitrypsin variants inhibit the proteolytic maturation of GP-C. Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of alpha(1-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells. Moreover, S1P-specific alpha(1-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus. Inhibition of viral replication correlated with the ability of the different alpha(1-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor. CONCLUSIONS/SIGNIFICANCE: Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies.

  8. Engineering D-Amino Acid Containing Collagen Like Peptide at the Cleavage Site of Clostridium histolyticum Collagenase for Its Inhibition.

    Directory of Open Access Journals (Sweden)

    Punitha Velmurugan

    Full Text Available Collagenase is an important enzyme which plays an important role in degradation of collagen in wound healing, cancer metastasis and even in embryonic development. However, the mechanism of this degradation has not yet been completely understood. In the field of biomedical and protein engineering, the design and development of new peptide based materials is of main concern. In the present work an attempt has been made to study the effect of DAla in collagen like peptide (imino-poor region of type I collagen on the structure and stability of peptide against enzyme hydrolysis. Effect of replacement of DAla in the collagen like peptide has been studied using circular dichroic spectroscopy (CD. Our findings suggest that, DAla substitution leads to conformational changes in the secondary structure and favours the formation of polyproline II conformation than its L-counterpart in the imino-poor region of collagen like peptides. Change in the chirality of alanine at the cleavage site of collagenase in the imino-poor region inhibits collagenolytic activity. This may find application in design of peptides and peptidomimics for enzyme-substrate interaction, specifically with reference to collagen and other extra cellular matrix proteins.

  9. Crystal structure of Bombyx mori arylphorins reveals a 3:3 heterohexamer with multiple papain cleavage sites.

    Science.gov (United States)

    Hou, Yong; Li, Jianwei; Li, Yi; Dong, Zhaoming; Xia, Qingyou; Yuan, Y Adam

    2014-06-01

    In holometabolous insects, the accumulation and utilization of storage proteins (SPs), including arylphorins and methionine-rich proteins, are critical for the insect metamorphosis. SPs function as amino acids reserves, which are synthesized in fat body, secreted into the larval hemolymph and taken up by fat body shortly before pupation. However, the detailed molecular mechanisms of digestion and utilization of SPs during development are largely unknown. Here, we report the crystal structure of Bombyx mori arylphorins at 2.8 Å, which displays a heterohexameric structural arrangement formed by trimerization of dimers comprising two structural similar arylphorins. Our limited proteolysis assay and microarray data strongly suggest that papain-like proteases are the major players for B. mori arylphorins digestion in vitro and in vivo. Consistent with the biochemical data, dozens of papain cleavage sites are mapped on the surface of the heterohexameric structure of B. mori arylphorins. Hence, our results provide the insightful information to understand the metamorphosis of holometabolous insects at molecular level.

  10. Disclosure of key stereoelectronic factors for efficient H2 binding and cleavage in the active site of [NiFe]-hydrogenases.

    Science.gov (United States)

    Bruschi, Maurizio; Tiberti, Matteo; Guerra, Alessandro; De Gioia, Luca

    2014-02-05

    A comparative analysis of a series of DFT models of [NiFe]-hydrogenases, ranging from minimal NiFe clusters to very large systems including both the first and second coordination sphere of the bimetallic cofactor, was carried out with the aim of unraveling which stereoelectronic properties of the active site of [NiFe]-hydrogenases are crucial for efficient H2 binding and cleavage. H2 binding to the Ni-SIa redox state is energetically favored (by 4.0 kcal mol(-1)) only when H2 binds to Ni, the NiFe metal cluster is in a low spin state, and the Ni cysteine ligands have a peculiar seesaw coordination geometry, which in the enzyme is stabilized by the protein environment. The influence of the Ni coordination geometry on the H2 binding affinity was then quantitatively evaluated and rationalized analyzing frontier molecular orbitals and populations. Several plausible reaction pathways leading to H2 cleavage were also studied. It turned out that a two-step pathway, where H2 cleavage takes place on the Ni-SIa redox state of the enzyme, is characterized by very low reaction barriers and favorable reaction energies. More importantly, the seesaw coordination geometry of Ni was found to be a key feature for facile H2 cleavage. The discovery of the crucial influence of the Ni coordination geometry on H2 binding and activation in the active site of [NiFe]-hydrogenases could be exploited in the design of novel biomimetic synthetic catalysts.

  11. Optimization of a multi-stage, multi-subunit malaria vaccine candidate for the production in Pichia pastoris by the identification and removal of protease cleavage sites.

    Science.gov (United States)

    Spiegel, Holger; Schinkel, Helga; Kastilan, Robin; Dahm, Pia; Boes, Alexander; Scheuermayer, Matthias; Chudobová, Ivana; Maskus, Dominika; Fendel, Rolf; Schillberg, Stefan; Reimann, Andreas; Fischer, Rainer

    2015-04-01

    We demonstrated the successful optimization of a recombinant multi-subunit malaria vaccine candidate protein for production in the methylotrophic yeast Pichia pastoris by the identification and subsequent removal of two protease cleavage sites. After observing protein degradation in the culture supernatant of a fed-batch fermentation, the predominant proteolytic fragment of the secreted recombinant protein was analyzed by mass spectrometry. The MS data indicated the cleavage of an amino acid sequence matching the yeast KEX2-protease consensus motif EKRE. The cleavage in this region was completely abolished by the deletion of the EKRE motif in a modified variant. This modified variant was produced, purified, and used for immunization of rabbits, inducing high antigen specific antibody titers (2 × 10(6) ). Total IgG from rabbit immune sera recognized different stages of Plasmodium falciparum parasites in immunofluorescence assays, indicating native folding of the vaccine candidate. However, the modified variant was still degraded, albeit into different fragments. Further analysis by mass spectrometry and N-terminal sequencing revealed a second cleavage site downstream of the motif PEVK. We therefore removed a 17-amino-acid stretch including the PEVK motif, resulting in the subsequent production of the full-length recombinant vaccine candidate protein without significant degradation, with a yield of 53 mg per liter culture volume. We clearly demonstrate that the proteolytic degradation of recombinant proteins by endogenous P. pastoris proteases can be prevented by the identification and removal of such cleavage sites. This strategy is particularly relevant for the production of recombinant subunit vaccines, where product yield and stability play a more important role than for the production of a stringently-defined native sequence which is necessary for most therapeutic molecules.

  12. Alternative Selection of β-Site APP-Cleaving Enzyme 1 (BACE1) Cleavage Sites in Amyloid β-Protein Precursor (APP) Harboring Protective and Pathogenic Mutations within the Aβ Sequence.

    Science.gov (United States)

    Kimura, Ayano; Hata, Saori; Suzuki, Toshiharu

    2016-11-11

    β-Site APP-cleaving enzyme 1 (BACE1) cleaves amyloid β-protein precursor (APP) at the bond between Met(671) and Asp(672) (β-site) to generate the carboxyl-terminal fragment (CTFβ/C99). BACE1 also cleaves APP at another bond between Thr(681) and Gln(682) (β'-site), yielding CTFβ'/C89. Cleavage of CTFβ/C99 by γ-secretase generates Aβ(1-XX), whereas cleavage of CTFβ'/C89 generates Aβ(11-XX). Thus, β'-site cleavage by BACE1 is amyloidolytic rather than amyloidogenic. β' cleavage of mouse APP is more common than the corresponding cleavage of human APP. We found that the H684R substitution within human Aβ, which replaces the histidine in the human protein with the arginine found at the corresponding position in mouse, facilitated β' cleavage irrespective of the species origin of BACE1, thereby significantly increasing the level of Aβ(11-XX) and decreasing the level of Aβ(1-XX). Thus, amino acid substitutions within the Aβ sequence influenced the selectivity of alternative β- or β'-site cleavage of APP by BACE1. In familial Alzheimer's disease (FAD), the APP gene harbors pathogenic variations such as the Swedish (K670N/M671L), Leuven (E682K), and A673V mutations, all of which decrease Aβ(11-40) generation, whereas the protective Icelandic mutation (A673T) increases generation of Aβ(11-40). Thus, A673T promotes β' cleavage of APP and protects subjects against AD. In addition, CTFβ/C99 was cleaved by excess BACE1 activity to generate CTFβ'/C89, followed by Aβ(11-40), even if APP harbored pathogenic mutations. The resultant Aβ(11-40) was more metabolically labile in vivo than Aβ(1-40). Our analysis suggests that some FAD mutations in APP are amyloidogenic and/or amyloidolytic via selection of alternative BACE1 cleavage sites.

  13. Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A.

    Science.gov (United States)

    Nguyen, G N; George, L A; Siner, J I; Davidson, R J; Zander, C B; Zheng, X L; Arruda, V R; Camire, R M; Sabatino, D E

    2017-01-01

    Essentials Factor (F) VIII is an inefficiently expressed protein. Furin deletion FVIII variants were purified and characterized using in vitro and in vivo assays. These minimally modified novel FVIII variants have enhanced function. These variants provide a strategy for increasing FVIII expression in hemophilia A gene therapy. Background The major challenge for developing gene-based therapies for hemophilia A is that human factor VIII (hFVIII) has intrinsic properties that result in inefficient biosynthesis. During intracellular processing, hFVIII is predominantly cleaved at a paired basic amino acid cleaving enzyme (PACE) or furin cleavage site to yield a heterodimer that is the major form of secreted protein. Previous studies with B-domain-deleted (BDD) canine FVIII and hFVIII-R1645H, both differing from hFVIII by a single amino acid at this site, suggested that these proteins are secreted mainly in a single polypeptide chain (SC) form and exhibit enhanced function. Objective We hypothesized that deletion(s) of the furin site modulates FVIII biology and may enhance its function. Methods A series of recombinant hFVIII-furin deletion variants were introduced into hFVIII-BDD [Δ1645, 1645-46(Δ2), 1645-47(Δ3), 1645-48(Δ4), or Δ1648] and characterized. Results In vitro, recombinant purified Δ3 and Δ4 were primarily SC and, interestingly, had 2-fold higher procoagulant activity compared with FVIII-BDD. In vivo, the variants also have improved hemostatic function. After adeno-associated viral (AAV) vector delivery, the expression of these variants is 2-4-fold higher than hFVIII-BDD. Protein challenges of each variant in mice tolerant to hFVIII-BDD showed no anti-FVIII immune response. Conclusions These data suggest that the furin deletion hFVIII variants are superior to hFVIII-BDD without increased immunogenicity. In the setting of gene-based therapeutics, these novel variants provide a unique strategy to increase FVIII expression, thus lowering the vector dose, a

  14. Determining the cleavage site for the mature antimicrobial peptide of Nile tilapia β-defensin using 2D electrophoresis, western blot, and mass spectrometry analysis.

    Science.gov (United States)

    Chang, Chin-I; Chen, Li-Hao; Hu, Yeh-Fang; Wu, Chia-Che; Tsai, Jyh-Ming

    2017-03-01

    Several proteomic techniques were used to determine the cleavage site of the mature antimicrobial peptide of Nile tilapia β-defensin. The computer-predicted Nile tilapia β-defensin ((25)ASFPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL(66)) composed of 42 amino acids was chemically synthesized and prepared to produce an antibody for Western blotting. Total proteins from the skin of the Nile tilapia were separated on two-dimensional electrophoresis, and the spot of Nile tilapia β-defensin was recognized using Western blot analysis. It was then excised and extracted from the gel. The precise molecular mass of this spot was determined by LC-MS/MS spectrometry. Four major peptides were discovered, with molecular weights of 4293.2 Da, 4306.5 Da, 4678.9 Da, and 4715.0 Da. The calculated mass of the 40-amino-acid sequence ((27)FPWSCLSLSGVCRKVCLPTELFFGPLGCGKGSLCCVSHFL(66)) of Nile tilapia β-defensin starting from Phe27 and ending with Leu66 was 4293.18 Da, which completely matched the 4293.2 Da peptide that was obtained from the mass spectrometry analysis. This result confirmed that the cleavage site for the mature C-terminal Nile tilapia β-defensin is at residue Ser26-Phe27, not at Ala24-25 as predicted by computer analysis. This study provides a simple but reliable model to determine the cleavage site for a mature antimicrobial peptide. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Scores of amino acid 0D-3D information as applied in cleavage site prediction and better specificity elucidation for human immunodeficiency virus type 1 protease

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    A new set of descriptors,namely score vectors of the zero dimension,one dimension,two dimensions and three dimensions(SZOTT),was derived from principle component analysis of a matrix of 1369 structural variables including 0D,1D,2D and 3D information for the 20 coded amino acids. SZOTT scales were then used in cleavage site prediction of human immunodeficiency virus type 1 protease. Linear discriminant analysis(LDA) and support vector machines(SVM) were applied to developing models to predict the cleavage sites. The results obtained by linear discriminant analysis(LDA) and support vector machines(SVM) are as follows. The Matthews correlation coefficients(MCC) by the resubstitution test,leave-one-out cross validation(LOOCV) and external validation are 0.879 and 0.911,0.849 and 0.901,0.822 and 0.846,respectively. The receiver operating characteristic(ROC) analysis showed that the SVM model possesses better simulative and predictive ability in comparison with the LDA model. Satisfactory results show that SZOTT descriptors can be further used to predict cleavage sites of human immunodeficiency virus type 1 protease.

  16. Scores of amino acid 0D-3D information as applied in cleavage site prediction and better specificity elucidation for human immunodeficiency virus type 1 protease

    Institute of Scientific and Technical Information of China (English)

    KANG LiFang; LIANG GuiZhao; SHU Mao; YANG ShanBin; LI ZhiLiang

    2008-01-01

    A new set of descriptors, namely score vectors of the zero dimension, one dimension, two dimensions and three dimensions (SZOTT), was derived from principle component analysis of a matrix of 1369 structural variables including 0D, 1D, 2D and 3D information for the 20 coded amino acids. SZOTT scales were then used in cleavage site prediction of human immunodeficiency virus type 1 protease. Linear discriminant analysis (LDA) and support vector machines (SVM) were applied to developing models to predict the cleavage sites. The results obtained by linear discriminant analysis (LDA) and support vector machines (SVM) are as follows. The Matthews correlation coefficients (MCC) by the resubstitution test, leave-one-out cross validation (LOOCV) and external validation are 0.879 and 0.911, 0.649 and 0.901, 0.822 and 0.846, respectively. The receiver operating characteristic (ROC) analysis showed that the SVM model possesses better simulative and predictive ability in comparison with the LDA model. Satisfactory results show that SZOTT descriptors can be further used to predict cleavage sites of human immunodeficiency virus type 1 protease.

  17. Fluoroquinolones stimulate the DNA cleavage activity of topoisomerase IV by promoting the binding of Mg2+ to the second metal binding site

    Science.gov (United States)

    Oppegard, Lisa M.; Schwanz, Heidi A.; Towle, Tyrell R.; Kerns, Robert J.; Hiasa, Hiroshi

    2016-01-01

    Background Fluoroquinolones target bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Fluoroquinolones trap a topoisomerase-DNA covalent complex as a topoisomerase-fluoroquinolone-DNA ternary complex and ternary complex formation is critical for their cytotoxicity. A divalent metal ion is required for type IIA topoisomerase-catalyzed strand breakage and religation reactions. Recent studies have suggested that type IIA topoisomerases use two metal ions, one structural and one catalytic, to carry out the strand breakage reaction. Methods We conducted a series of DNA cleavage assays to examine the effects of fluoroquinolones and quinazolinediones on Mg2+-, Mn2+-, or Ca2+-supported DNA cleavage activity of Esherichia coli Topo IV. Results In the absence of any drug, 20–30 mM Mg2+ was required for the maximum levels of the DNA cleavage activity of Topo IV, whereas approximately 1 mM of either Mn2+ or Ca2+ was sufficient to support the maximum levels of the DNA cleavage activity of Topo IV. Fluoroquinolones promoted the Topo IV-catalyzed strand breakage reaction at low Mg2+ concentrations where Topo IV alone could not efficiently cleave DNA. Conclusions and General Significance At low Mg2+ concentrations, fluoroquinolones may stimulate the Topo IV-catalyzed strand breakage reaction by promoting Mg2+ binding to metal binding site B through the structural distortion in DNA. As Mg2+ concentration increases, fluoroquinolones may inhibit the religation reaction by either stabilizing Mg2+ at site B or inhibition the binding of Mg2+ to site A. This study provides a molecular basis of how fluoroquinolones stimulate the Topo IV-catalyzed strand breakage reaction by modulating Mg2+ binding. PMID:26723176

  18. Fluoroquinolones stimulate the DNA cleavage activity of topoisomerase IV by promoting the binding of Mg(2+) to the second metal binding site.

    Science.gov (United States)

    Oppegard, Lisa M; Schwanz, Heidi A; Towle, Tyrell R; Kerns, Robert J; Hiasa, Hiroshi

    2016-03-01

    Fluoroquinolones target bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Fluoroquinolones trap a topoisomerase-DNA covalent complex as a topoisomerase-fluoroquinolone-DNA ternary complex and ternary complex formation is critical for their cytotoxicity. A divalent metal ion is required for type IIA topoisomerase-catalyzed strand breakage and religation reactions. Recent studies have suggested that type IIA topoisomerases use two metal ions, one structural and one catalytic, to carry out the strand breakage reaction. We conducted a series of DNA cleavage assays to examine the effects of fluoroquinolones and quinazolinediones on Mg(2+)-, Mn(2+)-, or Ca(2+)-supported DNA cleavage activity of Escherichia coli Topo IV. In the absence of any drug, 20-30 mM Mg(2+) was required for the maximum levels of the DNA cleavage activity of Topo IV, whereas approximately 1mM of either Mn(2+) or Ca(2+) was sufficient to support the maximum levels of the DNA cleavage activity of Topo IV. Fluoroquinolones promoted the Topo IV-catalyzed strand breakage reaction at low Mg(2+) concentrations where Topo IV alone could not efficiently cleave DNA. At low Mg(2+) concentrations, fluoroquinolones may stimulate the Topo IV-catalyzed strand breakage reaction by promoting Mg(2+) binding to metal binding site B through the structural distortion in DNA. As Mg(2+) concentration increases, fluoroquinolones may inhibit the religation reaction by either stabilizing Mg(2+) at site B or inhibition the binding of Mg(2+) to site A. This study provides a molecular basis of how fluoroquinolones stimulate the Topo IV-catalyzed strand breakage reaction by modulating Mg(2+) binding. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations.

    Science.gov (United States)

    van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D

    2015-12-02

    The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1-2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ∼30 bp.

  20. Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

    Science.gov (United States)

    van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D.

    2015-01-01

    The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1–2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ∼30 bp. PMID:26507855

  1. Residues in Conserved Loops of Intramembrane Metalloprotease SpoIVFB Interact with Residues near the Cleavage Site in Pro-σK

    Science.gov (United States)

    Zhang, Yang; Luethy, Paul M.

    2013-01-01

    Intramembrane metalloproteases (IMMPs) control critical biological processes by cleaving membrane-associated proteins within a transmembrane segment or at a site near the membrane surface. Phylogenetic analysis divides IMMPs into four groups. SpoIVFB is a group III IMMP that regulates Bacillus subtilis endospore formation by cleaving Pro-σK and releasing the active sigma factor from a membrane. To elucidate the enzyme-substrate interaction, single-cysteine versions of catalytically inactive SpoIVFB and C-terminally truncated Pro-σK(1-126) (which can be cleaved by active SpoIVFB) were coexpressed in Escherichia coli, and proximity was tested by disulfide cross-linking in vivo. As expected, the results provided evidence that catalytic residue Glu-44 of SpoIVFB is near the cleavage site in the substrate. Also near the cleavage site were two residues of SpoIVFB in predicted conserved loops; Pro-135 in a short loop and Val-70 in a longer loop. Pro-135 corresponds to Pro-399 of RseP, a group I IMMP, and Pro-399 was reported previously to interact with substrate near the cleavage site, suggesting a conserved interaction across IMMP subfamilies. Val-70 follows a newly recognized conserved motif, PXGG (X is a large hydrophobic residue), which is in a hydrophobic region predicted to be a membrane reentrant loop. Following the hydrophobic region is a negatively charged region that is conserved in IMMPs of groups I and III. At least two residues with a negatively charged side chain are required in this region for activity of SpoIVFB. The region exhibits other features in IMMPs of groups II and IV. Its possible roles, as well as that of the short loop, are discussed. New insights into IMMP-substrate interaction build toward understanding how IMMPs function and may facilitate manipulation of their activity. PMID:23995631

  2. Short communication: high natural polymorphism in the gag gene cleavage sites of non-B HIV type 1 isolates from Gabon.

    Science.gov (United States)

    Liégeois, Florian; Reteno, Dorine Gaëlle Ikanga; Mouinga-Ondémé, Augustin; Sica, Jeanne; Rouet, François

    2013-08-01

    The main goal of the present study was to determine the frequency of substitutions in the cleavage sites (CS) of gag gene among non-B HIV-1 isolates from Gabon. Fifty plasma specimens, collected in 2010-2011, from HIV-1-infected patients failing first-line antiretroviral (ARV) regimens (constituted of two nucleoside reverse transcriptase inhibitors+one nonnucleoside reverse transcriptase inhibitor) (n=38) and from HIV-1-infected individuals untreated with ARV (n=12) were analyzed in the gag and gag-pol cleavage sites. Compared to HXB2 reference sequence, the total median number of substitutions in gag and gag-pol CS was 10 (range, 5-18). The cleavage site p2/NC was the most variable of the four gag CS with 100% (50/50) isolates carrying at least 1 substitution (range, 1-9). The two gag-pol TFP/p6pol and p6pol/PR CS sites were also highly variable (at least one substitution, 50/50, 100% in both cases). Substitutions at position G381 (p2/NC), L449 (p1/p6gag), and K444 (TFP/p6pol) were significantly more frequent in CRF02_AG strains, compared to other non-B strains (30.4% vs. 3.7%, p=0.03; 87.0% vs. 59.3%, p=0.03; and 91.3% vs. 59.3%, p=0.01, respectively). Other non-B subtypes were significantly more likely to harbor substitutions at position N487 (p6pol) (70.4%) than CRF02_AG (39.1%) (p=0.02). In Gabon, gag and gag-pol cleavage sites were highly polymorphic in protease inhibitor-naive patients harboring non-B HIV-1 strains. In sub-Saharan Africa, further studies are definitively required to better understand the impact of gag mutations among subjects receiving second-line LPV/r-containing regimens (monotherapy or triple combinations).

  3. Site-specific O-Glycosylation on the MUC2 Mucin Protein Inhibits Cleavage by the Porphyromonas gingivalis Secreted Cysteine Protease (RgpB)

    DEFF Research Database (Denmark)

    van der Post, Sjoerd; Subramani, Durai B; Bäckström, Malin

    2013-01-01

    The colonic epithelial surface is protected by an inner mucus layer that the commensal microflora cannot penetrate. We previously demonstrated that Entamoeba histolytica secretes a protease capable of dissolving this layer that is required for parasite penetration. Here, we asked whether there ar......The colonic epithelial surface is protected by an inner mucus layer that the commensal microflora cannot penetrate. We previously demonstrated that Entamoeba histolytica secretes a protease capable of dissolving this layer that is required for parasite penetration. Here, we asked whether...... there are bacteria that can secrete similar proteases. We screened bacterial culture supernatants for such activity using recombinant fragments of the MUC2 mucin, the major structural component, and the only gel-forming mucin in the colonic mucus. MUC2 has two central heavily O-glycosylated mucin domains...... was isolated and identified as Arg-gingipain B (RgpB). Two cleavage sites were localized to IR↓TT and NR↓QA. IR↓TT cleavage will disrupt the MUC2 polymers. Because this site has two potential O-glycosylation sites, we tested whether recombinant GalNAc-transferases (GalNAc-Ts) could glycosylate a synthetic...

  4. Genomic DNA breakpoints in AML1/RUNX1 and ETO cluster with topoisomerase II DNA cleavage and DNase I hypersensitive sites in t(8;21) leukemia

    Science.gov (United States)

    Zhang, Yanming; Strissel, Pamela; Strick, Reiner; Chen, Jianjun; Nucifora, Giuseppina; Le Beau, Michelle M.; Larson, Richard A.; Rowley, Janet D.

    2002-01-01

    The translocation t(8;21)(q22;q22) is one of the most frequent chromosome translocations in acute myeloid leukemia (AML). AML1/RUNX1 at 21q22 is involved in t(8;21), t(3;21), and t(16;21) in de novo and therapy-related AML and myelodysplastic syndrome as well as in t(12;21) in childhood B cell acute lymphoblastic leukemia. Although DNA breakpoints in AML1 and ETO (at 8q22) cluster in a few introns, the mechanisms of DNA recombination resulting in t(8;21) are unknown. The correlation of specific chromatin structural elements, i.e., topoisomerase II (topo II) DNA cleavage sites, DNase I hypersensitive sites, and scaffold-associated regions, which have been implicated in chromosome recombination with genomic DNA breakpoints in AML1 and ETO in t(8;21) is unknown. The breakpoints in AML1 and ETO were clustered in the Kasumi 1 cell line and in 31 leukemia patients with t(8;21); all except one had de novo AML. Sequencing of the breakpoint junctions revealed no common DNA motif; however, deletions, duplications, microhomologies, and nontemplate DNA were found. Ten in vivo topo II DNA cleavage sites were mapped in AML1, including three in intron 5 and seven in intron 7a, and two were in intron 1b of ETO. All strong topo II sites colocalized with DNase I hypersensitive sites and thus represent open chromatin regions. These sites correlated with genomic DNA breakpoints in both AML1 and ETO, thus implicating them in the de novo 8;21 translocation. PMID:11867721

  5. Reversed DNA strand cleavage specificity in initiation of Cre-LoxP recombination induced by the His289Ala active-site substitution.

    Science.gov (United States)

    Gelato, Kathy A; Martin, Shelley S; Baldwin, Enoch P

    2005-11-25

    During the first steps of site-specific recombination, Cre protein cleaves and religates a specific homologous pair of LoxP strands to form a Holliday junction (HJ) intermediate. The HJ is resolved into recombination products through exchange of the second homologous strand pair. CreH289A, containing a His to Ala substitution in the conserved R-H-R catalytic motif, has a 150-fold reduced recombination rate and accumulates HJs. However, to produce these HJs, CreH289A exchanges the opposite set of strands compared to wild-type Cre (CreWT). To investigate how CreH289A and CreWT impose strand exchange order, we characterized their reactivities and strand cleavage preferences toward LoxP duplex and HJ substrates containing 8bp spacer substitutions. Remarkably, CreH289A had different and often opposite strand exchange preferences compared to CreWT with nearly all substrates. CreH289N was much less perturbed, implying that overall recombination rate and strand exchange depend more on His289 hydrogen bonding capability than on its acid/base properties. LoxP substitutions immediately 5' (S1 nucleotide) or 3' (S1' nucleotide) of the scissile phosphate had large effects on substrate utilization and strand exchange order. S1' substitutions, designed to alter base-unstacking events concomitant with Cre-induced LoxP bending, caused HJ accumulation and dramatically inverted the cleavage preferences. That pre-formed HJs were resolved via either strand in vitro suggests that inhibition of the "conformational switch" isomerization required to trigger the second strand exchange accounts for the observed HJ accumulation. Rather than reflecting CreWT behavior, CreH289A accumulates HJs of opposite polarity through a combination of its unique cleavage specificity and an HJ isomerization defect. The overall implication is that cleavage specificity is mediated by sequence-dependent DNA deformations that influence the scissile phosphate positioning and reactivity. A role of His289 may be to

  6. Enhanced Protective Efficacy of H5 Subtype Influenza Vaccine with Modification of the Multibasic Cleavage Site of Hemagglutinin in Retroviral Pseudotypes

    Institute of Scientific and Technical Information of China (English)

    Ling Tao; JianJun Chen; Jin Meng; Yao Chen; Hongxia Li; Yan Liu; Zhenhua Zheng

    2013-01-01

    Traditionally,the multibasic cleavage site (MBCS) of surface protein H5-hemagglutinin (HA) is converted to a monobasic one so as to weaken the virulence of recombinant H5N1 influenza viruses and to produce inactivated and live attenuated vaccines.Whether such modification benefits new candidate vaccines has not been adequately investigated.We previously used retroviral vectors to generate wtH5N1 pseudotypes containing the wild-type HA (wtH5) from A/swine/Anhui/ca/2004 (H5N1) virus.Here,we generated mtH5N1 pseudotypes,which contained a mutant-type HA (mtH5) with a modified monobasic cleavage site.Groups of mice were subcutaneously injected with the two types of influenza pseudotypes.Compared to the group immunized with wtH5N1 pseudotypes,the inoculation of mtH5N1 pseudotypes induced significantly higher levels of HA specific IgG and IFN-γ in immunized mice,and enhanced protection against the challenge of mouse-adapted avian influenza virus A/Chicken/Henan/12/2004 (H5N1).This study suggests modification of the H5-hemagglutinin MBCS in retroviral pseudotypes enhances protection efficacy in mice and this information may be helpful for development of vaccines from mammalian cells to fight against H5N1 influenza viruses.

  7. Requirement of helix P2.2 and nucleotide G1 for positioning the cleavage site and cofactor of the glmS ribozyme.

    Science.gov (United States)

    Klein, Daniel J; Wilkinson, Sara R; Been, Michael D; Ferré-D'Amaré, Adrian R

    2007-10-12

    The glmS ribozyme is a catalytic RNA that self-cleaves at its 5'-end in the presence of glucosamine 6-phosphate (GlcN6P). We present structures of the glmS ribozyme from Thermoanaerobacter tengcongensis that are bound with the cofactor GlcN6P or the inhibitor glucose 6-phosphate (Glc6P) at 1.7 A and 2.2 A resolution, respectively. The two structures are indistinguishable in the conformations of the small molecules and of the RNA. GlcN6P binding becomes apparent crystallographically when the pH is raised to 8.5, where the ribozyme conformation is identical with that observed previously at pH 5.5. A key structural feature of this ribozyme is a short duplex (P2.2) that is formed between sequences just 3' of the cleavage site and within the core domain, and which introduces a pseudoknot into the active site. Mutagenesis indicates that P2.2 is required for activity in cis-acting and trans-acting forms of the ribozyme. P2.2 formation in a trans-acting ribozyme was exploited to demonstrate that N1 of the guanine at position 1 contributes to GlcN6P binding by interacting with the phosphate of the cofactor. At neutral pH, RNAs with adenine, 2-aminopurine, dimethyladenine or purine substitutions at position 1 cleave faster with glucosamine than with GlcN6P. This altered cofactor preference provides biochemical support for the orientation of the cofactor within the active site. Our results establish two features of the glmS ribozyme that are important for its activity: a sequence within the core domain that selects and positions the cleavage-site sequence, and a nucleobase at position 1 that helps position GlcN6P.

  8. Fluorescence quenching of graphene oxide combined with the site-specific cleavage of restriction endonuclease for deoxyribonucleic acid demethylase activity assay

    Energy Technology Data Exchange (ETDEWEB)

    Ji, Lijuan; Qian, Yingdan; Wu, Ping; Zhang, Hui; Cai, Chenxin, E-mail: cxcai@njnu.edu.cn

    2015-04-15

    Highlights: • An approach for sensitive and selective DNA demethylase activity assay is reported. • This assay is based on the fluorescence quenching of GO and site-specific cleavage of endonuclease. • It can determine as low as 0.05 ng mL{sup −1} of MBD2 with a linear range of 0.2–300 ng mL{sup −1}. • It has an ability to recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. • It can avoid false signals, requiring no bisulfite conversion, PCR amplification, radioisotope-labeling. - Abstract: We report on the development of a sensitive and selective deoxyribonucleic acid (DNA) demethylase (using MBD2 as an example) activity assay by coupling the fluorescence quenching of graphene oxide (GO) with the site-specific cleavage of HpaII endonuclease to improve the selectivity. This approach was developed by designing a single-stranded probe (P1) that carries a binding region to facilitate the interaction with GO, which induces fluorescence quenching of the labeled fluorophore (FAM, 6-carboxyfluorescein), and a sensing region, which contains a hemi-methylated site of 5′-CmCGG-3′, to specifically recognize the target (T1, a 32-mer DNA from the promoter region of p53 gene) and hybridize with it to form a P1/T1 duplex. After demethylation with MBD2, the duplex can be specifically cleaved using HpaII, which releases the labeled FAM from the GO surface and results in the recovery of fluorescence. However, this cleavage is blocked by the hemi-methylation of this site. Thus, the magnitude of the recovered fluorescence signal is related to the MBD2 activity, which establishes the basis of the DNA demethylase activity assay. This assay can determine as low as ∼(0.05 ± 0.01) ng mL{sup −1} (at a signal/noise of 3) of MBD2 with a linear range of 0.2–300 ng mL{sup −1} and recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. The advantage of this assay is its ability to avoid false signals and no

  9. The vhs1 mutant form of herpes simplex virus virion host shutoff protein retains significant internal ribosome entry site-directed RNA cleavage activity.

    Science.gov (United States)

    Lu, P; Saffran, H A; Smiley, J R

    2001-01-01

    The virion host shutoff (vhs) protein of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated turnover of host and viral mRNAs during HSV infection. As well, it induces endoribonucleolytic cleavage of RNA substrates when produced in a rabbit reticulocyte lysate (RRL) in vitro translation system. The vhs1 point mutation (Thr 214-->Ile) eliminates vhs function during virus infection and in transiently transfected mammalian cells and was therefore previously considered to abolish vhs activity. Here we demonstrate that the vhs1 mutant protein induces readily detectable endoribonuclease activity on RNA substrates bearing the internal ribosome entry site of encephalomyocarditis virus in the RRL assay system. These data document that the vhs1 mutation does not eliminate catalytic activity and raise the possibility that the vhs-dependent endoribonuclease employs more than one mode of substrate recognition.

  10. Improving Proteolytic Cleavage at the 3A/3B Site of the Hepatitis A Virus Polyprotein Impairs Processing and Particle Formation, and the Impairment Can Be Complemented in trans by 3AB and 3ABC

    OpenAIRE

    Kusov, Yuri; Gauss-Müller, Verena

    1999-01-01

    The orchestrated liberation of viral proteins by 3Cpro-mediated proteolysis is pivotal for gene expression by picornaviruses. Proteolytic processing is regulated either by the amino acid sequence at the cleavage site of the substrate or by cofactors covalently or noncovalently linked to the viral proteinase. To determine the role of the amino acid sequence at cleavage sites 3A/3B and 3B/3C that are essential for the liberation of 3Cpro from its precursors and to assess the function of the sta...

  11. β-site amyloid precursor protein cleaving enzyme 1(BACE1) regulates Notch signaling by controlling the cleavage of Jagged 1 (Jag1) and Jagged 2 (Jag2) proteins.

    Science.gov (United States)

    He, Wanxia; Hu, Jinxuan; Xia, Yuxing; Yan, Riqiang

    2014-07-25

    BACE1 is a type I transmembrane aspartyl protease that cleaves amyloid precursor protein at the β-secretase site to initiate the release of β-amyloid peptide. As a secretase, BACE1 also cleaves additional membrane-bound molecules by exerting various cellular functions. In this study, we showed that BACE1 can effectively shed the membrane-anchored signaling molecule Jagged 1 (Jag1).Wealso mapped the cleavage sites of Jag1 by ADAM10 and ADAM17. Although Jag1 shares a high degree of homology with Jag2 in the ectodomain region, BACE1 fails to cleave Jag2 effectively, indicating a selective cleavage of Jag1. Abolished cleavage of Jag1 in BACE1-null mice leads to enhanced astrogenesis and, concomitantly, reduced neurogenesis. This characterization provides biochemical evidence that the Jag1-Notch pathway is under the control of BACE1 activity

  12. D-Isonucleotide (isoNA) incorporation around cleavage site of passenger strand promotes the vibration of Ago2-PAZ domain and enhances in vitro potency of siRNA.

    Science.gov (United States)

    Huang, Ye; Tian, Miao; Zhang, Yichao; Sheng, Gang; Chen, Zhuo; Ma, Yuan; Chen, Yue; Peng, Yihong; Zhao, Yi-Lei; Wang, Yanli; Zhang, Lihe; Yang, Zhenjun

    2015-11-28

    It has been demonstrated that passenger strand cleavage is important for the activation of RNA-induced silencing complex (RISC), which is a crucial step for siRNA-mediated gene silencing. Herein, we report that isonucleotide (isoNA) modification around the cleavage site of the passenger strand would affect the in vitro potency of modified siRNAs by altering the motion pattern of the Ago2-PAZ domain. According to western blotting, q-PCR and antiviral test results, we proved that D-isonucleotide (isoNA) modification at the position 8 of the passenger strand (siMek1-S08D), which is adjacent to the cleavage site, markedly improved the in vitro potency of the modified siRNA, whereas siRNAs with D-isoNA incorporation at position 9 (siMek1-S09D) or L-isoNA incorporation at positions 8 and 9 (siMek1-S08L, siMek1-S09L) displayed lower activity compared to native siRNA. Kinetics evaluation of passenger strand cleavage induced by T. thermophilus Ago (Tt-Ago) showed that D-isoNA modification at position 8 of the passenger strand had no significant influence on the cleavage rate, but L-isoNA modification at position 8 slowed the cleavage rate markedly. Moreover, the results of molecular dynamics simulations showed that D-isoNA modification at position 8 affected the open-close motion of the PAZ domain in the Ago/siRNA complex, which may promote the loading of RISC and release of a passenger strand cleavage product, and consequently accelerate the activation of RISC and enhance silencing activity. However, D-isoNA modification at position 9 or L-isoNA modification at position 8 or 9 exerted opposite influences on the motion of the Ago-PAZ domain.

  13. Beneficial effect of antibodies against β- secretase cleavage site of APP on Alzheimer's-like pathology in triple-transgenic mice.

    Directory of Open Access Journals (Sweden)

    Inna Rabinovich-Nikitin

    Full Text Available The toxicity of amyloid β and tau, the two hallmark proteins in Alzheimer's disease (AD, has been extensively studied individually. Recently new data suggest their possible interactions and synergistic effects in the disease. In this study, we investigate the ability of antibodies against the β secretase cleavage site on APP, named BBS1, to affect tau pathology, besides their well established effect on intracellular Aβ and amyloid load. For this purpose we treated the triple transgenic mice model of AD (3x Tg-AD with mAb BBS1 intracerebroventricularly, using mini osmotic pumps for one month. The experimental data demonstrated reduction in total and phosphorylated tau levels, explained by significant reduction in GSK3β which phosphorylates tau on sites recognized by antibodies against PHF1 and AT-8. The treatment increased the cognitive capabilities and reduced the brain inflammation levels which accompany AD pathology. The data showing that tau pathology was significantly reduced by BBS1 antibodies suggest a close interaction between tau and Aβ in the development of AD, and may serve as an efficient novel immunotherapy against both hallmarks of this disease.

  14. Swedish mutant APP-based BACE1 binding site peptide reduces APP β-cleavage and cerebral Aβ levels in Alzheimer's mice.

    Science.gov (United States)

    Li, Song; Hou, Huayan; Mori, Takashi; Sawmiller, Darrell; Smith, Adam; Tian, Jun; Wang, Yanjiang; Giunta, Brian; Sanberg, Paul R; Zhang, Sheqing; Tan, Jun

    2015-06-19

    BACE1 initiates amyloid-β (Aβ) generation and the resultant cerebral amyloidosis, as a characteristic of Alzheimer's disease (AD). Thus, inhibition of BACE1 has been the focus of a large body of research. The most recent clinical trials highlight the difficulty involved in this type of anti-AD therapy as evidenced by side effects likely due to the ubiquitous nature of BACE1, which cleaves multiple substrates. The human Swedish mutant form of amyloid protein precursor (APPswe) has been shown to possess a higher affinity for BACE1 compared to wild-type APP (APPwt). We pursued a new approach wherein harnessing this greater affinity to modulate BACE1 APP processing activity. We found that one peptide derived from APPswe, containing the β-cleavage site, strongly inhibits BACE1 activity and thereby reduces Aβ production. This peptide, termed APPswe BACE1 binding site peptide (APPsweBBP), was further conjugated to the fusion domain of the HIV-1 Tat protein (TAT) at the C-terminus to facilitate its biomembrane-penetrating activity. APPwt and APPswe over-expressing CHO cells treated with this TAT-conjugated peptide resulted in a marked reduction of Aβ and a significant increase of soluble APPα. Intraperitoneal administration of this peptide to 5XFAD mice markedly reduced β-amyloid deposits as well as improved hippocampal-dependent learning and memory.

  15. Ordered cleavage of myeloperoxidase ester bonds releases active site heme leading to inactivation of myeloperoxidase by benzoic acid hydrazide analogs.

    Science.gov (United States)

    Huang, Jiansheng; Smith, Forrest; Panizzi, Peter

    2014-04-15

    Myeloperoxidase (MPO) catalyzes the breakdown of hydrogen peroxide and the formation of the potent oxidant hypochlorous acid. We present the application of the fluorogenic peroxidase substrate 10-acetyl-3,7-dihydroxyphenoxazine (ADHP) in steady-state and transient kinetic studies of MPO function. Using initial kinetic parameters for the MPO system, we characterized under the same conditions a number of gold standards for MPO inhibition, namely 4-amino benzoic acid hydrazide (4-ABAH), isoniazid and NaN3 before expanding our focus to isomers of 4-ABAH and benzoic acid hydrazide analogs. We determined that in the presence of hydrogen peroxide that 4-ABAH and its isomer 2-ABAH are both slow-tight binding inhibitors of MPO requiring at least two steps, whereas NaN3 and isoniazid-based inhibition has a single observable step. We also determined that MPO inhibition by benzoic acid hydrazide and 4-(trifluoromethyl) benzoic acid hydrazide was due to hydrolysis of the ester bond between MPO heavy chain Glu 242 residue and the heme pyrrole A ring, freeing the light chain and heme b fragment from the larger remaining MPO heavy chain. This new mechanism would essentially indicate that the benzoic acid hydrazide analogs impart inhibition through initial ejection of the heme catalytic moiety without prior loss of the active site iron.

  16. Properties of a homogeneous C-lobe prepared by introduction of a TEV cleavage site between the lobes of human transferrin.

    Science.gov (United States)

    Steere, Ashley N; Roberts, Samantha E; Byrne, Shaina L; Dennis Chasteen, N; Bobst, Cedric E; Kaltashov, Igor A; Smith, Valerie C; MacGillivray, Ross T A; Mason, Anne B

    2010-07-01

    Essential to iron transport and delivery, human serum transferrin (hTF) is a bilobal glycoprotein capable of reversibly binding one ferric ion in each lobe (the N- and C-lobes). A complete description of iron release from hTF, as well as insight into the physiological significance of the bilobal structure, demands characterization of the isolated lobes. Although production of large amounts of isolated N-lobe and full-length hTF has been well documented, attempts to produce the C-lobe (by recombinant and/or proteolytic approaches) have met with more limited success. Our new strategy involves replacing the hepta-peptide, PEAPTDE (comprising the bridge between the lobes) with the sequence ENLYFQ/G in a His-tagged non-glycosylated monoferric hTF construct, designated Fe(C)hTF. The new bridge sequence of this construct, designated Fe(C)TEV hTF, is readily cleaved by the tobacco etch virus (TEV) protease yielding non-glycosylated C-lobe. Following nickel column chromatography (to remove the N-lobe and the TEV protease which are both His tagged), the homogeneity of the C-lobe has been confirmed by mass spectroscopy. Differing reactivity with a monoclonal antibody specific to the C-lobe indicates that introduction of the TEV cleavage site into the bridge alters its conformation. The spectral and kinetic properties of the isolated C-lobe differ significantly from those of the isolated N-lobe. Copyright 2010 Elsevier Inc. All rights reserved.

  17. Snake venom metalloproteinases.

    Science.gov (United States)

    Markland, Francis S; Swenson, Stephen

    2013-02-01

    Recent proteomic analyses of snake venoms show that metalloproteinases represent major components in most of the Crotalid and Viperid venoms. In this chapter we discuss the multiple activities of the SVMPs. In addition to hemorrhagic activity, members of the SVMP family also have fibrin(ogen)olytic activity, act as prothrombin activators, activate blood coagulation factor X, possess apoptotic activity, inhibit platelet aggregation, are pro-inflammatory and inactivate blood serine proteinase inhibitors. Clearly the SVMPs have multiple functions in addition to their well-known hemorrhagic activity. The realization that there are structural variations in the SVMPs and the early studies that led to their classification represents an important event in our understanding of the structural forms of the SVMPs. The SVMPs were subdivided into the P-I, P-II and P-III protein classes. The noticeable characteristic that distinguished the different classes was their size (molecular weight) differences and domain structure: Class I (P-I), the small SVMPs, have molecular masses of 20-30 kDa, contain only a pro domain and the proteinase domain; Class II (P-II), the medium size SVMPs, molecular masses of 30-60 kDa, contain the pro domain, proteinase domain and disintegrin domain; Class III (P-III), the large SVMPs, have molecular masses of 60-100 kDa, contain pro, proteinase, disintegrin-like and cysteine-rich domain structure. Another significant advance in the SVMP field was the characterization of the crystal structure of the first P-I class SVMP. The structures of other P-I SVMPs soon followed and the structures of P-III SVMPs have also been determined. The active site of the metalloproteinase domain has a consensus HEXXHXXGXXHD sequence and a Met-turn. The "Met-turn" structure contains a conserved Met residue that forms a hydrophobic basement for the three zinc-binding histidines in the consensus sequence. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Snake Venom Metalloproteinases

    Directory of Open Access Journals (Sweden)

    Gâz Florea Şerban Andrei

    2016-03-01

    Full Text Available As more data are generated from proteome and transcriptome analysis revealing that metalloproteinases represent most of the Viperid and Colubrid venom components authors decided to describe in a short review a classification and some of the multiple activities of snake venom metalloproteinases. SVMPs are classified in three major classes (P-I, P-II and P-III classes based on the presence of various domain structures and according to their domain organization. Furthermore, P-II and P-III classes were separated in subclasses based on distinctive post-translational modifications. SVMPs are synthesized in a latent form, being activated through a Cys-switch mechanism similar to matrix metalloproteinases. Most of the metalloproteinases of the snake venom are responsible for the hemorrhagic events but also have fibrinogenolytic activity, poses apoptotic activity, activate blood coagulation factor II and X, inhibit platelet aggregation, demonstrating that SVMPs have multiple functions in addition to well-known hemorrhagic function.

  19. Matrix metalloproteinase-2 (MMP-2) generates soluble HLA-G1 by cell surface proteolytic shedding.

    Science.gov (United States)

    Rizzo, Roberta; Trentini, Alessandro; Bortolotti, Daria; Manfrinato, Maria C; Rotola, Antonella; Castellazzi, Massimiliano; Melchiorri, Loredana; Di Luca, Dario; Dallocchio, Franco; Fainardi, Enrico; Bellini, Tiziana

    2013-09-01

    Human leukocyte antigen-G (HLA-G) molecules are non-classical HLA class I antigens with an important role in pregnancy immune regulation and inflammation control. Soluble HLA-G proteins can be generated through two mechanisms: alternative splicing and proteolytic release, which is known to be metalloprotease mediated. Among this class of enzymes, matrix metalloproteinases (MMPs) might be involved in the HLA-G1 membrane cleavage. Of particular interest are MMP-2 and MMP-9, which regulate the inflammatory process by cytokine and chemokine modulation. We evaluated the effect of MMP-9 and MMP-2 on HLA-G1 membrane shedding. In particular, we analyzed the in vitro effect of these two gelatinases on the secretion of HLA-G1 via proteolytic cleavage in 221-G1-transfected cell line, in JEG3 cell line, and in peripheral blood mononuclear cells. The results obtained by both cell lines showed the role of MMP-2 in HLA-G1 shedding. On the contrary, MMP-9 was not involved in this process. In addition, we identified three possible highly specific cleavage sites for MMP-2, whereas none were detected for MMP-9. This study suggests an effective link between MMP-2 and HLA-G1 shedding, increasing our knowledge on the regulatory machinery beyond HLA-G regulation in physiological and pathological conditions.

  20. Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage

    DEFF Research Database (Denmark)

    Goth, Christoffer K; Tuhkanen, Hanna E; Khan, Hamayun

    2017-01-01

    The β1-adrenergic receptor (β1AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for β-adrenergic antagonists, such as β-blockers, r......The β1-adrenergic receptor (β1AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for β-adrenergic antagonists, such as β......NAc-transferase 2 (GalNAc-T2) specifically O-glycosylates β1AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O-glycosylation and proteolytic cleavage assays, a cell line deficient in O......-glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of β1AR. Furthermore, we demonstrate that impaired O-glycosylation and enhanced proteolysis lead to attenuated receptor signaling...

  1. Shutoff of RNA polymerase II transcription by poliovirus involves 3C protease-mediated cleavage of the TATA-binding protein at an alternative site: incomplete shutoff of transcription interferes with efficient viral replication.

    Science.gov (United States)

    Kundu, Pallob; Raychaudhuri, Santanu; Tsai, Weimin; Dasgupta, Asim

    2005-08-01

    The TATA-binding protein (TBP) plays a crucial role in cellular transcription catalyzed by all three DNA-dependent RNA polymerases. Previous studies have shown that TBP is targeted by the poliovirus (PV)-encoded protease 3C(pro) to bring about shutoff of cellular RNA polymerase II-mediated transcription in PV-infected cells. The processing of the majority of viral precursor proteins by 3C(pro) involves cleavages at glutamine-glycine (Q-G) sites. We present evidence that suggests that the transcriptional inactivation of TBP by 3C(pro) involves cleavage at the glutamine 104-serine 105 (Q104-S105) site of TBP and not at the Q18-G19 site as previously thought. The TBP Q104-S105 cleavage by 3C(pro) is greatly influenced by the presence of an aliphatic amino acid at the P4 position, a hallmark of 3C(pro)-mediated proteolysis. To examine the importance of host cell transcription shutoff in the PV life cycle, stable HeLa cell lines were created that express recombinant TBP resistant to cleavage by the viral proteases, called GG rTBP. Transcription shutoff was significantly impaired and delayed in GG rTBP cells upon infection with poliovirus compared with the cells that express wild-type recombinant TBP (wt rTBP). Infection of GG rTBP cells with poliovirus resulted in small plaques, significantly reduced viral RNA synthesis, and lower viral yields compared to the wt rTBP cell line. These results suggest that a defect in transcription shutoff can lead to inefficient replication of poliovirus in cultured cells.

  2. Lectican proteoglycans, their cleaving metalloproteinases, and plasticity in the central nervous system extracellular microenvironment.

    Science.gov (United States)

    Howell, M D; Gottschall, P E

    2012-08-16

    The extracellular matrix (ECM) in the central nervous system actively orchestrates and modulates changes in neural structure and function in response to experience, after injury, during disease, and with changes in neuronal activity. A component of the multi-protein, ECM aggregate in brain, the chondroitin sulfate (CS)-bearing proteoglycans (PGs) known as lecticans, inhibit neurite outgrowth, alter dendritic spine shape, elicit closure of critical period plasticity, and block target reinnervation and functional recovery after injury as the major component of a glial scar. While removal of the CS chains from lecticans with chondroitinase ABC improves plasticity, proteolytic cleavage of the lectican core protein may change the conformation of the matrix aggregate and also modulate neural plasticity. This review centers on the roles of the lecticans and the endogenous metalloproteinase families that proteolytically cleave lectican core proteins, the matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs), in neural plasticity. These extracellular metalloproteinases modulate structural neural plasticity-including changes in neurite outgrowth and dendritic spine remodeling-and synaptic plasticity. Some of these actions have been demonstrated to occur via cleavage of the PG core protein. Other actions of the proteases include cleavage of non-matrix substrate proteins, whereas still other actions may occur directly at the cell surface without proteolytic cleavage. The data convincingly demonstrate that metalloproteinases modulate physiological and pathophysiological neural plasticity. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  3. Soluble Axl is generated by ADAM10-dependent cleavage and associates with Gas6 in mouse serum.

    Science.gov (United States)

    Budagian, Vadim; Bulanova, Elena; Orinska, Zane; Duitman, Erwin; Brandt, Katja; Ludwig, Andreas; Hartmann, Dieter; Lemke, Greg; Saftig, Paul; Bulfone-Paus, Silvia

    2005-11-01

    Axl receptor tyrosine kinase exists as a transmembrane protein and as a soluble molecule. We show that constitutive and phorbol 12-myristate 13-acetate-induced generation of soluble Axl (sAxl) involves the activity of disintegrin-like metalloproteinase 10 (ADAM10). Spontaneous and inducible Axl cleavage was inhibited by the broad-spectrum metalloproteinase inhibitor GM6001 and by hydroxamate GW280264X, which is capable of blocking ADAM10 and ADAM17. Furthermore, murine fibroblasts deficient in ADAM10 expression exhibited a significant reduction in constitutive and inducible Axl shedding, whereas reconstitution of ADAM10 restored sAxl production, suggesting that ADAM10-mediated proteolysis constitutes a major mechanism for sAxl generation in mice. Partially overlapping 14-amino-acid stretch deletions in the membrane-proximal region of Axl dramatically affected sAxl generation, indicating that these regions are involved in regulating the access of the protease to the cleavage site. Importantly, relatively high circulating levels of sAxl are present in mouse sera in a heterocomplex with Axl ligand Gas6. Conversely, two other family members, Tyro3 and Mer, were not detected in mouse sera and conditioned medium. sAxl is constitutively released by murine primary cells such as dendritic and transformed cell lines. Upon immobilization, sAxl promoted cell migration and induced the phosphorylation of Axl and phosphatidylinositol 3-kinase. Thus, ADAM10-mediated generation of sAxl might play an important role in diverse biological processes.

  4. Soluble Axl Is Generated by ADAM10-Dependent Cleavage and Associates with Gas6 in Mouse Serum†

    Science.gov (United States)

    Budagian, Vadim; Bulanova, Elena; Orinska, Zane; Duitman, Erwin; Brandt, Katja; Ludwig, Andreas; Hartmann, Dieter; Lemke, Greg; Saftig, Paul; Bulfone-Paus, Silvia

    2005-01-01

    Axl receptor tyrosine kinase exists as a transmembrane protein and as a soluble molecule. We show that constitutive and phorbol 12-myristate 13-acetate-induced generation of soluble Axl (sAxl) involves the activity of disintegrin-like metalloproteinase 10 (ADAM10). Spontaneous and inducible Axl cleavage was inhibited by the broad-spectrum metalloproteinase inhibitor GM6001 and by hydroxamate GW280264X, which is capable of blocking ADAM10 and ADAM17. Furthermore, murine fibroblasts deficient in ADAM10 expression exhibited a significant reduction in constitutive and inducible Axl shedding, whereas reconstitution of ADAM10 restored sAxl production, suggesting that ADAM10-mediated proteolysis constitutes a major mechanism for sAxl generation in mice. Partially overlapping 14-amino-acid stretch deletions in the membrane-proximal region of Axl dramatically affected sAxl generation, indicating that these regions are involved in regulating the access of the protease to the cleavage site. Importantly, relatively high circulating levels of sAxl are present in mouse sera in a heterocomplex with Axl ligand Gas6. Conversely, two other family members, Tyro3 and Mer, were not detected in mouse sera and conditioned medium. sAxl is constitutively released by murine primary cells such as dendritic and transformed cell lines. Upon immobilization, sAxl promoted cell migration and induced the phosphorylation of Axl and phosphatidylinositol 3-kinase. Thus, ADAM10-mediated generation of sAxl might play an important role in diverse biological processes. PMID:16227584

  5. Anacardic acid inhibits the catalytic activity of matrix metalloproteinase-2 and matrix metalloproteinase-9.

    Science.gov (United States)

    Omanakuttan, Athira; Nambiar, Jyotsna; Harris, Rodney M; Bose, Chinchu; Pandurangan, Nanjan; Varghese, Rebu K; Kumar, Geetha B; Tainer, John A; Banerji, Asoke; Perry, J Jefferson P; Nair, Bipin G

    2012-10-01

    Cashew nut shell liquid (CNSL) has been used in traditional medicine for the treatment of a wide variety of pathophysiological conditions. To further define the mechanism of CNSL action, we investigated the effect of cashew nut shell extract (CNSE) on two matrix metalloproteinases, MMP-2/gelatinase A and MMP-9/gelatinase B, which are known to have critical roles in several disease states. We observed that the major constituent of CNSE, anacardic acid, markedly inhibited the gelatinase activity of 3T3-L1 cells. Our gelatin zymography studies on these two secreted gelatinases, present in the conditioned media from 3T3-L1 cells, established that anacardic acid directly inhibited the catalytic activities of both MMP-2 and MMP-9. Our docking studies suggested that anacardic acid binds into the MMP-2/9 active site, with the carboxylate group of anacardic acid chelating the catalytic zinc ion and forming a hydrogen bond to a key catalytic glutamate side chain and the C15 aliphatic group being accommodated within the relatively large S1' pocket of these gelatinases. In agreement with the docking results, our fluorescence-based studies on the recombinant MMP-2 catalytic core domain demonstrated that anacardic acid directly inhibits substrate peptide cleavage in a dose-dependent manner, with an IC₅₀ of 11.11 μM. In addition, our gelatinase zymography and fluorescence data confirmed that the cardol-cardanol mixture, salicylic acid, and aspirin, all of which lack key functional groups present in anacardic acid, are much weaker MMP-2/MMP-9 inhibitors. Our results provide the first evidence for inhibition of gelatinase catalytic activity by anacardic acid, providing a novel template for drug discovery and a molecular mechanism potentially involved in CNSL therapeutic action.

  6. Functional study of elafin cleaved by Pseudomonas aeruginosa metalloproteinases.

    LENUS (Irish Health Repository)

    Guyot, Nicolas

    2010-06-01

    Elafin is a 6-kDa innate immune protein present at several epithelial surfaces including the pulmonary epithelium. It is a canonical protease inhibitor of two neutrophil serine proteases [neutrophil elastase (NE) and proteinase 3] with the capacity to covalently bind extracellular matrix proteins by transglutamination. In addition to these properties, elafin also possesses antimicrobial and immunomodulatory activities. The aim of the present study was to investigate the effect of Pseudomonas aeruginosa proteases on elafin function. We found that P. aeruginosa PAO1-conditioned medium and two purified Pseudomonas metalloproteases, pseudolysin (elastase) and aeruginolysin (alkaline protease), are able to cleave recombinant elafin. Pseudolysin was shown to inactivate the anti-NE activity of elafin by cleaving its protease-binding loop. Interestingly, antibacterial properties of elafin against PAO1 were found to be unaffected after pseudolysin treatment. In contrast to pseudolysin, aeruginolysin failed to inactivate the inhibitory properties of elafin against NE. Aeruginolysin cleaves elafin at the amino-terminal Lys6-Gly7 peptide bond, resulting in a decreased ability to covalently bind purified fibronectin following transglutaminase activity. In conclusion, this study provides evidence that elafin is susceptible to proteolytic cleavage at alternative sites by P. aeruginosa metalloproteinases, which can affect different biological functions of elafin.

  7. Functional study of elafin cleaved by Pseudomonas aeruginosa metalloproteinases

    Science.gov (United States)

    Guyot, Nicolas; Bergsson, Gudmundur; Butler, Marcus W.; Greene, Catherine M.; Weldon, Sinead; Kessler, Efrat; Levine, Rodney L.; O’Neill, Shane J.; Taggart, Clifford C.; McElvaney, Noel G.

    2012-01-01

    Elafin is a 6 kDa innate immune protein present at several epithelial surfaces including the pulmonary epithelium. It is a canonical protease inhibitor of two neutrophil serine proteases (neutrophil elastase (NE) and proteinase 3) with the capacity to covalently bind extracellular matrix proteins by transglutamination. In addition to these properties, elafin also possesses antimicrobial and immunomodulatory activities. The aim of the present study was to investigate the effect of Pseudomonas aeruginosa proteases on elafin function. We found that P. aeruginosa PAO1-conditioned medium and two purified Pseudomonas metalloproteases, pseudolysin (elastase) and aeruginolysin (alkaline protease), were able to cleave recombinant elafin. Pseudolysin was shown to inactivate the anti-NE activity of elafin by cleaving its protease-binding loop. Interestingly, antibacterial properties of elafin against PAO1 were found to be unaffected after pseudolysin treatment. In contrast to pseudolysin, aeruginolysin failed to inactivate the inhibitory properties of elafin against NE. Aeruginolysin cleaved elafin at the amino-terminal Lys6-Gly7 peptide bond resulting in a decreased ability to covalently bind purified fibronectin following transglutaminase activity. In conclusion, this study provides evidences that elafin is susceptible to proteolytic cleavage at alternative sites by P. aeruginosa metalloproteinases, which can affect different biological functions of elafin. PMID:20370321

  8. Monocyte ADAM17 promotes diapedesis during transendothelial migration: identification of steps and substrates targeted by metalloproteinases.

    Science.gov (United States)

    Tsubota, Yoshiaki; Frey, Jeremy M; Tai, Phillip W L; Welikson, Robert E; Raines, Elaine W

    2013-04-15

    Despite expanded definition of the leukocyte adhesion cascade and mechanisms underlying individual steps, very little is known about regulatory mechanisms controlling sequential shifts between steps. We tested the hypothesis that metalloproteinases provide a mechanism to rapidly transition monocytes between different steps. Our study identifies diapedesis as a step targeted by metalloproteinase activity. Time-lapse video microscopy shows that the presence of a metalloproteinase inhibitor results in a doubling of the time required for human monocytes to complete diapedesis on unactivated or inflamed human endothelium, under both static and physiological-flow conditions. Thus, diapedesis is promoted by metalloproteinase activity. In contrast, neither adhesion of monocytes nor their locomotion over the endothelium is altered by metalloproteinase inhibition. We further demonstrate that metalloproteinase inhibition significantly elevates monocyte cell surface levels of integrins CD11b/CD18 (Mac-1), specifically during transendothelial migration. Interestingly, such alterations are not detected for other endothelial- and monocyte-adhesion molecules that are presumed metalloproteinase substrates. Two major transmembrane metalloproteinases, a disintegrin and metalloproteinase (ADAM)17 and ADAM10, are identified as enzymes that control constitutive cleavage of Mac-1. We further establish that knockdown of monocyte ADAM17, but not endothelial ADAM10 or ADAM17 or monocyte ADAM10, reproduces the diapedesis delay observed with metalloproteinase inhibition. Therefore, we conclude that monocyte ADAM17 facilitates the completion of transendothelial migration by accelerating the rate of diapedesis. We propose that the progression of diapedesis may be regulated by spatial and temporal cleavage of Mac-1, which is triggered upon interaction with endothelium.

  9. The Multibasic Cleavage Site of the Hemagglutinin of Highly Pathogenic A/Vietnam/1203/2004 (H5N1) Avian Influenza Virus Acts as a Virulence Factor in a Host-Specific Manner in Mammals

    OpenAIRE

    2012-01-01

    Highly pathogenic avian influenza (HPAI) viruses of the H5 and H7 subtypes typically possess multiple basic amino acids around the cleavage site (MBS) of their hemagglutinin (HA) protein, a recognized virulence motif in poultry. To determine the importance of the H5 HA MBS as a virulence factor in mammals, recombinant wild-type HPAI A/Vietnam/1203/2004 (H5N1) viruses that possessed (H5N1) or lacked (ΔH5N1) the H5 HA MBS were generated and evaluated for their virulence in BALB/c mice, ferrets,...

  10. Centralspindlin in Rappaport's cleavage signaling.

    Science.gov (United States)

    Mishima, Masanori

    2016-05-01

    Cleavage furrow in animal cell cytokinesis is formed by cortical constriction driven by contraction of an actomyosin network activated by Rho GTPase. Although the role of the mitotic apparatus in furrow induction has been well established, there remain discussions about the detailed molecular mechanisms of the cleavage signaling. While experiments in large echinoderm embryos highlighted the role of astral microtubules, data in smaller cells indicate the role of central spindle. Centralspindlin is a constitutive heterotetramer of MKLP1 kinesin and the non-motor CYK4 subunit and plays crucial roles in formation of the central spindle and recruitment of the downstream cytokinesis factors including ECT2, the major activator of Rho during cytokinesis, to the site of division. Recent reports have revealed a role of this centralspindlin-ECT2 pathway in furrow induction both by the central spindle and by the astral microtubules. Here, a unified view of the stimulation of cortical contractility by this pathway is discussed. Cytokinesis, the division of the whole cytoplasm, is an essential process for cell proliferation and embryonic development. In animal cells, cytokinesis is executed using a contractile network of actin filaments driven by a myosin-II motor that constricts the cell cortex (cleavage furrow ingression) into a narrow channel between the two daughter cells, which is resolved by scission (abscission) [1-3]. The anaphase-specific organization of the mitotic apparatus (MA, spindle with chromosomes plus asters) positions the cleavage furrow and plays a major role in spatial coupling between mitosis and cytokinesis [4-6]. The nucleus and chromosomes are dispensable for furrow specification [7-10], although they contribute to persistent furrowing and robust completion in some cell types [11,12]. Likewise, centrosomes are not essential for cytokinesis, but they contribute to the general fidelity of cell division [10,13-15]. Here, classical models of cleavage furrow

  11. In vivo analysis of the Notch receptor S1 cleavage.

    Directory of Open Access Journals (Sweden)

    Robert J Lake

    Full Text Available A ligand-independent cleavage (S1 in the extracellular domain of the mammalian Notch receptor results in what is considered to be the canonical heterodimeric form of Notch on the cell surface. The in vivo consequences and significance of this cleavage on Drosophila Notch signaling remain unclear and contradictory. We determined the cleavage site in Drosophila and examined its in vivo function by a transgenic analysis of receptors that cannot be cleaved. Our results demonstrate a correlation between loss of cleavage and loss of in vivo function of the Notch receptor, supporting the notion that S1 cleavage is an in vivo mechanism of Notch signal control.

  12. Ectodomain cleavage of the EGF ligands HB-EGF, neuregulin1-beta, and TGF-alpha is specifically triggered by different stimuli and involves different PKC isoenzymes.

    Science.gov (United States)

    Herrlich, Andreas; Klinman, Eva; Fu, Jonathan; Sadegh, Cameron; Lodish, Harvey

    2008-12-01

    Metalloproteinase cleavage of transmembrane proteins (ectodomain cleavage), including the epidermal growth factor (EGF) ligands heparin-binding EGF-like growth factor (HB-EGF), neuregulin (NRG), and transforming growth factor-alpha (TGF-alpha), is important in many cellular signaling pathways and is disregulated in many diseases. It is largely unknown how physiological stimuli of ectodomain cleavage--hypertonic stress, phorbol ester, or activation of G-protein-coupled receptors [e.g., by lysophosphatidic acid (LPA)]--are molecularly connected to metalloproteinase activation. To study this question, we developed a fluorescence-activated cell sorting (FACS)- based assay that measures cleavage of EGF ligands in single living cells. EGF ligands expressed in mouse lung epithelial cells are differentially and specifically cleaved depending on the stimulus. Inhibition of protein kinase C (PKC) isoenzymes or metalloproteinase inhibition by batimastat (BB94) showed that different regulatory signals are used by different stimuli and EGF substrates, suggesting differential effects that act on the substrate, the metalloproteinase, or both. For example, hypertonic stress led to strong cleavage of HB-EGF and NRG but only moderate cleavage of TGF-alpha. HB-EGF, NRG, and TGF-alpha cleavage was not dependent on PKC, and only HB-EGF and NRG cleavage were inhibited by BB94. In contrast, phorbol 12-myristate-13-acetate (TPA) -induced cleavage of HB-EGF, NRG, and TGF-alpha was dependent on PKC and sensitive to BB94 inhibition. LPA led to significant cleavage of only NRG and TGF-alpha and was inhibited by BB94; only LPA-induced NRG cleavage required PKC. Surprisingly, specific inhibition of atypical PKCs zeta and iota [not activated by diacylglycerol (DAG) and calcium] significantly enhanced TPA-induced NRG cleavage. Employed in a high-throughput cloning strategy, our cleavage assay should allow the identification of candidate proteins involved in signal transduction of different

  13. Mutagenesis of the catalytic and cleavage site residues of the hypovirus papain-like proteases p29 and p48 reveals alternative processing and contributions to optimal viral RNA accumulation.

    Science.gov (United States)

    Jensen, Kenneth S; Nuss, Donald L

    2014-10-01

    The positive-stranded RNA genome of the prototypic virulence-attenuating hypovirus CHV-1/EP713 contains two open reading frames (ORF), each encoding an autocatalytic papain-like leader protease. Protease p29, derived from the N-terminal portion of ORF A, functions as a suppressor of RNA silencing, while protease p48, derived from the N-terminal portion of ORF B, is required for viral RNA replication. The catalytic and cleavage site residues required for autoproteolytic processing have been functionally mapped in vitro for both proteases but not confirmed in the infected fungal host. We report here the mutagenesis of the CHV-1/EP713 infectious cDNA clone to define the requirements for p29 and p48 cleavage and the role of autoproteolysis in the context of hypovirus replication. Mutation of the catalytic cysteine and histidine residues for either p29 or p48 was tolerated but reduced viral RNA accumulation to ca. 20 to 50% of the wild-type level. Mutation of the p29 catalytic residues caused an accumulation of unprocessed ORF A product p69. Surprisingly, the release of p48 from the ORF B-encoded polyprotein was not prevented by mutation of the p48 catalytic and cleavage site residues and was independent of p29. The results show that, while dispensable for hypovirus replication, the autocatalytic processing of the leader proteases p29 and p48 contributes to optimal virus RNA accumulation. The role of the predicted catalytic residues in autoproteolytic processing of p29 was confirmed in the infected host, while p48 was found to also undergo alternative processing independent of the encoded papain-like protease activities. Importance: Hypoviruses are positive-strand RNA mycoviruses that attenuate virulence of their pathogenic fungal hosts. The prototypic hypovirus CHV-1/EP713, which infects the chestnut bight fungus Cryphonetria parasitica, encodes two papain-like autocatalytic leader proteases, p29 and p48, that also have important functions in suppressing the RNA

  14. Resonance Raman spectroscopy reveals pH-dependent active site structural changes of lactoperoxidase compound 0 and its ferryl heme O-O bond cleavage products.

    Science.gov (United States)

    Mak, Piotr J; Thammawichai, Warut; Wiedenhoeft, Dennis; Kincaid, James R

    2015-01-14

    The first step in the enzymatic cycle of mammalian peroxidases, including lactoperoxidase (LPO), is binding of hydrogen peroxide to the ferric resting state to form a ferric-hydroperoxo intermediate designated as Compound 0, the residual proton temporarily associating with the distal pocket His109 residue. Upon delivery of this "stored" proton to the hydroperoxo fragment, it rapidly undergoes O-O bond cleavage, thereby thwarting efforts to trap it using rapid mixing methods. Fortunately, as shown herein, both the peroxo and the hydroperoxo (Compound 0) forms of LPO can be trapped by cryoradiolysis, with acquisition of their resonance Raman (rR) spectra now permitting structural characterization of their key Fe-O-O fragments. Studies were conducted under both acidic and alkaline conditions, revealing pH-dependent differences in relative populations of these intermediates. Furthermore, upon annealing, the low pH samples convert to two forms of a ferryl heme O-O bond-cleavage product, whose ν(Fe═O) frequencies reflect substantially different Fe═O bond strengths. In the process of conducting these studies, rR structural characterization of the dioxygen adduct of LPO, commonly called Compound III, has also been completed, demonstrating a substantial difference in the strengths of the Fe-O linkage of the Fe-O-O fragment under acidic and alkaline conditions, an effect most reasonably attributed to a corresponding weakening of the trans-axial histidyl imidazole linkage at lower pH. Collectively, these new results provide important insight into the impact of pH on the disposition of the key Fe-O-O and Fe═O fragments of intermediates that arise in the enzymatic cycles of LPO, other mammalian peroxidases, and related proteins.

  15. A caged substrate peptide for matrix metalloproteinases.

    Science.gov (United States)

    Decaneto, Elena; Abbruzzetti, Stefania; Heise, Inge; Lubitz, Wolfgang; Viappiani, Cristiano; Knipp, Markus

    2015-02-01

    Based on the widely applied fluorogenic peptide FS-6 (Mca-Lys-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2; Mca = methoxycoumarin-4-acetyl; Dpa = N-3-(2,4-dinitrophenyl)l-α,β-diaminopropionyl) a caged substrate peptide Ac-Lys-Pro-Leu-Gly-Lys*-Lys-Ala-Arg-NH2 (*, position of the cage group) for matrix metalloproteinases was synthesized and characterized. The synthesis implies the modification of a carbamidated lysine side-chain amine with a photocleavable 2-nitrobenzyl group. Mass spectrometry upon UV irradiation demonstrated the complete photolytic cleavage of the protecting group. Time-resolved laser-flash photolysis at 355 nm in combination with transient absorption spectroscopy determined the biphasic decomposition with τa = 171 ± 3 ms (79%) and τb = 2.9 ± 0.2 ms (21%) at pH 6.0 of the photo induced release of the 2-nitrobenzyl group. The recombinantly expressed catalytic domain of human membrane type I matrix metalloproteinase (MT1-MMP or MMP-14) was used to determine the hydrolysis efficiency of the caged peptide before and after photolysis. It turned out that the cage group sufficiently shields the peptide from peptidase activity, which can be thus controlled by UV light.

  16. Fabrication of peptide stabilized fluorescent gold nanocluster/graphene oxide nanocomplex and its application in turn-on detection of metalloproteinase-9.

    Science.gov (United States)

    Nguyen, Phuong-Diem; Cong, Vu Thanh; Baek, Changyoon; Min, Junhong

    2017-03-15

    This study introduces the double-ligands stabilizing gold nanoclusters and the fabrication of gold nanocluster/graphene nanocomplex as a "turn-on" fluorescent probe for the detection of cancer-related enzyme matrix metalloproteinase-9. A facile, one-step approach was developed for the synthesis of fluorescent gold nanoclusters using peptides and mercaptoundecanoic acid as co-templating ligands. The peptide was designed to possess a metalloproteinase-9 cleavage site and to act not only as a stabilizer but also as a targeting ligand for the enzyme detection. The prepared gold nanoclusters show an intense red fluorescence with a broad adsorption spectrum. In the presence of the enzyme, due to the excellent quenching properties and the negligible background of graphene oxide, the developed peptide-gold nanocluster/graphene nanocomplex yielded an intense "turn-on" fluorescent response, which strongly correlated with the enzyme concentration. The limit of detection of the nanocomplex was 0.15nM. The sensor was successfully applied for "turn-on" detection of metalloproteinase-9 secreted from human breast adenocarcinoma MCF-7 cells with high sensitivity, selectivity, significant improvement in terms of detection time and simplicity. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. H9N2 influenza virus acquires intravenous pathogenicity on the introduction of a pair of di-basic amino acid residues at the cleavage site of the hemagglutinin and consecutive passages in chickens

    Directory of Open Access Journals (Sweden)

    Sakoda Yoshihiro

    2011-02-01

    Full Text Available Abstract Background Outbreaks of avian influenza (AI caused by infection with low pathogenic H9N2 viruses have occurred in poultry, resulting in serious economic losses in Asia and the Middle East. It has been difficult to eradicate the H9N2 virus because of its low pathogenicity, frequently causing in apparent infection. It is important for the control of AI to assess whether the H9N2 virus acquires pathogenicity as H5 and H7 viruses. In the present study, we investigated whether a non-pathogenic H9N2 virus, A/chicken/Yokohama/aq-55/2001 (Y55 (H9N2, acquires pathogenicity in chickens when a pair of di-basic amino acid residues is introduced at the cleavage site of its HA molecule. Results rgY55sub (H9N2, which had four basic amino acid residues at the HA cleavage site, replicated in MDCK cells in the absence of trypsin after six consecutive passages in the air sacs of chicks, and acquired intravenous pathogenicity to chicken after four additional passages. More than 75% of chickens inoculated intravenously with the passaged virus, rgY55sub-P10 (H9N2, died, indicating that it is pathogenic comparable to that of highly pathogenic avian influenza viruses (HPAIVs defined by World Organization for Animal Health (OIE. The chickens inoculated with the virus via the intranasal route, however, survived without showing any clinical signs. On the other hand, an avirulent H5N1 strain, A/duck/Hokkaido/Vac-1/2004 (Vac1 (H5N1, acquired intranasal pathogenicity after a pair of di-basic amino acid residues was introduced into the cleavage site of the HA, followed by two passages by air sac inoculation in chicks. Conclusion The present results demonstrate that an H9N2 virus has the potential to acquire intravenous pathogenicity in chickens although the morbidity via the nasal route of infection is lower than that of H5N1 HPAIV.

  18. Deformability in the cleavage site of primary microRNA is not sensed by the double-stranded RNA binding domains in the microprocessor component DGCR8.

    Science.gov (United States)

    Quarles, Kaycee A; Chadalavada, Durga; Showalter, Scott A

    2015-06-01

    The prevalence of double-stranded RNA (dsRNA) in eukaryotic cells has only recently been appreciated. Of interest here, RNA silencing begins with dsRNA substrates that are bound by the dsRNA-binding domains (dsRBDs) of their processing proteins. Specifically, processing of microRNA (miRNA) in the nucleus minimally requires the enzyme Drosha and its dsRBD-containing cofactor protein, DGCR8. The smallest recombinant construct of DGCR8 that is sufficient for in vitro dsRNA binding, referred to as DGCR8-Core, consists of its two dsRBDs and a C-terminal tail. As dsRBDs rarely recognize the nucleotide sequence of dsRNA, it is reasonable to hypothesize that DGCR8 function is dependent on the recognition of specific structural features in the miRNA precursor. Previously, we demonstrated that noncanonical structural elements that promote RNA flexibility within the stem of miRNA precursors are necessary for efficient in vitro cleavage by reconstituted Microprocessor complexes. Here, we combine gel shift assays with in vitro processing assays to demonstrate that neither the N-terminal dsRBD of DGCR8 in isolation nor the DGCR8-Core construct is sensitive to the presence of noncanonical structural elements within the stem of miRNA precursors, or to single-stranded segments flanking the stem. Extending DGCR8-Core to include an N-terminal heme-binding region does not change our conclusions. Thus, our data suggest that although the DGCR8-Core region is necessary for dsRNA binding and recruitment to the Microprocessor, it is not sufficient to establish the previously observed connection between RNA flexibility and processing efficiency.

  19. Limited caspase cleavage of human BAP31.

    Science.gov (United States)

    Määttä, J; Hallikas, O; Welti, S; Hildén, P; Schröder, J; Kuismanen, E

    2000-11-10

    Human BAP31 was cleaved at both of its two identical caspase cleavage sites in two previously reported models of apoptosis. We show here that only the most carboxy-terminal site is cleaved during apoptosis induced in HeLa cells by tunicamycin, tumor necrosis factor and cycloheximide, or staurosporine. Similar results were obtained in HL-60 cells using Fas/APO-1 antibodies, or cycloheximide. This limited cleavage, which is inhibited by several caspase inhibitors, removes eight amino acids from human BAP31 including the KKXX coat protein I binding motif. Ectopic expression of the resulting cleavage product induces redistribution of mannosidase II from the Golgi and prevents endoplasmic reticulum to Golgi transport of virus glycoproteins.

  20. Demasking kinetics of peptide bond cleavage for whey protein isolate hydrolysed by Bacillus licheniformis protease

    NARCIS (Netherlands)

    Vorob'ev, Mikhail M.; Butré, Claire I.; Sforza, Stefano; Wierenga, Peter A.; Gruppen, Harry

    2017-01-01

    During enzymatic hydrolysis of proteins, some cleavage sites will be accessible in the intact protein. Others may only become accessible after demasking of the cleavage site, i.e. after cleavage of other bonds in the protein. Studies on demasking so far have used data of the release of few

  1. Matrix Metalloproteinases & Implication in Periodontitis- A Short Review

    Directory of Open Access Journals (Sweden)

    Hitesh Desarda

    2013-01-01

    Full Text Available Matrix metalloproteinases (MMPs are a group of enzymes which are responsible for the degradation of extracellular matrix during normal tissue turnover and also during inflammatory processes. The expression and activity of MMPs in adult tissues is normally quite low, but increases significantly in various pathological conditions that may lead into unwanted tissue destruction,such as inflammatory diseases, tumour growth and metastasis. The role of MMP-8 in periodontitis is the well-known example of the unwanted tissue destruction related to increased activity of MMPs. Degradation of the extracellular matrix may involve four distinct pathways. A body of evidence suggests that matrix components may be dissolved by extracellular matrix metalloproteinase (MMP-dependent or plasmin (Pln-dependent cleavage reactions and that larger fragment of matrix may be disposed by a phagocytic pathway by way of cleavage by lysosomal proteinases. Mineralized matrices appear to be degraded by a complex process mediated by osteoclasts which relies on degradation by lysosomal proteinases in a narrow pericellular compartment. Matrix metalloproteinases can specifically cleave and degrade collagens and connective tissue matrix at physiologic pH and temperature. The objective of this review article is to understand the complete mechanisms regulating the expression of MMPs and enzymatic activity is of great importance.Source for all the articles is electronic Pub-Med system, published in between 1997-2011 searched by using keywords like Matrix Metalloproteinases, Periodontitis, Extracellular Matrix, Collagen . Future trend should be directed towards the development of easy, reliable and fast diagnostic tools and the effective therapeutic strategies to reduce the levels of MMPs.

  2. Prion Fragment Peptides Are Digested with Membrane Type Matrix Metalloproteinases and Acquire Enzyme Resistance through Cu2+-Binding

    Directory of Open Access Journals (Sweden)

    Aya Kojima

    2014-05-01

    Full Text Available Prions are the cause of neurodegenerative disease in humans and other mammals. The structural conversion of the prion protein (PrP from a normal cellular protein (PrPC to a protease-resistant isoform (PrPSc is thought to relate to Cu2+ binding to histidine residues. In this study, we focused on the membrane-type matrix metalloproteinases (MT-MMPs such as MT1-MMP and MT3-MMP, which are expressed in the brain as PrPC-degrading proteases. We synthesized 21 prion fragment peptides. Each purified peptide was individually incubated with recombinant MT1-MMP or MT3-MMP in the presence or absence of Cu2+ and the cleavage sites determined by LC-ESI-MS analysis. Recombinant MMP-7 and human serum (HS were also tested as control. hPrP61-90, from the octapeptide-repeat region, was cleaved by HS but not by the MMPs tested here. On the other hand, hPrP92-168 from the central region was cleaved by MT1-MMP and MT3-MMP at various sites. These cleavages were inhibited by treatment with Cu2+. The C-terminal peptides had higher resistance than the central region. The data obtained from this study suggest that MT-MMPs expressed in the brain might possess PrPC-degrading activity.

  3. Exploring the active site of the Streptococcus pneumoniae topoisomerase IV-DNA cleavage complex with novel 7,8-bridged fluoroquinolones

    OpenAIRE

    Laponogov, I; Pan, X-S; Veselkov, DA; Cirz, RT; Wagman, A; Moser, HE; Fisher, LM; Sanderson

    2016-01-01

    As part of a programme of synthesizing and investigating the biological properties of new fluoroquinolone antibacterials and their targeting of topoisomerase IV from Streptococcus pneumoniae, we have solved the X-ray structure of the complexes of two new 7,8-bridged fluoroquinolones (with restricted C7 group rotation favouring tight binding) in complex with the topoisomerase IV from S. pneumoniae and an 18-base-pair DNA binding site-the E-site-found by our DNA mapping studies to bind drug str...

  4. Collagenolytic Matrix Metalloproteinase Activities toward Peptomeric Triple-Helical Substrates.

    Science.gov (United States)

    Stawikowski, Maciej J; Stawikowska, Roma; Fields, Gregg B

    2015-05-19

    Although collagenolytic matrix metalloproteinases (MMPs) possess common domain organizations, there are subtle differences in their processing of collagenous triple-helical substrates. In this study, we have incorporated peptoid residues into collagen model triple-helical peptides and examined MMP activities toward these peptomeric chimeras. Several different peptoid residues were incorporated into triple-helical substrates at subsites P3, P1, P1', and P10' individually or in combination, and the effects of the peptoid residues were evaluated on the activities of full-length MMP-1, MMP-8, MMP-13, and MMP-14/MT1-MMP. Most peptomers showed little discrimination between MMPs. However, a peptomer containing N-methyl Gly (sarcosine) in the P1' subsite and N-isobutyl Gly (NLeu) in the P10' subsite was hydrolyzed efficiently only by MMP-13 [nomenclature relative to the α1(I)772-786 sequence]. Cleavage site analysis showed hydrolysis at the Gly-Gln bond, indicating a shifted binding of the triple helix compared to the parent sequence. Favorable hydrolysis by MMP-13 was not due to sequence specificity or instability of the substrate triple helix but rather was based on the specific interactions of the P7' peptoid residue with the MMP-13 hemopexin-like domain. A fluorescence resonance energy transfer triple-helical peptomer was constructed and found to be readily processed by MMP-13, not cleaved by MMP-1 and MMP-8, and weakly hydrolyzed by MT1-MMP. The influence of the triple-helical structure containing peptoid residues on the interaction between MMP subsites and individual substrate residues may provide additional information about the mechanism of collagenolysis, the understanding of collagen specificity, and the design of selective MMP probes.

  5. Fibrillin degradation by matrix metalloproteinases: implications for connective tissue remodelling.

    Science.gov (United States)

    Ashworth, J L; Murphy, G; Rock, M J; Sherratt, M J; Shapiro, S D; Shuttleworth, C A; Kielty, C M

    1999-05-15

    Fibrillin is the principal structural component of the 10-12 nm diameter elastic microfibrils of the extracellular matrix. We have previously shown that both fibrillin molecules and assembled microfibrils are susceptible to degradation by serine proteases. In this study, we have investigated the potential catabolic effects of six matrix metalloproteinases (MMP-2, MMP-3, MMP-9, MMP-12, MMP-13 and MMP-14) on fibrillin molecules and on intact fibrillin-rich microfibrils isolated from ciliary zonules. Using newly synthesized recombinant fibrillin molecules, major cleavage sites within fibrillin-1 were identified. In particular, the six different MMPs generated a major degradation product of approximately 45 kDa from the N-terminal region of the molecule, whereas treatment of truncated, unprocessed and furin-processed C-termini also generated large degradation products. Introduction of a single ectopia lentis-causing amino acid substitution (E2447K; one-letter symbols for amino acids) in a calcium-binding epidermal growth factor-like domain, predicted to disrupt calcium binding, markedly altered the pattern of C-terminal fibrillin-1 degradation. However, the fragmentation pattern of a mutant fibrillin-1 with a comparable E-->K substitution in an upstream calcium-binding epidermal growth factor-like domain was indistinguishable from wild-type molecules. Ultrastructural examination highlighted that fibrillin-rich microfibrils isolated from ciliary zonules were grossly disrupted by MMPs. This is the first demonstration that fibrillin molecules and fibrillin-rich microfibrils are degraded by MMPs and that certain amino acid substitutions change the fragmentation patterns. These studies have important implications for physiological and pathological fibrillin catabolism and for loss of connective tissue elasticity in ageing and disease.

  6. Exploring the active site of the Streptococcus pneumoniae topoisomerase IV-DNA cleavage complex with novel 7,8-bridged fluoroquinolones.

    Science.gov (United States)

    Laponogov, Ivan; Pan, Xiao-Su; Veselkov, Dennis A; Cirz, Ryan T; Wagman, Allan; Moser, Heinz E; Fisher, L Mark; Sanderson, Mark R

    2016-09-01

    As part of a programme of synthesizing and investigating the biological properties of new fluoroquinolone antibacterials and their targeting of topoisomerase IV from Streptococcus pneumoniae, we have solved the X-ray structure of the complexes of two new 7,8-bridged fluoroquinolones (with restricted C7 group rotation favouring tight binding) in complex with the topoisomerase IV from S. pneumoniae and an 18-base-pair DNA binding site-the E-site-found by our DNA mapping studies to bind drug strongly in the presence of topoisomerase IV (Leo et al. 2005 J. Biol. Chem. 280, 14 252-14 263, doi:10.1074/jbc.M500156200). Although the degree of antibiotic resistance towards fluoroquinolones is much lower than that of β-lactams and a range of ribosome-bound antibiotics, there is a pressing need to increase the diversity of members of this successful clinically used class of drugs. The quinolone moiety of the new 7,8-bridged agents ACHN-245 and ACHN-454 binds similarly to that of clinafloxocin, levofloxacin, moxifloxacin and trovofloxacin but the cyclic scaffold offers the possibility of chemical modification to produce interactions with other topoisomerase residues at the active site.

  7. Metalloproteinases and metalloproteinase inhibitors in age-related diseases.

    Science.gov (United States)

    Gargiulo, Simona; Gamba, Paola; Poli, Giuseppe; Leonarduzzi, Gabriella

    2014-01-01

    Degradation of the extracellular matrix is an important feature of embryonic development, morphogenesis, angiogenesis, tissue repair and remodeling. It is precisely regulated under physiological conditions, but when dysregulated it becomes a cause of many diseases, including atherosclerosis, osteoarthritis, diabetic vascular complications, and neurodegeneration. Various types of proteinases are implicated in extracellular matrix degradation, but the major enzymes are considered to be metalloproteinases such as matrix metalloproteinases (MMPs) and disintegrin and metalloproteinase domain (ADAMs) that include ADAMs with a thrombospondin domain (ADAMTS). This review discusses involvement of the major metalloproteinases in some age-related chronic diseases, and examines what is currently known about the beneficial effects of their inhibitors, used as new therapeutic strategies for treating or preventing the development and progression of these diseases.

  8. Multiple sites of the cleavage of 21- and 25-mer encephalytogenic oligopeptides corresponding to human myelin basic protein (MBP by specific anti-MBP antibodies from patients with systemic lupus erythematosus.

    Directory of Open Access Journals (Sweden)

    Anna M Timofeeva

    Full Text Available IgGs from patients with multiple sclerosis and systemic lupus erythematosus (SLE purified on MBP-Sepharose in contrast to canonical proteases hydrolyze effectively only myelin basic protein (MBP, but not many other tested proteins. Here we have shown for the first time that anti-MBP SLE IgGs hydrolyze nonspecific tri- and tetrapeptides with an extreme low efficiency and cannot effectively hydrolyze longer 20-mer nonspecific oligopeptides corresponding to antigenic determinants (AGDs of HIV-1 integrase. At the same time, anti-MBP SLE IgGs efficiently hydrolyze oligopeptides corresponding to AGDs of MBP. All sites of IgG-mediated proteolysis of 21-and 25-mer encephalytogenic oligopeptides corresponding to two known AGDs of MBP were found by a combination of reverse-phase chromatography, TLC, and MALDI spectrometry. Several clustered major, moderate, and minor sites of cleavage were revealed in the case of 21- and 25-mer oligopeptides. The active sites of anti-MBP abzymes are localised on their light chains, while heavy chains are responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of abzymes provide high affinity to MBP and specificity of this protein hydrolysis. The affinity of anti-MBP abzymes for intact MBP is approximately 1000-fold higher than for the oligopeptides. The data suggest that all oligopeptides interact mainly with the light chains of different monoclonal abzymes of total pool of IgGs, which possesses a lower affinity for substrates, and therefore, depending on the oligopeptide sequences, their hydrolysis may be less specific than globular protein and can occur in several sites.

  9. Exploring the active site of the Streptococcus pneumoniae topoisomerase IV–DNA cleavage complex with novel 7,8-bridged fluoroquinolones

    Science.gov (United States)

    Laponogov, Ivan; Pan, Xiao-Su; Veselkov, Dennis A.; Cirz, Ryan T.; Wagman, Allan; Moser, Heinz E.

    2016-01-01

    As part of a programme of synthesizing and investigating the biological properties of new fluoroquinolone antibacterials and their targeting of topoisomerase IV from Streptococcus pneumoniae, we have solved the X-ray structure of the complexes of two new 7,8-bridged fluoroquinolones (with restricted C7 group rotation favouring tight binding) in complex with the topoisomerase IV from S. pneumoniae and an 18-base-pair DNA binding site—the E-site—found by our DNA mapping studies to bind drug strongly in the presence of topoisomerase IV (Leo et al. 2005 J. Biol. Chem. 280, 14 252–14 263, doi:10.1074/jbc.M500156200). Although the degree of antibiotic resistance towards fluoroquinolones is much lower than that of β-lactams and a range of ribosome-bound antibiotics, there is a pressing need to increase the diversity of members of this successful clinically used class of drugs. The quinolone moiety of the new 7,8-bridged agents ACHN-245 and ACHN-454 binds similarly to that of clinafloxocin, levofloxacin, moxifloxacin and trovofloxacin but the cyclic scaffold offers the possibility of chemical modification to produce interactions with other topoisomerase residues at the active site. PMID:27655731

  10. Specificity of the proteasome cleavage to the antigen protein

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    In the MHC classⅠmolecule binding antigenic peptides processing and presentation pathway,the ubiquitin-proteasome system plays a key role in degrading the protein substrate.For the purpose of studying the specificities of proteasomal cleavage sites,partial least squares method is used to predict the proteasomal cleavage sites,and the predictive accuracy of the model is 82.8%.The specificities of the cleavage sites and the adjacent positions come from the contribution of the amino acids of the samples to the cleavage sites,showing the information of proteasome interacting with antigen protein.It demonstrates that the proteasome cleaving to target protein is selective,but not random.

  11. Anhydride formation is not a valid mechanism for peptide cleavage by carboxypeptidase-A: a semiempirical reaction pathway study

    Science.gov (United States)

    Vardi-Kilshtain, Alexandra; Shoham, Gil; Goldblum, Amiram

    The mechanism of action of zinc metalloproteinases has been studied by following the direct nucleophilic pathway, which has been frequently suggested but not yet examined by computational methods, and comparing it to other pathways. We computed the reaction enthalpies for the direct nucleophilic attack by Glu270 in the active site model of carboxypeptidase-A on a model substrate's peptide carbonyl and followed this pathway through mixed anhydride formation and subsequent anhydride cleavage by water. The starting molecular coordinates originate in our own high-resolution crystal structure and the computations have been conducted with the minimal neglect of differential overlap (MNDO) Hamiltonian, modified to include the d-orbitals of zinc and the effects of multiple hydrogen bonding, thus labelled MNDO/d/H. Compared to our recent results for two other candidate pathways for this mechanism, both of the General-Acid-General-Base type, we conclude that the direct nucleophilic or 'anhydride' pathway has a much higher energy barrier at the rate determining step, which is a proton transfer, than previously calculated paths. We argue that the 'anhydride' pathway is thus not a valid one for the cleavage of peptides by carboxypeptidase-A.

  12. The Multibasic Cleavage Site of the Hemagglutinin of Highly Pathogenic A/Vietnam/1203/2004 (H5N1) Avian Influenza Virus Acts as a Virulence Factor in a Host-Specific Manner in Mammals

    Science.gov (United States)

    Suguitan, Amorsolo L.; Matsuoka, Yumiko; Lau, Yuk-Fai; Santos, Celia P.; Vogel, Leatrice; Cheng, Lily I.; Orandle, Marlene

    2012-01-01

    Highly pathogenic avian influenza (HPAI) viruses of the H5 and H7 subtypes typically possess multiple basic amino acids around the cleavage site (MBS) of their hemagglutinin (HA) protein, a recognized virulence motif in poultry. To determine the importance of the H5 HA MBS as a virulence factor in mammals, recombinant wild-type HPAI A/Vietnam/1203/2004 (H5N1) viruses that possessed (H5N1) or lacked (ΔH5N1) the H5 HA MBS were generated and evaluated for their virulence in BALB/c mice, ferrets, and African green monkeys (AGMs) (Chlorocebus aethiops). The presence of the H5 HA MBS was associated with lethality, significantly higher virus titers in the respiratory tract, virus dissemination to extrapulmonary organs, lymphopenia, significantly elevated levels of proinflammatory cytokines and chemokines, and inflammation in the lungs of mice and ferrets. In AGMs, neither H5N1 nor ΔH5N1 virus was lethal and neither caused clinical symptoms. The H5 HA MBS was associated with mild enhancement of replication and delayed virus clearance. Thus, the contribution of H5 HA MBS to the virulence of the HPAI H5N1 virus varies among mammalian hosts and is most significant in mice and ferrets and less remarkable in nonhuman primates. PMID:22205751

  13. Molecular models of the Mojave rattlesnake (Crotalus scutulatus scutulatus) venom metalloproteinases reveal a structural basis for differences in hemorrhagic activities.

    Science.gov (United States)

    Dagda, Ruben K; Gasanov, Sardar E; Zhang, Boris; Welch, William; Rael, Eppie D

    2014-03-01

    Rattlesnake venom can differ in composition and in metalloproteinase-associated activities. The molecular basis for this intra-species variation in Crotalus scutulatus scutulatus (Mojave rattlesnake) remains an enigma. To understand the molecular basis for intra-species variation of metalloproteinase-associated activities, we modeled the three-dimensional structures of four metalloproteinases based on the amino acid sequence of four variations of the proteinase domain of the C. s. scutulatus metalloproteinase gene (GP1, GP2, GP3, and GP4). For comparative purposes, we modeled the atrolysin metalloproteinases of C. atrox as well. All molecular models shared the same topology. While the atrolysin metalloproteinase molecular models contained highly conserved substrate binding sites, the Mojave rattlesnake metalloproteinases showed higher structural divergence when superimposed onto each other. The highest structural divergence among the four C. s. scutulatus molecular models was located at the northern cleft wall and the S'1-pocket of the substrate binding site, molecular regions that modulate substrate selectivity. Molecular dynamics and field potential maps for each C. s. scutulatus metalloproteinase model demonstrated that the non-hemorrhagic metalloproteinases (GP2 and GP3) contain highly basic molecular and field potential surfaces while the hemorrhagic metalloproteinases GP1 and atrolysin C showed extensive acidic field potential maps and shallow but less dynamic active site pockets. Hence, differences in the spatial arrangement of the northern cleft wall, the S'1-pocket, and the physico-chemical environment surrounding the catalytic site contribute to differences in metalloproteinase activities in the Mojave rattlesnake. Our results provide a structural basis for variation of metalloproteinase-associated activities in the rattlesnake venom of the Mojave rattlesnake.

  14. Correlations between papillary thyroid cancer and peripheral blood levels of matrix metalloproteinase-2, matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, and tissue inhibitor of metalloproteinase-2

    Institute of Scientific and Technical Information of China (English)

    ZHOU Shao-fei; HU San-yuan; MA Lei; MIAO Lei; MAO Wei-zheng

    2013-01-01

    Background The relationship between the presence of metalloproteinases and thyroid cancer remains unknown,and many controversies still exist in this field.The objective of this study was to investigate the correlations between papillary thyroid cancer and peripheral blood levels of matrix metalloproteinase-2,matrix metalloproteinase-9,tissue inhibitor of metalloproteinase-1,and tissue inhibitor of metalloproteinase-2.Methods The correlations were studied by detecting the levels of matrix metalloproteinase-2,matrix metalloproteinase-9,tissue inhibitor of metalloproteinase-1,and tissue inhibitor of metalloproteinase-2 by enzyme-linked immunosorbant assay and reverse-transcription polymerase chain reaction in the peripheral blood of 30 patients with papillary thyroid carcinoma,27 patients with benign thyroid disease,and 25 healthy volunteers.Results The levels of matrix metalloproteinase-2,matrix metalloproteinase-9,tissue inhibitor of metalloproteinase-1,and tissue inhibitor of metalloproteinase-2 in the peripheral blood of patients with papillary thyroid carcinoma were significantly higher than those in the peripheral blood of patients with benign thyroid disease and healthy volunteers (P <0.05).However,there were no significant differences between patients with benign thyroid disease and healthy volunteers (P >0.05).The accuracy of detection by both enzyme-linked immunosorbant assay and reverse-transcription polymerase chain reaction in the papillary thyroid cancer group was 83.33%.Conclusions The levels of matrix metalloproteinase-2,matrix metalloproteinase-9,tissue inhibitor of metalloproteinase-1,and tissue inhibitor of metalloproteinase-2 in the peripheral blood are helpful in identifying thyroid carcinoma and aid in preoperative assessment.

  15. Identification of accelerated evolution in the metalloproteinase ...

    African Journals Online (AJOL)

    U

    2016-02-24

    Feb 24, 2016 ... evolution of closely related proteins sequences including snake venom metalloproteinase sequences. (SVMPs) ... metalloproteinase also exists in the toxin/venom of ... diseases of the central nervous system such as bacterial.

  16. Identification of collagen binding domain residues that govern catalytic activities of matrix metalloproteinase-2 (MMP-2).

    Science.gov (United States)

    Mikhailova, Margarita; Xu, Xiaoping; Robichaud, Trista K; Pal, Sanjay; Fields, Gregg B; Steffensen, Bjorn

    2012-01-01

    An innovative approach to enhance the selectivity of matrix metalloproteinase (MMP) inhibitors comprises targeting these inhibitors to catalytically required substrate binding sites (exosites) that are located outside the catalytic cleft. In MMP-2, positioning of collagen substrate molecules occurs via a unique fibronectin-like domain (CBD) that contains three distinct modular collagen binding sites. To characterize the contributions of these exosites to gelatinolysis by MMP-2, seven MMP-2 variants were generated with single, or concurrent double and triple alanine substitutions in the three fibronectin type II modules of the CBD. Circular dichroism spectroscopy verified that recombinant MMP-2 wild-type (WT) and variants had the same fold. Moreover, the MMP-2 WT and variants had the same activity on a short FRET peptide substrate that is hydrolyzed independently of CBD binding. Among single-point variants, substitution in the module 3 binding site had greatest impact on the affinity of MMP-2 for gelatin. Simultaneous substitutions in two or three CBD modules further reduced gelatin binding. The rates of gelatinolysis of MMP-2 variants were reduced by 20-40% following single-point substitutions, by 60-75% after double-point modifications, and by >90% for triple-point variants. Intriguingly, the three CBD modules contributed differentially to cleavage of dissociated α-1(I) and α-2(I) collagen chains. Importantly, kinetic analyses (k(cat)/K(m)) revealed that catalysis of a triple-helical FRET peptide substrate by MMP-2 relied primarily on the module 3 binding site. Thus, we have identified three collagen binding site residues that are essential for gelatinolysis and constitute promising targets for selective inhibition of MMP-2.

  17. The Balance Between Metalloproteinases and TIMPs: Critical Regulator of Microvascular Endothelial Cell Function in Health and Disease.

    Science.gov (United States)

    Masciantonio, Marcello G; Lee, Christopher K S; Arpino, Valerie; Mehta, Sanjay; Gill, Sean E

    2017-01-01

    Endothelial cells (EC), especially the microvascular EC (MVEC), have critical functions in health and disease. For example, healthy MVEC provide a barrier between the fluid and protein found within the blood, and the surrounding tissue. Following tissue injury or infection, the microvascular barrier is often disrupted due to activation and dysfunction of the MVEC. Multiple mechanisms promote MVEC activation and dysfunction, including stimulation by cytokines, mechanical interaction with activated leukocytes, and exposure to harmful leukocyte-derived molecules, which collectively result in a loss of MVEC barrier function. However, MVEC activation is also critical to facilitate recruitment of inflammatory cells, such as neutrophils (PMNs) and monocytes, into the injured or infected tissue. Metalloproteinases, including the matrix metalloproteinases (MMPs) and the closely related, a disintegrin and metalloproteinases (ADAMs), have been implicated in regulating both MVEC barrier function, through cleavage of adherens and tight junctions proteins between adjacent MVEC and through degradation of the extracellular matrix, as well as PMN-MVEC interaction, through shedding of cell surface PMN receptors. Moreover, the tissue inhibitors of metalloproteinases (TIMPs), which collectively inhibit most MMPs and ADAMs, are critical regulators of MVEC activation and dysfunction through their ability to inhibit metalloproteinases and thereby promote MVEC stability. However, TIMPs have been also found to modulate MVEC function through metalloproteinase-independent mechanisms, such as regulation of vascular endothelial growth factor signaling. This chapter is focused on examining the role of the metalloproteinases and TIMPs in regulation of MVEC function in both health and disease. © 2017 Elsevier Inc. All rights reserved.

  18. Prediction of proprotein convertase cleavage sites

    DEFF Research Database (Denmark)

    Duckert, Peter; Brunak, Søren; Blom, Nikolaj

    2004-01-01

    by members of the subtilisin/kexin-like proprotein convertase (PC) family. In mammals, seven members have been identified, with furin being the one first discovered and best characterized. Recently, the involvement of furin in diseases ranging from Alzheimer's disease and cancer to anthrax and Ebola fever...

  19. Metalloproteinases: a Functional Pathway for Myeloid Cells.

    Science.gov (United States)

    Chou, Jonathan; Chan, Matilda F; Werb, Zena

    2016-04-01

    Myeloid cells have diverse roles in regulating immunity, inflammation, and extracellular matrix turnover. To accomplish these tasks, myeloid cells carry an arsenal of metalloproteinases, which include the matrix metalloproteinases and the adamalysins. These enzymes have diverse substrate repertoires, and are thus involved in mediating proteolytic cascades, cell migration, and cell signaling. Dysregulation of metalloproteinases contributes to pathogenic processes, including inflammation, fibrosis, and cancer. Metalloproteinases also have important nonproteolytic functions in controlling cytoskeletal dynamics during macrophage fusion and enhancing transcription to promote antiviral immunity. This review highlights the diverse contributions of metalloproteinases to myeloid cell functions.

  20. Cleavage of a specific bond in troponin C by thrombin.

    Science.gov (United States)

    Leavis, P C; Rosenfeld, S; Lu, R C

    1978-08-21

    Limited proteolysis of rabbit skeletal troponin C with bovine thrombin yielded two fragments, TH1 (Mr = 11000) containing Ca2+ binding regions I--III and TH2 (Mr = 6000) containing region IV. Determination of the partial sequences of the fragments established the site of cleavage at Arg120-Ala121. Secondary cleavage by thrombin at other arginyl or lysyl residues in troponin C was ruled out by the sequence data and by the amino acid compositions of the two fragments.

  1. Fine Tuning Cell Migration by a Disintegrin and Metalloproteinases

    Science.gov (United States)

    Theodorou, K.

    2017-01-01

    Cell migration is an instrumental process involved in organ development, tissue homeostasis, and various physiological processes and also in numerous pathologies. Both basic cell migration and migration towards chemotactic stimulus consist of changes in cell polarity and cytoskeletal rearrangement, cell detachment from, invasion through, and reattachment to their neighboring cells, and numerous interactions with the extracellular matrix. The different steps of immune cell, tissue cell, or cancer cell migration are tightly coordinated in time and place by growth factors, cytokines/chemokines, adhesion molecules, and receptors for these ligands. This review describes how a disintegrin and metalloproteinases interfere with several steps of cell migration, either by proteolytic cleavage of such molecules or by functions independent of proteolytic activity. PMID:28260841

  2. Matrix Metalloproteinases in Inflammatory Bowel Disease: An Update

    Directory of Open Access Journals (Sweden)

    Shane O’Sullivan

    2015-01-01

    Full Text Available Matrix metalloproteinases (MMPs are known to be upregulated in inflammatory bowel disease (IBD and other inflammatory conditions, but while their involvement is clear, their role in many settings has yet to be determined. Studies of the involvement of MMPs in IBD since 2006 have revealed an array of immune and stromal cells which release the proteases in response to inflammatory cytokines and growth factors. Through digestion of the extracellular matrix and cleavage of bioactive proteins, a huge diversity of roles have been revealed for the MMPs in IBD, where they have been shown to regulate epithelial barrier function, immune response, angiogenesis, fibrosis, and wound healing. For this reason, MMPs have been recognised as potential biomarkers for disease activity in IBD and inhibition remains a huge area of interest. This review describes new roles of MMPs in the pathophysiology of IBD and suggests future directions for the development of treatment strategies in this condition.

  3. Fine Tuning Cell Migration by a Disintegrin and Metalloproteinases.

    Science.gov (United States)

    Dreymueller, D; Theodorou, K; Donners, M; Ludwig, A

    2017-01-01

    Cell migration is an instrumental process involved in organ development, tissue homeostasis, and various physiological processes and also in numerous pathologies. Both basic cell migration and migration towards chemotactic stimulus consist of changes in cell polarity and cytoskeletal rearrangement, cell detachment from, invasion through, and reattachment to their neighboring cells, and numerous interactions with the extracellular matrix. The different steps of immune cell, tissue cell, or cancer cell migration are tightly coordinated in time and place by growth factors, cytokines/chemokines, adhesion molecules, and receptors for these ligands. This review describes how a disintegrin and metalloproteinases interfere with several steps of cell migration, either by proteolytic cleavage of such molecules or by functions independent of proteolytic activity.

  4. Myocardial structure and matrix metalloproteinases.

    Science.gov (United States)

    Aggeli, C; Pietri, P; Felekos, I; Rautopoulos, L; Toutouzas, K; Tsiamis, E; Stefanadis, C

    2012-01-01

    Metalloproteinases (MMPs) are enzymes which enhance proteolysis of extracellular matrix proteins. The pathophysiologic and prognostic role of MMPs has been demonstrated in numerous studies. The present review covers a wide a range of topics with regards to MMPs structural and functional properties, as well as their role in myocardial remodeling in several cardiovascular diseases. Moreover, the clinical and therapeutic implications from their assessment are highlighted.

  5. Vertebrate Embryonic Cleavage Pattern Determination.

    Science.gov (United States)

    Hasley, Andrew; Chavez, Shawn; Danilchik, Michael; Wühr, Martin; Pelegri, Francisco

    2017-01-01

    The pattern of the earliest cell divisions in a vertebrate embryo lays the groundwork for later developmental events such as gastrulation, organogenesis, and overall body plan establishment. Understanding these early cleavage patterns and the mechanisms that create them is thus crucial for the study of vertebrate development. This chapter describes the early cleavage stages for species representing ray-finned fish, amphibians, birds, reptiles, mammals, and proto-vertebrate ascidians and summarizes current understanding of the mechanisms that govern these patterns. The nearly universal influence of cell shape on orientation and positioning of spindles and cleavage furrows and the mechanisms that mediate this influence are discussed. We discuss in particular models of aster and spindle centering and orientation in large embryonic blastomeres that rely on asymmetric internal pulling forces generated by the cleavage furrow for the previous cell cycle. Also explored are mechanisms that integrate cell division given the limited supply of cellular building blocks in the egg and several-fold changes of cell size during early development, as well as cytoskeletal specializations specific to early blastomeres including processes leading to blastomere cohesion. Finally, we discuss evolutionary conclusions beginning to emerge from the contemporary analysis of the phylogenetic distributions of cleavage patterns. In sum, this chapter seeks to summarize our current understanding of vertebrate early embryonic cleavage patterns and their control and evolution.

  6. Circulating levels of matrix metalloproteinases and tissue inhibitors of metalloproteinases in patients with incisional hernia

    DEFF Research Database (Denmark)

    Henriksen, Nadia A; Sørensen, Lars T; Jorgensen, Lars N

    2013-01-01

    Incisional hernia formation is a common complication to laparotomy and possibly associated with alterations in connective tissue metabolism. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are closely involved in the metabolism of the extracellular matrix. Our...

  7. Metalloproteinases and tissue inhibitor of metalloproteinases in mesothelial cells. Cellular differentiation influences expression.

    Science.gov (United States)

    Marshall, B C; Santana, A; Xu, Q P; Petersen, M J; Campbell, E J; Hoidal, J R; Welgus, H G

    1993-04-01

    Mesothelial cells play a critical role in the remodeling process that follows serosal injury. Although mesothelial cells are known to synthesize a variety of extracellular matrix components including types I, III, and IV collagens, their potential to participate in matrix degradation has not been explored. We now report that human pleural and peritoneal mesothelial cells express interstitial collagenase, 72- and 92-kD gelatinases (type IV collagenases), and the counterregulatory tissue inhibitor of metalloproteinases (TIMP). Our initial characterization of the mesothelial cell metalloenzymes and TIMP has revealed: (a) they are likely identical to corresponding molecules secreted by other human cells; (b) they are secreted rather than stored in an intracellular pool; (c) a primary site of regulation occurs at a pretranslational level; (d) phorbol myristate acetate, via activation of protein kinase C, upregulates expression of collagenase, 92-kD gelatinase, and TIMP, but has no effect on expression of 72-kD gelatinase; and (e) lipopolysaccharide fails to upregulate the biosynthesis of either metalloproteinases or TIMP. Of particular interest is the observation that the state of cellular differentiation has a striking influence on the expression of metalloenzymes and TIMP, such that epitheloid cells display a more matrix-degradative phenotype (increased 92-kD gelatinase and decreased TIMP) than their fibroblastoid counterparts. We speculate that mesothelial cells directly participate in the extracellular matrix turnover that follows serosal injury via elaboration of metalloproteinases and TIMP. Additionally, the reactive cuboidal mesothelium which is characteristic of the early response to serosal injury may manifest a matrix-degenerative phenotype favoring normal repair rather than fibrosis.

  8. Deformation-dependent enzyme mechanokinetic cleavage of type I collagen.

    Science.gov (United States)

    Wyatt, Karla E-K; Bourne, Jonathan W; Torzilli, Peter A

    2009-05-01

    Collagen is a key structural protein in the extracellular matrix of many tissues. It provides biological tissues with tensile mechanical strength and is enzymatically cleaved by a class of matrix metalloproteinases known as collagenases. Collagen enzymatic kinetics has been well characterized in solubilized, gel, and reconstituted forms. However, limited information exists on enzyme degradation of structurally intact collagen fibers and, more importantly, on the effect of mechanical deformation on collagen cleavage. We studied the degradation of native rat tail tendon fibers by collagenase after the fibers were mechanically elongated to strains of epsilon=1-10%. After the fibers were elongated and the stress was allowed to relax, the fiber was immersed in Clostridium histolyticum collagenase and the decrease in stress (sigma) was monitored as a means of calculating the rate of enzyme cleavage of the fiber. An enzyme mechanokinetic (EMK) relaxation function T(E)(epsilon) in s(-1) was calculated from the linear stress-time response during fiber cleavage, where T(E)(epsilon) corresponds to the zero order Michaelis-Menten enzyme-substrate kinetic response. The EMK relaxation function T(E)(epsilon) was found to decrease with applied strain at a rate of approximately 9% per percent strain, with complete inhibition of collagen cleavage predicted to occur at a strain of approximately 11%. However, comparison of the EMK response (T(E) versus epsilon) to collagen's stress-strain response (sigma versus epsilon) suggested the possibility of three different EMK responses: (1) constant T(E)(epsilon) within the toe region (epsiloncollagen triple helix may be by a conformational change in the triple helix since the decrease in T(E)(epsilon) appeared concomitant with stretching of the collagen molecule.

  9. Immunohistochemical Studies of the Expression of Matrix Metalloproteinase-2 and Metalloproteinase-9 in Human Prostate Cancer

    Institute of Scientific and Technical Information of China (English)

    曾汉青; 肖亚军; 鲁功成; 陈勇

    2003-01-01

    To study the expression of matrix metalloproteinase-2 and -9 in human prostate cancer,matrix metalloproteinase-2 and -9 were immunohistochemically detected in tissues of prostate cancer and benign prostatic hyperplasia (BPH). Our results showed that matrix metalloproteinase-2 and -9 levels in prostate cancer were much higher than those in tissues of BPH, with the cancer invasion being positively correlated with the expression of the metalloproteinases. It is concluded that matrix metalloproteinase-2 and -9 are better molecular markers, which are of help in the diagnosis and prediction of prognosis of prostate cancer.

  10. Structural and functional diversity of metalloproteinases encoded by the Bacteroides fragilis pathogenicity island.

    Science.gov (United States)

    Shiryaev, Sergey A; Aleshin, Alexander E; Muranaka, Norihito; Kukreja, Muskan; Routenberg, David A; Remacle, Albert G; Liddington, Robert C; Cieplak, Piotr; Kozlov, Igor A; Strongin, Alex Y

    2014-06-01

    Bacteroides fragilis causes the majority of anaerobic infections in humans. The presence of a pathogenicity island in the genome discriminates pathogenic and commensal B. fragilis strains. The island encodes metalloproteinase II (MPII), a potential virulence protein, and one of three homologous fragilysin isozymes (FRA; also termed B. fragilis toxin or BFT). Here, we report biochemical data on the structural-functional characteristics of the B. fragilis pathogenicity island proteases by reporting the crystal structure of MPII at 2.13 Å resolution, combined with detailed characterization of the cleavage preferences of MPII and FRA3 (as a representative of the FRA isoforms), identified using a high-throughput peptide cleavage assay with 18 583 substrate peptides. We suggest that the evolution of the MPII catalytic domain can be traced to human and archaebacterial proteinases, whereas the prodomain fold is a feature specific to MPII and FRA. We conclude that the catalytic domain of both MPII and FRA3 evolved differently relative to the prodomain, and that the prodomain evolved specifically to fit the B. fragilis pathogenicity. Overall, our data provide insights into the evolution of cleavage specificity and activation mechanisms in the virulent metalloproteinases. © 2014 FEBS.

  11. A novel secreted metzincin metalloproteinase from Bacillus intermedius.

    Science.gov (United States)

    Sabirova, Albina R; Rudakova, Natalya L; Balaban, Nelly P; Ilyinskaya, Olga N; Demidyuk, Ilya V; Kostrov, Sergey V; Rudenskaya, Galina N; Sharipova, Margarita R

    2010-11-05

    The mprBi gene from Bacillus intermedius 3-19 encoding a novel secreted metalloproteinase was identified. The mpriBi gene was expressed in an extracellular proteinase-deficient Bacillus subtilis BG 2036 strain and the corresponding protein was characterized biochemically. The 19 kDa MprBi protein was purified to homogeneity and sequenced by mass spectroscopy and Edman degradation methods. Amino acid sequence analysis of MprBi identified an active site motif HEYGHNFGLPHD and a conserved structural component Met-turn, both of which are unique features of the metzincin clan. Furthermore, MprBi harbors a number of distinct sequence elements characteristic of proteinase domains in eukaryotic adamalysins. We conclude that MprBi and similar proteins from other Bacillus species form a novel group of metzincin metalloproteinases in prokaryotes.

  12. Identification of a mutation in the spike protein cleavage site in Brazilian strains of wild-type bovine coronavirus Identificação de uma mutação no sítio de clivagem da proteína da espícula em amostras brasileiras de coronavírus bovino

    Directory of Open Access Journals (Sweden)

    Elisabete Takiuchi

    2007-12-01

    Full Text Available The spike (S protein of coronaviruses, a type I membrane glycoprotein, is primarily responsible for entry into susceptible cells by binding with specific receptors on cells and mediating subsequent virus-cell fusion. The bovine coronavirus (BCoV S protein is cleaved into two subunits, the N-terminal S1 and the C-terminal S2. The proteolytic cleavage site of S protein is highly conserved among BCoV strains and is located between amino acids 763 and 768 (KRRSRR. This study describes a single mutation in the S protein cleavage site of three Brazilian strains of BCoV detected in diarrheic fecal samples from calves naturally infected. The sequenced PCR products revealed that amino acid sequence of the cleavage site of our strains was KRRSSR, indicating a mutation at amino acid position 767 (R ® S. This amino acid substitution occurred due to a single nucleotide substitution in the sequence of DNA corresponding to the proteolytic cleavage site, CGT to AGT. This is the first description of this nucleotide mutation (C to A, which resulted in the substitution of arginine to serine in the S cleavage site. In this study we speculated the probable effects of this mutation in the proteolytic cleavage site using the murine hepatitis coronavirus (MHV as a comparative model.A proteína da espícula (S, uma glicoproteína de membrana do tipo I, é primariamente responsável pela entrada do vírus em células susceptíveis por meio da interação inicial com receptores celulares específicos e subseqüente mediação da fusão vírus-célula. A proteína S do coronavírus bovino (BCoV é clivada em duas subunidades: a S1, na região N-terminal e a S2, na região C-terminal. O sítio de clivagem proteolítica da proteína S é altamente conservado entre as estirpes de BCoV e está situado entre os aminoácidos 763-768 (KRRSRR. Este estudo descreve uma mutação no sítio de clivagem da proteína S de três estirpes do BCoV detectadas em amostras fecais diarr

  13. Metalloproteinases and their regulators in colorectal cancer.

    NARCIS (Netherlands)

    Jagt, M.F.P. van der; Wobbes, T.; Strobbe, L.J.; Sweep, F.C.; Span, P.N.

    2010-01-01

    Metalloproteinases (MPs) such as the matrix metalloproteinases (MMPs) and adamalysins (ADAMs and ADAMTS) are expressed in various stages of colorectal cancer (CRC), and some correlate with survival and prognosis. The MPs are regulated by various factors including EMMPRIN, TIMPs, and RECK. In additio

  14. Matrix Metalloproteinases in patients with Multiple Sclerosis.

    Directory of Open Access Journals (Sweden)

    Rebeca A. Fernández Carriera

    2007-05-01

    Full Text Available Fundament: The proteolitic rupture of the extracellular matrix due to metalloproteinase 2 and 9 is one of the aspects that can influence in the alteration of the permeability of the blood-brain barrier (BBB in multiple sclerosis. Objective: To determine metalloproteinase activity with gelatinous activity in patients suffering from multiple sclerosis. Methods: the cerebrospinal fluid (CSF samples taken from 31 patients suffering from multiple sclerosis and a control group formed by 21 patients without neurological disease. The metalloproteinase 2 and 9 activities in the cerebrospinal fluid were determined by zimográfica technique through polyacrylamide gel electrophoresis. The bands were later analysed by their molecular weight and the relative metalloproteinase 9 activity was calculated. Total protein concentrations, albumin and immunoglobulin G (IgG, the IgG rate and the Q rate were assessed to evaluate the IgG intrathecal and the functional state of the blood-brain barrier. Results: metalloproteinase 2 activity was detected in the cerebrospinal fluid of all patients and control group. Metalloproteinase 9 activity was only found in the 61.3 % of the patients. The presence of relative metalloproteinase 9 activity was neither associated with the clinical variables nor the laboratory ones. An association was found between its presence and the oligoclonal bands in patients with multiple sclerosis. In those patients under immunomodular treatment it was presented with less frequency. Conclusions: There is a possible participation of Metalloproteinase 9 in the immunopathological mechanisms of the multiple sclerosis.

  15. Inflammatory effects of snake venom metalloproteinases

    Directory of Open Access Journals (Sweden)

    Catarina de Fátima Pereira Teixeira

    2005-03-01

    Full Text Available Metalloproteinases are abundant enzymes in crotaline and viperine snake venoms. They are relevant in the pathophysiology of envenomation, being responsible for local and systemic hemorrhage frequently observed in the victims. Snake venom metalloproteinases (SVMP are zinc-dependent enzymes of varying molecular weights having multidomain organization. Some SVMP comprise only the proteinase domain, whereas others also contain a disintegrin-like domain, cysteine-rich, and lectin domains. They have strong structural similarities with both mammalian matrix metalloproteinases (MMP and members of ADAMs (a disintegrin and metalloproteinase group. Besides hemorrhage, snake venom metalloproteinase induce local myonecrosis, skin damage, and inflammatory reaction in experimental models. Local inflammation is an important characteristic of snakebite envenomations inflicted by viperine and crotaline snake species. Thus, in the recent years there is a growing effort to understand the mechanisms responsible for SVMP-induced inflammatory reaction and the structural determinants of this effect. This short review focuses the inflammatory effects evoked by SVMP.

  16. Matrix metalloproteinases-2, -9 and tissue inhibitor of metallo-proteinase-1 in lung cancer invasion and metastasis

    Institute of Scientific and Technical Information of China (English)

    MING Shu-hong; SUN Tie-ying; XIAO Wei; XU Xiao-mao

    2005-01-01

    @@ Lung cancer is a major cause of death from malignant disease due to its high incidence, malignant behavior and lack of major advancements in treatment strategies. The ability to invade tissues and establish colonies at remote sites is a defining characteristic of malignant neoplasms. Matrix metalloproteinases (MMPs) are zinc proteinases that degrade compounds of extracellular matrix (ECM). These enzymes have been implicated in tumour invasion and metastasis through degrading many extracellular matrix proteins especially MMP-2 and MMP-9, which are regarded as markers of tumour invasion and metastasis.1 The purpose of this study is to examine the role of MMP-9, MMP-2, tissue inhibitor of metalloproteinase-1 (TIMP-1) and MMP-9/TIMP-1 in tumour invasion and metastasis as well as the relationships between the mRNA expression of MMP-9 in white blood cells and MMP-9 levels in the plasma.

  17. Natural soluble interleukin-15Ralpha is generated by cleavage that involves the tumor necrosis factor-alpha-converting enzyme (TACE/ADAM17).

    Science.gov (United States)

    Budagian, Vadim; Bulanova, Elena; Orinska, Zane; Ludwig, Andreas; Rose-John, Stefan; Saftig, Paul; Borden, Ernest C; Bulfone-Paus, Silvia

    2004-09-24

    This study shows that the high affinity alpha-chain of the interleukin (IL)-15 receptor exists not only in membrane-anchored but also in soluble form. Soluble IL-15Ralpha (sIL-15Ralpha) can be detected in mouse sera and cell-conditioned media by enzyme-linked immunosorbent assay and by immunoprecipitation and Western blotting. This protein has a molecular mass of about 30 kDa because of the presence of a single N-glycosylation site, which is reduced to 26 kDa after N-glycosidase treatment. Transmembrane IL-15Ralpha is constitutively converted into its soluble form by proteolytic cleavage that involves tumor necrosis factor-alpha-converting enzyme (TACE), and this process is further enhanced by phorbol 12-myristate 13-acetate (PMA) stimulation. The hydroxamate GW280264X, which is capable of blocking TACE and the closely related disintegrin-like metalloproteinase 10 (ADAM10), effectively inhibited both spontaneous and PMA-inducible cleavage of IL-15Ralpha, whereas GI254023X, which preferentially blocks ADAM10, was ineffective. Overexpression of TACE but not ADAM10 in COS-7 cells enhanced the constitutive and PMA-inducible cleavage of IL-15Ralpha. Moreover, murine fibroblasts deficient in TACE but not ADAM10 expression exhibited a significant reduction in the spontaneous and inducible IL-15Ralpha shedding, whereas a reconstitution of TACE in these cells restored the release of sIL-15Ralpha, thereby suggesting that TACE-mediated proteolysis may represent a major mechanism for sIL-15Ralpha generation in mice. The existence of natural sIL-15Ralpha offers novel insights into the complex biology of IL-15 and envisages a new level for therapeutic intervention.

  18. Matrix metalloproteinases-2/9-sensitive peptide-conjugated polymer micelles for site-specific release of drugs and enhancing tumor accumulation: preparation and in vitro and in vivo evaluation

    Directory of Open Access Journals (Sweden)

    Zhang X

    2016-04-01

    Full Text Available Xiaoyan Zhang, Xiaofei Wang, Weitong Zhong, Xiaoqing Ren, Xianyi Sha, Xiaoling FangKey Laboratory of Smart Drug Delivery, Ministry of Education and People’s Liberation Army of China, School of Pharmacy, Fudan University, Shanghai, People’s Republic of China Abstract: Since elevated expression of matrix metalloproteinase (MMP-2 and MMP-9 is commonly observed in several malignant tumors, MMPs have been widely reported as key factors in the design of drug delivery systems. Several strategies have been proposed to develop MMPs-responsive nanoparticles to deliver chemotherapeutics to malignant solid tumors. A stimuli-responsive drug delivery system, which could be cleaved by MMPs, was proposed in this study. By inserting an MMP-2/9 cleavable oligopeptide GPVGLIGK-NH2 (GK8 as spacer between α-tocopherol succinate (α-TOS and methoxy-polyethylene glycol molecular weight (MW 2000 Da activated by N-hydroxysuccinimide (mPEG2K-NHS, mPEG2K-GK8-α-TOS (TGK was synthesized as the primary ingredient for MMP-2/9-sensitive micelles composed of D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS and TGK (n:n =40:60, TGK micelles. mPEG2K-α-TOS (T2K was similarly synthesized as nonsensitive control. The TGK micelles showed better stability than nonsensitive micelles composed of TPGS and T2K (n:n =40:60, T2K micelles owing to the inserted peptide. Fluorescence resonance energy transfer results indicated that TGK micelles could be successfully cleaved by MMP-2/9. Effective drug release was demonstrated in the presence of collagenase type IV, a mixture of MMP-2 and MMP-9. Compared with nonsensitive micelles, docetaxel (DTX-loaded TGK micelles showed a fold higher cellular uptake in HT1080 cells. While the half-maximal inhibitory concentration (IC50 of TGK and T2K micelles were similar (P>0.05 in MCF-7 cells (MMP-2/9 underexpression, the IC50 values of the aforementioned micelles were 0.064±0.006 and 0.122±0.009 µg/mL, respectively, in HT1080 cells (MMP

  19. Imbalance of matrix metalloproteinase-9 and matrix metalloproteinase tissue inhibitor-1 may contribute to hemorrhage in cerebellar arteriovenous malformations

    Institute of Scientific and Technical Information of China (English)

    Fei Di; Tongyan Chen; Hongli Li; Jizong Zhao; Shuo Wang; Yuanli Zhao; Dong Zhang

    2012-01-01

    In this study,we determined the expression levels of matrix metalloproteinase-2 and -9 and matrix metalloproteinase tissue inhibitor-1 and -2 in brain tissues and blood plasma of patients undergoing surgery for cerebellar arteriovenous malformations or primary epilepsy (control group).Immunohistochemistry and enzyme-linked immunosorbent assay revealed that the expression of matrix metalloproteinase-9 and matrix metalloproteinase tissue inhibitor-1 was significantly higher in patients with cerebellar arteriovenous malformations than in patients with primary epilepsy.The ratio of matrix metalloproteinase-9 to matrix metalloproteinase tissue inhibitor-1 was significantly higher in patients with hemorrhagic cerebellar arteriovenous malformations compared with those with non-hemorrhagic malformations.Matrix metalloproteinase-2 and matrix metalloproteinase tissue inhibitor-2 levels were not significantly changed.These findings indicate that an imbalance of matrix metalloproteinase-9 and matrix metalloproteinase tissue inhibitor-1,resulting in a relative overabundance of matrix metalloproteinase-9,might be the underlying mechanism of hemorrhage of cerebellar arteriovenous malformations.

  20. Unexpected tolerance of alpha-cleavage of the prion protein to sequence variations.

    Directory of Open Access Journals (Sweden)

    José B Oliveira-Martins

    Full Text Available The cellular form of the prion protein, PrP(C, undergoes extensive proteolysis at the alpha site (109K [see text]H110. Expression of non-cleavable PrP(C mutants in transgenic mice correlates with neurotoxicity, suggesting that alpha-cleavage is important for PrP(C physiology. To gain insights into the mechanisms of alpha-cleavage, we generated a library of PrP(C mutants with mutations in the region neighbouring the alpha-cleavage site. The prevalence of C1, the carboxy adduct of alpha-cleavage, was determined for each mutant. In cell lines of disparate origin, C1 prevalence was unaffected by variations in charge and hydrophobicity of the region neighbouring the alpha-cleavage site, and by substitutions of the residues in the palindrome that flanks this site. Instead, alpha-cleavage was size-dependently impaired by deletions within the domain 106-119. Almost no cleavage was observed upon full deletion of this domain. These results suggest that alpha-cleavage is executed by an alpha-PrPase whose activity, despite surprisingly limited sequence specificity, is dependent on the size of the central region of PrP(C.

  1. Matrix metalloproteinases in lung biology

    Directory of Open Access Journals (Sweden)

    Parks William C

    2000-12-01

    Full Text Available Abstract Despite much information on their catalytic properties and gene regulation, we actually know very little of what matrix metalloproteinases (MMPs do in tissues. The catalytic activity of these enzymes has been implicated to function in normal lung biology by participating in branching morphogenesis, homeostasis, and repair, among other events. Overexpression of MMPs, however, has also been blamed for much of the tissue destruction associated with lung inflammation and disease. Beyond their role in the turnover and degradation of extracellular matrix proteins, MMPs also process, activate, and deactivate a variety of soluble factors, and seldom is it readily apparent by presence alone if a specific proteinase in an inflammatory setting is contributing to a reparative or disease process. An important goal of MMP research will be to identify the actual substrates upon which specific enzymes act. This information, in turn, will lead to a clearer understanding of how these extracellular proteinases function in lung development, repair, and disease.

  2. Riding the Metalloproteinase Roller Coaster.

    Science.gov (United States)

    Murphy, Gillian

    2017-03-15

    To many of us in the field, working on Matrix Metalloproteinases (MMPs) has felt like riding a roller coaster, traveling through times of both excitement and despair. I was fortunate to join the ride when it was a mere carousel of three activities thought to target the proteins that comprise the extracellular matrix (ECM). New technologies brought the thrills of discovery, as we uncovered specific proteinase genes and defined specialised activities in different cellular processes. The MMPs and the sister families of 'A Disintegrin And Metalloproteinase' (ADAMs), ADAMs with ThromboSpondin domains (ADAM-TS) and Astacins are now recognised as key signalling 'scissors' that drive rapid changes in a plethora of cellular pathways. My many excellent colleagues and collaborators and I were enthused to contribute to the early development of the field and continue to be amazed at its growth and sophistication. In contrast, the hype and failure of early inhibitor discovery have dogged our standing with the pharmaceutical industry and grant giving bodies. However, the true believers have kept going, and knowledge of particular functions of MMPs and their contributions to disease progression has progressed. Recognition of the strategic importance of proteinase function should inspire more work harnessing new technologies such as imaging, proteomics and gene editing to generate a more precise understanding of individual situations. New approaches to inhibitor design and assessment are possible and the consequent ability to precisely abrogate specific MMP activity could contribute to the fight against a number of pathologies with unmet needs. What a ride it could be!

  3. Structural differences of matrix metalloproteinases. Homology modeling and energy minimization of enzyme-substrate complexes

    DEFF Research Database (Denmark)

    Terp, G E; Christensen, I T; Jørgensen, Flemming Steen

    2000-01-01

    Matrix metalloproteinases are extracellular enzymes taking part in the remodeling of extracellular matrix. The structures of the catalytic domain of MMP1, MMP3, MMP7 and MMP8 are known, but structures of enzymes belonging to this family still remain to be determined. A general approach...... to the homology modeling of matrix metalloproteinases, exemplified by the modeling of MMP2, MMP9, MMP12 and MMP14 is described. The models were refined using an energy minimization procedure developed for matrix metalloproteinases. This procedure includes incorporation of parameters for zinc and calcium ions...... in the AMBER 4.1 force field, applying a non-bonded approach and a full ion charge representation. Energy minimization of the apoenzymes yielded structures with distorted active sites, while reliable three-dimensional structures of the enzymes containing a substrate in active site were obtained. The structural...

  4. Preliminary evidence for a matrix metalloproteinase-2 (MMP-2)-dependent shedding of soluble CD40 ligand (sCD40L) from activated platelets.

    Science.gov (United States)

    Reinboldt, Stephan; Wenzel, Folker; Rauch, Bernhard H; Hohlfeld, Thomas; Grandoch, Maria; Fischer, Jens W; Weber, Artur-Aron

    2009-09-01

    Platelets are the major source of soluble CD40 ligand (sCD40L) in the blood. It has been demonstrated that CD40L is cleaved from the surface of activated platelets to release sCD40L. However, the enzyme involved in sCD40L shedding has not been identified yet. Using a panel of pharmacological inhibitors of serine, cysteine, aspartate, or metalloproteinases, preliminary evidence is presented for the hypothesis that matrix metalloproteinase-2 (MMP-2) might be the protease, primarily responsible for CD40L cleavage from platelet surface.

  5. Matrix metalloproteinases in wound repair (review).

    Science.gov (United States)

    Ravanti, L; Kähäri, V M

    2000-10-01

    Wound repair is initiated with the aggregation of platelets, formation of a fibrin clot, and release of growth factors from the activated coagulation pathways, injured cells, platelets, and extracellular matrix (ECM), followed by migration of inflammatory cells to the wound site. Thereafter, keratinocytes migrate over the wound, angiogenesis is initiated, and fibroblasts deposit and remodel the granulation tissue. Cell migration, angiogenesis, degradation of provisional matrix, and remodeling of newly formed granulation tissue, all require controlled degradation of the ECM. Disturbance in the balance between ECM production and degradation leads to formation of chronic ulcers with excessive ECM degradation, or to fibrosis, for example hypertrophic scars or keloids characterized by excessive accumulation of ECM components. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases, which as a group can degrade essentially all ECM components. So far, 20 members of the human MMP family have been identified. Based on their structure and substrate specificity, they can be divided into subgroups of collagenases, stromelysins, stromelysin-like MMPs, gelatinases, membrane-type MMPs (MT-MMPs), and other MMPs. In this review, the role of MMPs in normal wound repair as well as in chronic ulcers is discussed. In addition, the role of signaling pathways, in particular, mitogen-activated protein kinases (MAPKs) in regulating MMP expression is discussed as possible therapeutical targets for wound healing disorders.

  6. Matrix metalloproteinase inhibition in atherosclerosis and stroke.

    Science.gov (United States)

    Roycik, M D; Myers, J S; Newcomer, R G; Sang, Q-X A

    2013-09-01

    Matrix metalloproteinases (MMPs) are a family of tightly regulated, zinc-dependent proteases that degrade extracellular matrix (ECM), cell surface, and intracellular proteins. Vascular remodeling, whether as a function of normal physiology or as a consequence of a myriad of pathological processes, requires degradation of the ECM. Thus, the expression and activity of many MMPs are up-regulated in numerous conditions affecting the vasculature and often exacerbate vascular dysfunction. A growing body of evidence supports the rationale of using MMP inhibitors for the treatment of cardiovascular diseases, stroke, and chronic vascular dementia. This manuscript will examine promising targets for MMP inhibition in atherosclerosis and stroke, reviewing findings in preclinical animal models and human patient studies. Strategies for MMP inhibition have progressed beyond chelating the catalytic zinc to functional blocking antibodies and peptides that target either the active site or exosites of the enzyme. While the inhibition of MMP activity presents a rational therapeutic avenue, the multiplicity of roles for MMPs and the non-selective nature of MMP inhibitors that cause unintended side-effects hinder full realization of MMP inhibition as therapy for vascular disease. For optimal therapeutic effects to be realized, specific targets for MMP inhibition in these pathologies must first be identified and then attacked by potent and selective agents during the most appropriate timepoint.

  7. The structure of the catalytic domain of Tannerella forsythia karilysin reveals it is a bacterial xenolog of animal matrix metalloproteinases

    Science.gov (United States)

    Cerdà-Costa, Núria; Guevara, Tibisay; Karim, Abdulkarim Y.; Ksiazek, Miroslaw; Nguyen, Ky-Anh; Arolas, Joan L.; Potempa, Jan; Gomis-Rüth, F. Xavier

    2010-01-01

    Metallopeptidases (MPs) are among virulence factors secreted by pathogenic bacteria at the site of infection. One such pathogen is Tannerella forsythia, a member of the microbial consortium that causes peridontitis, arguably the most prevalent infective chronic inflammatory disease known to mankind. The only reported MP secreted by T. forsythia is karilysin, a 52-kDa multidomain protein comprising a central 18-kDa catalytic domain (CD), termed Kly18, flanked by domains unrelated to any known protein. We analyzed the 3D structure of Kly18 in the absence and presence of Mg2+ or Ca2+, which are required for function and stability, and found that it evidences most of the structural features characteristic of the CDs of mammalian matrix metalloproteinases (MMPs). Unexpectedly, a peptide was bound to the active-site cleft of Kly18 mimicking a left-behind cleavage product, which revealed that the specificity pocket accommodates bulky hydrophobic side chains of substrates as in mammalian MMPs. In addition, Kly18 displayed a unique Mg2+ or Ca2+ binding site and two flexible segments that could play a role in substrate binding. Phylogenetic and sequence similarity studies revealed that Kly18 is evolutionarily much closer to winged-insect and mammalian MMPs than to potential bacterial counterparts found by genomic sequencing projects. Therefore, we conclude that this first structurally-characterized non-mammalian MMP is a xenolog co-opted through horizontal gene transfer during the intimate coexistence between T. forsythia and humans or other animals, in a very rare case of gene shuffling from eukaryotes to prokaryotes. Subsequently, this protein would have evolved in a bacterial environment to give rise to full-length karilysin that is furnished with unique flanking domains that do not conform to the general multidomain architecture of animal MMPs. PMID:21166898

  8. The structure of the catalytic domain of Tannerella forsythia karilysin reveals it is a bacterial xenologue of animal matrix metalloproteinases.

    Science.gov (United States)

    Cerdà-Costa, Núria; Guevara, Tibisay; Karim, Abdulkarim Y; Ksiazek, Miroslaw; Nguyen, Ky-Anh; Arolas, Joan L; Potempa, Jan; Gomis-Rüth, F Xavier

    2011-01-01

    Metallopeptidases (MPs) are among virulence factors secreted by pathogenic bacteria at the site of infection. One such pathogen is Tannerella forsythia, a member of the microbial consortium that causes peridontitis, arguably the most prevalent infective chronic inflammatory disease known to mankind. The only reported MP secreted by T. forsythia is karilysin, a 52 kDa multidomain protein comprising a central 18 kDa catalytic domain (CD), termed Kly18, flanked by domains unrelated to any known protein. We analysed the 3D structure of Kly18 in the absence and presence of Mg(2+) or Ca(2+) , which are required for function and stability, and found that it evidences most of the structural features characteristic of the CDs of mammalian matrix metalloproteinases (MMPs). Unexpectedly, a peptide was bound to the active-site cleft of Kly18 mimicking a left-behind cleavage product, which revealed that the specificity pocket accommodates bulky hydrophobic side-chains of substrates as in mammalian MMPs. In addition, Kly18 displayed a unique Mg(2+) or Ca(2+) binding site and two flexible segments that could play a role in substrate binding. Phylogenetic and sequence similarity studies revealed that Kly18 is evolutionarily much closer to winged-insect and mammalian MMPs than to potential bacterial counterparts found by genomic sequencing projects. Therefore, we conclude that this first structurally characterized non-mammalian MMP is a xenologue co-opted through horizontal gene transfer during the intimate coexistence between T. forsythia and humans or other animals, in a very rare case of gene shuffling from eukaryotes to prokaryotes. Subsequently, this protein would have evolved in a bacterial environment to give rise to full-length karilysin that is furnished with unique flanking domains that do not conform to the general multidomain architecture of animal MMPs.

  9. Metalloproteinases and their regulators in colorectal cancer.

    Science.gov (United States)

    van der Jagt, Michel F P; Wobbes, Theo; Strobbe, Luc J A; Sweep, Fred C G J; Span, Paul N

    2010-03-01

    Metalloproteinases (MPs) such as the matrix metalloproteinases (MMPs) and adamalysins (ADAMs and ADAMTS) are expressed in various stages of colorectal cancer (CRC), and some correlate with survival and prognosis. The MPs are regulated by various factors including EMMPRIN, TIMPs, and RECK. In addition, micro-RNAs are found to be relevant for both MP expression levels and CRC prognostication. Both MPs and their regulators could be potential targets for intervention and therapy in CRC.

  10. Requirements for efficient proteolytic cleavage of prelamin A by ZMPSTE24.

    Science.gov (United States)

    Barrowman, Jemima; Hamblet, Corinne; Kane, Megan S; Michaelis, Susan

    2012-01-01

    The proteolytic maturation of the nuclear protein lamin A by the zinc metalloprotease ZMPSTE24 is critical for human health. The lamin A precursor, prelamin A, undergoes a multi-step maturation process that includes CAAX processing (farnesylation, proteolysis and carboxylmethylation of the C-terminal CAAX motif), followed by ZMPSTE24-mediated cleavage of the last 15 amino acids, including the modified C-terminus. Failure to cleave the prelamin A "tail", due to mutations in either prelamin A or ZMPSTE24, results in a permanently prenylated form of prelamin A that underlies the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS) and related progeroid disorders. Here we have investigated the features of the prelamin A substrate that are required for efficient cleavage by ZMPSTE24. We find that the C-terminal 41 amino acids of prelamin A contain sufficient context to allow cleavage of the tail by ZMPSTE24. We have identified several mutations in amino acids immediately surrounding the cleavage site (between Y646 and L647) that interfere with efficient cleavage of the prelamin A tail; these mutations include R644C, L648A and N650A, in addition to the previously reported L647R. Our data suggests that 9 of the 15 residues within the cleaved tail that lie immediately upstream of the CAAX motif are not critical for ZMPSTE24-mediated cleavage, as they can be replaced by the 9 amino acid HA epitope. However, duplication of the same 9 amino acids (to increase the distance between the prenyl group and the cleavage site) impairs the ability of ZMPSTE24 to cleave prelamin A. Our data reveals amino acid preferences flanking the ZMPSTE24 cleavage site of prelamin A and suggests that spacing from the farnesyl-cysteine to the cleavage site is important for optimal ZMPSTE24 cleavage. These studies begin to elucidate the substrate requirements of an enzyme activity critical to human health and longevity.

  11. Sequence specific inhibition of DNA restriction enzyme cleavage by PNA

    DEFF Research Database (Denmark)

    Nielsen, P.E.; Egholm, M.; Berg, R.H.

    1993-01-01

    Plasmids containing double-stranded 10-mer PNA (peptide nucleic acid chimera) targets proximally flanked by two restriction enzyme sites were challenged with the complementary PNA or PNAs having one or two mismatches, and the effect on the restriction enzyme cleavage of the flanking sites...... was assayed. The following PNAs were used: T10-LysNH2, T5CT4-LysNH2 and T2CT2CT4-LysNH2 and the corresponding targets cloned into pUC 19 were flanked by BamH1, Sal1 or Pstl sites, respectively. In all cases it was found that complete inhibition of restriction enzyme cleavage was obtained...

  12. A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing

    OpenAIRE

    Fukuda, Masatora; Kurihara, Kei; Tanaka, Yasuyoshi; Deshimaru, Masanobu

    2012-01-01

    Engineered site-specific RNA cleavage is widely used for gene regulation, RNA mapping, and synthetic RNA production. Here the authors extend the range of engineered recognition selectivity to include cleavage of sequence motifs containing naturally occurring base modifications. They describe and implement a designer hammerhead ribozyme that cleaves a target sequence 1 nt from a site of adenosine to inosine (A-to-I) or cytosine to uracil (C-to-U) editing in synthetic or physiological mRNA cont...

  13. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in gingival crevicular fluid during orthodontic tooth movement

    NARCIS (Netherlands)

    Bildt, Miriam; Bloemen, M; Kuijpers-Jagtman, A.M.; Von Den Hoff, Johannes W

    2009-01-01

    Orthodontic tooth movement requires extensive re-modelling of the periodontium. Matrix metalloproteinases (MMPs) degrade the extracellular matrix during re-modelling, while their activity is regulated by the tissue inhibitors of metalloproteinases (TIMPs). The aim of this study was to investigate di

  14. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in gingival crevicular fluid during orthodontic tooth movement

    NARCIS (Netherlands)

    Bildt, Miriam; Bloemen, M; Kuijpers-Jagtman, A.M.; Von Den Hoff, Johannes W

    2009-01-01

    Orthodontic tooth movement requires extensive re-modelling of the periodontium. Matrix metalloproteinases (MMPs) degrade the extracellular matrix during re-modelling, while their activity is regulated by the tissue inhibitors of metalloproteinases (TIMPs). The aim of this study was to investigate

  15. Structure of matrix metalloproteinase-3 with a platinum-based inhibitor.

    Science.gov (United States)

    Belviso, Benny Danilo; Caliandro, Rocco; Siliqi, Dritan; Calderone, Vito; Arnesano, Fabio; Natile, Giovanni

    2013-06-18

    An X-ray investigation has been performed with the aim of characterizing the binding sites of a platinum-based inhibitor (K[PtCl3(DMSO)]) of matrix metalloproteinase-3 (stromelysin-1). The platinum complex targets His224 in the S1' specificity loop, representing the first step in the selective inhibition process (PDB ID code 4JA1).

  16. Ammodytagin, a heterodimeric metalloproteinase from Vipera ammodytes ammodytes venom with strong hemorrhagic activity.

    Science.gov (United States)

    Kurtović, Tihana; Brgles, Marija; Leonardi, Adrijana; Balija, Maja Lang; Križaj, Igor; Allmaier, Günter; Marchetti-Deschmann, Martina; Halassy, Beata

    2011-11-01

    Ammodytagin, a hemorrhagic Zn(2+)-dependent metalloproteinase from Vipera ammodytes ammodytes (Vaa) venom, is a glycosylated heterodimer of 108 kDa, as determined by MALDI mass spectrometry. Partial amino acid sequencing by Edman degradation and MS/MS analysis identified sequences belonging to metalloproteinase, disintegrin-like and cysteine-rich domains, which in addition to its heterodimeric nature allows classification into the P-IIIc group of snake venom metalloproteinases (SVMPs). Only few members of that group have been described so far. Ammodytagin possesses potent azocaseinolytic activity which can be inhibited by Na(2)EDTA, Zn(2+) and DTT. It cleaves insulin B-chain, hydrolysing it at positions Gln(4)-His(5), His(10)-Leu(11) and Tyr(16)-Leu(17). Furthermore, ammodytagin acts as a strong hemorrhagin in both rats and mice. Investigation of a substrate specificity revealed that the hemorrhagic activity of the novel SVMP might be the result of its involvement in cleavage of basal membrane components and depletion of fibrinogen, prothrombin and factor X in blood circulation. Finally, antiserum raised against ammodytagin was able to completely neutralise the hemorrhagic activity of the whole venom, suggesting it might be one of the key molecules towards which effective Vaa specific antivenom should be directed. Copyright © 2011 Elsevier Ltd. All rights reserved.

  17. Triacontyl p-coumarate: an inhibitor of snake venom metalloproteinases.

    Science.gov (United States)

    Mendes, M M; Vieira, S A P B; Gomes, M S R; Paula, V F; Alcântara, T M; Homsi-Brandeburgo, M I; dos Santos, J I; Magro, A J; Fontes, M R M; Rodrigues, V M

    2013-02-01

    Snake venom metalloproteinases (SVMPs) participate in a number of important biological, physiological and pathophysiological processes and are primarily responsible for the local tissue damage characteristic of viperid snake envenomations. The use of medicinal plant extracts as antidotes against animal venoms is an old practice, especially against snake envenomations. Such plants are sources of many pharmacologically active compounds and have been shown to antagonize the effects of some venoms and toxins. The present study explores the activity of triacontyl p-coumarate (PCT), an active compound isolated from root bark of Bombacopsis glabra vegetal extract (Bg), against harmful effects of Bothropoides pauloensis snake venom and isolated toxins (SVMPs or phospholipase A(2)). Before inhibition assays, Bg or PCT was incubated with venom or toxins at ratios of 1:1 and 1:5 (w/w; venom or isolated toxins/PCT) for 30 min at 37°C. Treatment conditions were also assayed to simulate snakebite with PCT inoculated at either the same venom or toxin site. PCT neutralized fibrinogenolytic activity and plasmatic fibrinogen depletion induced by B. pauloensis venom or isolated toxin. PCT also efficiently inhibited the hemorrhagic (3MDH - minimum hemorrhagic dose injected i.d into mice) and myotoxic activities induced by Jararhagin, a metalloproteinase from B. jararaca at 1:5 ratio (toxin: inhibitor, w/w) when it was previously incubated with PCT and injected into mice or when PCT was administered after toxin injection. Docking simulations using data on a metalloproteinase (Neuwiedase) structure suggest that the binding between the protein and the inhibitor occurs mainly in the active site region causing blockade of the enzymatic reaction by displacement of catalytic water. Steric hindrance may also play a role in the mechanism since the PCT hydrophobic tail was found to interact with the loop associated with substrate anchorage. Thus, PCT may provide a alternative to complement

  18. Metalloproteinase activity secreted by fibrogenic cells in the processing of prolysyl oxidase. Potential role of procollagen C-proteinase.

    Science.gov (United States)

    Panchenko, M V; Stetler-Stevenson, W G; Trubetskoy, O V; Gacheru, S N; Kagan, H M

    1996-03-22

    Lysyl oxidase is secreted from fibrogenic cells as a 50-kDa proenzyme that is proteolytically processed to the mature enzyme in the extracellular space. To characterize the secreted proteinase activity, a truncated, recombinant form of lysyl oxidase was prepared as a proteinase substrate containing the sequence of the propeptide cleavage region. The processing proteinase activity secreted by cultured fibrogenic cells resists inhibitors of serine or aspartyl proteinases as well as tissue inhibitor of matrix metalloproteinases-2 (MMP-2) but is completely inhibited by metal ion chelators. Known metalloproteinases were tested for their activity toward this substrate. Carboxyl-terminal procollagen proteinase (C-proteinase), MMP-2, and conditioned fibrogenic cell culture medium cleave the lysyl oxidase substrate to the size of the mature enzyme. The NH2-terminal sequence generated by arterial smooth muscle conditioned medium and the C-proteinase but not by MMP-2, i.e. Asp-Asp-Pro-Tyr, was identical to that previously identified in mature lysyl oxidase isolated from connective tissue. The C-proteinase activity against the model substrate was inhibited by a synthetic oligopeptide mimic of the cleavage sequence (Ac-Met-Val-Gly-Asp-Asp-Pro-Tyr-Asn-amide), whereas this peptide also inhibited the generation of lysyl oxidase activity in the medium of fetal rat lung fibroblasts in culture. In toto, these results identify a secreted metalloproteinase activity participating in the activation of prolysyl oxidase, identify inhibitors of the processing activity, and implicate procollagen C-proteinase in this role.

  19. Key metalloproteinases are expressed by specific cell types in experimental autoimmune encephalomyelitis

    DEFF Research Database (Denmark)

    Toft-Hansen, Henrik; Nuttall, Robert K; Edwards, Dylan R

    2004-01-01

    Metalloproteinases (MPs) include matrix metalloproteinases (MMPs) and metalloproteinase-disintegrins (ADAMs). Their physiological inhibitors are tissue inhibitor of metalloproteinases (TIMPs). MPs are thought to be mediators of cellular infiltration in the pathogenesis of multiple sclerosis and its...

  20. Cloning and molecular characterization of BumaMPs1, a novel metalloproteinases from the venom of scorpion Buthus martensi Karsch.

    Science.gov (United States)

    Xia, Xichao; Ma, Yuhong; Xue, Shipeng; Wang, Aimei; Tao, Junliang; Zhao, Yan; Zhang, Qingyuan; Liu, Rongzhi; Lu, Shaoe

    2013-12-15

    Scorpion venoms metalloproteinase is involved in a number of important biological, physiological and pathophysiological processes. In this work, a complete sequence of metalloproteinase was first obtained from venom of scorpion Buthus martensi and named as BumaMPs1. BumaMPs1 has 393 amino acid residues containing with a molecular mass of 44.53 kDa, showing an isoelectric point of 5.66. The primary sequence analysis indicated that the BumaMPs1 contains a zinc-binding motif (HELGHNLGISH), methionine-turn motif (YIM), disintegrin-like domain (ETCD) and N-glycosylation site. The multiple alignment of its deduced amino acid sequence and those of other metalloproteinase showed a high structural similarly, mainly among class reprolysin proteases. The phylogenetic analysis showed early divergence and independent evolution of BumaMPs1 from other metalloproteinase.

  1. Establishment of cell lines stably co-expressing Japanese encephalitis virus prM and E protein with a mutant in prM furin cleavage site%流行性乙型脑炎prM蛋白剪切位点突变的prM-E蛋白表达细胞系的建立

    Institute of Scientific and Technical Information of China (English)

    李业南; 陈振师; 步志高; 华荣虹

    2013-01-01

    In order to establish the BHK-21 cell lines stably co-expressing prM-E fusion protein of Japanese encephalitis virus (JEV) with a mutant furin site to disable the pre-peptide cleavage of prM, BHK-21 cells was transfected with the recombinant plasmid pCAG-JEV-prM(R89A)E, which was constructed by introducing a point mutation at the cleavage site of prM gene by PCR and cloned into eukaryotic vector pCAG-neo with E gene. Cells stably co-expressing prM-E fusion protein were selected in the present of G418 and identified by indirect immunofluorescence assay and western blot, In addition, the cells displaying high-level protein expression were purified through limiting dilution cloning. Eventfully, the results showed that the cell line was able to express the intact prM of prM-E fusion protein. The established cell line would provide a basis for further study the effect of the cleavage event on mature mechanism of JEV and the development of JEV subunit vaccine.%为建立稳定共表达乙型脑炎病-毒(JEV)完整M前体蛋白(prM)和E蛋白的BHK-21细胞系,本研究在prM蛋白furin蛋白酶剪切位点编码基因中引入突变,将突变后的prM基因克隆于质粒中构建重组表达质粒pCAG-JEV-prM(R89A)E.将重组质粒转染BHK-21细胞,转染48 h后细胞用含G418的选择性培养基选择培养,进一步经细胞克隆纯化制备表达剪切位点突变的JEV prM-E蛋白的稳定细胞系.经IFA和western blot鉴定表明,该细胞系能表达JEV prM与E蛋白,所表达的prM蛋白未发生剪切;细胞经多次传代后仍能够稳定地共表达prM与E蛋白.该细胞系的建立为研究prM蛋白剪切对JEV粒子形成的影响以及亚单位疫苗的制备奠定了基础.

  2. Multiple roles of metalloproteinases in neurological disorders.

    Science.gov (United States)

    Yang, Yi; Hill, Jeff W; Rosenberg, Gary A

    2011-01-01

    Once thought to mainly act in brain to remodel the extracellular matrix, the family of metalloproteinases is important in many normal and pathological processes in the nervous system. Matrix metalloproteinases (MMPs) and A disintegrin and metalloproteinases (ADAMs) are the two major families of metalloproteinases in the brain. MMPs are comprised of several related enzymes that act on extracellular molecules. Normally, they are important in angiogenesis and neurogenesis in development. In neuroinflammatory illnesses, they disrupt the basal lamina and tight junction proteins to open the blood-brain barrier (BBB). ADAMs are important in neuroinflammation through activation of tumor necrosis factor-α (TNF-α) and their action as secretases that modulate the action of receptors on the cell surface. Four tissue inhibitors of metalloproteinases (TIMPs) are the main inhibitors of the MMPs and ADAMs. Recently, MMPs were found to affect DNA repair processes by an unexpected intranuclear action. MMPs and ADAMs have been implicated in the pathophysiology of neurodegenerative diseases such as Alzheimer's disease and vascular cognitive impairment. Growing literature on the functions of MMPs and ADAMs in the central nervous system is opening up new and exciting areas of research that may lead to novel approaches to treatment of neurological diseases. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Glutamic Acid Selective Chemical Cleavage of Peptide Bonds.

    Science.gov (United States)

    Nalbone, Joseph M; Lahankar, Neelam; Buissereth, Lyssa; Raj, Monika

    2016-03-04

    Site-specific hydrolysis of peptide bonds at glutamic acid under neutral aqueous conditions is reported. The method relies on the activation of the backbone amide chain at glutamic acid by the formation of a pyroglutamyl (pGlu) imide moiety. This activation increases the susceptibility of a peptide bond toward hydrolysis. The method is highly specific and demonstrates broad substrate scope including cleavage of various bioactive peptides with unnatural amino acid residues, which are unsuitable substrates for enzymatic hydrolysis.

  4. Mitochondria localize to the cleavage furrow in mammalian cytokinesis.

    Science.gov (United States)

    Lawrence, Elizabeth J; Mandato, Craig A

    2013-01-01

    Mitochondria are dynamic organelles with multiple cellular functions, including ATP production, calcium buffering, and lipid biosynthesis. Several studies have shown that mitochondrial positioning is regulated by the cytoskeleton during cell division in several eukaryotic systems. However, the distribution of mitochondria during mammalian cytokinesis and whether the distribution is regulated by the cytoskeleton has not been examined. Using live spinning disk confocal microscopy and quantitative analysis of mitochondrial fluorescence intensity, we demonstrate that mitochondria are recruited to the cleavage furrow during cytokinesis in HeLa cells. After anaphase onset, the mitochondria are recruited towards the site of cleavage furrow formation, where they remain enriched as the furrow ingresses and until cytokinesis completion. Furthermore, we show that recruitment of mitochondria to the furrow occurs in multiple mammalian cells lines as well as in monopolar, bipolar, and multipolar divisions, suggesting that the mechanism of recruitment is conserved and robust. Using inhibitors of cytoskeleton dynamics, we show that the microtubule cytoskeleton, but not actin, is required to transport mitochondria to the cleavage furrow. Thus, mitochondria are specifically recruited to the cleavage furrow in a microtubule-dependent manner during mammalian cytokinesis. Two possible reasons for this could be to localize mitochondrial function to the furrow to facilitate cytokinesis and / or ensure accurate mitochondrial inheritance.

  5. Hyperphosphorylation and cleavage at D421 enhance tau secretion.

    Directory of Open Access Journals (Sweden)

    Vanessa Plouffe

    Full Text Available It is well established that tau pathology propagates in a predictable manner in Alzheimer's disease (AD. Moreover, tau accumulates in the cerebrospinal fluid (CSF of AD's patients. The mechanisms underlying the propagation of tau pathology and its accumulation in the CSF remain to be elucidated. Recent studies have reported that human tau was secreted by neurons and non-neuronal cells when it was overexpressed indicating that tau secretion could contribute to the spreading of tau pathology in the brain and could lead to its accumulation in the CSF. In the present study, we showed that the overexpression of human tau resulted in its secretion by Hela cells. The main form of tau secreted by these cells was cleaved at the C-terminal. Surprisingly, secreted tau was dephosphorylated at several sites in comparison to intracellular tau which presented a strong immunoreactivity to all phospho-dependent antibodies tested. Our data also revealed that phosphorylation and cleavage of tau favored its secretion by Hela cells. Indeed, the mimicking of phosphorylation at 12 sites known to be phosphorylated in AD enhanced tau secretion. A mutant form of tau truncated at D421, the preferential cleavage site of caspase-3, was also significantly more secreted than wild-type tau. Taken together, our results indicate that hyperphosphorylation and cleavage of tau by favoring its secretion could contribute to the propagation of tau pathology in the brain and its accumulation in the CSF.

  6. A systematic study of modulation of ADAM-mediated ectodomain shedding by site-specific O-glycosylation

    DEFF Research Database (Denmark)

    Goth, Christoffer K; Halim, Adnan; Khetarpal, Sumeet A;

    2015-01-01

    -glycosylation is often found and examples of crosstalk between shedding and O-glycosylation have been reported. Here, we systematically investigated the potential of site-specific O-glycosylation mediated by distinct polypeptide GalNAc-transferase (GalNAc-T) isoforms to coregulate ectodomain shedding mediated...... by the A Disintegrin And Metalloproteinase (ADAM) subfamily of proteases and in particular ADAM17. We analyzed 25 membrane proteins that are known to undergo ADAM17 shedding and where the processing sites included Ser/Thr residues within ± 4 residues that could represent O-glycosites. We used in vitro GalNAc-T enzyme...... and ADAM cleavage assays to demonstrate that shedding of at least 12 of these proteins are potentially coregulated by O-glycosylation. Using TNF-α as an example, we confirmed that shedding mediated by ADAM17 is coregulated by O-glycosylation controlled by the GalNAc-T2 isoform both ex vivo in isogenic cell...

  7. The role of the methyltransferase domain of bifunctional restriction enzyme RM.BpuSI in cleavage activity.

    Directory of Open Access Journals (Sweden)

    Arthur Sarrade-Loucheur

    Full Text Available Restriction enzyme (REase RM.BpuSI can be described as a Type IIS/C/G REase for its cleavage site outside of the recognition sequence (Type IIS, bifunctional polypeptide possessing both methyltransferase (MTase and endonuclease activities (Type IIC and endonuclease activity stimulated by S-adenosyl-L-methionine (SAM (Type IIG. The stimulatory effect of SAM on cleavage activity presents a major paradox: a co-factor of the MTase activity that renders the substrate unsusceptible to cleavage enhances the cleavage activity. Here we show that the RM.BpuSI MTase activity modifies both cleavage substrate and product only when they are unmethylated. The MTase activity is, however, much lower than that of M1.BpuSI and is thought not to be the major MTase for host DNA protection. SAM and sinefungin (SIN increase the Vmax of the RM.BpuSI cleavage activity with a proportional change in Km, suggesting the presence of an energetically more favorable pathway is taken. We further showed that RM.BpuSI undergoes substantial conformational changes in the presence of Ca(2+, SIN, cleavage substrate and/or product. Distinct conformers are inferred as the pre-cleavage/cleavage state (in the presence of Ca(2+, substrate or both and MTase state (in the presence of SIN and substrate, SIN and product or product alone. Interestingly, RM.BpuSI adopts a unique conformation when only SIN is present. This SIN-bound state is inferred as a branch point for cleavage and MTase activity and an intermediate to an energetically favorable pathway for cleavage, probably through increasing the binding affinity of the substrate to the enzyme under cleavage conditions. Mutation of a SAM-binding residue resulted in altered conformational changes in the presence of substrate or Ca(2+ and eliminated cleavage activity. The present study underscores the role of the MTase domain as facilitator of efficient cleavage activity for RM.BpuSI.

  8. Matrix metalloproteinase-9-mediated type III collagen degradation as a novel serological biochemical marker for liver fibrogenesis

    DEFF Research Database (Denmark)

    Veidal, Sanne S; Vassiliadis, Efstathios; Barascuk, Natasha

    2010-01-01

    During fibrogenesis in the liver, in which excessive remodelling of the extracellular matrix (ECM) occurs, both the quantity of type III collagen (CO3) and levels of matrix metalloproteinases (MMPs), including MMP-9, increase significantly. MMPs play major roles in ECM remodelling, via their acti......During fibrogenesis in the liver, in which excessive remodelling of the extracellular matrix (ECM) occurs, both the quantity of type III collagen (CO3) and levels of matrix metalloproteinases (MMPs), including MMP-9, increase significantly. MMPs play major roles in ECM remodelling, via...... their activity in the proteolytic degradation of extracellular macromolecules such as collagens, resulting in the generation of specific cleavage fragments. These neo-epitopes may be used as markers of fibrosis....

  9. Matrix metalloproteinase-2 and tissue inhibitor of metallo-proteinase-2 in colorectal carcinoma invasion and metastasis

    OpenAIRE

    Li, Bing-hui; zhao,Peng; Liu, Shi-Zheng; Yu, Yue-Ming; Han, Mei; Wen, Jin-kun

    2005-01-01

    AIM: To explore the relationship between matrix metallopr-oteinase-2 (MMP-2) and tissue inhibitor of metallopr-oteinase-2 (TIMP-2) in the development of colorectal carcinoma and to provide a valuable marker for clinical diagnosis.

  10. Molecular determinants of survival motor neuron (SMN protein cleavage by the calcium-activated protease, calpain.

    Directory of Open Access Journals (Sweden)

    Jennifer L Fuentes

    Full Text Available Spinal muscular atrophy (SMA is a leading genetic cause of childhood mortality, caused by reduced levels of survival motor neuron (SMN protein. SMN functions as part of a large complex in the biogenesis of small nuclear ribonucleoproteins (snRNPs. It is not clear if defects in snRNP biogenesis cause SMA or if loss of some tissue-specific function causes disease. We recently demonstrated that the SMN complex localizes to the Z-discs of skeletal and cardiac muscle sarcomeres, and that SMN is a proteolytic target of calpain. Calpains are implicated in muscle and neurodegenerative disorders, although their relationship to SMA is unclear. Using mass spectrometry, we identified two adjacent calpain cleavage sites in SMN, S192 and F193. Deletion of small motifs in the region surrounding these sites inhibited cleavage. Patient-derived SMA mutations within SMN reduced calpain cleavage. SMN(D44V, reported to impair Gemin2 binding and amino-terminal SMN association, drastically inhibited cleavage, suggesting a role for these interactions in regulating calpain cleavage. Deletion of A188, a residue mutated in SMA type I (A188S, abrogated calpain cleavage, highlighting the importance of this region. Conversely, SMA mutations that interfere with self-oligomerization of SMN, Y272C and SMNΔ7, had no effect on cleavage. Removal of the recently-identified SMN degron (Δ268-294 resulted in increased calpain sensitivity, suggesting that the C-terminus of SMN is important in dictating availability of the cleavage site. Investigation into the spatial determinants of SMN cleavage revealed that endogenous calpains can cleave cytosolic, but not nuclear, SMN. Collectively, the results provide insight into a novel aspect of the post-translation regulation of SMN.

  11. Features of Pro-σK Important for Cleavage by SpoIVFB, an Intramembrane Metalloprotease

    Science.gov (United States)

    Chen, Kangming; Xiang, Xianling; Gu, Liping

    2013-01-01

    Intramembrane proteases regulate diverse processes by cleaving substrates within a transmembrane segment or near the membrane surface. Bacillus subtilis SpoIVFB is an intramembrane metalloprotease that cleaves Pro-σK during sporulation. To elucidate features of Pro-σK important for cleavage by SpoIVFB, coexpression of the two proteins in Escherichia coli was used along with cell fractionation. In the absence of SpoIVFB, a portion of the Pro-σK was peripherally membrane associated. This portion was not observed in the presence of SpoIVFB, suggesting that it serves as the substrate. Deletion of Pro-σK residues 2 to 8, addition of residues at its N terminus, or certain single-residue substitutions near the cleavage site impaired cleavage. Certain multiresidue substitutions near the cleavage site changed the position of cleavage, revealing preferences for a small residue preceding the cleavage site N-terminally (i.e., at the P1 position) and a hydrophobic residue at the second position following the cleavage site C-terminally (i.e., P2′). These features appear to be conserved among Pro-σK orthologs. SpoIVFB did not tolerate an aromatic residue at P1 or P2′ of Pro-σK. A Lys residue at P3′ of Pro-σK could not be replaced with Ala unless a Lys was provided farther C-terminally (e.g., at P9′). α-Helix-destabilizing residues near the cleavage site were not crucial for SpoIVFB to cleave Pro-σK. The preferences and tolerances of SpoIVFB are somewhat different from those of other intramembrane metalloproteases, perhaps reflecting differences in the interaction of the substrate with the membrane and the enzyme. PMID:23585539

  12. Serine-selective aerobic cleavage of peptides and a protein using a water-soluble copper-organoradical conjugate.

    Science.gov (United States)

    Seki, Yohei; Tanabe, Kana; Sasaki, Daisuke; Sohma, Youhei; Oisaki, Kounosuke; Kanai, Motomu

    2014-06-16

    The site-specific cleavage of peptide bonds is an important chemical modification of biologically relevant macromolecules. The reaction is not only used for routine structural determination of peptides, but is also a potential artificial modulator of protein function. Realizing the substrate scope beyond the conventional chemical or enzymatic cleavage of peptide bonds is, however, a formidable challenge. Here we report a serine-selective peptide-cleavage protocol that proceeds at room temperature and near neutral pH value, through mild aerobic oxidation promoted by a water-soluble copper-organoradical conjugate. The method is applicable to the site-selective cleavage of polypeptides that possess various functional groups. Peptides comprising D-amino acids or sensitive disulfide pairs are competent substrates. The system is extendable to the site-selective cleavage of a native protein, ubiquitin, which comprises more than 70 amino acid residues.

  13. Fracto—emissions in Catastrophic Cleavage Process

    Institute of Scientific and Technical Information of China (English)

    HonglaiTAN; WeiYANG

    1996-01-01

    Fracto-emissions accompanying crack propagation are observed in the recent experiments.The energy impulses during and after fracture stimulate the fracto-emissions.Model concerning atomic scale cleavage processes is proposed to formulate a catastrophic fracure theory relevant to these phenomena.A criterion for catastrophic jump of the cleavage potential is applied to representative crystals.

  14. Microstructure and cleavage in lath martensitic steels

    Directory of Open Access Journals (Sweden)

    John W Morris Jr, Chris Kinney, Ken Pytlewski and Y Adachi

    2013-01-01

    Full Text Available In this paper we discuss the microstructure of lath martensitic steels and the mechanisms by which it controls cleavage fracture. The specific experimental example is a 9Ni (9 wt% Ni steel annealed to have a large prior austenite grain size, then examined and tested in the as-quenched condition to produce a relatively coarse lath martensite. The microstructure is shown to approximate the recently identified 'classic' lath martensite structure: prior austenite grains are divided into packets, packets are subdivided into blocks, and blocks contain interleaved laths whose variants are the two Kurjumov–Sachs relations that share the same Bain axis of the transformation. When the steel is fractured in brittle cleavage, the laths in the block share {100} cleavage planes and cleave as a unit. However, cleavage cracks deflect or blunt at the boundaries between blocks with different Bain axes. It follows that, as predicted, the block size governs the effective grain size for cleavage.

  15. Cleavage of galectin-3 by matrix metalloproteases induces angiogenesis in breast cancer

    Science.gov (United States)

    Nangia-Makker, Pratima; Wang, Yi; Raz, Tirza; Tait, Larry; Balan, Vitaly; Hogan, Victor; Raz, Avraham

    2012-01-01

    Galectin-3 cleavage is related to progression of human breast and prostate cancer and is partly responsible for tumor growth, angiogenesis and apoptosis resistance in mouse models. A functional polymorphism in galectin-3 gene, determining its susceptibility to cleavage by matrix metalloproteinases (MMPs)-2/-9 is related to racial disparity in breast cancer incidence in Asian and Caucasian women. The purpose of our study is to evaluate (i) if cleavage of galectin-3 could be related to angiogenesis during the progression of human breast cancer, (ii) the role of cleaved galectin-3 in induction of angiogenesis and (iii) determination of the galectin-3 domain responsible for induction of angiogenic response. Galectin-3 null breast cancer cells BT-459 were transfected with either cleavable full-length galectin-3 or its fragmented peptides. Chemotaxis, chemoinvasion, heterotypic aggregation, epithelial-endothelial cell interactions and angiogenesis were compared to noncleavable galectin-3. BT-549-H64 cells harboring cleavable galectin-3 exhibited increased chemotaxis, invasion and interactions with endothelial cells resulting in angiogenesis and 3D morphogenesis compared to BT-549-P64 cells harboring noncleavable galectin-3. BT-549-H64 cells induced increased migration and phosphorylation of focal adhesion kinase in migrating endothelial cells. Endothelial cells cocultured with BT-549 cells transfected with galectin-3 peptides indicate that amino acids 1–62 and 33–250 stimulate migration and morphogenesis of endothelial cells. Immunohistochemical analysis of blood vessel density and galectin-3 cleavage in a breast cancer progression tissue array support the in vitro findings. We conclude that the cleavage of the N terminus of galectin-3 followed by its release in the tumor microenvironment in part leads to breast cancer angiogenesis and progression. PMID:20162566

  16. Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg-Carbon Bond Cleavage and Metal Binding Specificity.

    Science.gov (United States)

    Wahba, Haytham M; Lecoq, Lauriane; Stevenson, Michael; Mansour, Ahmed; Cappadocia, Laurent; Lafrance-Vanasse, Julien; Wilkinson, Kevin J; Sygusch, Jurgen; Wilcox, Dean E; Omichinski, James G

    2016-02-23

    In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL.

  17. Targets and intracellular signaling mechanisms for deoxynivalenol-induced ribosomal RNA cleavage.

    Science.gov (United States)

    He, Kaiyu; Zhou, Hui-Ren; Pestka, James J

    2012-06-01

    The trichothecene mycotoxin deoxynivalenol (DON), a known translational inhibitor, induces ribosomal RNA (rRNA) cleavage. Here, we characterized this process relative to (1) specific 18S and 28S ribosomal RNA cleavage sites and (2) identity of specific upstream signaling elements in this pathway. Capillary electrophoresis indicated that DON at concentrations as low as 200 ng/ml evoked selective rRNA cleavage after 6 h and that 1000 ng/ml caused cleavage within 2 h. Northern blot analysis revealed that DON exposure induced six rRNA cleavage fragments from 28S rRNA and five fragments from 18S rRNA. When selective kinase inhibitors were used to identify potential upstream signals, RNA-activated protein kinase (PKR), hematopoietic cell kinase (Hck), and p38 were found to be required for rRNA cleavage, whereas c-Jun N-terminal kinase and extracellular signal-regulated kinase were not. Furthermore, rRNA fragmentation was suppressed by the p53 inhibitors pifithrin-α and pifithrin-μ as well as the pan caspase inhibitor Z-VAD-FMK. Concurrent apoptosis was confirmed by acridine orange/ethidium bromide staining and flow cytometry. DON activated caspases 3, 8, and 9, thus suggesting the possible coinvolvement of both extrinsic and intrinsic apoptotic pathways in rRNA cleavage. Satratoxin G (SG), anisomycin, and ricin also induced specific rRNA cleavage profiles identical to those of DON, suggesting that ribotoxins might share a conserved rRNA cleavage mechanism. Taken together, DON-induced rRNA cleavage is likely to be closely linked to apoptosis activation and appears to involve the sequential activation of PKR/Hck →p38→p53→caspase 8/9→caspase 3.

  18. Matrix metalloproteinase-12 (MMP-12) in osteoclasts

    DEFF Research Database (Denmark)

    Hou, Peng; Troen, Tine; Ovejero, Maria C

    2004-01-01

    Osteoclasts require matrix metalloproteinase (MMP) activity and cathepsin K to resorb bone, but the critical MMP has not been identified. Osteoclasts express MMP-9 and MMP-14, which do not appear limiting for resorption, and the expression of additional MMPs is not clear. MMP-12, also called...

  19. Functional characterization of the cleavage specificity of the sapovirus chymotrypsin-like protease.

    Science.gov (United States)

    Robel, Ivonne; Gebhardt, Julia; Mesters, Jeroen R; Gorbalenya, Alexander; Coutard, Bruno; Canard, Bruno; Hilgenfeld, Rolf; Rohayem, Jacques

    2008-08-01

    Sapovirus is a positive-stranded RNA virus with a translational strategy based on processing of a polyprotein precursor by a chymotrypsin-like protease. So far, the molecular mechanisms regulating cleavage specificity of the viral protease are poorly understood. In this study, the catalytic activities and substrate specificities of the predicted forms of the viral protease, the 3C-like protease (NS6) and the 3CD-like protease-polymerase (NS6-7), were examined in vitro. The purified NS6 and NS6-7 were able to cleave synthetic peptides (15 to 17 residues) displaying the cleavage sites of the sapovirus polyprotein, both NS6 and NS6-7 proteins being active forms of the viral protease. High-performance liquid chromatography and subsequent mass spectrometry analysis of digested products showed a specific trans cleavage of peptides bearing Gln-Gly, Gln-Ala, Glu-Gly, Glu-Pro, or Glu-Lys at the scissile bond. In contrast, peptides bearing Glu-Ala or Gln-Asp at the scissile bond (NS4-NS5 and NS5-NS6, or NS6-NS7 junctions, respectively) were resistant to trans cleavage by NS6 or NS6-7 proteins, whereas cis cleavage of the Glu-Ala scissile bond of the NS5-NS6 junction was evidenced. Interestingly, the presence of a Phe at position P4 overruled the resistance to trans cleavage of the Glu-Ala junction (NS5-NS6), whereas substitutions at the P1 and P2' positions altered the cleavage efficiency. The differential cleavage observed is supported by a model of the substrate-binding site of the sapovirus protease, indicating that the P4, P1, and P2' positions in the substrate modulate the cleavage specificity and efficiency of the sapovirus chymotrypsin-like protease.

  20. Functional Characterization of the Cleavage Specificity of the Sapovirus Chymotrypsin-Like Protease▿ †

    Science.gov (United States)

    Robel, Ivonne; Gebhardt, Julia; Mesters, Jeroen R.; Gorbalenya, Alexander; Coutard, Bruno; Canard, Bruno; Hilgenfeld, Rolf; Rohayem, Jacques

    2008-01-01

    Sapovirus is a positive-stranded RNA virus with a translational strategy based on processing of a polyprotein precursor by a chymotrypsin-like protease. So far, the molecular mechanisms regulating cleavage specificity of the viral protease are poorly understood. In this study, the catalytic activities and substrate specificities of the predicted forms of the viral protease, the 3C-like protease (NS6) and the 3CD-like protease-polymerase (NS6-7), were examined in vitro. The purified NS6 and NS6-7 were able to cleave synthetic peptides (15 to 17 residues) displaying the cleavage sites of the sapovirus polyprotein, both NS6 and NS6-7 proteins being active forms of the viral protease. High-performance liquid chromatography and subsequent mass spectrometry analysis of digested products showed a specific trans cleavage of peptides bearing Gln-Gly, Gln-Ala, Glu-Gly, Glu-Pro, or Glu-Lys at the scissile bond. In contrast, peptides bearing Glu-Ala or Gln-Asp at the scissile bond (NS4-NS5 and NS5-NS6, or NS6-NS7 junctions, respectively) were resistant to trans cleavage by NS6 or NS6-7 proteins, whereas cis cleavage of the Glu-Ala scissile bond of the NS5-NS6 junction was evidenced. Interestingly, the presence of a Phe at position P4 overruled the resistance to trans cleavage of the Glu-Ala junction (NS5-NS6), whereas substitutions at the P1 and P2′ positions altered the cleavage efficiency. The differential cleavage observed is supported by a model of the substrate-binding site of the sapovirus protease, indicating that the P4, P1, and P2′ positions in the substrate modulate the cleavage specificity and efficiency of the sapovirus chymotrypsin-like protease. PMID:18550673

  1. Cleavage and polyadenylation: Ending the message expands gene regulation

    Science.gov (United States)

    Neve, Jonathan

    2017-01-01

    ABSTRACT Cleavage and polyadenylation (pA) is a fundamental step that is required for the maturation of primary protein encoding transcripts into functional mRNAs that can be exported from the nucleus and translated in the cytoplasm. 3′end processing is dependent on the assembly of a multiprotein processing complex on the pA signals that reside in the pre-mRNAs. Most eukaryotic genes have multiple pA signals, resulting in alternative cleavage and polyadenylation (APA), a widespread phenomenon that is important to establish cell state and cell type specific transcriptomes. Here, we review how pA sites are recognized and comprehensively summarize how APA is regulated and creates mRNA isoform profiles that are characteristic for cell types, tissues, cellular states and disease. PMID:28453393

  2. Ostensible enzyme promiscuity: alkene cleavage by peroxidases.

    Science.gov (United States)

    Mutti, Francesco G; Lara, Miguel; Kroutil, Markus; Kroutil, Wolfgang

    2010-12-17

    Enzyme promiscuity is generally accepted as the ability of an enzyme to catalyse alternate chemical reactions besides the 'natural' one. In this paper peroxidases were shown to catalyse the cleavage of a C=C double bond adjacent to an aromatic moiety for selected substrates at the expense of molecular oxygen at an acidic pH. It was clearly shown that the reaction occurs due to the presence of the enzyme; furthermore, the reactivity was clearly linked to the hemin moiety of the peroxidase. Comparison of the transformations catalysed by peroxidase and by hemin chloride revealed that these two reactions proceed equally fast; additional experiments confirmed that the peptide backbone was not obligatory for the reaction and only a single functional group of the enzyme was required, namely in this case the prosthetic group (hemin). Consequently, we propose to define such a promiscuous activity as 'ostensible enzyme promiscuity'. Thus, we call an activity that is catalysed by an enzyme 'ostensible enzyme promiscuity' if the reactivity can be tracked back to a single catalytic site, which on its own can already perform the reaction equally well in the absence of the peptide backbone.

  3. MMP2-cleavage of DMP1 generates a bioactive peptide promoting differentiation of dental pulp stem/progenitor cell

    Directory of Open Access Journals (Sweden)

    C Chaussain

    2009-11-01

    Full Text Available Dentin Matrix Protein 1 (DMP1 plays a regulatory role in dentin mineralization and can also function as a signaling molecule. MMP-2 (matrix metalloproteinase-2 is a predominant protease in the dentin matrix that plays a prominent role in tooth formation and a potential role during the carious process. The possibility that MMP-2 can cleave DMP1 to release biologically active peptides was investigated in this study. DMP1, both in the recombinant form and in its native state within the dentin matrix, was shown to be a substrate for MMP-2. Proteolytic processing of DMP1 by MMP-2 produced two major peptides, one that contains the C-terminal region of the protein known to carry both the ASARM (aspartic acid and serine rich domain domain involved in biomineralization and the DNA binding site of DMP1. In vitro experiments with recombinant N- and C-terminal polypeptides mimicking the MMP-2 cleavage products of DMP1 demonstrated an effect of the C-polypeptide on the differentiation of dental pulp stem/progenitor cells to a putative odontoblast phenotype. In vivo implantation of this peptide in a rat injured pulp model induced a rapid formation of a homogeneous dentin bridge covered by a palisade of orientated cells expressing dentin sialoprotein (DSP and DMP1, attesting an efficient repair process. These data suggest that a peptide generated through the proteolytic processing of DMP1 by MMP-2 can regulate the differentiation of mesenchymal cells during dentinogenesis and thus sustain reparative dentin formation in pathological situations such as carious decay. In addition, these data open a new therapeutic possibility of using this peptide to regenerate dentin after an injury.

  4. Indirect DNA Sequence Recognition and Its Impact on Nuclease Cleavage Activity.

    Science.gov (United States)

    Lambert, Abigail R; Hallinan, Jazmine P; Shen, Betty W; Chik, Jennifer K; Bolduc, Jill M; Kulshina, Nadia; Robins, Lori I; Kaiser, Brett K; Jarjour, Jordan; Havens, Kyle; Scharenberg, Andrew M; Stoddard, Barry L

    2016-06-07

    LAGLIDADG meganucleases are DNA cleaving enzymes used for genome engineering. While their cleavage specificity can be altered using several protein engineering and selection strategies, their overall targetability is limited by highly specific indirect recognition of the central four base pairs within their recognition sites. In order to examine the physical basis of indirect sequence recognition and to expand the number of such nucleases available for genome engineering, we have determined the target sites, DNA-bound structures, and central four cleavage fidelities of nine related enzymes. Subsequent crystallographic analyses of a meganuclease bound to two noncleavable target sites, each containing a single inactivating base pair substitution at its center, indicates that a localized slip of the mutated base pair causes a small change in the DNA backbone conformation that results in a loss of metal occupancy at one binding site, eliminating cleavage activity.

  5. Relationship between uterine expression of matrix metalloproteinases and their inhibitors and endometrial receptivity

    Institute of Scientific and Technical Information of China (English)

    高飞; 魏鹏; 陈鑫磊; 张志宏; 刘以训

    2002-01-01

    In order to investigate the relationship between the endometrial receptivity and matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1,-3 (TIMP-1,-3) in the endometrium, we used early pregnant mice as the animal model and studied the expression of MMP-2, TIMP-1,-3 in the endometrium in relation to the number of implantation sites after RU486 treatment. The results indicated that RU486 could significantly inhibit embryo implantation and change the expression of MMP-2 and TIMP-1,-3 in a dose-dependent pattern. When the mice were treated with 12 mg/kg RU486, there were a few embryos implanted as compared with the control. The expression of matrix metalloproteinase MMP-2 was low during the period of "implantation window", while the tissue inhibitor of metalloproteinase in the endometrial cells was high, suggesting that the activity of the proteolytic enzyme was strictly controlled by its inhibitors. After RU486 treatment, the generation of TIMP-1,3 was decreased while the MMP-2 was significantly increased, indicating that the normal balance between the activators and their inhibitors in the tissue was broken and the extracellular matrix was excessively degraded, subsequently the embryo implantation was inhibited. Therefore, it is suggested that the anti-implantation effect of RU486 may be mediated by MMPs and their inhibitors TIMPs.

  6. Expression and in vitro cleavage activity of anti-caspase-7 hammerhead ribozymes

    Institute of Scientific and Technical Information of China (English)

    Wei Zhang; Qing Xie; Xia-Qiu Zhou; Shan Jiang; You-Xin Jin

    2004-01-01

    AIM: To prepare hammerhead ribozymes against mouse caspase-7 and identify their cleavage activityin vitro, in order to select a ribozyme with specific cleavage activity against mouse caspase-7 as a potential gene therapy for apoptosis-related diseases.METHODS: Anti-caspase-7 ribozymes targeting sites 333and 394 (named Rz333 and Rz394) were designed by computer software, and their DNA sequences encoding ribozymes were synthesized. Caspase-7 DNA sequence was acquired by RT-PCR. Ribozymes and caspase-7 DNA obtained byin vitro transcription were cloned into pBSKneo U6' and pGEM-T vectors, respectively. The cleavage activity of ribozymes against mouse caspase-7 was identified by cleavage experimentsin vitro.RESULTS: Rz333 and Rz394 were designed and their DNA sequences were synthesized respectively. The expression vector of caspase-7 and plasmids containing Rz333 and Rz394 were reconstructed successfully. Ribozymes and caspase-7 mRNA were expressed byin vitro transcription.In vitro cleavage experiment showed that 243-nt and 744-nt segments were produced after caspase-7 mRNA was mixed with Rz333 in equivalent, and the cleavage efficiency was 67.98%. No cleaved segment was observed when caspase-7 mRNA was mixed with Rz394.CONCLUSION: Rz333 can site-specific cleave mouse caspase-7 mRNA, and it shows a potential for gene therapy of apoptosis-related diseases by down-regulating gene expression of caspase-7.

  7. Matrix Metalloproteinases During Axonal Regeneration, a Multifactorial Role from Start to Finish.

    Science.gov (United States)

    Andries, Lien; Van Hove, Inge; Moons, Lieve; De Groef, Lies

    2017-04-01

    By proteolytic cleavage, matrix metalloproteinases (MMPs) not only remodel the extracellular matrix (ECM) but they also modify the structure and activity of other proteinases, growth factors, signaling molecules, cell surface receptors, etc. Their vast substrate repertoire adds a complex extra dimension of biological control and turns MMPs into important regulatory nodes in the protease web. In the central nervous system (CNS), the detrimental impact of elevated MMP activities has been well-described for traumatic injuries and many neurodegenerative diseases. Nonetheless, there is ample proof corroborating MMPs as fine regulators of CNS physiology, and well-balanced MMP activity is instrumental to development, plasticity, and repair. In this manuscript, we review the emerging evidence for MMPs as beneficial modulators of axonal regeneration in the mammalian CNS. By exploring the multifactorial causes underlying the inability of mature axons to regenerate, and describing how MMPs can help to overcome these hurdles, we emphasize the benign actions of these Janus-faced proteases.

  8. Dentin matrix degradation by host Matrix Metalloproteinases: inhibition and clinical perspectives towards regeneration.

    Directory of Open Access Journals (Sweden)

    Catherine eChaussain

    2013-11-01

    Full Text Available Bacterial enzymes have long been considered solely accountable for the degradation of the dentin matrix during the carious process. However, the emerging literature suggests that host-derived enzymes, and in particular the matrix metalloproteinases (MMPs contained in dentin and saliva can play a major role in this process by their ability to degrade the dentin matrix from within. These findings are important since they open new therapeutic options for caries prevention and treatment. The possibility of using MMP inhibitors to interfere with dentin caries progression is discussed. Furthermore, the potential release of bioactive peptides by the enzymatic cleavage of dentin matrix proteins by MMPs during the carious process is discussed. These peptides, once identified, may constitute promising therapeutical tools for tooth and bone regeneration.

  9. Metalloproteinases: potential therapeutic targets for rheumatoid arthritis.

    Science.gov (United States)

    Itoh, Yoshifumi

    2015-01-01

    In different inflammatory diseases, many metalloproteinases are over expressed and thought to promote progression of the disease. Understanding roles of these enzymes in disease progression as well as in normal homeostasis is crucial to identify target enzymes for the disease. Rheumatoid arthritis (RA) is one of the autoimmune inflammatory diseases in which around 1-2 % of the world populations are suffered from. Roles of metalloproteinases are well documented in RA, but so far none of them is proposed to be a target enzyme. However, there are at least three enzymes that can potentially be molecular targets to inhibit progression of RA. Understanding roles of these enzymes in more detail and developing highly selective inhibitors to these enzymes would be essential for novel antimetalloproteinase therapies in future.

  10. Activation of matrix metalloproteinase-2 (MMP-2) by membrane type 1 matrix metalloproteinase through an artificial receptor for proMMP-2 generates active MMP-2.

    Science.gov (United States)

    Nishida, Yuki; Miyamori, Hisashi; Thompson, Erik W; Takino, Takahisa; Endo, Yoshio; Sato, Hiroshi

    2008-11-01

    The suggested model for pro-matrix metalloproteinase-2 (proMMP-2) activation by membrane type 1 MMP (MT1-MMP) implicates the complex between MT1-MMP and tissue inhibitor of MMP-2 (TIMP-2) as a receptor for proMMP-2. To dissect this model and assess the pathologic significance of MMP-2 activation, an artificial receptor for proMMP-2 was created by replacing the signal sequence of TIMP-2 with cytoplasmic/transmembrane domain of type II transmembrane mosaic serine protease (MSP-T2). Unlike TIMP-2, MSP-T2 served as a receptor for proMMP-2 without inhibiting MT1-MMP, and generated TIMP-2-free active MMP-2 even at a low level of MT1-MMP. Thus, MSP-T2 did not affect direct cleavage of the substrate testican-1 by MT1-MMP, whereas TIMP-2 inhibited it even at the level that stimulates proMMP-2 processing. Expression of MSP-T2 in HT1080 cells enhanced MMP-2 activation by endogenous MT1-MMP and caused intensive hydrolysis of collagen gel. Expression of MSP-T2 in U87 glioma cells, which express a trace level of endogenous MT1-MMP, induced MMP-2 activation and enhanced cell-associated protease activity, activation of extracellular signal-regulated kinase, and metastatic ability into chick embryonic liver and lung. MT1-MMP can exert both maximum MMP-2 activation and direct cleavage of substrates with MSP-T2, which cannot be achieved with TIMP-2. These results suggest that MMP-2 activation by MT1-MMP potentially amplifies protease activity, and combination with direct cleavage of substrate causes effective tissue degradation and enhances tumor invasion and metastasis, which highlights the complex role of TIMP-2. MSP-T2 is a unique tool to analyze physiologic and pathologic roles of MMP-2 and MT1-MMP in comparison with TIMP-2.

  11. Matrix metalloproteinases in impaired wound healing

    OpenAIRE

    auf dem Keller, Ulrich; Sabino,Fabio

    2015-01-01

    Fabio Sabino, Ulrich auf dem Keller Institute of Molecular Health Sciences, Eidgenössische Technische Hochschule (ETH) Zürich, Zürich, Switzerland Abstract: Cutaneous wound healing is a complex tissue response that requires a coordinated interplay of multiple cells in orchestrated biological processes to finally re-establish the skin's barrier function upon injury. Proteolytic enzymes and in particular matrix metalloproteinases (MMPs) contribute to all phas...

  12. Matrix metalloproteinases in exercise and obesity

    OpenAIRE

    Jaoude J; Koh Y

    2016-01-01

    Jonathan Jaoude,1 Yunsuk Koh2 1Department of Biology, 2Department of Health, Human Performance, and Recreation, Baylor University, Waco, TX, USA Abstract: Matrix metalloproteinases (MMPs) are zinc- and calcium-dependent endoproteinases that have the ability to break down extracellular matrix. The large range of MMPs’ functions widens their spectrum of potential role as activators or inhibitors in tissue remodeling, cardiovascular diseases, and obesity. In particular, MMP-1, -2, and ...

  13. Scandium(iii) triflate-promoted serine/threonine-selective peptide bond cleavage.

    Science.gov (United States)

    Ni, Jizhi; Sohma, Youhei; Kanai, Motomu

    2017-02-01

    The site-selective cleavage of peptide bonds is an important chemical modification that is useful not only for the structural determination of peptides, but also as an artificial modulator of peptide/protein function and properties. Here we report site-selective hydrolysis of peptide bonds at the Ser and Thr positions with a high conversion yield. This chemical cleavage relies on Sc(iii)-promoted N,O-acyl rearrangement and subsequent hydrolysis. The method is applicable to a broad scope of polypeptides with various functional groups, including a post-translationally modified peptide that is unsuitable for enzymatic hydrolysis. The system was further extended to site-selective cleavage of a native protein, Aβ1-42, which is closely related to the onset of Alzheimer's disease.

  14. Neutrophil activator of matrix metalloproteinase-2 (NAM).

    Science.gov (United States)

    Rollo, Ellen E; Hymowitz, Michelle; Schmidt, Cathleen E; Montana, Steve; Foda, Hussein; Zucker, Stanley

    2006-01-01

    We have isolated a novel soluble factor(s), neutrophil activator of matrix metalloproteinases (NAM), secreted by unstimulated normal human peripheral blood neutrophils that causes the activation of cell secreted promatrix metalloproteinase-2 (proMMP-2). Partially purified preparations of NAM have been isolated from the conditioned media of neutrophils employing gelatin-Sepharose chromatography and differential membrane filter centrifugation. NAM activity, as assessed by exposing primary human umbilical vein endothelial cells (HUVEC) or HT1080 cells to NAM followed by gelatin zymography, was seen within one hour. Tissue inhibitor of metalloproteinase-2 (TIMP-2) and hydroxamic acid derived inhibitors of MMPs (CT1746 and BB94) abrogated the activation of proMMP-2 by NAM, while inhibitors of serine and cysteine proteases showed no effect. NAM also produced an increase in TIMP-2 binding to HUVEC and HT1080 cell surfaces that was inhibited by TIMP-2, CT1746, and BB94. Time-dependent increases in MT1-MMP protein and mRNA were seen following the addition of NAM to cells. These data support a role for NAM in cancer dissemination.

  15. A metalloproteinase mirolysin of Tannerella forsythia inhibits all pathways of the complement system

    Science.gov (United States)

    Jusko, Monika; Potempa, Jan; Mizgalska, Danuta; Bielecka, Ewa; Ksiazek, Miroslaw; Riesbeck, Kristian; Garred, Peter; Eick, Sigrun; Blom, Anna M.

    2015-01-01

    Recent reports focusing on virulence factors of periodontal pathogens implicated proteinases as major determinants of remarkable pathogenicity of these species, with special emphasis on their capacity to modulate complement activity. In particular, bacteria-mediated cleavage of C5 and subsequent release of C5a seems to be an important phenomenon in the manipulation of the local inflammatory response in periodontitis. Here we present mirolysin, a novel metalloproteinase secreted by Tannerella forsythia, a well-recognized pathogen strongly associated with periodontitis. Mirolysin exhibited a strong effect on all complement pathways. It inhibited the classical and lectin complement pathways due to efficient degradation of mannose-binding lectin, ficolin-2, ficolin-3 and C4, while inhibition of the alternative pathway was caused by degradation of C5. This specificity toward complement largely resembled the activity of a previously characterized metalloproteinase of T. forsythia, karilysin. Interestingly, mirolysin released the biologically active C5a peptide in human plasma and induced migration of neutrophils. Importantly, we demonstrated that combination of mirolysin with karilysin, as well as a cysteine proteinase of another periodontal pathogen, Prevotella intermedia, resulted in a strong synergistic effect on complement. Furthermore, mutant strains of T. forsythia, devoid of either mirolysin or karilysin, showed diminished survival in human serum, providing further evidence for the synergistic inactivation of complement by these metalloproteinases. Taken together, our findings on interactions of mirolysin with complement significantly add to the understanding of immune evasion strategies of T. forsythia, and expand the knowledge on molecular mechanisms driving pathogenic events in the infected periodontium. PMID:26209620

  16. A Metalloproteinase Mirolysin of Tannerella forsythia Inhibits All Pathways of the Complement System.

    Science.gov (United States)

    Jusko, Monika; Potempa, Jan; Mizgalska, Danuta; Bielecka, Ewa; Ksiazek, Miroslaw; Riesbeck, Kristian; Garred, Peter; Eick, Sigrun; Blom, Anna M

    2015-09-01

    Recent reports focusing on virulence factors of periodontal pathogens implicated proteinases as major determinants of remarkable pathogenicity of these species, with special emphasis on their capacity to modulate complement activity. In particular, bacteria-mediated cleavage of C5 and subsequent release of C5a seems to be an important phenomenon in the manipulation of the local inflammatory response in periodontitis. In this study, we present mirolysin, a novel metalloproteinase secreted by Tannerella forsythia, a well-recognized pathogen strongly associated with periodontitis. Mirolysin exhibited a strong effect on all complement pathways. It inhibited the classical and lectin complement pathways due to efficient degradation of mannose-binding lectin, ficolin-2, ficolin-3, and C4, whereas inhibition of the alternative pathway was caused by degradation of C5. This specificity toward complement largely resembled the activity of a previously characterized metalloproteinase of T. forsythia, karilysin. Interestingly, mirolysin released the biologically active C5a peptide in human plasma and induced migration of neutrophils. Importantly, we demonstrated that combination of mirolysin with karilysin, as well as a cysteine proteinase of another periodontal pathogen, Prevotella intermedia, resulted in a strong synergistic effect on complement. Furthermore, mutant strains of T. forsythia, devoid of either mirolysin or karilysin, showed diminished survival in human serum, providing further evidence for the synergistic inactivation of complement by these metalloproteinases. Taken together, our findings on interactions of mirolysin with complement significantly add to the understanding of immune evasion strategies of T. forsythia and expand the knowledge on molecular mechanisms driving pathogenic events in the infected periodontium.

  17. Requirements for efficient proteolytic cleavage of prelamin A by ZMPSTE24.

    Directory of Open Access Journals (Sweden)

    Jemima Barrowman

    Full Text Available BACKGROUND: The proteolytic maturation of the nuclear protein lamin A by the zinc metalloprotease ZMPSTE24 is critical for human health. The lamin A precursor, prelamin A, undergoes a multi-step maturation process that includes CAAX processing (farnesylation, proteolysis and carboxylmethylation of the C-terminal CAAX motif, followed by ZMPSTE24-mediated cleavage of the last 15 amino acids, including the modified C-terminus. Failure to cleave the prelamin A "tail", due to mutations in either prelamin A or ZMPSTE24, results in a permanently prenylated form of prelamin A that underlies the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS and related progeroid disorders. METHODOLOGY/PRINCIPAL FINDINGS: Here we have investigated the features of the prelamin A substrate that are required for efficient cleavage by ZMPSTE24. We find that the C-terminal 41 amino acids of prelamin A contain sufficient context to allow cleavage of the tail by ZMPSTE24. We have identified several mutations in amino acids immediately surrounding the cleavage site (between Y646 and L647 that interfere with efficient cleavage of the prelamin A tail; these mutations include R644C, L648A and N650A, in addition to the previously reported L647R. Our data suggests that 9 of the 15 residues within the cleaved tail that lie immediately upstream of the CAAX motif are not critical for ZMPSTE24-mediated cleavage, as they can be replaced by the 9 amino acid HA epitope. However, duplication of the same 9 amino acids (to increase the distance between the prenyl group and the cleavage site impairs the ability of ZMPSTE24 to cleave prelamin A. CONCLUSIONS/SIGNIFICANCE: Our data reveals amino acid preferences flanking the ZMPSTE24 cleavage site of prelamin A and suggests that spacing from the farnesyl-cysteine to the cleavage site is important for optimal ZMPSTE24 cleavage. These studies begin to elucidate the substrate requirements of an enzyme activity critical to human

  18. Functionally Expression of Metalloproteinase in Taenia solium Metacestode and Its Evaluation for Serodiagnosis of Cysticercosis

    Science.gov (United States)

    ZHANG, Ying; BAE, Young-An; ZONG, Hong-Ying; KONG, Yoon; CAI, Guo-Bin

    2016-01-01

    Background: Parasite proteases have important roles in cleavage of host proteins during the invasion of host tissues and participate in the parasite’s evasion from the host’s immune response. The aim of the present study was to estimate a metalloproteinase properties of Taenia solium metacestode (TsMP) during host-parasite interactions, and evaluate its potential as a serodiagnostic antigen for cysticercosis. Methods: The cDNA coding for the mature catalytic domain of TsMP was cloned into pGEX-6P-1 expression vector. A recombinant glutathione S-transferase and TsMP fusion protein was induced. After refolding and purification, enzymatic properties of the recombinant metalloproteinase were observed. Immunoblot assay was processed to evaluate its potential as a serodiagnostic antigen for cysticercosis. Results: The recombinant TsMP protein showed proteolytic activity, which preferred host extracellular matrix proteins such as collagen and fibronectin as degradable substrates. In immunoblot assay, 87.5% of sera from patients with cysticercosis showed strong reactivity. In sera from patients with other parasitic infections and from normal controls, it showed high specificity. Conclusions: TsMP might be involved in the processing of numerous host proteins and play an important role in the parasite life cycle. A single recombinant TsMP antigen could have a potential value for serodiagnosis of cysticercosis. PMID:27095967

  19. Trichonomas vaginalis metalloproteinase induces apoptosis of SiHa cells through disrupting the Mcl-1/Bim and Bcl-xL/Bim complexes.

    Science.gov (United States)

    Quan, Juan-Hua; Kang, Byung-Hun; Cha, Guang-Ho; Zhou, Wei; Koh, Young-Bok; Yang, Jung-Bo; Yoo, Heon-Jong; Lee, Min-A; Ryu, Jae-Sook; Noh, Heung-Tae; Kwon, Jaeyul; Lee, Young-Ha

    2014-01-01

    To elucidate the roles of metalloproteinases and the Bcl-2 family of proteins in Trichovaginalis. vaginalis-induced apoptosis in human cervical cancer cells (SiHa cells) and vaginal epithelial cells (MS74 cells), SiHa cells and MS74 cells were incubated with live T. vaginalis, T. vaginalis excretory and secretory products (ESP), and T. vaginalis lysates, either with or without the specific metalloproteinase inhibitor 1,10-phenanthroline (1,10-PT), and examined apoptotic events and Bcl-2 signaling. The live T. vaginalis and the T. vaginalis ESP induced the release of cytochrome c into the cytosol, the activation of caspase-3 and caspase-9, and the cleavage of PARP. Additionally, the live T. vaginalis, but not the T. vaginalis lysate, induced the cleavage of the proapoptotic Bim protein. The live T. vaginalis and the T. vaginalis ESP, but not the T. vaginalis lysate, induced the dose-dependent cleavage of the antiapoptotic Bcl-xL and Mcl-1 proteins and decreased the association levels of Bcl-xL/Bim and Mcl-1/Bim complexes. We performed gelatin zymography and casein-hydrolysis assays on the live T. vaginalis and the T. vaginalis ESP to identify the apoptosis-inducing factor. Both the live T. vaginalis and the ESP contained high levels of metalloproteinases, of which activities were significantly inhibited by 1,10-PT treatment. Furthermore, the 1,10-PT blocked the cleavage of Bcl-xL, Mcl-1, PARP, caspase-3, and caspase-9, as well as the release of cytochrome c into the cytosol, and it significantly increased the association levels of the Bcl-xL/Bim and Mcl-1/Bim protein complexes, returning them to normal levels. Our results demonstrate that T. vaginalis induces mitochondria-dependent apoptosis in SiHa cells through the dissociation of Bcl-xL/Bim and Mcl-1/Bim complexes and that the apoptosis is blocked by the metalloproteinase inhibitor 1,10-PT. These results expand our understanding of the role of metalloproteinases in T. vaginalis-induced apoptosis and the signaling

  20. Trichonomas vaginalis metalloproteinase induces apoptosis of SiHa cells through disrupting the Mcl-1/Bim and Bcl-xL/Bim complexes.

    Directory of Open Access Journals (Sweden)

    Juan-Hua Quan

    Full Text Available To elucidate the roles of metalloproteinases and the Bcl-2 family of proteins in Trichovaginalis. vaginalis-induced apoptosis in human cervical cancer cells (SiHa cells and vaginal epithelial cells (MS74 cells, SiHa cells and MS74 cells were incubated with live T. vaginalis, T. vaginalis excretory and secretory products (ESP, and T. vaginalis lysates, either with or without the specific metalloproteinase inhibitor 1,10-phenanthroline (1,10-PT, and examined apoptotic events and Bcl-2 signaling. The live T. vaginalis and the T. vaginalis ESP induced the release of cytochrome c into the cytosol, the activation of caspase-3 and caspase-9, and the cleavage of PARP. Additionally, the live T. vaginalis, but not the T. vaginalis lysate, induced the cleavage of the proapoptotic Bim protein. The live T. vaginalis and the T. vaginalis ESP, but not the T. vaginalis lysate, induced the dose-dependent cleavage of the antiapoptotic Bcl-xL and Mcl-1 proteins and decreased the association levels of Bcl-xL/Bim and Mcl-1/Bim complexes. We performed gelatin zymography and casein-hydrolysis assays on the live T. vaginalis and the T. vaginalis ESP to identify the apoptosis-inducing factor. Both the live T. vaginalis and the ESP contained high levels of metalloproteinases, of which activities were significantly inhibited by 1,10-PT treatment. Furthermore, the 1,10-PT blocked the cleavage of Bcl-xL, Mcl-1, PARP, caspase-3, and caspase-9, as well as the release of cytochrome c into the cytosol, and it significantly increased the association levels of the Bcl-xL/Bim and Mcl-1/Bim protein complexes, returning them to normal levels. Our results demonstrate that T. vaginalis induces mitochondria-dependent apoptosis in SiHa cells through the dissociation of Bcl-xL/Bim and Mcl-1/Bim complexes and that the apoptosis is blocked by the metalloproteinase inhibitor 1,10-PT. These results expand our understanding of the role of metalloproteinases in T. vaginalis-induced apoptosis

  1. Cleavage of desmin by cysteine proteases: Calpains and cathepsin B

    DEFF Research Database (Denmark)

    Baron, Caroline; Jacobsen, S.; Purslow, P.P.

    2004-01-01

    The intermediate filament protein, desmin, was purified from pork longissimus dorsi and incubated with either P-calpain, m-calpain or cathepsin B. Proteolysis of desmin was followed using SDS-PAGE and Western blotting. After incubation of desmin with the proteases, cleavage sites on the desmin...... a sequential C-terminal degradation pattern characteristic of this dipeptylpeptidase. The substrate primary structure was not found to be essential for regulation of the proteolytic activity of the cysteine peptidases studied. However, the degradation patterns obtained imply that calpains are involved...

  2. The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

    Science.gov (United States)

    Hilbert, Brendan J.; Hayes, Janelle A.; Stone, Nicholas P.; Xu, Rui-Gang

    2017-01-01

    Abstract Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA. PMID:28082398

  3. Differential cleavage of the norovirus polyprotein precursor by two active forms of the viral protease.

    Science.gov (United States)

    Scheffler, Ulrike; Rudolph, Wolfram; Gebhardt, Julia; Rohayem, Jacques

    2007-07-01

    Protein translation in noroviruses requires translational processing of a polyprotein precursor by the viral protease. So far, the molecular mechanisms of catalytic cleavage by the viral protease are poorly understood. In this study, the catalytic activities and substrate specificities of the viral protease were examined in vitro by using synthetic peptides (11-15 residues) corresponding to the cleavage sites of the norovirus polyprotein. Both predicted forms of the viral protease, the 3C-like protease (3C(pro)) and the 3CD-like protease polymerase protein (3CD(propol)), displayed a specific trans cleavage activity of peptides bearing Gln-Gly at the scissile bond. In contrast, peptides bearing Glu-Gly at the scissile bond (p20/VPg and 3C(pro)/3D(pol) junctions) were resistant to trans-cleavage by 3C(pro) and 3CD(propol). Interestingly, the VPg/3C(pro) scissile bond (Glu-Ala) was cleaved only by 3CD(propol), and examination of relative cleavage efficiencies revealed significant differences in processing of peptides, indicating differential cleavage patterns for 3C(pro) and 3CD(propol).

  4. Glycoprotein B cleavage is important for murid herpesvirus 4 to infect myeloid cells.

    Science.gov (United States)

    Glauser, Daniel L; Milho, Ricardo; Frederico, Bruno; May, Janet S; Kratz, Anne-Sophie; Gillet, Laurent; Stevenson, Philip G

    2013-10-01

    Glycoprotein B (gB) is a conserved herpesvirus virion component implicated in membrane fusion. As with many-but not all-herpesviruses, the gB of murid herpesvirus 4 (MuHV-4) is cleaved into disulfide-linked subunits, apparently by furin. Preventing gB cleavage for some herpesviruses causes minor infection deficits in vitro, but what the cleavage contributes to host colonization has been unclear. To address this, we mutated the furin cleavage site (R-R-K-R) of the MuHV-4 gB. Abolishing gB cleavage did not affect its expression levels, glycosylation, or antigenic conformation. In vitro, mutant viruses entered fibroblasts and epithelial cells normally but had a significant entry deficit in myeloid cells such as macrophages and bone marrow-derived dendritic cells. The deficit in myeloid cells was not due to reduced virion binding or endocytosis, suggesting that gB cleavage promotes infection at a postendocytic entry step, presumably viral membrane fusion. In vivo, viruses lacking gB cleavage showed reduced lytic spread in the lungs. Alveolar epithelial cell infection was normal, but alveolar macrophage infection was significantly reduced. Normal long-term latency in lymphoid tissue was established nonetheless.

  5. Predicting proteasomal cleavage sites: a comparison of available methods

    DEFF Research Database (Denmark)

    Saxova, P.; Buus, S.; Brunak, Søren

    2003-01-01

    The proteasome plays an essential role in the immune responses of vertebrates. By degrading intercellular proteins from self and non-self, the proteasome produces the majority of the peptides that are presented to cytotoxic T cells (CTL). There is accumulating evidence that the C......-terminal, in particular, of CTL epitopes is cleaved precisely by the proteasome, whereas the N-terminal is produced with an extension, and later trimmed by peptidases in the cytoplasm and in the endoplasmic reticulum. Recently, three publicly available methods have been developed for prediction of the specificity...... of the proteasome. Here, we compare the performance of these methods on a large set of CTL epitopes. The best method, NetChop at www.cbs.dtu.dk/Services/NetChop, can capture similar to70% of the C-termini correctly. This result suggests that the predictions can still be improved, particularly if more quantitative...

  6. Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

    Science.gov (United States)

    2016-09-07

    modification or cleavages of transcripts through various RNA editing mechanisms , including siRNA targeted mRNA cleavages. Currently, 5′-RACE (rapid ampli... transcription polymerase chain reaction (SLA-RT-PCR) approach to detect and verify the siRNA- mediated target mRNA cleavage sites by determining precise...Reverse Transcription kit (Life Technologies) in combination with an array of stem-loop RT primers. The 20 ml of RT reaction con- tained 50 ng of total RNA

  7. Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs using PICS with proteome-derived peptide libraries.

    Directory of Open Access Journals (Sweden)

    Olivier Barré

    Full Text Available BACKGROUND: Type II transmembrane serine proteases (TTSPs are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors. METHODOLOGY/PRINCIPAL FINDING: To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS. Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin to simultaneously determine sequence preferences on the N-terminal non-prime (P and C-terminal prime (P' sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1' position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived. CONCLUSIONS: Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1' positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.

  8. Synthesis and enzymatic cleavage of dual-ligand quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Sewell, Sarah L. [Department of Biomedical Engineering, Vanderbilt University, Nashville, TN (United States); Giorgio, Todd D., E-mail: todd.d.giorgio@vanderbilt.edu [Department of Biomedical Engineering, Vanderbilt University, Nashville, TN (United States); Department of Chemical and Biomolecular Engineering, Vanderbilt University, Nashville, TN (United States)

    2009-05-05

    Site directed therapy promises to minimize treatment-limiting systemic effects associated with cytotoxic agents that have no specificity for pathologic tissues. One general strategy is to target cell surface receptors uniquely presented on particular tissues. Highly specific in vivo targeting of an emerging neoplasm through a single molecular recognition mechanism has not generally been successful. Nonspecific binding and specific binding to non-target cells compromise the therapeutic index of small molecule, ubiquitous cancer targeting ligands. In this work, we have designed and fabricated a nanoparticle (NP) construct that could potentially overcome the current limitations of targeted in vivo delivery. Quantum dots (QDs) were functionalized with a poly(ethylene glycol) (PEG) modified to enable specific cleavage by matrix metalloprotease-7 (MMP-7). The QDs were further functionalized with folic acid, a ligand for a cell surface receptor that is overexpressed in many tumors, but also expressed in some normal tissues. The nanomolecular construct is designed so that the PEG initially conceals the folate ligand and construct binding to cells is inhibited. MMP-7 activated peptide cleavage and subsequent unmasking of the folate ligand occurs only near tumor tissue, resulting in a proximity activated (PA) targeting system. QDs functionalized with both the MMP-7 cleavable substrate and folic acid were successfully synthesized and characterized. The proteolytic capability of the dual ligand QD construct was quantitatively assessed by fluorometric analysis and compared to a QD construct functionalized with only the PA ligand. The dual ligand PA nanoparticles studied here exhibit significant susceptibility to cleavage by MMP-7 at physiologically relevant conditions. The capacity to autonomously convert a biopassivated nanostructure to a tissue-specific targeted delivery agent in vivo represents a paradigm change for site-directed therapies.

  9. Multiple-turnover cleavage of double-stranded DNA by sandwiched zinc-finger nuclease.

    Science.gov (United States)

    Mineta, Yusuke; Okamoto, Tomoyuki; Takenaka, Kosuke; Doi, Norio; Aoyama, Yasuhiro; Sera, Takashi

    2009-01-01

    To refine zinc-finger nuclease (ZFN) technology, we constructed a sandwiched ZFN, in which a DNA cleavage enzyme was sandwiched with two artificial zinc-finger proteins (AZPs). Because the sandwiched ZFN is designed to cleave the DNA between the two AZP-binding sites, the sandwiched ZFN is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To prove the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two 3-finger AZPs. The AZP-sandwiched SNase cleaved large amounts of dsDNA site-specifically. Such multiple-turnover cleavage was not observed with control nucleases that possess a single AZP.

  10. IRE1α nucleotide sequence cleavage specificity in the unfolded protein response.

    Science.gov (United States)

    Poothong, Juthakorn; Sopha, Pattarawut; Kaufman, Randal J; Tirasophon, Witoon

    2017-01-01

    Inositol-requiring enzyme 1 (IRE1) is a conserved sensor of the unfolded protein response that has protein kinase and endoribonuclease (RNase) enzymatic activities and thereby initiates HAC1/XBP1 splicing. Previous studies demonstrated that human IRE1α (hIRE1α) does not cleave Saccharomyces cerevisiae HAC1 mRNA. Using an in vitro cleavage assay, we show that adenine to cytosine nucleotide substitution at the +1 position in the 3' splice site of HAC1 RNA is required for specific cleavage by hIRE1α. A similar restricted nucleotide specificity in the RNA substrate was observed for XBP1 splicing in vivo. Together these findings underscore the essential role of cytosine nucleotide at +1 in the 3' splice site for determining cleavage specificity of hIRE1α.

  11. Cleavage-induced termination in U2 snRNA gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Nabavi, Sadeq [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (Canada); Nazar, Ross N., E-mail: rnnazar@uoguelph.ca [Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1 (Canada)

    2010-03-12

    The maturation of many small nuclear RNAs is dependent on RNase III-like endonuclease mediated cleavage, which generates a loading site for the exosome complex that trims the precursor at its 3' end. Using a temperature sensitive Pac1 nuclease, here we show that the endonuclease cleavage is equally important in terminating the transcription of the U2 snRNA in Schizosaccharomyces pombe. Using a temperature sensitive Dhp1p 5' {yields} 3' exonuclease, we demonstrate that it also is an essential component of the termination pathway. Taken together the results support a 'reversed torpedoes' model for the termination and maturation of the U2 snRNA; the Pac1 endonuclease cleavage provides entry sites for the 3' and 5' exonuclease activities, leading to RNA maturation in one direction and transcript termination in the other.

  12. Functional proteomics on zinc-dependent metalloproteinases using inhibitor probes

    NARCIS (Netherlands)

    Klein, Theo; Geurink, Paul P.; Overkleeft, Hermen S.; Kauffman, Henk K.; Bischoff, Rainer

    2009-01-01

    Metzincins ore a family of zinc(II)-dependent metolloproteinases with well known members such as the matrix metalloproteinases (MMPs) and A disintegrin and metalloproteinases (ADAMs). Metzincins are largely responsible for the modulation and regulation of the extracellular matrix by proteolytic degr

  13. Functional proteomics on zinc-dependent metalloproteinases using inhibitor probes

    NARCIS (Netherlands)

    Klein, Theo; Geurink, Paul P.; Overkleeft, Hermen S.; Kauffman, Henk K.; Bischoff, Rainer

    Metzincins ore a family of zinc(II)-dependent metolloproteinases with well known members such as the matrix metalloproteinases (MMPs) and A disintegrin and metalloproteinases (ADAMs). Metzincins are largely responsible for the modulation and regulation of the extracellular matrix by proteolytic

  14. [Structural properties and functional importance of metzincin metalloproteinases].

    Science.gov (United States)

    Balaban, N P; Rudakova, N L; Sharipova, M R

    2013-01-01

    Here wediscuss known properties of metzincin metalloproteinases, their structure, physiological roles in the cell and potential medical uses. We also present results describing a novel extracellular metzincin metalloproteinase from Bacillus pumilus with a unique combination of properties typical for both astacins and adamalysins.

  15. Tumorigenic Potential of Extracellular Matrix Metalloproteinase Inducer

    Science.gov (United States)

    Zucker, Stanley; Hymowitz, Michelle; Rollo, Ellen E.; Mann, Richard; Conner, Cathleen E.; Cao, Jian; Foda, Hussein D.; Tompkins, David C.; Toole, Bryan P.

    2001-01-01

    Extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein present on the cancer cell plasma membrane, enhances fibroblast synthesis of matrix metalloproteinases (MMPs). The demonstration that peritumoral fibroblasts synthesize most of the MMPs in human tumors rather than the cancer cells themselves has ignited interest in the role of EMMPRIN in tumor dissemination. In this report we have demonstrated a role for EMMPRIN in cancer progression. Human MDA-MB-436 breast cancer cells, which are tumorigenic but slow growing in vivo, were transfected with EMMPRIN cDNA and injected orthotopically into mammary tissue of female NCr nu/nu mice. Green fluorescent protein was used to visualize metastases. In three experiments, breast cancer cell clones transfected with EMMPRIN cDNA were considerably more tumorigenic and invasive than plasmid-transfected cancer cells. Increased gelatinase A and gelatinase B expression (demonstrated by in situ hybridization and gelatin substrate zymography) was demonstrated in EMMPRIN-enhanced tumors. In contrast to de novo breast cancers in humans, human tumors transplanted into mice elicited minimal stromal or inflammatory cell reactions. Based on these experimental studies and our previous demonstration that EMMPRIN is prominently displayed in human cancer tissue, we propose that EMMPRIN plays an important role in cancer progression by increasing synthesis of MMPs. PMID:11395366

  16. Complex role of matrix metalloproteinases in angiogenesis

    Institute of Scientific and Technical Information of China (English)

    SANGQINGXIANGAMY

    1998-01-01

    Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) play a significant role in regulating angiogenesis,the process of new blood vessel formation.Interstitial collagenase (MMP-1),72kDa gelatinase A/type IV collagenase (MMP-2),and 92 kDA gelatinase B/type IV collagenase (MMP-9) dissolve extracellular matrix (ECM) and may initiate and promote angiogenesis.TIMP-1,TIMP-2,TIMP-3,and possibly,TIMP-4 inhibit neovascularization.A new paradign is emerging that matrilysin (MMP-7),MMP-9,and metalloelastase (MMP-12) may block angiogenesis by converting plasminogen to angiostatin,which is one of the most potent angiogenesis antagonists.MMPs and TIMPs play a complex role in regulating angiogenesis.An understanding of the biochemical and cellular pathways and mechanisms of angiogenesis will provide important information to allow the control of angiogenesis,e.g.the stimulation of angiogenesis for coronary collateral circulation formation;while the inhibition for treating arthritis and cancer.

  17. Matrix metalloproteinase gene polymorphisms and oral cancer.

    Science.gov (United States)

    Pereira, Andresa C; Dias do Carmo, Elaine; Dias da Silva, Marco A; Blumer Rosa, Luiz E

    2012-12-01

    Since oral squamous cell carcinoma (OSCC) is the most prevalent malignant cancer in the oral cavity, several researches have been performed to study the role of important enzymes in this disease. Among them, the matrix metalloproteinases (MMPs) are highlighted, due to the fact that they are proteinases responsible to degrade many extra-cellular matrix components, making possible the invasion of neoplasic cells. Important tools in cancer prognosis have been utilized aiming to correlate high levels of MMPs and OSCC, such as immunohistochemical, zymographic and mRNA detection methods. However, these techniques are usually applied after cancer detection, characterizing a curative but not a preventive medicine. Trying to make interventions before the development of the disease and making possible the identification of people at high risk and, analysis of modifications in MMP genes has been a chance for modern medicine. Recently, polymorphisms in MMP genes have been related to different neoplasias, including OSCC. Despite investigation is beginning, MMP gene polymorphisms seems to have a promising future in oral cancer research and some of the present results have shown that there are MMP polymorphisms related to an increased risk for developing oral cancer. Key words:Oral cancer, polymorphism, matrix metalloproteinase.

  18. Enhanced cleavage of double-stranded DNA by artificial zinc-finger nuclease sandwiched between two zinc-finger proteins.

    Science.gov (United States)

    Mineta, Yusuke; Okamoto, Tomoyuki; Takenaka, Kosuke; Doi, Norio; Aoyama, Yasuhiro; Sera, Takashi

    2008-11-25

    To enhance DNA cleavage by zinc-finger nucleases (ZFNs), we sandwiched a DNA cleavage enzyme with two artificial zinc-finger proteins (AZPs). Because the DNA between the two AZP-binding sites is cleaved, the AZP-sandwiched nuclease is expected to bind preferentially to a DNA substrate rather than to cleavage products and thereby cleave it with multiple turnovers. To demonstrate the concept, we sandwiched a staphylococcal nuclease (SNase), which cleaves DNA as a monomer, between two three-finger AZPs. The AZP-sandwiched SNase cleaved large amounts of dsDNA site-specifically. Such multiple-turnover cleavage was not observed with nucleases that possess a single AZP. Thus, AZP-sandwiched nucleases will further refine ZFN technology.

  19. A new dress code for MMPs: cleavage optional.

    Science.gov (United States)

    Fingleton, Barbara; Lynch, Conor C

    2010-01-19

    Matrix metalloproteinases are well known for their key roles in the processing of extracellular matrix and nonmatrix molecules. Recently, a new theme of proteolysis-independent roles for metalloproteinases has emerged, with a paper from Gonzalo et al. in this issue of Developmental Cell providing an illustration. (c) 2010 Elsevier Inc. All rights reserved.

  20. Characterization of insulin-degrading enzyme-mediated cleavage of Aβ in distinct aggregation states.

    Science.gov (United States)

    Hubin, Ellen; Cioffi, Federica; Rozenski, Jef; van Nuland, Nico A J; Broersen, Kerensa

    2016-06-01

    To enhance our understanding of the potential therapeutic utility of insulin-degrading enzyme (IDE) in Alzheimer's disease (AD), we studied in vitro IDE-mediated degradation of different amyloid-beta (Aβ) peptide aggregation states. Our findings show that IDE activity is driven by the dynamic equilibrium between Aβ monomers and higher ordered aggregates. We identify Met(35)-Val(36) as a novel IDE cleavage site in the Aβ sequence and show that Aβ fragments resulting from IDE cleavage form non-toxic amorphous aggregates. These findings need to be taken into account in therapeutic strategies designed to increase Aβ clearance in AD patients by modulating IDE activity.

  1. Mutation G805R in the transmembrane domain of the LDL receptor gene causes familial hypercholesterolemia by inducing ectodomain cleavage of the LDL receptor in the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Thea Bismo Strøm

    2014-01-01

    Full Text Available More than 1700 mutations in the low density lipoprotein receptor (LDLR gene have been found to cause familial hypercholesterolemia (FH. These are commonly divided into five classes based upon their effects on the structure and function of the LDLR. However, little is known about the mechanism by which mutations in the transmembrane domain of the LDLR gene cause FH. We have studied how the transmembrane mutation G805R affects the function of the LDLR. Based upon Western blot analyses of transfected HepG2 cells, mutation G805R reduced the amounts of the 120 kDa precursor LDLR in the endoplasmic reticulum. This led to reduced amounts of the mature 160 kDa LDLR at the cell surface. However, significant amounts of a secreted 140 kDa G805R-LDLR ectodomain fragment was observed in the culture media. Treatment of the cells with the metalloproteinase inhibitor batimastat largely restored the amounts of the 120 and 160 kDa forms in cell lysates, and prevented secretion of the 140 kDa ectodomain fragment. Together, these data indicate that a metalloproteinase cleaved the ectodomain of the 120 kDa precursor G805R-LDLR in the endoplasmic reticulum. It was the presence of the polar Arg805 and not the lack of Gly805 which led to ectodomain cleavage. Arg805 also prevented γ-secretase cleavage within the transmembrane domain. It is conceivable that introducing a charged residue within the hydrophobic membrane lipid bilayer, results in less efficient incorporation of the 120 kDa G805R-LDLR in the endoplasmic reticulum membrane and makes it a substrate for metalloproteinase cleavage.

  2. Cleavage mediated by the catalytic domain of bacterial RNase P RNA.

    Science.gov (United States)

    Wu, Shiying; Kikovska, Ema; Lindell, Magnus; Kirsebom, Leif A

    2012-09-14

    Like other RNA molecules, RNase P RNA (RPR) is composed of domains, and these have different functions. Here, we provide data demonstrating that the catalytic (C) domain of Escherichia coli (Eco) RPR when separated from the specificity (S) domain mediates cleavage using various model RNA hairpin loop substrates. Compared to full-length Eco RPR, the rate constant, k(obs), of cleavage for the truncated RPR (CP RPR) was reduced 30- to 13,000-fold depending on substrate. Specifically, the structural architecture of the -1/+73 played a significant role where a C(-1)/G(+73) pair had the most dramatic effect on k(obs). Substitution of A(248) (E. coli numbering), positioned near the cleavage site in the RNase P-substrate complex, with G in the CP RPR resulted in 30-fold improvement in rate. In contrast, strengthening the interaction between the RPR and the 3' end of the substrate only had a modest effect. Interestingly, although deleting the S-domain gave a reduction in the rate, it resulted in a less erroneous RPR with respect to cleavage site selection. These data support and extend our understanding of the coupling between the distal interaction between the S-domain and events at the active site. Our findings will also be discussed with respect to the structure of RPR derived from different organisms.

  3. Matrix Metalloproteinases Expression in Choroidal Neovascular Membranes

    Institute of Scientific and Technical Information of China (English)

    Jun Zeng; Deyong Jiang; Xiangping Liu; Xiaohua Zhu; Luosheng Tang

    2004-01-01

    Purpose: To investigate the expression of matrix metalloproteinases (MMPs) in choroidal neovascular membranes with age-related macular degeneration (AMD).Methods: Seventeen choroidal neovascular membranes surgically removed from AMD patients with pars plana vitrectomy and subretinal membranes peeling were investigated.The expression of MMP-2 and MMP-9 was determined with immunohistochemical technique.Results: Immunohistochemistry staining in choroidal neovascular membranes for MMP2 and MMP-9 was observed in 17 specimens. There was no detective of MMP-2 and MMP-9 in normal retinas.Conclusions: MMP-2 and MMP-9 were found in choroidal neovascular membranes, may degrade the Bruch membrane and be associated with the perforation of new vessels into Bruch membrane, involving a basic pathogenic process of AMD.

  4. Matrix metalloproteinase-12 (MMP-12) in osteoclasts

    DEFF Research Database (Denmark)

    Hou, Peng; Troen, Tine; Ovejero, Maria C

    2004-01-01

    and resorption. Furthermore, we investigated the role of MMP-12 in bone resorption and osteoclast recruitment by comparing MMP-12 knockout and wild-type mice in specialized culture models known to depend on MMP activity, as well as in the ovariectomy model, and we did not find any indication for a limiting role......Osteoclasts require matrix metalloproteinase (MMP) activity and cathepsin K to resorb bone, but the critical MMP has not been identified. Osteoclasts express MMP-9 and MMP-14, which do not appear limiting for resorption, and the expression of additional MMPs is not clear. MMP-12, also called....... Northern blots show that highly purified rabbit osteoclasts in culture express MMP-12 at the same level as macrophages, whereas in situ hybridizations performed on rabbit bone do not show any MMP-12 expression in osteoclasts whatever the bone type. In contrast, in situ hybridizations performed on mouse...

  5. Role of metalloproteinases in rotator cuff tear.

    Science.gov (United States)

    Garofalo, Raffaele; Cesari, Eugenio; Vinci, Enzo; Castagna, Alessandro

    2011-09-01

    The role of matrix metalloproteinases (MMPs) and their inhibitors (TIMPS) in the pathophysiology of rotator cuff tears has not been established yet. Recent advances empathize about the role of MMPs and TIMPS in extracellular matrix (ECM) remodeling and degradation in rotator cuff tears pathogenesis and healing after surgical repair. An increase in MMPs synthesis and the resulting MMPs mediated alterations in the ECM of tendons have been implicated in the etiopathogenesis of tendinopathy, and there is an increase in the expression of MMPs and a decrease in TIMP messenger ribonucleic acid expression in tenocytes from degenerative or ruptured tendons. Importantly, MMPs are amenable to inhibition by cheap, safe, and widely available drugs such as the tetracycline antibiotics and bisphosphonates. A better understanding of relationship and activity of these molecules could provide better strategies to optimize outcomes of rotator cuff therapy.

  6. Matrix Metalloproteinases as Regulators of Periodontal Inflammation

    Science.gov (United States)

    Franco, Cavalla; Patricia, Hernández-Ríos; Timo, Sorsa; Claudia, Biguetti; Marcela, Hernández

    2017-01-01

    Periodontitis are infectious diseases characterized by immune-mediated destruction of periodontal supporting tissues and tooth loss. Matrix metalloproteinases (MMPs) are key proteases involved in destructive periodontal diseases. The study and interest in MMP has been fuelled by emerging evidence demonstrating the broad spectrum of molecules that can be cleaved by them and the myriad of biological processes that they can potentially regulate. The huge complexity of MMP functions within the ‘protease web’ is crucial for many physiologic and pathologic processes, including immunity, inflammation, bone resorption, and wound healing. Evidence points out that MMPs assemble in activation cascades and besides their classical extracellular matrix substrates, they cleave several signalling molecules—such as cytokines, chemokines, and growth factors, among others—regulating their biological functions and/or bioavailability during periodontal diseases. In this review, we provide an overview of emerging evidence of MMPs as regulators of periodontal inflammation. PMID:28218665

  7. Regioselectivity in the Reductive Bond Cleavage of Diarylalkylsulfonium Salts

    DEFF Research Database (Denmark)

    Kampmeier, Jack; Mansurul Hoque, AKM; D. Saeva, Franklin;

    2009-01-01

    This investigation was stimulated by reports that one-electron reductions of monoaryldialkylsulfonium salts never give aryl bond cleavage whereas reductions of diarylmonoalkylsulfonium salts preferentially give aryl bond cleavage. We studied the product ratios from the reductive cleavage of di-4-...

  8. Global identification of target recognition and cleavage by the Microprocessor in human ES cells.

    Science.gov (United States)

    Seong, Youngmo; Lim, Do-Hwan; Kim, Augustine; Seo, Jae Hong; Lee, Young Sik; Song, Hoseok; Kwon, Young-Soo

    2014-11-10

    The Microprocessor plays an essential role in canonical miRNA biogenesis by facilitating cleavage of stem-loop structures in primary transcripts to yield pre-miRNAs. Although miRNA biogenesis has been extensively studied through biochemical and molecular genetic approaches, it has yet to be addressed to what extent the current miRNA biogenesis models hold true in intact cells. To address the issues of in vivo recognition and cleavage by the Microprocessor, we investigate RNAs that are associated with DGCR8 and Drosha by using immunoprecipitation coupled with next-generation sequencing. Here, we present global protein-RNA interactions with unprecedented sensitivity and specificity. Our data indicate that precursors of canonical miRNAs and miRNA-like hairpins are the major substrates of the Microprocessor. As a result of specific enrichment of nascent cleavage products, we are able to pinpoint the Microprocessor-mediated cleavage sites per se at single-nucleotide resolution. Unexpectedly, a 2-nt 3' overhang invariably exists at the ends of cleaved bases instead of nascent pre-miRNAs. Besides canonical miRNA precursors, we find that two novel miRNA-like structures embedded in mRNAs are cleaved to yield pre-miRNA-like hairpins, uncoupled from miRNA maturation. Our data provide a framework for in vivo Microprocessor-mediated cleavage and a foundation for experimental and computational studies on miRNA biogenesis in living cells.

  9. The cleavage of phosphoenolpyruvate by vanadate.

    Science.gov (United States)

    Aureliano, M; Leta, J; Madeira, V M; de Meis, L

    1994-05-30

    Vanadate rapidly promotes the cleavage of phosphoenolpyruvate with phosphate liberation. This was not observed when ATP, glucose-6-phosphate and acetyl phosphate were incubated with vanadate. 51V NMR spectra shows that phosphoenolpyruvate and acetyl phosphate broadened and shifted upfield the monomeric vanadate signal at -561 ppm, indicative of vanadate/phosphate interactions. Comparatively, smaller changes were detected when glucose-6-phosphate was added to the vanadate solution. The shift behavior was not observed in the presence of ATP, ADP or pyruvate.

  10. Identification of Sequence CCACA as a Recognition Motif of Mitochondrial Transcripts Orf355-orf77 Cleavage Sites in CMS-S Maize%CCACA碱基序列是玉米CMS-S线粒体orf355-orf77转录本剪切识别位点

    Institute of Scientific and Technical Information of China (English)

    秦雅婷; 赵衍鑫; 岳兵; 肖海林

    2013-01-01

    玉米S型细胞质雄性不育系(CMS-S)及其近等基因恢复系是研究核-质互作机制的重要遗传资源和理想模式体系.目前认为,CMS-S花粉败育是由其线粒体内细胞质不育基因orf355-orf77表达的毒性蛋白引起,而核育性恢复基因f3可通过引发orf355-orf77转录本的降解而解除其毒性作用,使花粉育性得以恢复.本研究采用Northern杂交和3′RACE技术确定了orf355-orf77转录本的剪切位点,并发现在育性恢复的花粉中,orf355-orf77转录本被剪切成小片段之后聚合了poly(A)序列,推测这一过程加速了mRNA分子的降解,是育性恢复的关键环节.利用生物信息学方法分析了orf355-orf77转录本6个剪切位点的侧翼序列,发现在剪切位点下游10个碱基的位置均含有5′-CCACA-3′序列,推测该序列受到特定功能蛋白的识别,然后募集核酸内切酶对其进行剪切.研究结果可为揭示玉米CMS-S育性恢复机理提供重要的理论依据.%S-type cytoplasmic male sterility line (CMS-S) and its near isogenic restorer lines of maize are suitable materials for the study on nucleus-mitochondria transcript interactions.The toxicity product coded by orf355-orf77 is considered to be potentially inducible in CMS-S.Its fertility restorer,Rf3 gene can induce the degradation and removal of orf355-orf77 transcripts of its toxic effect,and protect the development of microspores in normal status.In this study,the cleaving sites of orf355-orf77 transcript was determined by Northern blotting and 3 RACE.We found that most of the small cleaved fragments from accelerated RNA degradation.were polyadenylated.Through bioinformatic analysis of the six flanking sequences of cleaved sites of orf355-orf77 transcripts,a conserved recognition pattern of CCACA was identified at the 10 nucleotides downstream of the cleavage sites.This sequence was predicted to be recognizable by a specific protein to cleave the RNAs by recruiting endonuclease

  11. Kinetic analysis of the inhibition of matrix metalloproteinases: lessons from the study of tissue inhibitors of metalloproteinases.

    Science.gov (United States)

    Willenbrock, Frances; Thomas, Daniel A; Amour, Augustin

    2010-01-01

    Tissue inhibitors of metalloproteinases (TIMPs) are a group of highly potent inhibitors of matrix metalloproteinases (MMPs) and disintegrin metalloproteinases (ADAMs). The high affinity and "tight-binding" nature of the inhibition of MMPs or ADAMs by TIMPs presents challenges for the determination of both equilibrium and dissociation rate constants of these inhibitory events. Methodologies that enable some of these challenges to be overcome are described in this chapter and represent valuable lessons for the in vitro assessment of MMP or ADAM inhibitors within a drug discovery context.

  12. Cobalt (III) complexes as novel matrix metalloproteinase-9 inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jiyoun [Sungshin Women' s Univ., Seoul (Korea, Republic of)

    2012-04-15

    We have synthesized a series of novel MMP-9 inhibitors containing cobalt(III) complexes. The synthesized cobalt(III) complexes are effective as enzyme inhibitors and the attachment of a biphenyl group enhanced the efficiency of enzyme inhibition up to 6-fold. When compared to the reported non-hydroxamate MMP inhibitors, the synthesized complexes showed comparable in vitro potency. The enzyme assay showed that the cobalt(III) complex can disrupt the zinc binding active site of MMP-9 and is proposed to work via a ligand exchange mechanism. Since histidine residues are essential for the catalytic activity of a large percentage of enzymes and zinc finger proteins, these cobalt(III) complexes can serve as a prototype inhibitor towards various zinc containing enzymes and proteins. Matrix metalloproteinases (MMPs) are a family of zinc binding endopeptidases that play crucial roles in various physiological processes and diseases such as embryogenic growth, angiogenesis, arthritis, skin ulceration, liver fibrosis and tumor metastasis. Because of their implications in a wide range of diseases, MMPs are considered as intriguing drug targets. The majority of MMP inhibitors are organic small molecules containing a hydroxamate functionality for the zinc binding group. This hydroxamate group binds to a zinc(II) center in a bidentate fashion and creates a distorted trigonal bipyramidal geometry.

  13. Photoinduced cleavage by a rhodium complex at G.U mismatches and exposed guanines in large and small RNAs.

    Science.gov (United States)

    Chow, Christine S; Cunningham, Philip R; Lee, KangSeok; Meroueh, May; SantaLucia, John; Varma, Shikha

    2002-09-01

    Photoinduced cleavage reactions by the rhodium complex tris(4,7-diphenyl-1,10-phenanthroline)rhodium(III) [Rh(DIP)(3)(3+)] with three RNA hairpins, r(GGGGU UCGCUC CACCA) (16 nucleotide, tetraloop(Ala2)), r(GGGGCUAUAGCUCUAGCUC CACCA) (24 nucleotide, microhelix(Ala)), and r(GGCGGUUAGAUAUCGCC) (17 nucleotide, 790 loop), and full-length (1542 nucleotide) 16S rRNA from Escherichia coli were investigated. The cleavage reactions were monitored by gel electrophoresis and the sites of cleavage by Rh(DIP)(3)(3+) were determined by comparisons with chemical or enzymatic sequencing reactions. In general, RNA backbone scission by the metal complex was induced at G.U mismatches and at exposed G residues. The cleavage activity was observed on the three small RNA hairpins as well as on the isolated 1542-nucleotide ribosomal RNA.

  14. Cleavage crystallography of liquid metal embrittled aluminum alloys

    Science.gov (United States)

    Reynolds, A. P.; Stoner, G. E.

    1991-01-01

    The crystallography of liquid metal-induced transgranular cleavage in six aluminum alloys having a variety of microstructures has been determined via Laue X-ray back reflection. The cleavage crystallography was independent of alloy microstructure, and the cleavage plane was 100-plane oriented in all cases. It was further determined that the cleavage crystallography was not influenced by alloy texture. Examination of the fracture surface indicated that there was not a unique direction of crack propagation. In addition, the existence of 100-plane cleavage on alloy 2024 fracture surfaces was inferred by comparison of secondary cleavage crack intersection geometry on the 2024 surfaces with the geometry of secondary cleavage crack intersections on the test alloys.

  15. Peptide-Based Selective Inhibitors of Matrix Metalloproteinase-Mediated Activities

    Directory of Open Access Journals (Sweden)

    Margaret W. Ndinguri

    2012-11-01

    Full Text Available The matrix metalloproteinases (MMPs exhibit a broad array of activities, some catalytic and some non-catalytic in nature. An overall lack of selectivity has rendered small molecule, active site targeted MMP inhibitors problematic in execution. Inhibitors that favor few or individual members of the MMP family often take advantage of interactions outside the enzyme active site. We presently focus on peptide-based MMP inhibitors and probes that do not incorporate conventional Zn2+ binding groups. In some cases, these inhibitors and probes function by binding only secondary binding sites (exosites, while others bind both exosites and the active site. A myriad of MMP mediated-activities beyond selective catalysis can be inhibited by peptides, particularly cell adhesion, proliferation, motility, and invasion. Selective MMP binding peptides comprise highly customizable, unique imaging agents. Areas of needed improvement for MMP targeting peptides include binding affinity and stability.

  16. Structural differences of matrix metalloproteinases. Homology modeling and energy minimization of enzyme-substrate complexes

    DEFF Research Database (Denmark)

    Terp, G E; Christensen, I T; Jørgensen, Flemming Steen

    2000-01-01

    in the AMBER 4.1 force field, applying a non-bonded approach and a full ion charge representation. Energy minimization of the apoenzymes yielded structures with distorted active sites, while reliable three-dimensional structures of the enzymes containing a substrate in active site were obtained. The structural...... differences between the eight enzyme-substrate complexes were studied with particular emphasis on the active site, and possible sites for obtaining selectivity among the MMP's are discussed. Differences in the P1' pocket are well-documented and have been extensively exploited in inhibitor design. The present......Matrix metalloproteinases are extracellular enzymes taking part in the remodeling of extracellular matrix. The structures of the catalytic domain of MMP1, MMP3, MMP7 and MMP8 are known, but structures of enzymes belonging to this family still remain to be determined. A general approach...

  17. Reappraising metalloproteinases in rheumatoid arthritis and osteoarthritis: destruction or repair?

    Science.gov (United States)

    Murphy, Gillian; Nagase, Hideaki

    2008-03-01

    Metalloproteinases such as the matrix metalloproteinases (MMPs) and disintegrin-metalloproteinases with thrombospondin motifs (ADAMTSs) have been implicated in the pathological destruction of joint tissues in rheumatoid arthritis and osteoarthritis. These enzymes degrade extracellular matrix macromolecules and modulate factors governing cell behavior. They may also be involved in tissue repair, but become a part of the destructive disease process due to overexpression. Studies investigating the roles of metalloproteinases have thrown light on the failure of the early clinical trials of MMP inhibitors as therapeutic agents in arthritic diseases. It is now clear that a more accurate knowledge of the enzymes in the different cells and their precise roles in the disease process is required for these approaches to be successful. The next generations of metalloproteinase inhibitors should have added specificity, gained from an understanding not only of the catalytic domain structures but the role of extracatalytic motifs in substrate binding, or by the generation of engineered tissue inhibitors of metalloproteinases. Inhibition of the enzymes by modulating gene expression or preventing protein activation could also be considered. Work on the development of effective biomarkers is also essential before an effective evaluation of the new generations of specific inhibitors can be made.

  18. Matrix Metalloproteinase-9 and Haemozoin: Wedding Rings for Human Host and Plasmodium falciparum Parasite in Complicated Malaria

    Directory of Open Access Journals (Sweden)

    Mauro Prato

    2011-01-01

    Full Text Available It is generally accepted that the combination of both Plasmodium falciparum parasite and human host factors is involved in the pathogenesis of complicated severe malaria, including cerebral malaria (CM. Among parasite products, the malarial pigment haemozoin (HZ has been shown to impair the functions of mononuclear and endothelial cells. Different CM models were associated with enhanced levels of matrix metalloproteinases (MMPs, a family of proteolytic enzymes able to disrupt subendothelial basement membrane and tight junctions and shed, activate, or inactivate cytokines, chemokines, and other MMPs through cleavage from their precursors. Among MMPs, a good candidate for targeted therapy might be MMP-9, whose mRNA and protein expression enhancement as well as direct proenzyme activation by HZ have been recently investigated in a series of studies by our group and others. In the present paper the role of HZ and MMP-9 in complicated malaria, as well as their interactions, will be discussed.

  19. Transcriptional down-regulation and rRNA cleavage in Dictyostelium discoideum mitochondria during Legionella pneumophila infection.

    Directory of Open Access Journals (Sweden)

    Chenyu Zhang

    Full Text Available Bacterial pathogens employ a variety of survival strategies when they invade eukaryotic cells. The amoeba Dictyostelium discoideum is used as a model host to study the pathogenic mechanisms that Legionella pneumophila, the causative agent of Legionnaire's disease, uses to kill eukaryotic cells. Here we show that the infection of D. discoideum by L. pneumophila results in a decrease in mitochondrial messenger RNAs, beginning more than 8 hours prior to detectable host cell death. These changes can be mimicked by hydrogen peroxide treatment, but not by other cytotoxic agents. The mitochondrial large subunit ribosomal RNA (LSU rRNA is also cleaved at three specific sites during the course of infection. Two LSU rRNA fragments appear first, followed by smaller fragments produced by additional cleavage events. The initial LSU rRNA cleavage site is predicted to be on the surface of the large subunit of the mitochondrial ribosome, while two secondary sites map to the predicted interface with the small subunit. No LSU rRNA cleavage was observed after exposure of D. discoideum to hydrogen peroxide, or other cytotoxic chemicals that kill cells in a variety of ways. Functional L. pneumophila type II and type IV secretion systems are required for the cleavage, establishing a correlation between the pathogenesis of L. pneumophila and D. discoideum LSU rRNA destruction. LSU rRNA cleavage was not observed in L. pneumophila infections of Acanthamoeba castellanii or human U937 cells, suggesting that L. pneumophila uses distinct mechanisms to interrupt metabolism in different hosts. Thus, L. pneumophila infection of D. discoideum results in dramatic decrease of mitochondrial RNAs, and in the specific cleavage of mitochondrial rRNA. The predicted location of the cleavage sites on the mitochondrial ribosome suggests that rRNA destruction is initiated by a specific sequence of events. These findings suggest that L. pneumophila specifically disrupts mitochondrial

  20. Computational analysis and modeling of cleavage by the immunoproteasome and the constitutive proteasome

    Directory of Open Access Journals (Sweden)

    Lafuente Esther M

    2010-09-01

    Full Text Available Abstract Background Proteasomes play a central role in the major histocompatibility class I (MHCI antigen processing pathway. They conduct the proteolytic degradation of proteins in the cytosol, generating the C-terminus of CD8 T cell epitopes and MHCI-peptide ligands (P1 residue of cleavage site. There are two types of proteasomes, the constitutive form, expressed in most cell types, and the immunoproteasome, which is constitutively expressed in mature dendritic cells. Protective CD8 T cell epitopes are likely generated by the immunoproteasome and the constitutive proteasome, and here we have modeled and analyzed the cleavage by these two proteases. Results We have modeled the immunoproteasome and proteasome cleavage sites upon two non-overlapping sets of peptides consisting of 553 CD8 T cell epitopes, naturally processed and restricted by human MHCI molecules, and 382 peptides eluted from human MHCI molecules, respectively, using N-grams. Cleavage models were generated considering different epitope and MHCI-eluted fragment lengths and the same number of C-terminal flanking residues. Models were evaluated in 5-fold cross-validation. Judging by the Mathew's Correlation Coefficient (MCC, optimal cleavage models for the proteasome (MCC = 0.43 ± 0.07 and the immunoproteasome (MCC = 0.36 ± 0.06 were obtained from 12-residue peptide fragments. Using an independent dataset consisting of 137 HIV1-specific CD8 T cell epitopes, the immunoproteasome and proteasome cleavage models achieved MCC values of 0.30 and 0.18, respectively, comparatively better than those achieved by related methods. Using ROC analyses, we have also shown that, combined with MHCI-peptide binding predictions, cleavage predictions by the immunoproteasome and proteasome models significantly increase the discovery rate of CD8 T cell epitopes restricted by different MHCI molecules, including A*0201, A*0301, A*2402, B*0702, B*2705. Conclusions We have developed models that are specific

  1. Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 expression in early focal cerebral infarction following urokinase thrombolysis in rats

    Institute of Scientific and Technical Information of China (English)

    Yuqiang Song; Hongli Zou; Guofeng Wang; Hongxia Yang; Zhaohong Xie; Jianzhong Bi

    2012-01-01

    Activity of matrix metalloproteinase-9 increases following cerebral ischemia/reperfusion, and is associated with cerebral microvascular permeability, blood-brain barrier destruction, inflammatory cell infiltration and brain edema. Matrix metalloproteinase-9 also likely participates in thrombolysis. A rat model of middle cerebral artery infarction was established by injecting autologous blood clots into the internal carotid artery. At 3 hours following model induction, urokinase was injected into the caudal vein. Decreased neurological severity score, reduced infarct volume, and increased expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 were observed in the cerebral cortex 24 hours after urokinase thrombolysis. These results suggest that urokinase can suppress damage in the acute-early stage of cerebral infarction.

  2. MicroRNA-mediated target mRNA cleavage and 3'-uridylation in human cells.

    Science.gov (United States)

    Xu, Kai; Lin, Jing; Zandi, Roza; Roth, Jack A; Ji, Lin

    2016-07-21

    MicroRNAs (miRNAs) play an important role in targeted gene silencing by facilitating posttranscriptional and translational repression. However, the precise mechanism of mammalian miRNA-mediated gene silencing remains to be elucidated. Here, we used a stem-loop array reverse-transcription polymerase chain reaction assay to analyse miRNA-induced mRNA recognition, cleavage, posttranscriptional modification, and degradation. We detected endogenous let-7 miRNA-induced and Argonaute-catalysed endonucleolytic cleavage on target mRNAs at various sites within partially paired miRNA:mRNA sequences. Most of the cleaved mRNA 5'-fragments were 3'-oligouridylated by activities of terminal uridylyl transferases (TUTases) in miRNA-induced silencing complexes and temporarily accumulated in the cytosol for 5'-3' degradation or other molecular fates. Some 3'-5' decayed mRNA fragments could also be captured by the miRNA-induced silencing complex stationed at the specific miRNA:mRNA target site and oligouridylated by other TUTases at its proximity without involving Argonaute-mediated RNA cleavage. Our findings provide new insights into the molecular mechanics of mammalian miRNA-mediated gene silencing by coordinated target mRNA recognition, cleavage, uridylation and degradation.

  3. A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing.

    Science.gov (United States)

    Fukuda, Masatora; Kurihara, Kei; Tanaka, Yasuyoshi; Deshimaru, Masanobu

    2012-09-01

    Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo.

  4. Efficient and specific internal cleavage of a retroviral palindromic DNA sequence by tetrameric HIV-1 integrase.

    Directory of Open Access Journals (Sweden)

    Olivier Delelis

    Full Text Available BACKGROUND: HIV-1 integrase (IN catalyses the retroviral integration process, removing two nucleotides from each long terminal repeat and inserting the processed viral DNA into the target DNA. It is widely assumed that the strand transfer step has no sequence specificity. However, recently, it has been reported by several groups that integration sites display a preference for palindromic sequences, suggesting that a symmetry in the target DNA may stabilise the tetrameric organisation of IN in the synaptic complex. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the ability of several palindrome-containing sequences to organise tetrameric IN and investigated the ability of IN to catalyse DNA cleavage at internal positions. Only one palindromic sequence was successfully cleaved by IN. Interestingly, this symmetrical sequence corresponded to the 2-LTR junction of retroviral DNA circles-a palindrome similar but not identical to the consensus sequence found at integration sites. This reaction depended strictly on the cognate retroviral sequence of IN and required a full-length wild-type IN. Furthermore, the oligomeric state of IN responsible for this cleavage differed from that involved in the 3'-processing reaction. Palindromic cleavage strictly required the tetrameric form, whereas 3'-processing was efficiently catalysed by a dimer. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the restriction-like cleavage of palindromic sequences may be a general physiological activity of retroviral INs and that IN tetramerisation is strongly favoured by DNA symmetry, either at the target site for the concerted integration or when the DNA contains the 2-LTR junction in the case of the palindromic internal cleavage.

  5. Metalloproteinase inhibition prevents inhibitory synapse reorganization and seizure genesis.

    Science.gov (United States)

    Pollock, Emily; Everest, Michelle; Brown, Arthur; Poulter, Michael O

    2014-10-01

    The integrity and stability of interneurons in a cortical network are essential for proper network function. Loss of interneuron synaptic stability and precise organization can lead to disruptions in the excitation/inhibition balance, a characteristic of epilepsy. This study aimed to identify alterations to the GABAergic interneuron network in the piriform cortex (PC: a cortical area believed to be involved in the development of seizures) after kindling-induced seizures. Immunohistochemistry was used to mark perineuronal nets (PNNs: structures in the extracellular matrix that provide synaptic stability and restrict reorganization of inhibitory interneurons) and interneuron nerve terminals in control and kindled tissues. We found that PNNs were significantly decreased around parvalbumin-positive interneurons after the induction of experimental epilepsy. Additionally, we found layer-specific increases in GABA release sites originating from calbindin, calretinin, and parvalbumin interneurons, implying that there is a re-wiring of the interneuronal network. This increase in release sites was matched by an increase in GABAergic post-synaptic densities. We hypothesized that the breakdown of the PNN could be due to the activity of matrix metalloproteinases (MMP) and that the prevention of PNN breakdown may reduce the rewiring of interneuronal circuits and suppress seizures. To test this hypothesis we employed doxycycline, a broad spectrum MMP inhibitor, to stabilize PNNs in kindled rats. We found that doxycycline prevented PNN breakdown, re-organization of the inhibitory innervation, and seizure genesis. Our observations indicate that PNN degradation may be necessary for the development of seizures by facilitating interneuron plasticity and increased GABAergic activity.

  6. Neutrophil Protease Cleavage of Von Willebrand Factor in Glomeruli - An Anti-thrombotic Mechanism in the Kidney.

    Science.gov (United States)

    Tati, Ramesh; Kristoffersson, Ann-Charlotte; Manea Hedström, Minola; Mörgelin, Matthias; Wieslander, Jörgen; van Kooten, Cees; Karpman, Diana

    2017-02-01

    Adequate cleavage of von Willebrand factor (VWF) prevents formation of thrombi. ADAMTS13 is the main VWF-cleaving protease and its deficiency results in development of thrombotic microangiopathy. Besides ADAMTS13 other proteases may also possess VWF-cleaving activity, but their physiological importance in preventing thrombus formation is unknown. This study investigated if, and which, proteases could cleave VWF in the glomerulus. The content of the glomerular basement membrane (GBM) was studied as a reflection of processes occurring in the subendothelial glomerular space. VWF was incubated with human GBMs and VWF cleavage was assessed by multimer structure analysis, immunoblotting and mass spectrometry. VWF was cleaved into the smallest multimers by the GBM, which contained ADAMTS13 as well as neutrophil proteases, elastase, proteinase 3 (PR3), cathepsin-G and matrix-metalloproteinase 9. The most potent components of the GBM capable of VWF cleavage were in the serine protease or metalloprotease category, but not ADAMTS13. Neutralization of neutrophil serine proteases inhibited GBM-mediated VWF-cleaving activity, demonstrating a marked contribution of elastase and/or PR3. VWF-platelet strings formed on the surface of primary glomerular endothelial cells, in a perfusion system, were cleaved by both elastase and the GBM, a process blocked by elastase inhibitor. Ultramorphological studies of the human kidney demonstrated neutrophils releasing elastase into the GBM. Neutrophil proteases may contribute to VWF cleavage within the subendothelium, adjacent to the GBM, and thus regulate thrombus size. This anti-thrombotic mechanism would protect the normal kidney during inflammation and could also explain why most patients with ADAMTS13 deficiency do not develop severe kidney failure.

  7. A peptide hydroxamate library for enrichment of metalloproteinases: towards an affinity-based metalloproteinase profiling protocol.

    Science.gov (United States)

    Geurink, Paul; Klein, Theo; Leeuwenburgh, Michiel; van der Marel, Gijs; Kauffman, Henk; Bischoff, Rainer; Overkleeft, Herman

    2008-04-07

    A compound library of 96 enantiopure N-terminal succinyl hydroxamate functionalized peptides was synthesized on solid phase. All compounds were tested for their inhibitory potential towards MMP-9, MMP-12 and ADAM-17, which led to the identification of both broad spectrum inhibitors and metalloproteinase-selective ones. Eight potent and less potent inhibitors were immobilized on Sepharose beads and evaluated in solid-phase enrichment of active MMP-9, MMP-12 and ADAM-17. In addition, one of these inhibitors was used for solid-phase enrichment of endogenous ADAM-17 from a complex proteome (a lysate prepared from cultured A549 cells).

  8. Computational prediction of cleavage using proteasomal in vitro digestion and MHC I ligand data

    Institute of Scientific and Technical Information of China (English)

    Yu-feng LU; Hao SHENG; Yi ZHANG; Zhi-yang LI

    2013-01-01

    Proteasomes are responsible for the production of the majority of cytotoxic T lymphocyte (CTL) epitopes.Hence,it is important to identify correctly which peptides will be generated by proteasomes from an unknown protein.However,the pool of proteasome cleavage data used in the prediction algorithms,whether from major histocompatibility complex (MHC) I ligand or in vitro digestion data,is not identical to in vivo proteasomal digestion products.Therefore,the accuracy and reliability of these models still need to be improved.In this paper,three types of proteasomal cleavage data,constitutive proteasome (cCP),immunoproteasome (iCP) in vitro cleavage,and MHC I ligandv data,were used for training cleave-site predictive methods based on the kernel-function stabilized matrix method (KSMM).The predictive accuracies of the KSMM+pair coefficients were 75.0%,72.3%,and 83.1% for cCP,iCP,and MHC I ligand data,respectively,which were comparable to the results from support vector machine (SVM).The three proteasomal cleavage methods were combined in turn with MHC I-peptide binding predictions to model MHC I-peptide processing and the presentation pathway.These integrations markedly improved MHC I peptide identification,increasing area under the receiver operator characteristics (ROC) curve (AUC) values from 0.82 to 0.91.The results suggested that both MHC I ligand and proteasomal in vitro degradation data can give an exact simulation of in vivo processed digestion.The information extracted from cCP and iCP in vitro cleavage data demonstrated that both cCP and iCP are selective in their usage of peptide bonds for cleavage.

  9. Abyssal fiction: common shares, colonial cleavages

    Directory of Open Access Journals (Sweden)

    Alexandre Montaury

    2016-12-01

    Full Text Available The paper aims to develop a reflection on the interaction between the legacies of colonialism and traditional symbolic and cultural practices in African Portuguese-speaking spaces. From a preliminary analysis of fictional texts of wide circulation in Brazil, aims to examine the cleavages, or “abyssal lines” that constitute experiences printed in the daily life of the former Portuguese colony of Cape Verde, Mozambique and Angola.---DOI: http://dx.doi.org/10.21881/abriluff.2016n17a378

  10. Computational redesign of endonuclease DNA binding and cleavage specificity

    Science.gov (United States)

    Ashworth, Justin; Havranek, James J.; Duarte, Carlos M.; Sussman, Django; Monnat, Raymond J.; Stoddard, Barry L.; Baker, David

    2006-06-01

    The reprogramming of DNA-binding specificity is an important challenge for computational protein design that tests current understanding of protein-DNA recognition, and has considerable practical relevance for biotechnology and medicine. Here we describe the computational redesign of the cleavage specificity of the intron-encoded homing endonuclease I-MsoI using a physically realistic atomic-level forcefield. Using an in silico screen, we identified single base-pair substitutions predicted to disrupt binding by the wild-type enzyme, and then optimized the identities and conformations of clusters of amino acids around each of these unfavourable substitutions using Monte Carlo sampling. A redesigned enzyme that was predicted to display altered target site specificity, while maintaining wild-type binding affinity, was experimentally characterized. The redesigned enzyme binds and cleaves the redesigned recognition site ~10,000 times more effectively than does the wild-type enzyme, with a level of target discrimination comparable to the original endonuclease. Determination of the structure of the redesigned nuclease-recognition site complex by X-ray crystallography confirms the accuracy of the computationally predicted interface. These results suggest that computational protein design methods can have an important role in the creation of novel highly specific endonucleases for gene therapy and other applications.

  11. Inhibitory effects of caffeic acid phenethyl ester on cancer cell metastasis mediated by the down-regulation of matrix metalloproteinase expression in human HT1080 fibrosarcoma cells.

    Science.gov (United States)

    Hwang, Hye Jin; Park, Hyen Joo; Chung, Hwa-Jin; Min, Hye-Young; Park, Eun-Jung; Hong, Ji-Young; Lee, Sang Kook

    2006-05-01

    Caffeic acid phenethyl ester (CAPE) derived from honeybee propolis has been used as a folk medicine. Recent study also revealed that CAPE has several biological activities including antioxidation, anti-inflammation and inhibition of tumor growth. The present study investigated the effect of CAPE on tumor invasion and metastasis by determining the regulation of matrix metalloproteinases (MMPs). Matrix metalloproteinases, which are zinc-dependent proteolytic enzymes, play a pivotal role in tumor metastasis by cleavage of extracellular matrix (ECM) as well as nonmatrix substrates. On this line, we examined the influence of CAPE on the gene expression of MMPs (MMP-2, MMP-9, MT1-MMP), tissue inhibitor of metalloproteinase-2 (TIMP-2) and in vitro invasiveness of human fibrosarcoma cells. Dose-dependent decreases in MMP and TIMP-2 mRNA levels were observed in CAPE-treated HT1080 human fibrosarcoma cells as detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Gelatin zymography analysis also exhibited a significant down-regulation of MMP-2 and MMP-9 expression in HT1080 cells treated with CAPE compared to controls. In addition, CAPE inhibited the activated MMP-2 activity as well as invasion, motility, cell migration and colony formation of tumor cells. These data therefore provide direct evidence for the role of CAPE as a potent antimetastatic agent, which can markedly inhibit the metastatic and invasive capacity of malignant cells.

  12. QM/MM Studies of the Matrix Metalloproteinase 2 (MMP2) Inhibition Mechanism of (S)-SB-3CT and its Oxirane Analogue.

    Science.gov (United States)

    Zhou, Jia; Tao, Peng; Fisher, Jed F; Shi, Qicun; Mobashery, Shahriar; Schlegel, H Bernhard

    2010-11-09

    SB-3CT, (4-phenoxyphenylsulfonyl)methylthiirane, is a potent, mechanism-based inhibitor of the gelatinase sub-class of the matrix metalloproteinase (MMP) family of zinc proteases. The gelatinase MMPs are unusual in that there are several examples where both enantiomers of a racemic inhibitor have comparable inhibitory abilities. SB-3CT is one such example. Here, the inhibition mechanism of the MMP2 gelatinase by the (S)-SB-3CT enantiomer and its oxirane analogue is examined computationally, and compared to the mechanism of (R)-SB-3CT. Inhibition of MMP2 by (R)-SB-3CT was shown previously to involve enzyme-catalyzed C-H deprotonation adjacent to the sulfone, with concomitant opening by β-elimination of the sulfur of the three-membered thiirane ring. Similarly to the R enantiomer, (S)-SB-3CT was docked into the active site of MMP2, followed by molecular dynamics simulation to prepare the complex for combined quantum mechanics and molecular mechanics (QM/MM) calculations. QM/MM calculations with B3LYP/6-311+G(d,p) for the QM part (46 atoms) and the AMBER force field for the MM part were used to compare the reaction of (S)-SB-3CT and its oxirane analogue in the active site of MMP2 (9208 atoms). These calculations show that the barrier for the proton abstraction coupled ring opening reaction of (S)-SB-3CT in the MMP2 active site is 4.4 kcal/mol lower than its oxirane analogue, and the ring opening reaction energy of (S)-SB-3CT is only 1.6 kcal/mol less exothermic than its oxirane analogue. Calculations also show that the protonation of the ring-opened products by water is thermodynamically much more favorable for the alkoxide obtained from the oxirane, than for the thiolate obtained from the thiirane. In contrast to (R)-SB-3CT and the R-oxirane analogue, the double bonds of the ring-opened products of (S)-SB-3CT and its S-oxirane analogue have the cis-configuration. Vibrational frequency and intrinsic reaction path calculations on a reduced size QM/MM model (2747 atoms

  13. Characterization of the prohormone complement in cattle using genomic libraries and cleavage prediction approaches

    Directory of Open Access Journals (Sweden)

    Rodriguez-Zas Sandra L

    2009-05-01

    Full Text Available Abstract Background Neuropeptides are cell to cell signalling molecules that regulate many critical biological processes including development, growth and reproduction. These peptides result from the complex processing of prohormone proteins, making their characterization both challenging and resource demanding. In fact, only 42 neuropeptide genes have been empirically confirmed in cattle. Neuropeptide research using high-throughput technologies such as microarray and mass spectrometry require accurate annotation of prohormone genes and products. However, the annotation and associated prediction efforts, when based solely on sequence homology to species with known neuropeptides, can be problematic. Results Complementary bioinformatic resources were integrated in the first survey of the cattle neuropeptide complement. Functional neuropeptide characterization was based on gene expression profiles from microarray experiments. Once a gene is identified, knowledge of the enzymatic processing allows determination of the final products. Prohormone cleavage sites were predicted using several complementary cleavage prediction models and validated against known cleavage sites in cattle and other species. Our bioinformatics approach identified 92 cattle prohormone genes, with 84 of these supported by expressed sequence tags. Notable findings included an absence of evidence for a cattle relaxin 1 gene and evidence for a cattle galanin-like peptide pseudogene. The prohormone processing predictions are likely accurate as the mammalian proprotein convertase enzymes, except for proprotein convertase subtilisin/kexin type 9, were also identified. Microarray analysis revealed the differential expression of 21 prohormone genes in the liver associated with nutritional status and 8 prohormone genes in the placentome of embryos generated using different reproductive techniques. The neuropeptide cleavage prediction models had an exceptional performance, correctly

  14. Direct measurement of matrix metalloproteinase activity in 3D cellular microenvironments using a fluorogenic peptide substrate.

    Science.gov (United States)

    Leight, Jennifer L; Alge, Daniel L; Maier, Andrew J; Anseth, Kristi S

    2013-10-01

    Incorporation of degradable moieties into synthetic hydrogels has greatly increased the utility of these three-dimensional matrices for in vitro cell culture as well as tissue engineering applications. A common method for introducing degradability is the inclusion of oligopeptides sensitive to cleavage by matrix metalloproteinases (MMPs), enabling cell-mediated remodeling and migration within the material. While this strategy has been effective, characterization and measurement of cell-mediated degradation in these materials has remained challenging. There are 20+ MMP family members whose activity is regulated in space and time by a number of biochemical and biophysical cues. Thus, the typical approach of characterizing cleavage of degradable moieties in solution with recombinant enzymes does not easily translate to three-dimensional cell-mediated matrix remodeling. To address this challenge, we report here the synthesis of a cell-laden hydrogel matrix functionalized with a fluorogenic peptide substrate to provide real-time, quantitative monitoring of global MMP activity. Using this system, stimulation of MMP activity was observed with growth factor treatment in mammary epithelial cells and compared to classical zymography results. Further, the effect of biophysical cues on MMP activity of human mesenchymal stem cells was also investigated where more rigid hydrogels were observed to increase MMP activity. The regulation of MMP activity by these biochemical and biophysical cues highlights the need for in situ, real-time measurement of hydrogel degradation, and use of these functionalized hydrogels will aid in future rational design of degradable synthetic hydrogels for in vitro cell studies and tissue engineering applications. Copyright © 2013 Elsevier Ltd. All rights reserved.

  15. Matrix metalloproteinases in exercise and obesity.

    Science.gov (United States)

    Jaoude, Jonathan; Koh, Yunsuk

    2016-01-01

    Matrix metalloproteinases (MMPs) are zinc- and calcium-dependent endoproteinases that have the ability to break down extracellular matrix. The large range of MMPs' functions widens their spectrum of potential role as activators or inhibitors in tissue remodeling, cardiovascular diseases, and obesity. In particular, MMP-1, -2, and -9 may be associated with exercise and obesity. Thus, the current study reviewed the effects of different types of exercise (resistance and aerobic) on MMP-1, -2, and -9. Previous studies report that the response of MMP-2 and -9 to resistance exercise is dependent upon the length of exercise training, since long-term resistance exercise training increased both MMP-2 and -9, whereas acute bout of resistance exercise decreased these MMPs. Aerobic exercise produces an inconsistent result on MMPs, although some studies showed a decrease in MMP-1. Obesity is related to a relatively lower level of MMP-9, indicating that an exercise-induced increase in MMP-9 may positively influence obesity. A comprehensive understanding of the relationship between exercise, obesity, and MMPs does not exist yet. Future studies examining the acute and chronic responses of these MMPs using different subject models may provide a better understanding of the molecular mechanisms that are associated with exercise, obesity, and cardiovascular disease.

  16. The matrix metalloproteinase in larynx cancer

    Directory of Open Access Journals (Sweden)

    Weronika Lucas Grzelczyk

    2016-12-01

    Full Text Available One of the most common carcinoma occurring in the head and neck is laryngeal cancer. Despite the rapid scientific advances in medicine the prognosis for patients with such type of disease is not satisfying. In the last few years matrix metalloproteinases ‑ MMPs and their tissue inhibitors – TIMPs, mostly MMP‑2 and MMP‑9, arouses a great interest, especially in the process of carcinogenesis. It seems that their impact in the formation and development of laryngeal cancer is significant. MMPs a group of zinc‑ and calcium‑ dependent endopeptidases play crucial role extracellular matrix collagen degradation. That are enzymes, that degrade and the basement membrane by facilitating tumor growth, cell migration and tumor invasion. They are implicated in metastasis and angiogenesis potentiate within the tumor. Clear tendency was observed towards the higher MMPs and TIMPs expression in larynx cancer than in the stroma. Recent studies show correlations between increased MMP‑2 gene expression in the tumor tissue and clinical status, histopathological grading and metastases occurrence. The similar MMP2 over expression dependence were found on tumor recurrence and survival. Many authors pointed out, significant higher MMP‑2 expression as a potential marker of tumor invasiveness and worse prognosis in patients with larynx cancer. However, association of MMP 9 gene expression with laryngeal cancer clinicopathological features and survival of patients are ambiguous. Although, numerous researches show that this relationship does exists. Similar correlations could be found in TIMPs, but further studies are necessary because of small amount of literature.

  17. Matrix metalloproteinases in exercise and obesity

    Directory of Open Access Journals (Sweden)

    Jaoude J

    2016-07-01

    Full Text Available Jonathan Jaoude,1 Yunsuk Koh2 1Department of Biology, 2Department of Health, Human Performance, and Recreation, Baylor University, Waco, TX, USA Abstract: Matrix metalloproteinases (MMPs are zinc- and calcium-dependent endoproteinases that have the ability to break down extracellular matrix. The large range of MMPs’ functions widens their spectrum of potential role as activators or inhibitors in tissue remodeling, cardiovascular diseases, and obesity. In particular, MMP-1, -2, and -9 may be associated with exercise and obesity. Thus, the current study reviewed the effects of different types of exercise (resistance and aerobic on MMP-1, -2, and -9. Previous studies report that the response of MMP-2 and -9 to resistance exercise is dependent upon the length of exercise training, since long-term resistance exercise training increased both MMP-2 and -9, whereas acute bout of resistance exercise decreased these MMPs. Aerobic exercise produces an inconsistent result on MMPs, although some studies showed a decrease in MMP-1. Obesity is related to a relatively lower level of MMP-9, indicating that an exercise-induced increase in MMP-9 may positively influence obesity. A comprehensive understanding of the relationship between exercise, obesity, and MMPs does not exist yet. Future studies examining the acute and chronic responses of these MMPs using different subject models may provide a better understanding of the molecular mechanisms that are associated with exercise, obesity, and cardiovascular disease. Keywords: cardiovascular disease, gelatinases, collagenases, TIMP

  18. Matrix metalloproteinase imbalance in muscle disuse atrophy.

    Science.gov (United States)

    Giannelli, G; De Marzo, A; Marinosci, F; Antonaci, S

    2005-01-01

    Muscle atrophy commonly occurs as a consequence of prolonged muscle inactivity, as observed after cast immobilization, bed rest or space flights. The molecular mechanisms responsible for muscle atrophy are still unknown, but a role has been proposed for altered permeability of the sarcolemma and of the surrounding connective tissue. Matrix metallo-proteinases (MMPs) are a family of enzymes with proteolytic activity toward a number of extracellular matrix (ECM) components; they are inhibited by tissue inhibitors of MMPs (TIMPs). In a rat tail-suspension experimental model, we show that after fourteen days of non-weight bearing there is increased expression of MMP-2 in the atrophic soleus and gastrocnemius and decreased expression of TIMP-2. In the same experimental model the expression of Collagen I and Collagen IV, two main ECM components present in the muscles, was reduced and unevenly distributed in unloaded animals. The difference was more evident in the soleus than in the gastrocnemius muscle. This suggests that muscle disuse induces a proteolytic imbalance, which could be responsible for the breakdown of basal lamina structures such as Collagen I and Collagen IV, and that this leads to an altered permeability with consequent atrophy. In conclusion, an MMP-2/TIMP-2 imbalance could have a role in the mechanism underlying muscle disuse atrophy; more studies are needed to expand our molecular knowledge on this issue and to explore the possibility of targeting the proteolytic imbalance with MMP inhibitors.

  19. Matrix Metalloproteinases-7 and Kidney Fibrosis

    Science.gov (United States)

    Ke, Ben; Fan, Chuqiao; Yang, Liping; Fang, Xiangdong

    2017-01-01

    Matrix metalloproteinase-7 (MMP-7) is a secreted zinc- and calcium-dependent endopeptidase that degrades a broad range of extracellular matrix substrates and additional substrates. MMP-7 playsa crucial role in a diverse array of cellular processes and appears to be a key regulator of fibrosis in several diseases, including pulmonary fibrosis, liver fibrosis, and cystic fibrosis. In particular, the relationship between MMP-7 and kidney fibrosis has attracted significant attention in recent years. Growing evidence indicates that MMP-7 plays an important role in the pathogenesis of kidney fibrosis. Here, we summarize the recent progress in the understanding of the role of MMP-7 in kidney fibrosis. In particular, we discuss how MMP-7 contributes to kidney fibrotic lesions via the following three pathways: epithelial-mesenchymal transition (EMT), transforming growth factor-beta (TGF-β) signaling, and extracellular matrix (ECM) deposition. Further dissection of the crosstalk among and regulation of these pathways will help clinicians and researchers develop effective therapeutic approaches for treating chronic kidney disease.

  20. MMP-12 catalytic domain recognizes and cleaves at multiple sites in human skin collagen type I and type III.

    Science.gov (United States)

    Taddese, Samuel; Jung, Michael C; Ihling, Christian; Heinz, Andrea; Neubert, Reinhard H H; Schmelzer, Christian E H

    2010-04-01

    Collagens of either soft connective or mineralized tissues are subject to continuous remodeling and turnover. Undesired cleavage can be the result of an imbalance between proteases and their inhibitors. Owing to their superhelical structure, collagens are resistant to many proteases and matrix metalloproteinases (MMPs) are required to initiate further degradation by other enzymes. Several MMPs are known to degrade collagens, but the action of MMP-12 has not yet been studied in detail. In this work, the potential of MMP-12 in recognizing sites in human skin collagen types I and III has been investigated. The catalytic domain of MMP-12 binds to the triple helix and cleaves the typical sites -Gly(775)-Leu(776)- in alpha-2 type I collagen and -Gly(775)-Ile(776)- in alpha-1 type I and type III collagens and at multiple other sites in both collagen types. Moreover, it was observed that the region around these typical sites contains comparatively less prolines, of which some have been proven to be only partially hydroxylated. This is of relevance since partial hydroxylation in the vicinity of a potential scissile bond may have a local effect on the conformational thermodynamics with probable consequences on the collagenolysis process. Taken together, the results of the present work confirm that the catalytic domain of MMP-12 alone binds and degrades collagens I and III. Copyright 2009 Elsevier B.V. All rights reserved.

  1. Localization and possible role of membrane type metallo-proteinase and tissue inhibitors of metalloproteinase-1 in early stages of placentation

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Human placental tissues from the first and second trimesters of gestation have been investigated using riboprobe in situ hybridisation of mRNA sequences coding for membrane type metalloproteinase (MT-1-MMP) and tissue inhibitors of metalloproteinase-1 (TIMP-1). Results show that (i) both mRNAs express at a relatively high level in the chorion laeve trophoblast cells and the adjacent decidual cells of fetal membrane; (ii) the most abundant expression of the two mRNAs was found in the extravillous trophoblast between Rohrs and Nitabuch striae of basal plate, trophoblast shell and gland cells of the decidua; (iii) isolated or small groups of cytotrophoblast cells in the chorionic villi and in the cells lining arterioles in decidua and stem villi also expressed both MT-1-MMP and TIMP-1 at defferent extents. The data suggest that the coordinated expression of the MT-MMP and its inhibitor TIMP in defferent cells of the placental tissue may play an essential role in trophoblast invasion and angiogenesis related to placentation in the first two trimesters of gestation. They may also have an ability to effect separation of fetal from material tissue at a favorable junctional site during parturition.

  2. Plasma matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 as biomarkers of ulcerative colitis activity

    Institute of Scientific and Technical Information of China (English)

    Alicja Wiercinska-Drapalo; Jerzy Jaroszewicz; Robert Flisiak; Danuta Prokopowicz

    2003-01-01

    AIM: Overexpression of mucosal metalloproteinases (MMP)has been demonstrated recently in inflammatory boweldisease. Their activity can be counterbalanced by the tissueinhibitor of metalloproteinases (TIMP). The aim of this studywas to evaluate the effect of ulcerative colitis (UC) on MMP-1 and TTMP-1 plasma concentrations, as two possiblebiomarkers of the disease activity.METHODS: MMP-1 and TIMP-1 plasma concentrations weremeasured with an enzyme immunoassay in 16 patients withendoscopically confirmed active UC.RESULTS: Plasma concentrations of both MMP-11 (13.7±0.2ng/ml) and TIMP-L (799±140 ng/ml) were significantlyelevated in UC patients in comparison to healthy controls(11.9±0.9 ng/ml and 220±7 ng/ml respectively). There wasno correlation between TIMP-1 and MMP-1 concentrations(r=-0.02). TIMP-1 levels revealed significant positivecorrelations with scored endoscopic degree of mucosai injury,disease activity index and clinical activity index values aswell as C-reactive protein concentration. There was nocorrelation between MMP-1 and laboratory, clinical orendoscopic indices of the disease activity.CONCLUSION: These results confirm the role of both MMP-1 and TIMP-1 in the pathogenesis of ulcerative colitis.However only TIMP-1 can be useful as a biomarker of thedisease activity, demonstrating association with clinical andendoscopic pictures.

  3. Investigation of Matrix Metalloproteinase -1, 2 and tissue inhibitor of matrix metalloproteinases-1 in Endometriosis

    Institute of Scientific and Technical Information of China (English)

    Rong Liu; Demin Pu; Dan Yang

    2007-01-01

    Objective: To explore the role of matrix metalloproteinase-1,2 (MMP-1, MMP-2) and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1) in endometriosis. Methods: The eutopic and ectopic endometria from 40 subjects suffering from endometriosis and regular.endometria from 40 subjects (excluding endometriosis) were collected and examined by in situ hybridization technology and western blot assay. Results: Both expressions of MMP-1 and -2 were stronger in ectopic endometrium and eutopic endometrium than in normal endometrium. On the contrary, the expression of TIMP-1 in ectopic endometrium and eutopic endometrium was lower. The differences were significant (P < 0.01). Moreover, there was no relationship among the expressions of MMP-1, 2 and TIMP-1 in ectopic endometrium. Conclusion: The expressions of MMP-1, 2 and TIMP-1 lose balance and lack of periodic changes in ectopic endometrium , which explains the biological invasive behavior of endometriosis. It was suggested that regulating the balance between the MMPs and TIMP-1 should be an ideal therapeutic target to endometriosis.

  4. Time-dependent matrix metalloproteinases and tissue inhibitor of metalloproteinases expression change in fusarium solani keratitis.

    Science.gov (United States)

    Li, Qian; Gao, Xin-Rui; Cui, Hong-Ping; Lang, Li-Li; Xie, Xiu-Wen; Chen, Qun

    2016-01-01

    To investigate matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) expression during the progress of fusarium solani (F.solani) keratitis in a rat model. A rat model of F.solani keratitis was produced using corneal scarification and a hand-made contact lens. MMPs and TIMPs expressiond were explored in this rat model of F.solani keratitis using real-time polymerase chain reaction (PCR) and DIF. GM6001 (400 µmol/mL) was used to treat infected corneas. The keratitis duration, amount and area of corneal neovascularization (CNV) were evaluated. MMP-3 expression was 66.3 times higher in infected corneas compared to normal corneas. MMP-8, -9, and -13 expressions were significantly upregulated in the mid-period of the infection, with infected-to-normal ratios of 4.03, 39.86, and 5.94, respectively. MMP-2 and -7 expressions increased in the late period, with the infected-to-normal ratios of 5.94 and 16.22, respectively. TIMP-1 expression was upregulated in the early period, and it was 43.17 times higher in infected compared to normal corneas, but TIMP-2, -3, and -4 expressions were mildly downregulated or unchanged. The results of DIF were consistent with the result of real-time PCR. GM6001, a MMPs inhibitor, decreased the duration of F.solani infection and the amount and area of CNV. MMPs and TIMPs contributed into the progress of F.solani keratitis.

  5. Matrix metalloproteinase-9 and -2 and tissue inhibitor of matrix metalloproteinase-2 in invasive pituitary adenomas

    Science.gov (United States)

    Liu, Hong-Yan; Gu, Wei-Jun; Wang, Cheng-Zhi; Ji, Xiao-Jian; Mu, Yi-Ming

    2016-01-01

    Abstract The extracellular matrix is important for tumor invasion and metastasis. Normal function of the extracellular matrix depends on the balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The objective of this meta-analysis was to assess the relationship between expression of MMP-9, MMP-2, and TIMP-2 and invasion of pituitary adenomas. We searched Pubmed, Embase, and the Chinese Biomedical Database up to October 2015. RevMan 5.1 software (Cochrane Collaboration, Copenhagen, Denmark) was used for statistical analysis. We calculated the standardized mean difference (SMD) for data expressed as mean ± standard deviation because of the difference in the detection method. Twenty-four studies (1320 patients) were included. MMP-9 expression was higher in the patients with invasive pituitary adenomas (IPAs) than patients with noninvasive pituitary adenomas (NIPAs) with detection methods of IHC [odds ratio (OR) = 5.48, 95% confidence interval (CI) = 2.61–11.50, P prolactinomas and nonfunctioning pituitary adenomas was also no difference (OR = 1.03, 95% CI = 0.48–2.20, P = 0.95). The results indicated that MMP-9 and -2 may be correlated with invasiveness of pituitary adenomas, although their relationship with functional status of pituitary adenomas is still not clear. TIMP-2 expression in IPAs needs to be investigated further. PMID:27310993

  6. Intratree Variability of Cleavage Resistance of Chinese Fir from Plantation

    Institute of Scientific and Technical Information of China (English)

    XU Ming; REN Haiqing; LUO Xiuqin; YIN Yafang

    2006-01-01

    This paper studied the variation of cleavage resistance of Chinese fir wood from plantation.Six trees of 36 years old were investigated,and the cleavage resistance properties for 672 samples made of the trees were tested.The samples were cut from the sapwood and heartwood at different directions (south and north) and heights (1.3,3.3,5.3 and 7.3 m) of the trees.The result showed that:tangential cleavage resistance was higher than radial one, and cleavage resistance of sapwood was higher than that of heartwood,but there was no significant difference in cleavage resistances between sections of the north and the south of the trees.There was a little variation in cleavage resistance between the radial and tangential from butt to top log,which shows alittle decrease with the height from 1.3 to 5.3 m,but a rise in the top of the trees.

  7. Efficient second strand cleavage during Holliday junction resolution by RuvC requires both increased junction flexibility and an exposed 5' phosphate.

    Directory of Open Access Journals (Sweden)

    Fekret Osman

    Full Text Available BACKGROUND: Holliday junction (HJ resolution is a critical step during homologous recombination. In Escherichia coli this job is performed by a member of the RNase H/Integrase superfamily called RuvC, whereas in Schizosaccharomyces pombe it has been attributed to the XPF family member Mus81-Eme1. HJ resolution is achieved through the sequential cleavage of two strands of like polarity at or close to the junction crossover point. RuvC functions as a dimer, whereas Mus81-Eme1 is thought to function as a dimer of heterodimers. However, in both cases the multimer contains two catalytic sites, which act independently and sequentially during the resolution reaction. To ensure that both strands are cleaved before the nuclease dissociates from the junction, the rate of second strand cleavage is greatly enhanced compared to that of the first. The enhancement of second strand cleavage has been attributed to the increased flexibility of the nicked HJ, which would facilitate rapid engagement of the second active site and scissile bond. Here we have investigated whether other properties of the nicked HJ are important for enhancing second strand cleavage. PRINCIPAL FINDINGS: A comparison of the efficiency of cleavage of nicked HJs with and without a 5' phosphate at the nick site shows that a 5' phosphate is required for most of the enhancement of second strand cleavage by RuvC. In contrast Mus81-Eme1 cleaves nicked HJs with and without a 5' phosphate with equal efficiency, albeit there are differences in cleavage site selection. CONCLUSIONS: Our data show that efficient HJ resolution by RuvC depends on the 5' phosphate revealed by incision of the first strand. This is a hitherto unappreciated factor in promoting accelerated second strand cleavage. However, a 5' phosphate is not a universal requirement since efficient cleavage by Mus81-Eme1 appears to depend solely on the increased junction flexibility that is developed by the first incision.

  8. Efficient second strand cleavage during Holliday junction resolution by RuvC requires both increased junction flexibility and an exposed 5' phosphate.

    Science.gov (United States)

    Osman, Fekret; Gaskell, Louise; Whitby, Matthew C

    2009-01-01

    Holliday junction (HJ) resolution is a critical step during homologous recombination. In Escherichia coli this job is performed by a member of the RNase H/Integrase superfamily called RuvC, whereas in Schizosaccharomyces pombe it has been attributed to the XPF family member Mus81-Eme1. HJ resolution is achieved through the sequential cleavage of two strands of like polarity at or close to the junction crossover point. RuvC functions as a dimer, whereas Mus81-Eme1 is thought to function as a dimer of heterodimers. However, in both cases the multimer contains two catalytic sites, which act independently and sequentially during the resolution reaction. To ensure that both strands are cleaved before the nuclease dissociates from the junction, the rate of second strand cleavage is greatly enhanced compared to that of the first. The enhancement of second strand cleavage has been attributed to the increased flexibility of the nicked HJ, which would facilitate rapid engagement of the second active site and scissile bond. Here we have investigated whether other properties of the nicked HJ are important for enhancing second strand cleavage. A comparison of the efficiency of cleavage of nicked HJs with and without a 5' phosphate at the nick site shows that a 5' phosphate is required for most of the enhancement of second strand cleavage by RuvC. In contrast Mus81-Eme1 cleaves nicked HJs with and without a 5' phosphate with equal efficiency, albeit there are differences in cleavage site selection. Our data show that efficient HJ resolution by RuvC depends on the 5' phosphate revealed by incision of the first strand. This is a hitherto unappreciated factor in promoting accelerated second strand cleavage. However, a 5' phosphate is not a universal requirement since efficient cleavage by Mus81-Eme1 appears to depend solely on the increased junction flexibility that is developed by the first incision.

  9. Association of a peptoid ligand with the apical loop of pri-miR-21 inhibits cleavage by Drosha.

    Science.gov (United States)

    Diaz, Jason P; Chirayil, Rachel; Chirayil, Sara; Tom, Martin; Head, Katie J; Luebke, Kevin J

    2014-04-01

    We have found a small molecule that specifically inhibits cleavage of a precursor to the oncogenic miRNA, miR-21, by the microprocessor complex of Drosha and DGCR8. We identified novel ligands for the apical loop of this precursor from a screen of 14,024 N-substituted oligoglycines (peptoids) in a microarray format. Eight distinct compounds with specific affinity were obtained, three having affinities for the targeted loop in the low micromolar range and greater than 15-fold discrimination against a closely related hairpin. One of these compounds completely inhibits microprocessor cleavage of a miR-21 primary transcript at concentrations at which cleavage of another miRNA primary transcript, pri-miR-16, is little affected. The apical loop of pri-miR-21, placed in the context of pri-miR-16, is sufficient for inhibition of microprocessor cleavage by the peptoid. This compound also inhibits cleavage of pri-miR-21 containing the pri-miR-16 apical loop, suggesting an additional site of association within pri-miR-21. The reported peptoid is the first example of a small molecule that inhibits microprocessor cleavage by binding to the apical loop of a pri-miRNA.

  10. Structures of a CRISPR-Cas9 R-loop complex primed for DNA cleavage.

    Science.gov (United States)

    Jiang, Fuguo; Taylor, David W; Chen, Janice S; Kornfeld, Jack E; Zhou, Kaihong; Thompson, Aubri J; Nogales, Eva; Doudna, Jennifer A

    2016-02-19

    Bacterial adaptive immunity and genome engineering involving the CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) protein Cas9 begin with RNA-guided DNA unwinding to form an RNA-DNA hybrid and a displaced DNA strand inside the protein. The role of this R-loop structure in positioning each DNA strand for cleavage by the two Cas9 nuclease domains is unknown. We determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions, in turn, position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation.

  11. Social cleavages and electoral support in Turkey -- toward convergence?

    OpenAIRE

    Hazama, Yasushi

    2003-01-01

    Studies on Western democracies have shown that deep-seated social cleavages stabilize the electoral behavior and thus reduce electoral volatility. But how do social cleavages affect a party system that is undergoing democratic consolidation, such as in Turkey? In this study, investigations were carried out on long- and short-term relationships between social cleavages (religiosity, ethnicity, and sectarism) and electoral volatility in Turkey during the 1961-2002 period. Cross-sectional multip...

  12. Phylotranscriptomic analysis uncovers a wealth of tissue inhibitor of metalloproteinases variants in echinoderms.

    Science.gov (United States)

    Clouse, Ronald M; Linchangco, Gregorio V; Kerr, Alexander M; Reid, Robert W; Janies, Daniel A

    2015-12-01

    Tissue inhibitors of metalloproteinases (TIMPs) help regulate the extracellular matrix (ECM) in animals, mostly by inhibiting matrix metalloproteinases (MMPs). They are important activators of mutable collagenous tissue (MCT), which have been extensively studied in echinoderms, and the four TIMP copies in humans have been studied for their role in cancer. To understand the evolution of TIMPs, we combined 405 TIMPs from an echinoderm transcriptome dataset built from 41 specimens representing all five classes of echinoderms with variants from protostomes and chordates. We used multiple sequence alignment with various stringencies of alignment quality to cull highly divergent sequences and then conducted phylogenetic analyses using both nucleotide and amino acid sequences. Phylogenetic hypotheses consistently recovered TIMPs as diversifying in the ancestral deuterostome and these early lineages continuing to diversify in echinoderms. The four vertebrate TIMPs diversified from a single copy in the ancestral chordate, all other copies being lost. Consistent with greater MCT needs owing to body wall liquefaction, evisceration, autotomy and reproduction by fission, holothuroids had significantly more TIMPs and higher read depths per contig. Ten cysteine residues, an HPQ binding site and several other residues were conserved in at least 70% of all TIMPs. The conservation of binding sites and the placement of echinoderm TIMPs involved in MCT modification suggest that ECM regulation remains the primary function of TIMP genes, although within this role there are a large number of specialized copies.

  13. Invasiveness of Hepatocellular Carcinoma Cell Lines: Contribution of Membrane-Type 1 Matrix Metalloproteinase

    Directory of Open Access Journals (Sweden)

    Koji Murakami

    1999-11-01

    Full Text Available Intrahepatic metastasis is one of the malignant features of hepatocellular carcinoma (HCC. Matrix metalloproteoinases (MMPs and urokinase-type plasminogen activator (u-PA/plasmin, are known to be associated with the invasive properties of various types of tumor cells. In this study, we examined which proteinases play a role in the metastatic invasion of human HCC cell lines. JHH-5 and JHH-6 cells constitutively expressed mRNAs for both membrane-type 1 matrix metalloproteinase (MT1-MMP and u-PA and invaded through reconstituted MATRIGEL in vitro, whereas JHH-7 cells expressed u-PA mRNA but not MT1-MMP and did not invade. However, hepatocyte growth factor (HGF induced MT1-MMP expression on the surface of JHH-7 cells and markedly increased invasiveness of JHH-7 in a concentration-dependent manner. Moreover, cleavage activity for pro-MMP-2 was induced in HGF-treated JHH7 cells. MMP inhibitor, rather than serine proteinase inhibitor, potently inhibited HCC cell invasion. Intrahepatic injection of HCC cell lines into athymic nude mice caused visible intrahepatic metastases in vivo. Moreover, JHH-7 tumors showed expression of MT1-MMP mRNA, while in vitro cultured JHH-7 cells did not. These findings suggest that MTi-MMP plays an important role in the invasive properties of HCC cells, and that HGF modifies the invasive properties of noninvasive HCC cells.

  14. The parasite Entamoeba histolytica exploits the activities of human matrix metalloproteinases to invade colonic tissue.

    Science.gov (United States)

    Thibeaux, Roman; Avé, Patrick; Bernier, Michèle; Morcelet, Marie; Frileux, Pascal; Guillén, Nancy; Labruyère, Elisabeth

    2014-10-07

    Intestinal invasion by the protozoan parasite Entamoeba histolytica is characterized by remodelling of the extracellular matrix (ECM). The parasite cysteine proteinase A5 (CP-A5) is thought to cooperate with human matrix metalloproteinases (MMPs) involved in ECM degradation. Here, we investigate the role CP-A5 plays in the regulation of MMPs upon mucosal invasion. We use human colon explants to determine whether CP-A5 activates human MMPs. Inhibition of the MMPs' proteolytic activities abolishes remodelling of the fibrillar collagen structure and prevents trophozoite invasion of the mucosa. In the presence of trophozoites, MMPs-1 and -3 are overexpressed and are associated with fibrillar collagen remodelling. In vitro, CP-A5 performs the catalytic cleavage needed to activate pro-MMP-3, which in turn activates pro-MMP-1. Ex vivo, incubation with recombinant CP-A5 was enough to rescue CP-A5-defective trophozoites. Our results suggest that MMP-3 and/or CP-A5 inhibitors may be of value in further studies aiming to treat intestinal amoebiasis.

  15. Structural and functional basis for RNA cleavage by Ire1

    Directory of Open Access Journals (Sweden)

    Stroud Robert M

    2011-07-01

    Full Text Available Abstract Background The unfolded protein response (UPR controls the protein folding capacity of the endoplasmic reticulum (ER. Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1. The endoribonuclease (RNase domain of Ire1 initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The mRNA splicing reaction is an obligatory step of Ire1 signaling, yet its mechanism has remained poorly understood due to the absence of substrate-bound crystal structures of Ire1, the lack of structural similarity between Ire1 and other RNases, and a scarcity of quantitative enzymological data. Here, we experimentally define the active site of Ire1 RNase and quantitatively evaluate the contribution of the key active site residues to catalysis. Results This analysis and two new crystal structures suggest that Ire1 RNase uses histidine H1061 and tyrosine Y1043 as the general acid-general base pair contributing ≥ 7.6 kcal/mol and 1.4 kcal/mol to transition state stabilization, respectively, and asparagine N1057 and arginine R1056 for coordination of the scissile phosphate. Investigation of the stem-loop recognition revealed that additionally to the stem-loops derived from the classic Ire1 substrates HAC1 and Xbp1 mRNA, Ire1 can site-specifically and rapidly cleave anticodon stem-loop (ASL of unmodified tRNAPhe, extending known substrate specificity of Ire1 RNase. Conclusions Our data define the catalytic center of Ire1 RNase and suggest a mechanism of RNA cleavage: each RNase monomer apparently contains a separate catalytic apparatus for RNA cleavage, whereas two RNase subunits contribute to RNA stem-loop docking. Conservation of the key residues among Ire1 homologues suggests that the mechanism elucidated here for yeast Ire1 applies to Ire1 in metazoan cells, and to the only known Ire1 homologue RNase L.

  16. Sequence/structure selective thermal and photochemical cleavage of yeast-tRNA(Phe) by UO(2)2+

    DEFF Research Database (Denmark)

    Nielsen, Peter E.; Møllegaard, N E

    1997-01-01

    The uranyl(VI) ion, UO(2)2+, cleaves yeast tRNA(Phe) both thermally and photochemically. Photochemical cleavage takes place at all positions but exhibits maxima at G10, G18, G30, A38, C49 and A62. Furthermore, in the presence of stoichiometric concentrations of citrate, the cleavage is generally...... suppressed except that strong cleavage at positions G10 and C48-U50 persists, indicating the presence of a high-affinity metal-ion binding site. It is proposed that these photocleavage sites reflect the tertiary structure of the yeast tRNA(Phe) molecule in terms of D-loop/T-loop interaction and anticodon...

  17. The N-terminal domain allosterically regulates cleavage and activation of the epithelial sodium channel.

    Science.gov (United States)

    Kota, Pradeep; Buchner, Ginka; Chakraborty, Hirak; Dang, Yan L; He, Hong; Garcia, Guilherme J M; Kubelka, Jan; Gentzsch, Martina; Stutts, M Jackson; Dokholyan, Nikolay V

    2014-08-15

    The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr(370) in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation.

  18. The N-terminal Domain Allosterically Regulates Cleavage and Activation of the Epithelial Sodium Channel*

    Science.gov (United States)

    Kota, Pradeep; Buchner, Ginka; Chakraborty, Hirak; Dang, Yan L.; He, Hong; Garcia, Guilherme J. M.; Kubelka, Jan; Gentzsch, Martina; Stutts, M. Jackson; Dokholyan, Nikolay V.

    2014-01-01

    The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP2), we found that PIP2 increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP2 or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr370 in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation. PMID:24973914

  19. Antisense oligonucleotide-mediated exon skipping as a strategy to reduce proteolytic cleavage of ataxin-3.

    Science.gov (United States)

    Toonen, Lodewijk J A; Schmidt, Iris; Luijsterburg, Martijn S; van Attikum, Haico; van Roon-Mom, Willeke M C

    2016-10-12

    Spinocerebellar ataxia type-3 (SCA3) is a neurodegenerative disorder caused by a polyglutamine repeat expansion in the ataxin-3 protein. Cleavage of mutant ataxin-3 by proteolytic enzymes yields ataxin-3 fragments containing the polyglutamine stretch. These shorter ataxin-3 fragments are thought to be involved in SCA3 pathogenesis due to their increased cellular toxicity and their involvement in formation of the characteristic neuronal aggregates. As a strategy to prevent formation of toxic cleavage fragments, we investigated an antisense oligonucleotide-mediated modification of the ataxin-3 pre-mRNA through exon skipping of exon 8 and 9, resulting in the removal of a central 88 amino acid region of the ataxin-3 protein. This removed protein region contains several predicted cleavage sites and two ubiquitin-interacting motifs. In contrast to unmodified mutant ataxin-3, the internally truncated ataxin-3 protein did not give rise to potentially toxic cleavage fragments when incubated with caspases. In vitro experiments did not show cellular toxicity of the modified ataxin-3 protein. However, the modified protein was incapable of binding poly-ubiquitin chains, which may interfere with its normal deubiquitinating function. Low exon skipping efficiencies combined with reduction in important ataxin-3 protein functions suggest that skipping of exon 8 and 9 is not a viable therapeutic option for SCA3.

  20. Quercetin-Iron Complex: Synthesis, Characterization, Antioxidant, DNA Binding, DNA Cleavage, and Antibacterial Activity Studies.

    Science.gov (United States)

    Raza, Aun; Xu, Xiuquan; Xia, Li; Xia, Changkun; Tang, Jian; Ouyang, Zhen

    2016-11-01

    Quercetin-iron (II) complex was synthesized and characterized by elemental analysis, ultraviolet-visible spectrophotometry, fourier transform infrared spectroscopy, mass spectrometry, proton nuclear magnetic resonance spectroscopy, thermogravimetry and differential scanning calorimetry, scanning electron micrography and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal:ligand) of the complex. Antioxidant study of the quercetin and its metal complex against 2, 2-di-phenyl-1-picryl hydrazyl radical showed that the complex has much more radical scavenging activity than free quercetin. The interaction of quercetin-iron (II) complex with DNA was determined using ultraviolet visible spectra, fluorescence spectra and agarose gel electrophoresis. The results showed that quercetin-iron (II) complex can intercalate moderately with DNA, quench a strong intercalator ethidium bromide and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form to nicked circular form and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was an oxidative cleavage pathway. These results revealed the potential nuclease activity of complex to cleave DNA. In addition, antibacterial activity of complex on E.coli and S. aureus was also investigated. The results showed that complex has higher antibacterial activity than ligand.

  1. Rutin-Nickel Complex: Synthesis, Characterization, Antioxidant, DNA Binding, and DNA Cleavage Activities.

    Science.gov (United States)

    Raza, Aun; Bano, Shumaila; Xu, Xiuquan; Zhang, Rong Xian; Khalid, Haider; Iqbal, Furqan Muhammad; Xia, Changkun; Tang, Jian; Ouyang, Zhen

    2016-12-17

    The rutin-nickel (II) complex (RN) was synthesized and characterized by elemental analysis, UV-visible spectroscopy, IR, mass spectrometry, (1)H NMR, TG-DSC, SEM, and molar conductivity. The low molar conductivity value investigates the non-electrolyte nature of the complex. The elemental analysis and other physical and spectroscopic methods reveal the 1:2 stoichiometric ratio (metal/ligand) of the complex. An antioxidant study of rutin and its metal complex against DPPH radical showed that the complex has more radical scavenging activity than free rutin. The interaction of complex RN with DNA was determined using fluorescence spectra and agarose gel electrophoresis. The results showed that RN can intercalate moderately with DNA, quench a strong intercalator ethidium bromide (EB), and compete for the intercalative binding sites. The complex showed significant cleavage of pBR 322 DNA from supercoiled form (SC) to nicked circular form (NC), and these cleavage effects were dose-dependent. Moreover, the mechanism of DNA cleavage indicated that it was a hydrolytic cleavage pathway. These results revealed the potential nuclease activity of the complex to cleave DNA.

  2. Single-molecule analysis of RAG-mediated V(D)J DNA cleavage.

    Science.gov (United States)

    Lovely, Geoffrey A; Brewster, Robert C; Schatz, David G; Baltimore, David; Phillips, Rob

    2015-04-07

    The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. We have developed single-molecule assays to examine RSS binding by RAG1/2 and their cofactor high-mobility group-box protein 1 (HMGB1) as they proceed through the steps of this reaction. These assays allowed us to observe in real time the individual molecular events of RAG-mediated cleavage. As a result, we are able to measure the binding statistics (dwell times) and binding energies of the initial RAG binding events and characterize synapse formation at the single-molecule level, yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage on forming the synapse. Interestingly, we find that the synaptic complex has a mean lifetime of roughly 400 s and that its formation is readily reversible, with only ∼40% of observed synapses resulting in cleavage at consensus RSS binding sites.

  3. The DNA sequence specificity of bleomycin cleavage in a systematically altered DNA sequence.

    Science.gov (United States)

    Gautam, Shweta D; Chen, Jon K; Murray, Vincent

    2017-08-01

    Bleomycin is an anti-tumour agent that is clinically used to treat several types of cancers. Bleomycin cleaves DNA at specific DNA sequences and recent genome-wide DNA sequencing specificity data indicated that the sequence 5'-RTGT*AY (where T* is the site of bleomycin cleavage, R is G/A and Y is T/C) is preferentially cleaved by bleomycin in human cells. Based on this DNA sequence, we constructed a plasmid clone to explore this bleomycin cleavage preference. By systematic variation of single nucleotides in the 5'-RTGT*AY sequence, we were able to investigate the effect of nucleotide changes on bleomycin cleavage efficiency. We observed that the preferred consensus DNA sequence for bleomycin cleavage in the plasmid clone was 5'-YYGT*AW (where W is A/T). The most highly cleaved sequence was 5'-TCGT*AT and, in fact, the seven most highly cleaved sequences conformed to the consensus sequence 5'-YYGT*AW. A comparison with genome-wide results was also performed and while the core sequence was similar in both environments, the surrounding nucleotides were different.

  4. HMGB1/2 can target DNA for illegitimate cleavage by the RAG1/2 complex

    Directory of Open Access Journals (Sweden)

    Swanson Patrick C

    2009-03-01

    Full Text Available Abstract Background V(DJ recombination is initiated in antigen receptor loci by the pairwise cleavage of recombination signal sequences (RSSs by the RAG1 and RAG2 proteins via a nick-hairpin mechanism. The RSS contains highly conserved heptamer (consensus: 5'-CACAGTG and nonamer (consensus: 5'-ACAAAAACC motifs separated by either 12- or 23-base pairs of poorly conserved sequence. The high mobility group proteins HMGB1 and HMGB2 (HMGB1/2 are highly abundant architectural DNA binding proteins known to promote RAG-mediated synapsis and cleavage of consensus recombination signals in vitro by facilitating RSS binding and bending by the RAG1/2 complex. HMGB1/2 are known to recognize distorted DNA structures such as four-way junctions, and damaged or modified DNA. Whether HMGB1/2 can promote RAG-mediated DNA cleavage at sites lacking a canonical RSS by targeting or stabilizing structural distortions is unclear, but is important for understanding the etiology of chromosomal translocations involving antigen receptor genes and proto-oncogene sequences that do not contain an obvious RSS-like element. Results Here we identify a novel DNA breakpoint site in the plasmid V(DJ recombination substrate pGG49 (bps6197 that is cleaved by the RAG proteins via a nick-hairpin mechanism. The bps6197 sequence lacks a recognizable heptamer at the breakpoint (5'-CCTGACG-3' but contains a nonamer-like element (5'-ACATTAACC-3' 30 base pairs from the cleavage site. We find that RAG-mediated bps6197 cleavage is promoted by HMGB1/2, requiring both HMG-box domains to be intact to facilitate RAG-mediated cleavage, and is stimulated by synapsis with a 12-RSS. A dyad-symmetric inverted repeat sequence lying 5' to the breakpoint is implicated as a target for HMGB1/2 activity. Conclusion We have identified a novel DNA sequence, called bps6197, that supports standard V(DJ-type cleavage despite the absence of an apparent heptamer motif. Efficient RAG-mediated bps6197 cleavage requires

  5. HMGB1/2 can target DNA for illegitimate cleavage by the RAG1/2 complex.

    Science.gov (United States)

    Zhang, Ming; Swanson, Patrick C

    2009-03-24

    V(D)J recombination is initiated in antigen receptor loci by the pairwise cleavage of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins via a nick-hairpin mechanism. The RSS contains highly conserved heptamer (consensus: 5'-CACAGTG) and nonamer (consensus: 5'-ACAAAAACC) motifs separated by either 12- or 23-base pairs of poorly conserved sequence. The high mobility group proteins HMGB1 and HMGB2 (HMGB1/2) are highly abundant architectural DNA binding proteins known to promote RAG-mediated synapsis and cleavage of consensus recombination signals in vitro by facilitating RSS binding and bending by the RAG1/2 complex. HMGB1/2 are known to recognize distorted DNA structures such as four-way junctions, and damaged or modified DNA. Whether HMGB1/2 can promote RAG-mediated DNA cleavage at sites lacking a canonical RSS by targeting or stabilizing structural distortions is unclear, but is important for understanding the etiology of chromosomal translocations involving antigen receptor genes and proto-oncogene sequences that do not contain an obvious RSS-like element. Here we identify a novel DNA breakpoint site in the plasmid V(D)J recombination substrate pGG49 (bps6197) that is cleaved by the RAG proteins via a nick-hairpin mechanism. The bps6197 sequence lacks a recognizable heptamer at the breakpoint (5'-CCTGACG-3') but contains a nonamer-like element (5'-ACATTAACC-3') 30 base pairs from the cleavage site. We find that RAG-mediated bps6197 cleavage is promoted by HMGB1/2, requiring both HMG-box domains to be intact to facilitate RAG-mediated cleavage, and is stimulated by synapsis with a 12-RSS. A dyad-symmetric inverted repeat sequence lying 5' to the breakpoint is implicated as a target for HMGB1/2 activity. We have identified a novel DNA sequence, called bps6197, that supports standard V(D)J-type cleavage despite the absence of an apparent heptamer motif. Efficient RAG-mediated bps6197 cleavage requires the presence of HMGB1/2, is stimulated by

  6. Matrix metalloproteinases and gastrointestinal cancers: Impacts of dietary antioxidants

    Institute of Scientific and Technical Information of China (English)

    Sugreev; Verma; Kousik; Kesh; Nilanjan; Ganguly; Sayantan; Jana; Snehasikta; Swarnakar

    2014-01-01

    The process of carcinogenesis is tightly regulated by antioxidant enzymes and matrix degrading enzymes, namely, matrix metalloproteinases(MMPs). Degradation of extracellular matrix(ECM) proteins like collagen, proteoglycan, laminin, elastin and fibronectin is considered to be the prerequisite for tumor invasion and metastasis. MMPs can degrade essentially all of the ECM components and, most MMPs also substantially contribute to angiogenesis, differentiation, proliferation and apoptosis. Hence, MMPs are important regulators of tumor growth both at the primary site and in distant metastases; thus the enzymes are considered as important targets for cancer therapy. The implications of MMPs in cancers are no longer mysterious; however, the mechanism of action is yet to be explained. Herein, our major interest is to clarify how MMPs are tied up with gastrointestinal cancers. Gastrointestinal cancer is a variety of cancer types, including the cancers of gastrointestinal tract and organs, i.e., esophagus, stomach, biliary system, pancreas, small intestine, large intestine, rectum and anus. The activity of MMPs is regulated by its endogenous inhibitor tissue inhibitor of metallopro-teinase(TIMP) which bind MMPs with a 1:1 stoichiometry. In addition, RECK(reversion including cysteinerich protein with kazal motifs) is a membrane bound glycoprotein that inhibits MMP-2,-9 and-14. Moreover, α2-macroglobulin mediates the uptake of several MMPs thereby inhibit their activity. Cancerous conditions increase intrinsic reactive oxygen species(ROS) through mitochondrial dysfunction leading to altered protease/anti-protease balance. ROS, an index of oxidative stress is also involved in tumorigenesis by activation of different MAP kinase pathways including MMP induction. Oxidative stress is involved in cancer by changing the activity and expression of regulatory proteins especially MMPs. Epidemiological studies have shown that high intake of fruits that rich in antioxidants is

  7. Unsaturated Fatty Acids Drive Disintegrin and Metalloproteinase (ADAM)-dependent Cell Adhesion, Proliferation, and Migration by Modulating Membrane Fluidity*

    Science.gov (United States)

    Reiss, Karina; Cornelsen, Isabell; Husmann, Matthias; Gimpl, Gerald; Bhakdi, Sucharit

    2011-01-01

    The disintegrin-metalloproteinases ADAM10 and ADAM17 mediate the release of several cell signaling molecules and cell adhesion molecules such as vascular endothelial cadherin or L-selectin affecting endothelial permeability and leukocyte transmigration. Dysregulation of ADAM activity may contribute to the pathogenesis of vascular diseases, but the mechanisms underlying the control of ADAM functions are still incompletely understood. Atherosclerosis is characterized by lipid plaque formation and local accumulation of unsaturated free fatty acids (FFA). Here, we show that unsaturated FFA increase ADAM-mediated substrate cleavage. We demonstrate that these alterations are not due to genuine changes in enzyme activity, but correlate with changes in membrane fluidity as revealed by measurement of 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy and fluorescence recovery after photobleaching analyses. ELISA and immunoblot experiments conducted with granulocytes, endothelial cells, and keratinocytes revealed rapid increase of ectodomain shedding of ADAM10 and ADAM17 substrates upon membrane fluidization. Large amounts of unsaturated FFA may be liberated from cholesteryl esters in LDL that is entrapped in atherosclerotic lesions. Incubation of cells with thus modified LDL resulted in rapid cleavage of ADAM substrates with corresponding functional consequences on cell proliferation, cell migration, and endothelial permeability, events of high significance in atherogenesis. We propose that FFA represent critical regulators of ADAM function that may assume relevance in many biological settings through their influence on mobility of enzyme and substrate in lipid bilayers. PMID:21642425

  8. Unsaturated fatty acids drive disintegrin and metalloproteinase (ADAM)-dependent cell adhesion, proliferation, and migration by modulating membrane fluidity.

    Science.gov (United States)

    Reiss, Karina; Cornelsen, Isabell; Husmann, Matthias; Gimpl, Gerald; Bhakdi, Sucharit

    2011-07-29

    The disintegrin-metalloproteinases ADAM10 and ADAM17 mediate the release of several cell signaling molecules and cell adhesion molecules such as vascular endothelial cadherin or L-selectin affecting endothelial permeability and leukocyte transmigration. Dysregulation of ADAM activity may contribute to the pathogenesis of vascular diseases, but the mechanisms underlying the control of ADAM functions are still incompletely understood. Atherosclerosis is characterized by lipid plaque formation and local accumulation of unsaturated free fatty acids (FFA). Here, we show that unsaturated FFA increase ADAM-mediated substrate cleavage. We demonstrate that these alterations are not due to genuine changes in enzyme activity, but correlate with changes in membrane fluidity as revealed by measurement of 1,6-diphenyl-1,3,5-hexatriene fluorescence anisotropy and fluorescence recovery after photobleaching analyses. ELISA and immunoblot experiments conducted with granulocytes, endothelial cells, and keratinocytes revealed rapid increase of ectodomain shedding of ADAM10 and ADAM17 substrates upon membrane fluidization. Large amounts of unsaturated FFA may be liberated from cholesteryl esters in LDL that is entrapped in atherosclerotic lesions. Incubation of cells with thus modified LDL resulted in rapid cleavage of ADAM substrates with corresponding functional consequences on cell proliferation, cell migration, and endothelial permeability, events of high significance in atherogenesis. We propose that FFA represent critical regulators of ADAM function that may assume relevance in many biological settings through their influence on mobility of enzyme and substrate in lipid bilayers.

  9. Hydrogel-Framed Nanofiber Matrix Integrated with a Microfluidic Device for Fluorescence Detection of Matrix Metalloproteinases-9.

    Science.gov (United States)

    Han, Sang Won; Koh, Won-Gun

    2016-06-21

    Matrix metalloproteinases (MMPs) play a pivotal role in regulating the composition of the extracellular matrix and have a critical role in vascular disease, cancer progression, and bone disorders. This paper describes the design and fabrication of a microdevice as a new platform for highly sensitive MMP-9 detection. In this sensing platform, fluorescein isocyanate (FITC)-labeled MMP-9 specific peptides were covalently immobilized on an electrospun nanofiber matrix to utilize an enzymatic cleavage strategy. Prior to peptide immobilization, the nanofiber matrix was incorporated into hydrogel micropatterns for easy size control and handling of the nanofiber matrix. The resultant hydrogel-framed nanofiber matrix immobilizing the peptides was inserted into microfluidic devices consisting of reaction chambers and detection zones. The immobilized peptides were reacted with the MMP-9-containing solution in a reaction chamber, which resulted in the cleavage of the FITC-containing peptide fragments and subsequently generated fluorescent flow at the detection zone. As higher concentrations of the MMP-9 solution were introduced or larger peptide-immobilizing nanofiber areas were used, more peptides were cleaved, and a stronger fluorescence signal was observed. Due to the huge surface area of the nanofiber and small dimensions of the microsystem, a faster response time (30 min) and lower detection limit (10 pM) could be achieved in this study. The hydrogel-framed nanofiber matrix is disposable and can be replaced with new ones immobilizing either the same or different biomolecules for various bioassays, while the microfluidic system can be continuously reused.

  10. Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2.

    Science.gov (United States)

    Reinke, Lennart Michel; Spiegel, Martin; Plegge, Teresa; Hartleib, Anika; Nehlmeier, Inga; Gierer, Stefanie; Hoffmann, Markus; Hofmann-Winkler, Heike; Winkler, Michael; Pöhlmann, Stefan

    2017-01-01

    The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated.

  11. Potential matrix metalloproteinase inhibitors from edible marine algae: a review.

    Science.gov (United States)

    Thomas, Noel Vinay; Manivasagan, Panchanathan; Kim, Se-Kwon

    2014-05-01

    Matrix metalloproteinases are endopeptidases which belong to the group of metalloproteinases that contribute for the extra-cellular matrix degradation, and several tissue remodeling processes. An imbalance in the regulation of these endopeptidases eventually leads to several severe pathological complications like cancers, cardiac, cartilage, and neurological related diseases. Hence inhibitory substances of metalloproteinases (MMPIs) could prove beneficial in the management of above specified pathological conditions. The available synthetic MMPIs that have been reported until now have few shortcomings and thus many of them could not make to the final clinical trials. Hence a growing interest among researchers on screening of MMPIs from different natural resources is evident and especially natural products from marine origin. As there has been an unparalleled contribution of several biologically active compounds from marine resources that have shown profound applications in nutraceuticals, cosmeceuticals, and pharmaceuticals, we have attempted to discuss the various MMPIs from edible sea-weeds. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Fucoidans from marine algae as potential matrix metalloproteinase inhibitors.

    Science.gov (United States)

    Thomas, Noel Vinay; Kim, Se-Kwon

    2014-01-01

    Matrix metalloproteinases are endopeptidases which belong to the group of metalloproteinases that contribute for the extracellular matrix degradation and several tissue remodeling processes. An imbalance in the regulation of these endopeptidases eventually leads to several severe pathological complications like cancers, cardiac, cartilage, and neurological-related diseases. Hence, inhibitory substances of metalloproteinases (MMPIs) could prove beneficial in the management of above specified pathological conditions. The available synthetic MMPIs that have been reported until now have few shortcomings, and thus many of them could not make to the final clinical trials. Hence, a growing interest among researchers on screening of MMPIs from different natural resources is evident and especially natural products from marine origin. As there has been an unparalleled contribution of several biologically active compounds from marine resources that have shown a profound applications in nutraceuticals, cosmeceuticals, and pharmaceuticals, we have attempted to discuss the various MMPIs from edible seaweeds. © 2014 Elsevier Inc. All rights reserved.

  13. [Role of matrix metalloproteinases and tissue inhibitors of metalloproteinases in hypertension. Pathogenesis of hypertension and obesity].

    Science.gov (United States)

    Trojanek, Joanna B

    2015-01-01

    Hypertension (HT), obesity and related metabolic disorders are increasing cause diseases with risk of premature death in western societies. Both hypertension and obesity are characterized by similar disorders such as chronic low systemic inflammation, changes in the vessel wall, abdominal obesity, insulin-resistance or dyslipidemia. Chronic, untreated HT leads to adverse changes in internal organs like kidney damage, arterial remodeling and hypertrophy of the left ventricle. The important role metalloproteinases and their inhibitors (TIMPs) in the pathophysiology of hypertension is associated with the degradation of vascular wall components, especially collagen and elastin. The activated RAAS system (renin-angiotensin-aldosterone) is displaying direct impact in the pathogenesis and progress of hypertension. Angiotensin II affects the expression and activation of many growth factors, cytokines and MMPs. The fat tissue of obese people is in the state of low intensity chronic inflammation and undergoes continual process of remodeling. Obesity is one of the direct cause of hypertension.

  14. Agonist-regulated Cleavage of the Extracellular Domain of Parathyroid Hormone Receptor Type 1*

    Science.gov (United States)

    Klenk, Christoph; Schulz, Stefan; Calebiro, Davide; Lohse, Martin J.

    2010-01-01

    The receptor for parathyroid hormone (PTHR) is a main regulator of calcium homeostasis and bone maintenance. As a member of class B of G protein-coupled receptors, it harbors a large extracellular domain, which is required for ligand binding. Here, we demonstrate that the PTHR extracellular domain is cleaved by a protease belonging to the family of extracellular metalloproteinases. We show that the cleavage takes place in a region of the extracellular domain that belongs to an unstructured loop connecting the ligand-binding parts and that the N-terminal 10-kDa fragment is connected to the receptor core by a disulfide bond. Cleaved receptor revealed reduced protein stability compared with noncleaved receptor, suggesting degradation of the whole receptor. In the presence of the agonistic peptides PTH(1–34), PTH(1–14), or PTH(1–31), the processing of the PTHR extracellular domain was inhibited, and receptor protein levels were stabilized. A processed form of the PTHR was also detected in human kidney. These findings suggest a new model of PTHR processing and regulation of its stability. PMID:20080964

  15. Agonist-regulated cleavage of the extracellular domain of parathyroid hormone receptor type 1.

    Science.gov (United States)

    Klenk, Christoph; Schulz, Stefan; Calebiro, Davide; Lohse, Martin J

    2010-03-19

    The receptor for parathyroid hormone (PTHR) is a main regulator of calcium homeostasis and bone maintenance. As a member of class B of G protein-coupled receptors, it harbors a large extracellular domain, which is required for ligand binding. Here, we demonstrate that the PTHR extracellular domain is cleaved by a protease belonging to the family of extracellular metalloproteinases. We show that the cleavage takes place in a region of the extracellular domain that belongs to an unstructured loop connecting the ligand-binding parts and that the N-terminal 10-kDa fragment is connected to the receptor core by a disulfide bond. Cleaved receptor revealed reduced protein stability compared with noncleaved receptor, suggesting degradation of the whole receptor. In the presence of the agonistic peptides PTH(1-34), PTH(1-14), or PTH(1-31), the processing of the PTHR extracellular domain was inhibited, and receptor protein levels were stabilized. A processed form of the PTHR was also detected in human kidney. These findings suggest a new model of PTHR processing and regulation of its stability.

  16. PARP-1 cleavage fragments: signatures of cell-death proteases in neurodegeneration

    Directory of Open Access Journals (Sweden)

    Alexander Jonathan S

    2010-12-01

    Full Text Available Abstract The normal function of poly (ADP-ribose polymerase-1 (PARP-1 is the routine repair of DNA damage by adding poly (ADP ribose polymers in response to a variety of cellular stresses. Recently, it has become widely appreciated that PARP-1 also participates in diverse physiological and pathological functions from cell survival to several forms of cell death and has been implicated in gene transcription, immune responses, inflammation, learning, memory, synaptic functions, angiogenesis and aging. In the CNS, PARP inhibition attenuates injury in pathologies like cerebral ischemia, trauma and excitotoxicity demonstrating a central role of PARP-1 in these pathologies. PARP-1 is also a preferred substrate for several 'suicidal' proteases and the proteolytic action of suicidal proteases (caspases, calpains, cathepsins, granzymes and matrix metalloproteinases (MMPs on PARP-1 produces several specific proteolytic cleavage fragments with different molecular weights. These PARP-1 signature fragments are recognized biomarkers for specific patterns of protease activity in unique cell death programs. This review focuses on specific suicidal proteases active towards PARP-1 to generate signature PARP-1 fragments that can identify key proteases and particular forms of cell death involved in pathophysiology. The roles played by some of the PARP-1 fragments and their associated binding partners in the control of different forms of cell death are also discussed.

  17. Matrix metalloproteinases during and outside of migraine attacks without aura

    DEFF Research Database (Denmark)

    Ashina, M.; Tvedskov, J.F.; Thiesen, Kerstin Lipka

    2010-01-01

    Ashina M, Tvedskov JF, Lipka K, Bilello J, Penkowa M & Olesen J. Matrix metalloproteinases during and outside of migraine attacks without aura. Cephalalgia 2009. London. ISSN 0333-1024To test the hypothesis that permeability of the blood-brain barrier (BBB) is altered during migraine attack due...... to enhanced activation of matrix metalloproteinases (MMPs), we investigated MMP-3, MMP-9 and tissue inhibitor of metalloproteases (TIMP)-1 in the external jugular vein during and outside of migraine attacks in 21 patients with migraine without aura. In addition, we measured plasma levels of several other...

  18. Matrix metalloproteinase-8 overexpression prevents proper tissue repair

    DEFF Research Database (Denmark)

    Danielsen, Patricia Louise; Holst, Anders V; Maltesen, Henrik R

    2011-01-01

    The collagenolytic matrix metalloproteinase-8 (MMP-8) is essential for normal tissue repair but is often overexpressed in wounds with disrupted healing. Our aim was to study the impact of a local excess of this neutrophil-derived proteinase on wound healing using recombinant adenovirus-driven tra......The collagenolytic matrix metalloproteinase-8 (MMP-8) is essential for normal tissue repair but is often overexpressed in wounds with disrupted healing. Our aim was to study the impact of a local excess of this neutrophil-derived proteinase on wound healing using recombinant adenovirus...

  19. Tissue inhibitor of metalloproteinases-1 in breast cancer

    DEFF Research Database (Denmark)

    Würtz, Sidse Ørnbjerg; Rasmussen, Anne-Sofie Schrohl; Sørensen, Nanna Møller

    2005-01-01

    parameters a significant proportion of patients are over-treated. Thus, in order to improve stratification of breast cancer patients, additional prognostic factors need to be identified. Tissue inhibitor of metalloproteinases-1 (TIMP-1) is one of the promising candidates for new prognostic markers in breast...... is inhibition of the activity of various matrix metalloproteinases (MMPs). The association between high levels of protease inhibitor and poor prognosis may be somewhat surprising, as proteolytic activity plays a pivotal role in cancer cell invasion and metastasis. However, the recent discovery of other...

  20. Presence of Meiotic Spindles Indicates Early Cleavage of Embryos

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To assess whether the detection of the meiotic spindle could anticipate the appearance of early cleavage.Methods Oocytes were obtained from stimulated ovaries of consenting patients undergoing oocytes retrieval for ICSI.Spindles were imaged with the Polscope.After ICSI,oocytes with or without spindles were cultured for examination of early cleavage and embryo development.A total of 328 oocytes from 50 cycles were examined with the Polscope and inseminated by ICSI.Results Spindles were imaged in 81.7% of oocytes.After ICSI,more oocytes with spindles (78.4%) fertilized normally than oocytes without spindles (53.3%)(P<0.001).At 25-27 h post ICSI.more fertilized oocytes developed from oocytes with spindles (81.9%) were detected early cleavage than those from oocytes without spindles(28.1%)(P<0.001).Significantly more embryos with early cleavage (82.2%) developed to high quality embryos at d 3 compared with the embryos without early cleavage(48.3%)(P=0.001).The value of rs related to the relationship between spindles and early cleavage was 0.420(P<0.0001).Conclusion The existing of the early cleavage may have a predictive value on the opportunity of high quality embryos and the existing of the spindle may have a predictive value in the appearance of early cleavage.

  1. Amino Acid Derivatives as New Zinc Binding Groups for the Design of Selective Matrix Metalloproteinase Inhibitors

    Directory of Open Access Journals (Sweden)

    Mariateresa Giustiniano

    2013-01-01

    Full Text Available A number of matrix metalloproteinases (MMPs are important medicinal targets for conditions ranging from rheumatoid arthritis to cardiomyopathy, periodontal disease, liver cirrhosis, multiple sclerosis, and cancer invasion and metastasis, where they showed to have a dual role, inhibiting or promoting important processes involved in the pathology. MMPs contain a zinc (II ion in the protein active site. Small-molecule inhibitors of these metalloproteins are designed to bind directly to the active site metal ions. In an effort to devise new approaches to selective inhibitors, in this paper, we describe the synthesis and preliminary biological evaluation of amino acid derivatives as new zinc binding groups (ZBGs. The incorporation of selected metal-binding functions in more complex biphenyl sulfonamide moieties allowed the identification of one compound able to interact selectively with different MMP enzymatic isoforms.

  2. The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors.

    Science.gov (United States)

    Janek, Katharina; Niewienda, Agathe; Wöstemeyer, Johannes; Voigt, Jürgen

    2016-11-15

    Particular peptides generated from the vicilin-class(7S) globulin of the cocoa beans by acid-induced proteolysis during cocoa fermentation are essential precursors of the cocoa-specific aroma notes. As revealed by in vitro studies, the formation of the cocoa-specific aroma precursors depends on the particular cleavage specificity of the cocoa aspartic protease, which cannot be substituted by pepsin. Therefore, we have investigated the effects of aspartic protease inhibitors on both enzymes and comparatively studied their cleavage specificities using different protein substrates and MALDI-TOF mass spectrometric analyses of the generated oligopeptides. Three classes of cleavage sites have been identified and characterized: (I) sequences exclusively cleaved by the cocoa enzyme, (II) sequences cleaved by both pepsin and the cocoa enzyme, and (III) those cleaved exclusively by pepsin. In contrast to most aspartic proteases from other origins, basic amino acid residues, particularly lysine, were found to be abundant in the specific cleavage sites of the cocoa enzyme.

  3. The Generation of Dehydroalanine Residues in Protonated Polypeptides: Ion/Ion Reactions for Introducing Selective Cleavages

    Science.gov (United States)

    Peng, Zhou; Bu, Jiexun; McLuckey, Scott A.

    2017-05-01

    We examine a gas-phase approach for converting a subset of amino acid residues in polypeptide cations to dehydroalanine (Dha). Subsequent activation of the modified polypeptide ions gives rise to specific cleavage N-terminal to the Dha residue. This process allows for the incorporation of selective cleavages in the structural characterization of polypeptide ions. An ion/ion reaction within the mass spectrometer between a multiply protonated polypeptide and the sulfate radical anion introduces a radical site into the multiply protonated polypeptide reactant. Subsequent collisional activation of the polypeptide radical cation gives rise to radical side chain loss from one of several particular amino acid side chains (e.g., leucine, asparagine, lysine, glutamine, and glutamic acid) to yield a Dha residue. The Dha residues facilitate preferential backbone cleavages to produce signature c- and z-ions, demonstrated with cations derived from melittin, mechano growth factor (MGF), and ubiquitin. The efficiencies for radical side chain loss and for subsequent generation of specific c- and z-ions have been examined as functions of precursor ion charge state and activation conditions using cations of ubiquitin as a model for a small protein. It is noted that these efficiencies are not strongly dependent on ion trap collisional activation conditions but are sensitive to precursor ion charge state. Moderate to low charge states show the greatest overall yields for the specific Dha cleavages, whereas small molecule losses (e.g., water/ammonia) dominate at the lowest charge states and proton catalyzed amide bond cleavages that give rise to b- and y-ions tend to dominate at high charge states.

  4. Endoproteolytic cleavage of TUG protein regulates GLUT4 glucose transporter translocation.

    Science.gov (United States)

    Bogan, Jonathan S; Rubin, Bradley R; Yu, Chenfei; Löffler, Michael G; Orme, Charisse M; Belman, Jonathan P; McNally, Leah J; Hao, Mingming; Cresswell, James A

    2012-07-06

    To promote glucose uptake into fat and muscle cells, insulin causes the translocation of GLUT4 glucose transporters from intracellular vesicles to the cell surface. Previous data support a model in which TUG traps GLUT4-containing vesicles and tethers them intracellularly in unstimulated cells and in which insulin mobilizes this pool of vesicles by releasing this tether. Here we show that TUG undergoes site-specific endoproteolytic cleavage, which separates a GLUT4-binding, N-terminal region of TUG from a C-terminal region previously suggested to bind an intracellular anchor. Cleavage is accelerated by insulin stimulation in 3T3-L1 adipocytes and is highly dependent upon adipocyte differentiation. The N-terminal TUG cleavage product has properties of a novel 18-kDa ubiquitin-like modifier, which we call TUGUL. The C-terminal product is observed at the expected size of 42 kDa and also as a 54-kDa form that is released from membranes into the cytosol. In transfected cells, intact TUG links GLUT4 to PIST and also binds Golgin-160 through its C-terminal region. PIST is an effector of TC10α, a GTPase previously shown to transmit an insulin signal required for GLUT4 translocation, and we show using RNAi that TC10α is required for TUG proteolytic processing. Finally, we demonstrate that a cleavage-resistant form of TUG does not support highly insulin-responsive GLUT4 translocation or glucose uptake in 3T3-L1 adipocytes. Together with previous results, these data support a model whereby insulin stimulates TUG cleavage to liberate GLUT4 storage vesicles from the Golgi matrix, which promotes GLUT4 translocation to the cell surface and enhances glucose uptake.

  5. The Generation of Dehydroalanine Residues in Protonated Polypeptides: Ion/Ion Reactions for Introducing Selective Cleavages

    Science.gov (United States)

    Peng, Zhou; Bu, Jiexun; McLuckey, Scott A.

    2017-09-01

    We examine a gas-phase approach for converting a subset of amino acid residues in polypeptide cations to dehydroalanine (Dha). Subsequent activation of the modified polypeptide ions gives rise to specific cleavage N-terminal to the Dha residue. This process allows for the incorporation of selective cleavages in the structural characterization of polypeptide ions. An ion/ion reaction within the mass spectrometer between a multiply protonated polypeptide and the sulfate radical anion introduces a radical site into the multiply protonated polypeptide reactant. Subsequent collisional activation of the polypeptide radical cation gives rise to radical side chain loss from one of several particular amino acid side chains (e.g., leucine, asparagine, lysine, glutamine, and glutamic acid) to yield a Dha residue. The Dha residues facilitate preferential backbone cleavages to produce signature c- and z-ions, demonstrated with cations derived from melittin, mechano growth factor (MGF), and ubiquitin. The efficiencies for radical side chain loss and for subsequent generation of specific c- and z-ions have been examined as functions of precursor ion charge state and activation conditions using cations of ubiquitin as a model for a small protein. It is noted that these efficiencies are not strongly dependent on ion trap collisional activation conditions but are sensitive to precursor ion charge state. Moderate to low charge states show the greatest overall yields for the specific Dha cleavages, whereas small molecule losses (e.g., water/ammonia) dominate at the lowest charge states and proton catalyzed amide bond cleavages that give rise to b- and y-ions tend to dominate at high charge states. [Figure not available: see fulltext.

  6. Intein-Promoted Cyclization of Aspartic Acid Flanking the Intein Leads to Atypical N-Terminal Cleavage.

    Science.gov (United States)

    Minteer, Christopher J; Siegart, Nicolle M; Colelli, Kathryn M; Liu, Xinyue; Linhardt, Robert J; Wang, Chunyu; Gomez, Alvin V; Reitter, Julie N; Mills, Kenneth V

    2017-02-28

    Protein splicing is a post-translational reaction facilitated by an intein, or intervening protein, which involves the removal of the intein and the ligation of the flanking polypeptides, or exteins. A DNA polymerase II intein from Pyrococcus abyssi (Pab PolII intein) can promote protein splicing in vitro on incubation at high temperature. Mutation of active site residues Cys1, Gln185, and Cys+1 to Ala results in an inactive intein precursor, which cannot promote the steps of splicing, including cleavage of the peptide bond linking the N-extein and intein (N-terminal cleavage). Surprisingly, coupling the inactivating mutations to a change of the residue at the C-terminus of the N-extein (N-1 residue) from the native Asn to Asp reactivates N-terminal cleavage at pH 5. Similar "aspartic acid effects" have been observed in other proteins and peptides but usually only occur at lower pH values. In this case, however, the unusual N-terminal cleavage is abolished by mutations to catalytic active site residues and unfolding of the intein, indicating that this cleavage effect is mediated by the intein active site and the intein fold. We show via mass spectrometry that the reaction proceeds through cyclization of Asp resulting in anhydride formation coupled to peptide bond cleavage. Our results add to the richness of the understanding of the mechanism of protein splicing and provide insight into the stability of proteins at moderately low pH. The results also explain, and may help practitioners avoid, a side reaction that may complicate intein applications in biotechnology.

  7. Increased expression of matrix metalloproteinase-9 associated with gastric ulcer recurrence.

    Science.gov (United States)

    Li, Sen-Lin; Zhao, Jing-Run; Ren, Xiao-Yan; Xie, Jia-Ping; Ma, Qing-Zhu; Rong, Qiu-Hua

    2013-07-28

    To compare matrix metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 in gastric ulcer (GU) and chronic superficial gastritis (CSG). This study enrolled 63 patients with GU and 25 patients with CSG. During upper gastroduodenal endoscopy, we took samples of gastric mucosa from the antrum and ulcer site from patients with GU, and samples of antral mucosa from patients with CSG. Mucosal biopsy tissues were cultured for 24 h, and the culture supernatant was measured for levels of MMP-9 and TIMP-1. After receiving eradication therapy for Helicobacter pylori (H. pylori) and 8 wk proton-pump inhibitor therapy for GU, follow-up endoscopy examination was performed after 6 mo and whenever severe symptoms occurred. Levels of MMP-9 and TIMP-1 at the ulcer site or in the antrum were significantly higher in GU than CSG patients. MMP-9 levels at the ulcer site were significantly higher than in the antrum in GU patients, and had a significantly positive correlation with TIMP-1. MMP-9 levels were significantly higher in H. pylori-positive than H. pylori-negative GU and CSG patients. Levels of MMP-9 or TIMP-1 at the ulcer site were associated with the histological severity of activity and inflammation. About 57 GU patients were followed up, and seven had GU recurrence. H. pyloriinfection and MMP-9 levels were risk factors for the recurrence of GU adjusted for age and sex by multiple logistic regression analysis. MMP-9 may perform an important function in gastric ulcer formation and recurrence.

  8. Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue-damaging activities

    Science.gov (United States)

    Watanabe, Leandra; Shannon, John D.; Valente, Richard H.; Rucavado, Alexandra; Alape-Girón, Alberto; Kamiguti, Aura S.; Theakston, R. David G.; Fox, Jay W.; Gutiérrez, José María; Arni, Raghuvir K.

    2003-01-01

    BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated from the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117–Cys 197, Cys 159–Cys 181, Cys 157–Cys 164). It has the consensus sequence H142E143XXH146XXGXXH152, as well as the sequence C164I165M166, which characterize the “metzincin” superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1–152), comprising four α-helices and a five-stranded β-sheet, from the minor subdomain, which is formed by a single α-helix and several loops. The catalytic zinc ion is coordinated by the Nɛ2 nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active-site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix. PMID:14500885

  9. Yeast SREBP cleavage activation requires the Golgi Dsc E3 ligase complex.

    Science.gov (United States)

    Stewart, Emerson V; Nwosu, Christine C; Tong, Zongtian; Roguev, Assen; Cummins, Timothy D; Kim, Dong-Uk; Hayles, Jacqueline; Park, Han-Oh; Hoe, Kwang-Lae; Powell, David W; Krogan, Nevan J; Espenshade, Peter J

    2011-04-22

    Mammalian lipid homeostasis requires proteolytic activation of membrane-bound sterol regulatory element binding protein (SREBP) transcription factors through sequential action of the Golgi Site-1 and Site-2 proteases. Here we report that while SREBP function is conserved in fungi, fission yeast employs a different mechanism for SREBP cleavage. Using genetics and biochemistry, we identified four genes defective for SREBP cleavage, dsc1-4, encoding components of a transmembrane Golgi E3 ligase complex with structural homology to the Hrd1 E3 ligase complex involved in endoplasmic reticulum-associated degradation. The Dsc complex binds SREBP and cleavage requires components of the ubiquitin-proteasome pathway: the E2-conjugating enzyme Ubc4, the Dsc1 RING E3 ligase, and the proteasome. dsc mutants display conserved aggravating genetic interactions with components of the multivesicular body pathway in fission yeast and budding yeast, which lacks SREBP. Together, these data suggest that the Golgi Dsc E3 ligase complex functions in a post-ER pathway for protein degradation.

  10. Foot-and-mouth disease virus leader proteinase: structural insights into the mechanism of intermolecular cleavage.

    Science.gov (United States)

    Steinberger, Jutta; Grishkovskaya, Irina; Cencic, Regina; Juliano, Luiz; Juliano, Maria A; Skern, Tim

    2014-11-01

    Translation of foot-and-mouth disease virus RNA initiates at one of two start codons leading to the synthesis of two forms of leader proteinase L(pro) (Lab(pro) and Lb(pro)). These forms free themselves from the viral polyprotein by intra- and intermolecular self-processing and subsequently cleave the cellular eukaryotic initiation factor (eIF) 4 G. During infection, Lb(pro) removes six residues from its own C-terminus, generating sLb(pro). We present the structure of sLb(pro) bound to the inhibitor E64-R-P-NH2, illustrating how sLb(pro) can cleave between Lys/Gly and Gly/Arg pairs. In intermolecular cleavage on polyprotein substrates, Lb(pro) was unaffected by P1 or P1' substitutions and processed a substrate containing nine eIF4GI cleavage site residues whereas sLb(pro) failed to cleave the eIF4GI containing substrate and cleaved appreciably more slowly on mutated substrates. Introduction of 70 eIF4GI residues bearing the Lb(pro) binding site restored cleavage. These data imply that Lb(pro) and sLb(pro) may have different functions in infected cells.

  11. Porcine deltacoronavirus nsp5 inhibits interferon-β production through the cleavage of NEMO.

    Science.gov (United States)

    Zhu, Xinyu; Fang, Liurong; Wang, Dang; Yang, Yuting; Chen, Jiyao; Ye, Xu; Foda, Mohamed Frahat; Xiao, Shaobo

    2017-02-01

    Porcine deltacoronavirus (PDCoV) causes acute enteric disease and mortality in seronegative neonatal piglets. Previously we have demonstrated that PDCoV infection suppresses the production of interferon-beta (IFN-β), while the detailed mechanisms are poorly understood. Here, we demonstrate that nonstructural protein 5 (nsp5) of PDCoV, the 3C-like protease, significantly inhibits Sendai virus (SEV)-induced IFN-β production by targeting the NF-κB essential modulator (NEMO), confirmed by the diminished function of NEMO cleaved by PDCoV. The PDCoV nsp5 cleavage site in the NEMO protein was identified as glutamine 231, and was identical to the porcine epidemic diarrhea virus nsp5 cleavage site, revealing the likelihood of a common target in NEMO for coronaviruses. Furthermore, this cleavage impaired the ability of NEMO to activate the IFN response and downstream signaling. Taken together, our findings reveal PDCoV nsp5 to be a newly identified IFN antagonist and enhance the understanding of immune evasion by deltacoronaviruses. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Hemorrhage Caused by Snake Venom Metalloproteinases: A Journey of Discovery and Understanding.

    Science.gov (United States)

    Gutiérrez, José María; Escalante, Teresa; Rucavado, Alexandra; Herrera, Cristina

    2016-03-26

    The historical development of discoveries and conceptual frames for understanding the hemorrhagic activity induced by viperid snake venoms and by hemorrhagic metalloproteinases (SVMPs) present in these venoms is reviewed. Histological and ultrastructural tools allowed the identification of the capillary network as the main site of action of SVMPs. After years of debate, biochemical developments demonstrated that all hemorrhagic toxins in viperid venoms are zinc-dependent metalloproteinases. Hemorrhagic SVMPs act by initially hydrolyzing key substrates at the basement membrane (BM) of capillaries. This degradation results in the weakening of the mechanical stability of the capillary wall, which becomes distended owing of the action of the hemodynamic biophysical forces operating in the circulation. As a consequence, the capillary wall is disrupted and extravasation occurs. SVMPs do not induce rapid toxicity to endothelial cells, and the pathological effects described in these cells in vivo result from the mechanical action of these hemodynamic forces. Experimental evidence suggests that degradation of type IV collagen, and perhaps also perlecan, is the key event in the onset of microvessel damage. It is necessary to study this phenomenon from a holistic, systemic perspective in which the action of other venom components is also taken into consideration.

  13. Snake venom metalloproteinases and disintegrins: interactions with cells

    Directory of Open Access Journals (Sweden)

    Kamiguti A.S.

    1998-01-01

    Full Text Available Metalloproteinases and disintegrins are important components of most viperid and crotalid venoms. Large metalloproteinases referred to as MDC enzymes are composed of an N-terminal Metalloproteinase domain, a Disintegrin-like domain and a Cys-rich C-terminus. In contrast, disintegrins are small non-enzymatic RGD-containing cysteine-rich polypeptides. However, the disintegrin region of MDC enzymes bears a high degree of structural homology to that of the disintegrins, although it lacks the RGD motif. Despite these differences, both components share the property of being able to recognize integrin cell surface receptors and thereby to inhibit integrin-dependent cell reactions. Recently, several membrane-bound MDC enzymes, closely related to soluble venom MDC enzymes, have been described in mammalian cells. This group of membrane-anchored mammalian enzymes is also called the ADAM family of proteins due to the structure revealing A Disintegrin And Metalloproteinase domains. ADAMs are involved in the shedding of molecules from the cell surface, a property which is also shared by some venom MDC enzymes.

  14. Venous aneurysm complicating arteriovenous fistula access and matrix metalloproteinases

    Directory of Open Access Journals (Sweden)

    Serra Raffaele

    2015-01-01

    Full Text Available Introduction: An arteriovenous fistula (AVF for placed for hemodialysis may be burdened by one particular complication-the formation of a venous aneurysm. It has been shown that matrix metalloproteinases (MMPs and neutrophil gelatinase-associated lipocalin (NGAL could represent markers of disease in both venous and arterial vessels.

  15. Plasma tissue inhibitor of metalloproteinases-1 as a biological marker?

    DEFF Research Database (Denmark)

    Lomholt, Anne F.; Frederiksen, Camilla B.; Christensen, Ib J.;

    2007-01-01

    Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) may be a valuable biological marker in Colorectal Cancer (CRC). However, prospective validation of TIMP-1 as a biological marker should include a series of pre-analytical considerations. TIMP-1 is stored in platelets, which may degranulate during ...

  16. Beneficial Regulation of Matrix Metalloproteinases for Skin Health

    Science.gov (United States)

    Philips, Neena; Auler, Susan; Hugo, Raul; Gonzalez, Salvador

    2011-01-01

    Matrix metalloproteinases (MMPs) are essential to the remodeling of the extracellular matrix. While their upregulation facilitates aging and cancer, they are essential to epidermal differentiation and the prevention of wound scars. The pharmaceutical industry is active in identifying products that inhibit MMPs to prevent or treat aging and cancer and products that stimulate MMPs to prevent epidermal hyperproliferative diseases and wound scars. PMID:21423679

  17. Synthetic tools to illuminate matrix metalloproteinase and proteasome activities

    NARCIS (Netherlands)

    Geurink, Paulus Petrus

    2010-01-01

    This thesis describes the design, synthesis and application of chemical tools for the activity-based protein profiling of proteases, with the main focus on matrix metalloproteinases (MMPs) and the proteasome. The use of photoaffinity labeling is described and the thesis starts with an extensive

  18. Spontaneous metastasis in matrix metalloproteinase 3-deficient mice

    DEFF Research Database (Denmark)

    Juncker-Jensen, Anna; Rømer, John; Pennington, Caroline J

    2009-01-01

    Matrix metalloproteinases (MMPs) have been linked to the metastatic potential of tumor cells due to their ability to degrade the extracellular matrix. MMP-3 (stromelysin-1) is upregulated in a wide variety of human tumors. We used the MMTV-PyMT breast cancer model to determine if MMP-3 is involved...

  19. Unraveling the processing and activation of snake venom metalloproteinases.

    Science.gov (United States)

    Portes-Junior, José A; Yamanouye, Norma; Carneiro, Sylvia M; Knittel, Paloma S; Sant'Anna, Sávio S; Nogueira, Fabio C S; Junqueira, Magno; Magalhães, Geraldo S; Domont, Gilberto B; Moura-da-Silva, Ana M

    2014-07-03

    Snake venom metalloproteinases (SVMPs) are zinc-dependent enzymes responsible for most symptoms of human envenoming. Like matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase (ADAM) proteins, SVMPs are synthesized as zymogens, and enzyme activation is regulated by hydrolysis of their prodomain, but the processing of SVMPs is still unclear. In this study, we attempted to identify the presence of prodomain in different compartments of snake venom glands as zymogens or in the free form to elucidate some mechanism involved in SVMP activation. Using antibodies obtained by immunization with a recombinant prodomain, bands of zymogen molecular mass and prodomain peptides were detected mostly in gland extracts all along the venom production cycle and in the venom collected from the lumen at the peak of venom production. Prodomain was detected in secretory cells mostly in the secretory vesicles near the Golgi. We hypothesize that the processing of SVMPs starts within secretory vesicles and continues in the lumen of the venom gland just after enzyme secretion and involves different steps compared to ADAMs and MMPs but can be used as a model for studying the relevance of peptides resulting from prodomain processing and degradation for controlling the activity of metalloproteinases.

  20. Synthetic tools to illuminate matrix metalloproteinase and proteasome activities

    NARCIS (Netherlands)

    Geurink, Paulus Petrus

    2010-01-01

    This thesis describes the design, synthesis and application of chemical tools for the activity-based protein profiling of proteases, with the main focus on matrix metalloproteinases (MMPs) and the proteasome. The use of photoaffinity labeling is described and the thesis starts with an extensive outl

  1. Isolation and characterization of chicken bile matrix metalloproteinase

    Science.gov (United States)

    Avian bile is rich in matrix metalloproteinases (MMP), the enzymes that cleave extracellular matrix (ECM) proteins such as collagens and proteoglycans. Changes in bile MMP expression have been correlated with hepatic and gall bladder pathologies but the significance of their expression in normal, he...

  2. Chicken bile Matrix metalloproteinase; its characterization and significance

    Science.gov (United States)

    Previous studies from our lab had shown that the avian bile was rich in matrix metalloproteinase (MMP), enzymes implicated in the degradation of extracellular matrices (ECM) such as collagens and proteoglycans. We hypothesized that bile MMP may be evolutionarily associated with the digestion of ECM ...

  3. Mercury Detoxification by Bacteria: Simulations of Transcription Activation and Mercury-Carbon Bond Cleavage

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Hao-Bo [ORNL; Parks, Jerry M [ORNL; Johs, Alexander [ORNL; Smith, Jeremy C [ORNL

    2011-01-01

    In this chapter, we summarize recent work from our laboratory and provide new perspective on two important aspects of bacterial mercury resistance: the molecular mechanism of transcriptional regulation by MerR, and the enzymatic cleavage of the Hg-C bond in methylmercury by the organomercurial lyase, MerB. Molecular dynamics (MD) simulations of MerR reveal an opening-and-closing dynamics, which may be involved in initiating transcription of mercury resistance genes upon Hg(II) binding. Density functional theory (DFT) calculations on an active-site model of the enzyme reveal how MerB catalyzes the Hg-C bond cleavage using cysteine coordination and acid-base chemistry. These studies provide insight into the detailed mechanisms of microbial gene regulation and defense against mercury toxicity.

  4. Structural basis for duplex RNA recognition and cleavage by Archaeoglobus fulgidus C3PO

    Science.gov (United States)

    Parizotto, Eneida A; Lowe, Edward D; Parker, James S

    2013-01-01

    Oligomeric complexes of Trax and Translin proteins, known as C3POs, participate in a variety of eukaryotic nucleic acid metabolism pathways including RNAi and tRNA processing. In RNAi in humans and Drosophila, C3PO activates pre-RISC by removing the passenger strand of the siRNA precursor duplex using nuclease activity present in Trax. It is not known how C3POs engage with nucleic acid substrates. Here we identify a single protein from Archaeoglobus fulgidus that assembles into an octamer with striking similarity to human C3PO. The structure in complex with duplex RNA reveals that the octamer entirely encapsulates a single thirteen base-pair RNA duplex inside a large inner cavity. Trax-like subunit catalytic sites target opposite strands of the duplex for cleavage, separated by seven base pairs. The structure provides insight into the mechanism of RNA recognition and cleavage by an archaeal C3PO-like complex. PMID:23353787

  5. Apoptosis Mediated by HIV Protease is Preceded by Cleavage of Bcl-2

    Science.gov (United States)

    Strack, Peter R.; West Frey, Michelle; Rizzo, Christopher J.; Cordova, Beverly; George, Henry J.; Meade, Raymond; Ho, Siew Peng; Corman, Jeanne; Tritch, Radonna; Korant, Bruce D.

    1996-09-01

    Expression of the human immunodeficiency virus type 1 (HIV) protease in cultured cells leads to apoptosis, preceded by cleavage of bcl-2, a key negative regulator of cell death. In contrast, a high level of bcl-2 protects cells in vitro and in vivo from the viral protease and prevents cell death following HIV infection of human lymphocytes, while reducing the yields of viral structural proteins, infectivity, and tumor necrosis factor α . We present a model for HIV replication in which the viral protease depletes the infected cells of bcl-2, leading to oxidative stress-dependent activation of NFkappa B, a cellular factor required for HIV transcription, and ultimately to cell death. Purified bcl-2 is cleaved by HIV protease between phenylalanine 112 and alanine 113. The results suggest a new option for HIV gene therapy; bcl-2 muteins that have noncleavable alterations surrounding the HIV protease cleavage site.

  6. A cleavage toughness master curve model

    Science.gov (United States)

    Odette, G. R.; He, M. Y.

    2000-12-01

    Development of fusion power will require a fracture toughness database, derived largely from small specimen tests, closely integrated with methods to assess first wall and blanket structural integrities. A master curve-shift (MC-ΔT) method has been proposed as an engineering expedient to treat the effects of structural geometry, irradiation, loading rates and safety margins. However, a number of issues related to the MC-ΔT method remain to be resolved, including the universality of MC shapes. A new micromechanical model of fracture toughness in the cleavage transition regime is proposed that combines analytical representations of finite element analysis simulations of crack-tip stress fields with a local critical stress-critical stressed area (σ∗-A∗) fracture criterion. This model, has been successful in predicting geometry effects, as well as high loading rate and irradiation hardening-induced Charpy shifts. By incorporating a modest temperature dependence in σ∗(T), an inconsistency between model predictions and an observed universal-type MC shape is resolved.

  7. ZFN-Site searches genomes for zinc finger nuclease target sites and off-target sites

    Directory of Open Access Journals (Sweden)

    Iseli Christian

    2011-05-01

    Full Text Available Abstract Background Zinc Finger Nucleases (ZFNs are man-made restriction enzymes useful for manipulating genomes by cleaving target DNA sequences. ZFNs allow therapeutic gene correction or creation of genetically modified model organisms. ZFN specificity is not absolute; therefore, it is essential to select ZFN target sites without similar genomic off-target sites. It is important to assay for off-target cleavage events at sites similar to the target sequence. Results ZFN-Site is a web interface that searches multiple genomes for ZFN off-target sites. Queries can be based on the target sequence or can be expanded using degenerate specificity to account for known ZFN binding preferences. ZFN off-target sites are outputted with links to genome browsers, facilitating off-target cleavage site screening. We verified ZFN-Site using previously published ZFN half-sites and located their target sites and their previously described off-target sites. While we have tailored this tool to ZFNs, ZFN-Site can also be used to find potential off-target sites for other nucleases, such as TALE nucleases. Conclusions ZFN-Site facilitates genome searches for possible ZFN cleavage sites based on user-defined stringency limits. ZFN-Site is an improvement over other methods because the FetchGWI search engine uses an indexed search of genome sequences for all ZFN target sites and possible off-target sites matching the half-sites and stringency limits. Therefore, ZFN-Site does not miss potential off-target sites.

  8. Efficacy of a metalloproteinase inhibitor in spinal cord injured dogs.

    Science.gov (United States)

    Levine, Jonathan M; Cohen, Noah D; Heller, Michael; Fajt, Virginia R; Levine, Gwendolyn J; Kerwin, Sharon C; Trivedi, Alpa A; Fandel, Thomas M; Werb, Zena; Modestino, Augusta; Noble-Haeusslein, Linda J

    2014-01-01

    Matrix metalloproteinase-9 is elevated within the acutely injured murine spinal cord and blockade of this early proteolytic activity with GM6001, a broad-spectrum matrix metalloproteinase inhibitor, results in improved recovery after spinal cord injury. As matrix metalloproteinase-9 is likewise acutely elevated in dogs with naturally occurring spinal cord injuries, we evaluated efficacy of GM6001 solubilized in dimethyl sulfoxide in this second species. Safety and pharmacokinetic studies were conducted in naïve dogs. After confirming safety, subsequent pharmacokinetic analyses demonstrated that a 100 mg/kg subcutaneous dose of GM6001 resulted in plasma concentrations that peaked shortly after administration and were sustained for at least 4 days at levels that produced robust in vitro inhibition of matrix metalloproteinase-9. A randomized, blinded, placebo-controlled study was then conducted to assess efficacy of GM6001 given within 48 hours of spinal cord injury. Dogs were enrolled in 3 groups: GM6001 dissolved in dimethyl sulfoxide (n = 35), dimethyl sulfoxide (n = 37), or saline (n = 41). Matrix metalloproteinase activity was increased in the serum of injured dogs and GM6001 reduced this serum protease activity compared to the other two groups. To assess recovery, dogs were a priori stratified into a severely injured group and a mild-to-moderate injured group, using a Modified Frankel Scale. The Texas Spinal Cord Injury Score was then used to assess long-term motor/sensory function. In dogs with severe spinal cord injuries, those treated with saline had a mean motor score of 2 (95% CI 0-4.0) that was significantly (Pinjured cord.

  9. Quantification of DNA cleavage specificity in Hi-C experiments.

    Science.gov (United States)

    Meluzzi, Dario; Arya, Gaurav

    2016-01-01

    Hi-C experiments produce large numbers of DNA sequence read pairs that are typically analyzed to deduce genomewide interactions between arbitrary loci. A key step in these experiments is the cleavage of cross-linked chromatin with a restriction endonuclease. Although this cleavage should happen specifically at the enzyme's recognition sequence, an unknown proportion of cleavage events may involve other sequences, owing to the enzyme's star activity or to random DNA breakage. A quantitative estimation of these non-specific cleavages may enable simulating realistic Hi-C read pairs for validation of downstream analyses, monitoring the reproducibility of experimental conditions and investigating biophysical properties that correlate with DNA cleavage patterns. Here we describe a computational method for analyzing Hi-C read pairs to estimate the fractions of cleavages at different possible targets. The method relies on expressing an observed local target distribution downstream of aligned reads as a linear combination of known conditional local target distributions. We validated this method using Hi-C read pairs obtained by computer simulation. Application of the method to experimental Hi-C datasets from murine cells revealed interesting similarities and differences in patterns of cleavage across the various experiments considered.

  10. A Subset of Membrane-Altering Agents and γ-Secretase Modulators Provoke Nonsubstrate Cleavage by Rhomboid Proteases

    Directory of Open Access Journals (Sweden)

    Siniša Urban

    2014-09-01

    Full Text Available Rhomboid proteases are integral membrane enzymes that regulate cell signaling, adhesion, and organelle homeostasis pathways, making substrate specificity a key feature of their function. Interestingly, we found that perturbing the membrane pharmacologically in living cells had little effect on substrate processing but induced inappropriate cleavage of nonsubstrates by rhomboid proteases. A subclass of drugs known to modulate γ-secretase activity acted on the membrane directly and induced nonsubstrate cleavage by rhomboid proteases but left true substrate cleavage sites unaltered. These observations highlight an active role for the membrane in guiding rhomboid selectivity and caution that membrane-targeted drugs should be evaluated for cross-activity against membrane-resident enzymes that are otherwise unrelated to the intended drug target. Furthermore, some γ-secretase-modulating activity or toxicity could partly result from global membrane effects.

  11. Expression of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in ulcerative colitis

    Institute of Scientific and Technical Information of China (English)

    Ying-De Wang; Pei-Yun Yan

    2006-01-01

    AIM: To examine the expression of metalloproteinase-1(MMP-1) and tissue inhibitor of metalloproteinase-1(TIMP-1) in the colonic mucosa of patients with ulcerative colitis (UC).METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry were used to study the expression of MMP-1 and TTMP-1 at both mRNA and protein levels in patients with UC and controls. The relationship between MMP-1 mRNA, TIMP-1mRNA, MMP-1 mRNA/TIMP-1 mRNA ratio and the severity of clinical symptoms of the patients with UC were also analyzed.RESULTS: The expression of MMP-1 mRNA and TIMP-1mRNA in the ulcerated and inflamed colonic mucosa was significantly higher than that in the non-inflamed colonic mucosa (P < 0.001), but there was no statistically significant difference in the non-inflamed colcnic mucosa of UC patients and normal controls (P > 0.05). The mRNA expression of MMP-1 and TIMP-1 in ulcerated colonic mucosa of UC patients was increased by 80-fold and 2.2-fold,respectively when compared with the normal controls. In the inflamed colonic mucosa, the increase was 30-fold and 1.6-fold, respectively. Tmmunohistochemical analysis showed that among the ulcerated, inflamed, and non-inflamed colonic mucosae of UC patients and the normal controls, the positive rate of MMP-1 expression was 87%, 87%, 40% and 35% respectively, and the positive rate of TIMP-1 expression was 89%, 89%, 80%and 75%, respectively. Furthermore, the expression of MMP-1 mRNA, TIMP-1 mRNA and the MMP-1 mRNA/TIMP-1 mRNA ratio were correlated with the severity of clinical symptoms (P <0.05).CONCLUSION: Excessive expression of MMP-1 in the diseased colonic mucosa causes excessive hydrolysis of the extracellular matrix (ECM) and ulceration in UC patients. MMP-1 mRNA, TIMP-1 mRNA and MMP-1 mRNA/TIMP-1 mRNA ratio can be used as biomarkers to judge the severity of clinical symptoms in patients with UC.Exogenous TIMP-1 or MMP-1 inhibitor therapy is a novel treatment for patients with UC.

  12. Matrix metalloproteinase 2 and membrane type 1 matrix metalloproteinase co-regulate axonal outgrowth of mouse retinal ganglion cells

    DEFF Research Database (Denmark)

    Gaublomme, Djoere; Buyens, Tom; De Groef, Lies

    2014-01-01

    regenerative therapies, an improved understanding of axonal outgrowth and the various molecules influencing it, is highly needed. Matrix metalloproteinases (MMPs) constitute a family of zinc-dependent proteases that were sporadically reported to influence axon outgrowth. Using an ex vivo retinal explant model...

  13. The caspase-generated cleavage product of Ets-1 p51 and Ets-1 p27, Cp17, induces apoptosis.

    Science.gov (United States)

    Choul-Li, Souhaila; Tulasne, David; Aumercier, Marc

    2016-11-04

    The transcription factor Ets-1 is involved in various physiological processes and invasive pathologies. Human Ets-1 exists under three isoforms: p51, the predominant full-length isoform, p42 and p27, shorter alternatively spliced isoforms. We have previously demonstrated that Ets-1 p51, but not the spliced variant Ets-1 p42, is processed by caspases in vitro and during apoptosis. However, the caspase cleavage of the second spliced variant Ets-1 p27 remains to investigate. In the present study, we demonstrate that Ets-1 p27 is a cleavage substrate of caspases. We show that Ets-1 p27 is processed in vitro by caspase-3, resulting in three C-terminal fragments Cp20, Cp17 and Cp14. Similarly, Ets-1 p27 was cleaved during apoptotic cell death induced by anisomycin, producing fragments consistent with those observed in in vitro cleavage assay. These fragments are generated by cleavage at three sites located in the exon VII-encoded region of Ets-1 p27. As a functional consequences, Cp17 fragment, the major cleavage product generated during apoptosis, induced itself apoptosis when transfected into cells. Our results show that Ets-1 p27 is cleaved in the same manner as Ets-1 p51 within the exon VII-encoded region, thus generating a stable C-terminal fragment that induces cell death by initiating apoptosis. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Effects of S1 Cleavage on the Structure, Surface Export, and Signaling Activity of Human Notch1 and Notch2

    Science.gov (United States)

    Gordon, Wendy R.; Vardar-Ulu, Didem; L'Heureux, Sarah; Ashworth, Todd; Malecki, Michael J.; Sanchez-Irizarry, Cheryll; McArthur, Debbie G.; Histen, Gavin; Mitchell, Jennifer L.; Aster, Jon C.; Blacklow, Stephen C.

    2009-01-01

    Background Notch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR) of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD) that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia. Principal Findings The X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity. Conclusions/Significance S1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with

  15. Effects of S1 cleavage on the structure, surface export, and signaling activity of human Notch1 and Notch2.

    Directory of Open Access Journals (Sweden)

    Wendy R Gordon

    Full Text Available Notch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia.The X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity.S1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with models for receptor activation in which

  16. Effects of S1 Cleavage on the Structure, Surface Export, and Signaling Activity of Human Notch1 and Notch2

    Energy Technology Data Exchange (ETDEWEB)

    Gordon, Wendy R.; Vardar-Ulu, Didem; L' Heureux, Sarah; Ashworth, Todd; Malecki, Michael J.; Sanchez-Irizarry, Cheryll; McArthur, Debbie G.; Histen, Gavin; Mitchell, Jennifer L.; Aster, Jon C.; Blacklow, Stephen C.; (BWH); (Wellesley)

    2009-09-25

    Notch receptors are normally cleaved during maturation by a furin-like protease at an extracellular site termed S1, creating a heterodimer of non-covalently associated subunits. The S1 site lies within a key negative regulatory region (NRR) of the receptor, which contains three highly conserved Lin12/Notch repeats and a heterodimerization domain (HD) that interact to prevent premature signaling in the absence of ligands. Because the role of S1 cleavage in Notch signaling remains unresolved, we investigated the effect of S1 cleavage on the structure, surface trafficking and ligand-mediated activation of human Notch1 and Notch2, as well as on ligand-independent activation of Notch1 by mutations found in human leukemia. The X-ray structure of the Notch1 NRR after furin cleavage shows little change when compared with that of an engineered Notch1 NRR lacking the S1-cleavage loop. Likewise, NMR studies of the Notch2 HD domain show that the loop containing the S1 site can be removed or cleaved without causing a substantial change in its structure. However, Notch1 and Notch2 receptors engineered to resist S1 cleavage exhibit unexpected differences in surface delivery and signaling competence: S1-resistant Notch1 receptors exhibit decreased, but detectable, surface expression and ligand-mediated receptor activation, whereas S1-resistant Notch2 receptors are fully competent for cell surface delivery and for activation by ligands. Variable dependence on S1 cleavage also extends to T-ALL-associated NRR mutations, as common class 1 mutations display variable decrements in ligand-independent activation when introduced into furin-resistant receptors, whereas a class 2 mutation exhibits increased signaling activity. S1 cleavage has distinct effects on the surface expression of Notch1 and Notch2, but is not generally required for physiologic or pathophysiologic activation of Notch proteins. These findings are consistent with models for receptor activation in which ligand-binding or

  17. An effective system for detecting protein-protein interaction based on in vivo cleavage by PPV NIa protease.

    Science.gov (United States)

    Zheng, Nuoyan; Huang, Xiahe; Yin, Bojiao; Wang, Dan; Xie, Qi

    2012-12-01

    Detection of protein-protein interaction can provide valuable information for investigating the biological function of proteins. The current methods that applied in protein-protein interaction, such as co-immunoprecipitation and pull down etc., often cause plenty of working time due to the burdensome cloning and purification procedures. Here we established a system that characterization of protein-protein interaction was accomplished by co-expression and simply purification of target proteins from one expression cassette within E. coli system. We modified pET vector into co-expression vector pInvivo which encoded PPV NIa protease, two cleavage site F and two multiple cloning sites that flanking cleavage sites. The target proteins (for example: protein A and protein B) were inserted at multiple cloning sites and translated into polyprotein in the order of MBP tag-protein A-site F-PPV NIa protease-site F-protein B-His(6) tag. PPV NIa protease carried out intracellular cleavage along expression, then led to the separation of polyprotein components, therefore, the interaction between protein A-protein B can be detected through one-step purification and analysis. Negative control for protein B was brought into this system for monitoring interaction specificity. We successfully employed this system to prove two cases of reported protien-protein interaction: RHA2a/ANAC and FTA/FTB. In conclusion, a convenient and efficient system has been successfully developed for detecting protein-protein interaction.

  18. Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation.

    Science.gov (United States)

    Ginster, Stefanie; Bardet, Maureen; Unterreiner, Adeline; Malinverni, Claire; Renner, Florian; Lam, Stephen; Freuler, Felix; Gerrits, Bertran; Voshol, Johannes; Calzascia, Thomas; Régnier, Catherine H; Renatus, Martin; Nikolay, Rainer; Israël, Laura; Bornancin, Frédéric

    2017-01-01

    The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed that TRAF6 can activate MALT1 independently of the CBM. Intramolecular cleavage at R781/R770 removes a C-terminal TRAF6-binding site in both MALT1 isoforms, leaving MALT1B devoid of the two key interaction sites with TRAF6. A previously identified auto-proteolysis site of MALT1 at R149 leads to deletion of the death-domain, thereby abolishing interaction with BCL10. By using MALT1 isoforms and cleaved fragments thereof, as well as TRAF6 WT and mutant forms, this work shows that TRAF6 induces N-terminal auto-proteolytic cleavage of MALT1 at R149 and accelerates MALT1 protein turnover. The MALT1 fragment generated by N-terminal self-cleavage at R149 was labile and displayed enhanced signaling properties that required an intact K644 residue, previously shown to be a site for mono-ubiquitination of MALT1. Conversely, C-terminal self-cleavage at R781/R770 hampered the ability for self-cleavage at R149 and stabilized MALT1 by hindering interaction with TRAF6. C-terminal self-cleavage had limited impact on MALT1A but severely reduced MALT1B proteolytic and signaling functions. It also abrogated NF-κB activation by N-terminally cleaved MALT1A. Altogether, this study provides further insights into mechanisms that regulate the scaffolding and activation cycle of MALT1. It also emphasizes the reduced functional capacity of MALT1B as compared to MALT1A.

  19. Two Antagonistic MALT1 Auto-Cleavage Mechanisms Reveal a Role for TRAF6 to Unleash MALT1 Activation

    Science.gov (United States)

    Renner, Florian; Lam, Stephen; Freuler, Felix; Gerrits, Bertran; Voshol, Johannes; Calzascia, Thomas; Régnier, Catherine H.; Renatus, Martin; Nikolay, Rainer; Israël, Laura; Bornancin, Frédéric

    2017-01-01

    The paracaspase MALT1 has arginine-directed proteolytic activity triggered by engagement of immune receptors. Recruitment of MALT1 into activation complexes is required for MALT1 proteolytic function. Here, co-expression of MALT1 in HEK293 cells, either with activated CARD11 and BCL10 or with TRAF6, was used to explore the mechanism of MALT1 activation at the molecular level. This work identified a prominent self-cleavage site of MALT1 isoform A (MALT1A) at R781 (R770 in MALT1B) and revealed that TRAF6 can activate MALT1 independently of the CBM. Intramolecular cleavage at R781/R770 removes a C-terminal TRAF6-binding site in both MALT1 isoforms, leaving MALT1B devoid of the two key interaction sites with TRAF6. A previously identified auto-proteolysis site of MALT1 at R149 leads to deletion of the death-domain, thereby abolishing interaction with BCL10. By using MALT1 isoforms and cleaved fragments thereof, as well as TRAF6 WT and mutant forms, this work shows that TRAF6 induces N-terminal auto-proteolytic cleavage of MALT1 at R149 and accelerates MALT1 protein turnover. The MALT1 fragment generated by N-terminal self-cleavage at R149 was labile and displayed enhanced signaling properties that required an intact K644 residue, previously shown to be a site for mono-ubiquitination of MALT1. Conversely, C-terminal self-cleavage at R781/R770 hampered the ability for self-cleavage at R149 and stabilized MALT1 by hindering interaction with TRAF6. C-terminal self-cleavage had limited impact on MALT1A but severely reduced MALT1B proteolytic and signaling functions. It also abrogated NF-κB activation by N-terminally cleaved MALT1A. Altogether, this study provides further insights into mechanisms that regulate the scaffolding and activation cycle of MALT1. It also emphasizes the reduced functional capacity of MALT1B as compared to MALT1A. PMID:28052131

  20. Expression of matrix metalloproteinase-7 and tissue inhibitor of metalloproteinase-2 in human endometrial carcinoma

    Institute of Scientific and Technical Information of China (English)

    Chen Mei; Bo Nai-xiu; Huang Ya-jun; Dai Qi; Gong Li-mei

    2010-01-01

    Objective: To investigate the expression of matrix metalloproteinase-7 (MMP-7) and its tissue inhibitor (TIMP-2) in endometrial carcinoma and analyze their significance in endometrial cancer′s invasion and metastasis. Methods: Endometrial tissues were collected from 64 patients with endometrial carcinoma, 20 patients with endometrial hyperplasia and 20 normal women. The expressions of MMP-7, TIMP-2 in endometrium were measured by immuohistochemistry. Results: Expressions of MMP-7, TIMP-2 in endometrium of patients with endometrial carcinoma were significantly higher than those in normal endometrium (P<0.05). MMP-7 expression increased with surgical-pathological staging, depth of myometrial invasion, histologic grades and lymph node metastasis (P<0.05), while TIMP-2 expression was related to lymph node metastasis (P<0.05). TIMP-2 expression in endometrial cancer was significantly higher than that in hyperplastic endometrium (P<0.05). Expressions of TIMP-2 and MMP-7 in endometrium of patients with endometrial carcinoma were positively correlated (r=0.654, P<0.001). Conclusion: Highly expressed MMP-7 and TIMP-2 in endometrium may be related to development, invasion and metastasis of endometrial cancers.

  1. Implementation of a combinatorial cleavage and deprotection scheme

    DEFF Research Database (Denmark)

    Nielsen, John; Rasmussen, Palle H.

    1996-01-01

    Phthalhydrazide libraries are synthesized in solution from substituted hydrazines and phthalimides in several different library formats including single compounds, indexed sub-libraries and a full library. When carried out during solid-phase synthesis, this combinatorial cleavage and deprotection...

  2. Synthesis and Cleavage Activity of Artifical Minic Polypeptides

    Institute of Scientific and Technical Information of China (English)

    Yong YE; Xiao Lian HU; Ping LI; Ming Yu NIU; Li Feng CAO; Yu Fen ZHAO

    2006-01-01

    Two artificial minic polypeptides which are synthetic analogues of natural products with DNA affinity were synthesized, and theirs cleavage activity with DNA were examined. The structures of these compounds was confirmed by 1H NMR, MS and IR.

  3. Distinct interactions with cellular E-cadherin of the two virulent metalloproteinases encoded by a Bacteroides fragilis pathogenicity island.

    Science.gov (United States)

    Remacle, Albert G; Shiryaev, Sergey A; Strongin, Alex Y

    2014-01-01

    Bacteroides fragilis causes the majority of Gram-negative anaerobic infections in the humans. The presence of a short, 6-kb, pathogenicity island in the genome is linked to enterotoxigenic B. fragilis (ETBF). The role of the enterotoxin in B. fragilis virulence, however, remains to be determined, as the majority of clinical isolates lack ETBF genes and healthy individuals carry enterotoxin-positive B. fragilis. The island encodes secretory metalloproteinase II (MPII) and one of three homologous enterotoxigenic fragilysin isoenzymes (FRA; also termed B. fragilis toxin or BFT). The secretory metalloproteinases expressed from the genes on the B. fragilis pathogenicity island may have pathological importance within the gut, not linked to diarrhea. MPII and FRA are counter-transcribed in the bacterial genome, implying that regardless of their structural similarity and overlapping cleavage preferences these proteases perform distinct and highly specialized functions in the course of B. fragilis infection. The earlier data by us and others have demonstrated that FRA cleaves cellular E-cadherin, an important adherens junction protein, and weakens cell-to-cell contacts. Using E-cadherin-positive and E-cadherin-deficient cancer cells, and the immunostaining, direct cell binding and pull-down approaches, we, however, demonstrated that MPII via its catalytic domain efficiently binds, rather than cleaves, E-cadherin. According to our results, E-cadherin is an adherens junction cellular receptor, rather than a proteolytic target, of the B. fragilis secretory MPII enzyme. As a result of the combined FRA and MPII proteolysis, cell-to-cell contacts and adherens junctions are likely to weaken further.

  4. Distinct interactions with cellular E-cadherin of the two virulent metalloproteinases encoded by a Bacteroides fragilis pathogenicity island.

    Directory of Open Access Journals (Sweden)

    Albert G Remacle

    Full Text Available Bacteroides fragilis causes the majority of Gram-negative anaerobic infections in the humans. The presence of a short, 6-kb, pathogenicity island in the genome is linked to enterotoxigenic B. fragilis (ETBF. The role of the enterotoxin in B. fragilis virulence, however, remains to be determined, as the majority of clinical isolates lack ETBF genes and healthy individuals carry enterotoxin-positive B. fragilis. The island encodes secretory metalloproteinase II (MPII and one of three homologous enterotoxigenic fragilysin isoenzymes (FRA; also termed B. fragilis toxin or BFT. The secretory metalloproteinases expressed from the genes on the B. fragilis pathogenicity island may have pathological importance within the gut, not linked to diarrhea. MPII and FRA are counter-transcribed in the bacterial genome, implying that regardless of their structural similarity and overlapping cleavage preferences these proteases perform distinct and highly specialized functions in the course of B. fragilis infection. The earlier data by us and others have demonstrated that FRA cleaves cellular E-cadherin, an important adherens junction protein, and weakens cell-to-cell contacts. Using E-cadherin-positive and E-cadherin-deficient cancer cells, and the immunostaining, direct cell binding and pull-down approaches, we, however, demonstrated that MPII via its catalytic domain efficiently binds, rather than cleaves, E-cadherin. According to our results, E-cadherin is an adherens junction cellular receptor, rather than a proteolytic target, of the B. fragilis secretory MPII enzyme. As a result of the combined FRA and MPII proteolysis, cell-to-cell contacts and adherens junctions are likely to weaken further.

  5. Mechanisms for ribotoxin-induced ribosomal RNA cleavage

    Energy Technology Data Exchange (ETDEWEB)

    He, Kaiyu [Department of Microbiology and Molecular Genetics (United States); Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Zhou, Hui-Ren [Food Science and Human Nutrition (United States); Pestka, James J., E-mail: pestka@msu.edu [Department of Microbiology and Molecular Genetics (United States); Food Science and Human Nutrition (United States); Center for Integrative Toxicology, Michigan State University, East Lansing, MI 48824 (United States)

    2012-11-15

    The Type B trichothecene deoxynivalenol (DON), a ribotoxic mycotoxin known to contaminate cereal-based foods, induces ribosomal RNA (rRNA) cleavage in the macrophage via p38-directed activation of caspases. Here we employed the RAW 264.7 murine macrophage model to test the hypothesis that this rRNA cleavage pathway is similarly induced by other ribotoxins. Capillary electrophoresis confirmed that the antibiotic anisomycin (≥ 25 ng/ml), the macrocylic trichothecene satratoxin G (SG) (≥ 10 ng/ml) and ribosome-inactivating protein ricin (≥ 300 ng/ml) induced 18s and 28s rRNA fragmentation patterns identical to that observed for DON. Also, as found for DON, inhibition of p38, double-stranded RNA-activated kinase (PKR) and hematopoietic cell kinase (Hck) suppressed MAPK anisomycin-induced rRNA cleavage, while, in contrast, their inhibition did not affect SG- and ricin-induced rRNA fragmentation. The p53 inhibitor pifithrin-μ and pan caspase inhibitor Z-VAD-FMK suppressed rRNA cleavage induced by anisomycin, SG and ricin, indicating that these ribotoxins shared with DON a conserved downstream pathway. Activation of caspases 8, 9 and 3 concurrently with apoptosis further suggested that rRNA cleavage occurred in parallel with both extrinsic and intrinsic pathways of programmed cell death. When specific inhibitors of cathepsins L and B (lysosomal cysteine cathepsins active at cytosolic neutral pH) were tested, only the former impaired anisomycin-, SG-, ricin- and DON-induced rRNA cleavage. Taken together, the data suggest that (1) all four ribotoxins induced p53-dependent rRNA cleavage via activation of cathepsin L and caspase 3, and (2) activation of p53 by DON and anisomycin involved p38 whereas SG and ricin activated p53 by an alternative mechanism. Highlights: ► Deoxynivalenol (DON) anisomycin, satratoxin G (SG) and ricin are ribotoxins. ► Ribotoxins induce 18s and 28s rRNA cleavage in the RAW 264.7 macrophage model. ► Ribotoxins induce rRNA cleavage via

  6. Matrix metalloproteinases in the brain and blood-brain barrier: Versatile breakers and makers.

    Science.gov (United States)

    Rempe, Ralf G; Hartz, Anika Ms; Bauer, Björn

    2016-09-01

    Matrix metalloproteinases are versatile endopeptidases with many different functions in the body in health and disease. In the brain, matrix metalloproteinases are critical for tissue formation, neuronal network remodeling, and blood-brain barrier integrity. Many reviews have been published on matrix metalloproteinases before, most of which focus on the two best studied matrix metalloproteinases, the gelatinases MMP-2 and MMP-9, and their role in one or two diseases. In this review, we provide a broad overview of the role various matrix metalloproteinases play in brain disorders. We summarize and review current knowledge and understanding of matrix metalloproteinases in the brain and at the blood-brain barrier in neuroinflammation, multiple sclerosis, cerebral aneurysms, stroke, epilepsy, Alzheimer's disease, Parkinson's disease, and brain cancer. We discuss the detrimental effects matrix metalloproteinases can have in these conditions, contributing to blood-brain barrier leakage, neuroinflammation, neurotoxicity, demyelination, tumor angiogenesis, and cancer metastasis. We also discuss the beneficial role matrix metalloproteinases can play in neuroprotection and anti-inflammation. Finally, we address matrix metalloproteinases as potential therapeutic targets. Together, in this comprehensive review, we summarize current understanding and knowledge of matrix metalloproteinases in the brain and at the blood-brain barrier in brain disorders.

  7. Investigation and improvement of DNA cleavage models of polyamide + Cu(II nuclease + OOH- ligands bound to DNA

    Directory of Open Access Journals (Sweden)

    Wang Yan

    2010-10-01

    Full Text Available Abstract Background Copper nucleases as a famous class of artificial metallonucleases have attracted considerable interest in relation to their diverse potentials not only as therapeutic agents but also in genomic researches. Copper nucleases present high efficient oxidative cleavage of DNA, in which DNA strand scission occurs generally after hydrogen atom abstracted from a sugar moiety. In order to achieve the selective cleavage of DNA sequences by copper nucleases, the DNA specific recognition agents of the Dervan-type hairpin and cyclic polyamides can be considered as proper carriers of copper nucleases. Investigation of the DNA cleavage selectivity of copper nucleases assisted by the hairpin and cyclic polyamides at the molecular level has not yet been elucidated. Results We carried out a series of molecular dynamics simulations for the nuclease [Cu(BPA]2+ or [Cu(IDB]2+ bound to the hairpin/cyclic polyamide and associated with DNA to investigate the selective DNA cleavage properties of Cu(II-based artificial nucleases. The simulated results demonstrate that the DNA cleavage selectivity of the two nucleases assisted by the hairpin polyamide is improved efficiently. The [Cu(BPA]2+ or [Cu(IDB]2+ nuclease with a substrate OOH- bound to the hairpin polyamide can be stably located at the minor groove of DNA, and possibly abstracts H atom from the sugar of DNA. However, the DNA cleavage properties of the two nucleases assisted by the cyclic polyamide are significantly poor due to the rigidity of linking region between the cyclic polyamide and nuclease. With introduction of the flexible linker -CH2CH2CH2NH2, the modified cyclic polyamide can assist the two copper nucleases to improve the selective DNA cleavage properties efficiently. Conclusion A flexible linker and a proper binding site of the polyamide-type recognition agents play an important role in improving the DNA cleavage selectivity of copper nucleases. Current investigations provide an

  8. Cell-surface acceleration of urokinase-catalyzed receptor cleavage

    DEFF Research Database (Denmark)

    Høyer-Hansen, G; Ploug, M; Behrendt, N;

    1997-01-01

    937 cell lysates, had the same amino termini as uPAR(2+3), generated by uPA in a purified system. In both cases cleavage had occurred at two positions in the hinge region connecting domain 1 and 2, between Arg83-Ala84 and Arg89-Ser90, respectively. The uPA-catalyzed cleavage of uPAR is a new negative...

  9. Effect of Guanxinshutong capsule on the expression of matrix metalloproteinases-9 and tissue matrix metalloproteinase inhibitor-1 of atherosclerotic plaque in apoE-/- mice

    Institute of Scientific and Technical Information of China (English)

    霍煜

    2014-01-01

    Objective To approach the possible mechanism of Guanxinshutong capsule on the progression and stability of atherosclerotic plaque through observing the effects of Guanxinshutong capsule on pathologic morphology and expression of tissue matrix metalloproteinase inhibitor-1(TIMP-1),matrix metalloproteinases-9(MMP-9)of atherosclerotic plaque in Apo E-/-mice model with experimental atherosclerosis.Methods The animals were fed

  10. Enhanced cerebrovascular expression of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 via the MEK/ERK pathway during cerebral ischemia in the rat

    DEFF Research Database (Denmark)

    Maddahi, Aida; Chen, Qingwen; Edvinsson, Lars

    2009-01-01

    of metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1). RESULTS: Here, we found an infarct volume of 24.8 +/- 2% and a reduced neurological function after two hours of middle cerebral artery occlusion (MCAO), followed by 48 hours of recirculation in rat. Immunocytochemistry and confocal...

  11. Adult Vascular Wall Resident Multipotent Vascular Stem Cells, Matrix Metalloproteinases, and Arterial Aneurysms

    Directory of Open Access Journals (Sweden)

    Bruno Amato

    2015-01-01

    Full Text Available Evidences have shown the presence of multipotent stem cells (SCs at sites of arterial aneurysms: they can differentiate into smooth muscle cells (SMCs and are activated after residing in a quiescent state in the vascular wall. Recent studies have implicated the role of matrix metalloproteinases in the pathogenesis of arterial aneurysms: in fact the increased synthesis of MMPs by arterial SMCs is thought to be a pivotal mechanism in aneurysm formation. The factors and signaling pathways involved in regulating wall resident SC recruitment, survival, proliferation, growth factor production, and differentiation may be also related to selective expression of different MMPs. This review explores the relationship between adult vascular wall resident multipotent vascular SCs, MMPs, and arterial aneurysms.

  12. Cleavage events and sperm dynamics in chick intrauterine embryos.

    Directory of Open Access Journals (Sweden)

    Hyung Chul Lee

    Full Text Available This study was undertaken to elucidate detailed event of early embryogenesis in chicken embryos using a noninvasive egg retrieval technique before oviposition. White Leghorn intrauterine eggs were retrieved from 95 cyclic hens aged up to 54-56 weeks and morphogenetic observation was made under both bright field and fluorescent image in a time course manner. Differing from mammals, asymmetric cleavage to yield preblastodermal cells was observed throughout early embryogenesis. The first two divisions occurred synchronously and four polarized preblastodermal cells resulted after cruciform cleavage. Then, asynchronous cleavage continued in a radial manner and overall cell size in the initial cleavage region was smaller than that in the distal area. Numerous sperms were visible, regardless of zygotic nuclei formation. Condensed sperm heads were present mainly in the perivitelline space and cytoplasm, and rarely in the yolk region, while decondensed sperm heads were only visible in the yolk. In conclusion, apparent differences in sperm dynamics and early cleavage events compared with mammalian embryos were detected in chick embryo development, which demonstrated polarized cleavage with penetrating supernumerary sperm into multiple regions.

  13. Structural Basis for Accelerated Cleavage of Bovine Pancreatic Trypsin Inhibitor (BPTI) by Human Mesotrypsin

    Energy Technology Data Exchange (ETDEWEB)

    Salameh,M.; Soares, A.; Hockla, A.; Radisky, E.

    2008-01-01

    Human mesotrypsin is an isoform of trypsin that displays unusual resistance to polypeptide trypsin inhibitors and has been observed to cleave several such inhibitors as substrates. Whereas substitution of arginine for the highly conserved glycine 193 in the trypsin active site has been implicated as a critical factor in the inhibitor resistance of mesotrypsin, how this substitution leads to accelerated inhibitor cleavage is not clear. Bovine pancreatic trypsin inhibitor (BPTI) forms an extremely stable and cleavage-resistant complex with trypsin, and thus provides a rigorous challenge of mesotrypsin catalytic activity toward polypeptide inhibitors. Here, we report kinetic constants for mesotrypsin and the highly homologous (but inhibitor sensitive) human cationic trypsin, describing inhibition by, and cleavage of BPTI, as well as crystal structures of the mesotrypsin-BPTI and human cationic trypsin-BPTI complexes. We find that mesotrypsin cleaves BPTI with a rate constant accelerated 350-fold over that of human cationic trypsin and 150,000-fold over that of bovine trypsin. From the crystal structures, we see that small conformational adjustments limited to several side chains enable mesotrypsin-BPTI complex formation, surmounting the predicted steric clash introduced by Arg-193. Our results show that the mesotrypsin-BPTI interface favors catalysis through (a) electrostatic repulsion between the closely spaced mesotrypsin Arg-193 and BPTI Arg-17, and (b) elimination of two hydrogen bonds between the enzyme and the amine leaving group portion of BPTI. Our model predicts that these deleterious interactions accelerate leaving group dissociation and deacylation.

  14. Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations

    Science.gov (United States)

    Zuo, Zhicheng; Liu, Jin

    2016-11-01

    The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

  15. Determinants beyond both complementarity and cleavage govern microR159 efficacy in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Junyan Li

    2014-03-01

    Full Text Available Plant microRNAs (miRNAs are critical regulators of gene expression, however little attention has been given to the principles governing miRNA silencing efficacy. Here, we utilize the highly conserved Arabidopsis miR159-MYB33/MYB65 regulatory module to explore these principles. Firstly, we show that perfect central complementarity is not required for strong silencing. Artificial miR159 variants with two cleavage site mismatches can potently silence MYB33/MYB65, fully complementing a loss-of-function mir159 mutant. Moreover, these miR159 variants can cleave MYB33/MYB65 mRNA, however cleavage appears attenuated, as the ratio of cleavage products to full length transcripts decreases with increasing central mismatches. Nevertheless, high levels of un-cleaved MYB33/MYB65 transcripts are strongly silenced by a non-cleavage mechanism. Contrary to MIR159a variants that strongly silenced endogenous MYB33/MYB65, artificial MYB33 variants with central mismatches to miR159 are not efficiently silenced. We demonstrate that differences in the miRNA:target mRNA stoichiometry underlie this paradox. Increasing miR159 abundance in the MYB33 variants results in a strong silencing outcome, whereas increasing MYB33 transcript levels in the MIR159a variants results in a poor silencing outcome. Finally, we identify highly conserved nucleotides that flank the miR159 binding site in MYB33, and demonstrate that they are critical for efficient silencing, as mutation of these flanking nucleotides attenuates silencing at a level similar to that of central mismatches. This implies that the context in which the miRNA binding site resides is a key determinant in controlling the degree of silencing and that a miRNA "target site" encompasses sequences that extend beyond the miRNA binding site. In conclusion, our findings dismiss the notion that miRNA:target complementarity, underpinned by central matches, is the sole dictator of the silencing outcome.

  16. Molecular design of a highly selective and strong protein inhibitor against matrix metalloproteinase-2 (MMP-2).

    Science.gov (United States)

    Higashi, Shouichi; Hirose, Tomokazu; Takeuchi, Tomoka; Miyazaki, Kaoru

    2013-03-29

    Synthetic inhibitors of matrix metalloproteinases (MMPs), designed previously, as well as tissue inhibitors of metalloproteinases (TIMPs) lack enzyme selectivity, which has been a major obstacle for developing inhibitors into safe and effective MMP-targeted drugs. Here we designed a fusion protein named APP-IP-TIMP-2, in which the ten amino acid residue sequence of APP-derived MMP-2 selective inhibitory peptide (APP-IP) is added to the N terminus of TIMP-2. The APP-IP and TIMP-2 regions of the fusion protein are designed to interact with the active site and the hemopexin-like domain of MMP-2, respectively. The reactive site of the TIMP-2 region, which has broad specificity against MMPs, is blocked by the APP-IP adduct. The recombinant APP-IP-TIMP-2 showed strong inhibitory activity toward MMP-2 (Ki(app) = 0.68 pm), whereas its inhibitory activity toward MMP-1, MMP-3, MMP-7, MMP-8, MMP-9, or MT1-MMP was six orders of magnitude or more weaker (IC50 > 1 μm). The fusion protein inhibited the activation of pro-MMP-2 in the concanavalin A-stimulated HT1080 cells, degradation of type IV collagen by the cells, and the migration of stimulated cells. Compared with the decapeptide APP-IP (t½ = 30 min), APP-IP-TIMP-2 (t½ ≫ 96 h) showed a much longer half-life in cultured tumor cells. Therefore, the fusion protein may be a useful tool to evaluate contributions of proteolytic activity of MMP-2 in various pathophysiological processes. It may also be developed as an effective anti-tumor drug with restricted side effects.

  17. Matrix metalloproteinase-20 mediates dental enamel biomineralization by preventing protein occlusion inside apatite crystals.

    Science.gov (United States)

    Prajapati, Saumya; Tao, Jinhui; Ruan, Qichao; De Yoreo, James J; Moradian-Oldak, Janet

    2016-01-01

    Reconstruction of enamel-like materials is a central topic of research in dentistry and material sciences. The importance of precise proteolytic mechanisms in amelogenesis to form a hard tissue with more than 95% mineral content has already been reported. A mutation in the Matrix Metalloproteinase-20 (MMP-20) gene results in hypomineralized enamel that is thin, disorganized and breaks from the underlying dentin. We hypothesized that the absence of MMP-20 during amelogenesis results in the occlusion of amelogenin in the enamel hydroxyapatite crystals. We used spectroscopy and electron microscopy techniques to qualitatively and quantitatively analyze occluded proteins within the isolated enamel crystals from MMP-20 null and Wild type (WT) mice. Our results showed that the isolated enamel crystals of MMP-20 null mice had more organic macromolecules occluded inside them than enamel crystals from the WT. The crystal lattice arrangements of MMP-20 null enamel crystals analyzed by High Resolution Transmission Electron Microscopy (HRTEM) were found to be significantly different from those of the WT. Raman studies indicated that the crystallinity of the MMP-20 null enamel crystals was lower than that of the WT. In conclusion, we present a novel functional mechanism of MMP-20, specifically prevention of unwanted organic material entrapped in the forming enamel crystals, which occurs as the result of precise amelogenin cleavage. MMP-20 action guides the growth morphology of the forming hydroxyapatite crystals and enhances their crystallinity. Elucidating such molecular mechanisms can be applied in the design of novel biomaterials for future clinical applications in dental restoration or repair.

  18. Early upregulation of matrix metalloproteinases following reperfusion triggers neuroinflammatory mediators in brain ischemia in rat.

    Science.gov (United States)

    Amantea, Diana; Russo, Rossella; Gliozzi, Micaela; Fratto, Vincenza; Berliocchi, Laura; Bagetta, G; Bernardi, G; Corasaniti, M Tiziana

    2007-01-01

    Abnormal expression of matrix metalloproteinases (MMPs) has been implicated in the pathophysiology of neuroinflammatory processes that accompany most central nervous system disease. In particular, early upregulation of the gelatinases MMP-2 and MMP-9 has been shown to contribute to disruption of the blood-brain barrier and to death of neurons in ischemic stroke. In situ zymography reveals a significant increase in gelatinolytic MMPs activity in the ischemic brain hemisphere after 2-h middle cerebral artery occlusion (MCAo) followed by 2-h reperfusion in rat. Accordingly, gel zymography demonstrates that expression and activity of MMP-2 and MMP-9 are enhanced in cortex and striatum ipsilateral to the ischemic insult. The latter effect appears to be instrumental for development of delayed brain damage since administration of a broad spectrum, highly specific MMPs inhibitor, GM6001, but not by its negative control, results in a significant (50%) reduction in ischemic brain volume. Increased gelatinase activity in the ischemic cortex coincides with elevation (166% vs sham) of mature interleukin-1beta (IL-1beta) after 2-h reperfusion and this does not appear to implicate a caspase-1-dependent processing of pro(31kDa)-IL-1beta to yield mature (17kDa) IL-1beta. More importantly, when administered at a neuroprotective dose GM6001 abolishes the early IL-1beta increase in the ischemic cortex and reduces the cleavage of the cytokine proform supporting the deduction that MMPs may initiate IL-1beta processing. In conclusion, development of tissue damage that follows transient ischemia implicates a crucial interplay between MMPs and mediators of neuroinflammation (e.g., IL-1beta), and this further underscores the therapeutic potential of MMPs inhibitors in the treatment of stroke.

  19. Upconversion fluorescence resonance energy transfer based biosensor for ultrasensitive detection of matrix metalloproteinase-2 in blood.

    Science.gov (United States)

    Wang, Yuhui; Shen, Pei; Li, Chunya; Wang, Yanying; Liu, Zhihong

    2012-02-01

    Matrix metalloproteinase-2 (MMP-2) is a very important biomarker in blood. Presently, sensitive and selective determination of MMP-2 directly in blood samples is still a challenging job because of the high complexity of the sample matrix. In this work, we reported a new homogeneous biosensor for MMP-2 based on fluorescence resonance energy transfer (FRET) from upconversion phosphors (UCPs) to carbon nanoparticles (CNPs). A polypeptide chain (NH(2)-GHHYYGPLGVRGC-COOH) comprising both the specific MMP-2 substrate domain (PLGVR) and a π-rich motif (HHYY) was designed and linked to the surface of UCPs at the C terminus. The FRET process was initiated by the π-π interaction between the peptide and CNPs, which thus quenched the fluorescence of the donor. Upon the cleavage of the substrate by the protease at the amide bond between Gly and Val, the donor was separated from the acceptor while the π-rich motif stayed on the acceptor. As a result, the fluorescence of the donor was restored. The fluorescence recovery was found to be proportional to the concentration of MMP-2 within the range from 10-500 pg/mL in an aqueous solution. The quantification limit of this sensor was at least 1 order of magnitude lower than that of other reported assays for MMP-2. The sensor was used to determine the MMP-2 level directly in human plasma and whole blood samples with satisfactory results obtained. Owing to the hypersensitivity of the method, clinical samples of only less than 1 μL were needed for accurate quantification, which can be meaningful in MMP-2-related clinical and bioanalytical applications.

  20. Significance of matrix metalloproteinases in norepinephrine-induced remodelling of rat hearts.

    Science.gov (United States)

    Briest, Wilfried; Hölzl, Alexander; Rassler, Beate; Deten, Alexander; Baba, Hideo A; Zimmer, Heinz-Gerd

    2003-02-01

    Norepinephrine (NE) induced hypertrophy and remodelling of the extracellular matrix (ECM) in the left ventricle (LV) of the rat heart with resulting fibrosis. However, there was no increased collagen deposition in the right ventricle (RV). To test the hypothesis that lack of RV fibrosis is the result of elevated cleavage of collagens we inhibited the activity of matrix metalloproteinases (MMP) by doxycycline (Doxy) and then measured function and collagen metabolism in the RV as compared to the LV. Female Sprague-Dawley rats were treated with 30 mg/kg per day doxycycline alone or in combination with i.v. infusion of NE (0.1 mg/kg per h). The activity of MMP-2 was increased both in the LV and RV after 3 days of NE infusion and reduced after concomitant doxycycline treatment which also caused inhibition when given alone. After 14 days of NE infusion in combination with doxycycline there was an additional increase in the NE-induced elevation of collagen accumulation in the LV (interstitial collagen fraction: NE-Doxy 1.797%, PDoxy: 36.4 mmHg) and RV dP/dt(max) (NE: 5500 mmHg/s, PDoxy: 2550 mmHg/s). Also in the NE-stimulated LV, the doxycycline-induced collagen accumulation was associated with reduced LV dP/dt(max) (NE-Doxy: 13169 mmHg/s; NE: 18849 mmHg/s, P<0.05). MMP inhibition leads to myocardial stiffness with negative functional consequences for the RV and LV in NE-treated rat hearts.

  1. Assessment of Synthetic Matrix Metalloproteinase Inhibitors by Fluorogenic Substrate Assay.

    Science.gov (United States)

    Lively, Ty J; Bosco, Dale B; Khamis, Zahraa I; Sang, Qing-Xiang Amy

    2016-01-01

    Matrix metalloproteinases (MMPs) are a family of metzincin enzymes that act as the principal regulators and remodelers of the extracellular matrix (ECM). While MMPs are involved in many normal biological processes, unregulated MMP activity has been linked to many detrimental diseases, including cancer, neurodegenerative diseases, stroke, and cardiovascular disease. Developed as tools to investigate MMP function and as potential new therapeutics, matrix metalloproteinase inhibitors (MMPIs) have been designed, synthesized, and tested to regulate MMP activity. This chapter focuses on the use of enzyme kinetics to characterize inhibitors of MMPs. MMP activity is measured via fluorescence spectroscopy using a fluorogenic substrate that contains a 7-methoxycoumarin-4-acetic acid N-succinimidyl ester (Mca) fluorophore and a 2,4-dinitrophenyl (Dpa) quencher separated by a scissile bond. MMP inhibitor (MMPI) potency can be determined from the reduction in fluorescent intensity when compared to the absence of the inhibitor. This chapter describes a technique to characterize a variety of MMPs through enzyme inhibition assays.

  2. Matrix metalloproteinases and their expression in mammary gland

    Institute of Scientific and Technical Information of China (English)

    URIAJOSEA; ZENAWERB

    1998-01-01

    The matrix metalloproteinases (MMPs) are a family of zine-dependent endopeptidases that play a key role in both normal and pathological processes involving tissue remodeling events.The expression of these proteolytic enzymes is highly regulated by a balance between extracellular matrix (ECM) deposition and its degradation,and is controlled by growth factors,cytokines,hormones,as well as interactions with the ECM macromolecules.Furthermore,the activity of the MMPs is regulated by their natural endogenous inhibitors,which are members of the tissue inhibitor of metalloproteinases (TIMP) family.In the normal mammary gland,MMPs are expressed during ductal development,lobulo-alveolar development in pregnancy and involution after lactation.Under pathological conditions,such as tumorigenesis,the dysregulated expression of MMPs play a role in tumor initiation,progression and malignant conversion as well as facilitating invasion and metastasis of malignant cells through degradation of the ECM and basement membranes.

  3. Osteopontin is highly susceptible to cleavage in bovine milk and the proteolytic fragments bind the αVβ3-integrin receptor

    DEFF Research Database (Denmark)

    Christensen, Brian Søndergaard; Sørensen, Esben Skipper

    2014-01-01

    Site-specific and partial proteolysis of milk proteins can both alter and increase their biological activity. The milk protein osteopontin (OPN) is a highly phosphorylated integrin-binding molecule present in most tissues and body fluids. Osteopontin is a biological substrate for matrix...... a substantial effect on the functionality of OPN. Bovine milk OPN (bOPN) exists in both intact full-length and cleaved forms, and in this study, 6 N-terminal bOPN fragments originating from proteolytic cleavage were purified and characterized by mass spectrometry. These fragments were generated by cleavage...... at the Lys145-Ser146, Arg147-Ser148, Lys149-Lys150, Phe151-Arg152, Arg152-Arg153, and Arg153-Ser154 peptide bonds. The principal protease in milk, plasmin, appeared to cleave 3 of these sites. However, the major cleavage site was observed to be at the Phe151-Arg152 bond, which does not match the specificity...

  4. Cleavage of serum response factor mediated by enteroviral protease 2A contributes to impaired cardiac function

    Institute of Scientific and Technical Information of China (English)

    Jerry Wong; Jingchun Zhang; Bobby Yanagawa; Zongshu Luo; Xiangsheng Yang; Jiang Chang; Bruce McManus; Honglin Luo

    2012-01-01

    Enteroviral infection can lead to dilated cardiomyopathy (DCM),which is a major cause of cardiovascular mortality worldwide.However,the pathogenetic mechanisms have not been fully elucidated.Serum response factor (SRF) is a cardiac-enriched transcription regulator controlling the expression of a variety of target genes,including those involved in the contractile apparatus and immediate early response,as well as microRNAs that silence the expression of cardiac regulatory factors.Knockout of SRF in the heart results in downregulation of cardiac contractile gene expression and development of DCM.The goal of this study is to understand the role of SRF in enterovirus-induced cardiac dysfunction and progression to DCM.Here we report that SRF is cleaved following enteroviral infection of mouse heart and cultured cardiomyocytes.This cleavage is accompanied by impaired cardiac function and downregulation of cardiac-specific contractile and regulatory genes.Further investigation by antibody epitope mapping and site-directed mutagenesis demonstrates that SRF cleavage occurs at the region of its transactivation domain through the action of virus-encoded protease 2A.Moreover,we demonstrate that cleavage of SRF dissociates its transactivation domain from DNA-binding domain,resulting in the disruption of SRF-mediated gene transactivation.In addition to loss of functional SRF,finally we report that the N-terminal fragment of SRF cleavage products can also act as a dominant-negative transcription factor,which likely competes with the native SRF for DNA binding.Our results suggest a mechanism by which virus infection impairs heart function and may offer a new therapeutic strategy to ameliorate myocardial damage and progression to DCM.

  5. Mechanistic Insights into Ring Cleavage and Contraction of Benzene over a Titanium Hydride Cluster.

    Science.gov (United States)

    Kang, Xiaohui; Luo, Gen; Luo, Lun; Hu, Shaowei; Luo, Yi; Hou, Zhaomin

    2016-09-14

    Carbon-carbon bond cleavage of benzene by transition metals is of great fundamental interest and practical importance, as this transformation is involved in the production of fuels and other important chemicals in the industrial hydrocracking of naphtha on solid catalysts. Although this transformation is thought to rely on cooperation of multiple metal sites, molecular-level information on the reaction mechanism has remained scarce to date. Here, we report the DFT studies of the ring cleavage and contraction of benzene by a molecular trinuclear titanium hydride cluster. Our studies suggest that the reaction is initiated by benzene coordination, followed by H2 release, C6H6 hydrometalation, repeated C-C and C-H bond cleavage and formation to give a MeC5H4 unit, and insertion of a Ti atom into the MeC5H4 unit with release of H2 to give a metallacycle product. The C-C bond cleavage and ring contraction of toluene can also occur in a similar fashion, though some details are different due to the presence of the methyl substituent. Obviously, the facile release of H2 from the metal hydride cluster to provide electrons and to alter the charge population at the metal centers, in combination with the flexible metal-hydride connections and dynamic redox behavior of the trimetallic framework, has enabled this unusual transformation to occur. This work has not only provided unprecedented insights into the activation and transformation of benzene over a multimetallic framework but it may also offer help in the design of new molecular catalysts for the activation and transformation of inactive aromatics.

  6. Beneficial Regulation of Matrix Metalloproteinases for Skin Health

    Directory of Open Access Journals (Sweden)

    Neena Philips

    2011-01-01

    Full Text Available Matrix metalloproteinases (MMPs are essential to the remodeling of the extracellular matrix. While their upregulation facilitates aging and cancer, they are essential to epidermal differentiation and the prevention of wound scars. The pharmaceutical industry is active in identifying products that inhibit MMPs to prevent or treat aging and cancer and products that stimulate MMPs to prevent epidermal hyperproliferative diseases and wound scars.

  7. Update of human and mouse matrix metalloproteinase families

    OpenAIRE

    Jackson Brian C; Nebert Daniel W; Vasiliou Vasilis

    2010-01-01

    Abstract Matrix metalloproteinases (MMPs) are a family of zinc proteases that degrade most of the components of the extracellular matrix (ECM). MMPs also have a number of non-traditional roles in processing factors related to cell growth/proliferation, inflammation and more. There are 23 human MMPs and 23 mouse MMPs, most of which share orthology among most vertebrates; other examples have been found in invertebrates and plants. MMPs are named in order of discovery, but also have been grouped...

  8. Matrix Metalloproteinases And Their Role In Oral Diseases: A Review

    Directory of Open Access Journals (Sweden)

    Anna P. joseph

    2012-01-01

    Full Text Available Matrix metalloproteinases (MMPs are zinc dependent endopeptidases that are capable of degrading extra cellular matrix proteins. The activity of MMPs is seen not only during normal organogenesis and wound healing, but also in pathological conditions like inflammatory diseases and tumor invasion. This review describes the structure, function and regulation of MMPs and also highlights their role in certain oral diseases like oral cancer, periodontitis and dental caries.

  9. Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism.

    Directory of Open Access Journals (Sweden)

    Annie Nguyen

    Full Text Available Cystatin C (Cys C is a small, potent, cysteine protease inhibitor. An Ala25Thr (A25T polymorphism in Cys C has been associated with both macular degeneration and late-onset Alzheimer's disease. Previously, studies have suggested that this polymorphism may compromise the secretion of Cys C. Interestingly, we found that untagged A25T, A25T tagged C-terminally with FLAG, or A25T FLAG followed by green fluorescent protein (GFP, were all secreted as efficiently from immortalized human cells as their wild-type (WT counterparts (e.g., 112%, 100%, and 88% of WT levels from HEK-293T cells, respectively. Supporting these observations, WT and A25T Cys C variants also showed similar intracellular steady state levels. Furthermore, A25T Cys C did not activate the unfolded protein response and followed the same canonical endoplasmic reticulum (ER-Golgi trafficking pathway as WT Cys C. WT Cys C has been shown to undergo signal sequence cleavage between residues Gly26 and Ser27. While the A25T polymorphism did not affect Cys C secretion, we hypothesized that it may alter where the Cys C signal sequence is preferentially cleaved. Under normal conditions, WT and A25T Cys C have the same signal sequence cleavage site after Gly26 (referred to as 'site 2' cleavage. However, in particular circumstances when the residues around site 2 are modified (such as by the presence of an N-terminal FLAG tag immediately after Gly26, or by a Gly26Lys (G26K mutation, A25T has a significantly higher likelihood than WT Cys C of alternative signal sequence cleavage after Ala20 ('site 1' or even earlier in the Cys C sequence. Overall, our results indicate that the A25T polymorphism does not cause a significant reduction in Cys C secretion, but instead predisposes the protein to be cleaved at an alternative signal sequence cleavage site if site 2 is hindered. Additional N-terminal amino acids resulting from alternative signal sequence cleavage may, in turn, affect the protease

  10. Scyphozoan jellyfish venom metalloproteinases and their role in the cytotoxicity.

    Science.gov (United States)

    Lee, Hyunkyoung; Jung, Eun-sun; Kang, Changkeun; Yoon, Won Duk; Kim, Jong-Shu; Kim, Euikyung

    2011-09-01

    The present study, for the first time, comparatively investigated the enzymatic activities (proteases and hyaluronidases) in the venoms of four Scyphozoan jellyfish species, including Nemopilema nomurai, Rhopilema esculenta, Cyanea nozakii, and Aurelia aurita. For this, various zymographic analyses were performed using assay specific substrates. Interestingly, all the four jellyfish venoms showed gelatinolytic, caseinolytic, and fibrinolytic activities, each of which contains a multitude of enzyme components with molecular weights between 17 and 130 kDa. These four jellyfish venoms demonstrated a huge variation in their proteolytic activities in quantitative and qualitative manner depending on the species. Most of these enzymatic activities were disappeared by the treatment of 1,10-phenanthroline, suggesting they might be belonged to metalloproteinases. Toxicological significance of these venom proteases was examined by comparing their proteolytic activity and the cytotoxicity in NIH 3T3 cells. The relative cytotoxic potency was C. nozakii > N. nomurai > A. aurita > R. esculenta. The cytotoxicity of jellyfish venom shows a positive correlation with its overall proteolytic activity. The metalloproteinases appear to play an important role in the induction of jellyfish venom toxicities. In conclusion, the present report proposes a novel finding of Scyphozoan jellyfish venom metalloproteinases and their potential role in the cytotoxicity.

  11. The presence of outer arm fucose residues on the N-glycans of tissue inhibitor of metalloproteinases-1 reduces its activity.

    Science.gov (United States)

    Kim, Han Ie; Saldova, Radka; Park, Jun Hyoung; Lee, Young Hun; Harvey, David J; Wormald, Mark R; Wynne, Kieran; Elia, Giuliano; Kim, Hwa-Jung; Rudd, Pauline M; Lee, Seung-Taek

    2013-08-02

    Tissue inhibitor of metalloproteinases-1 (TIMP-1) inhibits matrix metalloproteinases (MMPs) by binding at a 1:1 stoichiometry. Here we have shown the involvement of N-glycosylation in the MMP inhibitory ability of TIMP-1. TIMP-1, purified from HEK 293 cells overexpressing TIMP-1 (293 TIMP-1), showed less binding and inhibitory abilities to MMPs than TIMP-1 purified from fibroblasts or SF9 insect cells infected with TIMP-1 baculovirus. Following deglycosylation of TIMP-1, all forms of TIMP-1 showed similar levels of MMP binding and inhibition, suggesting that glycosylation is involved in the regulation of these TIMP-1 activities. Analysis of the N-glycan structures showed that SF9 TIMP-1 has the simplest N-glycan structures, followed by fibroblast TIMP-1 and 293 TIMP-1, in order of increasing complexity in their N-glycan structures. Further analyses showed that cleavage of outer arm fucose residues from the N-glycans of 293 TIMP-1 or knockdown of both FUT4 and FUT7 (which encode for fucosyltransferases that add outer arm fucose residues to N-glycans) enhanced the MMP-binding and catalytic abilities of 293 TIMP-1, bringing them up to the levels of the other TIMP-1. These results demonstrate that the ability of TIMP-1 to inhibit MMPs is at least in part regulated by outer arm fucosylation of its N-glycans.

  12. Targeting of matrix metalloproteinase activation for noninvasive detection of vulnerable atherosclerotic lesions

    Energy Technology Data Exchange (ETDEWEB)

    Hartung, Dagmar [University of California, School of Medicine, Irvine, CA (United States); School of Medicine, Department of Radiology, Hannover (Germany); Schaefers, Michael; Kopka, Klaus [University of Muenster, Department of Nuclear Medicine, Muenster (Germany); Fujimoto, Shinichiro; Narula, Navneet; Petrov, Artiom; Narula, Jagat [University of California, School of Medicine, Irvine, CA (United States); Levkau, Bodo [University of Duisburg-Essen, Institute of Pathophysiology, Duisburg (Germany); Virmani, Renu; Kolodgie, Frank D. [Cardiovascular Pathology, Gaithersburg, MD (United States); Reutelingsperger, Chris; Hofstra, Leo [Cardiovascular Research Institute, Maastricht (Netherlands)

    2007-06-15

    Inflammation plays an important role in vulnerability of atherosclerotic plaques to rupture and hence acute coronary events. The monocyte-macrophage infiltration in plaques leads to upregulation of cytokines and metalloproteinase enzymes. Matrix metalloproteinases result in matrix dissolution and consequently expansive remodeling of the vessel. They also contribute to attenuation of fibrous cap and hence susceptibility to rupture. Assessment of metalloproteinase expression and activity should provide information about plaque instability. (orig.)

  13. The presence of promatrix metalloproteinase-3 and its relation with different categories of coal workers' pneumoconiosis

    Directory of Open Access Journals (Sweden)

    Remzi Altin

    2004-01-01

    Full Text Available Extracellular matrix formation (ECM and remodeling are critical events related to the pathogenesis of pulmonary fibrosis. Matrix metalloproteinases play an essential role in degrading and remodeling the ECM. In this study, we tried to show the presence and correlation of promatrix metalloproteinase-3 (proMMP-3 (the inactive form of metalloproteinase-3 levels in coal workers' pneumoconiosis (CWP with different categories.

  14. Membrane Type-1 Matrix Metalloproteinases and Tissue Inhibitor of Metalloproteinases-2 RNA Levels Mimic Each Other during Xenopus laevis Metamorphosis

    OpenAIRE

    Walsh, Logan A.; Deanna A Carere; Cooper, Colin A.; Sashko Damjanovski

    2007-01-01

    Matrix metalloproteinases (MMPs) and their endogenous inhibitors TIMPs (tissue inhibitors of MMPs), are two protein families that work together to remodel the extracellular matrix (ECM). TIMPs serve not only to inhibit MMP activity, but also aid in the activation of MMPs that are secreted as inactive zymogens. Xenopus laevis metamorphosis is an ideal model for studying MMP and TIMP expression levels because all tissues are remodeled under the control of one molecule, thyroid hormone. Here, us...

  15. Transition State Charge Stabilization and Acid-Base Catalysis of mRNA Cleavage by the Endoribonuclease RelE.

    Science.gov (United States)

    Dunican, Brian F; Hiller, David A; Strobel, Scott A

    2015-12-01

    The bacterial toxin RelE is a ribosome-dependent endoribonuclease. It is part of a type II toxin-antitoxin system that contributes to antibiotic resistance and biofilm formation. During amino acid starvation, RelE cleaves mRNA in the ribosomal A-site, globally inhibiting protein translation. RelE is structurally similar to microbial RNases that employ general acid-base catalysis to facilitate RNA cleavage. The RelE active site is atypical for acid-base catalysis, in that it is enriched with positively charged residues and lacks the prototypical histidine-glutamate catalytic pair, making the mechanism of mRNA cleavage unclear. In this study, we use a single-turnover kinetic analysis to measure the effect of pH and phosphorothioate substitution on the rate constant for cleavage of mRNA by wild-type RelE and seven active-site mutants. Mutation and thio effects indicate a major role for stabilization of increased negative change in the transition state by arginine 61. The wild-type RelE cleavage rate constant is pH-independent, but the reaction catalyzed by many of the mutants is strongly dependent on pH, suggestive of general acid-base catalysis. pH-rate curves indicate that wild-type RelE operates with the pK(a) of at least one catalytic residue significantly downshifted by the local environment. Mutation of any single active-site residue is sufficient to disrupt this microenvironment and revert the shifted pK(a) back above neutrality. pH-rate curves are consistent with K54 functioning as a general base and R81 as a general acid. The capacity of RelE to effect a large pK(a) shift and facilitate a common catalytic mechanism by uncommon means furthers our understanding of other atypical enzymatic active sites.

  16. Matrix metalloproteinase 2 and tissue inhibitor of matrix metalloproteinases 2 in the diagnosis of colorectal adenoma and cancer patients.

    Directory of Open Access Journals (Sweden)

    Barbara Mroczko

    2011-04-01

    Full Text Available The aim of the study was to assess the importance of the measurement of matrix metalloproteinase 2 (MMP-2 and tissue inhibitor of matrix metalloproteinases 2 (TIMP-2 in patients with colorectal cancer (CRC in relation to clinicopathological features of tumor and patients' survival. Additionally, we determined serum MMP-2 and TIMP-2 in colorectal adenoma (CA patients and healthy controls and compared them with tumor markers, CEA and CA 19-9. The serum levels of MMP-2 and TIMP-2 in 91 CRC patients, 28 CA subjects and 91 healthy controls were determined by ELISA method, but concentrations of CEA and CA 19-9 using MEIA method. Nonparametric statistical analyses were used. Serum levels of MMP-2 and TIMP-2 were significantly lower in CRC patients than in healthy subjects and decreased with tumor stage. Additionally, MMP-2 concentrations were significantly lower in patients with CRC than in CA group. Diagnostic sensitivity of TIMP-2 (59% was the highest among biomarkers tested and increased in combined use with CEA (79%. Moreover, the area under ROC curve (AUC of TIMP-2 was larger than AUC of MMP-2 in differentiation between CRC and healthy subjects, but lower than AUC of matrix metalloproteinase 2 in differentiation between colorectal cancer and adenoma. Our findings suggest clinical usefulness of TIMP-2 as a biomarker in the diagnosis of CRC, especially in combination with CEA. However, further investigation is necessary.

  17. Identification of an imino group indispensable for cleavage by a small ribozyme.

    Science.gov (United States)

    Spitale, Robert C; Volpini, Rosaria; Heller, Moriah G; Krucinska, Jolanta; Cristalli, Gloria; Wedekind, Joseph E

    2009-05-06

    The hairpin ribozyme is a small, noncoding RNA (ncRNA) that catalyzes a site-specific phosphodiester bond cleavage reaction. Prior biochemical and structural analyses pinpointed the amidine moiety of base Ade38 as a key functional group in catalysis, but base changes designed to probe function resulted in localized misfolding of the active site. To define the requirements for chemical activity using a conservative modification, we synthesized and incorporated N1-deazaadenosine into the full-length ribozyme construct. This single-atom variant severely impairs activity, although the active-site fold remains intact in the accompanying crystal structures. The results demonstrate the essentiality of the imino moiety as well as the importance of its interaction with the substrate in the precatalytic and transition-state conformations. This work demonstrates the efficacy of single-atom approaches in the analysis of ncRNA structure-function relationships.

  18. Cleavage entropy as quantitative measure of protease specificity.

    Science.gov (United States)

    Fuchs, Julian E; von Grafenstein, Susanne; Huber, Roland G; Margreiter, Michael A; Spitzer, Gudrun M; Wallnoefer, Hannes G; Liedl, Klaus R

    2013-04-01

    A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases) and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity.

  19. Cleavage entropy as quantitative measure of protease specificity.

    Directory of Open Access Journals (Sweden)

    Julian E Fuchs

    2013-04-01

    Full Text Available A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity.

  20. Identification of Rbd2 as a candidate protease for sterol regulatory element binding protein (SREBP) cleavage in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jinsil; Ha, Hye-Jeong [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Kim, Sujin [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Department of Functional Genomics, University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113 (Korea, Republic of); Choi, Ah-Reum [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Lee, Sook-Jeong [Department of New Drug Discovery and Development, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134 (Korea, Republic of); Hoe, Kwang-Lae, E-mail: kwanghoe@cnu.ac.kr [Department of New Drug Discovery and Development, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134 (Korea, Republic of); Kim, Dong-Uk, E-mail: kimdongu@kribb.re.kr [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of)

    2015-12-25

    Lipid homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP) transcription factors that are activated through sequential cleavage by Golgi Site-1 and Site-2 proteases. Fission yeast SREBP, Sre1, engages a different mechanism involving the Golgi Dsc E3 ligase complex, but it is not clearly understood exactly how Sre1 is proteolytically cleaved and activated. In this study, we screened the Schizosaccharomyces pombe non-essential haploid deletion collection to identify missing components of the Sre1 cleavage machinery. Our screen identified an additional component of the SREBP pathway required for Sre1 proteolysis named rhomboid protein 2 (Rbd2). We show that an rbd2 deletion mutant fails to grow under hypoxic and hypoxia-mimetic conditions due to lack of Sre1 activity and that this growth phenotype is rescued by Sre1N, a cleaved active form of Sre1. We found that the growth inhibition phenotype under low oxygen conditions is specific to the strain with deletion of rbd2, not any other fission yeast rhomboid-encoding genes. Our study also identified conserved residues of Rbd2 that are required for Sre1 proteolytic cleavage. All together, our results suggest that Rbd2 is a functional SREBP protease with conserved residues required for Sre1 cleavage and provide an important piece of the puzzle to understand the mechanisms for Sre1 activation and the regulation of various biological and pathological processes involving SREBPs. - Highlights: • An rbd2-deleted yeast strain shows defects in growth in response to low oxygen levels. • rbd2-deficient cells fail to generate cleaved Sre1 (Sre1N) under hypoxic conditions. • Expression of Sre1N rescues the rbd2 deletion mutant growth phenotype. • Rbd2 contains conserved residues potentially critical for catalytic activity. • Mutation of the conserved Rbd2 catalytic residues leads to defects in Sre1 cleavage.

  1. A new cultural cleavage in post-modern society

    Directory of Open Access Journals (Sweden)

    Jan-Erik Lane

    2007-09-01

    Full Text Available The attitudes towards gender and homosexuality tend to be linked at the micro level (individuals, which explains the political saliency of this newly emerging cleavage. At the macro level (country, the main finding is that the value orientations towards gender and homosexuality are strongly embedded in the basic cultural or civilisation differences among countries. As developing countries modernise and enter post-modernity, they will also experience the gender cleavage, especially when they adhere to an individualistic culture. Cultural cleavages in the post-modern society, whether in rich or developing countries, can only be properly researched by the survey method. It opens up a large area for both micro and macro analyses in the social sciences.

  2. Photochemical cleavage of DNA by nitrobenzamides linked to 9-aminoacridine

    Energy Technology Data Exchange (ETDEWEB)

    Nielsen, P.E.; Jeppesen, C.; Egholm, M.; Buchardt, O.

    1988-08-23

    Nitrobenzamido ligands linked to the DNA intercalator 9-aminoacridine via poly(methylene) chains induce single-strand nicks in DNA upon irradiation with long-wavelength ultraviolet light (lambda greater than or equal to 300 nm). Optimal photocleavage activity was found for the reagent 9-((6-(4-nitrobenzamido)hexyl)amino)-acridine. Removal of the acridinyl ligand or changing the position of the nitro group from the 4- to the 2-position caused a 10-fold decrease in photocleavage efficiency, whereas a change to the 3-position caused a 30-fold reduction. The DNA cleavage was 5-fold enhanced by subsequent piperidine treatment and showed some sequence dependency with predominant cleavage at G and T residues. Furthermore, significant differences in cleavage preference were observed when the poly(methylene) linker length was changed.

  3. Effect of Supracervical Apposition and Spontaneous Labour on Apoptosis and Matrix Metalloproteinases in Human Fetal Membranes

    Directory of Open Access Journals (Sweden)

    Mahalia Chai

    2013-01-01

    Full Text Available Background. Apoptosis and matrix metalloproteinase (MMP-9 are capable of hydrolysing components of the extracellular matrix and weakening the fetal membranes which leads to eventual rupture, a key process of human parturition. The aim of this study was to determine the effect of supracervical apposition and spontaneous labour on apoptosis and MMP-9 in human fetal membranes at term. Methods. Fetal membranes were obtained from term non-labouring supracervical site (SCS and compared to (i a paired distal site (DS or (ii site of rupture (SOR after spontaneous labour onset. Results. The expression of the proapoptotic markers Bax, Smac, Fas, FasL, caspase-3, and PARP, was significantly higher in the non-labouring SCS chorion compared to paired DS. Bax, Smac, FasL, caspase-3, and PARP staining was higher in the non-labouring SCS fetal membranes than that in the post-labour SOR. MMP-9 expression and activity were higher in the post-labour SOR fetal membranes compared to non-labouring SCS fetal membranes. Conclusion. Components of the apoptotic signalling pathways and MMP-9 may play a role in rupture and labour. Non-labouring SCS fetal membranes display altered morphology and altered apoptotic biochemical characteristics in preparation for labour, while the laboured SOR displays unique MMP characteristics.

  4. New Insight into the Cleavage Reaction of Nostoc sp. Strain PCC 7120 Carotenoid Cleavage Dioxygenase in Natural and Nonnatural Carotenoids

    Science.gov (United States)

    Heo, Jinsol; Kim, Se Hyeuk

    2013-01-01

    Carotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preferences of NSC3, a CCD of Nostoc sp. strain PCC 7120, in vivo and in vitro using natural and nonnatural carotenoid structures. NSC3 cleaved β-apo-8′-carotenal at 3 positions, C-13C-14, C-15C-15′, and C-13′C-14′, revealing a unique cleavage pattern. NSC3 cleaves the natural structure of carotenoids 4,4′-diaponeurosporene, 4,4′-diaponeurosporen-4′-al, 4,4′-diaponeurosporen-4′-oic acid, 4,4′-diapotorulene, and 4,4′-diapotorulen-4′-al to generate novel cleavage products (apo-14′-diaponeurosporenal, apo-13′-diaponeurosporenal, apo-10′-diaponeurosporenal, apo-14′-diapotorulenal, and apo-10′-diapotorulenal, respectively). The study of carotenoids with natural or nonnatural structures produced by using synthetic modules could provide information valuable for understanding the cleavage reactions or substrate preferences of other CCDs in vivo and in vitro. PMID:23524669

  5. New insight into the cleavage reaction of Nostoc sp. strain PCC 7120 carotenoid cleavage dioxygenase in natural and nonnatural carotenoids.

    Science.gov (United States)

    Heo, Jinsol; Kim, Se Hyeuk; Lee, Pyung Cheon

    2013-06-01

    Carotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preferences of NSC3, a CCD of Nostoc sp. strain PCC 7120, in vivo and in vitro using natural and nonnatural carotenoid structures. NSC3 cleaved β-apo-8'-carotenal at 3 positions, C-13 C-14, C-15 C-15', and C-13' C-14', revealing a unique cleavage pattern. NSC3 cleaves the natural structure of carotenoids 4,4'-diaponeurosporene, 4,4'-diaponeurosporen-4'-al, 4,4'-diaponeurosporen-4'-oic acid, 4,4'-diapotorulene, and 4,4'-diapotorulen-4'-al to generate novel cleavage products (apo-14'-diaponeurosporenal, apo-13'-diaponeurosporenal, apo-10'-diaponeurosporenal, apo-14'-diapotorulenal, and apo-10'-diapotorulenal, respectively). The study of carotenoids with natural or nonnatural structures produced by using synthetic modules could provide information valuable for understanding the cleavage reactions or substrate preferences of other CCDs in vivo and in vitro.

  6. Caspase-3-dependent cleavage of Akt modulates tau phosphorylation via GSK3β kinase: implications for Alzheimer's disease.

    Science.gov (United States)

    Chu, J; Lauretti, E; Praticò, D

    2017-01-31

    The pathological hallmark of Alzheimer's disease (AD) is accumulation of misfolded amyloid-β peptides and hyperphosphorylated tau protein in the brain. Increasing evidence suggests that serine-aspartyl proteases-caspases are activated in the AD brain. Previous studies identified a caspase-3 cleavage site within the amyloid-β precursor protein, and a caspase-3 cleavage of tau as the mechanisms involved in the development of Aβ and tau neuropathology, respectively. However, the potential role that caspase-3 could have on tau metabolism remains unknown. In the current studies, we provide experimental evidence that caspase-3 directly and specifically regulates tau phosphorylation, and demonstrate that this effect is mediated by the GSK3β kinase pathway via a caspase-3-dependent cleavage of the protein kinase B (also known as Akt). In addition, we confirm these results in vivo by using a transgenic mouse model of AD. Collectively, our findings demonstrate a new role for caspase-3 in the neurobiology of tau, and suggest that therapeutic strategies aimed at inhibiting this protease-dependent cleavage of Akt may prove beneficial in preventing tau hyperphosphorylation and subsequent neuropathology in AD and related tauopathies.Molecular Psychiatry advance online publication, 31 January 2017; doi:10.1038/mp.2016.214.

  7. Effects of Secondary Metabolites from the Fungus Septofusidium berolinense on DNA Cleavage Mediated by Human Topoisomerase IIα.

    Science.gov (United States)

    Vann, Kendra R; Ekiz, Güner; Zencir, Sevil; Bedir, Erdal; Topcu, Zeki; Osheroff, Neil

    2016-03-21

    Two metabolites from the ascomycete fungus Septofusidium berolinense were recently identified as having antineoplastic activity [Ekiz et al. (2015) J. Antibiot. , DOI: 10.1038/ja.2015.84]. However, the basis for this activity is not known. One of the compounds [3,6-dihydroxy-2-propylbenzaldehyde (GE-1)] is a hydroquinone, and the other [2-hydroxymethyl-3-propylcyclohexa-2,5-diene-1,4-dione (GE-2)] is a quinone. Because some hydroquinones and quinones act as topoisomerase II poisons, the effects of GE-1 and GE-2 on DNA cleavage mediated by human topoisomerase IIα were assessed. GE-2 enhanced DNA cleavage ∼4-fold and induced scission with a site specificity similar to that of the anticancer drug etoposide. Similar to other quinone-based topoisomerase II poisons, GE-2 displayed several hallmark characteristics of covalent topoisomerase II poisons, including (1) the inability to poison a topoisomerase IIα construct that lacks the N-terminal domain, (2) the inhibition of DNA cleavage when the compound was incubated with the enzyme prior to the addition of plasmid, and (3) the loss of poisoning activity in the presence of a reducing agent. In contrast to GE-2, GE-1 did not enhance DNA cleavage mediated by topoisomerase IIα except at very high concentrations. However, the activity and potency of the metabolite were dramatically enhanced under oxidizing conditions. These results suggest that topoisomerase IIα may play a role in mediating the cytotoxic effects of these fungal metabolites.

  8. Hydrolytic protein cleavage mediated by unusual mononuclear copper(II) complexes: X-ray structures and solution studies.

    Science.gov (United States)

    de Oliveira, Mauricio C B; Scarpellini, Marciela; Neves, Ademir; Terenzi, Hernán; Bortoluzzi, Adailton J; Szpoganics, Bruno; Greatti, Alessandra; Mangrich, Antônio S; de Souza, Emanuel M; Fernandez, Pablo M; Soares, Marcia R

    2005-02-21

    The crystal structures and redox and UV-vis/EPR spectroscopic properties of two new mononuclear copper(II) complexes, [Cu(HL1)Cl2] (1) and [Cu(L1)Cl] (2), prepared through the reaction between copper(II) chloride and the ligand 2-[(bis(pyridylmethyl)amino)methyl]-4-methyl-6-formylphenol (HL1) under distinct base conditions, are reported along with solution studies. Also, we demonstrate that these CuII complexes are able to cleave unactivated peptide bonds from bovine serum albumin (BSA) and the thermostable enzyme Taq DNA polymerase at micromolar concentration, under mild pH and temperature conditions. The cleavage activity seems to be specific with defined proteolytic fragments appearing after protein treatment. The location of the specific cleavage sites was tentatively assigned to solvent-accessible portions of the protein. These are two of the most active Cu(II) complexes described to date, since their cleavage activity is detected in minutes and evidence is here presented for a hydrolytic mechanism mediating protein cleavage by these complexes.

  9. A reduced graphene oxide-based fluorescence resonance energy transfer sensor for highly sensitive detection of matrix metalloproteinase 2.

    Science.gov (United States)

    Xi, Gaina; Wang, Xiaoping; Chen, Tongsheng

    2016-01-01

    A novel fluorescence nanoprobe (reduced nano-graphene oxide [nrGO]/fluorescein isothiocyanate-labeled peptide [Pep-FITC]) for ultrasensitive detection of matrix metalloproteinase 2 (MMP2) has been developed by engineering the Pep-FITC comprising the specific MMP2 substrate domain (PLGVR) onto the surface of nrGO particles through non-covalent linkage. The nrGO was obtained by water bathing nano-graphene oxide under 90°C for 4 hours. After mixing the nrGO and Pep-FITC for 30 seconds, the fluorescence from Pep-FITC was almost completely quenched due to the fluorescence resonance energy transfer between fluorescein isothiocyanate (FITC) and nrGO. Upon cleavage of the amide bond between Leu and Gly in the Pep-FITC by protease-MMP2, the FITC bound to nrGO was separated from nrGO surface, disrupting the fluorescence resonance energy transfer process and resulting in fluorescence recovery of FITC. Under optimal conditions, the fluorescence recovery of nrGO/Pep-FITC was found to be directly proportional to the concentration of MMP2 within 0.02-0.1 nM. The detection limit of the nrGO/Pep-FITC was determined to be 3 pM, which is approximately tenfold lower than that of the unreduced carboxylated nano-graphene oxide/Pep-FITC probe.

  10. THE CLINICAL SIGNIFICANCE OF MATRIX METALLOPROTEINASES IN RHEUMATOID ARTHRITIS PATIENTS (REVIEW OF THE LITERATURE AND OUR OWN DATA

    Directory of Open Access Journals (Sweden)

    Anastasiya S Avdeeva

    2014-01-01

    Full Text Available Matrix metalloproteinases (MMPs are a group of over 20 proteolytic enzymes responsible for cleavage of protein components of the extracellular matrix. Three types of MMPs play an important role in the development of joint damage in patients with rheumatoid arthritis (RA: collagenases (MMP1, 8 and 13, stromelysins (MMP3, and gelatinases (MMP9. MMP3 is considered to be one of the key mediators of joint damage. Increased serum level of MMP is not specific for RA and may be registered in other rheumatic diseases (osteoarthritis, psoriatic arthritis, gout, ankylosing spondylitis, systemic lupus erythematosus; however, monitoring of the level of MMP is of particular clinical importance in patients with RA. MMP3 serum level may be a useful marker of disease activity. Several studies have shown a correlation of MMP3 concentration with clinical and laboratorial parameters of inflammatory activity (ESR and C-reactive protein – CRP in RA patients. The elevated level of MMP3 is associated with radiological changes in joints and can also be a predictor of severe destructive lesions in RA patients. Evaluation of the MMP3 level can also be useful for monitoring the therapy effectiveness using both standard disease-modifying antirheumatic drugs (DMARDs and genetically engineered biological drugs (GEBD. Thus, evaluation of MMP3 concentration is useful for assessing disease activity and efficacy of treatment with DMARDs and GEBD, as well as for predicting the severity of destructive changes in joints.

  11. Metal compound-mediated hydrolytic cleavage of oxidized insulin B chain: Regioselectivity and influence of peptide secondary structure

    Institute of Scientific and Technical Information of China (English)

    罗雪梅; 何卫江; 张宇; 郭子建; 朱龙根

    2000-01-01

    The interaction of oxidized insulin B chain (B) with cis-[Pd-(en) Cl2] (en= ethylendiamine), cis-[Pd-(dtco-3-OH)Cl2](dtc o-3-OH= dithiacyclooctan-3-ol) and CuCl2 was studied by electrospray mass spectrometry. It is discovered that the binding of Pd(Ⅱ) complexes and the sites of cleavage are highly dependent on the secondary structure and local e nvironment of B. The hydrolytic cleavage of denatured B by Pd (Ⅱ) complexes was monitored by HPLC. The reaction is regioselective and follows first order kinetics with half-life of 4.8 days at 40°C. Two a mide bonds, i.e. at Leu6-Cys7 and at Gly8-Ser9, which are close to the two potential Pd(Ⅱ) binding sites His5 and His10, are selectively cleaved. In the case of Cu(Ⅱ) ion as promoter, only one cleavage site was observed which is located at Gly8-Ser9 bond. These results provide improved understanding on the deign of artificial metallopeptidase.

  12. Impaired cleavage of preproinsulin signal peptide linked to autosomal-dominant diabetes.

    Science.gov (United States)

    Liu, Ming; Lara-Lemus, Roberto; Shan, Shu-ou; Wright, Jordan; Haataja, Leena; Barbetti, Fabrizio; Guo, Huan; Larkin, Dennis; Arvan, Peter

    2012-04-01

    Recently, missense mutations upstream of preproinsulin's signal peptide (SP) cleavage site were reported to cause mutant INS gene-induced diabetes of youth (MIDY). Our objective was to understand the molecular pathogenesis using metabolic labeling and assays of proinsulin export and insulin and C-peptide production to examine the earliest events of insulin biosynthesis, highlighting molecular mechanisms underlying β-cell failure plus a novel strategy that might ameliorate the MIDY syndrome. We find that whereas preproinsulin-A(SP23)S is efficiently cleaved, producing authentic proinsulin and insulin, preproinsulin-A(SP24)D is inefficiently cleaved at an improper site, producing two subpopulations of molecules. Both show impaired oxidative folding and are retained in the endoplasmic reticulum (ER). Preproinsulin-A(SP24)D also blocks ER exit of coexpressed wild-type proinsulin, accounting for its dominant-negative behavior. Upon increased expression of ER-oxidoreductin-1, preproinsulin-A(SP24)D remains blocked but oxidative folding of wild-type proinsulin improves, accelerating its ER export and increasing wild-type insulin production. We conclude that the efficiency of SP cleavage is linked to the oxidation of (pre)proinsulin. In turn, impaired (pre)proinsulin oxidation affects ER export of the mutant as well as that of coexpressed wild-type proinsulin. Improving oxidative folding of wild-type proinsulin may provide a feasible way to rescue insulin production in patients with MIDY.

  13. Proteolytic cleavage of protein kinase Cmu upon induction of apoptosis in U937 cells.

    Science.gov (United States)

    Häussermann, S; Kittstein, W; Rincke, G; Johannes, F J; Marks, F; Gschwendt, M

    1999-12-03

    Treatment of U937 cells with various apoptosis-inducing agents, such as TNFalpha and beta-D-arabinofuranosylcytosine (ara-C) alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), bryostatin 1 or cycloheximide, causes proteolytic cleavage of protein kinase Cmu (PKCmu) between the regulatory and catalytic domain, generating a 62 kDa catalytic fragment of the kinase. The formation of this fragment is effectively suppressed by the caspase-3 inhibitor Z-DEVD-FMK. In accordance with these in vivo data, treatment of recombinant PKCmu with caspase-3 in vitro results also in the generation of a 62 kDa fragment (p62). Treatment of several aspartic acid to alanine mutants of PKCmu with caspase-3 resulted in an unexpected finding. PKCmu is not cleaved at one of the typical cleavage sites containing the motif DXXD but at the atypical site CQND378/S379. The respective fragment (amino acids 379-912) was expressed in bacteria as a GST fusion protein (GST-p62) and partially purified. In contrast to the intact kinase, the fragment does not respond to the activating cofactors TPA and phosphatidylserine and is thus unable to phosphorylate substrates effectively.

  14. of Matrix Metalloproteinase-9 and Neutrophil Gelatinase-Associated Lipocalin

    Directory of Open Access Journals (Sweden)

    De Caridi Giovanni

    2015-01-01

    Full Text Available The association of an axillary artery aneurysm and an abdominal aortic aneurysm is extremely rare. In this study, we describe this association in a 69 year-old-man. We measured this patient’s metalloproteinases (MMPs and Neutrophil Gelatinase - Associated Lipocalin (NGAL levels over a three years period before the abdominal aortic aneurysm rupture. We speculate that high serium levels of MMPs and NGAL may have a prognostic role and may predict aneurysm rupture in patients with an uncommon association of arterial aneurysms.

  15. Comparison of matrix metalloproteinase-3 and tissue inhibitor of matrix metalloproteinase-1 levels in gingival crevicular fluid in periodontal health, disease and after treatment: A clinico biochemical study

    Directory of Open Access Journals (Sweden)

    P Mohan Kumar

    2013-01-01

    Full Text Available Background: Considering the role of matrix metalloproteinase-3 (MMP-3 and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1 in the pathogenesis of periodontitis, the present study is to estimate the levels of MMP-3 and TIMP-1 in gingival crevicular fluid (GCF in periodontal health, disease and to evaluate the effect of periodontal therapy on MMP-3 and TIMP-1 concentrations in GCF. Materials and Methods: A periodontal examination and collection of GCF by extra-crevicular method was performed in 30 subjects selected randomly and categorized into 3 groups. Group I consists of 10 subjects Group II consists of 20 patients and Group III consists of 20 patients of Group II. Non surgical periodontal therapy was performed, and GCF was collected after 8 weeks from the same site of 20 chronic periodontitis patients who are considered as Group III. MMP- 3 and TIMP-1 levels were estimated in GCF-samples by using enzyme-linked immunosorbent assay. The findings were analyzed using the software and descriptive statistical methods such as Mann- Whitney U-test and Kruskal-Wallis test. P value < 0.001 was considered significant. Results: MMP-3 and TIMP-1 was detected in all samples. Highest mean MMP-3 concentrations in GCF were obtained for Group II (7.490 ng/ml while the lowest concentrations were seen in Group I (0.344 ng/ml and Group III (2.129 ng/ml. This suggests that MMP-3 levels in GCF increases proportionally with the progression of periodontal disease and decreases after treatment. Lowest mean TIMP-1 concentrations in GCF were obtained for Group-II (1.592 ng/ml, while the highest concentrations were seen in Group-I (8.78 ng/ml and Group-III (6.40 ng/ml. This suggests that TIMP-1 levels in GCF decreases proportionally with progression of periodontal disease and increases after treatment. Conclusion: There is a substantial increase in the concentrations of MMP-3 and decrease in TIMP-1 as periodontal disease progress. Since MMP-3 and TIMP-1 levels

  16. Effect of topical fluoroquinolones on the expression of matrix metalloproteinases in the cornea

    OpenAIRE

    O'Brien Terrence P; Song Jae K; Hakim Melinda A; Reviglio Victor E

    2003-01-01

    Abstract Background Matrix metalloproteinases play an important role in extracellular matrix deposition and degradation. Based on previous clinical observations of corneal perforations during topical fluoroquinolone treatment, we decided to evaluate the comparative effects of various fluoroquinolone eye drops on the expression of matrix metalloproteinases (MMPs) in cornea. Methods Eighty female Lewis rats were divided into two experimental groups: intact and wounded corneal epithelium. Unifor...

  17. Matrix metalloproteinases in gastric inflammation and cancer : clinical relevance and prognostic impact

    NARCIS (Netherlands)

    Kubben, Francois Jozef Gerard Marie

    2007-01-01

    The studies in this thesis describe the clinical impact of several matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in H. pylori-induced gastritis and gastric cancer. In patients with H. pylori-induced gastritis, significantly increased mucosal MMP-9 levels were f

  18. Matrix metalloproteinases in gastric inflammation and cancer : clinical relevance and prognostic impact

    NARCIS (Netherlands)

    Kubben, Francois Jozef Gerard Marie

    2007-01-01

    The studies in this thesis describe the clinical impact of several matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in H. pylori-induced gastritis and gastric cancer. In patients with H. pylori-induced gastritis, significantly increased mucosal MMP-9 levels were

  19. Quantification of Tissue Inhibitor of Metalloproteinases 2 in Plasma from Healthy Donors and Cancer Patients

    DEFF Research Database (Denmark)

    Larsen, M B; Stephens, R W; Brünner, Nils

    2005-01-01

    Tissue inhibitor of metalloproteinases (TIMP)-2 is a highly conserved molecule, which binds both active and latent matrix metalloproteinase (MMP)-2. TIMP-2 is also involved in the activation of MMP-2 on the cell surface. A quantitative enzyme-linked immunosorbent assay (ELISA) was established...

  20. Profibrinolytic effects of metalloproteinases during skin wound healing in the absence of plasminogen

    DEFF Research Database (Denmark)

    Green, Kirsty A; Almholt, Kasper; Ploug, Michael;

    2008-01-01

    Genetic ablation of plasminogen (Plg) and pharmacological inhibition of metalloproteinase activity by galardin delay skin wound healing in mice, whereas the combined inhibition of these two enzyme systems completely prevents healing. In this study, the impact of plasmin and metalloproteinases as ...

  1. The effect of subgingival antimicrobial therapy on the levels of stromelysin and tissue inhibitor of metalloproteinases in gingival crevicular fluid.

    Science.gov (United States)

    Pourtaghi, N; Radvar, M; Mooney, J; Kinane, D F

    1996-09-01

    Recent investigations imply that a key mechanism in the pathogenesis of periodontal disease may be the ability of oral microorganisms to induce production and/or activation of matrix metalloproteinases (MMPs) in the host tissues. It has been suggested that the pharmacologic inhibition of MMP activity could play an important role in achieving a desirable outcome in periodontal therapy. The efficacy of locally delivered antibiotics on the level of gingival crevicular fluid (GCF) stromelysin (SL) and tissue inhibitor of metalloproteinases (TIMP) on sites with a history of a poor response to mechanical treatment was studied. Fifty-two patients with 4 periodontal pockets > or = 5 mm and bleeding on probing were randomized into four groups of 13 patients. One group received scaling and root planing alone and the other three groups received scaling and root planing plus a locally delivered antimicrobial system. These included 25% tetracycline fiber, 2% minocycline gel, and 25% metronidazole gel. The GCF samples taken at baseline and 6 weeks after treatments were analyzed using an enzyme linked immunosorbent assay (ELISA). GCF SL levels significantly decreased after adjunctive tetracycline fiber (paired t-test, P = 0.020) and minocycline gel (paired t-test, P = 0.023) treatments whereas it remained almost unchanged in the other two groups. While the GCF TIMP level did not change significantly in the scaling and root planing alone group, it significantly increased for all three adjunctive antimicrobial treatments (for tetracycline fiber P family, may offer an advantage in changing the metalloproteinase profile of the GCF to one more compatible with periodontal health.

  2. Structural and biochemical characterisation of VaF1, a P-IIIa fibrinogenolytic metalloproteinase from Vipera ammodytes ammodytes venom.

    Science.gov (United States)

    Leonardi, Adrijana; Sajevic, Tamara; Latinović, Zorica; Pungerčar, Jože; Balija, Maja Lang; Bakija, Alenka Trampuš; Vidmar, Robert; Halassy, Beata; Križaj, Igor

    2015-02-01

    A high molecular mass metalloproteinase with α-fibrinogenolytic activity, termed VaF1, was purified from nose-horned viper (Vipera ammodytes ammodytes) venom. Subcutaneous injection of 9 μg of VaF1 did not induce bleeding in rats. Nevertheless, in vitro it degraded collagen IV, nidogen and fibronectin, components of the extracellular matrix, although with low efficacy and narrow specificity. VaF1 would be expected to exert anti-coagulant action, due to its hydrolysis of fibrinogen, factor X, prothrombin and plasminogen, plasma proteins involved in blood coagulation. The enzyme is a single-chain glycoprotein with a molecular mass of 49.7 kDa, as determined by mass spectrometry, and multiple isoelectric points centred at pH 5.8. The complete amino acid sequence of the precursor of VaF1 was deduced by cloning and sequencing its cDNA. Composed of metalloproteinase, disintegrin-like and cysteine-rich domains, VaF1 is a typical P-IIIa subclass snake venom metalloproteinase. Although it possesses a collagen-binding sequence in its disintegrin-like domain, VaF1 displayed no effect on collagen-induced platelet aggregation in vitro. Two consensus N-glycosylation sites are present in the sequence of VaF1, however, the extent of its glycosylation is low, only 5.2% of the total molecular mass. Interestingly, in standard experimental conditions VaF1 is not recognised by antiserum against the whole venom, so it can contribute to post-serotherapy complications, such as ineffective blood coagulation, in the envenomed patient. Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

  3. Feline Calicivirus Infection Disrupts Assembly of Cytoplasmic Stress Granules and Induces G3BP1 Cleavage.

    Science.gov (United States)

    Humoud, Majid N; Doyle, Nicole; Royall, Elizabeth; Willcocks, Margaret M; Sorgeloos, Frederic; van Kuppeveld, Frank; Roberts, Lisa O; Goodfellow, Ian G; Langereis, Martijn A; Locker, Nicolas

    2016-07-15

    In response to stress such as virus infection, cells can stall translation by storing mRNAs away in cellular compartments called stress granules (SGs). This defense mechanism favors cell survival by limiting the use of energy and nutrients until the stress is resolved. In some cases it may also block viral propagation as viruses are dependent on the host cell resources to produce viral proteins. Human norovirus is a member of the Caliciviridae family responsible for gastroenteritis outbreaks worldwide. Previous studies on caliciviruses have identified mechanisms by which they can usurp the host translational machinery, using the viral protein genome-linked VPg, or regulate host protein synthesis through the mitogen-activated protein kinase (MAPK) pathway. Here, we examined the effect of feline calicivirus (FCV) infection on SG accumulation. We show that FCV infection impairs the assembly of SGs despite an increased phosphorylation of eukaryotic initiation factor eIF2α, a hallmark of stress pathway activation. Furthermore, SGs did not accumulate in FCV-infected cells that were stressed with arsenite or hydrogen peroxide. FCV infection resulted in the cleavage of the SG-nucleating protein Ras-GTPase activating SH3 domain-binding protein (G3BP1), which is mediated by the viral 3C-like proteinase NS6(Pro) Using mutational analysis, we identified the FCV-induced cleavage site within G3BP1, which differs from the poliovirus 3C proteinase cleavage site previously identified. Finally, we showed that NS6(Pro)-mediated G3BP1 cleavage impairs SG assembly. In contrast, murine norovirus (MNV) infection did not impact arsenite-induced SG assembly or G3BP1 integrity, suggesting that related caliciviruses have distinct effects on the stress response pathway. Human noroviruses are a major cause of viral gastroenteritis, and it is important to understand how they interact with the infected host cell. Feline calicivirus (FCV) and murine norovirus (MNV) are used as models to

  4. Induction of matrix metalloproteinase-9 and -2 activity in mouse blastocyst by fibronectin-integrin interaction

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Fibronectin, a major extracellular matrix, plays an important role in embryo implantation by mediating embryo adhesion and outgrowth. In this work, mouse blastocysts produced pro-matrix metalloproteinase-9, pro-matrix metalloproteinase-2 and 64 ku matrix metalloproteinase-2 when they were co-cultured with fibronectin. In contrast, mouse blastocysts did not produce these proteinases without fibronectin. Focal adhesion kinase is a fundamental molecule of integrin signaling pathway and its antisense oligodeoxynucleiotide inhibited blastocyst matrix metalloproteinases expression induced by fibronectin. The results indicated that fibronectin triggered matrix metalloproteinase-9 and -2 expression in mouse blastocyst through its integrin receptors and subsequent signaling pathway, which enhanced the synchronization of blastocyst invasiveness and uterine receptivity and ensured the accuracy of events relative to implantation in timing and spatiality.

  5. Kinetics of phycocyanobilin cleavage from C-phycocyanin by methanolysis

    DEFF Research Database (Denmark)

    Malwade, Chandrakant Ramkrishna; Roda Serrat, Maria Cinta; Christensen, Knud Villy

    2016-01-01

    Phycocyanobilin (PCB) is an important linear tetrapyrrolic molecule for food as well as pharmaceutical industry. It is obtained from blue-green algae, where it is attached covalently to phycobiliproteins (C-PC and APC) present in the light harvesting complexes. In this work, cleavage of PCB from...

  6. Synthesis, Characterization and DNA Cleavage of Copper(II ...

    African Journals Online (AJOL)

    (UV) light. Results: ATR-FTIR confirmed the formation of copper(II) complex with DTT by binding through thiol group based on the .... DNA cleavage detection ... The infrared spectra of pure DTT and its Cu(II) .... and iron complexes. J Phys Conf ...

  7. Sensitive and fast mutation detection by solid phase chemical cleavage

    DEFF Research Database (Denmark)

    Hansen, Lise Lotte; Justesen, Just; Kruse, Torben A

    1996-01-01

    We have developed a solid phase chemical cleavage method (SpCCM) for screening large DNA fragments for mutations. All reactions can be carried out in microtiterwells from the first amplification of the patient (or test) DNA through the search for mutations. The reaction time is significantly...

  8. Kinetic analysis demonstrates a requirement for the Rat1 exonuclease in cotranscriptional pre-rRNA cleavage.

    Directory of Open Access Journals (Sweden)

    Konstantin Axt

    Full Text Available During yeast ribosome synthesis, three early cleavages generate the 20S precursor to the 18S rRNA component of the 40S subunits. These cleavages can occur either on the nascent transcript (nascent transcript cleavage; NTC or on the 35S pre-rRNA that has been fully transcribed and released from the rDNA (released transcript cleavage; RTC. These alternative pathways cannot be assessed by conventional RNA analyses, since the pre-rRNA products of NTC and RTC are identical. They can, however, be distinguished kinetically by metabolic labeling and quantified by modeling of the kinetic data. The aim of this work was to use these approaches as a practical tool to identify factors that mediate the decision between utilization of NTC and RTC. The maturation pathways of the 40S and 60S ribosomal subunits are largely distinct. However, depletion of some early-acting 60S synthesis factors, including the 5'-exonuclease Rat1, leads to accumulation of the 35S pre-rRNA and delayed 20S pre-rRNA synthesis. We speculated that this might reflect the loss of NTC. Rat1 acts catalytically in 5.8S and 25S rRNA processing but binds to the pre-rRNA prior to these activities. Kinetic data for strains depleted of Rat1 match well with the modeled effects of strongly reduced NTC. This was confirmed by EM visualization of "Miller" chromatin spreads of nascent pre-rRNA transcripts. Modeling further indicates that NTC takes place in a limited time window, when the polymerase has transcribed ∼ 1.5 Kb past the A2 cleavage site. We speculate that assembly of early-acting 60S synthesis factors is monitored as a quality control system prior to NTC.

  9. Amlodipine inhibits matrix metalloproteinases expression and secretion in mouse macrophage

    Institute of Scientific and Technical Information of China (English)

    Yamin CAO; Shiwen WANG; Haiyun WU

    2005-01-01

    To investigate whether the calcium channel blocker amlodipine could inhibit macrophage matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) expression and secretion. Methods Peritoneal macrophages were isolated from BALB/C mice and incubated with low (5μg/L), middle (15μg/L) and high (305μg/L) concentrations of amlodipine, or in the medium alone (controls) for 24 hours, and the expression and secretion of MMP-2 and MM-9 of the cells were analyzed by RT-PCR and gelatin zymography. Results Compared with controls, amlodipine at low concentration had no significant effects on the expression and secretion of either MMP-2 and MMP-9 (P>0.05);at middle concentrationit it could inhibited MMP-2 and MMP-9 expressions completely and significantly reduced the secretion of MMP-9 (P<0.05); but it had no effect on the secretion of MMP-2. At high concentration it also inhibited MMP-2 and MMP-9 expression completely. Conclusion Amlodipine at 15 ìg/L inhibited the expression of MMP-2 and MMP-9 and reduced the secretion of MMP-9, suggesting that amlodipine may stabilize atherosclerotic plaque.

  10. The regulation of matrix metalloproteinases and their inhibitors.

    Science.gov (United States)

    Clark, Ian M; Swingler, Tracey E; Sampieri, Clara L; Edwards, Dylan R

    2008-01-01

    The matrix metalloproteinases (MMP) are a family of 23 enzymes in man. These enzymes were originally described as cleaving extracellular matrix (ECM) substrates with a predominant role in ECM homeostasis, but it is now clear that they have much wider functionality. Control over MMP and/or tissue inhibitor of metalloproteinases (TIMP) activity in vivo occurs at different levels and involves factors such as regulation of gene expression, activation of zymogens and inhibition of active enzymes by specific inhibitors. Whilst these enzymes and inhibitors have clear roles in physiological tissue turnover and homeostasis, if control of their expression or activity is lost, they contribute to a number of pathologies including e.g. cancer, arthritis and cardiovascular disease. The expression of many MMPs and TIMPs is regulated at the level of transcription by a variety of growth factors, cytokines and chemokines, though post-transcriptional pathways may contribute to this regulation in specific cases. The contribution of epigenetic modifications has also been uncovered in recent years. The promoter regions of many of these genes have been, at least partly, characterised including the role of identified single nucleotide polymorphisms. This article aims to review current knowledge across these gene families and use a bioinformatic approach to fill the gaps where no functional data are available.

  11. Regulation of ovarian function by the matrix metalloproteinase system

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    @@ In most organs of mammals, cyclic remodelling of tissues after morphogenesis is minimal; however, repro-ductive tissues of female animals including endometrium, mammary gland, ovarian follicle and corpus luteum un-dergo growth, maturation and involution at various stages in the reproductive cycle or lifespan of the animal. Recon-struction of the extracellular matrix (ECM) is required for the dynamic tissue reorganization characteristic of these tissues. The ECM consists of proteinaceous and nonpro-teinaceous molecules that provide the tissue-specific, ex-tracellular architecture to which cells attach. Furthermore, interaction of cellular receptors with proteins of the ECM can regulate cellular structure, second messenger genera-tion and gene expression. Maintenance of ECM homeo-stasis depends largely on coordinated action of matrix metalloproteinases (MMPs) and tissue inhibitors of met-alloproteinases (TIMPs)-- an important proteinase sys-tem responsible for degradating and remodelling of ECM[1]. MMPs/TIMPs have been recognized as the cru-cial role players in regulating follicular and luteal function for their extensive involvements in the cyclic changes of dynamic ovarian tissues. In recent years, literature that MMP system has important roles in ovary is accumulating. The focus of this review is on the effects of MMPs and their inhibitors, TIMPs on follicular growth, atresia, ovu-lation, luteal development, and luteolysis. Emphasis has been given to the recent progress in the new field when-ever possible.

  12. Interaction of Bothrops jararaca venom metalloproteinases with protein inhibitors.

    Science.gov (United States)

    Asega, Amanda F; Oliveira, Ana K; Menezes, Milene C; Neves-Ferreira, Ana Gisele C; Serrano, Solange M T

    2014-03-01

    Snake venom metalloproteinases (SVMPs) play important roles in the local and systemic hemorrhage observed upon envenomation. In a previous study on the structural elements important for the activities of HF3 (highly hemorrhagic, P-III-SVMP), bothropasin (hemorrhagic, P-III-SVMP) and BJ-PI (non-hemorrhagic, P-I-SVMP), from Bothrops jararaca, it was demonstrated that they differ in their proteolysis profile of plasma and extracellular matrix proteins. In this study, we evaluated the ability of proteins DM43 and α2-macroglobulin to interfere with the proteolytic activity of these SVMPs on fibrinogen and collagen VI and with their ability to induce hemorrhage. DM43 inhibited the proteolytic activity of bothropasin and BJ-PI but not that of HF3, and was not cleaved the three proteinases. On the other hand, α2-macroglobulin did not inhibit any of the proteinases and was rather cleaved by them. In agreement with these findings, binding analysis showed interaction of bothropasin and BJ-PI but not HF3 to DM43 while none of the proteinases bound to α2-macroglobulin. Moreover, DM43 promoted partial inhibition of the hemorrhagic activity of bothropasin but not that of HF3. Our results demonstrate that metalloproteinases of B. jararaca venom showing different domain composition, glycosylation level and hemorrhagic potency show variable susceptibilities to protein inhibitors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Acknowledged Signatures of Matrix Metalloproteinases in Takayasu’s Arteritis

    Directory of Open Access Journals (Sweden)

    Gang Wu

    2014-01-01

    Full Text Available Takayasu’s arteritis (TA was reported as an eye disease in the year 1905 and later was confirmed as a vasculitis. Since then, the etiology of the disease remains unknown; however, characteristic clinical features suggest multiple causative factors. Recent progress in vascular biology and other disciplines enlightens the pathophysiology of TA and demonstrated induction of various nonspecific inflammatory symptoms and destruction of the arterial wall, which leads to aneurysms and rupture of the affected arteries. Matrix metalloproteinases (MMPs as an enzyme family have well-established roles in several vascular pathologies including intima formation, atherosclerosiss and aneurysms. MMPs have been proposed to be one of the molecules with a potential of having dual role in the course of TA, first as an active participant in pathophysiology and secondly as a diagnostic biomarker for TA disease. The desire to improve our understanding of the importance of MMPs and their endogenous inhibitors (TIMPs in TA disease and for the development of therapeutic agents has inspired basic and clinical scientists for over a decade. In the present paper, we summarized the scientific rationale which highlights the signatures of matrix metalloproteinases and their endogenous inhibitors in pathophysiology as well as their being a potential candidate as biomarker for Takayasu’s arteritis.

  14. Dimerization of matrix metalloproteinase-2 (MMP-2): functional implication in MMP-2 activation.

    Science.gov (United States)

    Koo, Bon-Hun; Kim, Yeon Hyang; Han, Jung Ho; Kim, Doo-Sik

    2012-06-29

    Matrix metalloproteinase-2 (MMP-2) functions in diverse biological processes through the degradation of extracellular and non-extracellular matrix molecules. Because of its potential for tissue damage, there are several ways to regulate MMP-2 activity, including gene expression, compartmentalization, zymogen activation, and enzyme inactivation by extracellular inhibitors. Enzyme regulation through zymogen activation is important for the regulation of MMP-2 activity. In our previous studies, we showed that thrombin directly cleaved the propeptide of MMP-2 at specific sites for enzyme activation. We also demonstrated that heparan sulfate was required for thrombin-mediated activation of pro-MMP-2 by binding to thrombin, presumably through conformational changes at the active site of the enzyme. This suggests a regulatory mechanism for thrombin-mediated activation of pro-MMP-2. In this study, we found that MMP-2 formed a reduction-sensitive homodimer in a controlled manner and that Ca(2+) ion was essential for homodimerization of MMP-2. Homodimerization was not associated with protein kinase C-mediated phosphorylation of MMP-2. MMP-2 formed a homodimer through an intermolecular disulfide bond between Cys(102) and the neighboring Cys(102). Homodimerization of MMP-2 enhanced thrombin-mediated activation of pro-MMP-2. Moreover, the MMP-2 homodimer could cleave a small peptide substrate without removal of the propeptide. Taken together, our experimental data suggest a novel regulatory mechanism for pro-MMP-2 activation that is modulated through homodimerization of MMP-2.

  15. Identification of GPR65, a novel regulator of matrix metalloproteinases using high through-put screening

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Hongbo; Chen, Xiaohong; Huang, Junwei [Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Key Laboratory of Otolaryngology Head and Neck Surgery, Beijing (China); Deng, Weiwei [Functional Genomics Group, Chinese National Human Genome Center (CHGB) at Beijing (China); Zhong, Qi [Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Key Laboratory of Otolaryngology Head and Neck Surgery, Beijing (China); Yue, Changli [Department of Pathology, Beijing Tongren Hospital, Capital Medical University, Beijing (China); Wang, Pingzhang, E-mail: wangpzh@bjmu.edu.cn [Department of Immunology, School of Basic Medical Sciences, Peking University Health Science Center, Peking University Center for Human Disease Genomics, Key Laboratory of Medical Immunology, Ministry of Health (China); Functional Genomics Group, Chinese National Human Genome Center (CHGB) at Beijing (China); Huang, Zhigang, E-mail: enthuangzhigang@sohu.com [Department of Otolaryngology-Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Key Laboratory of Otolaryngology Head and Neck Surgery, Beijing (China)

    2013-06-21

    Highlights: •A novel mechanism of MMP3 regulation by proton-sensing G-protein-coupled receptors was defined. •GPR65 was identified to induce the MMP3 expression. •GPR65 mediated MMP induction under acidic conditions. •AP-1 binding site in MMP3 promoter was crucial for MMP3 induction. •GPR65 overexpression can accelerate the invision of A549 cells. -- Abstract: Matrix metalloproteinases (MMPs) are over-expressed in nearly all cancers. To study novel regulatory factors of MMP expression in head and neck cancer (HNC), we screened a total of 636 candidate genes encoding putative human transmembrane proteins using MMP promoter reporter in a dual luciferase assay system. Three genes GPR65, AXL and TNFRSF10B dramatically activated the induction of MMP3 expression. The induction of MMP expression by GPR65 was further confirmed in A549 and/or FaDu cells. GPR65 mediated MMP induction under acidic conditions. The AP-1 binding site in MMP3 promoter was crucial for MMP3 induction. Moreover, the A549 cells infected by recombinant adenovirus of GPR65 showed accelerated cell invasion. In conclusion, we validate that GPR65 is vital regulatory genes upstream of MMP3, and define a novel mechanism of MMP3 regulation by proton-sensing G-protein-coupled receptors.

  16. Thermodynamic Basis of Selectivity in the Interactions of Tissue Inhibitors of Metalloproteinases N-domains with Matrix Metalloproteinases-1, -3, and -14.

    Science.gov (United States)

    Zou, Haiyin; Wu, Ying; Brew, Keith

    2016-05-20

    The four tissue inhibitors of metalloproteinases (TIMPs) are potent inhibitors of the many matrixins (MMPs), except that TIMP1 weakly inhibits some MMPs, including MMP14. The broad-spectrum inhibition of MMPs by TIMPs and their N-domains (NTIMPs) is consistent with the previous isothermal titration calorimetric finding that their interactions are entropy-driven but differ in contributions from solvent and conformational entropy (ΔSsolv, ΔSconf), estimated using heat capacity changes (ΔCp). Selective engineered NTIMPs have potential applications for treating MMP-related diseases, including cancer and cardiomyopathy. Here we report isothermal titration calorimetric studies of the effects of selectivity-modifying mutations in NTIMP1 and NTIMP2 on the thermodynamics of their interactions with MMP1, MMP3, and MMP14. The weak inhibition of MMP14 by NTIMP1 reflects a large conformational entropy penalty for binding. The T98L mutation, peripheral to the NTIMP1 reactive site, enhances binding by increasing ΔSsolv but also reduces ΔSconf However, the same mutation increases NTIMP1 binding to MMP3 in an interaction that has an unusual positive ΔCp This indicates a decrease in solvent entropy compensated by increased conformational entropy, possibly reflecting interactions involving alternative conformers. The NTIMP2 mutant, S2D/S4A is a selective MMP1 inhibitor through electrostatic effects of a unique MMP-1 arginine. Asp-2 increases reactive site polarity, reducing ΔCp, but increases conformational entropy to maintain strong binding to MMP1. There is a strong negative correlation between ΔSsolv and ΔSconf for all characterized interactions, but the data for each MMP have characteristic ranges, reflecting intrinsic differences in the structures and dynamics of their free and inhibitor-bound forms.

  17. Vesicle-associated membrane protein (VAMP) cleavage by a new metalloprotease from the Brazilian scorpion Tityus serrulatus.

    Science.gov (United States)

    Fletcher, Paul L; Fletcher, Maryann D; Weninger, Keith; Anderson, Trevor E; Martin, Brian M

    2010-03-01

    We present evidence that venom from the Brazilian scorpion Tityus serrulatus and a purified fraction selectively cleave essential SNARE proteins within exocrine pancreatic tissue. Western blotting for vesicle-associated membrane protein type v-SNARE proteins (or synaptobrevins) reveals characteristic alterations to venom-treated excised pancreatic lobules in vitro. Immunocytochemistry by electron microscopy confirms both the SNARE identity as VAMP2 and the proteolysis of VAMP2 as a marked decrease in secondary antibody-conjugated colloidal gold particles that are predominantly associated with mature zymogen granules. Studies with recombinant SNARE proteins were used to determine the specific cleavage site in VAMP2 and the susceptibility of VAMP8 (endobrevin). The VAMP2 cleavage site is between the transmembrane anchor and the SNARE motif that assembles into the ternary SNARE complex. Inclusion of divalent chelating agents (EDTA) with fraction nu, an otherwise active purified component from venom, eliminates SNARE proteolysis, suggesting the active protein is a metalloprotease. The unique cleavages of VAMP2 and VAMP8 may be linked to pancreatitis that develops following scorpion envenomation as both of these v-SNARE proteins are associated with zymogen granule membranes in pancreatic acinar cells. We have isolated antarease, a metalloprotease from fraction nu that cleaves VAMP2, and report its amino acid sequence.

  18. Vesicle-associated Membrane Protein (VAMP) Cleavage by a New Metalloprotease from the Brazilian Scorpion Tityus serrulatus*

    Science.gov (United States)

    Fletcher, Paul L.; Fletcher, Maryann D.; Weninger, Keith; Anderson, Trevor E.; Martin, Brian M.

    2010-01-01

    We present evidence that venom from the Brazilian scorpion Tityus serrulatus and a purified fraction selectively cleave essential SNARE proteins within exocrine pancreatic tissue. Western blotting for vesicle-associated membrane protein type v-SNARE proteins (or synaptobrevins) reveals characteristic alterations to venom-treated excised pancreatic lobules in vitro. Immunocytochemistry by electron microscopy confirms both the SNARE identity as VAMP2 and the proteolysis of VAMP2 as a marked decrease in secondary antibody-conjugated colloidal gold particles that are predominantly associated with mature zymogen granules. Studies with recombinant SNARE proteins were used to determine the specific cleavage site in VAMP2 and the susceptibility of VAMP8 (endobrevin). The VAMP2 cleavage site is between the transmembrane anchor and the SNARE motif that assembles into the ternary SNARE complex. Inclusion of divalent chelating agents (EDTA) with fraction ν, an otherwise active purified component from venom, eliminates SNARE proteolysis, suggesting the active protein is a metalloprotease. The unique cleavages of VAMP2 and VAMP8 may be linked to pancreatitis that develops following scorpion envenomation as both of these v-SNARE proteins are associated with zymogen granule membranes in pancreatic acinar cells. We have isolated antarease, a metalloprotease from fraction ν that cleaves VAMP2, and report its amino acid sequence. PMID:20026600

  19. Cleavage of recombinant proteins at poly-His sequences by Co(II) and Cu(II)

    Science.gov (United States)

    Andberg, Martina; Jäntti, Jussi; Heilimo, Sara; Pihkala, Päivi; Paananen, Arja; Koskinen, Ari M.P.; Söderlund, Hans; Linder, Markus B.

    2007-01-01

    Improved ways to cleave peptide chains at engineered sites easily and specifically would form useful tools for biochemical research. Uses of such methods include the activation or inactivation of enzymes or the removal of tags for enhancement of recombinant protein expression or tags used for purification of recombinant proteins. In this work we show by gel electrophoresis and mass spectroscopy that salts of Co(II) and Cu(II) can be used to cleave fusion proteins specifically at sites where sequences of His residues have been introduced by protein engineering. The His residues could be either consecutive or spaced with other amino acids in between. The cleavage reaction required the presence of low concentrations of ascorbate and in the case of Cu(II) also hydrogen peroxide. The amount of metal ions required for cleavage was very low; in the case of Cu(II) only one to two molar equivalents of Cu(II) to protein was required. In the case of Co(II), 10 molar equivalents gave optimal cleavage. The reaction occurred within minutes, at a wide pH range, and efficiently at temperatures ranging from 0°C to 70°C. The work described here can also have implications for understanding protein stability in vitro and in vivo. PMID:17600148

  20. Mutations in the human immunodeficiency virus type 1 polypurine tract (PPT) reduce the rate of PPT cleavage and plus-strand DNA synthesis.

    Science.gov (United States)

    McWilliams, M J; Julias, J G; Hughes, S H

    2008-05-01

    Previously, we analyzed the effects of point mutations in the human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT) and found that some mutations affected both titer and cleavage specificity. We used HIV-1 vectors containing two PPTs and the D116N integrase active-site mutation in a cell-based assay to measure differences in the relative rates of PPT processing and utilization. The relative rates were measured by determining which of the two PPTs in the vector is used to synthesize viral DNA. The results indicate that mutations that have subtle effects on titer and cleavage specificity can have dramatic effects on rates of PPT generation and utilization.

  1. Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus infected cells

    DEFF Research Database (Denmark)

    Kristensen, Thea; Normann, Preben; Gullberg, Maria

    2016-01-01

    The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been des...... have implications for the testing of potential antiviral agents targeting the FMDV 3C protease....

  2. Expressions of matrix metalloproteinases and tissue inhibitor of metalloproteinases after bare and magnetic stent implantation in rabbits

    Institute of Scientific and Technical Information of China (English)

    Xinhong Guo; Guoliang Jia; Anlin Lu; Xinguo Zhao; Fei Li; Rongqing Zhang

    2008-01-01

    Objective We aimed to investigate whether magnetic stent has preventive effect on in-stent restenosis by observing expressions of matrix metalioproteinase (MMP)2,MMP9,tissue inhibitor of matrix metalloproteinase (TIMP)1 and TIMP2 after balloon angioplasty,bare and magnetic stent implantation in rabbits.Methods Rabbits underwent balloon angioplasty,bare and magnetic stent implantation in the left iliac arteries.The changes of MMPs and TIMPs were examined at various time points in the injured arteries using the methods of zymography,Western blot analysis,reverse transcription-polymerase chain reaction (RT-PCR) and morphometric analysis.Results Balloon angioplasty group (BA) and magnetic stent group (MS) showed lower intrinsic gelatinolytic activity and higher expression of TIMPs with less intimae hyperplasia;Whereas bare stent (BS) group exhibited higher intrinsic gelatinolytic activity and lower expression of TIMPs with significant intimae hyperplasia.Conclusion Magnetic stent probably has preventive effect on in-stent restenosis by changing intrinsic matrix metalloproteinases activity and expression of TIMPs.

  3. Association of metalloproteinases, tissue inhibitors of matrix metalloproteinases, and proteoglycans with development, aging, and osteoarthritis processes in mouse temporomandibular joint.

    Science.gov (United States)

    Gepstein, Amira; Arbel, Gil; Blumenfeld, Israel; Peled, Micha; Livne, Erella

    2003-07-01

    The temporomandibular joint (TMJ) is an important growth and articulation center in the craniofacial complex. In aging it develops spontaneous degenerative osteoarthritic (OA) lesions. Metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPS) play key roles in extracellular matrix remodeling and degradation. Gelatinase activities and immunohistochemical localization of MMP-2, -3, -8, -9, and -13 and TIMP-1 and -2 were examined in mandibular condyle cartilage of neonatal mice up to 18 months old. The most intense immunostaining for all enzymes and TIMPs and the peak of gelatinase activities were found in animals in the stages of early growth (1 week to 3 months) followed by a decrease during maturation and aging. However, clusters of positively immunoreactive chondrocytes were detected in cartilages of old animals displaying OA lesions. Positive safranin-O staining, indicative of sulfated proteoglycans (PGs), was prominent in the TMJ of newborn mice up to 3 months old followed by reduction during maturation and aging, except in regions displaying OA lesions. Temporal codistribution of PGs, MMPs, and TIMPs during skeletal maturation reflected an active growth phase, whereas their reduction coincided with the more quiescent articulating and maintenance phase in the joint cartilage. Osteoarthritic lesions were associated with both increased PG synthesis and MMP immunoreactivity, indicating limited repair activity during initial stages of osteoarthritis.

  4. Fractional Excretion of Survivin, Extracellular Matrix Metalloproteinase Inducer, and Matrix Metalloproteinase 7 in Children with Chronic Kidney Disease

    Directory of Open Access Journals (Sweden)

    Agnieszka Bargenda

    2016-07-01

    Full Text Available Background: Epithelial–mesenchymal transition (EMT is defined as a transformation of tubular epithelial cells into mesenchymal ones. These cells migrate through the extracellular matrix and change into active myofibroblasts, which are responsible for excessive matrix deposition. Such changes may lead to tubular dysfunction and fibrosis of the renal parenchyma, characteristic of chronic kidney disease (CKD. However, there are no data on potential EMT markers in children with CKD. The aim of our study was to assess the usefulness of fractional excretion (FE of survivin, E-cadherin, extracellular matrix metalloproteinase inducer (EMMPRIN, matrix metalloproteinase (MMP7, and transforming growth factor beta 1 (TGF-β1 as potential markers of CKD-related complications such as tubular damage and fibrosis. Methods: Forty-one pre-dialysis children with CKD Stages 3–5 and 23 age-matched controls were enrolled in the study. The serum and urine concentrations of analysed parameters were assessed by an enzyme-linked immunosorbent assay test. Results: Tubular reabsorption of all analysed parameters was >99% in the control group. All FE values rose significantly in children with CKD, yet they remained 1%. Conclusions: FE of the examined markers may become a useful tool in the assessment of tubular dysfunction during the course of CKD. The FE of survivin, EMMPRIN, and MMP7 warrant further research as potential independent markers of kidney-specific EMT.

  5. Immunohistochemical Correlation of Matrix Metalloproteinase-2 and Tissue Inhibitors of Metalloproteinase-2 in Tobacco Associated Epithelial Dysplasia

    Directory of Open Access Journals (Sweden)

    Dipshikha Bajracharya

    2014-01-01

    Full Text Available Aim. To study the immunohistochemical expression of matrix metalloproteinase and tissue inhibitors of matrix metalloproteinase-2 in different histological grades of tobacco associated epithelial dysplasia and correlate the association between these proteases. Potentially malignant oral disorders (PMODs progressing to oral cancer are related to the severity of epithelial dysplasia. Methods. A retrospective immunohistochemical study was carried out on 30 clinically and histologically proven cases of leukoplakia with dysplasia and 10 cases of normal buccal mucosa using anti-MMP-2 and anti-TIMP-2 monoclonal antibodies. Results. Mann Whitney U test, for comparing the expression of both MMP-2 and TIMP-2 in normal mucosa with dysplasia, was highly significant (P<0.001. Kruskal-Wallis test to compare the median score of MMP-2 and TIMP-2 in different grades of dysplasia showed statistical significance (P<0.001, and a Spearman’s correlation between MMP-2 and TIMP-2 through different grades of dysplasia and cells observed showed positive correlation. Conclusion. Concomitant increase in the expression of both MMP-2 and TIMP-2 suggested that the activation of MMP-2 is dependent on TIMP-2 acting as a cofactor. Changes in TIMP-2 levels are considered important because they directly affect the level of MMP-2 activity.

  6. Relationship between Serum Levels of Metalloproteinase-8 and Tissue Inhibitor of Metalloproteinases-1 and Exercise Test Results in Postmenopausal Women

    Directory of Open Access Journals (Sweden)

    J. Mieczkowska

    2016-01-01

    Full Text Available Physical activity as a part of the lifestyle is a significant factor influencing health condition. Exercises that require stamina are of particular importance. Oxygen metabolism, which is a significant part of all longer training processes, has an influence on cardiovascular and respiratory system functioning as well as all the processes taking part in maintenance of efficient homeostasis. Presentation of the correlation between exercise test results and MMP-8 (metalloproteinase-8 and TIMP-1 (tissue inhibitor of metalloproteinases-1 levels was attempted in this work. MMP-8 is a proteolytic enzyme taking part in progression of diseases related to process of ageing. 62 healthy women in postmenopausal period were qualified for the study (mean age: 54±3.6. There was exercise test on the treadmill according to Bruce’s protocol performed. MMP-8 and TIMP-1 serum levels were measured. There was statistically important correlation between increased level of MMP-8 and increased level of TIMP-1 with lower results of exercise test observed. The conducted study provides further biochemical arguments for prophylactic role of physical activity, which lowers the risk of noninfectious diseases, typical for middle adulthood, by influencing physical capacity.

  7. Metalloproteinases control brain inflammation induced by pertussis toxin in mice overexpressing the chemokine CCL2 in the central nervous system

    DEFF Research Database (Denmark)

    Toft-Hansen, Henrik; Buist, Richard; Sun, Xue-Jun

    2006-01-01

    symptoms. Metalloproteinase (MPs) enzymes are implicated in leukocyte infiltration in neuroinflammation. Unmanipulated Tg mice had elevated expression of tissue inhibitor of metalloproteinase-1, matrix metalloproteinase (MMP)-10, and -12 mRNA in the brain. PTx further induced expression of tissue inhibitor...... of metalloproteinase-1, metalloproteinase disintegrins-12, MMP-8, and -10 in brains of Tg mice. Levels of the microglial-associated MP MMP-15 were not affected in control or PTx-treated Tg mice. PTx also up-regulated expression of proinflammatory cytokines IL-1beta and TNF-alpha mRNA in Tg CNS. Weight loss...

  8. Delayed-type hypersensitivity lesions in the central nervous system are prevented by inhibitors of matrix metalloproteinases.

    Science.gov (United States)

    Matyszak, M K; Perry, V H

    1996-09-01

    We have studied the effect of an inhibitor of matrix metalloproleinases, BB-1101, on a delayed-type hypersensitivity (DTH) response in the CNS. We used a recently described model in which heat-killed bacillus Calmette-Guérin (BCG) sequestered behind the blood-brain barrier (BBB) is targeted by a T-cell mediated response after subcutaneous injection of BCG (Matyszak and Perry, 1995). The DTH lesions are characterised by breakdown of the BBB, macrophage and lymphocyte infiltration and tissue damage including myelin loss. Treatment with BB-1101, which is not only a potent inhibitor of matrix metalloproteinases but also strongly inhibits TNF-alpha release, dramatically attenuated the CNS lesions. Breakdown of the BBB and the recruitment of T-cells into the site of the lesion were significantly reduced. There were many fewer inflammatory macrophages in DTH lesions than in comparable lesions from untreated animals. There was also significantly less myelin damage (assessed by staining with anti-MBP antibody). The DTH response in animals treated with dexamethasone was also reduced, but to a lesser degree. No significant effect was seen after administration of pentoxifylline, a phosphodiesterase inhibitor with effects including the inhibition of TNF-alpha production. Our results suggest that inhibitors of matrix metalloproteinases may be of considerable therapeutic benefit in neuroinflammatory diseases.

  9. Matrix Metalloproteinase-20 Over-Expression Is Detrimental to Enamel Development: A Mus musculus Model

    Science.gov (United States)

    Shin, Masashi; Hu, Yuanyuan; Tye, Coralee E.; Guan, Xiaomu; Deagle, Craig C.; Antone, Jerry V.; Smith, Charles E.; Simmer, James P.; Bartlett, John D.

    2014-01-01

    Background Matrix metalloproteinase-20 (Mmp20) ablated mice have enamel that is thin and soft with an abnormal rod pattern that abrades from the underlying dentin. We asked if introduction of transgenes expressing Mmp20 would revert this Mmp20 null phenotype back to normal. Unexpectedly, for transgenes expressing medium or high levels of Mmp20, we found opposite enamel phenotypes depending on the genetic background (Mmp20−/− or Mmp20+/+) in which the transgenes were expressed. Methodology/Principal Findings Amelx-promoter-Mmp20 transgenic founder mouse lines were assessed for transgene expression and those expressing low, medium or high levels of Mmp20 were selected for breeding into the Mmp20 null background. Regardless of expression level, each transgene brought the null enamel back to full thickness. However, the high and medium expressing Mmp20 transgenes in the Mmp20 null background had significantly harder more mineralized enamel than did the low transgene expresser. Strikingly, when the high and medium expressing Mmp20 transgenes were present in the wild-type background, the enamel was significantly less well mineralized than normal. Protein gel analysis of enamel matrix proteins from the high and medium expressing transgenes present in the wild-type background demonstrated that greater than normal amounts of cleavage products and smaller quantities of higher molecular weight proteins were present within their enamel matrices. Conclusions/Significance Mmp20 expression levels must be within a specific range for normal enamel development to occur. Creation of a normally thick enamel layer may occur over a wider range of Mmp20 expression levels, but acquisition of normal enamel hardness has a narrower range. Since over-expression of Mmp20 results in decreased enamel hardness, this suggests that a balance exists between cleaved and full-length enamel matrix proteins that are essential for formation of a properly hardened enamel layer. It also suggests that

  10. DIDS prevents ischemic membrane degradation in cultured hippocampal neurons by inhibiting matrix metalloproteinase release.

    Science.gov (United States)

    Pamenter, Matthew E; Ryu, Julie; Hua, Serena T; Perkins, Guy A; Mendiola, Vincent L; Gu, Xiang Q; Ellisman, Mark H; Haddad, Gabriel G

    2012-01-01

    During stroke, cells in the infarct core exhibit rapid failure of their permeability barriers, which releases ions and inflammatory molecules that are deleterious to nearby tissue (the penumbra). Plasma membrane degradation is key to penumbral spread and is mediated by matrix metalloproteinases (MMPs), which are released via vesicular exocytosis into the extracellular fluid in response to stress. DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) preserves membrane integrity in neurons challenged with an in vitro ischemic penumbral mimic (ischemic solution: IS) and we asked whether this action was mediated via inhibition of MMP activity. In cultured murine hippocampal neurons challenged with IS, intracellular proMMP-2 and -9 expression increased 4-10 fold and extracellular latent and active MMP isoform expression increased 2-22 fold. MMP-mediated extracellular gelatinolytic activity increased ∼20-50 fold, causing detachment of 32.1±4.5% of cells from the matrix and extensive plasma membrane degradation (>60% of cells took up vital dyes and >60% of plasma membranes were fragmented or blebbed). DIDS abolished cellular detachment and membrane degradation in neurons and the pathology-induced extracellular expression of latent and active MMPs. DIDS similarly inhibited extracellular MMP expression and cellular detachment induced by the pro-apoptotic agent staurosporine or the general proteinase agonist 4-aminophenylmercuric acetate (APMA). Conversely, DIDS-treatment did not impair stress-induced intracellular proMMP production, nor the intracellular cleavage of proMMP-2 to the active form, suggesting DIDS interferes with the vesicular extrusion of MMPs rather than directly inhibiting proteinase expression or activation. In support of this hypothesis, an antagonist of the V-type vesicular ATPase also inhibited extracellular MMP expression to a similar degree as DIDS. In addition, in a proteinase-independent model of vesicular exocytosis, DIDS prevented stimulus

  11. DIDS prevents ischemic membrane degradation in cultured hippocampal neurons by inhibiting matrix metalloproteinase release.

    Directory of Open Access Journals (Sweden)

    Matthew E Pamenter

    Full Text Available During stroke, cells in the infarct core exhibit rapid failure of their permeability barriers, which releases ions and inflammatory molecules that are deleterious to nearby tissue (the penumbra. Plasma membrane degradation is key to penumbral spread and is mediated by matrix metalloproteinases (MMPs, which are released via vesicular exocytosis into the extracellular fluid in response to stress. DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid preserves membrane integrity in neurons challenged with an in vitro ischemic penumbral mimic (ischemic solution: IS and we asked whether this action was mediated via inhibition of MMP activity. In cultured murine hippocampal neurons challenged with IS, intracellular proMMP-2 and -9 expression increased 4-10 fold and extracellular latent and active MMP isoform expression increased 2-22 fold. MMP-mediated extracellular gelatinolytic activity increased ∼20-50 fold, causing detachment of 32.1±4.5% of cells from the matrix and extensive plasma membrane degradation (>60% of cells took up vital dyes and >60% of plasma membranes were fragmented or blebbed. DIDS abolished cellular detachment and membrane degradation in neurons and the pathology-induced extracellular expression of latent and active MMPs. DIDS similarly inhibited extracellular MMP expression and cellular detachment induced by the pro-apoptotic agent staurosporine or the general proteinase agonist 4-aminophenylmercuric acetate (APMA. Conversely, DIDS-treatment did not impair stress-induced intracellular proMMP production, nor the intracellular cleavage of proMMP-2 to the active form, suggesting DIDS interferes with the vesicular extrusion of MMPs rather than directly inhibiting proteinase expression or activation. In support of this hypothesis, an antagonist of the V-type vesicular ATPase also inhibited extracellular MMP expression to a similar degree as DIDS. In addition, in a proteinase-independent model of vesicular exocytosis, DIDS

  12. Analysis of X-ray Structures of Matrix Metalloproteinases via Chaotic Map Clustering

    Directory of Open Access Journals (Sweden)

    Gargano Gianfranco

    2010-10-01

    Full Text Available Abstract Background Matrix metalloproteinases (MMPs are well-known biological targets implicated in tumour progression, homeostatic regulation, innate immunity, impaired delivery of pro-apoptotic ligands, and the release and cleavage of cell-surface receptors. With this in mind, the perception of the intimate relationships among diverse MMPs could be a solid basis for accelerated learning in designing new selective MMP inhibitors. In this regard, decrypting the latent molecular reasons in order to elucidate similarity among MMPs is a key challenge. Results We describe a pairwise variant of the non-parametric chaotic map clustering (CMC algorithm and its application to 104 X-ray MMP structures. In this analysis electrostatic potentials are computed and used as input for the CMC algorithm. It was shown that differences between proteins reflect genuine variation of their electrostatic potentials. In addition, the analysis has been also extended to analyze the protein primary structures and the molecular shapes of the MMP co-crystallised ligands. Conclusions The CMC algorithm was shown to be a valuable tool in knowledge acquisition and transfer from MMP structures. Based on the variation of electrostatic potentials, CMC was successful in analysing the MMP target family landscape and different subsites. The first investigation resulted in rational figure interpretation of both domain organization as well as of substrate specificity classifications. The second made it possible to distinguish the MMP classes, demonstrating the high specificity of the S1' pocket, to detect both the occurrence of punctual mutations of ionisable residues and different side-chain conformations that likely account for induced-fit phenomena. In addition, CMC demonstrated a potential comparable to the most popular UPGMA (Unweighted Pair Group Method with Arithmetic mean method that, at present, represents a standard clustering bioinformatics approach. Interestingly, CMC and

  13. Amplified voltammetric characterization of cleavage of the biotinylated peptide by BACE1 and screening of BACE1 inhibitors.

    Science.gov (United States)

    Yi, Xinyao; Han, Hongxing; Zhang, Yu; Wang, Jianxiu; Zhang, Yi; Zhou, Feimeng

    2013-12-15

    Cleavage of amyloid precursor protein (APP) by the β-site APP cleaving enzyme 1 (BACE1) is a key step in the formation of amyloid beta (Aβ) peptide, the main component of amyloid plaques in Alzheimer's disease (AD). Suppression of BACE1 activity has thus become an efficient way for the treatment of AD. In this study, BACE1 in the absence or presence of BACE1 inhibitors was exposed to the biotinylated peptide substrate-modified electrode. This step was followed by the attachment of ferrocene (Fc)-capped gold nanoparticle/streptavidin conjugates. Due to the blockage of the BACE1 activity by select inhibitors, well-defined voltammetric peaks of high signal intensity were obtained. However, featureless voltammogram was obtained upon initiating the cleavage reaction. The proposed method is simple, sensitive, and suitable for monitoring of BACE1 activity and screening of BACE1 inhibitors.

  14. Post-translational processing of progastrin: inhibition of cleavage, phosphorylation and sulphation by brefeldin A.

    Science.gov (United States)

    Varro, A; Dockray, G J

    1993-11-01

    The precursor for the acid-stimulating hormone gastrin provides a useful model for studies of post-translational processing because defined sites of cleavage, amidation, sulphation and phosphorylation occur within a dodecapeptide sequence. The factors determining these post-translational processing events are still poorly understood. We have used brefeldin A, which disrupts transport from rough endoplasmic reticulum to the Golgi complex, to examine the mechanisms of cleavage, phosphorylation and sulphation of rat progastrin-derived peptides. Biosynthetic products were detected after immunoprecipitation using antibodies specific for the extreme C-terminus of progastrin, followed by reversed-phase and ion-exchange h.p.l.c. Gastrin cells incorporated [3H]tyrosine, [32P]phosphate and [35S]sulphate into both progastrin and its extreme C-terminal tryptic (nona-) peptide. Ion-exchange chromatography resolved four forms of the C-terminal tryptic fragment of progastrin which differed in whether they were phosphorylated at Ser96, sulphated at Tyr103, both or neither. The specific activity of [3H]tyrosine in the peak that was both phosphorylated and sulphated was higher than in the others. Brefeldin A inhibited the appearance of [3H]tyrosine-labelled C-terminal tryptic fragment but there was an accumulation of labelled progastrin and a peptide corresponding to the C-terminal 46 residues of progastrin. Brefeldin A also inhibited incorporation of 32P and 35S into both progastrin and its C-terminal fragment. Thus phosphorylation of Ser96, sulphation of Tyr103 and cleavage at Arg94-Arg95 depend on passage of newly synthesized progastrin along the secretory pathway; as brefeldin A is thought to act proximal to the trans-Golgi, these processing steps would appear to occur distal to this point. The data also indicate that the stores of unphosphorylated C-terminal tryptic fragment are not available for phosphorylation, implying that this modification occurs proximal to the secretory

  15. Structural Characterization of the Ectodomain of a Disintegrin and Metalloproteinase-22 (ADAM22), a Neural Adhesion Receptor Instead of Metalloproteinase INSIGHTS ON ADAM FUNCTION

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Heli; Shim, Ann H.R.; He, Xiaolin; (NWU)

    2009-12-01

    ADAMs (adisintegrin and metalloproteinases) are a family of multidomain transmembrane glycoproteins with diverse roles in physiology and diseases, with several members being drug targets for cancer and inflammation therapies. The spatial organization of the ADAM extracellular segment and its influence on the function of ADAMs have been unclear. Although most members of the ADAM family are active zinc metalloproteinases, 8 of 21 ADAMs lack functional metalloproteinase domains and are implicated in protein-protein interactions instead of membrane protein ectodomain shedding. One of such non-proteinase ADAMs, ADAM22, acts as a receptor on the surface of the postsynaptic neuron to regulate synaptic signal transmission. The crystal structure of the full ectodomain of mature human ADAM22 shows that it is a compact four-leaf clover with the metalloproteinase-like domain held in the concave face of a rigid module formed by the disintegrin, cysteine-rich, and epidermal growth factor-like domains. The loss of metalloproteinase activity is ensured by the absence of critical catalytic residues, the filling of the substrate groove, and the steric hindrance by the cysteine-rich domain. The structure, combined with calorimetric experiments, suggests distinct roles of three putative calcium ions bound to ADAM22, with one in the metalloproteinase-like domain being regulatory and two in the disintegrin domain being structural. The metalloproteinase-like domain contacts the rest of ADAM22 with discontinuous, hydrophilic, and poorly complemented interactions, suggesting the possibility of modular movement of ADAM22 and other ADAMs. The ADAM22 structure provides a framework for understanding how different ADAMs exert their adhesive function and shedding activities.

  16. Meganuclease-mediated virus self-cleavage facilitates tumor-specific virus replication.

    Science.gov (United States)

    Gürlevik, Engin; Schache, Peter; Goez, Anneliese; Kloos, Arnold; Woller, Norman; Armbrecht, Nina; Manns, Michael P; Kubicka, Stefan; Kühnel, Florian

    2013-09-01

    Meganucleases can specifically cleave long DNA sequence motifs, a feature that makes them an ideal tool for gene engineering in living cells. In a proof-of-concept study, we investigated the use of the meganuclease I-Sce I for targeted virus self-disruption to generate high-specific oncolytic viruses. For this purpose, we provided oncolytic adenoviruses with a molecular circuit that selectively responds to p53 activation by expression of I-Sce I subsequently leading to self-disruption of the viral DNA via heterologous I-Sce I recognition sites within the virus genome. We observed that virus replication and cell lysis was effectively impaired in p53-normal cells, but not in p53-dysfunctional tumor cells. I-Sce I activity led to effective intracellular processing of viral DNA as confirmed by detection of specific cleavage products. Virus disruption did not interfere with E1A levels indicating that reduction of functional virus genomes was the predominant cause for conditional replication. Consequently, tumor-specific replication was further enhanced when E1A expression was additionally inhibited by targeted tr