WorldWideScience

Sample records for proteolytic cleavage products

  1. Regulation of proteolytic cleavage of brain-derived neurotrophic factor precursor by antidepressants in human neuroblastoma cells

    Directory of Open Access Journals (Sweden)

    Lin PY

    2015-10-01

    Full Text Available Pao-Yen Lin1,2 1Department of Psychiatry, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, 2Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan Abstract: Evidence has supported the role of brain-derived neurotrophic factor (BDNF in antidepressant effect. The precursor of BDNF (proBDNF often exerts opposing biological effects on mature BDNF (mBDNF. Hence, the balance between proBDNF and mBDNF might be critical in total neurotrophic effects, leading to susceptibility to or recovery from depression. In the current study, we measured the protein expression levels of proBDNF, and its proteolytic products, truncated BDNF, and mBDNF, in human SH-SY5Y cells treated with different antidepressants. We found that the treatment significantly increased the production of mBDNF, but decreased the production of truncated BDNF and proBDNF. These results support that antidepressants can promote proBDNF cleavage. Further studies are needed to clarify whether proBDNF cleavage plays a role in antidepressant mechanisms. Keywords: antidepressant, mature BDNF, neurotrophic effect, proBDNF cleavage 

  2. In-capillary self-assembly and proteolytic cleavage of polyhistidine peptide capped quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jianhao; Li, Jingyan; Li, Jinchen; Liu, Feifei [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); Zhou, Xiang; Yao, Yi [Changzhou Qianhong Bio-pharma Co. Ltd, Changzhou 213164, Jiangsu (China); Wang, Cheli [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); Qiu, Lin, E-mail: linqiupjj@gmail.com [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); Jiang, Pengju, E-mail: pengju.jiang@gmail.com [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); State Key Laboratory of Pharmaceutical Biotechnology, Nanjing, Jiangsu (China)

    2015-10-01

    A new method using fluorescence coupled capillary electrophoresis (CE-FL) for monitoring self-assembly and proteolytic cleavage of hexahistidine peptide capped quantum dots (QDs) inside a capillary has been developed in this report. QDs and the ATTO 590-labeled hexahistidine peptide (H6-ATTO) were injected into a capillary, sequentially. Their self-assembly inside the capillary was driven by a metal-affinity force which yielded a new fluorescence signal due to Förster resonance energy transfer (FRET). The highly efficient separation of fluorescent complexes and the FRET process were analyzed using CE-FL. The self-assembly of QDs and biomolecules was found to effectively take place inside the capillary. The kinetics of the assembly was monitored by CE-FL, and the approach was extended to the study of proteolytic cleavage of surface conjugated peptides. Being the first in-depth analysis of in-capillary nanoparticle–biomolecule assembly, the novel approach reported here provides inspiration to the development of QD-based FRET probes for biomedical applications. - Highlights: • We examined the self-assembly QDs with H6-ATTO inside a capillary. • We prove CE-FL to be a powerful method to resolve QDs-H6-ATTO complex. • We achieve chromatographic separation of QDs-H6-ATTO complex. • We discovered a novel strategy for the online detection of thrombin. • This technique integrated “injection, mixing, reaction, separation and detection”.

  3. Unconjugated Bilirubin Inhibits Proteolytic Cleavage of von Willebrand Factor by ADAMTS13 Protease

    Science.gov (United States)

    Lu, Rui-Nan; Yang, Shangbin; Wu, Haifeng M.; Zheng, X. Long

    2015-01-01

    Summary Background Bilirubin is a yellow breakdown product of heme catabolism. Increased serum levels of unconjugated bilirubin are conditions commonly seen in premature neonates and adults with acute hemolysis including thrombotic microangiopathy. Previous studies have shown that unconjugated bilirubin lowers plasma ADAMTS13 activity, but the mechanism is not fully understood. Objectives The study is to determine whether unconjugated bilirubin directly inhibits the cleavage of von Willebrand factor (VWF) and its analogs by ADAMTS13. Methods Fluorogenic, SELDI-TOF mass spectrometric assay, and Western blotting analyses were employed to address this question. Results Unconjugated bilirubin inhibits the cleavage of F485-rVWF73-H, D633-rVWF73-H, and GST-rVWF71-11K by ADAMTS13 in a concentration-dependent manner with a half-maximal inhibitory concentration (IC50) of ~13 μM, ~70 μM, and ~17 μM, respectively. Unconjugated bilirubin also dose-dependently inhibits the cleavage of multimeric VWF by ADAMTS13 under denaturing conditions. The inhibitory activity of bilirubin on the cleavage of D633-rVWF73-H and multimeric VWF, but not F485-rVWF73-H, was eliminated after incubation with bilirubin oxidase that converts bilirubin to biliverdin. Furthermore, plasma ADAMTS13 activity in patients with hyperbilirubinemia is lower prior to than after treatment with bilirubin oxidase. Conclusions unconjugated bilirubin directly inhibits ADAMTS13’s ability to cleave both peptidyl and native VWF substrates in addition to its interference with certain fluorogenic assays. Our findings may help proper interpretation of ADAMTS13 results under pathological conditions. Whether elevated serum unconjugated bilirubin has an adverse effect in vivo remains to be determined in our future study. PMID:25782102

  4. Proteolytic receptor cleavage in the pathogenesis of blood rheology and co-morbidities in metabolic syndrome. Early forms of autodigestion.

    Science.gov (United States)

    Mazor, Rafi; Schmid-Schönbein, Geert W

    2015-01-01

    Abnormal blood rheological properties seldom occur in isolation and instead are accompanied by other complications, often designated as co-morbidities. In the metabolic syndrome with complications like hypertension, diabetes and lack of normal microvascular blood flow, the underlying molecular mechanisms that simultaneously lead to elevated blood pressure and diabetes as well as abnormal microvascular rheology and other cell dysfunctions have remained largely unknown. In this review, we propose a new hypothesis for the origin of abnormal cell functions as well as multiple co-morbidities. Utilizing experimental models for the metabolic disease with diverse co-morbidities we summarize evidence for the presence of an uncontrolled extracellular proteolytic activity that causes ectodomain receptor cleavage and loss of their associated cell function. We summarize evidence for unchecked degrading proteinase activity, e.g. due to matrix metalloproteases, in patients with hypertension, Type II diabetes and obesity, in addition to evidence for receptor cleavage in the form of receptor fragments and decreased extracellular membrane expression levels. The evidence suggest that a shift in blood rheological properties and other co-morbidities may in fact be derived from a common mechanism that is due to uncontrolled proteolytic activity, i.e. an early form of autodigestion. Identification of the particular proteases involved and the mechanisms of their activation may open the door to treatment that simultaneously targets multiple co-morbidities in the metabolic syndrome.

  5. Sequence motif upstream of the Hendra virus fusion protein cleavage site is not sufficient to promote efficient proteolytic processing

    International Nuclear Information System (INIS)

    Craft, Willie Warren; Dutch, Rebecca Ellis

    2005-01-01

    The Hendra virus fusion (HeV F) protein is synthesized as a precursor, F 0 , and proteolytically cleaved into the mature F 1 and F 2 heterodimer, following an HDLVDGVK 109 motif. This cleavage event is required for fusogenic activity. To determine the amino acid requirements for processing of the HeV F protein, we constructed multiple mutants. Individual and simultaneous alanine substitutions of the eight residues immediately upstream of the cleavage site did not eliminate processing. A chimeric SV5 F protein in which the furin site was substituted for the VDGVK 109 motif of the HeV F protein was not processed but was expressed on the cell surface. Another chimeric SV5 F protein containing the HDLVDGVK 109 motif of the HeV F protein underwent partial cleavage. These data indicate that the upstream region can play a role in protease recognition, but is neither absolutely required nor sufficient for efficient processing of the HeV F protein

  6. Deletion of the thrombin cleavage domain of osteopontin mediates breast cancer cell adhesion, proteolytic activity, tumorgenicity, and metastasis

    International Nuclear Information System (INIS)

    Beausoleil, Michel S; Schulze, Erika B; Goodale, David; Postenka, Carl O; Allan, Alison L

    2011-01-01

    Osteopontin (OPN) is a secreted phosphoprotein often overexpressed at high levels in the blood and primary tumors of breast cancer patients. OPN contains two integrin-binding sites and a thrombin cleavage domain located in close proximity to each other. To study the role of the thrombin cleavage site of OPN, MDA-MB-468 human breast cancer cells were stably transfected with either wildtype OPN (468-OPN), mutant OPN lacking the thrombin cleavage domain (468-ΔTC) or an empty vector (468-CON) and assessed for in vitro and in vivo functional differences in malignant/metastatic behavior. All three cell lines were found to equivalently express thrombin, tissue factor, CD44, αvβ5 integrin and β1 integrin. Relative to 468-OPN and 468-CON cells, 468-ΔTC cells expressing OPN with a deleted thrombin cleavage domain demonstrated decreased cell adhesion (p < 0.001), decreased mRNA expression of MCAM, maspin and TRAIL (p < 0.01), and increased uPA expression and activity (p < 0.01) in vitro. Furthermore, injection of 468-ΔTC cells into the mammary fat pad of nude mice resulted in decreased primary tumor latency time (p < 0.01) and increased primary tumor growth and lymph node metastatic burden (p < 0.001) compared to 468-OPN and 468-CON cells. The results presented here suggest that expression of thrombin-uncleavable OPN imparts an early tumor formation advantage as well as a metastatic advantage for breast cancer cells, possibly due to increased proteolytic activity and decreased adhesion and apoptosis. Clarification of the mechanisms responsible for these observations and the translation of this knowledge into the clinic could ultimately provide new therapeutic opportunities for combating breast cancer

  7. Transcript levels, alternative splicing and proteolytic cleavage of TFIIIA control 5S rRNA accumulation during Arabidopsis thaliana development.

    Science.gov (United States)

    Layat, Elodie; Cotterell, Sylviane; Vaillant, Isabelle; Yukawa, Yasushi; Tutois, Sylvie; Tourmente, Sylvette

    2012-07-01

    Ribosome biogenesis is critical for eukaryotic cells and requires coordinated synthesis of the protein and rRNA moieties of the ribosome, which are therefore highly regulated. 5S ribosomal RNA, an essential component of the large ribosomal subunit, is transcribed by RNA polymerase III and specifically requires transcription factor IIIA (TFIIIA). To obtain insight into the regulation of 5S rRNA transcription, we have investigated the expression of 5S rRNA and the exon-skipped (ES) and exon-including (EI) TFIIIA transcripts, two transcript isoforms that result from alternative splicing of the TFIIIA gene, and TFIIIA protein amounts with respect to requirements for 5S rRNA during development. We show that 5S rRNA quantities are regulated through distinct but complementary mechanisms operating through transcriptional and post-transcriptional control of TFIIIA transcripts as well as at the post-translational level through proteolytic cleavage of the TFIIIA protein. During the reproductive phase, high expression of the TFIIIA gene together with low proteolytic cleavage contributes to accumulation of functional, full-length TFIIIA protein, and results in 5S rRNA accumulation in the seed. In contrast, just after germination, the levels of TFIIIA-encoding transcripts are low and stable. Full-length TFIIIA protein is undetectable, and the level of 5S rRNA stored in the embryo progressively decreases. After day 4, in correlation with the reorganization of 5S rDNA chromatin to a mature state, full-length TFIIIA protein with transcriptional activity accumulates and permits de novo transcription of 5S rRNA. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

  8. Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis.

    Science.gov (United States)

    Senger, Moritz; Stripp, Sven T; Soboh, Basem

    2017-07-14

    Metalloenzymes catalyze complex and essential processes, such as photosynthesis, respiration, and nitrogen fixation. For example, bacteria and archaea use [NiFe]-hydrogenases to catalyze the uptake and release of molecular hydrogen (H 2 ). [NiFe]-hydrogenases are redox enzymes composed of a large subunit that harbors a NiFe(CN) 2 CO metallo-center and a small subunit with three iron-sulfur clusters. The large subunit is synthesized with a C-terminal extension, cleaved off by a specific endopeptidase during maturation. The exact role of the C-terminal extension has remained elusive; however, cleavage takes place exclusively after assembly of the [NiFe]-cofactor and before large and small subunits form the catalytically active heterodimer. To unravel the functional role of the C-terminal extension, we used an enzymatic in vitro maturation assay that allows synthesizing functional [NiFe]-hydrogenase-2 of Escherichia coli from purified components. The maturation process included formation and insertion of the NiFe(CN) 2 CO cofactor into the large subunit, endoproteolytic cleavage of the C-terminal extension, and dimerization with the small subunit. Biochemical and spectroscopic analysis indicated that the C-terminal extension of the large subunit is essential for recognition by the maturation machinery. Only upon completion of cofactor insertion was removal of the C-terminal extension observed. Our results indicate that endoproteolytic cleavage is a central checkpoint in the maturation process. Here, cleavage temporally orchestrates cofactor insertion and protein assembly and ensures that only cofactor-containing protein can continue along the assembly line toward functional [NiFe]-hydrogenase. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Analysis of the in vitro cleavage products of the tomato black ring virus RNA-1-encoded 250K polyprotein.

    OpenAIRE

    Demangeat, Gerard; Greif, Charles; Hemmer, O; Fritsch, C

    1990-01-01

    Tomato black ring virus RNA-1 was translated in a rabbit reticulocyte lysate. The primary translation product of Mr 250K, which corresponds to its whole coding capacity, was synthesized within 45 min and, during further incubation in the translation medium, was proteolytically processed. Essentially, four cleavage products (P190, P120, P60 and P50) were detected and located within P250 by pulse-chase and immunoprecipitation experiments. P190 is an intermediate cleavage product which is furthe...

  10. 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.

  11. Proteolytic Cleavage of ProBDNF into Mature BDNF in the Basolateral Amygdala Is Necessary for Defeat-Induced Social Avoidance

    Science.gov (United States)

    Dulka, Brooke N.; Ford, Ellen C.; Lee, Melissa A.; Donnell, Nathaniel J.; Goode, Travis D.; Prosser, Rebecca; Cooper, Matthew A.

    2016-01-01

    Brain-derived neurotrophic factor (BDNF) is essential for memory processes. The present study tested whether proteolytic cleavage of proBDNF into mature BDNF (mBDNF) within the basolateral amygdala (BLA) regulates the consolidation of defeat-related memories. We found that acute social defeat increases the expression of mBDNF, but not proBDNF, in…

  12. Direct proteolytic cleavage of NLRP1B is necessary and sufficient for inflammasome activation by anthrax lethal factor.

    Directory of Open Access Journals (Sweden)

    Joseph Chavarría-Smith

    Full Text Available Inflammasomes are multimeric protein complexes that respond to infection by recruitment and activation of the Caspase-1 (CASP1 protease. Activated CASP1 initiates immune defense by processing inflammatory cytokines and by causing a rapid and lytic cell death called pyroptosis. Inflammasome formation is orchestrated by members of the nucleotide-binding domain and leucine-rich repeat (NLR or AIM2-like receptor (ALR protein families. Certain NLRs and ALRs have been shown to function as direct receptors for specific microbial ligands, such as flagellin or DNA, but the molecular mechanism responsible for activation of most NLRs is still poorly understood. Here we determine the mechanism of activation of the NLRP1B inflammasome in mice. NLRP1B, and its ortholog in rats, is activated by the lethal factor (LF protease that is a key virulence factor secreted by Bacillus anthracis, the causative agent of anthrax. LF was recently shown to cleave mouse and rat NLRP1 directly. However, it is unclear if cleavage is sufficient for NLRP1 activation. Indeed, other LF-induced cellular events have been suggested to play a role in NLRP1B activation. Surprisingly, we show that direct cleavage of NLRP1B is sufficient to induce inflammasome activation in the absence of LF. Our results therefore rule out the need for other LF-dependent cellular effects in activation of NLRP1B. We therefore propose that NLRP1 functions primarily as a sensor of protease activity and thus could conceivably detect a broader spectrum of pathogens than just B. anthracis. By adding proteolytic cleavage to the previously established ligand-receptor mechanism of NLR activation, our results illustrate the remarkable flexibility with which the NLR architecture can be deployed for the purpose of pathogen-detection and host defense.

  13. Angiogenic activity of bFGF and VEGF suppressed by proteolytic cleavage by neutrophil elastase

    International Nuclear Information System (INIS)

    Ai, Shingo; Cheng Xianwu; Inoue, Aiko; Nakamura, Kae; Okumura, Kenji; Iguchi, Akihisa; Murohara, Toyoaki; Kuzuya, Masafumi

    2007-01-01

    Neutrophil elastase (NE), a serine protease released from the azurophil granules of activated neutrophil, proteolytically cleaves multiple cytokines, and cell surface proteins. In the present study, we examined whether NE affects the biological abilities of angiogenic growth factors such as basic-fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). NE degraded bFGF and VEGF in a time- and concentration-dependent manner, and these degradations were suppressed by sivelestat, a synthetic inhibitor of NE. The bFGF- or VEGF-mediated proliferative activity of human umbilical vein endothelial cells was inhibited by NE, and the activity was recovered by sivelestat. Furthermore, NE reduced the bFGF- or VEGF-induced tubulogenic response of the mice aortas, ex vivo angiogenesis assay, and these effects were also recovered by sivelestat. Neutrophil-derived NE degraded potent angiogenic factors, resulting in loss of their angiogenic activity. These findings provide additional insight into the role played by neutrophils in the angiogenesis process at sites of inflammation

  14. Production of extracellular proteolytic enzymes by Beauveria bassiana

    Directory of Open Access Journals (Sweden)

    Józefa Chrzanowska

    2014-08-01

    Full Text Available The production of proteolytic enzymes by two strains of Beauveria bassiana 278, B. bassiana 446 and one strain of Ascosphera apis 496 was analysed. It was demonstrated that the strain of B. bassiana 278 proved to be the best producer of basic and acid proteases. The influence of different environmental factors such as nitrogen and carbon sources on the production of extracellular hydrolytic enzymes was assessed. In addition the acid protease from B. bassiana was partially characterized.

  15. A novel immune evasion strategy of candida albicans: proteolytic cleavage of a salivary antimicrobial peptide.

    Directory of Open Access Journals (Sweden)

    Timothy F Meiller

    Full Text Available Oropharyngeal candidiasis is an opportunistic infection considered to be a harbinger of AIDS. The etiologic agent Candida albicans is a fungal species commonly colonizing human mucosal surfaces. However, under conditions of immune dysfunction, colonizing C. albicans can become an opportunistic pathogen causing superficial or even life-threatening infections. The reasons behind this transition, however, are not clear. In the oral cavity, salivary antimicrobial peptides are considered to be an important part of the host innate defense system in the prevention of microbial colonization. Histatin-5 specifically has exhibited potent activity against C. albicans. Our previous studies have shown histatin-5 levels to be significantly reduced in the saliva of HIV+ individuals, indicating an important role for histatin-5 in keeping C. albicans in its commensal stage. The versatility in the pathogenic potential of C. albicans is the result of its ability to adapt through the regulation of virulence determinants, most notably of which are proteolytic enzymes (Saps, involved in tissue degradation. In this study, we show that C. albicans cells efficiently and rapidly degrade histatin-5, resulting in loss of its anti-candidal potency. In addition, we demonstrate that this cellular activity is due to proteolysis by a member of the secreted aspartic proteases (Sap family involved in C. albicans pathogenesis. Specifically, the proteolysis was attributed to Sap9, in turn identifying histatin-5 as the first host-specific substrate for that isoenzyme. These findings demonstrate for the first time the ability of a specific C. albicans enzyme to degrade and deactivate a host antimicrobial peptide involved in the protection of the oral mucosa against C. albicans, thereby providing new insights into the factors directing the transition of C. albicans from commensal to pathogen, with important clinical implications for alternative therapy. This report characterizes the

  16. Combined Quantification of the Global Proteome, Phosphoproteome, and Proteolytic Cleavage to Characterize Altered Platelet Functions in the Human Scott Syndrome.

    Science.gov (United States)

    Solari, Fiorella A; Mattheij, Nadine J A; Burkhart, Julia M; Swieringa, Frauke; Collins, Peter W; Cosemans, Judith M E M; Sickmann, Albert; Heemskerk, Johan W M; Zahedi, René P

    2016-10-01

    The Scott syndrome is a very rare and likely underdiagnosed bleeding disorder associated with mutations in the gene encoding anoctamin-6. Platelets from Scott patients are impaired in various Ca 2+ -dependent responses, including phosphatidylserine exposure, integrin closure, intracellular protein cleavage, and cytoskeleton-dependent morphological changes. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared with control platelets. Therefore, we applied an iTRAQ-based multi-pronged strategy to quantify changes in (1) the global proteome, (2) the phosphoproteome, and (3) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we confirmed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1 by parallel reaction monitoring. The quantification of 1566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. In Scott platelets, phosphorylation levels of proteins regulating cytoskeletal or signaling events were increased. Finally, we quantified 1596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N termini was caspase-regulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was downregulated, in accordance with an increased phosphorylation state. Thus, multipronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca 2+ -dependent changes that are normally associated with phosphatidylserine exposure. © 2016 by The American Society for Biochemistry and Molecular

  17. Drosophila acetylcholinesterase: demonstration of a glycoinositol phospholipid anchor and an endogenous proteolytic cleavage

    International Nuclear Information System (INIS)

    Haas, R.; Marshall, T.L.; Rosenberry, T.L.

    1988-01-01

    The presence of a glycoinositol phospholipid anchor Drosophila acetylcholinesterase (AChE) was shown by several criteria. Chemical analysis of highly purified Drosophila AChE demonstrated approximately one residue of inositol per enzyme subunit. Selective cleavage by Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) was tested with Drosophila AChE radiolabeled by the photoactivatable affinity probe 3-(trifluoromethyl)-3-(m-[ 125 I]iodophenyl)diazirine ([ 125 I]TID), a reagent that specifically labels the lipid moiety of glycoinositol phospholipid-anchored proteins. Digestion with PI-PLC released 75% of this radiolabel from the protein. Gel electrophoresis of Drosophila AChE in sodium dodecyl sulfate indicated prominent 55- and 16-kDa bands and a faint 70-kDa band. The [ 125 ]I]TID label was localized on the 55-kDa fragment, suggesting that this fragment is the C-terminal portion of the protein. In support of this conclusion, a sensitive microsequencing procedure that involved manual Edman degradation combined with radiomethylation was used to determine residues 2-5 of the 16-kDa fragment. Comparison with the Drosophila AChE cDNA sequence confirmed that the 16-kDa fragment includes the N-terminus of AChE. Furthermore, the position of the N-terminal amino acid of the mature Drosophila AChE is closely homologous to that of Torpedo AChE. The presence of radiomethylatable ethanolamine in both 16- and 55-kDa fragments was also confirmed. Thus, Drosophila AChE may include a second posttranslational modification involving ethanolamine

  18. Patterns of proteolytic cleavage and carbodiimide derivatization in sarcoplasmic reticulum adenosinetriphosphatase

    International Nuclear Information System (INIS)

    de Ancos, J.G.; Inesi, G.

    1988-01-01

    Two series of experiments were carried out to characterize (a) peptide fragments of sarcoplasmic reticulum (SR) ATPase, based on proteolysis with different enzymes and distribution of known labels, and (b) specific labeling and functional inactivation patterns, following ATPase derivatization with dicyclohexylcarbodiimide (DCCD) under various conditions. Digestion with trypsin or chymotrypsin results in the initial cleavage of the SR ATPase in two fragments of similar size and then into smaller fragments, while subtilisin and thermolysin immediately yield smaller fragments. Peptide fragments were assigned to segments of the protein primary structure and to functionally relevant domains, such as those containing the 32 P at the active site and the fluorescein isothiocyanate at the nucleotide site. ATPase derivatization with [ 14 C]DCCD under mild conditions produced selective inhibition of ATPase hydrolytic catalysis without significant incorporation of the 14 C radioactive label. This effect is attributed to blockage of catalytically active residues by reaction of the initial DCCD adduct with endogenous or exogenous nucleophiles. ATPase derivatization with [ 14 C]DCCD under more drastic conditions produced inhibition of calcium binding, 14 C radioactive labeling of tryptic fragments A 1 and A 2 (but not of B), and extensive cross-linking. The presence of calcium during derivatization prevented functional inactivation, radioactive labeling of fragment A 2 , and internal cross-linking of fragment A 1 . It is proposed that both A 1 and A 2 fragments participate in formation of the calcium binding domain and that the labeled residues of fragment A 2 are directly involved in calcium complexation. A diagram is constructed, representing the relative positions of labels and functional domains within the ATPase protein

  19. Proteolytic processing of poliovirus polypeptides: antibodies to polypeptide P3-7c inhibit cleavage at glutamine-glycine pairs

    International Nuclear Information System (INIS)

    Hanecak, R.; Semler, B.L.; Anderson, C.W.; Wimmer, E.

    1982-01-01

    Proteolytic processing of poliovirus polypeptides was examined by the addition of antibodies directed against the viral proteins P3-7c and P2-X to a cell-free translation extract prepared from infected HeLa cells. Antisera to P3-7c specifically inhibited in vitro processing at Gln-Gly pairs. Partial amino acid sequence analysis revealed a second Tyr-Gly pair that is utilized in protein processing. Neither Tyr-Gly cleavage is affected by antibody to P3-7C. Anti-P3-7c antibodies react not only with P3-7c but also with P3-6a and P3-2, two viral polypeptides NH 2 -coterminal with P3-7c. Preimmune and anti-P2-X antibodies had no effect on the processing of poliovirus proteins in vitro. The authors conclude that the activity responsible for processing poliovirus polypeptides at Gln-Gly pairs resides in the primary structure of P3-7c and not in P2-X

  20. Bifunctional alkylating agent-mediated MGMT-DNA cross-linking and its proteolytic cleavage in 16HBE cells

    International Nuclear Information System (INIS)

    Cheng, Jin; Ye, Feng; Dan, Guorong; Zhao, Yuanpeng; Wang, Bin; Zhao, Jiqing; Sai, Yan; Zou, Zhongmin

    2016-01-01

    Nitrogen mustard (NM), a bifunctional alkylating agent (BAA), contains two alkyl arms and can act as a cross-linking bridge between DNA and protein to form a DNA-protein cross-link (DPC). O 6 -methylguanine–DNA methyltransferase (MGMT), a DNA repair enzyme for alkyl adducts removal, is found to enhance cell sensitivity to BAAs and to promote damage, possibly due to its stable covalent cross-linking with DNA mediated by BAAs. To investigate MGMT-DNA cross-link (mDPC) formation and its possible dual roles in NM exposure, human bronchial epithelial cell line 16HBE was subjected to different concentrations of HN2, a kind of NM, and we found mDPC was induced by HN2 in a concentration-dependent manner, but the mRNA and total protein of MGMT were suppressed. As early as 1 h after HN2 treatment, high mDPC was achieved and the level maintained for up to 24 h. Quick total DPC (tDPC) and γ-H2AX accumulation were observed. To evaluate the effect of newly predicted protease DVC1 on DPC cleavage, we applied siRNA of MGMT and DVC1, MG132 (proteasome inhibitor), and NMS-873 (p97 inhibitor) and found that proteolysis plays a role. DVC1 was proven to be more important in the cleavage of mDPC than tDPC in a p97-dependent manner. HN2 exposure induced DVC1 upregulation, which was at least partially contributed to MGMT cleavage by proteolysis because HN2-induced mDPC level and DNA damage was closely related with DVC1 expression. Homologous recombination (HR) was also activated. Our findings demonstrated that MGMT might turn into a DNA damage promoter by forming DPC when exposed to HN2. Proteolysis, especially DVC1, plays a crucial role in mDPC repair. - Highlights: • Nitrogen mustard-induced MGMT-DNA cross-linking was detected in a living cell. • Concentration- and time-dependent manners of MGMT-DNA cross-linking were revealed. • Proteolysis played an important role in protein (MGMT)-DNA cross-linking repair. • DVC1 acts as a proteolytic enzyme in cross-linking repair in a p

  1. Bacterial and Fungal Proteolytic Enzymes: Production, Catalysis and Potential Applications.

    Science.gov (United States)

    da Silva, Ronivaldo Rodrigues

    2017-09-01

    Submerged and solid-state bioprocesses have been extensively explored worldwide and employed in a number of important studies dealing with microbial cultivation for the production of enzymes. The development of these production technologies has facilitated the generation of new enzyme-based products with applications in pharmaceuticals, food, bioactive peptides, and basic research studies, among others. The applicability of microorganisms in biotechnology is potentiated because of their various advantages, including large-scale production, short time of cultivation, and ease of handling. Currently, several studies are being conducted to search for new microbial peptidases with peculiar biochemical properties for industrial applications. Bioprospecting, being an important prerequisite for research and biotechnological development, is based on exploring the microbial diversity for enzyme production. Limited information is available on the production of specific proteolytic enzymes from bacterial and fungal species, especially on the subgroups threonine and glutamic peptidases, and the seventh catalytic type, nonhydrolytic asparagine peptide lyase. This gap in information motivated the present study about these unique biocatalysts. In this study, the biochemical and biotechnological aspects of the seven catalytic types of proteolytic enzymes, namely aspartyl, cysteine, serine, metallo, glutamic, and threonine peptidase, and asparagine peptide lyase, are summarized, with an emphasis on new studies, production, catalysis, and application of these enzymes.

  2. Proteolytic cleavage and PKA phosphorylation of α1C subunit are not required for adrenergic regulation of CaV1.2 in the heart.

    Science.gov (United States)

    Katchman, Alexander; Yang, Lin; Zakharov, Sergey I; Kushner, Jared; Abrams, Jeffrey; Chen, Bi-Xing; Liu, Guoxia; Pitt, Geoffrey S; Colecraft, Henry M; Marx, Steven O

    2017-08-22

    Calcium influx through the voltage-dependent L-type calcium channel (Ca V 1.2) rapidly increases in the heart during "fight or flight" through activation of the β-adrenergic and protein kinase A (PKA) signaling pathway. The precise molecular mechanisms of β-adrenergic activation of cardiac Ca V 1.2, however, are incompletely known, but are presumed to require phosphorylation of residues in α 1C and C-terminal proteolytic cleavage of the α 1C subunit. We generated transgenic mice expressing an α 1C with alanine substitutions of all conserved serine or threonine, which is predicted to be a potential PKA phosphorylation site by at least one prediction tool, while sparing the residues previously shown to be phosphorylated but shown individually not to be required for β-adrenergic regulation of Ca V 1.2 current (17-mutant). A second line included these 17 putative sites plus the five previously identified phosphoregulatory sites (22-mutant), thus allowing us to query whether regulation requires their contribution in combination. We determined that acute β-adrenergic regulation does not require any combination of potential PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse α 1C subunits. We separately generated transgenic mice with inducible expression of proteolytic-resistant α 1C Prevention of C-terminal cleavage did not alter β-adrenergic stimulation of Ca V 1.2 in the heart. These studies definitively rule out a role for all conserved consensus PKA phosphorylation sites in α 1C in β-adrenergic stimulation of Ca V 1.2, and show that phosphoregulatory sites on α 1C are not redundant and do not each fractionally contribute to the net stimulatory effect of β-adrenergic stimulation. Further, proteolytic cleavage of α 1C is not required for β-adrenergic stimulation of Ca V 1.2.

  3. Analysis of the in vitro cleavage products of the tomato black ring virus RNA-1-encoded 250K polyprotein.

    Science.gov (United States)

    Demangeat, G; Greif, C; Hemmer, O; Fritsch, C

    1990-08-01

    Tomato black ring virus RNA-1 was translated in a rabbit reticulocyte lysate. The primary translation product of Mr 250K, which corresponds to its whole coding capacity, was synthesized within 45 min and, during further incubation in the translation medium, was proteolytically processed. Essentially, four cleavage products (P190, P120, P60 and P50) were detected and located within P250 by pulse-chase and immunoprecipitation experiments. P190 is an intermediate cleavage product which is further cleaved to form P60 and P120. P120, which contains the region that has been assigned to the virus protease and the virus polymerase, was not further cleaved in vitro.

  4. Proteolytic processing of the cilium adhesin MHJ_0194 (P123J ) in Mycoplasma hyopneumoniae generates a functionally diverse array of cleavage fragments that bind multiple host molecules.

    Science.gov (United States)

    Raymond, Benjamin B A; Jenkins, Cheryl; Seymour, Lisa M; Tacchi, Jessica L; Widjaja, Michael; Jarocki, Veronica M; Deutscher, Ania T; Turnbull, Lynne; Whitchurch, Cynthia B; Padula, Matthew P; Djordjevic, Steven P

    2015-03-01

    Mycoplasma hyopneumoniae, the aetiological agent of porcine enzootic pneumonia, regulates the presentation of proteins on its cell surface via endoproteolysis, including those of the cilial adhesin P123 (MHJ_0194). These proteolytic cleavage events create functional adhesins that bind to proteoglycans and glycoproteins on the surface of ciliated and non-ciliated epithelial cells and to the circulatory host molecule plasminogen. Two dominant cleavage events of the P123 preprotein have been previously characterized; however, immunoblotting studies suggest that more complex processing events occur. These extensive processing events are characterized here. The functional significance of the P97 cleavage fragments is also poorly understood. Affinity chromatography using heparin, fibronectin and plasminogen as bait and peptide arrays were used to expand our knowledge of the adhesive capabilities of P123 cleavage fragments and characterize a novel binding motif in the C-terminus of P123. Further, we use immunohistochemistry to examine in vivo, the biological significance of interactions between M. hyopneumoniae and fibronectin and show that M. hyopneumoniae induces fibronectin deposition at the site of infection on the ciliated epithelium. Our data supports the hypothesis that M. hyopneumoniae possesses the molecular machinery to influence key molecular communication pathways in host cells. © 2014 John Wiley & Sons Ltd.

  5. Monoclonal antibodies to the reactive centre loop (RCL) of human corticosteroid-binding globulin (CBG) can protect against proteolytic cleavage.

    Science.gov (United States)

    Lewis, John G; Elder, Peter A

    2017-07-01

    Corticosteroid-binding globulin (CBG) binds most of the cortisol in circulation and is a non-functional member of the family of serine protease inhibitors (serpins) with an exposed elastase sensitive reactive centre loop (RCL). The RCL can be cleaved by human neutrophil elastase, released from activated neutrophils, and can also be cleaved at nearby site(s) by elastase released by Pseudomonas aeruginosa, and at two further sites, also within the RCL, by bovine chymotrypsin. Cleavage of the RCL results in a conformational change accompanied by a marked decrease in affinity for cortisol and hence its release at the site of proteolysis. These cleavages are irreversible and the similar half-lives of cleaved and intact CBG could mean that there may be some advantage in slowing the rate of CBG cleavage in acute inflammation thereby increasing the proportion of intact CBG in circulation. Here we show, for the first time, that pre-incubation of tethered human CBG with two monoclonal antibodies to the RCL of CBG protects against cleavage by all three enzymes. Furthermore, in plasma, pre-incubation with both RCL monoclonal antibodies delays neutrophil elastase cleavage of the RCL and one of these RCL monoclonal antibodies also delays bovine chymotrypsin cleavage of the RCL. These findings may provide a basis and rationale for the concept of the use of RCL antibodies as therapeutic agents to effectively increase the proportion of intact CBG in circulation which may be of benefit in acute inflammation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Picking up the pieces: a generic porous Si biosensor for probing the proteolytic products of enzymes.

    Science.gov (United States)

    Shtenberg, Giorgi; Massad-Ivanir, Naama; Moscovitz, Oren; Engin, Sinem; Sharon, Michal; Fruk, Ljiljana; Segal, Ester

    2013-02-05

    A multifunctional porous Si biosensor that can both monitor the enzymatic activity of minute samples and allow subsequent retrieval of the entrapped proteolytic products for mass spectrometry analysis is described. The biosensor is constructed by DNA-directed/reversible immobilization of enzymes onto a Fabry-Pérot thin film. We demonstrate high enzymatic activity levels of the immobilized enzymes (more than 80%), while maintaining their specificity. Mild dehybridization conditions allow enzyme recycling and facile surface regeneration for consecutive biosensing analysis. The catalytic activity of the immobilized enzymes is monitored in real time by reflective interferometric Fourier transform spectroscopy. The real-time analysis of minute quantities of enzymes (concentrations at least 1 order of magnitude lower, 0.1 mg mL(-1), in comparison to previous reports, 1 mg mL(-1)), in particular proteases, paves the way for substrate profiling and the identification of cleavage sites. The biosensor configuration is compatible with common proteomic methods and allows for a successful downstream mass spectrometry analysis of the reaction products.

  7. Coupling fibroblast growth factor 23 production and cleavage: iron deficiency, rickets, and kidney disease.

    Science.gov (United States)

    Wolf, Myles; White, Kenneth E

    2014-07-01

    High levels of fibroblast growth factor 23 (FGF23) cause the rare disorders of hypophosphatemic rickets and are a risk factor for cardiovascular disease and death in patients with chronic kidney disease (CKD). Despite major advances in understanding FGF23 biology, fundamental aspects of FGF23 regulation in health and in CKD remain mostly unknown. Autosomal dominant hypophosphatemic rickets (ADHR) is caused by gain-of-function mutations in FGF23 that prevent its proteolytic cleavage, but affected individuals experience a waxing and waning course of phosphate wasting. This led to the discovery that iron deficiency is an environmental trigger that stimulates FGF23 expression and hypophosphatemia in ADHR. Unlike osteocytes in ADHR, normal osteocytes couple increased FGF23 production with commensurately increased FGF23 cleavage to ensure that normal phosphate homeostasis is maintained in the event of iron deficiency. Simultaneous measurement of FGF23 by intact and C-terminal assays supported these breakthroughs by providing minimally invasive insight into FGF23 production and cleavage in bone. These findings also suggest a novel mechanism of FGF23 elevation in patients with CKD, who are often iron deficient and demonstrate increased FGF23 production and decreased FGF23 cleavage, consistent with an acquired state that mimics the molecular pathophysiology of ADHR. Iron deficiency stimulates FGF23 production, but normal osteocytes couple increased FGF23 production with increased cleavage to maintain normal circulating levels of biologically active hormone. These findings uncover a second level of FGF23 regulation within osteocytes, failure of which culminates in elevated levels of biologically active FGF23 in ADHR and perhaps CKD.

  8. Tyrosine phosphorylation and proteolytic cleavage of Notch are required for non-canonical Notch/Abl signaling in Drosophila axon guidance.

    Science.gov (United States)

    Kannan, Ramakrishnan; Cox, Eric; Wang, Lei; Kuzina, Irina; Gu, Qun; Giniger, Edward

    2018-01-17

    Notch signaling is required for the development and physiology of nearly every tissue in metazoans. Much of Notch signaling is mediated by transcriptional regulation of downstream target genes, but Notch controls axon patterning in Drosophila by local modulation of Abl tyrosine kinase signaling, via direct interactions with the Abl co-factors Disabled and Trio. Here, we show that Notch-Abl axonal signaling requires both of the proteolytic cleavage events that initiate canonical Notch signaling. We further show that some Notch protein is tyrosine phosphorylated in Drosophila , that this form of the protein is selectively associated with Disabled and Trio, and that relevant tyrosines are essential for Notch-dependent axon patterning but not for canonical Notch-dependent regulation of cell fate. Based on these data, we propose a model for the molecular mechanism by which Notch controls Abl signaling in Drosophila axons. © 2018. Published by The Company of Biologists Ltd.

  9. Functional role of proteolytic cleavage at arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis

    International Nuclear Information System (INIS)

    Tate, K.M.; Higgins, D.L.; Holmes, W.E.; Winkler, M.E.; Heyneker, H.L.; Vehar, G.A.

    1987-01-01

    Activation of the zymogen form of a serine protease is associated with a conformational change that follows proteolysis at a specific site. Tissue-type plasminogen activator (t-PA) is homologous to mammalian serine proteases and contains an apparent activation cleavage site at arginine-275. To clarify the functional consequences of cleavage at arginine-275 of t-PA, site-specific mutagenesis was performed to convert arginine-275 to a glutamic acid. The mutant enzyme (designated Arg-275 → Glu t-PA) could be converted to the two-chain form by Staphylococcus aureus V8 protease but not by plasmin. The one-chain form was 8 times less active against the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine-rho-nitroanilide (S-2288), and the ability of the enzyme to activate plasminogen in the absence of fibrinogen was reduced 20-50 times compared to the two-chain form. In contrast, one-chain Arg-275 → Glu t-PA has equal activity to the two-chain form when assayed in the presence of physiological levels of fibrinogen and plasminogen. Fibrin bound significantly more of the one-chain form of t-PA than the two-chain form for both the wild-type and mutated enzymes. One- and two-chain forms of the wild-type and mutated plasminogen activators slowly formed complexes with plasma protease inhibitors, although the one-chain forms showed decreased complex formation with → 2 -macroglobulin. The one-chain form of t-PA therefore is fully functional under physiologic conditions and has a increased fibrin binding compared to the two-chain form

  10. Characterization of a bioactive 15 kDa fragment produced by proteolytic cleavage of chicken growth hormone.

    Science.gov (United States)

    Arámburo, C; Carranza, M; Reyes, M; Luna, M; Martinez-Coria, H; Berúmen, L; Scanes, C G

    2001-07-01

    There is evidence for a cleaved form of GH in the chicken pituitary gland. A 25 kDa band of immunoreactive-(ir-)GH, as well as the 22 kDa monomeric form and some oligomeric forms were observed when purified GH or fresh pituitary extract were subjected to SDS-PAGE under nonreducing conditions. Under reducing conditions, the 25 kDa ir-GH was no longer observed, being replaced by a 15 kDa band, consistent with reduction of the disulfide bridges of the cleaved form. The type of protease involved was investigated using exogenous proteases and monomeric cGH. Cleaved forms of chicken GH were generated by thrombin or collagenase. The site of cleavage was found in position Arg133-Gly134 as revealed by sequencing the fragments produced. The NH2-terminal sequence of 40 amino acid residues in the 15 kDa form was identical to that of the rcGH and analysis of the remaining 7 kDa fragment showed an exact identity with positions 134-140 of cGH structure. The thrombin cleaved GH and the 15 kDa form showed reduced activity (0.8% and 0.5% of GH, respectively) in a radioreceptor assay employing a chicken liver membrane preparation. However, this fragment had a clear bioactivity in an angiogenic bioassay and was capable to inhibit the activity of deiodinase type III in the chicken liver.

  11. Implications of caspase-dependent proteolytic cleavage of cyclin A1 in DNA damage-induced cell death

    Energy Technology Data Exchange (ETDEWEB)

    Woo, Sang Hyeok; Seo, Sung-Keum [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); An, Sungkwan; Choe, Tae-Boo [Department of Microbiological Engineering, Kon-Kuk University, Gwangjin-gu, Seoul (Korea, Republic of); Hong, Seok-Il [Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); Lee, Yun-Han, E-mail: yhlee87@yuhs.ac [Department of Radiation Oncology, College of Medicine, Yonsei University, 250 Seongsan-no, Seodaemun-gu, Seoul (Korea, Republic of); Park, In-Chul, E-mail: parkic@kcch.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of)

    2014-10-24

    Highlights: • Caspase-1 mediates doxorubicin-induced downregulation of cyclin A1. • Active caspase-1 effectively cleaved cyclin A1 at D165. • Cyclin A1 expression is involved in DNA damage-induced cell death. - Abstract: Cyclin A1 is an A-type cyclin that directly binds to CDK2 to regulate cell-cycle progression. In the present study, we found that doxorubicin decreased the expression of cyclin A1 at the protein level in A549 lung cancer cells, while markedly downregulating its mRNA levels. Interestingly, doxorubicin upregulated caspase-1 in a concentration-dependent manner, and z-YAVD-fmk, a specific inhibitor of caspase-1, reversed the doxorubicin-induced decrease in cyclin A1 in A549 lung cancer and MCF7 breast cancer cells. Active caspase-1 effectively cleaved cyclin A1 at D165 into two fragments, which in vitro cleavage assays showed were further cleaved by caspase-3. Finally, we found that overexpression of cyclin A1 significantly reduced the cytotoxicity of doxorubicin, and knockdown of cyclin A1 by RNA interference enhanced the sensitivity of cells to ionizing radiation. Our data suggest a new mechanism for the downregulation of cyclin A1 by DNA-damaging stimuli that could be intimately involved in the cell death induced by DNA damage-inducing stimuli, including doxorubicin and ionizing radiation.

  12. A Comparative Study of New Aspergillus Strains for Proteolytic Enzymes Production by Solid State Fermentation

    Directory of Open Access Journals (Sweden)

    Gastón Ezequiel Ortiz

    2016-01-01

    Full Text Available A comparative study of the proteolytic enzymes production using twelve Aspergillus strains previously unused for this purpose was performed by solid state fermentation. A semiquantitative and quantitative evaluation of proteolytic activity were carried out using crude enzymatic extracts obtained from the fermentation cultures, finding seven strains with high and intermediate level of protease activity. Biochemical, thermodynamics, and kinetics features such as optimum pH and temperature values, thermal stability, activation energy (Ea, quotient energy (Q10, Km, and Vmax were studied in four enzymatic extracts from the selected strains that showed the highest productivity. Additionally, these strains were evaluated by zymogram analysis obtaining protease profiles with a wide range of molecular weight for each sample. From these four strains with the highest productivity, the proteolytic extract of A. sojae ATCC 20235 was shown to be an appropriate biocatalyst for hydrolysis of casein and gelatin substrates, increasing its antioxidant activities in 35% and 125%, respectively.

  13. Transthyretin protects against A-beta peptide toxicity by proteolytic cleavage of the peptide: a mechanism sensitive to the Kunitz protease inhibitor.

    Directory of Open Access Journals (Sweden)

    Rita Costa

    Full Text Available Alzheimer's disease (AD is a neurodegenerative disorder characterized by the deposition of amyloid beta-peptide (A-Beta in the brain. Transthyretin (TTR is a tetrameric protein of about 55 kDa mainly produced in the liver and choroid plexus of the brain. The known physiological functions of TTR are the transport of thyroid hormone T(4 and retinol, through binding to the retinol binding protein. TTR has also been established as a cryptic protease able to cleave ApoA-I in vitro. It has been described that TTR is involved in preventing A-Beta fibrilization, both by inhibiting and disrupting A-Beta fibrils, with consequent abrogation of toxicity. We further characterized the nature of the TTR/A-Beta interaction and found that TTR, both recombinant or isolated from human sera, was able to proteolytically process A-Beta, cleaving the peptide after aminoacid residues 1, 2, 3, 10, 13, 14,16, 19 and 27, as determined by mass spectrometry, and reversed phase chromatography followed by N-terminal sequencing. A-Beta peptides (1-14 and (15-42 showed lower amyloidogenic potential than the full length counterpart, as assessed by thioflavin binding assay and ultrastructural analysis by transmission electron microscopy. A-Beta cleavage by TTR was inhibited in the presence of an alphaAPP peptide containing the Kunitz Protease Inhibitor (KPI domain but not in the presence of the secreted alphaAPP derived from the APP isoform 695 without the KPI domain. TTR was also able to degrade aggregated forms of A-Beta peptide. Our results confirmed TTR as a protective molecule in AD, and prompted A-Beta proteolysis by TTR as a protective mechanism in this disease. TTR may prove to be a useful therapeutic agent for preventing or retarding the cerebral amyloid plaque formation implicated in AD pathology.

  14. Evidence for proteolytic cleavage of brevican by the ADAMTSs in the dentate gyrus after excitotoxic lesion of the mouse entorhinal cortex

    Directory of Open Access Journals (Sweden)

    Gottschall Paul E

    2005-08-01

    Full Text Available Abstract Background Brevican is a member of the lectican family of aggregating extracellular matrix (ECM proteoglycans that bear chondroitin sulfate (CS chains. It is highly expressed in the central nervous system (CNS and is thought to stabilize synapses and inhibit neural plasticity and as such, neuritic or synaptic remodeling would be less likely to occur in regions with intact and abundant, lectican-containing, ECM complexes. Neural plasticity may occur more readily when these ECM complexes are broken down by endogenous proteases, the ADAMTSs (adisintegrin and metalloproteinase with thrombospondin motifs, that selectively cleave the lecticans. The purpose of these experiments was to determine whether the production of brevican or the ADAMTS-cleaved fragments of brevican were altered after deafferentation and reinnervation of the dentate gyrus via entorhinal cortex lesion (ECL. Results In the C57Bl6J mouse, synaptic density in the molecular layer of the dentate gyrus, as measured by synaptophysin levels in ELISA, was significantly attenuated 2 days (nearly 50% of contralateral and 7 days after lesion and returned to levels not different from the contralateral region at 30 days. Immunoreactive brevican in immunoblot was elevated 2 days after lesion, whereas there was a significant increase in the proteolytic product at 7, but not 30 days post-lesion. ADAMTS activity, estimated using the ratio of the specific ADAMTS-derived brevican fragment and intact brevican levels was increased at 7 days, but was not different from the contralateral side at 2 or 30 days after deafferentation. Conclusion These findings indicate that ADAMTS activity in the dentate outer molecular layer (OML is elevated during the initial synaptic reinnervation period (7 days after lesion. Therefore, proteolytic processing of brevican appears to be a significant extracellular event in the remodeling of the dentate after EC lesion, and may modulate the process of sprouting and

  15. Characterization of SNARE Cleavage Products Generated by Formulated Botulinum Neurotoxin Type-A Drug Products

    Directory of Open Access Journals (Sweden)

    Jack Xie

    2010-08-01

    Full Text Available The study evaluated substrate cleavage product(s generated by three botulinum neurotoxin serotype A (BoNT/A medicinal drug products utilizing a novel and highly specific, light-chain activity, high-performance liquid chromatography (LCA-HPLC method. Samples were reacted with a commercially available BoNT/A fluorescent substrate derived from the SNAP-25 sequence. Reaction products were separated by reversed-phase HPLC. The method detected an atypical cleavage pattern by one of the formulated drug products. IncobotulinumtoxinA produced two cleavage fragments rather than the single fragment typically generated by BoNT/A. Identification confirmed the secondary cleavage at a position corresponding to SNAP-25 Arg198–Ala199 (normal BoNT/A cleavage is Gln197–Arg198. Arg198–Ala199 is also the cleavage site for trypsin and serotype C toxin. Normal cleavage was observed for all other BoNT/A drug product samples, as well as 900-kD and 150-kD bulk toxin BoNT/A. The reason for this unexpected secondary cleavage pattern by one formulated BoNT/A drug product is unknown. Possible explanations include a contaminating protease and/or damage to the 150-kD type-A toxin causing nonspecific substrate recognition and subsequent cleavage uncharacteristic of type-A toxin. The BoNT/A drug products were also analyzed via the LCA-HPLC assay using a commercial BoNT/C fluorescent substrate derived from the syntaxin sequence. Cleavage of the serotype C substrate by incobotulinumtoxinA was also confirmed whilst neither of the other drug products cleaved the syntaxin substrate.

  16. Determination of proteolytic activity using L-[4,5-3H]leucine-labelled globin as a substrate

    International Nuclear Information System (INIS)

    Maliopoulou, T.B.; Dionyssiou-Asteriou, A.; Loucopoulos, D.

    1980-01-01

    A simple and sensitive method for the assay of proteolytic enzyme activity is described. This is based on the digestion of L-[4,5- 3 H]leucine globin by proteolytic enzymes and radioactivity measurement of the trichloroacetic acid soluble cleavage products. (Auth.)

  17. Effect of Cultured Celery Juice, Temperature, and Product Composition on the Inhibition of Proteolytic Clostridium botulinum Toxin Production.

    Science.gov (United States)

    Golden, Max C; Wanless, Brandon J; David, Jairus R D; Kottapalli, Bala; Lineback, D Scott; Talley, Ryan J; Glass, Kathleen A

    2017-08-01

    Clostridium botulinum may be of concern in prepared refrigerated meals, for which strict cold chain management cannot be guaranteed. This study evaluated the effect of temperature, product composition, and cultured celery juice powder (CCJP) as a source of nitrite on the inhibition of botulinum toxin formation in two experimental (meat- and vegetable-based) prepared meals. Data obtained from the challenge study were compared with a published mathematical model to determine whether the model is fail-safe with regard to the tested meals. Treatments were inoculated with proteolytic C. botulinum, vacuum packaged, cooked at 90°C for 10 min, and assayed for botulinum toxin at appropriate intervals in samples stored at 10, 15, or 20°C for up to 8 weeks. None of the treatments stored at 10°C for 8 weeks supported toxin production by proteolytic C. botulinum. The addition of CCJP delayed toxin production by 1 and 3 weeks in cauliflower potatoes and in Dijon pork, respectively, stored at 15°C. Toxin production was delayed by 1 week at 20°C when CCJP was added to the cauliflower potatoes. This study found that the predictive model was fail-safe but was overly conservative for the experimental meals described. Finally, this study confirms that product composition, the addition of nitrite via CCJP, storage time, and temperature play important roles in the inhibition of toxin formation by proteolytic C. botulinum.

  18. Serum disposition of bovine lactoferrin after oral and anal administration and its proteolytic cleavage by gastric transit in rainbow trout (Oncorhynchus mykiss W.).

    Science.gov (United States)

    Cecchini, Stefano; Caputo, Anna R

    2009-01-01

    Several studies have shown an immunomodulatory effect of orally administered bovine lactoferrin (LF) in fish, but the process of digestion was not characterized. In the present study, we investigated the fate of bovine LF after oral and anal administration, and studied the appearance of intact LF in the bloodstream and its proteolytic attack during the gastric transit in rainbow trout (Oncorhynchus mykiss) held at 9 degrees C and 18 degrees C. Data obtained showed the presence of intact bovine LF in the bloodstream only after anal administration in fish held at 18 degrees C and the presence of several peptides derived from bovine LF in the gastric content. Immunoblotting analysis showed that only a part of bovine LF-derived peptides reacted with the applied anti-bovine LF antibody. The concentration of intact bovine LF, after 30 min of administration, in the gastric content of fish reared at 18 degrees C, being extremely low, if any, led us to suspect that the immunoregulatory effect of dietary bovine LF shown in fish by several authors is not due to the intact form but to bioactive fragments, originated by the proteolytic attack during the gastric transit, as demonstrated in higher vertebrates.

  19. Differential production of proteolytic enzymes by normal human fibroblasts and their counterparts transformed by treatment with 60Co gamma rays

    International Nuclear Information System (INIS)

    Nishitani, Koji; Namba, Masayoshi; Ohkubo, Shigeki; Kimoto, Tetsuo

    1985-01-01

    Production of proteolytic enzymes by human fibroblasts in the process of transformation was investigated. The cells used were normal human fibroblasts (KSM-6) and their in vitro counterparts transformed by treatment with 60 Co gamma rays (KMST-6). Cells seeded by treatment with EDTA were cultured in a serum free medium. Proteolytic enzymes in the culture medium of cells were assayed using a synthetic substrate, N-α-(p-tosyl)-L-arginine ( 3 H) methyl ester hydrochloride. The transformed cells (KMST-6) produced a larger amount of enzymes than normal cells (KMS-6). The enzyme production in both cell lines was high in the exponential growth stage and then decreased as the cells reached confluency. The proteolytic enzymes produced by these cells were trypsin- and thrombin-like enzymes. Cell growth of KMST-6 or KMS-6 was not inhibited by the addition of protease inhibitors to the culture medium. (author)

  20. Amyloid-β production via cleavage of amyloid-β protein precursor is modulated by cell density.

    Science.gov (United States)

    Zhang, Can; Browne, Andrew; Divito, Jason R; Stevenson, Jesse A; Romano, Donna; Dong, Yuanlin; Xie, Zhongcong; Tanzi, Rudolph E

    2010-01-01

    Mounting evidence suggests that Alzheimer's disease (AD) is caused by the accumulation of the small peptide, amyloid-β (Aβ), a proteolytic cleavage product of amyloid-β protein precursor (AβPP). Aβ is generated through a serial cleavage of AβPP by β- and γ-secretase. Aβ40 and Aβ42 are the two main components of amyloid plaques in AD brains, with Aβ42 being more prone to aggregation. AβPP can also be processed by α-secretase, which cleaves AβPP within the Aβ sequence, thereby preventing the generation of Aβ. Little is currently known regarding the effects of cell density on AβPP processing and Aβ generation. Here we assessed the effects of cell density on AβPP processing in neuronal and non-neuronal cell lines, as well as mouse primary cortical neurons. We found that decreased cell density significantly increases levels of Aβ40, Aβ42, total Aβ, and the ratio of Aβ42: Aβ40. These results also indicate that cell density is a significant modulator of AβPP processing. Overall, these findings carry profound implications for both previous and forthcoming studies aiming to assess the effects of various conditions and genetic/chemical factors, e.g., novel drugs on AβPP processing and Aβ generation in cell-based systems. Moreover, it is interesting to speculate whether cell density changes in vivo may also affect AβPP processing and Aβ levels in the AD brain.

  1. Recovery of Proteolytic and Collagenolytic Activities from Viscera By-products of Rayfish (Raja clavata

    Directory of Open Access Journals (Sweden)

    José Antonio Vázquez

    2009-12-01

    Full Text Available The aim of this work was to study the recovery of proteolytic and collagenolytic activities from rayfish (Raja clavata viscera wastes. Initially, different parts of the gastrointestinal tract by-products (stomach, duodenum section including pancreas, final intestine were evaluated. The extracts from proximal intestine yielded the highest values of both enzymatic activities. Optimal conditions for protease activity quantification were established at pH = 6, T = 40 °C and incubation time ≤20 min. The mathematical equation used to model the joint effect of pH and temperature led to maximum activity at pH = 8.66 and 59.4 °C, respectively. Overcooled acetone was found to be best option for recovery of enzymatic activities in comparison with ethanol, PEG-4000, ammonium sulphate and ultrafiltration system. Finally, a simple and systematic protocol of partial purification and total recovery of proteases and collagenases was defined.

  2. Production of Proteolytic Enzymes by a Keratin-Degrading Aspergillus niger

    Directory of Open Access Journals (Sweden)

    Fernanda Cortez Lopes

    2011-01-01

    Full Text Available A fungal isolate with capability to grow in keratinous substrate as only source of carbon and nitrogen was identified as Aspergillus niger using the sequencing of the ITS region of the rDNA. This strain produced a slightly acid keratinase and an acid protease during cultivation in feather meal. The peak of keratinolytic activity occurred in 48 h and the maximum proteolytic activity in 96 h. These enzymes were partly characterized as serine protease and aspartic protease, respectively. The effects of feather meal concentration and initial pH on enzyme production were evaluated using a central composite design combined with response surface methodology. The optimal conditions were determined as pH 5.0 for protease and 7.8 for keratinase and 20 g/L of feather meal, showing that both models were predictive. Production of keratinases by A. niger is a less-exploited field that might represent a novel and promising biotechnological application for this microorganism.

  3. Endogenous proteolytic cleavage of disease-associated prion protein to produce C2 fragments is strongly cell- and tissue-dependent.

    Science.gov (United States)

    Dron, Michel; Moudjou, Mohammed; Chapuis, Jérôme; Salamat, Muhammad Khalid Farooq; Bernard, Julie; Cronier, Sabrina; Langevin, Christelle; Laude, Hubert

    2010-04-02

    The abnormally folded form of the prion protein (PrP(Sc)) accumulating in nervous and lymphoid tissues of prion-infected individuals can be naturally cleaved to generate a N-terminal-truncated fragment called C2. Information about the identity of the cellular proteases involved in this process and its possible role in prion biology has remained limited and controversial. We investigated PrP(Sc) N-terminal trimming in different cell lines and primary cultured nerve cells, and in the brain and spleen tissue from transgenic mice infected by ovine and mouse prions. We found the following: (i) the full-length to C2 ratio varies considerably depending on the infected cell or tissue. Thus, in primary neurons and brain tissue, PrP(Sc) accumulated predominantly as untrimmed species, whereas efficient trimming occurred in Rov and MovS cells, and in spleen tissue. (ii) Although C2 is generally considered to be the counterpart of the PrP(Sc) proteinase K-resistant core, the N termini of the fragments cleaved in vivo and in vitro can actually differ, as evidenced by a different reactivity toward the Pc248 anti-octarepeat antibody. (iii) In lysosome-impaired cells, the ratio of full-length versus C2 species dramatically increased, yet efficient prion propagation could occur. Moreover, cathepsin but not calpain inhibitors markedly inhibited C2 formation, and in vitro cleavage by cathepsins B and L produced PrP(Sc) fragments lacking the Pc248 epitope, strongly arguing for the primary involvement of acidic hydrolases of the endolysosomal compartment. These findings have implications on the molecular analysis of PrP(Sc) and cell pathogenesis of prion infection.

  4. Endogenous Proteolytic Cleavage of Disease-associated Prion Protein to Produce C2 Fragments Is Strongly Cell- and Tissue-dependent*

    Science.gov (United States)

    Dron, Michel; Moudjou, Mohammed; Chapuis, Jérôme; Salamat, Muhammad Khalid Farooq; Bernard, Julie; Cronier, Sabrina; Langevin, Christelle; Laude, Hubert

    2010-01-01

    The abnormally folded form of the prion protein (PrPSc) accumulating in nervous and lymphoid tissues of prion-infected individuals can be naturally cleaved to generate a N-terminal-truncated fragment called C2. Information about the identity of the cellular proteases involved in this process and its possible role in prion biology has remained limited and controversial. We investigated PrPSc N-terminal trimming in different cell lines and primary cultured nerve cells, and in the brain and spleen tissue from transgenic mice infected by ovine and mouse prions. We found the following: (i) the full-length to C2 ratio varies considerably depending on the infected cell or tissue. Thus, in primary neurons and brain tissue, PrPSc accumulated predominantly as untrimmed species, whereas efficient trimming occurred in Rov and MovS cells, and in spleen tissue. (ii) Although C2 is generally considered to be the counterpart of the PrPSc proteinase K-resistant core, the N termini of the fragments cleaved in vivo and in vitro can actually differ, as evidenced by a different reactivity toward the Pc248 anti-octarepeat antibody. (iii) In lysosome-impaired cells, the ratio of full-length versus C2 species dramatically increased, yet efficient prion propagation could occur. Moreover, cathepsin but not calpain inhibitors markedly inhibited C2 formation, and in vitro cleavage by cathepsins B and L produced PrPSc fragments lacking the Pc248 epitope, strongly arguing for the primary involvement of acidic hydrolases of the endolysosomal compartment. These findings have implications on the molecular analysis of PrPSc and cell pathogenesis of prion infection. PMID:20154089

  5. Characterisation of proteolytic activity of excretory-secretory products from adult Strongylus vulgaris.

    Science.gov (United States)

    Caffrey, C R; Ryan, M F

    1994-04-01

    An excretory-secretory (ES) preparation derived from adult Strongylus vulgaris in vitro was assessed for proteolytic activity using azocasein and synthetic, fluorogenic, peptide substrates. Fractionation was by molecular sieve fast protein liquid chromatography (molecular sieve FPLC) and resolution by gelatin-substrate sodium dodecyl sulphate-polyacrylamide gel electrophoresis (gelatin-substrate SDS-PAGE). The cysteine proteinase activator, dithiothreitol (DTT), enhanced azocaseinolysis and hydrolysis of carbobenzoxy-phenylalanyl-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-NMec) by the ES preparation and was a requirement for the detection of carbobenzoxy-arginyl-arginine-7-amido-4-methylcoumarin (Z-Arg-Arg-NMec) hydrolysis. Assays of FPLC-eluted fractions, with DTT, detected a broad peak of azocaseinolytic activity (22-24 kDa) and two peaks (24 and 18 kDa) of hydrolysis using the synthetic substrates. Hydrolysis by these peaks of Z-Phe-Arg-NMec was 50-fold greater than that of Z-Arg-Arg-NMec suggesting that their specificities are more like papain or cathepsin L rather than cathepsin B. In gelatin-substrate SDS-PAGE, DTT was required to detect proteolysis by the ES preparation which was optimal at pH 6.0 and resolved into eight bands (87-29 kDa). Cysteine proteinase inhibitors were the most effective in all assays. Collectively, these data indicate that cysteine-class proteolytic activity predominates in the ES preparation of adult S. vulgaris.

  6. Site-specific proteolytic degradation of IgG monoclonal antibodies expressed in tobacco plants.

    Science.gov (United States)

    Hehle, Verena K; Lombardi, Raffaele; van Dolleweerd, Craig J; Paul, Mathew J; Di Micco, Patrizio; Morea, Veronica; Benvenuto, Eugenio; Donini, Marcello; Ma, Julian K-C

    2015-02-01

    Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high-yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti-HIV mAb 2G12 and a tumour-targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the VL /CL interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant-produced mAbs and raise the possibility of predicting antibody degradation patterns 'a priori' and designing novel stabilization strategies by site-specific mutagenesis. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  7. 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.

  8. Elastinolytic and proteolytic enzymes.

    Science.gov (United States)

    Kessler, Efrat; Safrin, Mary

    2014-01-01

    Pseudomonas aeruginosa secretes into its environment at least seven extracellular proteases: pseudolysin (LasB protease; elastase), aeruginolysin (alkaline proteinase), staphylolysin (staphylolytic endopeptidase; LasA protease), lysyl endopeptidase (protease IV; PrpL), PASP (P. aeruginosa small protease), LepA (Large ExoProtease A), and an aminopeptidase. Their action on host proteins, both individually and synergistically, plays important roles in pathogenesis of P. aeruginosa infections. Methods to measure/detect their activities are fundamental for understanding their physiological functions, roles in pathogenesis, mechanisms of action, regulation, and secretion. Most assays for determination/detection of proteolytic activity employ modified/non-modified casein or gelatin as substrates. In the quantitative assay, fragments generated from azocasein are separated from undigested substrate by trichloroacetic acid precipitation and their absorbance is measured. In non-quantitative assays, proteolytic activity is detected as clearing zones around bacterial growth or samples of culture supernatants on casein containing solid media formed due to local casein degradation. In zymography, individual proteases are detected as clear bands in gelatin/casein containing gels after SDS-PAGE separation, renaturation and protein staining. The elastinolytic capacity of P. aeruginosa is reflected by clearing zones on nutrient agar plates containing insoluble elastin instead of casein. Mueller-Hinton agar plates on which S. aureus cells are grown as a lawn are used to assess the susceptibility of S. aureus isolates to staphylolysin. A clear zone around a staphylolysin-containing sample indicates inhibition of S. aureus growth. Methods for measuring the activity of individual proteases are based on their cleavage specificity. These include assays of elastinolytic activity of pseudolysin and/or staphylolysin using elastin-Congo red as a substrate, a method for determination of

  9. MODELS FOR MOUSE CHIMERA PRODUCTION: AGGREGATION OF ES CELLS WITH CLEAVAGE STAGE EMBRYOS

    Directory of Open Access Journals (Sweden)

    STANCA CLAUDIA

    2007-01-01

    Full Text Available In a mutant ES cells↔ wild-type embryo chimera, ES cells behave more like epiblastcells. They can contribute to the primitive ectoderm layers, which give rise to all theembryonic tissues and some extraembryonic tissues (Beddington and Robertson,1989, but not to trophectoderm or primitive endoderm. Using transgenic ES celllines, aggregated with cleavage stage host embryo, ES cells can integrate randomlyin the embryo proper. If they will be take part in the formation of ICM (inner cellmass, it will be possible to obtain germline chimera animals. To generate ES cells↔ cleavage stage host embryo chimeras, we used (CD-1 mice as donors of hostembryos as well as recipients of manipulated embryos. For chimera production, weused fluorescent-labeled ES cell line (CD1/EGFP, because in this case we canfollow the fate of ES cells during the embryonic development. We produced thechimers using “aggregation chimera technique”. 8 cells stage zona pellucida free,mouse embryos were aggregated in an aggregation plates, with a clump of ES cells(10 – 15 cells. The chimera embryos were cultivated for 24 hours in the incubator(at 37 °C, 5% CO2 in air. The chimera blastocysts resulted after cultivation, weretransferred to the uterus of the 2.5-dpc pseudo pregnant females.

  10. MODELS FOR MOUSE CHIMERA PRODUCTION: AGGREGATION OF ES CELLS WITH CLEAVAGE STAGE EMBRYOS

    Directory of Open Access Journals (Sweden)

    CLAUDIA STANCA

    2007-05-01

    Full Text Available In a mutant ES cells↔ wild-type embryo chimera, ES cells behave more like epiblastcells. They can contribute to the primitive ectoderm layers, which give rise to all theembryonic tissues and some extraembryonic tissues (Beddington and Robertson,1989, but not to trophectoderm or primitive endoderm. Using transgenic ES celllines, aggregated with cleavage stage host embryo, ES cells can integrate randomlyin the embryo proper. If they will be take part in the formation of ICM (inner cellmass, it will be possible to obtain germline chimera animals. To generate ES cells↔ cleavage stage host embryo chimeras, we used (CD-1 mice as donors of hostembryos as well as recipients of manipulated embryos. For chimera production, weused fluorescent-labeled ES cell line (CD1/EGFP, because in this case we canfollow the fate of ES cells during the embryonic development. We produced thechimers using “aggregation chimera technique”. 8 cells stage zona pellucida free,mouse embryos were aggregated in an aggregation plates, with a clump of ES cells(10 – 15 cells. The chimera embryos were cultivated for 24 hours in the incubator(at 37 °C, 5% CO2 in air. The chimera blastocysts resulted after cultivation, weretransferred to the uterus of the 2.5-dpc pseudo pregnant females.

  11. Evolutionarily distinct carotenoid cleavage dioxygenases are responsible for crocetin production in Buddleja davidii.

    Science.gov (United States)

    Ahrazem, Oussama; Diretto, Gianfranco; Argandoña, Javier; Rubio-Moraga, Ángela; Julve, José Manuel; Orzáez, Diego; Granell, Antonio; Gómez-Gómez, Lourdes

    2017-07-20

    Crocetin, one of the few colored apocarotenoids known in nature, is present in flowers and fruits and has long been used medicinally and as a colorant. Saffron is the main source of crocetin, although a few other plants produce lower amounts of this apocarotenoid. Notably, Buddleja davidii accumulates crocetin in its flowers. Recently, a carotenoid dioxygenase cleavage enzyme, CCD2, has been characterized as responsible for crocetin production in Crocus species. We searched for CCD2 homologues in B. davidii and identified several CCD enzymes from the CCD1 and CCD4 subfamilies. Unexpectedly, two out of the three CCD4 enzymes, namely BdCCD4.1 and BdCCD4.3, showed 7,8;7',8' activity in vitro and in vivo over zeaxanthin. In silico analyses of these enzymes and CCD2 allowed the determination of key residues for this activity. Both BdCCD4 genes are highly expressed during flower development and transcripts levels parallel the accumulation of crocins in the petals. Phylogenetic analysis showed that BdCCD4.2 grouped with almost all the characterized CCD4 enzymes, while BdCCD4.1 and BdCCD4.3 form a new sub-cluster together with CCD4 enzymes from certain Lamiales species. The present study indicates that convergent evolution led to the acquisition of 7,8;7',8' apocarotenoid cleavage activity in two separate CCD enzyme families. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  12. Proteolytic enzymes of lactic acid bacteria

    NARCIS (Netherlands)

    Law, J; Haandrikman, A

    The proteolytic system of lactic acid bacteria is essential for their growth in milk and contributes significantly to flavour development in fermented milk products where these microorganisms are used as starter cultures. The proteolytic system is composed of proteinases which initially cleave the

  13. Capillary gas chromatographic analysis of mycolic acid cleavage products, cellular fatty acids, and alcohols of Mycobacterium xenopi.

    OpenAIRE

    Luquin, M; Lopez, F; Ausina, V

    1989-01-01

    The fatty acids, alcohols, and mycolic acids of 26 strains of Mycobacterium xenopi were studied by capillary gas chromatography and thin-layer chromatography. All strains contained alpha-, keto-, and omega-carboxymycolates. The primary mycolic acid cleavage product was hexacosanoic acid. The fatty acid patterns and, especially, the presence of 2-docosanol are characteristic markers of M. xenopi.

  14. Polycystin-1 Cleavage and the Regulation of Transcriptional Pathways

    Science.gov (United States)

    Merrick, David; Bertuccio, Claudia A.; Chapin, Hannah C.; Lal, Mark; Chauvet, Veronique; Caplan, Michael J.

    2013-01-01

    Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end stage renal disease, affecting ~1 in 1,000 people. The disease is characterized by the development of numerous large fluid filled renal cysts over the course of decades. These cysts compress the surrounding renal parenchyma and impair its function. Mutations in two genes are responsible for ADPKD. The protein products of both of these genes, polycystin-1 and polycystin-2, localize to the primary cilium and participate in a wide variety of signaling pathways. Polycystin-1 undergoes several proteolytic cleavages that produce fragments that manifest biological activities. Recent results suggest that the production of polycystin-1 cleavage fragments is necessary and sufficient to account for at least some, although certainly not all, of the physiological functions of the parent protein. PMID:23824180

  15. The epithelial sodium channel γ-subunit is processed proteolytically in human kidney

    DEFF Research Database (Denmark)

    Langkilde, Rikke Zachar; Skjødt, Karsten; Marcussen, Niels

    2015-01-01

    The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the gamma-subunit and putative release of a 43-amino acid inhibitory tract from the gamma-subunit ectodomain. We......ENaC was detected consistently only in tissue from patients with proteinuria and observed in collecting ducts. In conclusion, human kidney gammaENaC is subject to proteolytic cleavage, yielding fragments compatible with furin cleavage, and proteinuria is associated with cleavage at the putative prostasin...

  16. A comparative immunogenicity study in rabbits of disulfide-stabilized, proteolytically cleaved, soluble trimeric human immunodeficiency virus type 1 gp140, trimeric cleavage-defective gp140 and monomeric gp120

    International Nuclear Information System (INIS)

    Beddows, Simon; Franti, Michael; Dey, Antu K.; Kirschner, Marc; Iyer, Sai Prasad N.; Fisch, Danielle C.; Ketas, Thomas; Yuste, Eloisa; Desrosiers, Ronald C.; Klasse, Per Johan; Maddon, Paul J.; Olson, William C.; Moore, John P.

    2007-01-01

    The human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein (Env) complex, a homotrimer containing gp120 surface glycoprotein and gp41 transmembrane glycoprotein subunits, mediates the binding and fusion of the virus with susceptible target cells. The Env complex is the target for neutralizing antibodies (NAbs) and is the basis for vaccines intended to induce NAbs. Early generation vaccines based on monomeric gp120 subunits did not confer protection from infection; one alternative approach is therefore to make and evaluate soluble forms of the trimeric Env complex. We have directly compared the immunogenicity in rabbits of two forms of soluble trimeric Env and monomeric gp120 based on the sequence of HIV-1 JR-FL . Both protein-only and DNA-prime, protein-boost immunization formats were evaluated, DNA-priming having little or no influence on the outcome. One form of trimeric Env was made by disrupting the gp120-gp41 cleavage site by mutagenesis (gp140 UNC ), the other contains an intramolecular disulfide bond to stabilize the cleaved gp120 and gp41 moieties (SOSIP.R6 gp140). Among the three immunogens, SOSIP.R6 gp140 most frequently elicited neutralizing antibodies against the homologous, neutralization-resistant strain, HIV-1 JR-FL . All three proteins induced NAbs against more sensitive strains, but the breadth of activity against heterologous primary isolates was limited. When antibodies able to neutralize HIV-1 JR-FL were detected, antigen depletion studies showed they were not directed at the V3 region but were targeted at other, undefined gp120 and also non-gp120 epitopes

  17. Endogenous proteolytic enzymes--a study of their impact on cod (Gadus morhua) muscle proteins and textural properties in a fermented product.

    Science.gov (United States)

    Yang, Fang; Rustad, Turid; Xu, Yanshun; Jiang, Qixing; Xia, Wenshui

    2015-04-01

    The aim of this study was to investigate endogenous proteolytic activities in a cod product and their impact on muscle proteins and textural properties during fermentation and storage. The result of specific proteolytic activities showed that cathepsins, especially cathepsin B, had the highest activities during fermentation and storage. SDS-PAGE indicated more degradation of myofibrillar proteins by cathepsin L than other proteases and that the hydrolysis by cathepsins was pronounced in the last stage of fermentation. Texture analysis showed that cathepsins had a negative impact on gel strength and this impact increased in the last stage of fermentation. However the product still had a firm texture. During storage (4 °C) for one week, no significant changes were seen in the gel strength. In conclusion, cathepsins had more impact on muscle proteins and textural properties than other proteases during fermentation but had little impact on gel strength during storage at 4 °C. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Production of barley endoprotease B2 in Pichia pastoris and its proteolytic activity against native and recombinant hordeins

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach

    2014-01-01

    -terminal truncated version (HvEPB2ΔC) and a proteolytic resistant His6 tag. Maximum yield was obtained after 4 days of induction. Recombinant HvEPB2ΔC (r-HvEPB2ΔC) was purified using a single step of Ni2+-affinity chromatography. Purified protein was evaluated by SDS–PAGE, Western blotting and activity assays...... was 60 °C, thermal stability T50 value was 44 °C and the pH optimum was 4.5. r-HvEPB2ΔC was incubated with native purified barley seed storage proteins for up to 48 h. After 12 h, r-HvEPB2ΔC efficiently reduced the C and D hordeins almost completely, as evaluated by SDS–PAGE. The intensities of the B...... and γ hordein bands decreased continuously over the 48 h. No degradation occurred in the presence of E64. Recombinant hordeins (B1, B3 and γ1) were expressed in Escherichia coli. After 2 h of incubation with r-HvEPB2ΔC, an almost complete degradation of γ1 and partial digests of hordein B1 and B3 were...

  19. The Progranulin Cleavage Products, Granulins, Exacerbate TDP-43 Toxicity and Increase TDP-43 Levels.

    Science.gov (United States)

    Salazar, Dominique A; Butler, Victoria J; Argouarch, Andrea R; Hsu, Tsung-Yuan; Mason, Amanda; Nakamura, Ayumi; McCurdy, Helen; Cox, David; Ng, Rachel; Pan, Gloria; Seeley, William W; Miller, Bruce L; Kao, Aimee W

    2015-06-24

    Mutations in the human progranulin gene resulting in protein haploinsufficiency cause frontotemporal lobar degeneration with TDP-43 inclusions. Although progress has been made in understanding the normal functions of progranulin and TDP-43, the molecular interactions between these proteins remain unclear. Progranulin is proteolytically processed into granulins, but the role of granulins in the pathogenesis of neurodegenerative disease is unknown. We used a Caenorhabditis elegans model of neuronal TDP-43 proteinopathy to specifically interrogate the contribution of granulins to the neurodegenerative process. Complete loss of the progranulin gene did not worsen TDP-43 toxicity, whereas progranulin heterozygosity did. Interestingly, expression of individual granulins alone had little effect on behavior. In contrast, when granulins were coexpressed with TDP-43, they exacerbated its toxicity in a variety of behaviors including motor coordination. These same granulins increased TDP-43 levels via a post-translational mechanism. We further found that in human neurodegenerative disease subjects, granulin fragments accumulated specifically in diseased regions of brain. To our knowledge, this is the first demonstration of a toxic role for granulin fragments in a neurodegenerative disease model. These studies suggest that presence of cleaved granulins, rather than or in addition to loss of full-length progranulin, may contribute to disease in TDP-43 proteinopathies. Copyright © 2015 the authors 0270-6474/15/359315-14$15.00/0.

  20. Effect of Equilibrated pH and Indigenous Spoilage Microorganisms on the Inhibition of Proteolytic Clostridium botulinum Toxin Production in Experimental Meals under Temperature Abuse.

    Science.gov (United States)

    Golden, Max C; Wanless, Brandon J; David, Jairus R D; Lineback, D Scott; Talley, Ryan J; Kottapalli, Bala; Glass, Kathleen A

    2017-08-01

    Clostridium botulinum is a foreseeable biological hazard in prepared refrigerated meals that needs to be addressed in food safety plans. The objective of this study was to evaluate the effect of product composition and storage temperature on the inhibition of botulinum toxin formation in nine experimental meals (meat, vegetable, or carbohydrate based). Treatments were inoculated with proteolytic C. botulinum, vacuum packaged, cooked at 90°C for 10 min, and assayed for botulinum toxin in samples stored at 25°C for up to 96 h for phase 1, or at 25°C for 12 h and then transferred to 12.5°C for up to 12 and 6 weeks in phases 1 and 2, respectively. For phase 1, none of the treatments (equilibrated pH 5.8) supported toxin production when stored at 25°C for 48 h, but toxin production was observed in all treatments at 72 h. For the remaining experiments with storage at 12.5°C, toxin production was dependent on equilibrated pH, storage time, and growth of indigenous spoilage microorganisms. In phase 1, no gross spoilage and no botulinum toxin was detected for any treatment (pH ≤5.8) stored at 12.5°C for 12 weeks. In phase 2, gross spoilage varied by commodity, with the brussels sprouts meal with pH 6.5 showing the most rapid spoilage within 2 weeks and botulinum toxin detected at 5 and 6 weeks for the control and cultured celery juice treatments, respectively. In contrast, spoilage microbes decreased the pH of a pH 5.9 beef treatment by 1.0 unit, potentially inhibiting C. botulinum through 6 weeks at 12.5°C. None of the other treatments with pH 5.8 or below supported toxin production or spoilage. This study provides validation for preventive controls in refrigerated meals. These include equilibrated product pH and storage temperature and time to inhibit toxin formation by proteolytic C. botulinum, but the impact of indigenous microflora on safety and interpretation of challenge studies is also highlighted.

  1. A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L

    International Nuclear Information System (INIS)

    Pager, Cara Theresia; Craft, Willie Warren; Patch, Jared; Dutch, Rebecca Ellis

    2006-01-01

    The Nipah virus fusion (F) protein is proteolytically processed to F 1 + F 2 subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsins can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form

  2. SKI2 mediates degradation of RISC 5′-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis

    Science.gov (United States)

    Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

    2015-01-01

    Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20–24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5′, but not 3′-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5′ to the cleavage site, but several examples of 3′-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5′-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5′-cleavage fragments. PMID:26464441

  3. SKI2 mediates degradation of RISC 5'-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis.

    Science.gov (United States)

    Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

    2015-12-15

    Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20-24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5', but not 3'-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5' to the cleavage site, but several examples of 3'-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5'-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5'-cleavage fragments. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Hydrogen production through microheterogeneous photocatalysis of hydrogen sulfide cleavage. The thiosulfate cycle

    Energy Technology Data Exchange (ETDEWEB)

    Borgarello, E; Serpone, N; Graetzel, M; Pelizzetti, E

    1985-01-01

    Cleavage of hydrogen sulfide into hydrogen and sulfur occurs in alkaline aqueous CdS dispersions under visible light illumination. Small quantities of a noble metal catalyst (RuO/sub 2/) loaded onto 'naked' CdS particles markedly improve the yield of hydrogen formation. The effect of RuO/sub 2/ is ascribed to catalysis of electron transfer to proton. Simultaneous and efficient photogeneration of hydrogen and thiosulfate occurs in CdS dispersions containing both sulfite and bisulfide (or sulfide) ions. Electron transfer from the conduction band of CdS to that of TiO/sub 2/ particles occurs in alkaline suspensions containing these HS/sup -/ ions and has been exploited to improve the performance of a system achieving decomposition of H/sub 2/S by visible light. Equally important is a recent finding that the performance of a system containing 'naked' CdS in combination with RuO/sub 2/-loaded TiO/sub 2/ particles is far better than that of CdS/RuO/sub 2/ alone. Additionally, conduction band electrons produced by bandgap excitation of TiO/sub 2/ particles efficiently reduce thiosulfate to sulfide and sulfite. The valence band process in alklaine TiO/sub 2/ dispersions is thought to involve oxidation of S/sub 2/O/sub 3//sup 2 -/ to tetrathionate, S/sub 4/O/sub 6//sup 2 -/, which quantitatively dismutates into sulfite and thiosulfate. The photodriven disproportionation of thiosulfate into sulfide and sulfate is of great interest in systems that photochemically cleave hydrogen sulfide into hydrogen and sulfur. (author).

  5. Influence of the mycelium growth conditions on the production of amylolytic, proteolytic and pectinolytic enzymes by Aspergillus niger C

    Energy Technology Data Exchange (ETDEWEB)

    Fiedurek, J.; Ilczuk, Z.; Lobarzewski, J. (Uniwersytet Marii Curie-Sklodowskiej, Lublin (Poland). Inst. Mikrobiologii i Biochemii)

    1989-01-01

    Various nitrogen and carbon sources, as well as natural products, were examined as inducers of the production of amylases, proteases and pectinases by A. niger C. A. niger C grown on wheat bran extract medium provided culture supernatants with the highest enzymatic activities. Some culture conditions, e.g. pH, medium temperature and time period of cultivation, were optimalized to improve the growth and enzyme biosynthesis by A. niger C. (orig.).

  6. Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.

    Directory of Open Access Journals (Sweden)

    Artur Kaul

    2009-08-01

    Full Text Available Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV, a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence

  7. Essential role of cyclophilin A for hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics.

    Science.gov (United States)

    Kaul, Artur; Stauffer, Sarah; Berger, Carola; Pertel, Thomas; Schmitt, Jennifer; Kallis, Stephanie; Zayas, Margarita; Lopez, Margarita Zayas; Lohmann, Volker; Luban, Jeremy; Bartenschlager, Ralf

    2009-08-01

    Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV

  8. Seneca Valley Virus Suppresses Host Type I Interferon Production by Targeting Adaptor Proteins MAVS, TRIF, and TANK for Cleavage.

    Science.gov (United States)

    Qian, Suhong; Fan, Wenchun; Liu, Tingting; Wu, Mengge; Zhang, Huawei; Cui, Xiaofang; Zhou, Yun; Hu, Junjie; Wei, Shaozhong; Chen, Huanchun; Li, Xiangmin; Qian, Ping

    2017-08-15

    Seneca Valley virus (SVV) is an oncolytic RNA virus belonging to the Picornaviridae family. Its nucleotide sequence is highly similar to those of members of the Cardiovirus genus. SVV is also a neuroendocrine cancer-selective oncolytic picornavirus that can be used for anticancer therapy. However, the interaction between SVV and its host is yet to be fully characterized. In this study, SVV inhibited antiviral type I interferon (IFN) responses by targeting different host adaptors, including mitochondrial antiviral signaling (MAVS), Toll/interleukin 1 (IL-1) receptor domain-containing adaptor inducing IFN-β (TRIF), and TRAF family member-associated NF-κB activator (TANK), via viral 3C protease (3C pro ). SVV 3C pro mediated the cleavage of MAVS, TRIF, and TANK at specific sites, which required its protease activity. The cleaved MAVS, TRIF, and TANK lost the ability to regulate pattern recognition receptor (PRR)-mediated IFN production. The cleavage of TANK also facilitated TRAF6-induced NF-κB activation. SVV was also found to be sensitive to IFN-β. Therefore, SVV suppressed antiviral IFN production to escape host antiviral innate immune responses by cleaving host adaptor molecules. IMPORTANCE Host cells have developed various defenses against microbial pathogen infection. The production of IFN is the first line of defense against microbial infection. However, viruses have evolved many strategies to disrupt this host defense. SVV, a member of the Picornavirus genus, is an oncolytic virus that shows potential functions in anticancer therapy. It has been demonstrated that IFN can be used in anticancer therapy for certain tumors. However, the relationship between oncolytic virus and innate immune response in anticancer therapy is still not well known. In this study, we showed that SVV has evolved as an effective mechanism to inhibit host type I IFN production by using its 3C pro to cleave the molecules MAVS, TRIF, and TANK directly. These molecules are crucial for

  9. Aerobic biotransformation of 3-methylindole to ring cleavage products by Cupriavidus sp. strain KK10.

    Science.gov (United States)

    Fukuoka, Kimiko; Ozeki, Yasuhiro; Kanaly, Robert A

    2015-09-01

    3-Methylindole, also referred to as skatole, is a pollutant of environmental concern due to its persistence, mobility and potential health impacts. Petroleum refining, intensive livestock production and application of biosolids to agricultural lands result in releases of 3-methylindole to the environment. Even so, little is known about the aerobic biodegradation of 3-methylindole and comprehensive biotransformation pathways have not been established. Using glycerol as feedstock, the soil bacterium Cupriavidus sp. strain KK10 biodegraded 100 mg/L of 3-methylindole in 24 h. Cometabolic 3-methylindole biodegradation was confirmed by the identification of biotransformation products through liquid chromatography electrospray ionization tandem mass spectrometry analyses. In all, 14 3-methylindole biotransformation products were identified which revealed that biotransformation occurred through different pathways that included carbocyclic aromatic ring-fission of 3-methylindole to single-ring pyrrole carboxylic acids. This work provides first comprehensive evidence for the aerobic biotransformation mechanisms of 3-methylindole by a soil bacterium and expands our understanding of the biodegradative capabilities of members of the genus Cupriavidus towards heteroaromatic pollutants.

  10. The Ups and Downs of Repeated Cleavage and Internal Fragment Production in Top-Down Proteomics

    Science.gov (United States)

    Lyon, Yana A.; Riggs, Dylan; Fornelli, Luca; Compton, Philip D.; Julian, Ryan R.

    2018-01-01

    Analysis of whole proteins by mass spectrometry, or top-down proteomics, has several advantages over methods relying on proteolysis. For example, proteoforms can be unambiguously identified and examined. However, from a gas-phase ion-chemistry perspective, proteins are enormous molecules that present novel challenges relative to peptide analysis. Herein, the statistics of cleaving the peptide backbone multiple times are examined to evaluate the inherent propensity for generating internal versus terminal ions. The raw statistics reveal an inherent bias favoring production of terminal ions, which holds true regardless of protein size. Importantly, even if the full suite of internal ions is generated by statistical dissociation, terminal ions are predicted to account for at least 50% of the total ion current, regardless of protein size, if there are three backbone dissociations or fewer. Top-down analysis should therefore be a viable approach for examining proteins of significant size. Comparison of the purely statistical analysis with actual top-down data derived from ultraviolet photodissociation (UVPD) and higher-energy collisional dissociation (HCD) reveals that terminal ions account for much of the total ion current in both experiments. Terminal ion production is more favored in UVPD relative to HCD, which is likely due to differences in the mechanisms controlling fragmentation. Importantly, internal ions are not found to dominate from either the theoretical or experimental point of view. [Figure not available: see fulltext.

  11. Petunia hybrida CAROTENOID CLEAVAGE DIOXYGENASE7 is involved in the production of negative and positive branching signals in petunia.

    Science.gov (United States)

    Drummond, Revel S M; Martínez-Sánchez, N Marcela; Janssen, Bart J; Templeton, Kerry R; Simons, Joanne L; Quinn, Brian D; Karunairetnam, Sakuntala; Snowden, Kimberley C

    2009-12-01

    One of the key factors that defines plant form is the regulation of when and where branches develop. The diversity of form observed in nature results, in part, from variation in the regulation of branching between species. Two CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes, CCD7 and CCD8, are required for the production of a branch-suppressing plant hormone. Here, we report that the decreased apical dominance3 (dad3) mutant of petunia (Petunia hybrida) results from the mutation of the PhCCD7 gene and has a less severe branching phenotype than mutation of PhCCD8 (dad1). An analysis of the expression of this gene in wild-type, mutant, and grafted petunia suggests that in petunia, CCD7 and CCD8 are coordinately regulated. In contrast to observations in Arabidopsis (Arabidopsis thaliana), ccd7ccd8 double mutants in petunia show an additive phenotype. An analysis using dad3 or dad1 mutant scions grafted to wild-type rootstocks showed that when these plants produce adventitious mutant roots, branching is increased above that seen in plants where the mutant roots are removed. The results presented here indicate that mutation of either CCD7 or CCD8 in petunia results in both the loss of an inhibitor of branching and an increase in a promoter of branching.

  12. Proteolytic activity of alkaliphilic, salt-tolerant actinomycetes from ...

    African Journals Online (AJOL)

    tolerant alkaliphilic. All the isolates need to be further studied for the ability of their potential protease enzyme production. Key words: Alkaliphilic actinomycetes, salt tolerant actinomycetes, desert soil, isolation, proteolytic activity.

  13. Bicarbonate-dependent secretion and proteolytic processing of recombinant myocilin.

    Directory of Open Access Journals (Sweden)

    José-Daniel Aroca-Aguilar

    Full Text Available Myocilin is an extracellular glycoprotein of poorly understood function. Mutations of this protein are involved in glaucoma, an optic neuropathy characterized by a progressive and irreversible visual loss and frequently associated with elevated intraocular pressure. We previously showed that recombinant myocilin undergoes an intracellular proteolytic processing by calpain II which cleaves the central region of the protein, releasing one N- and one C-terminal fragment. Myocilin cleavage is reduced by glaucoma mutations and it has been proposed to participate in intraocular pressure modulation. To identify possible factors regulating the proteolytic processing of recombinant myocilin, we used a cellular model in which we analyzed how different culture medium parameters (i.e., culture time, cell density, pH, bicarbonate concentration, etc. affect the presence of the extracellular C-terminal fragment. Extracellular bicarbonate depletion associated with culture medium acidification produced a reversible intracellular accumulation of full-length recombinant myocilin and incremented its intracellular proteolytic processing, raising the extracellular C-terminal fragment percentage. It was also determined that myocilin intracellular accumulation depends on its N-terminal region. These data suggest that aqueous humor bicarbonate variations could also modulate the secretion and cleavage of myocilin present in ocular tissues.

  14. Alzheimer's disease against peptides products of enzymatic cleavage APP protein: Biological, pathobiological and physico-chemical properties of fibrillating peptides.

    Science.gov (United States)

    Marszałek, Małgorzata

    2017-05-17

    Various peptides products of enzymatic cleavage of key for Alzheimer's disease Amyloid Precursor Protein (APP) are well known, but still are matter of scientific debate. The Aβ type products are especially challenging for experimental and medical research. This paper outlines several, still poorly known, biological and medical processes such as peptides biology, i.e., formation, biodistribution, translocation, transport and finally removal from brain compartments and body fluids like Intracellular Fluid (ICF), Cerebrospinal Fluid (CSF), Interstitial Fluid (ISF), blood serum or urine. In addition, the following studies concerning AD patients might prove challenging and simultaneously promising: peptides translocation through Blood-Brain - Barrier (BBB) and Blood-Cerebrospinal Fluid Barrier (BCSFB) and their removal from the brain according to a new concept of glymphatic system; - diagnostic difficulties that stem from physico-chemical properties and the nature of proteins or fibrillating peptides itself like low concentration, short half-live and from experimental-technical problems as well like high adsorption or low solubility of Aβ, tau or amylin. The study of diagnostic parameters is very important, as it may better reflect early changes before the disease develops; one such parameter is the Aβ42/Aβ40 ratio, or the ratio with the total tau concentration combination and other new biomarkers like Aβ1-38; other factors include oxidative stress and inflammation process proteins, complement factor H, alpha-2-macroglobulin, or clusterin. The study of various forms of pathological amyloid deposits that emerge in different but specific brain regions AD patients seems to be crucial as well. The composition of the first initial pathological, pre-fibrillating monomers of fibrillating peptides and their role in AD development and disease progression have been described as well. They are even more challenging for science and simultaneously might be more promising in

  15. Soybean P34 Probable Thiol Protease Probably Has Proteolytic Activity on Oleosins.

    Science.gov (United States)

    Zhao, Luping; Kong, Xiangzhen; Zhang, Caimeng; Hua, Yufei; Chen, Yeming

    2017-07-19

    P34 probable thiol protease (P34) and Gly m Bd 30K (30K) show high relationship with the protease of 24 kDa oleosin of soybean oil bodies. In this study, 9 day germinated soybean was used to separate bioprocessed P34 (P32) from bioprocessed 30K (28K). Interestingly, P32 existed as dimer, whereas 28K existed as monomer; a P32-rich sample had proteolytic activity and high cleavage site specificity (Lys-Thr of 24 kDa oleosin), whereas a 28K-rich sample showed low proteolytic activity; the P32-rich sample contained one thiol protease. After mixing with purified oil bodies, all P32 dimers were dissociated and bound to 24 kDa oleosins to form P32-24 kDa oleosin complexes. By incubation, 24 kDa oleosin was preferentially hydrolyzed, and two hydrolyzed products (HPs; 17 and 7 kDa) were confirmed. After most of 24 kDa oleosin was hydrolyzed, some P32 existed as dimer, and the other as P32-17 kDa HP. It was suggested that P32 was the protease.

  16. Calsyntenin-1 shelters APP from proteolytic processing during anterograde axonal transport

    Directory of Open Access Journals (Sweden)

    Martin Steuble

    2012-06-01

    Endocytosis of amyloid-β precursor protein (APP is thought to represent the major source of substrate for the production of the amyloidogenic Aβ peptide by the β-secretase BACE1. The irreversible nature of proteolytic cleavage implies the existence of an efficient replenishment route for APP from its sites of synthesis to the cell surface. We recently found that APP exits the trans-Golgi network in intimate association with calsyntenin-1, a transmembrane cargo-docking protein for Kinesin-1-mediated vesicular transport. Here we characterized the function of calsyntenin-1 in neuronal APP transport using selective immunoisolation of intracellular trafficking organelles, immunocytochemistry, live-imaging, and RNAi. We found that APP is co-transported with calsyntenin-1 along axons to early endosomes in the central region of growth cones in carriers that exclude the α-secretase ADAM10. Intriguingly, calsyntenin-1/APP organelles contained BACE1, suggesting premature cleavage of APP along its anterograde path. However, we found that APP contained in calsyntenin-1/APP organelles was stable. We further analyzed vesicular trafficking of APP in cultured hippocampal neurons, in which calsyntenin-1 was reduced by RNAi. We found a markedly increased co-localization of APP and ADAM10 in axons and growth cones, along with increased proteolytic processing of APP and Aβ secretion in these neurons. This suggested that the reduced capacity for calsyntenin-1-dependent APP transport resulted in mis-sorting of APP into additional axonal carriers and, therefore, the premature encounter of unprotected APP with its ectodomain proteases. In combination, our results characterize calsyntenin-1/APP organelles as carriers for sheltered anterograde axonal transport of APP.

  17. Proteolytic processing of the vitellogenin precursor in the boll weevil, Anthonomus grandis.

    Science.gov (United States)

    Heilmann, L J; Trewitt, P M; Kumaran, A K

    1993-01-01

    The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M(r)s of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M(r) vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M(r) honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M(r) boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10-15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein.

  18. Research Applications of Proteolytic Enzymes in Molecular Biology

    Directory of Open Access Journals (Sweden)

    József Tőzsér

    2013-11-01

    Full Text Available Proteolytic enzymes (also termed peptidases, proteases and proteinases are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications require their use, including production of Klenow fragments, peptide synthesis, digestion of unwanted proteins during nucleic acid purification, cell culturing and tissue dissociation, preparation of recombinant antibody fragments for research, diagnostics and therapy, exploration of the structure-function relationships by structural studies, removal of affinity tags from fusion proteins in recombinant protein techniques, peptide sequencing and proteolytic digestion of proteins in proteomics. The aim of this paper is to review the molecular biological aspects of proteolytic enzymes and summarize their applications in the life sciences.

  19. Incorporation of 12-methoxydodecanoate into the human immunodeficiency virus 1 gag polyprotein precursor inhibits its proteolytic processing and virus production in a chronically infected human lymphoid cell line.

    OpenAIRE

    Bryant, M L; Ratner, L; Duronio, R J; Kishore, N S; Devadas, B; Adams, S P; Gordon, J I

    1991-01-01

    Covalent linkage of myristate (tetradecanoate; 14:0) to the NH2-terminal glycine residue of the human immunodeficiency virus 1 (HIV-1) 55-kDa gag polyprotein precursor (Pr55gag) is necessary for its proteolytic processing and viral assembly. We have shown recently that several analogs of myristate in which a methylene group is replaced by a single oxygen or sulfur atom are substrates for Saccharomyces cerevisiae and mammalian myristoyl-CoA:protein N-myristoyltransferase (EC 2.3.1.97; NMT) des...

  20. Kallistatin Ameliorates Influenza Virus Pathogenesis by Inhibition of Kallikrein-Related Peptidase 1-Mediated Cleavage of Viral Hemagglutinin

    Science.gov (United States)

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

    2015-01-01

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

  1. Poly(ADP-ribose) polymerase-1 and its cleavage products differentially modulate cellular protection through NF-kB-dependent signaling

    Science.gov (United States)

    Castri, Paola; Lee, Yang-ja; Ponzio, Todd; Maric, Dragan; Spatz, Maria; Bembry, Joliet; Hallenbeck, John

    2014-01-01

    Poly(ADP-ribose) polymerase-1 (PARP-1) and its cleavage products regulate cell viability and NF-kB activity when expressed in neurons. PARP-1 cleavage generates a 24kDa (PARP-124) and an 89kDa fragment (PARP-189). Compared to WT (PARP-1WT), the expression of an uncleavable PARP-1 (PARP-1UNCL) or of PARP-124 conferred protection from oxygen/glucose deprivation (OGD) or OGD/restoration of oxygen and glucose (ROG) damage in vitro, whereas expression of PARP-189 was cytotoxic. Viability experiments were performed in SH-SY5Y, a human neuroblastoma cell line, as well as in rat primary cortical neurons. Following OGD, the higher viability in the presence of PARP-1UNCL or PARP-124 was not accompanied with decreased formation of poly(ADP-riboses) or higher NAD levels. PARP-1 is a known cofactor for NF-kB, hence we investigated whether PARP-1 cleavage influences the inflammatory response. All PARP-1 constructs mimicked PARP-1WT in regards to induction of NF-kB translocation into the nucleus and its increased activation during ischemic challenge. However, expression of PARP-189 construct induced significantly higher NF-kB activity than PARP-1WT; and the same was true for NF-kB-dependent iNOS promoter binding activity. At a protein level, PARP-1UNCL and PARP-124 decreased iNOS (and lower levels of iNOS transcript) and COX-2, and increased Bcl-xL. The increased levels of NF-kB and iNOS transcriptional activities, seen with cytotoxic PARP-189, were accompanied by higher protein expression of COX-2 and iNOS (and higher levels of iNOS transcript) and lower protein expression of Bcl-xL. Taken together, these findings suggest that PARP-1 cleavage products may regulate cellular viability and inflammatory responses in opposing ways during in vitro models of “ischemia”. PMID:24333653

  2. Analysis of proteolytic processes and enzymatic activities in the generation of huntingtin n-terminal fragments in an HEK293 cell model.

    Directory of Open Access Journals (Sweden)

    Andrew T N Tebbenkamp

    Full Text Available N-terminal fragments of mutant huntingtin (htt that terminate between residues 90-115, termed cleavage product A or 1 (cp-A/1, form intracellular and intranuclear inclusion bodies in the brains of patients with Huntington's disease (HD. These fragments appear to be proteolytic products of the full-length protein. Here, we use an HEK293 cell culture model to investigate huntingtin proteolytic processing; previous studies of these cells have demonstrated cleavage of htt to cp-A/1 like htt fragments.Recombinant N-terminal htt fragments, terminating at residue 171 (also referred to as cp-B/2 like, were efficiently cleaved to produce cp-A/1 whereas fragments representing endogenous caspase, calpain, and metalloproteinase cleavage products, terminating between residues 400-600, were inefficiently cleaved. Using cysteine-labeling techniques and antibody binding mapping, we localized the C-terminus of the cp-A/1 fragments produced by HEK293 cells to sequences minimally limited by cysteine 105 and an antibody epitope composed of residues 115-124. A combination of genetic and pharmacologic approaches to inhibit potential proteases, including γ-secretase and calpain, proved ineffective in preventing production of cp-A/1.Our findings indicate that HEK293 cells express a protease that is capable of efficiently cleaving cp-B/2 like fragments of htt with normal or expanded glutamine repeats. For reasons that remain unclear, this protease cleaves longer htt fragments, with normal or expanded glutamine expansions, much less efficiently. The protease in HEK293 cells that is capable of generating a cp-A/1 like htt fragment may be a novel protease with a high preference for a cp-B/2-like htt fragment as substrate.

  3. Toxin production of non-proteolytic Cl. botulinum type B in radurized fish. Part of a coordinated programme on the wholesomeness of the process of food irradiation

    International Nuclear Information System (INIS)

    Suhadi, F.

    1981-02-01

    Toxin formation by proteolytic and nonproteolytic strains of C. botulinum type B in radurized raw fish and in radurized Pindang fish was investigated. In radurized Pindang fish samples, inoculation was done either before or after cooking. Radurization process with 2 and 3 kGy caused the extension of storage life of Rastrelliger sp., Euthynnus sp., and Scomberomorus sp. by factors of 2 and 2.5 at storage temperatures between 5 and 10 0 C. In general at 10.5 +- 0.3 0 C, no toxin was formed before the samples were spoiled both in irradiated and unirradiated samples. At 5.6 +- 0.5 0 C no toxin was formed until after the samples were spoiled. The earliest toxin formation in unirradiated Pindang samples stored at ambient temperature was detected after the samples were spoiled. In irradiated Pindang samples inoculated with C. botulinum spores after cooking and stored at ambient temperature the toxin formation was detected before the samples were spoiled. However, if the inoculation was done before the fish was processed into Pindang, the toxin was always detected after the samples were spoiled regardless of the irradiation dose, strain and inoculation level. As fish may be contaminated - if at all - with spores of C. botulinum in its raw state, processing of fish into Pindang and irradiation would not contribute to the health hazard concerning botulism even if the samples are stored at ambient temperature

  4. Mechanisms for lymphocytic choriomeningitis virus glycoprotein cleavage, transport, and incorporation into virions

    International Nuclear Information System (INIS)

    Kunz, Stefan; Edelmann, Kurt H.; Torre, Juan-Carlos de la; Gorney, Robert; Oldstone, Michael B.A.

    2003-01-01

    The glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) serves as virus attachment protein to its receptor on host cells and is a key determinant for cell tropism, pathogenesis, and epidemiology of the virus. The GP of LCMV is posttranslationally cleaved by the subtilase SKI-1/S1P into two subunits, the peripheral GP1, which is implicated in receptor binding, and the transmembrane GP2 that is structurally similar to the fusion active membrane proximal portions of the glycoproteins of other enveloped viruses. The present study shows that cleavage by SKI-1/S1P is not required for cell surface expression of LCMVGP on infected cells but is essential for its incorporation into virions and for the production of infectious virus particles. In absence of SKI-1/S1P cleavage, cell-to-cell propagation of the virus was markedly reduced. Further, proteolytic processing of LCMVGP depends on the presence of a cluster of basic amino acids at the C-terminus of the cytoplasmic domain of GP2, a structural motif that is conserved in Old World arenaviruses. The effect of the truncation of the cytoplasmic tail on cleavage suggests a structural interdependence between the cytoplasmic domain and the ectodomains of LCMVGP

  5. Myc-nick: a cytoplasmic cleavage product of Myc that promotes alpha-tubulin acetylation and cell differentiation.

    Science.gov (United States)

    Conacci-Sorrell, Maralice; Ngouenet, Celine; Eisenman, Robert N

    2010-08-06

    The Myc oncoprotein family comprises transcription factors that control multiple cellular functions and are widely involved in oncogenesis. Here we report the identification of Myc-nick, a cytoplasmic form of Myc generated by calpain-dependent proteolysis at lysine 298 of full-length Myc. Myc-nick retains conserved Myc box regions but lacks nuclear localization signals and the bHLHZ domain essential for heterodimerization with Max and DNA binding. Myc-nick induces alpha-tubulin acetylation and altered cell morphology by recruiting histone acetyltransferase GCN5 to microtubules. During muscle differentiation, while the levels of full-length Myc diminish, Myc-nick and acetylated alpha-tubulin levels are increased. Ectopic expression of Myc-nick accelerates myoblast fusion, triggers the expression of myogenic markers, and permits Myc-deficient fibroblasts to transdifferentiate in response to MyoD. We propose that the cleavage of Myc by calpain abrogates the transcriptional inhibition of differentiation by full-length Myc and generates Myc-nick, a driver of cytoplasmic reorganization and differentiation. Copyright 2010 Elsevier Inc. All rights reserved.

  6. Efficient production of a bioactive Bevacizumab monoclonal antibody using the 2A self-cleavage peptide in transgenic rice callus

    Directory of Open Access Journals (Sweden)

    Lei Chen

    2016-08-01

    Full Text Available Bevacizumab, a humanized monoclonal antibody (mAb targeting to the vascular endothelial growth factor (VEGF, has been widely used in clinical practice for the treatment of multiple cancers. Bevacizumab was mostly produced by the mammalian cell expression system. We here reported the first plant-derived Bevacizumab by using transgenic rice callus as an alternative gene expression system. Codon-optimized Bevacizumab light chain (BLC and heavy chain (BHC genes were designed, synthesized as a polyprotein with a 2A self-cleavage linker peptide from the Foot-and-mouth disease virus (FMDV, cloned into a plant binary vector under a constitutive maize ubiquitin promoter, and transformed into rice nuclear genome through Agrobacterium-mediated transformation. Southern blot and western blot analyses confirmed the integration and expression of BLC and BHC genes in transgenic rice callus. Enzyme linked immunosorbent assay (ELISA analysis indicated that the rice-derived Bevacizumab mAb was biologically active and the recombinant mAb was expressed at high levels (160.7-242.8 mg kg-1FW in transgenic rice callus. The mAb was purified by using protein A affinity chromatography and the purified antibody was tested for its binding affinity with its target hVEGF antigen by ELISA. Rice callus produced Bevacizumab and a commercial Bevacizumab (Avastin were shown to have similar binding affinity to hVEGF. These results indicated that rice callus produced Bevacizumab could have similar biological activity and might potentially be used as a cost-effective biosimilar molecule in future cancer treatment.

  7. The multifaceted nature of amyloid precursor protein and its proteolytic fragments: friends and foes.

    Science.gov (United States)

    Nhan, Hoang S; Chiang, Karen; Koo, Edward H

    2015-01-01

    The amyloid precursor protein (APP) has occupied a central position in Alzheimer's disease (AD) pathophysiology, in large part due to the seminal role of amyloid-β peptide (Aβ), a proteolytic fragment derived from APP. Although the contribution of Aβ to AD pathogenesis is accepted by many in the research community, recent studies have unveiled a more complicated picture of APP's involvement in neurodegeneration in that other APP-derived fragments have been shown to exert pathological influences on neuronal function. However, not all APP-derived peptides are neurotoxic, and some even harbor neuroprotective effects. In this review, we will explore this complex picture by first discussing the pleiotropic effects of the major APP-derived peptides cleaved by multiple proteases, including soluble APP peptides (sAPPα, sAPPβ), various C- and N-terminal fragments, p3, and APP intracellular domain fragments. In addition, we will highlight two interesting sequences within APP that likely contribute to this duality in APP function. First, it has been found that caspase-mediated cleavage of APP in the cytosolic region may release a cytotoxic peptide, C31, which plays a role in synapse loss and neuronal death. Second, recent studies have implicated the -YENPTY- motif in the cytoplasmic region as a domain that modulates several APP activities through phosphorylation and dephosphorylation of the first tyrosine residue. Thus, this review summarizes the current understanding of various APP proteolytic products and the interplay among them to gain deeper insights into the possible mechanisms underlying neurodegeneration and AD pathophysiology.

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

    International Nuclear Information System (INIS)

    Selmer, B.L.; 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

  9. The cleavage product of amyloid-β protein precursor sAβPPα modulates BAG3-dependent aggresome formation and enhances cellular proteasomal activity.

    Science.gov (United States)

    Renziehausen, Jana; Hiebel, Christof; Nagel, Heike; Kundu, Arpita; Kins, Stefan; Kögel, Donat; Behl, Christian; Hajieva, Parvana

    2015-01-01

    Alzheimer's disease (AD) is the major age-associated form of dementia characterized by gradual cognitive decline. Aberrant cleavage of the amyloid-β protein precursor (AβPP) is thought to play an important role in the pathology of this disease. Two principal AβPP processing pathways exist: amyloidogenic cleavage of AβPP resulting in production of the soluble N-terminal fragment sAβPPβ, amyloid-β (Aβ), which accumulates in AD brain, and the AβPP intracellular domain (AICD) sAβPPα, p3 and AICD are generated in the non-amyloidogenic pathway. Prevalence of amyloidogenic versus non-amyloidogenic processing leads to depletion of sAβPPα and an increase in Aβ. Although sAβPPα is a well-accepted neurotrophic protein, molecular effects of this fragment remains unknown. Different studies reported impaired protein degradation pathways in AD brain, pointing to a role of disturbed proteasomal activity in the pathogenesis of this disease. Here we studied the possible role of sAβPPα in Bag3-mediated selective macroautophagy and proteasomal degradation. Employing human IMR90 cells, HEK 293 cells, and primary neurons, we demonstrate that sAβPPα prevents the proteotoxic stress-induced increase of Bag3 at the protein and at the mRNA level indicating a transcriptional regulation. Intriguingly, p62 and LC3, two other key players of autophagy, were not affected. Moreover, the formation and the accumulation of disease-related protein aggregates were significantly reduced by sAβPPα. Interestingly, there was a significant increase of proteasomal activity by sAβPPα as demonstrated by using various proteasome substrates. Our findings demonstrate that sAβPPα modulates Bag3 expression, aggresome formation, and proteasomal activity, thereby providing first evidence for a function of sAβPPα in the regulation of proteostasis.

  10. TAILS N-Terminomics and Proteomics Show Protein Degradation Dominates over Proteolytic Processing by Cathepsins in Pancreatic Tumors

    Directory of Open Access Journals (Sweden)

    Anna Prudova

    2016-08-01

    Full Text Available Deregulated cathepsin proteolysis occurs across numerous cancers, but in vivo substrates mediating tumorigenesis remain ill-defined. Applying 8-plex iTRAQ terminal amine isotopic labeling of substrates (TAILS, a systems-level N-terminome degradomics approach, we identified cathepsin B, H, L, S, and Z in vivo substrates and cleavage sites with the use of six different cathepsin knockout genotypes in the Rip1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis. Among 1,935 proteins and 1,114 N termini identified by TAILS, stable proteolytic products were identified in wild-type tumors compared with one or more different cathepsin knockouts (17%–44% of 139 cleavages. This suggests a lack of compensation at the substrate level by other cathepsins. The majority of neo-N termini (56%–83% for all cathepsins was consistent with protein degradation. We validated substrates, including the glycolytic enzyme pyruvate kinase M2 associated with the Warburg effect, the ER chaperone GRP78, and the oncoprotein prothymosin-alpha. Thus, the identification of cathepsin substrates in tumorigenesis improves the understanding of cathepsin functions in normal physiology and cancer.

  11. Low Proteolytic Clipping of Histone H3 in Cervical Cancer

    Science.gov (United States)

    Sandoval-Basilio, Jorge; Serafín-Higuera, Nicolás; Reyes-Hernandez, Octavio D.; Serafín-Higuera, Idanya; Leija-Montoya, Gabriela; Blanco-Morales, Magali; Sierra-Martínez, Monica; Ramos-Mondragon, Roberto; García, Silvia; López-Hernández, Luz Berenice; Yocupicio-Monroy, Martha; Alcaraz-Estrada, Sofia L.

    2016-01-01

    Chromatin in cervical cancer (CC) undergoes chemical and structural changes that alter the expression pattern of genes. Recently, a potential mechanism, which regulates gene expression at transcriptional levels is the proteolytic clipping of histone H3. However, until now this process in CC has not been reported. Using HeLa cells as a model of CC and human samples from patients with CC, we identify that the H3 cleavage was lower in CC compared with control tissue. Additionally, the histone H3 clipping was performed by serine and aspartyl proteases in HeLa cells. These results suggest that histone H3 clipping operates as part of post-translational modification system in CC. PMID:27698925

  12. 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

    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...

  13. The proteolytic system of Lactococcus lactis

    NARCIS (Netherlands)

    Kunji, Edmundus Richardus Stephanus

    1997-01-01

    The bacterium Lactococcus lactis usues an extencive proteolytic system to utilize milk proteins (caseins) in orde to meet its need for amino acids. The genetic and biochemical properties of the putative components of the proteolytic pathway are well-described. However, little is known about the role

  14. Testosterone stimulates progesterone production and STAR, P450 cholesterol side-chain cleavage and LH receptor mRNAs expression in hen (Gallus domesticus) granulosa cells.

    Science.gov (United States)

    Rangel, P L; Rodríguez, A; Rojas, S; Sharp, P J; Gutierrez, C G

    2009-12-01

    The chicken ovary is organized into a hierarchy of yellow yolky follicles that ovulate on successive days. Active or passive immunization of laying hens against testosterone blocks ovulation without affecting follicle development. Testosterone may play a role in pre-ovulatory follicle maturation by stimulating granulosa progesterone production. We assessed whether this stimulus is dose-related and depends on the maturity of the donor follicle, and if it does so by stimulating granulosa cell STAR, P450 cholesterol side-chain cleavage (P450scc), and LH receptor (LHCGR) mRNAs expression. Progesterone production by granulosa cells from F1, F3, and F4 follicles, cultured for 3 h without testosterone was greater in cells collected 11-14 h than 1-4 h after ovulation. These differences in progesterone production were less pronounced after granulosa cells had been cultured for 24 h. Culture of granulosa cells for 3 or 24 h with testosterone (1-100 ng/ml) stimulated progesterone production in cells collected from F4, F3, or F1 follicles 1-4, or 11-14 h after ovulation. Testosterone (0-4000 ng/ml) alone or in combination with LH (0-100 ng/ml) increased progesterone production by F1 granulosa cells, collected 1-4 and 11-14 h after ovulation and cultured for 3 h. Finally, testosterone (10 or 100 ng/ml) increased STAR, P450scc, and LHCGR mRNAs, when added to 3 h cultures of F1 granulosa cells. In conclusion, testosterone stimulates granulosa cell progesterone production in hen pre-ovulatory hierarchical follicles irrespective of maturational state, acting alone or additively with LH. We propose that testosterone promotes granulosa cell maturation to facilitate the pre-ovulatory release of LH.

  15. CleavPredict: A Platform for Reasoning about Matrix Metalloproteinases Proteolytic Events.

    Directory of Open Access Journals (Sweden)

    Sonu Kumar

    Full Text Available CleavPredict (http://cleavpredict.sanfordburnham.org is a Web server for substrate cleavage prediction for matrix metalloproteinases (MMPs. It is intended as a computational platform aiding the scientific community in reasoning about proteolytic events. CleavPredict offers in silico prediction of cleavage sites specific for 11 human MMPs. The prediction method employs the MMP specific position weight matrices (PWMs derived from statistical analysis of high-throughput phage display experimental results. To augment the substrate cleavage prediction process, CleavPredict provides information about the structural features of potential cleavage sites that influence proteolysis. These include: secondary structure, disordered regions, transmembrane domains, and solvent accessibility. The server also provides information about subcellular location, co-localization, and co-expression of proteinase and potential substrates, along with experimentally determined positions of single nucleotide polymorphism (SNP, and posttranslational modification (PTM sites in substrates. All this information will provide the user with perspectives in reasoning about proteolytic events. CleavPredict is freely accessible, and there is no login required.

  16. Top-down proteomics for the analysis of proteolytic events - Methods, applications and perspectives.

    Science.gov (United States)

    Tholey, Andreas; Becker, Alexander

    2017-11-01

    Mass spectrometry based proteomics is an indispensable tool for almost all research areas relevant for the understanding of proteolytic processing, ranging from the identification of substrates, products and cleavage sites up to the analysis of structural features influencing protease activity. The majority of methods for these studies are based on bottom-up proteomics performing analysis at peptide level. As this approach is characterized by a number of pitfalls, e.g. loss of molecular information, there is an ongoing effort to establish top-down proteomics, performing separation and MS analysis both at intact protein level. We briefly introduce major approaches of bottom-up proteomics used in the field of protease research and highlight the shortcomings of these methods. We then discuss the present state-of-the-art of top-down proteomics. Together with the discussion of known challenges we show the potential of this approach and present a number of successful applications of top-down proteomics in protease research. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Identification of the products of the cleavage of the galactans of red seaweeds in the form of pyridylamino derivatives

    International Nuclear Information System (INIS)

    Usov, A.I.; Dobkina, I.M.

    1986-01-01

    It has been shown that pyridylamino derivatives are convenient for the identification of various products of the galactans of red algae. Reductive amination with 2-aminopyridine and sodium thiohydroborate has been investigated for the case of several monosaccharides that are common components of the polysaccharide fractions of red seaweeds and also for carrabiose, agarobiose, and oligosaccharides of the neoagarobiose series. Various forms of chromatography and high-voltage paper electrophoresis were used to separate the pyridylamino derivatives, and NMR spectroscopy and mass spectroscopy of the full acetates for structural characterization

  18. Inhibition of Cellular Adhesion by Immunological Targeting of Osteopontin Neoepitopes Generated through Matrix Metalloproteinase and Thrombin Cleavage.

    Science.gov (United States)

    Jürets, Alexander; Le Bras, Marie; Staffler, Günther; Stein, Gesine; Leitner, Lukas; Neuhofer, Angelika; Tardelli, Matteo; Turkof, Edvin; Zeyda, Maximilian; Stulnig, Thomas M

    2016-01-01

    Osteopontin (OPN), a secreted protein involved in inflammatory processes and cancer, induces cell adhesion, migration, and activation of inflammatory pathways in various cell types. Cells bind OPN via integrins at a canonical RGD region in the full length form as well as to a contiguous cryptic site that some have shown is unmasked upon thrombin or matrix metalloproteinase cleavage. Thus, the adhesive capacity of osteopontin is enhanced by proteolytic cleavage that may occur in inflammatory conditions such as obesity, atherosclerosis, rheumatoid arthritis, tumor growth and metastasis. Our aim was to inhibit cellular adhesion to recombinant truncated proteins that correspond to the N-terminal cleavage products of thrombin- or matrix metalloproteinase-cleaved OPN in vitro. We specifically targeted the cryptic integrin binding site with monoclonal antibodies and antisera induced by peptide immunization of mice. HEK 293 cells adhered markedly stronger to truncated OPN proteins than to full length OPN. Without affecting cell binding to the full length form, the raised monoclonal antibodies specifically impeded cellular adhesion to the OPN fragments. Moreover, we show that the peptides used for immunization were able to induce antisera, which impeded adhesion either to all OPN forms, including the full-length form, or selectively to the corresponding truncated recombinant proteins. In conclusion, we developed immunological tools to selectively target functional properties of protease-cleaved OPN forms, which could find applications in treatment and prevention of various inflammatory diseases and cancers.

  19. Blocking of proteolytic processing and deletion of glycosaminoglycan side chain of mouse DMP1 by substituting critical amino acid residues.

    Science.gov (United States)

    Peng, Tao; Huang, Bingzhen; Sun, Yao; Lu, Yongbo; Bonewald, Lynda; Chen, Shuo; Butler, William T; Feng, Jerry Q; D'Souza, Rena N; Qin, Chunlin

    2009-01-01

    Dentin matrix protein 1 (DMP1) is present in the extracellular matrix (ECM) of dentin and bone as processed NH(2)- and COOH-terminal fragments, resulting from proteolytic cleavage at the NH(2) termini of 4 aspartic acid residues during rat DMP1 processing. One cleavage site residue, Asp(181) (corresponding to Asp(197) of mouse DMP1), and its flanking region are highly conserved across species. We speculate that cleavage at the NH(2) terminus of Asp(197) of mouse DMP1 represents an initial, first-step scission in the whole cascade of proteolytic processing. To test if Asp(197) is critical for initiating the proteolytic processing of mouse DMP1, we substituted Asp(197) with Ala(197) by mutating the corresponding nucleotides of mouse cDNA that encode this amino acid residue. This mutant DMP1 cDNA was cloned into a pcDNA3.1 vector. Data from transfection experiments indicated that this single substitution blocked the proteolytic processing of mouse DMP1 in HEK-293 cells, indicating that cleavage at the NH(2) terminus of Asp(197) is essential for exposing other cleavage sites for the conversion of DMP1 to its fragments. The NH(2)-terminal fragment of DMP1 occurs as a proteoglycan form (DMP1-PG) that contains a glycosaminoglycan (GAG) chain. Previously, we showed that a GAG chain is linked to Ser(74) in rat DMP1 (Ser(89) in mouse DMP1). To confirm that mouse DMP1-PG possesses a single GAG chain attached to Ser(89), we substituted Ser(89) by Gly(89). Data from transfection analysis indicated that this substitution completely prevented formation of the GAG-containing form, confirming that DMP1-PG contains a single GAG chain attached to Ser(89) in mouse DMP1. Copyright 2008 S. Karger AG, Basel.

  20. Structural and functional characterization of cleavage and inactivation of human serine protease inhibitors by the bacterial SPATE protease EspPα from enterohemorrhagic E. coli.

    Directory of Open Access Journals (Sweden)

    André Weiss

    Full Text Available EspPα and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins by EspPα and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspPα cleaved the serpins α1-protease inhibitor (α1-PI, α1-antichymotrypsin, angiotensinogen, and α2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory serpins such as α2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspPα-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition.

  1. Regioselectivity in the Reductive Bond Cleavage of Diarylalkylsulfonium Salts

    DEFF Research Database (Denmark)

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

    2009-01-01

    products vary from regiospecific alkyl cleavage to predominant aryl cleavage as a function of the potential of the reducing agent. We conclude that differences between the reductive cleavages of mono- and diarylsulfonium salts are direct consequences of the structures of the sulfuranyl radical......- tolylethylsulfonium and di-4-tolyl-2-phenylethylsulfonium salts by a variety of one-electron reducing agents ranging in potential from -0.77 to +2.5 eV (vs SCE) and including thermal reductants, indirect electrolyses mediated by a series of cyanoaromatics, and excited singlet states. We report that the cleavage...... intermediates and the bond dissociation energies of the alkyl and aryl bonds. Competitions between the rates of cleavage and oxidation of the intermediate sulfuranyl radicals and between concerted and stepwise mechanisms are discussed to explain the variations in bond cleavage products as a function...

  2. The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions.

    Science.gov (United States)

    Pettit, S C; Moody, M D; Wehbie, R S; Kaplan, A H; Nantermet, P V; Klein, C A; Swanstrom, R

    1994-12-01

    The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p2 site, however, was cleaved approximately 20-fold faster when the adjacent downstream p2/NC site was blocked from cleavage or when the p2 domain of Gag was deleted. These results suggest that the presence of a C-terminal p2 tail on processing intermediates slows cleavage at the upstream CA/p2 site. We also found that lower pH selectively accelerated cleavage of the CA/p2 processing site in the full-length precursor and as a peptide primarily by a sequence-based mechanism rather than by a change in protein conformation. Deletion of the p2 domain of Gag results in released virions that are less infectious despite the presence of the processed final products of Gag. These findings suggest that the p2 domain of HIV-1 Gag regulates the rate of cleavage at the CA/p2 processing site during sequential processing in vitro and in infected cells and that p2 may function in the proper assembly of virions.

  3. Unusual reactions of diazocarbonyl compounds with α,β-unsaturated δ-amino esters: Rh(II-catalyzed Wolff rearrangement and oxidative cleavage of N–H-insertion products

    Directory of Open Access Journals (Sweden)

    Valerij A. Nikolaev

    2016-08-01

    Full Text Available Rh(II-сatalyzed reactions of aroyldiazomethanes, diazoketoesters and diazodiketones with α,β-unsaturated δ-aminoesters, in contrast to reactions of diazomalonates and other diazoesters, give rise to the Wolff rearrangement and/or oxidative cleavage of the initially formed N–H-insertion products. These oxidation processes are mediated by Rh(II catalysts possessing perfluorinated ligands. The formation of pyrrolidine structures, characteristic for catalytic reactions of diazoesters, was not observed in these processes at all.

  4. Proteolytic activation transforms heparin cofactor II into a host defense molecule.

    Science.gov (United States)

    Kalle, Martina; Papareddy, Praveen; Kasetty, Gopinath; Tollefsen, Douglas M; Malmsten, Martin; Mörgelin, Matthias; Schmidtchen, Artur

    2013-06-15

    The abundant serine proteinase inhibitor heparin cofactor II (HCII) has been proposed to inhibit extravascular thrombin. However, the exact physiological role of this plasma protein remains enigmatic. In this study, we demonstrate a previously unknown role for HCII in host defense. Proteolytic cleavage of the molecule induced a conformational change, thereby inducing endotoxin-binding and antimicrobial properties. Analyses employing representative peptide epitopes mapped these effects to helices A and D. Mice deficient in HCII showed increased susceptibility to invasive infection by Pseudomonas aeruginosa, along with a significantly increased cytokine response. Correspondingly, decreased levels of HCII were observed in wild-type animals challenged with bacteria or endotoxin. In humans, proteolytically cleaved HCII forms were detected during wounding and in association with bacteria. Thus, the protease-induced uncovering of cryptic epitopes in HCII, which transforms the molecule into a host defense factor, represents a previously unknown regulatory mechanism in HCII biology and innate immunity.

  5. Fibrinogen cleavage products and Toll-like receptor 4 promote the generation of programmed cell death 1 ligand 2-positive dendritic cells in allergic asthma.

    Science.gov (United States)

    Cho, Minkyoung; Lee, Jeong-Eun; Lim, Hoyong; Shin, Hyun-Woo; Khalmuratova, Roza; Choi, Garam; Kim, Hyuk Soon; Choi, Wahn Soo; Park, Young-Jun; Shim, Inbo; Kim, Byung-Seok; Kang, Chang-Yuil; Kim, Jae-Ouk; Tanaka, Shinya; Kubo, Masato; Chung, Yeonseok

    2017-10-14

    Inhaled protease allergens preferentially trigger T H 2-mediated inflammation in allergic asthma. The role of dendritic cells (DCs) on induction of T H 2 cell responses in allergic asthma has been well documented; however, the mechanism by which protease allergens induce T H 2-favorable DCs in the airway remains unclear. We sought to determine a subset of DCs responsible for T H 2 cell responses in allergic asthma and the mechanism by which protease allergens induce the DC subset in the airway. Mice were challenged intranasally with protease allergens or fibrinogen cleavage products (FCPs) to induce allergic airway inflammation. DCs isolated from mediastinal lymph nodes were analyzed for surface phenotype and T-cell stimulatory function. Anti-Thy1.2 and Mas-TRECK mice were used to deplete innate lymphoid cells and mast cells, respectively. Adoptive cell transfer, bone marrow DC culture, anti-IL-13, and Toll-like receptor (TLR) 4-deficient mice were used for further mechanistic studies. Protease allergens induced a remarkable accumulation of T H 2-favorable programmed cell death 1 ligand 2 (PD-L2) + DCs in mediastinal lymph nodes, which was significantly abolished in mice depleted of mast cells and, to a lesser extent, innate lymphoid cells. Mechanistically, FCPs generated by protease allergens triggered IL-13 production from wild-type mast cells but not from TLR4-deficient mast cells, which resulted in an increase in the number of PD-L2 + DCs. Intranasal administration of FCPs induced an increase in numbers of PD-L2 + DCs in the airway, which was significantly abolished in TLR4- and mast cell-deficient mice. Injection of IL-13 restored the PD-L2 + DC population in mice lacking mast cells. Our findings unveil the "protease-FCP-TLR4-mast cell-IL-13" axis as a molecular mechanism for generation of T H 2-favorable PD-L2 + DCs in allergic asthma and suggest that targeting the PD-L2 + DC pathway might be effective in suppressing allergic T-cell responses in the airway

  6. Alzheimer’s disease against peptides products of enzymatic cleavage APP protein: Biological, pathobiological and physico-chemical properties of fibrillating peptides

    Directory of Open Access Journals (Sweden)

    Małgorzata Marszałek

    2017-05-01

    Full Text Available Various peptides products of enzymatic cleavage of key for Alzheimer’s disease Amyloid Precursor Protein (APP are well known, but still are matter of scientific debate. The Aβ type products are especially challenging for experimental and medical research. This paper outlines several, still poorly known, biological and medical processes such as peptides biology, i.e., formation, biodistribution, translocation, transport and finally removal from brain compartments and body fluids like Intracellular Fluid (ICF, Cerebrospinal Fluid (CSF, Interstitial Fluid (ISF, blood serum or urine. In addition, the following studies concerning AD patients might prove challenging and simultaneously promising: peptides translocation through Blood-Brain – Barrier (BBB and Blood–Cerebrospinal Fluid Barrier (BCSFB and their removal from the brain according to a new concept of glymphatic system; – diagnostic difficulties that stem from physico-chemical properties and the nature of proteins or fibrillating peptides itself like low concentration, short half-live and from experimental-technical problems as well like high adsorption or low solubility of Aβ, tau or amylin. The study of diagnostic parameters is very important, as it may better reflect early changes before the disease develops; one such parameter is the Aβ42/Aβ40 ratio, or the ratio with the total tau concentration combination and other new biomarkers like Aβ1-38; other factors include oxidative stress and inflammation process proteins, complement factor H, alpha-2-macroglobulin, or clusterin. The study of various forms of pathological amyloid deposits that emerge in different but specific brain regions AD patients seems to be crucial as well. The composition of the first initial pathological, pre-fibrillating monomers of fibrillating peptides and their role in AD development and disease progression have been described as well. They are even more challenging for science and simultaneously might be

  7. High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage.

    Science.gov (United States)

    Kim, Jung Min; Jang, Su A; Yu, Byung Jo; Sung, Bong Hyun; Cho, Ju Hyun; Kim, Sun Chang

    2008-02-01

    Direct expression of an antimicrobial peptide (AMP) in Escherichia coli causes several problems such as the toxicity of AMP to the host cell, its susceptibility to proteolytic degradation, and decreased antimicrobial activity due to the additional residue(s) introduced after cleavage of AMPs from fusion partners. To overcome these problems and produce a large quantity of a potent AMP histonin (RAGLQFPVGKLLKKLLKRLKR) in E. coli, an efficient expression system was developed, in which the toxicity of histonin was neutralized by a fusion partner F4 (a truncated fragment of PurF protein) and the productivity was increased by a multimeric expression of a histonin gene. The expression level of the fusion proteins reached a maximum with a 12-mer of a histonin gene. In addition, because of the RLKR residues present at the C terminus of histonin, furin cleavage of the multimeric histonin expressed produces an intact, natural histonin. The AMP activity of the histonin produced in E. coli was identical to that of a synthetic histonin. With our expression system, 167 mg of histonin was obtained from 1 l of E. coli culture. These results may lead to a cost-effective solution for the mass production of AMPs that are toxic to a host.

  8. Proteolytic cleavage of the Chlamydia pneumoniae major outer membrane protein in the absence of Pmp10

    DEFF Research Database (Denmark)

    Juul, Nicolai Stefan; Timmerman, E; Gevaert, K

    2007-01-01

    The genome of the obligate intracellular bacteria Chlamydia pneumoniae contains 21 genes encoding polymorphic membrane proteins (Pmp). While no function has yet been attributed to the Pmps, they may be involved in an antigenic variation of the Chlamydia surface. It has previously been demonstrated...

  9. Effect of follicular diameter, time of first cleavage and H3K4 methylation on embryo production rates of Bos indicus cattle

    Directory of Open Access Journals (Sweden)

    Paula Alvares Lunardelli

    2016-10-01

    Full Text Available This study aimed investigate the relationship between epigenetics, follicular diameter and cleavage speed, by evaluating the developmental potential and occurence of H3K4 monomethylation of early-, intermediate- and late-cleaving Bos indicus embryos from in vitro fertilized oocytes originating from follicles up to 2 mm in diameter or between 4 and 8 mm in diameter. Oocytes (n = 699 from small follicles (? 2 mm and 639 oocytes from large follicles (4-8 mm were punched from 1,982 Bos indicus’ slaughterhouse ovaries. After maturation and in vitro fertilization (IVF, the cultured embryos were separated into early (? 28 h post-IVF, intermediate (> 28 h and ? 34 h post-IVF and late (> 34 h and ? 54 h post-IVF cleavage groups. Blastocysts were subjected to an immunofluorescence assessment for H3K4me investigation. The blastocyst rate for large follicles (36.3% was higher than that for small follicles (22.9%, P < 0.05. In addition, blastocyst rates for early and intermediate cleavage groups (45.3% and 33.8%, respectively were higher than that for late cleavage group (13.5%, P < 0.05. The blastocysts from all groups displayed H3K4me staining by immunofluorescence, particularly intense in what seemed to be trophectoderm cells and weak or absent in cells seemingly from the inner cell mass. For the first time for indicus embryos, data from this study demonstrate that higher blastocyst embryo rates are obtained from embryos that cleave within 34 h after fertilization and from those produced from follicles of 4-8 mm in diameter, indicating a greater ability of these embryos to develop to the stage of embryonic preimplantation. This is the first article demonstrating the occurrence of H3K4me in cattle embryos; its presence in all the evaluated blastocysts suggests that this histone modification plays a key role in maintaining embryo viability at preimplantation stage.

  10. The proteolytic systems of lactic acid bacteria

    NARCIS (Netherlands)

    Kunji, Edmund R.S.; Mierau, Igor; Hagting, Anja; Poolman, Bert; Konings, Wil N.

    1996-01-01

    Proteolysis in dairy lactic acid bacteria has been studied in great detail by genetic, biochemical and ultrastructural methods. From these studies the picture emerges that the proteolytic systems of lactococci and lactobacilli are remarkably similar in their components and mode of action. The

  11. Detection of proteolytic signatures for Parkinson's disease

    DEFF Research Database (Denmark)

    Jordal, Peter Lüttge; Dyrlund, Thomas F.; Winge, Kristian

    2016-01-01

    Aim: To investigate if idiopathic Parkinson's disease (IPD) is associated with distinct proteolytic signatures relative to non-neurodegenerative controls (NND) and patients with multiple system atrophy (MSA). Materials & methods: A subtiligase-based N-terminomics screening method was exploited...

  12. Preventing cleavage of Mer promotes efferocytosis and suppresses acute lung injury in bleomycin treated mice

    International Nuclear Information System (INIS)

    Lee, Ye-Ji; Lee, Seung-Hae; Youn, Young-So; Choi, Ji-Yeon; Song, Keung-Sub; Cho, Min-Sun; Kang, Jihee Lee

    2012-01-01

    Mer receptor tyrosine kinase (Mer) regulates macrophage activation and promotes apoptotic cell clearance. Mer activation is regulated through proteolytic cleavage of the extracellular domain. To determine if membrane-bound Mer is cleaved during bleomycin-induced lung injury, and, if so, how preventing the cleavage of Mer enhances apoptotic cell uptake and down-regulates pulmonary immune responses. During bleomycin-induced acute lung injury in mice, membrane-bound Mer expression decreased, but production of soluble Mer and activity as well as expression of disintegrin and metalloproteinase 17 (ADAM17) were enhanced . Treatment with the ADAM inhibitor TAPI-0 restored Mer expression and diminished soluble Mer production. Furthermore, TAPI-0 increased Mer activation in alveolar macrophages and lung tissue resulting in enhanced apoptotic cell clearance in vivo and ex vivo by alveolar macrophages. Suppression of bleomycin-induced pro-inflammatory mediators, but enhancement of hepatocyte growth factor induction were seen after TAPI-0 treatment. Additional bleomycin-induced inflammatory responses reduced by TAPI-0 treatment included inflammatory cell recruitment into the lungs, levels of total protein and lactate dehydrogenase activity in bronchoalveolar lavage fluid, as well as caspase-3 and caspase-9 activity and alveolar epithelial cell apoptosis in lung tissue. Importantly, the effects of TAPI-0 on bleomycin-induced inflammation and apoptosis were reversed by coadministration of specific Mer-neutralizing antibodies. These findings suggest that restored membrane-bound Mer expression by TAPI-0 treatment may help resolve lung inflammation and apoptosis after bleomycin treatment. -- Highlights: ►Mer expression is restored by TAPI-0 treatment in bleomycin-stimulated lung. ►Mer signaling is enhanced by TAPI-0 treatment in bleomycin-stimulated lung. ►TAPI-0 enhances efferocytosis and promotes resolution of lung injury.

  13. Proteolytic activity regarding Sarconesiopsis magellanica (Diptera: Calliphoridae) larval excretions and secretions.

    Science.gov (United States)

    Pinilla, Yudi T; Moreno-Pérez, Darwin A; Patarroyo, Manuel A; Bello, Felio J

    2013-12-01

    Sarconesiopsis magellanica (Diptera: Calliphoridae) is a medically important necrophagous fly which is used for establishing the post-mortem interval. Diptera maggots release proteolytic enzymes contained in larval excretion and secretion (ES) products playing a key role in digestion. Special interest in proteolytic enzymes has also been aroused regarding understanding their role in wound healing since they degrade necrotic tissue during larval therapy. This study was thus aimed at identifying and characterising S. magellanica proteolytic enzyme ES products for the first time. These products were obtained from first-, second- and third-instar larvae taken from a previously-established colony. ES proteins were separated by SDS-PAGE and their proteolytic activity was characterised by zymograms and inhibition assays involving BAPNA (Nα-benzoyl-dl-Arg-p-nitroanilide) and SAPNA substrates, using synthetic inhibitors. The protein profile ranged from ∼69kDa to ∼23kDa; several of them coincided with the Lucilia sericata ES protein profile. Serine-protease hydrolysis activity (measured by zymogram) was confirmed when a ∼25kDa band disappeared upon ES incubation with PMSF inhibitor at pH 7.8. Analysis of larval ES proteolytic activity on BAPNA and SAPNA substrates (determined by using TLCK and TPCK specific inhibitors) suggested a greater amount of trypsin-like protease. These results support the need for further experiments aimed at validating S. magellanica use in larval therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Proteolytic enzymes involved in MHC class I antigen processing: A guerrilla army that partners with the proteasome.

    Science.gov (United States)

    Lázaro, Silvia; Gamarra, David; Del Val, Margarita

    2015-12-01

    Major histocompatibility complex class I proteins (MHC-I) load short peptides derived from proteolytic cleavage of endogenous proteins in any cell of the body, in a process termed antigen processing and presentation. When the source proteins are altered self or encoded by a pathogen, recognition of peptide/MHC-I complexes at the plasma membrane leads to CD8(+) T-lymphocyte responses that clear infections and probably underlie tumor immune surveillance. On the other hand, presentation of self peptides may cause some types of autoimmunity. The peptides that are presented determine the specificity and efficiency of pathogen clearance or, conversely, of immunopathology. In this review we highlight the growing number of peptidases which, as a by-product of their regular activity, can generate peptide epitopes for immune surveillance. These ∼20 peptidases collectively behave as a guerrilla army partnering with the regular proteasome army in generating a variety of peptides for presentation by MHC-I and thus optimally signaling infection. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. Intrinsic Tau Acetylation Is Coupled to Auto-Proteolytic Tau Fragmentation.

    Directory of Open Access Journals (Sweden)

    Todd J Cohen

    Full Text Available Tau proteins are abnormally aggregated in a range of neurodegenerative tauopathies including Alzheimer's disease (AD. Recently, tau has emerged as an extensively post-translationally modified protein, among which lysine acetylation is critical for normal tau function and its pathological aggregation. Here, we demonstrate that tau isoforms have different propensities to undergo lysine acetylation, with auto-acetylation occurring more prominently within the lysine-rich microtubule-binding repeats. Unexpectedly, we identified a unique intrinsic property of tau in which auto-acetylation induces proteolytic tau cleavage, thereby generating distinct N- and C-terminal tau fragments. Supporting a catalytic reaction-based mechanism, mapping and mutagenesis studies showed that tau cysteines, which are required for acetyl group transfer, are also essential for auto-proteolytic tau processing. Further mass spectrometry analysis identified the C-terminal 2nd and 4th microtubule binding repeats as potential sites of auto-cleavage. The identification of acetylation-mediated auto-proteolysis provides a new biochemical mechanism for tau self-regulation and warrants further investigation into whether auto-catalytic functions of tau are implicated in AD and other tauopathies.

  16. PROTEOLYTIC PROCESSING OF VON WILLEBRAND FACTOR BY ADAMTS13 AND LEUKOCYTE PROTEASES

    Directory of Open Access Journals (Sweden)

    Stefano Lancellotti

    2013-09-01

    Full Text Available ADAMTS13 is a 190 kDa zinc protease encoded by a gene located on chromosome 9q34.   This protease specifically hydrolyzes von Willebrand factor (VWF multimers, thus causing VWF size reduction. ADAMTS13 belongs to the A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS family, involved in proteolytic processing of many matrix proteins. ADAMTS13 consists of numerous domains including a metalloprotease domain, a disintegrin domain, several thrombospondin type 1 (TSP1 repeats, a cysteine-rich domain, a spacer domain and 2 CUB (Complement c1r/c1s, sea Urchin epidermal growth factor, and Bone morphogenetic protein domains. ADAMTS13 cleaves a single peptide bond (Tyr1605-Met1606 in the central A2 domain of the VWF molecule. This proteolytic cleavage is essential to reduce the size of ultra-large VWF polymers, which, when exposed to high shear stress in the microcirculation, are prone to form with platelets clumps, which cause severe syndromes called thrombotic microangiopathies (TMAs. In this review, we a discuss the current knowledge of structure-function aspects of ADAMTS13 and its involvement in the pathogenesis of TMAs, b address the recent findings concerning proteolytic processing of VWF multimers by different proteases, such as the leukocyte-derived serine and metallo-proteases and c indicate the direction of future investigations

  17. [Proteolytic activity of IgG-antibodies of mice, immunized by calf thymus histones].

    Science.gov (United States)

    Kit, Iu Ia; Korniĭ, N; Kril', I Ĭ; Mahorivs'ka, I B; Tkachenko, V; Bilyĭ, R O; Stoĭka, R S

    2014-01-01

    The main goal of the study was to determine the ability of histones to induce production of the proteolytically active IgG-antibodies in BALB/c mice. In order to perform this study 8 mice were immunized with the fraction of total calf thymus histones. IgGs were isolated from the serum of the immunized and not immunized animals by means of precipitation with 33% ammonium sulfate, followed by affinity chromatography on protein G-Sepharose column. Histones, myelin basic protein (MBP), lysozyme, BSA, ovalbumin, macroglobulin, casein and cytochrome c served as substrates for determining the proteolytic activity. It was found that IgGs from the blood serum of immunized mice are capable of hydrolyzing histone H1, core histone and MBP. On the contrary, the proteolytic activity of IgGs from the blood serum of not immunized mice was not detected. The absence of proteolytical enzymes in the fraction of IgGs was proven by HPLC chromatography. High levels of proteolytic activity toward histones have been also detected in affinity purified IgGs from blood serum of patients with rheumatoid arthritis, but not in healthy donors. These data indicate that eukaryotic histones may induce production of protabzymes in mammals. The possible origin of these protabzymes and their potential biological role in mammalians is discussed.

  18. Measurement of separase proteolytic activity in single living cells by a fluorogenic flow cytometry assay.

    Directory of Open Access Journals (Sweden)

    Wiltrud Haaß

    Full Text Available ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML. Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110 as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90-180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic

  19. Proteolytically modified human beta 2-microglobulin augments the specific cytotoxic activity in murine mixed lymphocyte culture

    DEFF Research Database (Denmark)

    Nissen, Mogens Holst; Claësson, M H

    1987-01-01

    the endogenous production of interleukin 2 in the MLC culture; monoclonal antibody which reacts with both the native beta 2-m and M-beta 2-m molecule blocks the augmentation of cytotoxic T lymphocyte production induced by M-beta 2-m; murine as well as human MLC responder cells can proteolytically modify native......A proteolytically modified form of beta 2-microglobulin (beta 2-m) present in the serum of patients suffering from autoimmune, immunodeficient diseases and cancer has been reported in the literature. In the present study we show that human beta 2-m as well as the proteolytically modified human form...... (M-beta 2-m) bind to murine lymphocytes expressing H-2 class I antigens; M-beta 2-m, when added at day 0 and 1 of culture in nanomolar concentrations to a one-way murine allogeneic mixed lymphocyte culture (MLC) augments the generation of specific cytotoxic T lymphocytes; M-beta 2-m increases...

  20. Proteolytic crosstalk in multi-protease networks

    Science.gov (United States)

    Ogle, Curtis T.; Mather, William H.

    2016-04-01

    Processive proteases, such as ClpXP in E. coli, are conserved enzyme assemblies that can recognize and rapidly degrade proteins. These proteases are used for a number of purposes, including degrading mistranslated proteins and controlling cellular stress response. However, proteolytic machinery within the cell is limited in capacity and can lead to a bottleneck in protein degradation, whereby many proteins compete (‘queue’) for proteolytic resources. Previous work has demonstrated that such queueing can lead to pronounced statistical relationships between different protein counts when proteins compete for a single common protease. However, real cells contain many different proteases, e.g. ClpXP, ClpAP, and Lon in E. coli, and it is not clear how competition between proteins for multiple classes of protease would influence the dynamics of cellular networks. In the present work, we theoretically demonstrate that a multi-protease proteolytic bottleneck can substantially couple the dynamics for both simple and complex (oscillatory) networks, even between substrates with substantially different affinities for protease. For these networks, queueing often leads to strong positive correlations between protein counts, and these correlations are strongest near the queueing theoretic point of balance. Furthermore, we find that the qualitative behavior of these networks depends on the relative size of the absolute affinity of substrate to protease compared to the cross affinity of substrate to protease, leading in certain regimes to priority queue statistics.

  1. Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin

    International Nuclear Information System (INIS)

    Hill-Eubanks, D.C.; Parker, C.G.; Lollar, P.

    1989-01-01

    Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. The authors isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO 4 /PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated 125 I-fVIII to function as a cofactor in the activation of factor X by factor IXa, they found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function

  2. Proteolytic Activity in Reduced-Fat Cheddar Cheese Made with Lactic Acid Bacteria and Camel Chymosin

    DEFF Research Database (Denmark)

    Børsting, Mette Winther

    be the need of an extended ripening period to reach a similar cheese structure as in cheeses produced with BC. The aim of this project was to compensate for the lower proteolytic activity in cheese produced with CC compared to BC. Selection of dairy lactic acid bacteria (LAB) for cheese production with high....... lactis subsp lactis, 10 thermophilic Lactobacillus strains and 15 frozen direct vat set strains of thermopholic Lactobacillus) to hydrolyse αS1-CN, candidates were selected for cheese-making experiments. None of the selected proteolytic strains contributed significantly to softening the cheese structure...

  3. 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.

  4. Identification of two auto-cleavage products of nonstructural protein 1 (nsp1) in porcine reproductive and respiratory syndrome virus infected cells: nsp1 function as interferon antagonist

    International Nuclear Information System (INIS)

    Chen, Z.; Lawson, S.; Sun, Z.; Zhou, X.; Guan, X.; Christopher-Hennings, J.; Nelson, E.A.; Fang, Y.

    2010-01-01

    The porcine reproductive and respiratory syndrome virus nsp1 is predicted to be auto-cleaved from the replicase polyprotein into nsp1α and nsp1β subunits. In infected cells, we detected the actual existence of nsp1α and nsp1β. Cleavage sites between nsp1α/nsp1β and nsp1β/nsp2 were identified by protein microsequencing analysis. Time course study showed that nsp1α and nsp1β mainly localize into the cell nucleus after 10 h post infection. Further analysis revealed that both proteins dramatically inhibited IFN-β expression. The nsp1β was observed to significantly inhibit expression from an interferon-stimulated response element promoter after Sendai virus infection or interferon treatment. It was further determined to inhibit nuclear translocation of STAT1 in the JAK-STAT signaling pathway. These results demonstrated that nsp1β has ability to inhibit both interferon synthesis and signaling, while nsp1α alone strongly inhibits interferon synthesis. These findings provide important insights into mechanisms of nsp1 in PRRSV pathogenesis and its impact in vaccine development.

  5. The γ-secretase cleavage product of Polycystin-1 regulates TCF and CHOP-mediated transcriptional activation through a p300-dependent mechanism

    Science.gov (United States)

    Merrick, David; Chapin, Hannah; Baggs, Julie E.; Yu, Zhiheng; Somlo, Stefan; Sun, Zhaoxia; Hogenesch, John B.; Caplan, Michael

    2011-01-01

    Summary Mutations in Pkd1, encoding polycystin-1 (PC1), cause Autosomal Dominant Polycystic Kidney Disease (ADPKD). We show that the carboxy-terminal tail (CTT) of PC1 is released by γ-secretase-mediated cleavage and regulates the Wnt and CHOP pathways by binding the transcription factors TCF and CHOP, disrupting their interaction with the common transcriptional co-activator p300. Loss of PC1 causes increased proliferation and apoptosis, while reintroducing PC1-CTT into cultured Pkd1 null cells reestablishes normal growth rate, suppresses apoptosis, and prevents cyst formation. Inhibition of γ-secretase activity impairs the ability of PC1 to suppress growth and apoptosis, and leads to cyst formation in cultured renal epithelial cells. Expression of the PC1-CTT is sufficient to rescue the dorsal body curvature phenotype in zebrafish embryos resulting from either γ-secretase inhibition or suppression of Pkd1 expression. Thus, γ-secretase-dependent release of the PC1-CTT creates a protein fragment whose expression is sufficient to suppress ADPKD-related phenotypes in vitro and in vivo. PMID:22178500

  6. Characterization of proteolytic and anti-proteolytic activity involvement in sterlet spermatozoon maturation

    Czech Academy of Sciences Publication Activity Database

    Dzyuba, V.; Słowińska, M.; Cosson, J.; Ciereszko, A.; Boryshpolets, S.; Štěrba, Ján; Rodina, M.; Linhart, O.; Dzyuba, B.

    2016-01-01

    Roč. 42, č. 6 (2016), s. 1755-1766 ISSN 0920-1742 R&D Projects: GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : amidase activity * anti-proteolytic activity * casein and gelatin zymography * siberian sturgeon sperm * spermatozoon maturation * sterlet sperm Subject RIV: ED - Physiology Impact factor: 1.647, year: 2016

  7. Seasonal variation in the temperature sensitivity of proteolytic enzyme activity in temperate forest soils

    Science.gov (United States)

    Brzostek, Edward R.; Finzi, Adrien C.

    2012-03-01

    Increasing soil temperature has the potential to alter the activity of the extracellular enzymes that mobilize nitrogen (N) from soil organic matter (SOM) and ultimately the availability of N for primary production. Proteolytic enzymes depolymerize N from proteinaceous components of SOM into amino acids, and their activity is a principal driver of the within-system cycle of soil N. The objectives of this study were to investigate whether the soils of temperate forest tree species differ in the temperature sensitivity of proteolytic enzyme activity over the growing season and the role of substrate limitation in regulating temperature sensitivity. Across species and sampling dates, proteolytic enzyme activity had relatively low sensitivity to temperature with a mean activation energy (Ea) of 33.5 kJ mol-1. Ea declined in white ash, American beech, and eastern hemlock soils across the growing season as soils warmed. By contrast, Eain sugar maple soil increased across the growing season. We used these data to develop a species-specific empirical model of proteolytic enzyme activity for the 2009 calendar year and studied the interactive effects of soil temperature (ambient or +5°C) and substrate limitation (ambient or elevated protein) on enzyme activity. Declines in substrate limitation had a larger single-factor effect on proteolytic enzyme activity than temperature, particularly in the spring. There was, however, a large synergistic effect of increasing temperature and substrate supply on proteolytic enzyme activity. Our results suggest limited increases in N availability with climate warming unless there is a parallel increase in the availability of protein substrates.

  8. Developing a capillary electrophoresis based method for dynamically monitoring enzyme cleavage activity using quantum dots-peptide assembly.

    Science.gov (United States)

    Wang, Jianhao; Fan, Jie; Liu, Li; Ding, Shumin; Liu, Xiaoqian; Wang, Jianpeng; Gao, Liqian; Chattopadhaya, Souvik; Miao, Peng; Xia, Jiang; Qiu, Lin; Jiang, Pengju

    2017-10-01

    Herein, a novel assay has been developed for monitoring PreScission protease (His-PSP) mediated enzyme cleavage of ATTO 590 labeled peptide substrate (ATTO-LEV). This novel method is based on combining the use of capillary electrophoresis and fluorescence detection (CE-FL) to dynamically monitor the enzyme cleavage activity. A multivalent peptide substrate was first constructed by immobilizing His-tagged ATTO 590 labeled peptide substrate (ATTO-LEVH6) onto the surface of CdSe/ZnS quantum dots (QDs). Once successfully immobilized, the novel multivalent peptide substrate resulted in the Förster resonance energy transfer (FRET) from QDs to ATTO 590. The ATTO-LEVH6-QD assembly was then incubated with His-PSP to study the proteolytic cleavage of surface bound ATTO-LEVH6 by CE-FL. Our data suggests that PreScission-mediated proteolytic cleavage is enzyme concentration- and incubation time-dependent. By combining capillary electrophoresis, QDs and FRET, our study herein not only provides a new method for the detection and dynamically monitoring of PSP enzyme cleavage activity, but also can be extended to the detection of many other enzymes and proteases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Intermolecular cleavage by UmuD-like mutagenesis proteins

    Science.gov (United States)

    McDonald, John P.; Frank, Ekaterina G.; Levine, Arthur S.; Woodgate, Roger

    1998-01-01

    The activity of a number of proteins is regulated by self-processing reactions. Elegant examples are the cleavage of the prokaryotic LexA and λCI transcriptional repressors and the UmuD-like mutagenesis proteins. Various studies support the hypothesis that LexA and λCI cleavage reactions are predominantly intramolecular in nature. The recently described crystal structure of the Escherichia coli UmuD′ protein (the posttranslational cleavage product of the UmuD protein) suggests, however, that the region of the protein corresponding to the cleavage site is at least 50 Å away from the catalytic active site. We considered the possibility, therefore, that the UmuD-like proteins might undergo self-processing that, in contrast to LexA and λCI, occurs via an intermolecular rather than intramolecular reaction. To test this hypothesis, we introduced into E. coli compatible plasmids with mutations at either the cleavage or the catalytic site of three UmuD-like proteins. Cleavage of these proteins only occurs in the presence of both plasmids, indicating that the reaction is indeed intermolecular in nature. Furthermore, this intermolecular reaction is completely dependent upon the multifunctional RecA protein and leads to the restoration of cellular mutagenesis in nonmutable E. coli strains. Intermolecular cleavage of a biotinylated UmuD active site mutant was also observed in vitro in the presence of the wild-type UmuD′ protein, indicating that in addition to the intact UmuD protein, the normal cleavage product (UmuD′) can also act as a classical enzyme. PMID:9465040

  10. Sequencing Lys-N Proteolytic Peptides by ESI and MALDI Tandem Mass Spectrometry

    Science.gov (United States)

    Dupré, Mathieu; Cantel, Sonia; Verdié, Pascal; Martinez, Jean; Enjalbal, Christine

    2011-02-01

    In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion, as a specific cleavage at the amide X-Lys chain is obtained that provides N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a lysine or arginine residue solely at the C-terminal position, and are thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with the ESI-QqTOF and MALDI-TOF/TOF mass spectrometers commonly used in proteomic bottom-up experiments were investigated.

  11. Dominant negative umuD mutations decreasing RecA-mediated cleavage suggest roles for intact UmuD in modulation of SOS mutagenesis

    International Nuclear Information System (INIS)

    Battista, J.R.; Ohta, Toshihiro; Nohmi, Takehiko; Sun, W.; Walker, G.C.

    1990-01-01

    The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli. The UmuD protein shares homology with a family of proteins that includes LexA and several bacteriophage repressors. UmuD is posttranslationally activated for its role n mutagenesis by a RecA-mediated proteolytic cleavage that yields UmuD'. A set of missense mutants of umuD was isolated and shown to encode mutant UmuD proteins that are deficient in RecA-mediated cleavage in vivo. Most of these mutations are dominant to umuD + with respect to UV mutagenesis yet do not interfere with SOS induction. Although both UmuD and UmuD' form homodimers, the authors provide evidence that they preferentially form heterodimers. The relationship of UmuD to LexA, λ repressor, and other members of the family of proteins is discussed and possible roles intact UmuD in modulating SOS mutagenesis are discussed

  12. MHC molecules protect T cell epitopes against proteolytic destruction

    DEFF Research Database (Denmark)

    Mouritsen, S; Meldal, M; Werdelin, O

    1992-01-01

    There is a subtle duality in the role of proteolytic enzymes in Ag processing. They are required to fragment protein Ag ingested by APC. However, prolonged exposure to proteolytic enzymes may lead to a complete degradation of the Ag, leaving nothing for the T cell system to recognize. What ensures...

  13. Multiplex profiling of tumor-associated proteolytic activity in serum of colorectal cancer patients.

    Science.gov (United States)

    Yepes, Diego; Costina, Victor; Pilz, Lothar R; Hofheinz, Ralf; Neumaier, Michael; Findeisen, Peter

    2014-06-01

    The monitoring of tumor-associated protease activity in blood specimens has recently been proposed as new diagnostic tool in cancer research. In this paper, we describe the screening of a peptide library for identification of reporter peptides (RPs) that are selectively cleaved in serum specimens from colorectal cancer patients and investigate the benefits of RP multiplexing. A library of 144 RPs was constructed that contained amino acid sequences of abundant plasma proteins. Proteolytic cleavage of RPs was monitored with MS. Five RPs that were selectively cleaved in serum specimens from tumor patients were selected for further validation in serum specimens of colorectal tumor patients (n = 30) and nonmalignant controls (n = 60). RP spiking and subsequent quantification of proteolytic fragments with LC-MS showed good reproducibility with CVs always below 26%. The linear discriminant analysis and PCA revealed that a combination of RPs for diagnostic classification is superior to single markers. Classification accuracy reached 88% (79/90) when all five markers were combined. Functional protease profiling with RPs might improve the laboratory-based diagnosis, monitoring and prognosis of malignant disease, and has to be evaluated thoroughly in future studies. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. EFFECT OF MARINATION WITH PROTEOLYTIC ENZYMES ON QUALITY OF BEEF MUSCLE

    Directory of Open Access Journals (Sweden)

    Daniela Istrati

    2012-03-01

    Full Text Available During storage and thermal treatment meat suffers a number of biochemical and physical-chemical changes in the substrate protein, changes that take place with varying intensity depending on the method of preservation utilized and temperature of thermal treatment applied. Application of different treatments aimed to influence the proteolytic activity as is the case of enzymatic tenderization of beef.Improving the meat tenderness with proteolytic enzymes is promising, but current legislation restricting the use of proteolytic enzymes from bacterial origin and recommended tenderizers salts containing papain, ficin and bromelain. Recent research revealed that meat marinating before grilling results in a reduction of heterocyclic amine content after thermal treatment. Also, the addition of fruit pulp, garlic or other spices contributes to decreased production of heterocyclic amines because of their antioxidant activity. In the present study was aimed influence of exogenous proteolytic enzymes on adult beef tenderness. To increase the tenderness of adult beef were used exogenous enzymes preparations (papain and bromelain and natural sources of enzymes using pineapple and papaya fruit. It was intended to establish the correlation between enzymatic activity of enzymes used in the study, the processing technology and changes in the physical-chemical and biochemical characteristics that occur during storage in refrigerated conditions (evolution of the rigidity index and water holding capacity, cooking losses and cooking yield of the samples injected/marinated with enzymes.

  15. Proteolytic and antimicrobial activity of lactic acid bacteria grown in goat milk.

    Science.gov (United States)

    Atanasova, Jivka; Moncheva, Penka; Ivanova, Iskra

    2014-11-02

    We examined 62 strains and 21 trade starter cultures from the collection of LB Bulgaricum PLC for proteolytic and antimicrobial activity of lactic acid bacteria (LAB) grown in goat milk. The aim of this study was to investigate the fermentation of caseins, α-lactalbumin and β-lactoglobulin by LAB, using the o -phthaldialdehyde (OPA) spectrophotometric assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The proteolysis targeted mainly caseins, especially β-casein. Whey proteins were proteolyzed, essentially β-lactoglobulin. The proteolytic activity of Lactococcus lactis l598, Streptococcus thermophilus t3D1, Dt1, Lactobacillus lactis 1043 and L. delbrueckii subsp. bulgaricus b38, b122 and b24 was notably high. The proteolysis process gave rise to medium-sized peptide populations. Most of the examined strains showed antimicrobial activity against some food pathogens, such as Escherichia coli , Staphylococcus aureus , Salmonella cholere enteridis , Listeria monocytogenes , Listeria innocua and Enterobacter aerogenes . The most active producers of antimicrobial-active peptides were strains of L. delbrueckii subsp. bulgaricus and S. thermophilus , which are of practical importance. The starter cultures containing the examined species showed high proteolytic and antimicrobial activity in skimmed goat milk. The greatest antimicrobial activity of the cultures was detected against E. aerogenes . The obtained results demonstrated the significant proteolytic potential of the examined strains in goat milk and their potential for application in the production of dairy products from goat's milk. The present results could be considered as the first data on the proteolytic capacity of strains and starter cultures in goat milk for the purposes of trade interest of LB Bulgaricum PLC.

  16. In vitro measurement of beta-carotene cleavage activity : methodological considerations and the effect of other carotenoids on beta-carotene cleavage

    NARCIS (Netherlands)

    Vliet, T. van; Schaik, F. van; Schreurs, W.H.P.; Berg, H. van den

    1996-01-01

    In view of controversies about assessment of the β-carotene cleavage activity, methodological aspects and problems of the dioxygenase assay are described. Using rat and hamster intestinal preparations the method was optimized on retinal formation, the only cleavage product we could demonstrate. It

  17. Fe-Catalyzed Oxidative Cleavage of Unsaturated Fatty Acids

    NARCIS (Netherlands)

    Spannring, P.

    2013-01-01

    The oxidative cleavage of unsaturated fatty acids into aldehydes or carboxylic acids gives access to valuable products. The products can be used as chemical building blocks, as emulsifiers or in the paint or polymer industry. Ozonolysis is applied industrially to cleave the fatty acid oleic acid

  18. 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/.

  19. Proteolytic activity of prostate-specific antigen (PSA towards protein substrates and effect of peptides stimulating PSA activity.

    Directory of Open Access Journals (Sweden)

    Johanna M Mattsson

    Full Text Available Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3 exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.

  20. Proteolytic Activity of Prostate-Specific Antigen (PSA) towards Protein Substrates and Effect of Peptides Stimulating PSA Activity

    Science.gov (United States)

    Mattsson, Johanna M.; Ravela, Suvi; Hekim, Can; Jonsson, Magnus; Malm, Johan; Närvänen, Ale; Stenman, Ulf-Håkan; Koistinen, Hannu

    2014-01-01

    Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA. PMID:25237904

  1. Functional characterization of the proteolytic activity of the tomato black ring nepovirus RNA-1-encoded polyprotein.

    Science.gov (United States)

    Hemmer, O; Greif, C; Dufourcq, P; Reinbolt, J; Fritsch, C

    1995-01-10

    Translation of tomato black ring virus (TBRV) RNA-1 in a rabbit reticulocyte lysate leads to the synthesis of a 250K polyprotein which cleaves itself into smaller proteins of 50, 60, 120, and 190K. Polypeptides synthesized from synthetic transcripts corresponding to different regions of TBRV RNA-1 are processed only when they encode the 23K protein delimited earlier by sequence homology with the cowpea mosaic virus 24K protease. The proteolytic activity of this protein is completely lost by mutating residues C170 (to I) or L188 (to H), residues which align with conserved residues of the viral serine-like proteases. The 120K protein is generated by cleavage of the dipeptide K/A localized in front of the VPg but is not further cleaved in vitro at the K/S site (at the C terminus of the VPg) or between the protease and polymerase domains. However, both the protein VPgProPol (120K) and the protein ProPol (117K) produced in vitro from synthetic transcripts can cleave in trans the RNA-2-encoded 150K polyprotein, but they cannot cleave in trans polypeptides containing a cleavage site expressed from RNA-1 transcripts in which the protease cistron is absent or modified.

  2. Pripper: prediction of caspase cleavage sites from whole proteomes

    Directory of Open Access Journals (Sweden)

    Salmi Jussi

    2010-06-01

    Full Text Available Abstract Background Caspases are a family of proteases that have central functions in programmed cell death (apoptosis and inflammation. Caspases mediate their effects through aspartate-specific cleavage of their target proteins, and at present almost 400 caspase substrates are known. There are several methods developed to predict caspase cleavage sites from individual proteins, but currently none of them can be used to predict caspase cleavage sites from multiple proteins or entire proteomes, or to use several classifiers in combination. The possibility to create a database from predicted caspase cleavage products for the whole genome could significantly aid in identifying novel caspase targets from tandem mass spectrometry based proteomic experiments. Results Three different pattern recognition classifiers were developed for predicting caspase cleavage sites from protein sequences. Evaluation of the classifiers with quality measures indicated that all of the three classifiers performed well in predicting caspase cleavage sites, and when combining different classifiers the accuracy increased further. A new tool, Pripper, was developed to utilize the classifiers and predict the caspase cut sites from an arbitrary number of input sequences. A database was constructed with the developed tool, and it was used to identify caspase target proteins from tandem mass spectrometry data from two different proteomic experiments. Both known caspase cleavage products as well as novel cleavage products were identified using the database demonstrating the usefulness of the tool. Pripper is not restricted to predicting only caspase cut sites, but it gives the possibility to scan protein sequences for any given motif(s and predict cut sites once a suitable cut site prediction model for any other protease has been developed. Pripper is freely available and can be downloaded from http://users.utu.fi/mijopi/Pripper. Conclusions We have developed Pripper, a tool for

  3. PROTEOLYTIC AND FIBRINOLYTIC ACTIVITIES OF HALOPHILIC LACTIC ACID BACTERIA FROM TWO INDONESIAN FERMENTED FOODS

    Directory of Open Access Journals (Sweden)

    Asep A. Prihanto

    2013-04-01

    Full Text Available Exploration of fermented foods as sources of fibrinolytic enzymes is increased in the last decades. Terasi and Jambal roti is Indonesian traditional fermented fish products, which were famous in Java Island. Both are important products in Indonesian dishes, especially in Java. Investigation on halophilic lactic acid bacteria using MRS and M-17 agar obtained seventy four isolated strains. Their proteolytic and fibrinolytic activities were determined using skim milk agar and plasminogen-free fibrin plate. Twenty five isolates showed protease activities, while only four of them secreted fibrinolitic enzyme. The highest proteolytic and fibrinolytic activity was shown by TB1 strain, which is identified as Bacillus coagulans. The 16s rDNA is still in investigating to confirm the TB1 strain identity.

  4. Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2 element

    Directory of Open Access Journals (Sweden)

    Kurth Reinhard

    2011-05-01

    Full Text Available Abstract Background The human genome harbors several largely preserved HERV-K(HML-2 elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2 virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2 Gag proteins and the exact position of the cleavage sites have until now remained unknown. Results By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs. The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2 is processed to yield p15-MA (matrix, SP1 (spacer peptide of 14 amino acids, p15, p27-CA (capsid, p10-NC (nucleocapsid and two

  5. Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2) element.

    Science.gov (United States)

    George, Maja; Schwecke, Torsten; Beimforde, Nadine; Hohn, Oliver; Chudak, Claudia; Zimmermann, Anja; Kurth, Reinhard; Naumann, Dieter; Bannert, Norbert

    2011-05-09

    The human genome harbors several largely preserved HERV-K(HML-2) elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs) for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2) virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2) Gag proteins and the exact position of the cleavage sites have until now remained unknown. By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs). The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2) is processed to yield p15-MA (matrix), SP1 (spacer peptide of 14 amino acids), p15, p27-CA (capsid), p10-NC (nucleocapsid) and two C-terminally encoded glutamine- and

  6. Activation of human monocytes by proteolytic fragments of gliadin

    Czech Academy of Sciences Publication Activity Database

    Jelínková, Lenka; Tučková, Ludmila; Cinová, Jana; Cimburek, Zdeněk; Tlaskalová, Helena

    2003-01-01

    Roč. 87, č. 1 (2003), s. 08 ISSN 0165-2478 Institutional research plan: CEZ:AV0Z5020903 Keywords : proteolytic * fragments * gliadin Subject RIV: EE - Microbiology, Virology Impact factor: 1.710, year: 2003

  7. [Inhibitors of proteolytic enzymes under abiotic stresses in plants (review)].

    Science.gov (United States)

    Mosolov, V V; Valueva, T A

    2011-01-01

    Data on the role of proteolytic enzyme inhibitors in plant adaptation to various unfavorable environmental abiotic factors--water deficiency, salinization of soil, extreme temperatures, etc.--and also probable functions of proteinases inhibitors in natural plant senescense are considered.

  8. Research Applications of Proteolytic Enzymes in Molecular Biology

    OpenAIRE

    Mótyán, János András; Tóth, Ferenc; Tőzsér, József

    2013-01-01

    Proteolytic enzymes (also termed peptidases, proteases and proteinases) are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications ...

  9. Proteolytic and lipolytic microbiota of refrigerated raw milk from northeast and southern regions of Brazil

    Directory of Open Access Journals (Sweden)

    Jose Carlos Ribeiro Junior

    2015-12-01

    Full Text Available The shelf life of milk and milk derivatives is directly related to the microbiological quality of refrigerated raw milk. Spoilage microorganisms with proteolytic and/or lipolytic properties are primarily responsible for the decrease in the quality of milk, which is reflected in the shelf life of pasteurized milk and all derivatives. The aim of this study was to determine the spoilage microbial load of refrigerated raw milk from the northeast and southern regions of Brazil, which have different climatic and technological conditions of production. We evaluated 46 samples of milk from the state of Paraná in the southern region, and 10 samples of milk from the state of Maranhão in the northeast region, totaling 56 samples collected from November 2013 to November 2014. The producers of Paraná were divided into large (20 or small (26 according to the average daily production. All producers of Maranhão were considered small (<500L/day. The proteolytic and lipolytic microorganism counts were conducted in milk agar and tributyrin agar, respectively. Milk from the large producers of Paraná had average counts of 1.4 × 104 CFU/mL for proteolytic microorganisms and 1.2 × 103 CFU/mL for lipolytics microorganisms, significantly (p <0.05 lower than the small producers in the same state, and the producers of Maranhão. Producers of Maranhao had counts of 1.1 × 105 CFU/mL for proteolytic microorganisms and 2 × 105 CFU/mL for lipolytic microorganisms, with the proteolytic count significantly lower than that of small Paraná producers. The amount of proteolytic and lipolytic spoilage microorganisms in milk is influenced by the adaptation of the microorganisms to cold, promoted by the cooling of milk, which is practiced less frequently in the country’s northeastern region. The amount of spoilage microorganisms is also affected by the implementation of milking hygiene practices, which reduce contamination. Such practices are more frequently and efficiently

  10. Neuron-Derived ADAM10 Production Stimulates Peripheral Nerve Injury-Induced Neuropathic Pain by Cleavage of E-Cadherin in Satellite Glial Cells.

    Science.gov (United States)

    Li, Jian; Ouyang, Qing; Chen, Cheng-Wen; Chen, Qian-Bo; Li, Xiang-Nan; Xiang, Zheng-Hua; Yuan, Hong-Bin

    2017-09-01

    Increasing evidence suggests the potential involvement of metalloproteinase family proteins in the pathogenesis of neuropathic pain, although the underlying mechanisms remain elusive. Using the spinal nerve ligation model, we investigated whether ADAM10 proteins participate in pain regulation. By implementing invitro methods, we produced a purified culture of satellite glial cells to study the underlying mechanisms of ADAM10 in regulating neuropathic pain. Results showed that the ADAM10 protein was expressed in calcitonin gene-related peptide (CGRP)-containing neurons of the dorsal root ganglia, and expression was upregulated following spinal nerve ligation surgery invivo. Intrathecal administration of GI254023X, an ADAM10 selective inhibitor, to the rats one to three days after spinal nerve ligation surgery attenuated the spinal nerve ligation-induced mechanical allodynia and thermal hyperalgesia. Intrathecal injection of ADAM10 recombinant protein simulated pain behavior in normal rats to a similar extent as those treated by spinal nerve ligation surgery. These results raised a question about the relative contribution of ADAM10 in pain regulation. Further results showed that ADAM10 might act by cleaving E-cadherin, which is mainly expressed in satellite glial cells. GI254023X reversed spinal nerve ligation-induced downregulation of E-cadherin and activation of cyclooxygenase 2 after spinal nerve ligation. β-catenin, which creates a complex with E-cadherin in the membranes of satellite glial cells, was also downregulated by spinal nerve ligation surgery in satellite glial cells. Finally, knockdown expression of β-catenin by lentiviral infection in purified satellite glial cells increased expression of inducible nitric oxide synthase and cyclooxygenase 2. Our findings indicate that neuron-derived ADAM10 production stimulates peripheral nerve injury-induced neuropathic pain by cleaving E-cadherin in satellite glial cells. © 2017 American Academy of Pain Medicine

  11. Physical-chemical and biocatalytic properties of a proteolytic complex of the preparation "Protepsin"

    Directory of Open Access Journals (Sweden)

    L. V. Antipova

    2016-01-01

    Full Text Available Enzymatic technologies were included strongly into practical activities of the person, the volume of the world market constantly grows and is updated. However the domestic production of enzymatic preparations very lags behind world level that is in many respects connected with insufficient scientific and technical base for a wide circulation of technologies in large-scale production. At the same time there were Russian producers of enzymatic preparations from animal fabrics and bodies for processing of raw materials of an animal origin, according to forecasts, of interest in rational use of resources of an animal origin. In article data on research of properties of the enzymatic preparation "Protepsin" and an assessment of prospects of application are provided in processing of raw materials of an animal origin. The enzymatic preparation "Protepsin" made in the conditions of JSC Plant of Endocrine Enzymes (Rzhavki, Moscow region activity at action on proteins of meat shows, including the strengthened structure, has milk-clotting effect, is active in the field of pH 4,0-6,0 and temperature 20-45zs. The proteinaceous complex includes 4 fractions, 2 from which possess the general proteolytic activity. One of them shows the general proteolytic and milk-clotting activity. Enzymes differ in an amino-acid set and molecular weight. The method of a disk electrophoresis determined molecular-mass structure of "Protepsin". The preparation inactivation conditions guaranteeing its safety in the production technology of foodstuff as active proteolytic enzymes in the course of digestion can cause violations of integrity of fabrics and corresponding diseases are shown. Thus, conditions of use of a perspective proteolytic preparation in technology of a wide range of food of an animal origin are in a complex proved and picked up.

  12. Autoactivation of mouse trypsinogens is regulated by chymotrypsin C via cleavage of the autolysis loop.

    Science.gov (United States)

    Németh, Balázs Csaba; Wartmann, Thomas; Halangk, Walter; Sahin-Tóth, Miklós

    2013-08-16

    Chymotrypsin C (CTRC) is a proteolytic regulator of trypsinogen autoactivation in humans. CTRC cleavage of the trypsinogen activation peptide stimulates autoactivation, whereas cleavage of the calcium binding loop promotes trypsinogen degradation. Trypsinogen mutations that alter these regulatory cleavages lead to increased intrapancreatic trypsinogen activation and cause hereditary pancreatitis. The aim of this study was to characterize the regulation of autoactivation of mouse trypsinogens by mouse Ctrc. We found that the mouse pancreas expresses four trypsinogen isoforms to high levels, T7, T8, T9, and T20. Only the T7 activation peptide was cleaved by mouse Ctrc, causing negligible stimulation of autoactivation. Surprisingly, mouse Ctrc poorly cleaved the calcium binding loop in all mouse trypsinogens. In contrast, mouse Ctrc readily cleaved the Phe-150-Gly-151 peptide bond in the autolysis loop of T8 and T9 and inhibited autoactivation. Mouse chymotrypsin B also cleaved the same peptide bond but was 7-fold slower. T7 was less sensitive to chymotryptic regulation, which involved slow cleavage of the Leu-149-Ser-150 peptide bond in the autolysis loop. Modeling indicated steric proximity of the autolysis loop and the activation peptide in trypsinogen, suggesting the cleaved autolysis loop may directly interfere with activation. We conclude that autoactivation of mouse trypsinogens is under the control of mouse Ctrc with some notable differences from the human situation. Thus, cleavage of the trypsinogen activation peptide or the calcium binding loop by Ctrc is unimportant. Instead, inhibition of autoactivation via cleavage of the autolysis loop is the dominant mechanism that can mitigate intrapancreatic trypsinogen activation.

  13. 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

  14. The tryptic cleavage product of the mature form of the bovine desmoglein 1 ectodomain is one of the antigen moieties immunoprecipitated by all sera from symptomatic patients affected by a new variant of endemic pemphigus.

    Science.gov (United States)

    Abréu-Vélez, Ana María; Javier Patiño, Pablo; Montoya, Fernando; Bollag, Wendy B

    2003-01-01

    Multiple antigens are recognized by sera from patients with pemphigus foliaceus (PF). Several have been identified including keratin 59, desmocollins, envoplakin, periplakin, and desmogleins 1 and 3 (Dsg1 and Dsg3). In addition, an 80 kDa antigen was identified as the N-terminal fragment of Dsg1 using as antigen source an insoluble epidermal cell envelope preparation. However, still unsolved was the identity of the most important antigenic moiety, a 45 kDa tryptic fragment which is recognized by all sera from patients with fogo selvagem, pemphigus foliaceus, by half of pemphigus vulgaris sera and by a new variant of endemic pemphigus in E1 Bagre, Colombia that resembles Senear-Usher syndrome. Here, we report the identification of the 45 kDa conformational epitope of a soluble tryptic cleavage product from viable bovine epidermis. To elucidate the nature of this peptide, viable bovine epidermis was trypsin-digested, and glycosylated peptides were partially purified on a concanavalin A (Con-A) affinity column. This column fraction was then used as an antigen source for further immunoaffinity purification. A PF patient's serum covalently coupled to a Staphylococcus aureus protein A column was incubated with the Con-A eluted products and the immuno-isolated antigen was separated by SDS-PAGE, transferred to a membrane, and visualized with Coomassie blue, silver and amido black stains. The 45 kD band was subjected to amino acid sequence analysis revealing the sequence, EXIKFAAAXREGED, which matched the mature form of the extracellular domain of bovine Dsg1. This study confirms the biological importance of the ectodomain of Dsg1 as well as the relevance of conformational epitopes in various types of pemphigus.

  15. Influence of autoclaved saprotrophic fungal mycelia on proteolytic activity in ectomycorrhizal fungi.

    Science.gov (United States)

    Mucha, Joanna; Dahm, Hanna; Werner, Antoni

    2007-07-01

    The production of proteolytic enzymes by several strains of ectomycorrhizal fungi i.e., Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), Suillus bovinus (15-3), Suillus luteus (14-7) on mycelia of Trichoderma harzianum, Trichoderma virens and Mucor hiemalis and sodium caseinate, yeast extract was evaluated. The strains of A. muscaria (16-3) and L. laccata (9-12) were characterized by the highest activity of the acidic and neutral proteases. Taking the mycelia of saprotrophic fungi into consideration, the mycelium of M. hiemalis was the best inductor for proteolytic activity. The examined ectomycorrhizal fungi exhibited higher activity of acidic proteases than neutral ones on the mycelia of saprotrophic fungi, which may imply the participation of acidic proteases in nutrition.

  16. Extracellular proteolytic enzymes produced by human pathogenic Vibrio species

    Directory of Open Access Journals (Sweden)

    Shin-Ichi eMiyoshi

    2013-11-01

    Full Text Available Bacteria in the genus Vibrio produce extracellular proteolytic enzymes to obtain nutrients via digestion of various protein substrates. However, the enzymes secreted by human pathogenic species have been documented to modulate the bacterial virulence. Several species including Vibrio cholerae and V. vulnificus are known to produce thermolysin-like metalloproteases termed vibriolysin. The vibriolysin from V. vulnificus, a causative agent of serious systemic infection, is a major toxic factor eliciting the secondary skin damage characterized by formation of the hemorrhagic brae. The vibriolysin from intestinal pathogens may play indirect roles in pathogenicity because it can activate protein toxins and hemagglutinin by the limited proteolysis and can affect the bacterial attachment to or detachment from the intestinal surface by degradation of the mucus layer. Two species causing wound infections, V. alginolyticus and V. parahaemolyticus, produce another metalloproteases so-called collagenases. Although the detailed pathological roles have not been studied, the collagenase is potent to accelerate the bacterial dissemination through digestion of the protein components of the extracellular matrix. Some species produce cymotrypsin-like serine proteases, which may also affect the bacterial virulence potential. The intestinal pathogens produce sufficient amounts of the metalloprotease at the small intestinal temperature; however, the metalloprotease production by extra-intestinal pathogens is much higher around the body surface temperature. On the other hand, the serine protease is expressed only in the absence of the metalloprotease.

  17. Screening for Proteolytic Activities in Snake Venom by Means of a Multiplexing ESI-MS Assay Scheme

    NARCIS (Netherlands)

    Liesener, A.; Perchuc, Anna-Maria; Schöni, Reto; Wilmer, Marianne; Karst, U.

    2005-01-01

    A multiplexed mass spectrometry based assay scheme for the simultaneous determination of five different substrate/product pairs was developed as a tool for screening of proteolytic activities in snake venom fractions from Bothrops moojeni. The assay scheme was employed in the functional

  18. The Zymogen-Enteropeptidase System: A Practical Approach to Study the Regulation of Enzyme Activity by Proteolytic Cleavage

    Science.gov (United States)

    Pizauro, Joao M., Jr.; Ferro, Jesus A.; de Lima, Andrea C. F.; Routman, Karina S.; Portella, Maria Celia

    2004-01-01

    The present research describes an efficient procedure to obtain high levels of trypsinogen and chymotrypsinogen by using a simple, rapid, and easily reproducible method. The extraction process and the time-course of activation of zymogens can be carried out in a single laboratory period, without sophisticated equipment. The main objective was to…

  19. 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.

  20. Cleavage and creep fracture of rock salt

    International Nuclear Information System (INIS)

    Chan, K.S.; Munson, D.E.; Bodner, S.R.

    1996-01-01

    The dominant failure mechanism in rock salt at ambient temperature is either cleavage or creep fracture. Since the transition of creep fracture to cleavage in a compressive stress field is not well understood, failure of rock salt by cleavage and creep fracture is analyzed in this paper to elucidate the effect of stress state on the competition between these two fracture mechanisms. For cleavage fracture, a shear crack is assumed to cause the formation and growth of a symmetric pair of wing cracks in a predominantly compressive stress field. The conditions for wing-crack instability are derived and presented as the cleavage fracture boundary in the fracture mechanism map. Using an existing creep fracture model, stress conditions for the onset of creep fracture and isochronous failure curves of specified times-to-rupture are calculated and incorporated into the fracture mechanism map. The regimes of dominance by cleavage and creep fracture are established and compared with experimental data. The result indicates that unstable propagation of cleavage cracks occurs only in the presence of tensile stress. The onset of creep fracture is promoted by a tensile stress, but can be totally suppressed by a high confining pressure. Transition of creep fracture to cleavage occurs when critical conditions of stress difference and tensile stress for crack instability are exceeded

  1. Post-transcription cleavage generates the 3' end of F17R transcripts in vaccinia virus

    International Nuclear Information System (INIS)

    D'Costa, Susan M.; Antczak, James B.; Pickup, David J.; Condit, Richard C.

    2004-01-01

    Most vaccinia virus intermediate and late mRNAs possess 3' ends that are extremely heterogeneous in sequence. However, late mRNAs encoding the cowpox A-type inclusion protein (ATI), the second largest subunit of the RNA polymerase, and the late telomeric transcripts possess homogeneous 3' ends. In the case of the ATI mRNA, it has been shown that the homogeneous 3' end is generated by a post-transcriptional endoribonucleolytic cleavage event. We have determined that the F17R gene also produces homogeneous transcripts generated by a post-transcriptional cleavage event. Mapping of in vivo mRNA shows that the major 3' end of the F17R transcript maps 1262 nt downstream of the F17R translational start site. In vitro transcripts spanning the in vivo 3' end are cleaved in an in vitro reaction using extracts from virus infected cells, and the site of cleavage is the same both in vivo and in vitro. Cleavage is not observed using extract from cells infected in the presence of hydroxyurea; therefore, the cleavage factor is either virus-coded or virus-induced during the post-replicative phase of virus replication. The cis-acting sequence responsible for cleavage is orientation specific and the factor responsible for cleavage activity has biochemical properties similar to the factor required for cleavage of ATI transcripts. Partially purified cleavage factor generates cleavage products of expected size when either the ATI or F17R substrates are used in vitro, strongly suggesting that cleavage of both transcripts is mediated by the same factor

  2. 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

  3. Microstructure and cleavage in lath martensitic steels

    International Nuclear Information System (INIS)

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

    2013-01-01

    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. (paper)

  4. Proteolytic fragmentation of inositol 1,4,5-trisphosphate receptors: a novel mechanism regulating channel activity?

    Science.gov (United States)

    Wang, Liwei; Alzayady, Kamil J; Yule, David I

    2016-06-01

    Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are a family of ubiquitously expressed intracellular Ca(2+) release channels. Regulation of channel activity by Ca(2+) , nucleotides, phosphorylation, protein binding partners and other cellular factors is thought to play a major role in defining the specific spatiotemporal characteristics of intracellular Ca(2+) signals. These properties are, in turn, believed pivotal for the selective and specific physiological activation of Ca(2+) -dependent effectors. IP3 Rs are also substrates for the intracellular cysteine proteases, calpain and caspase. Cleavage of the IP3 R has been proposed to play a role in apoptotic cell death by uncoupling regions important for IP3 binding from the channel domain, leaving an unregulated leaky Ca(2+) pore. Contrary to this hypothesis, we demonstrate following proteolysis that N- and C-termini of IP3 R1 remain associated, presumably through non-covalent interactions. Further, we show that complementary fragments of IP3 R1 assemble into tetrameric structures and retain their ability to be regulated robustly by IP3 . While peptide continuity is clearly not necessary for IP3 -gating of the channel, we propose that cleavage of the IP3 R peptide chain may alter other important regulatory events to modulate channel activity. In this scenario, stimulation of the cleaved IP3 R may support distinct spatiotemporal Ca(2+) signals and activation of specific effectors. Notably, in many adaptive physiological events, the non-apoptotic activities of caspase and calpain are demonstrated to be important, but the substrates of the proteases are poorly defined. We speculate that proteolytic fragmentation may represent a novel form of IP3 R regulation, which plays a role in varied adaptive physiological processes. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  5. Beta-scission of alkoxyl radicals on peptides and proteins can give rise to backbone cleavage and loss of side-chains

    International Nuclear Information System (INIS)

    Headlam, H.A.; Davies, M.J.; Mortimer, A.; Easton, C.J.

    2000-01-01

    Full text: Exposure of proteins to radicals in the presence of O 2 brings about multiple changes including side-chain oxidation, backbone fragmentation, cross-linking, unfolding, changes in hydrophobicity and conformation, altered susceptibility to proteolytic enzymes and formation of new reactive groups (e.g. hydroperoxides and 3,4-dihydroxyphenylalanine). All of these processes can result in loss of structural or enzymatic activity. The mechanisms that give rise to backbone cleavage are only partly understood. Whilst it is known that direct hydrogen atom abstraction at a-carbon sites gives backbone cleavages it has also been proposed that initial attack at side-chain sites might also give rise to backbone cleavage. In this study we have examined whether initial attack at the β- (C-3) position can give rise to α-carbon radicals (and hence backbone cleavage) via the formation, and subsequent β- scission, of C-3 alkoxyl radicals. This process has been observed previously with protected amino acids in organic solvents (J. Chem. Soc. Perkin Trans. 2, 1997, 503-507) but the occurrence of such reactions with proteins in aqueous solution has not been explored. Alkoxyl radicals were generated at the C-3 position of a variety of protected amino acids and small peptides by two methods: metal-ion catalysed decomposition of hydroperoxides formed as a result of γ-radiolysis in the presence of O 2 , and UV photolysis of nitrate esters. In most cases radicals have been detected by EPR spectroscopy using nitroso and nitrone spin traps, which can be assigned by comparison with literature data to α-carbon radicals; in some case assignments were confirmed by the generation of the putative species by other routes. With Ala peptide hydroperoxides and nitrate esters, and MNP as the spin trap, the major radical detected in each case has been assigned to the adduct of an α-carbon radical with partial structure - NH- . CH-C(O) - consistent with the rapid occurrence of the above

  6. Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity

    International Nuclear Information System (INIS)

    Du, Lanying; Kao, Richard Y.; Zhou, Yusen; He, Yuxian; Zhao, Guangyu; Wong, Charlotte; Jiang, Shibo; Yuen, Kwok-Yung; Jin, Dong-Yan; Zheng, Bo-Jian

    2007-01-01

    The spike (S) protein of SARS coronavirus (SARS-CoV) has been known to recognize and bind to host receptors, whose conformational changes then facilitate fusion between the viral envelope and host cell membrane, leading to viral entry into target cells. However, other functions of SARS-CoV S protein such as proteolytic cleavage and its implications to viral infection are incompletely understood. In this study, we demonstrated that the infection of SARS-CoV and a pseudovirus bearing the S protein of SARS-CoV was inhibited by a protease inhibitor Ben-HCl. Also, the protease Factor Xa, a target of Ben-HCl abundantly expressed in infected cells, was able to cleave the recombinant and pseudoviral S protein into S1 and S2 subunits, and the cleavage was inhibited by Ben-HCl. Furthermore, this cleavage correlated with the infectivity of the pseudovirus. Taken together, our study suggests a plausible mechanism by which SARS-CoV cleaves its S protein to facilitate viral infection

  7. Effects of Sunphenon and Polyphenon 60 on proteolytic pathways ...

    Indian Academy of Sciences (India)

    2016-08-26

    Aug 26, 2016 ... The effect of Sunphenon and Polyphenon 60 in oxidative stress response, myogenic regulatory factors, inflammatory cytokines, apoptotic and proteolytic pathways on H2O2-induced myotube atrophy was addressed. Cellular responses of H2O2-induced C2C12cells were examined, including mRNA ...

  8. Sirtuins and Proteolytic Systems: Implications for Pathogenesis of Synucleinopathies

    Directory of Open Access Journals (Sweden)

    Belém Sampaio-Marques

    2015-05-01

    Full Text Available Insoluble and fibrillar forms of α-synuclein are the major components of Lewy bodies, a hallmark of several sporadic and inherited neurodegenerative diseases known as synucleinopathies. α-Synuclein is a natural unfolded and aggregation-prone protein that can be degraded by the ubiquitin-proteasomal system and the lysosomal degradation pathways. α-Synuclein is a target of the main cellular proteolytic systems, but it is also able to alter their function further, contributing to the progression of neurodegeneration. Aging, a major risk for synucleinopathies, is associated with a decrease activity of the proteolytic systems, further aggravating this toxic looping cycle. Here, the current literature on the basic aspects of the routes for α-synuclein clearance, as well as the consequences of the proteolytic systems collapse, will be discussed. Finally, particular focus will be given to the sirtuins’s role on proteostasis regulation, since their modulation emerged as a promising therapeutic strategy to rescue cells from α-synuclein toxicity. The controversial reports on the potential role of sirtuins in the degradation of α-synuclein will be discussed. Connection between sirtuins and proteolytic systems is definitely worth of further studies to increase the knowledge that will allow its proper exploration as new avenue to fight synucleinopathies.

  9. Genetics of the proteolytic system of lactic acid bacteria

    NARCIS (Netherlands)

    Kok, Jan

    1990-01-01

    The proteolytic system of lactic acid bacteria is of eminent importance for the rapid growth of these organisms in protein-rich media. The combined action of proteinases and peptidases provides the cell with small peptides and essential amino acids. The amino acids and peptides thus liberated have

  10. Factors affecting proteolytic action of Lactococcus lactis in cheese

    NARCIS (Netherlands)

    Youssef, Y.B.

    1992-01-01

    Model cheeses were developed to study the behaviour of proteolytic agents involved in cheese maturation under conditions that closely resemble those in normal cheese. The models were applied to study protein breakdown by Lactococcus lactis ssp. cremoris HP , as a

  11. Proteolytic activation of proapoptotic kinase protein kinase Cδ by tumor necrosis factor α death receptor signaling in dopaminergic neurons during neuroinflammation

    Directory of Open Access Journals (Sweden)

    Gordon Richard

    2012-04-01

    Full Text Available Abstract Background The mechanisms of progressive dopaminergic neuronal loss in Parkinson’s disease (PD remain poorly understood, largely due to the complex etiology and multifactorial nature of disease pathogenesis. Several lines of evidence from human studies and experimental models over the last decade have identified neuroinflammation as a potential pathophysiological mechanism contributing to disease progression. Tumor necrosis factor α (TNF has recently emerged as the primary neuroinflammatory mediator that can elicit dopaminergic cell death in PD. However, the signaling pathways by which TNF mediates dopaminergic cell death have not been completely elucidated. Methods In this study we used a dopaminergic neuronal cell model and recombinant TNF to characterize intracellular signaling pathways activated during TNF-induced dopaminergic neurotoxicity. Etanercept and neutralizing antibodies to tumor necrosis factor receptor 1 (TNFR1 were used to block TNF signaling. We confirmed the results from our mechanistic studies in primary embryonic mesencephalic cultures and in vivo using the stereotaxic lipopolysaccharide (LPS model of nigral dopaminergic degeneration. Results TNF signaling in dopaminergic neuronal cells triggered the activation of protein kinase Cδ (PKCδ, an isoform of the novel PKC family, by caspase-3 and caspase-8 dependent proteolytic cleavage. Both TNFR1 neutralizing antibodies and the soluble TNF receptor Etanercept blocked TNF-induced PKCδ proteolytic activation. Proteolytic activation of PKCδ was accompanied by translocation of the kinase to the nucleus. Notably, inhibition of PKCδ signaling by small interfering (siRNA or overexpression of a PKCδ cleavage-resistant mutant protected against TNF-induced dopaminergic neuronal cell death. Further, primary dopaminergic neurons obtained from PKCδ knockout (−/− mice were resistant to TNF toxicity. The proteolytic activation of PKCδ in the mouse substantia nigra in the

  12. Drosha regulates gene expression independently of RNA cleavage function

    DEFF Research Database (Denmark)

    Gromak, Natalia; Dienstbier, Martin; Macias, Sara

    2013-01-01

    Drosha is the main RNase III-like enzyme involved in the process of microRNA (miRNA) biogenesis in the nucleus. Using whole-genome ChIP-on-chip analysis, we demonstrate that, in addition to miRNA sequences, Drosha specifically binds promoter-proximal regions of many human genes in a transcription......-dependent manner. This binding is not associated with miRNA production or RNA cleavage. Drosha knockdown in HeLa cells downregulated nascent gene transcription, resulting in a reduction of polyadenylated mRNA produced from these gene regions. Furthermore, we show that this function of Drosha is dependent on its N......-terminal protein-interaction domain, which associates with the RNA-binding protein CBP80 and RNA Polymerase II. Consequently, we uncover a previously unsuspected RNA cleavage-independent function of Drosha in the regulation of human gene expression....

  13. Characterization of sodium dodecyl sulfate-resistant proteolytic activity in the hyperthermophilic archaebacterium Pyrococcus furiosus

    Energy Technology Data Exchange (ETDEWEB)

    Blumentals, I.I.; Robinson, A.S.; Kelly, R.M. (Johns Hopkins Univ., Baltimore, MD (USA))

    1990-07-01

    Cell extracts from Pyrococcus furiosus were found to contain five proteases, two of which (S66 and S102) are resistant to sodium dodecyl sulfate (SDS) denaturation. Cell extracts incubated at 98{degree}C in the presence of 1% SDS for 24 h exhibited substantial cellular proteolysis such that only four proteins could be visualized by amido black-Coomassie brilliant blue staining of SDS-polyacrylamide gels. The SDS-treated extract retained 19% of the initial proteolytic activity as represented by two proteases, S66 (66 kilodaltons (kDa)) and S102 (102 kDa). Immunoblot analysis with guinea pig sera containing antibodies against protease S66 indicated that S66 is related neither to S102 nor to the other proteases. The results of this analysis also suggest that S66 might be the hydrolysis product of a 200-kDa precursor which does not have proteolytic activity. The 24-h SDS-treated extract showed unusually thermostable proteolytic activity; the measured half-life at 98{degree}C was found to be 33 h. Proteases S66 and S102 were also resistant to denaturation by 8 M urea, 80 mM dithiothreitol, and 5% {beta}-mercaptoethanol. Purified protease S66 was inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate but not by EDTA, ethylene glycol-bis({beta}-aminoethyl ether)-N,N,N{prime},N{prime}-tetraacetic acid, or iodoacetic acid. These results indicate that S66 is a serine protease. Amino acid ester hydrolysis studies showed that protease S66 was hydrolytically active towards N-benzoyl-L-arginine ethyl ester.

  14. Inhibition of proteolytic ferments by taurin in radiation sickness

    International Nuclear Information System (INIS)

    Dokshina, G.A.; Klimova, A.D.; Yartsev, E.I.; Yakovlev, V.G.

    1975-01-01

    The application of taurin potassium phosphate (TKPh) as radioprotective remedy resulted in a considerable influence on the activity of the proteolytic ferments in the irradiated organism. White male rats with a weight of 160 to 180 g being kept in cages without any food for 12 hours before this experiment were irradiated with a gamma unit GUM-Co-60 with a dose of 700 rad (LDsub(70/30)). A 4% solution of the preparation was injected in an amount of 40 mg per rat 1, 3, 5, 7 days after irradiation. Under the effect of ionizing radiation there was a progredient increase of proteolytic ferment activity of liver and spleen which was detected already 30 min after irradiation with maximum rate of proteolysis on the 21st day. After injection of the preparation a two-phase reaction developed: on the 7th to 12th day an increased activity of cathepsins in the tissue and in the following time up to the 30th day of observation an inhibition of ferment activity was demonstrated. Simultaneously it was found that the radiation-induced corticosteroid level was prevented by the preparation. A similar effect was also shown by TKPh inhibiting proteolytic ferment activity in experiments with rats with preceding application of hydrocortisone in high doses. The obtained results permit the assumption that the radioprotective activity of taurin comes about by its effect in the direction of structural integrity of the cell membranes leading to a normalization of the hormonal and fermentative events

  15. 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.

  16. Dopaminergic neurotoxicant 6-OHDA induces oxidative damage through proteolytic activation of PKCδ in cell culture and animal models of Parkinson's disease

    International Nuclear Information System (INIS)

    Latchoumycandane, Calivarathan; Anantharam, Vellareddy; Jin, Huajun; Kanthasamy, Anumantha; Kanthasamy, Arthi

    2011-01-01

    The neurotoxicant 6-hydroxydopamine (6-OHDA) is used to investigate the cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in Parkinson's disease (PD). Oxidative stress and caspase activation contribute to the 6-OHDA-induced apoptotic cell death of dopaminergic neurons. In the present study, we sought to systematically characterize the key downstream signaling molecule involved in 6-OHDA-induced dopaminergic degeneration in cell culture and animal models of PD. Treatment of mesencephalic dopaminergic neuronal N27 cells with 6-OHDA (100 μM) for 24 h significantly reduced mitochondrial activity and increased cytosolic cytochrome c, followed by sequential activation of caspase-9 and caspase-3. Co-treatment with the free radical scavenger MnTBAP (10 μM) significantly attenuated 6-OHDA-induced caspase activities. Interestingly, 6-OHDA induced proteolytic cleavage and activation of protein kinase C delta (PKCδ) was completely suppressed by treatment with a caspase-3-specific inhibitor, Z-DEVD-FMK (50 μM). Furthermore, expression of caspase-3 cleavage site-resistant mutant PKCδ D327A and kinase dead PKCδ K376R or siRNA-mediated knockdown of PKCδ protected against 6-OHDA-induced neuronal cell death, suggesting that caspase-3-dependent PKCδ promotes oxidative stress-induced dopaminergic degeneration. Suppression of PKCδ expression by siRNA also effectively protected N27 cells from 6-OHDA-induced apoptotic cell death. PKCδ cleavage was also observed in the substantia nigra of 6-OHDA-injected C57 black mice but not in control animals. Viral-mediated delivery of PKCδ D327A protein protected against 6-OHDA-induced PKCδ activation in mouse substantia nigra. Collectively, these results strongly suggest that proteolytic activation of PKCδ is a key downstream event in dopaminergic degeneration, and these results may have important translational value for development of novel treatment strategies for PD.

  17. Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw

    Directory of Open Access Journals (Sweden)

    Ravely Casarotti Orlandelli

    2015-06-01

    Full Text Available Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter suggests that these endophytes have the potential to produce enzymes using agricultural wastes.

  18. Dual regulatory switch confers tighter control on HtrA2 proteolytic activity.

    Science.gov (United States)

    Singh, Nitu; D'Souza, Areetha; Cholleti, Anuradha; Sastry, G Madhavi; Bose, Kakoli

    2014-05-01

    High-temperature requirement protease A2 (HtrA2), a multitasking serine protease that is involved in critical biological functions and pathogenicity, such as apoptosis and cancer, is a potent therapeutic target. It is established that the C-terminal post-synaptic density protein, Drosophila disc large tumor suppressor, zonula occludens-1 protein (PDZ) domain of HtrA2 plays pivotal role in allosteric modulation, substrate binding and activation, as commonly reported in other members of this family. Interestingly, HtrA2 exhibits an additional level of functional modulation through its unique N-terminus, as is evident from 'inhibitor of apoptosis proteins' binding and cleavage. This phenomenon emphasizes multiple activation mechanisms, which so far remain elusive. Using conformational dynamics, binding kinetics and enzymology studies, we addressed this complex behavior with respect to defining its global mode of regulation and activity. Our findings distinctly demonstrate a novel N-terminal ligand-mediated triggering of an allosteric switch essential for transforming HtrA2 to a proteolytically competent state in a PDZ-independent yet synergistic activation process. Dynamic analyses suggested that it occurs through a series of coordinated structural reorganizations at distal regulatory loops (L3, LD, L1), leading to a population shift towards the relaxed conformer. This precise synergistic coordination among different domains might be physiologically relevant to enable tighter control upon HtrA2 activation for fostering its diverse cellular functions. Understanding this complex rheostatic dual switch mechanism offers an opportunity for targeting various disease conditions with tailored site-specific effector molecules. © 2014 FEBS.

  19. Urinary Proteolytic Activation of Renal Epithelial Na+ Channels in Chronic Heart Failure.

    Science.gov (United States)

    Zheng, Hong; Liu, Xuefei; Sharma, Neeru M; Li, Yulong; Pliquett, Rainer U; Patel, Kaushik P

    2016-01-01

    One of the key mechanisms involved in renal Na(+) retention in chronic heart failure (CHF) is activation of epithelial Na(+) channels (ENaC) in collecting tubules. Proteolytic cleavage has an important role in activating ENaC. We hypothesized that enhanced levels of proteases in renal tubular fluid activate ENaC, resulting in renal Na(+) retention in rats with CHF. CHF was produced by left coronary artery ligation in rats. By immunoblotting, we found that several urinary serine proteases were significantly increased in CHF rats compared with sham rats (fold increases: furin 6.7, prostasin 23.6, plasminogen 2.06, and plasmin 3.57 versus sham). Similar increases were observed in urinary samples from patients with CHF. Whole-cell patch clamp was conducted in cultured renal collecting duct M-1 cells to record Na(+) currents. Protease-rich urine (from rats and patients with CHF) significantly increased the Na(+) inward current in M-1 cells. Two weeks of protease inhibitor treatment significantly abrogated the enhanced diuretic and natriuretic responses to ENaC inhibitor benzamil in rats with CHF. Increased podocyte lesions were observed in the kidneys of rats with CHF by transmission electron microscopy. Consistent with these results, podocyte damage markers desmin and podocin expressions were also increased in rats with CHF (increased ≈2-folds). These findings suggest that podocyte damage may lead to increased proteases in the tubular fluid, which in turn contributes to the enhanced renal ENaC activity, providing a novel mechanistic insight for Na(+) retention commonly observed in CHF. © 2015 American Heart Association, Inc.

  20. Urinary proteolytic activation of renal epithelial Na+ channels in chronic heart failure

    Science.gov (United States)

    Zheng, Hong; Liu, Xuefei; Sharma, Neeru M.; Li, Yulong; Pliquett, Rainer U; Patel, Kaushik P.

    2015-01-01

    One of the key mechanisms involved in renal Na+ retention in chronic heart failure (CHF) is activation of epithelial Na+ channels (ENaC) in collecting tubules. Proteolytic cleavage has an important role in activating ENaC. We hypothesized that enhanced levels of proteases in renal tubular fluid activate ENaC resulting in renal Na+ retention in rats with CHF. CHF was produced by left coronary artery ligation in rats. By immunoblotting, we found that several urinary serine proteases were significantly increased in CHF rats compared to sham rats (fold increases: furin 6.7, prostasin 23.6, plasminogen 2.06 and plasmin 3.57 vs. sham). Similar increases were observed in urinary samples from patients with CHF. Whole-cell patch-clamp was conducted in cultured renal collecting duct M-1 cells to record Na+ currents. Protease-rich urine (from rats and patients with CHF) significantly increased the Na+ inward current in M-1 cells. Two weeks of protease inhibitor treatment significantly abrogated the enhanced diuretic and natriuretic responses to ENaC inhibitor benzamil in rats with CHF. Increased podocyte lesions were observed in the kidneys of rats with CHF by transmission electron microscopy. Consistent with these results, podocyte damage markers desmin and podocin expressions were also increased in rats with CHF (increased ~2 folds). These findings suggest that podocyte damage may lead to increased proteases in the tubular fluid which in turn contributes to the enhanced renal ENaC activity, providing a novel mechanistic insight for Na+ retention commonly observed in CHF. PMID:26628676

  1. Carotenoid-cleavage activities of crude enzymes from Pandanous amryllifolius.

    Science.gov (United States)

    Ningrum, Andriati; Schreiner, Matthias

    2014-11-01

    Carotenoid degradation products, known as norisoprenoids, are aroma-impact compounds in several plants. Pandan wangi is a common name of the shrub Pandanus amaryllifolius. The genus name 'Pandanus' is derived from the Indonesian name of the tree, pandan. In Indonesia, the leaves from the plant are used for several purposes, e.g., as natural colorants and flavor, and as traditional treatments. The aim of this study was to determine the cleavage of β-carotene and β-apo-8'-carotenal by carotenoid-cleavage enzymes isolated from pandan leaves, to investigate dependencies of the enzymatic activities on temperature and pH, to determine the enzymatic reaction products by using Headspace Solid Phase Microextraction Gas Chromatography/Mass Spectrophotometry (HS-SPME GC/MS), and to investigate the influence of heat treatment and addition of crude enzyme on formation of norisoprenoids. Crude enzymes from pandan leaves showed higher activity against β-carotene than β-apo-8'-carotenal. The optimum temperature of crude enzymes was 70°, while the optimum pH value was 6. We identified β-ionone as the major volatile reaction product from the incubations of two different carotenoid substrates, β-carotene and β-apo-8'-carotenal. Several treatments, e.g., heat treatment and addition of crude enzymes in pandan leaves contributed to the norisoprenoid content. Our findings revealed that the crude enzymes from pandan leaves with carotenoid-cleavage activity might provide a potential application, especially for biocatalysis, in natural-flavor industry. Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.

  2. 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. Keywords: Aspartic protease, Cleavage sites, Cocoa, In-vitro proteolysis, Mass spectrometry, Peptides

  3. Characterization of an extensin-modifying metalloprotease: N-terminal processing and substrate cleavage pattern of Pectobacterium carotovorum Prt1.

    Science.gov (United States)

    Feng, Tao; Nyffenegger, Christian; Højrup, Peter; Vidal-Melgosa, Silvia; Yan, Kok-Phen; Fangel, Jonatan Ulrik; Meyer, Anne S; Kirpekar, Finn; Willats, William G; Mikkelsen, Jørn D

    2014-12-01

    Compared to other plant cell wall-degrading enzymes, proteases are less well understood. In this study, the extracellular metalloprotease Prt1 from Pectobacterium carotovorum (formerly Erwinia carotovora) was expressed in Escherichia coli and characterized with respect to N-terminal processing, thermal stability, substrate targets, and cleavage patterns. Prt1 is an autoprocessing protease with an N-terminal signal pre-peptide and a pro-peptide which has to be removed in order to activate the protease. The sequential cleavage of the N-terminus was confirmed by mass spectrometry (MS) fingerprinting and N-terminus analysis. The optimal reaction conditions for the activity of Prt1 on azocasein were at pH 6.0, 50 °C. At these reaction conditions, K M was 1.81 mg/mL and k cat was 1.82 × 10(7) U M(-1). The enzyme was relatively stable at 50 °C with a half-life of 20 min. Ethylenediaminetetraacetic acid (EDTA) treatment abolished activity; Zn(2+) addition caused regain of the activity, but Zn(2+)addition decreased the thermal stability of the Prt1 enzyme presumably as a result of increased proteolytic autolysis. In addition to casein, the enzyme catalyzed degradation of collagen, potato lectin, and plant extensin. Analysis of the cleavage pattern of different substrates after treatment with Prt1 indicated that the protease had a substrate cleavage preference for proline in substrate residue position P1 followed by a hydrophobic residue in residue position P1' at the cleavage point. The activity of Prt1 against plant cell wall structural proteins suggests that this enzyme might become an important new addition to the toolbox of cell-wall-degrading enzymes for biomass processing.

  4. The potato carotenoid cleavage dioxygenase 4 catalyzes a single cleavage of β-ionone ring-containing carotenes and non-epoxidated xanthophylls

    KAUST Repository

    Bruno, Mark; Beyer, Peter D.; Al-Babili, Salim

    2015-01-01

    amounts. This phenotype indicates a role of this enzyme in tuber development, which may be exerted by a cleavage product. In this work, we investigated the enzymatic activity of StCCD4, by expressing the corresponding cDNA in carotenoid accumulating

  5. Atorvastatin prevents Aβ oligomer-induced neurotoxicity in cultured rat hippocampal neurons by inhibiting Tau cleavage

    Science.gov (United States)

    Sui, Hai-juan; Zhang, Ling-ling; Liu, Zhou; Jin, Ying

    2015-01-01

    Aim: The proteolytic cleavage of Tau is involved in Aβ-induced neuronal dysfunction and cell death. In this study, we investigated whether atorvastatin could prevent Tau cleavage and hence prevent Aβ1–42 oligomer (AβO)-induced neurotoxicity in cultured cortical neurons. Methods: Cultured rat hippocampal neurons were incubated in the presence of AβOs (1.25 μmol/L) with or without atorvastatin pretreatment. ATP content and LDH in the culture medium were measured to assess the neuronal viability. Caspase-3/7 and calpain protease activities were detected. The levels of phospho-Akt, phospho-Erk1/2, phospho-GSK3β, p35 and Tau proteins were measured using Western blotting. Results: Treatment of the neurons with AβO significantly decreased the neuronal viability, induced rapid activation of calpain and caspase-3/7 proteases, accompanied by Tau degradation and relatively stable fragments generated in the neurons. AβO also suppressed Akt and Erk1/2 kinase activity, while increased GSK3β and Cdk5 activity in the neurons. Pretreatment with atorvastatin (0.5, 1, 2.5 μmol/L) dose-dependently inhibited AβO-induced activation of calpain and caspase-3/7 proteases, and effectively diminished the generation of Tau fragments, attenuated synaptic damage and increased neuronal survival. Atorvastatin pretreatment also prevented AβO-induced decreases in Akt and Erk1/2 kinase activity and the increases in GSK3β and Cdk5 kinase activity. Conclusion: Atorvastatin prevents AβO-induced neurotoxicity in cultured rat hippocampal neurons by inhibiting calpain- and caspase-mediated Tau cleavage. PMID:25891085

  6. Perturbation with intrabodies reveals that calpain cleavage is required for degradation of huntingtin exon 1.

    Directory of Open Access Journals (Sweden)

    Amber L Southwell

    2011-01-01

    Full Text Available Proteolytic processing of mutant huntingtin (mHtt, the protein that causes Huntington's disease (HD, is critical for mHtt toxicity and disease progression. mHtt contains several caspase and calpain cleavage sites that generate N-terminal fragments that are more toxic than full-length mHtt. Further processing is then required for the degradation of these fragments, which in turn, reduces toxicity. This unknown, secondary degradative process represents a promising therapeutic target for HD.We have used intrabodies, intracellularly expressed antibody fragments, to gain insight into the mechanism of mutant huntingtin exon 1 (mHDx-1 clearance. Happ1, an intrabody recognizing the proline-rich region of mHDx-1, reduces the level of soluble mHDx-1 by increasing clearance. While proteasome and macroautophagy inhibitors reduce turnover of mHDx-1, Happ1 is still able to reduce mHDx-1 under these conditions, indicating Happ1-accelerated mHDx-1 clearance does not rely on these processes. In contrast, a calpain inhibitor or an inhibitor of lysosomal pH block Happ1-mediated acceleration of mHDx-1 clearance. These results suggest that mHDx-1 is cleaved by calpain, likely followed by lysosomal degradation and this process regulates the turnover rate of mHDx-1. Sequence analysis identifies amino acid (AA 15 as a potential calpain cleavage site. Calpain cleavage of recombinant mHDx-1 in vitro yields fragments of sizes corresponding to this prediction. Moreover, when the site is blocked by binding of another intrabody, V(L12.3, turnover of soluble mHDx-1 in living cells is blocked.These results indicate that calpain-mediated removal of the 15 N-terminal AAs is required for the degradation of mHDx-1, a finding that may have therapeutic implications.

  7. Persistence of bacterial proteolytic enzymes in lake ecosystems.

    Science.gov (United States)

    Kiersztyn, Bartosz; Siuda, Waldemar; Chróst, Ryszard J

    2012-04-01

    This study analyzes proteolytic enzyme persistence and the role of dead (or metabolically inactive) aquatic bacteria in organic matter cycling. Samples from four lakes of different trophic status were used. Irrespective of the trophic status of the examined lakes, bacterial aminopeptidases remained active even 72 h after the death of the bacteria that produced them. The total pool of proteolytic enzymes in natural lake water samples was also stable. We found that the rates of amino acid enzymatic release from proteinaceous matter added to preserved lake water sample were constant for at least 96 h (r(2)  = 0.99, n = 17, P ≤ 0.0001, V(max)  = 84.6 nM h(-1) ). We also observed that proteases built into bacterial cell debris fragments remained active for a long time, even after the total destruction of cells. Moreover, during 24 h of incubation time, about 20% of these enzymatically active fragments adsorbed onto natural seston particles, becoming a part of the 'attached enzymes system' that is regarded as the 'hot-spot' of protein degradation in aquatic ecosystems. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  8. Calicivirus 3C-like proteinase inhibits cellular translation by cleavage of poly(A)-binding protein.

    Science.gov (United States)

    Kuyumcu-Martinez, Muge; Belliot, Gaël; Sosnovtsev, Stanislav V; Chang, Kyeong-Ok; Green, Kim Y; Lloyd, Richard E

    2004-08-01

    Caliciviruses are single-stranded RNA viruses that cause a wide range of diseases in both humans and animals, but little is known about the regulation of cellular translation during infection. We used two distinct calicivirus strains, MD145-12 (genus Norovirus) and feline calicivirus (FCV) (genus Vesivirus), to investigate potential strategies used by the caliciviruses to inhibit cellular translation. Recombinant 3C-like proteinases (r3CL(pro)) from norovirus and FCV were found to cleave poly(A)-binding protein (PABP) in the absence of other viral proteins. The norovirus r3CL(pro) PABP cleavage products were indistinguishable from those generated by poliovirus (PV) 3C(pro) cleavage, while the FCV r3CL(pro) products differed due to cleavage at an alternate cleavage site 24 amino acids downstream of one of the PV 3C(pro) cleavage sites. All cleavages by calicivirus or PV proteases separated the C-terminal domain of PABP that binds translation factors eIF4B and eRF3 from the N-terminal RNA-binding domain of PABP. The effect of PABP cleavage by the norovirus r3CL(pro) was analyzed in HeLa cell translation extracts, and the presence of r3CL(pro) inhibited translation of both endogenous and exogenous mRNAs. Translation inhibition was poly(A) dependent, and replenishment of the extracts with PABP restored translation. Analysis of FCV-infected feline kidney cells showed that the levels of de novo cellular protein synthesis decreased over time as virus-specific proteins accumulated, and cleavage of PABP occurred in virus-infected cells. Our data indicate that the calicivirus 3CL(pro), like PV 3C(pro), mediates the cleavage of PABP as part of its strategy to inhibit cellular translation. PABP cleavage may be a common mechanism among certain virus families to manipulate cellular translation.

  9. Intracellular Cleavage of the Cx43 C-Terminal Domain by Matrix-Metalloproteases: A Novel Contributor to Inflammation?

    Directory of Open Access Journals (Sweden)

    Marijke De Bock

    2015-01-01

    Full Text Available The coordination of tissue function is mediated by gap junctions (GJs that enable direct cell-cell transfer of metabolic and electric signals. GJs are formed by connexin (Cx proteins of which Cx43 is most widespread in the human body. Beyond its role in direct intercellular communication, Cx43 also forms nonjunctional hemichannels (HCs in the plasma membrane that mediate the release of paracrine signaling molecules in the extracellular environment. Both HC and GJ channel function are regulated by protein-protein interactions and posttranslational modifications that predominantly take place in the C-terminal domain of Cx43. Matrix metalloproteases (MMPs are a major group of zinc-dependent proteases, known to regulate not only extracellular matrix remodeling, but also processing of intracellular proteins. Together with Cx43 channels, both GJs and HCs, MMPs contribute to acute inflammation and a small number of studies reports on an MMP-Cx43 link. Here, we build further on these reports and present a novel hypothesis that describes proteolytic cleavage of the Cx43 C-terminal domain by MMPs and explores possibilities of how such cleavage events may affect Cx43 channel function. Finally, we set out how aberrant channel function resulting from cleavage can contribute to the acute inflammatory response during tissue injury.

  10. Kininogen Cleavage Assay: Diagnostic Assistance for Kinin-Mediated Angioedema Conditions.

    Directory of Open Access Journals (Sweden)

    Rémi Baroso

    Full Text Available Angioedema without wheals (AE is a symptom characterised by localised episodes of oedema presumably caused by kinin release from kininogen cleavage. It can result from a hereditary deficiency in C1 Inhibitor (C1Inh, but it can present with normal level of C1Inh. These forms are typically difficult to diagnose although enhanced kinin production is suspected or demonstrated in some cases.We wanted to investigate bradykinin overproduction in all AE condition with normal C1Inh, excluding cases with enhanced kinin catabolism, and to propose this parameter as a disease biomarker.We retrospectively investigated high molecular weight kininogen (HK cleavage pattern, using gel electrophoresis and immunorevelation. Plasma samples were drawn using the same standardised procedure from blood donors or AE patients with normal C1Inh conditions, normal kinin catabolism, and without prophylaxis.Circulating native HK plasma concentrations were similar in the healthy men (interquartile range: 98-175μg/mL, n = 51 and in healthy women (90-176μg/mL, n = 74, while HK cleavage was lower (p14.4% HK cleavage for men; 33.0% HK cleavage for women, with >98% specificity achieved for all parameters. In plasma from patients undergoing recovery two months after oestrogen/progestin combination withdrawal (n = 13 or two weeks after AE attack (n = 2, HK cleavage was not fully restored, suggesting its use as a post-attack assay.As a diagnostic tool, HK cleavage can offer physicians supportive arguments for kinin production in suspected AE cases and improve patient follow-up in clinical trials or prophylactic management.

  11. Trichomonas vaginalis Cysteine Proteinases: Iron Response in Gene Expression and Proteolytic Activity

    Science.gov (United States)

    Cárdenas-Guerra, Rosa Elena; Figueroa-Angulo, Elisa Elvira; Puente-Rivera, Jonathan; Zamudio-Prieto, Olga; Ortega-López, Jaime

    2015-01-01

    We focus on the iron response of Trichomonas vaginalis to gene family products such as the cysteine proteinases (CPs) involved in virulence properties. In particular, we examined the effect of iron on the gene expression regulation and function of cathepsin L-like and asparaginyl endopeptidase-like CPs as virulence factors. We addressed some important aspects about CPs genomic organization and we offer possible explanations to the fact that only few members of this large gene family are expressed at the RNA and protein levels and the way to control their proteolytic activity. We also summarized all known iron regulations of CPs at transcriptional, posttranscriptional, and posttranslational levels along with new insights into the possible epigenetic and miRNA processes. PMID:26090464

  12. Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin

    LENUS (Irish Health Repository)

    Boehm, Manja

    2012-04-25

    AbstractBackgroundCampylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear.ResultsIn the present study, we characterized the serine protease HtrA (high-temperature requirement A) of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni’s HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa) of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori’s HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER) as seen with Salmonella, Shigella, Listeria or Neisseria.ConclusionThese results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.

  13. Rapid paracellular transmigration of Campylobacter jejuni across polarized epithelial cells without affecting TER: role of proteolytic-active HtrA cleaving E-cadherin but not fibronectin.

    Science.gov (United States)

    Boehm, Manja; Hoy, Benjamin; Rohde, Manfred; Tegtmeyer, Nicole; Bæk, Kristoffer T; Oyarzabal, Omar A; Brøndsted, Lone; Wessler, Silja; Backert, Steffen

    2012-04-25

    Campylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear. In the present study, we characterized the serine protease HtrA (high-temperature requirement A) of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni's HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa) of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori's HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER) as seen with Salmonella, Shigella, Listeria or Neisseria. These results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.

  14. Controllable laser thermal cleavage of sapphire wafers

    Science.gov (United States)

    Xu, Jiayu; Hu, Hong; Zhuang, Changhui; Ma, Guodong; Han, Junlong; Lei, Yulin

    2018-03-01

    Laser processing of substrates for light-emitting diodes (LEDs) offers advantages over other processing techniques and is therefore an active research area in both industrial and academic sectors. The processing of sapphire wafers is problematic because sapphire is a hard and brittle material. Semiconductor laser scribing processing suffers certain disadvantages that have yet to be overcome, thereby necessitating further investigation. In this work, a platform for controllable laser thermal cleavage was constructed. A sapphire LED wafer was modeled using the finite element method to simulate the thermal and stress distributions under different conditions. A guide groove cut by laser ablation before the cleavage process was observed to guide the crack extension and avoid deviation. The surface and cross section of sapphire wafers processed using controllable laser thermal cleavage were characterized by scanning electron microscopy and optical microscopy, and their morphology was compared to that of wafers processed using stealth dicing. The differences in luminous efficiency between substrates prepared using these two processing methods are explained.

  15. Bortezomib-induced sensitization of malignant human glioma cells to vorinostat-induced apoptosis depends on reactive oxygen species production, mitochondrial dysfunction, Noxa upregulation, Mcl-1 cleavage, and DNA damage.

    Science.gov (United States)

    Premkumar, Daniel R; Jane, Esther P; Agostino, Naomi R; DiDomenico, Joseph D; Pollack, Ian F

    2013-02-01

    Glioblastomas are invasive tumors with poor prognosis despite current therapies. Histone deacetylase inhibitors (HDACIs) represent a class of agents that can modulate gene expression to reduce tumor growth, and we and others have noted some antiglioma activity from HDACIs, such as vorinostat, although insufficient to warrant use as monotherapy. We have recently demonstrated that proteasome inhibitors, such as bortezomib, dramatically sensitized highly resistant glioma cells to apoptosis induction, suggesting that proteasomal inhibition may be a promising combination strategy for glioma therapeutics. In this study, we examined whether bortezomib could enhance response to HDAC inhibition in glioma cells. Although primary cells from glioblastoma multiforme (GBM) patients and established glioma cell lines did not show significant induction of apoptosis with vorinostat treatment alone, the combination of vorinostat plus bortezomib significantly enhanced apoptosis. The enhanced efficacy was due to proapoptotic mitochondrial injury and increased generation of reactive oxygen species. Our results also revealed that combination of bortezomib with vorinostat enhanced apoptosis by increasing Mcl-1 cleavage, Noxa upregulation, Bak and Bax activation, and cytochrome c release. Further downregulation of Mcl-1 using shRNA enhanced cell killing by the bortezomib/vorinostat combination. Vorinostat induced a rapid and sustained phosphorylation of histone H2AX in primary GBM and T98G cells, and this effect was significantly enhanced by co-administration of bortezomib. Vorinostat/bortezomib combination also induced Rad51 downregulation, which plays an important role in the synergistic enhancement of DNA damage and apoptosis. The significantly enhanced antitumor activity that results from the combination of bortezomib and HDACIs offers promise as a novel treatment for glioma patients. Copyright © 2011 Wiley Periodicals, Inc.

  16. Caspase activation increases beta-amyloid generation independently of caspase cleavage of the beta-amyloid precursor protein (APP).

    Science.gov (United States)

    Tesco, Giuseppina; Koh, Young Ho; Tanzi, Rudolph E

    2003-11-14

    The amyloid precursor protein (APP) undergoes "alternative" proteolysis mediated by caspases. Three major caspase recognition sites have been identified in the APP, i.e. one at the C terminus (Asp720) and two at the N terminus (Asp197 and Asp219). Caspase cleavage at Asp720 has been suggested as leading to increased production of Abeta. Thus, we set out to determine which putative caspase sites in APP, if any, are cleaved in Chinese hamster ovary cell lines concurrently with the increased Abeta production that occurs during apoptosis. We found that cleavage at Asp720 occurred concurrently with caspase 3 activation and the increased production of total secreted Abeta and Abeta1-42 in association with staurosporine- and etoposide-induced apoptosis. To investigate the contribution of caspase cleavage of APP to Abeta generation, we expressed an APP mutant truncated at Asp720 that mimics APP caspase cleavage at the C-terminal site. This did not increase Abeta generation but, in contrast, dramatically decreased Abeta production in Chinese hamster ovary cells. Furthermore, the ablation of caspase-dependent cleavage at Asp720, Asp197, and Asp219 (by site-directed mutagenesis) did not prevent enhanced Abeta production following etoposide-induced apoptosis. These findings indicate that the enhanced Abeta generation associated with apoptosis does not require cleavage of APP at its C-terminal (Asp720) and/or N-terminal caspase sites.

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

    Directory of Open Access Journals (Sweden)

    Robert J Lake

    2009-08-01

    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.

  18. Role of Proteolytic Enzymes in the Interaction of Phytopathogenic Microorganisms with Plants.

    Science.gov (United States)

    Valueva, T A; Zaichik, B Ts; Kudryavtseva, N N

    2016-12-01

    Various forms of participation of proteolytic enzymes in pathogenesis and defense in plants are reviewed. Along with extracellular proteinases, phytopathogenic microorganisms produce specific effectors having proteolytic activity and capable of acting on proteins inside plant cells. In turn, for defense against pathogens, plants use both extracellular and intracellular proteinases.

  19. Isolation and removal of proteolytic enzymes with magnetic cross-linked erythrocytes

    International Nuclear Information System (INIS)

    Safarik, I. Ivo; Safarikova, Mirka

    2001-01-01

    New magnetic adsorbents for batch isolation and removal of various proteolytic enzymes were prepared by glutaraldehyde cross-linking of bovine, porcine and human erythrocytes in the presence of fine magnetic particles. Trypsin, chymotrypsin, alkaline bacterial protease and proteases present in various commercial enzyme preparations were efficiently adsorbed on these adsorbents; on the contrary, proteins without proteolytic activity were not adsorbed

  20. Host cell killing by the West Nile Virus NS2B-NS3 proteolytic complex: NS3 alone is sufficient to recruit caspase-8-based apoptotic pathway

    International Nuclear Information System (INIS)

    Ramanathan, Mathura P.; Chambers, Jerome A.; Pankhong, Panyupa; Chattergoon, Michael; Attatippaholkun, Watcharee; Dang, Kesen; Shah, Neelima; Weiner, David B.

    2006-01-01

    The West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B-NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B-NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B-NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B-NS3-transfected cells revealed ultra-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the apoptotic pathways involving caspases-8 and -3. Experimental results from the use of caspase-specific inhibitors and caspase-8 siRNA demonstrated that the activation of caspase-8 was essential to initiate apoptotic signaling in NS3-expressing cells. Downstream of caspase-3 activation, we observed nuclear membrane ruptures and cleavage of the DNA-repair enzyme, PARP in NS3-expressing cells. Nuclear herniations due to NS3 expression were absent in the cells treated with a caspase-3 inhibitor. Expression of protease and helicase domains themselves was sufficient to trigger apoptosis generating insight into the apoptotic pathways triggered by NS3 from WNV

  1. Microbiological Monitoring and Proteolytic Study of Clinical Samples From Burned and Burned Wounded Patients

    International Nuclear Information System (INIS)

    Toema, M.A.; El-Bazza, Z.E.; El-Hifnawi, H.N.; Abd-El-Hakim, E.E.

    2013-01-01

    In this study, clinical samples were collected from 100 patients admitted to Burn and Plastic Surgery Department, Faculty of Medicine, Ain Shams University, Egypt, over a period of 12 months. The proteolytic activity of 110 clinical samples taken from surfaces swabs which taken from burned and burned wounded patients with different ages and gender was examined. Screening for the proteolytic activity produced by pathogenic bacteria isolated from burned and burned wounded patients was evaluated as gram positive Bacilli and gram negative bacilli showed high proteolytic activity (46.4%) while 17.9% showed no activity. The isolated bacteria proved to have proteolytic activity were classified into high, moderate and weak. The pathogenic bacteria isolated from burned and burned wounded patients and showing proteolytic activity were identified as Pseudomonas aeruginosa, Proteus mirabilis, Proteus vulgaris, Bacillus megaterium, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella ozaeanae, Klebsiella oxytoca, Klebsiella pneumoniae and Pseudomonas fluoresces.

  2. Caspase-2 cleavage of tau reversibly impairs memory.

    Science.gov (United States)

    Zhao, Xiaohui; Kotilinek, Linda A; Smith, Benjamin; Hlynialuk, Chris; Zahs, Kathleen; Ramsden, Martin; Cleary, James; Ashe, Karen H

    2016-11-01

    In Alzheimer's disease (AD) and other tauopathies, the tau protein forms fibrils, which are believed to be neurotoxic. However, fibrillar tau has been dissociated from neuron death and network dysfunction, suggesting the involvement of nonfibrillar species. Here we describe a novel pathological process in which caspase-2 cleavage of tau at Asp314 impairs cognitive and synaptic function in animal and cellular models of tauopathies by promoting the missorting of tau to dendritic spines. The truncation product, Δtau314, resists fibrillation and is present at higher levels in brains from cognitively impaired mice and humans with AD. The expression of tau mutants that resisted caspase-2 cleavage prevented tau from infiltrating spines, dislocating glutamate receptors and impairing synaptic function in cultured neurons, and it prevented memory deficits and neurodegeneration in mice. Decreasing the levels of caspase-2 restored long-term memory in mice that had existing deficits. Our results suggest an overall treatment strategy for re-establishing synaptic function and restoring memory in patients with AD by preventing tau from accumulating in dendritic spines.

  3. Influence of the austenitizing temperature in the cleavage facet size of Niocor 2

    International Nuclear Information System (INIS)

    Darwish, F.A.I.; Teixeira, J.C.G.; Fernandes, R.A.; Juer, S.

    1983-01-01

    Convetional Charpy specimens of Niocor 2 steel cooled in air from various austenitizing temperatures were fractured at -196 0 C so as to insure failure by cleavage. The cleavage facet size distribution was determined and then correlated with the grain size and other aspects of the microstructure. The results that the average facet size can be increased through a coarsening of the microstructure. For the case where the γ→α transformation products are predominantely acicular, the facet size is shown to depend on substructural aspects primarily the lath packet size. (Author) [pt

  4. Di-µ-hydroxo Bridge Cleavage Reactions between [Co(nta)(µ-OH)] 2 ...

    African Journals Online (AJOL)

    NJD

    2004-04-22

    Apr 22, 2004 ... subsequent rate determining steps to form presumably a ligand-substituted, mono-bridged complex, [(nta)(OH)Co-µ-. OH-Co(nta)(L)]2– (L = py/dmap). The latter decomposes rapidly to form the products. The preferred pathway for these bridge cleavage seemed to be the reaction of the mono-µ-hydroxo-.

  5. Can laccases catalyze bond cleavage in lignin?

    DEFF Research Database (Denmark)

    Munk, Line; Sitarz, Anna Katarzyna; Kalyani, Dayanand

    2015-01-01

    illustrations of the putative laccase catalyzed reactions, including the possible reactions of the reactive radical intermediates taking place after the initial oxidation of the phenol-hydroxyl groups, we show that i) Laccase activity is able to catalyze bond cleavage in low molecular weight phenolic lignin......-substituted phenols, benzenethiols, polyphenols, and polyamines, which may be oxidized. In addition, the currently available analytical methods that can be used to detect enzyme catalyzed changes in lignin are summarized, and an improved nomenclature for unequivocal interpretation of the action of laccases on lignin...

  6. 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

  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

    2009-06-01

    Full Text Available 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.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.Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies.

  8. Proteolytic activities in yeast after UV irradiation. Pt. 1

    International Nuclear Information System (INIS)

    Schwencke, J.; Moustacchi, E.

    1982-01-01

    Specific proteolytic activities are known to be induced in Escherichia coli following irradiation. Consequently it seemed of interest to investigate whether variations in proteinase activities occur in yeast. Among the five most well known proteinases of Saccharomyces cerevisiae, we have found that proteinase B activity increases up to three times in wild-type RAD + yeast cells after a dose of 50 Jm -2 of 254 nm ultraviolet light (40% survival). Carboxypeptidase Y and aminopeptidase I (leucin aminopeptidase) activities were only moderately increased. Proteinase A activity was only slightly enhanced, while aminopeptidase II (lysin aminopeptidase) was unaffected in both RAD + strains studied. The observed post UV-increase in proteinase B activity was inhibited by cycloheximide and was dose dependent. Increases in proteinase B levels were independent of the activation method used to destroy the proteinase B-inhibitor complex present in the crude yeast extracts. A standard method for comparison of the postirradiation levels among different proteinases, strains and methods of activation is presented. (orig.)

  9. [Cell-derived microparticles unveil their fibrinolytic and proteolytic function].

    Science.gov (United States)

    Doeuvre, Loïc; Angles-Cano, Eduardo

    2009-01-01

    Cell-derived microparticles (MP) are membrane microvesicles, 0.1-1 microm in size, shed by cells following activation or during apoptosis in a variety of pathological conditions. MPs released by blood cells or by vascular endothelial cells display molecular signatures that allow their identification and functional characterization. In addition, they provide tissue factor (TF) and a procoagulant phospholipid surface. Therefore, at present, the most strongly established applied research on MPs is their procoagulant activity as a determinant of thrombotic risk in various clinical conditions. Previous studies have indicated that MPs derived from malignant cells express matrix metalloproteinases, urokinase and its receptor (uPA/uPAR) that, in the presence of plasminogen, may act in concert to degrade extracellular matrix proteins. Recently, it was shown that MPs from TNFa-stimulated endothelial cells served as a surface for interaction with plasminogen and its conversion into plasmin by the uPA/uPAR system expressed at their surface. This capacity of MPs to promote plasmin generation confers them a new profibrinolytic and proteolytic function that may be of relevance in fibrinolysis, cell migration, angiogenesis, dissemination of malignant cells, cell detachment and apoptosis.

  10. Proteolytic activities in yeast after UV irradiation. Pt. 1

    Energy Technology Data Exchange (ETDEWEB)

    Schwencke, J.; Moustacchi, E.

    1982-04-01

    Specific proteolytic activities are known to be induced in Escherichia coli following irradiation. Consequently it seemed of interest to investigate whether variations in proteinase activities occur in yeast. Among the five most well known proteinases of Saccharomyces cerevisiae, we have found that proteinase B activity increases up to three times in wild-type RAD/sup +/ yeast cells after a dose of 50 Jm/sup -2/ of 254 nm ultraviolet light (40% survival). Carboxypeptidase Y and aminopeptidase I (leucin aminopeptidase) activities were only moderately increased. Proteinase A activity was only slightly enhanced, while aminopeptidase II (lysin aminopeptidase) was unaffected in both RAD/sup +/ strains studied. The observed post UV-increase in proteinase B activity was inhibited by cycloheximide and was dose dependent. Increases in proteinase B levels were independent of the activation method used to destroy the proteinase B-inhibitor complex present in the crude yeast extracts. A standard method for comparison of the postirradiation levels among different proteinases, strains and methods of activation is presented.

  11. Dopaminergic neurotoxicant 6-OHDA induces oxidative damage through proteolytic activation of PKC{delta} in cell culture and animal models of Parkinson's disease

    Energy Technology Data Exchange (ETDEWEB)

    Latchoumycandane, Calivarathan; Anantharam, Vellareddy; Jin, Huajun; Kanthasamy, Anumantha; Kanthasamy, Arthi, E-mail: arthik@iastate.edu

    2011-11-15

    The neurotoxicant 6-hydroxydopamine (6-OHDA) is used to investigate the cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in Parkinson's disease (PD). Oxidative stress and caspase activation contribute to the 6-OHDA-induced apoptotic cell death of dopaminergic neurons. In the present study, we sought to systematically characterize the key downstream signaling molecule involved in 6-OHDA-induced dopaminergic degeneration in cell culture and animal models of PD. Treatment of mesencephalic dopaminergic neuronal N27 cells with 6-OHDA (100 {mu}M) for 24 h significantly reduced mitochondrial activity and increased cytosolic cytochrome c, followed by sequential activation of caspase-9 and caspase-3. Co-treatment with the free radical scavenger MnTBAP (10 {mu}M) significantly attenuated 6-OHDA-induced caspase activities. Interestingly, 6-OHDA induced proteolytic cleavage and activation of protein kinase C delta (PKC{delta}) was completely suppressed by treatment with a caspase-3-specific inhibitor, Z-DEVD-FMK (50 {mu}M). Furthermore, expression of caspase-3 cleavage site-resistant mutant PKC{delta}{sup D327A} and kinase dead PKC{delta}{sup K376R} or siRNA-mediated knockdown of PKC{delta} protected against 6-OHDA-induced neuronal cell death, suggesting that caspase-3-dependent PKC{delta} promotes oxidative stress-induced dopaminergic degeneration. Suppression of PKC{delta} expression by siRNA also effectively protected N27 cells from 6-OHDA-induced apoptotic cell death. PKC{delta} cleavage was also observed in the substantia nigra of 6-OHDA-injected C57 black mice but not in control animals. Viral-mediated delivery of PKC{delta}{sup D327A} protein protected against 6-OHDA-induced PKC{delta} activation in mouse substantia nigra. Collectively, these results strongly suggest that proteolytic activation of PKC{delta} is a key downstream event in dopaminergic degeneration, and these results may have important translational value for

  12. Diversity of Neuropeptide Cell-Cell Signaling Molecules Generated by Proteolytic Processing Revealed by Neuropeptidomics Mass Spectrometry

    Science.gov (United States)

    Hook, Vivian; Lietz, Christopher B.; Podvin, Sonia; Cajka, Tomas; Fiehn, Oliver

    2018-04-01

    Neuropeptides are short peptides in the range of 3-40 residues that are secreted for cell-cell communication in neuroendocrine systems. In the nervous system, neuropeptides comprise the largest group of neurotransmitters. In the endocrine system, neuropeptides function as peptide hormones to coordinate intercellular signaling among target physiological systems. The diversity of neuropeptide functions is defined by their distinct primary sequences, peptide lengths, proteolytic processing of pro-neuropeptide precursors, and covalent modifications. Global, untargeted neuropeptidomics mass spectrometry is advantageous for defining the structural features of the thousands to tens of thousands of neuropeptides present in biological systems. Defining neuropeptide structures is the basis for defining the proteolytic processing pathways that convert pro-neuropeptides into active peptides. Neuropeptidomics has revealed that processing of pro-neuropeptides occurs at paired basic residues sites, and at non-basic residue sites. Processing results in neuropeptides with known functions and generates novel peptides representing intervening peptide domains flanked by dibasic residue processing sites, identified by neuropeptidomics. While very short peptide products of 2-4 residues are predicted from pro-neuropeptide dibasic processing sites, such peptides have not been readily identified; therefore, it will be logical to utilize metabolomics to identify very short peptides with neuropeptidomics in future studies. Proteolytic processing is accompanied by covalent post-translational modifications (PTMs) of neuropeptides comprising C-terminal amidation, N-terminal pyroglutamate, disulfide bonds, phosphorylation, sulfation, acetylation, glycosylation, and others. Neuropeptidomics can define PTM features of neuropeptides. In summary, neuropeptidomics for untargeted, global analyses of neuropeptides is essential for elucidation of proteases that generate diverse neuropeptides for cell

  13. Isolation and Characterization of Proteolytic Ruminal Bacteria from Sheep and Goats Fed the Tannin-Containing Shrub Legume Calliandra calothyrsus

    Science.gov (United States)

    McSweeney, Christopher S.; Palmer, Brian; Bunch, Rowan; Krause, Denis O.

    1999-01-01

    Tannins in forages complex with protein and reduce the availability of nitrogen to ruminants. Ruminal bacteria that ferment protein or peptides in the presence of tannins may benefit digestion of these diets. Bacteria from the rumina of sheep and goats fed Calliandra calothyrsus (3.6% N and 6% condensed tannin) were isolated on proteinaceous agar medium overlaid with either condensed (calliandra tannin) or hydrolyzable (tannic acid) tannin. Fifteen genotypes were identified, based on 16S ribosomal DNA-restriction fragment length polymorphism analysis, and all were proteolytic and fermented peptides to ammonia. Ten of the isolates grew to high optical density (OD) on carbohydrates (glucose, cellobiose, xylose, xylan, starch, and maltose), while the other isolates did not utilize or had low growth on these substrates. In pure culture, representative isolates were unable to ferment protein that was present in calliandra or had been complexed with tannin. One isolate, Lp1284, had high protease activity (80 U), a high specific growth rate (0.28), and a high rate of ammonia production (734 nmol/min/ml/OD unit) on Casamino Acids and Trypticase Peptone. Phylogenetic analysis of the 16S ribosomal DNA sequence showed that Lp1284 was related (97.6%) to Clostridium botulinum NCTC 7273. Purified plant protein and casein also supported growth of Lp1284 and were fermented to ammonia. This is the first report of a proteolytic, ammonia-hyperproducing bacterium from the rumen. In conclusion, a diverse group of proteolytic and peptidolytic bacteria were present in the rumen, but the isolates could not digest protein that was complexed with condensed tannin. PMID:10388706

  14. Diversity of Neuropeptide Cell-Cell Signaling Molecules Generated by Proteolytic Processing Revealed by Neuropeptidomics Mass Spectrometry

    Science.gov (United States)

    Hook, Vivian; Lietz, Christopher B.; Podvin, Sonia; Cajka, Tomas; Fiehn, Oliver

    2018-05-01

    Neuropeptides are short peptides in the range of 3-40 residues that are secreted for cell-cell communication in neuroendocrine systems. In the nervous system, neuropeptides comprise the largest group of neurotransmitters. In the endocrine system, neuropeptides function as peptide hormones to coordinate intercellular signaling among target physiological systems. The diversity of neuropeptide functions is defined by their distinct primary sequences, peptide lengths, proteolytic processing of pro-neuropeptide precursors, and covalent modifications. Global, untargeted neuropeptidomics mass spectrometry is advantageous for defining the structural features of the thousands to tens of thousands of neuropeptides present in biological systems. Defining neuropeptide structures is the basis for defining the proteolytic processing pathways that convert pro-neuropeptides into active peptides. Neuropeptidomics has revealed that processing of pro-neuropeptides occurs at paired basic residues sites, and at non-basic residue sites. Processing results in neuropeptides with known functions and generates novel peptides representing intervening peptide domains flanked by dibasic residue processing sites, identified by neuropeptidomics. While very short peptide products of 2-4 residues are predicted from pro-neuropeptide dibasic processing sites, such peptides have not been readily identified; therefore, it will be logical to utilize metabolomics to identify very short peptides with neuropeptidomics in future studies. Proteolytic processing is accompanied by covalent post-translational modifications (PTMs) of neuropeptides comprising C-terminal amidation, N-terminal pyroglutamate, disulfide bonds, phosphorylation, sulfation, acetylation, glycosylation, and others. Neuropeptidomics can define PTM features of neuropeptides. In summary, neuropeptidomics for untargeted, global analyses of neuropeptides is essential for elucidation of proteases that generate diverse neuropeptides for cell

  15. Where do the immunostimulatory effects of oral proteolytic enzymes ('systemic enzyme therapy') come from? Microbial proteolysis as a possible starting point.

    Science.gov (United States)

    Biziulevicius, Gediminas A

    2006-01-01

    Enteric-coated proteolytic enzyme preparations like Wobenzym and Phlogenzym are widely used for the so-called 'systemic enzyme therapy' both in humans and animals. Numerous publications reveal that oral proteolytic enzymes are able to stimulate directly the activity of immune competent cells as well as to increase efficiency of some of their products. But origins of the immunostimulatory effects of oral proteolytic enzymes are still unclear. The hypothesis described here suggests that it may be proteolysis of intestinal microorganisms that makes the immune competent cells to work in the immunostimulatory manner. The hypothesis was largely formed by several scientific observations: First, microbial lysis products (lipopolysaccharides, muropeptides and other peptidoglycan fragments, beta-glucans, etc.) are well known for their immunostimulatory action. Second, a normal human being hosts a mass of intestinal microorganisms equivalent to about 1 kg. The biomass (mainly due to naturally occurring autolysis) continuously supplies the host's organism with immunostimulatory microbial cell components. Third, the immunostimulatory effects resulting from the oral application of exogenously acting antimicrobial (lytic) enzyme preparations, such as lysozyme and lysosubtilin, are likely to be a result of the action of microbial lysis products. Fourth, cell walls of most microorganisms contain a considerable amount of proteins/peptides, a possible target for exogenous proteolytic enzymes. In fact, several authors have already shown that a number of proteases possess an ability to lyse the microbial cells in vitro. Fifth, the pretreatment of microbial cells (at least of some species) in vitro with proteolytic enzymes makes them more sensitive to the lytic action of lysozyme and, otherwise, pretreatment with lysozyme makes them more susceptible to proteolytic degradation. Sixth, exogenous proteases, when in the intestines, may participate in final steps of food-protein digestion

  16. Proteolytic activity and cooperative hemolytic effect of dermatophytes with different species of bacteria

    Directory of Open Access Journals (Sweden)

    Keyvan Pakshir

    2016-12-01

    Conclusion: This study indicated that hemolysin and proteolytic enzymes potentially play a role in dermatophyte pathogenesis and S. aureus could be considered as a main bacterium for creation of co-hemolytic effect in association with dermatophyte species.

  17. The influence of radiation on bacterial cells and their proteolytic properties

    International Nuclear Information System (INIS)

    Szulc, M.; Stefaniakowa, A.; Stanczak, B.; Peconek, J.

    1980-01-01

    The suspension of bacterial cells and their spores were exposed to X rays in the environment with and without protein. The doses of radiation ranged from 1 to 100 Gy and in case of spores of B. subtilis from 50 to 1000 Gy. It was found that irradiation to Proteus vulgaris, Pseudomonas fluorescens and Ps. aeruginosa caused an inconsiderable decrease of proteolytic properties of the generation originated from irradiated bacteria. Irradiation of B. subtilis spores did not influence the proteolytic activity of bacterial cells derived from the exposed spores. The degree of wasting away of bacteria exposed to the same radiation was higher than the rate of proteolytic properties decrease. The presence of protein in the surroundings had no influence on proteolytic characteristics of new generations. (author)

  18. New radionuclide method for determination of gastric content proteolytic activity. (Experiments)

    International Nuclear Information System (INIS)

    Sergienko, V.B.; Popova, L.V.; Khodarev, N.N.; Astashenkova, K.Yu.

    1988-01-01

    A possibility of the use of a radionuclide tubeless rapid method for measuring gastric content proteolytic activity (GCPA) with the help of a protein (gelatin) RP containing capsule was demonstrated in experiments in vitro. There was correlation between the time of dissolution of RP containing capsules and GCPA determined after Mett's method. Reference time intervals were established for normal, raised and lowered proteolytic activity. The method was shown to be physiological, simple and timesaving

  19. Imaging proteolytic activity in live cells and animal models.

    Directory of Open Access Journals (Sweden)

    Stefanie Galbán

    Full Text Available In addition to their degradative role in protein turnover, proteases play a key role as positive or negative regulators of signal transduction pathways and therefore their dysregulation contributes to many disease states. Regulatory roles of proteases include their hormone-like role in triggering G protein-coupled signaling (Protease-Activated-Receptors; their role in shedding of ligands such as EGF, Notch and Fas; and their role in signaling events that lead to apoptotic cell death. Dysregulated activation of apoptosis by the caspase family of proteases has been linked to diseases such as cancer, autoimmunity and inflammation. In an effort to better understand the role of proteases in health and disease, a luciferase biosensor is described which can quantitatively report proteolytic activity in live cells and mouse models. The biosensor, hereafter referred to as GloSensor Caspase 3/7 has a robust signal to noise (50-100 fold and dynamic range such that it can be used to screen for pharmacologically active compounds in high throughput campaigns as well as to study cell signaling in rare cell populations such as isolated cancer stem cells. The biosensor can also be used in the context of genetically engineered mouse models of human disease wherein conditional expression using the Cre/loxP technology can be implemented to investigate the role of a specific protease in living subjects. While the regulation of apoptosis by caspase's was used as an example in these studies, biosensors to study additional proteases involved in the regulation of normal and pathological cellular processes can be designed using the concepts presented herein.

  20. Proteolytic enzymes in seawater: contribution of prokaryotes and protists

    Science.gov (United States)

    Obayashi, Y.; Suzuki, S.

    2016-02-01

    Proteolytic enzyme is one of the major catalysts of microbial processing of organic matter in biogeochemical cycle. Here we summarize some of our studies about proteases in seawater, including 1) distribution of protease activities in coastal and oceanic seawater, 2) responses of microbial community and protease activities in seawater to organic matter amending, and 3) possible contribution of heterotrophic protists besides prokaryotes to proteases in seawater, to clarify cleared facts and remaining questions. Activities of aminopeptidases, trypsin-type and chymotrypsin-type proteases were detected from both coastal and oceanic seawater by using MCA-substrate assay. Significant activities were detected from not only particulate (cell-associated) fraction but also dissolved fraction of seawater, especially for trypsin-type and chymotrypsin-type proteases. Hydrolytic enzymes in seawater have been commonly thought to be mainly derived from heterotrophic prokaryotes; however, it was difficult to determine actual source organisms of dissolved enzymes in natural seawater. Our experiment with addition of dissolved protein to subtropical oligotrophic Pacific water showed drastically enhancement of the protease activities especially aminopeptidases in seawater, and the prokaryotic community structure simultaneously changed to be dominant of Bacteroidetes, indicating that heterotrophic bacteria were actually one of the sources of proteases in seawater. Another microcosm experiment with free-living marine heterotrophic ciliate Paranophrys marina together with an associated bacterium showed that extracellular trypsin-type activity was mainly attributed to the ciliate. The protist seemed to work in organic matter digestion in addition to be a grazer. From the results, we propose a system of organic matter digestion by prokaryotes and protists in aquatic environments, although their actual contribution in natural environments should be estimated in future studies.

  1. Ultrarapid mutation detection by multiplex, solid-phase chemical cleavage

    Energy Technology Data Exchange (ETDEWEB)

    Rowley, G.; Saad, S.; Giannelli, F.; Green, P.M. [Guy`s & St. Thomas`s Hospitals, London (United Kingdom)

    1995-12-10

    The chemical cleavage of mismatches in heteroduplexes formed by probe and test DNA detects and locates any sequence change in long DNA segments ({approximately}1.8 kb), and its efficiency has been well tested in the analysis of both average (e.g., coagulation factor IX) and large, complex genes (e.g., coagulation factor VIII and dystrophin). In the latter application RT/PCR products allow the examination of all essential sequences of the gene in a minimum number of reactions. We use two specific chemical reactants (hydroxylamine and osmium tetroxide) and piperidine cleavage of the above procedure to develop a very fast mutation screening method. This is based on: (1) 5{prime} or internal fluorescent labeling to allow concurrent screening of three to four DNA fragments and (2) solid-phase chemistry to use a microliter format and reduce the time required for the procedure, from amplification of sequence to gel loading inclusive, to one person-working-day. We test the two variations of the method, one entailing 5{prime} labeling of probe DNA and the other uniform labeling of both probe and target DNA, by detecting 114 known hemophilia B (coagulation factor IX) mutations and by analyzing 129 new patients. Uniform labeling of both probe and target DNA prior to formation of the heteroduplexes leads to almost twofold redundancy in the ability to detect mutations. Alternatively, the latter procedure may offer very efficient though less than 100% screening for sequence changes with only hydroxylamine. The full method with two chemical reactions (hydroxylamine and osmium tetroxide) should allow one person to screen with virtually 100% accuracy more than 300 kb of sequence in three ABI 373 gels in 1 day. 26 refs., 7 figs., 1 tab.

  2. Use of Proteolytic Enzymes in the Treatment of Proteinaceous Esophageal Food Impaction.

    Science.gov (United States)

    Morse, Christopher R; Wang, Howard; Donahue, Dean M; Garrity, Julie M; Allan, James S

    2016-01-01

    Proteinaceous esophageal food impaction typically requires endoscopic intervention. An alternative approach is the use of proteolytic enzymes. Concerns regarding the use of proteolytic enzymes include the risk of perforation and aspiration pneumonitis. We retrospectively reviewed our series of 69 patients treated with papain to determine the safety and efficacy of proteolytic enzymes. Patients were retrospectively reviewed if treated for an esophageal food impaction from 1999 through 2008. Median age was 56 years (range 19-91 years), with 46 male and 23 female patients. In 27 patients (39%) this was their first presentation, in 14 (20%) it was the second, and 28 (41%) had multiple previous episodes. Meat was the cause in 49 (71%), chicken in 6 (9%), fish in 3 (4%), and unspecified in 11 (16%). All patients presented with dysphagia for solids, 56 (81%) could not tolerate liquids. Papain solution, 1 tsp in 8 oz of water, was given to patients in an unlimited quantity. Papain was successful in relieving the obstruction in 60 patients (87%). The remaining 9 patients (13%) underwent endoscopy with successful retrieval. No patient suffered a perforation, either with papain ingestion or endoscopy. There were no episodes of pneumonitis or pneumonia. We have used proteolytic enzymes with a high success rate and with minimal complication. Further, if proteolytic enzymes fail, endoscopy can be performed safely and effectively. We recommend the use of proteolytic enzymes as the initial management in all patients with proteinaceous food impaction of the esophagus. Published by Elsevier Inc.

  3. Full Length Research Paper Curcumin induces cleavage of -catenin ...

    African Journals Online (AJOL)

    β-Catenin/Tcf-4 signaling pathway plays important roles in colorectal tumorigenesis. RT-PCR, western blotting and immunoprecipitation were used to study the effects of curcumin on β-catenin/Tcf-4 signaling pathway in HT-29 cells. Treatment of curcumin could induce cleavage of β-catenin and the cleavage could be ...

  4. Modeling and inferring cleavage patterns in proliferating epithelia.

    Directory of Open Access Journals (Sweden)

    Ankit B Patel

    2009-06-01

    Full Text Available The regulation of cleavage plane orientation is one of the key mechanisms driving epithelial morphogenesis. Still, many aspects of the relationship between local cleavage patterns and tissue-level properties remain poorly understood. Here we develop a topological model that simulates the dynamics of a 2D proliferating epithelium from generation to generation, enabling the exploration of a wide variety of biologically plausible cleavage patterns. We investigate a spectrum of models that incorporate the spatial impact of neighboring cells and the temporal influence of parent cells on the choice of cleavage plane. Our findings show that cleavage patterns generate "signature" equilibrium distributions of polygonal cell shapes. These signatures enable the inference of local cleavage parameters such as neighbor impact, maternal influence, and division symmetry from global observations of the distribution of cell shape. Applying these insights to the proliferating epithelia of five diverse organisms, we find that strong division symmetry and moderate neighbor/maternal influence are required to reproduce the predominance of hexagonal cells and low variability in cell shape seen empirically. Furthermore, we present two distinct cleavage pattern models, one stochastic and one deterministic, that can reproduce the empirical distribution of cell shapes. Although the proliferating epithelia of the five diverse organisms show a highly conserved cell shape distribution, there are multiple plausible cleavage patterns that can generate this distribution, and experimental evidence suggests that indeed plants and fruitflies use distinct division mechanisms.

  5. 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 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.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.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.

  6. Maximizing Selective Cleavages at Aspartic Acid and Proline Residues for the Identification of Intact Proteins

    Science.gov (United States)

    Foreman, David J.; Dziekonski, Eric T.; McLuckey, Scott A.

    2018-04-01

    A new approach for the identification of intact proteins has been developed that relies on the generation of relatively few abundant products from specific cleavage sites. This strategy is intended to complement standard approaches that seek to generate many fragments relatively non-selectively. Specifically, this strategy seeks to maximize selective cleavage at aspartic acid and proline residues via collisional activation of precursor ions formed via electrospray ionization (ESI) under denaturing conditions. A statistical analysis of the SWISS-PROT database was used to predict the number of arginine residues for a given intact protein mass and predict a m/z range where the protein carries a similar charge to the number of arginine residues thereby enhancing cleavage at aspartic acid residues by limiting proton mobility. Cleavage at aspartic acid residues is predicted to be most favorable in the m/z range of 1500-2500, a range higher than that normally generated by ESI at low pH. Gas-phase proton transfer ion/ion reactions are therefore used for precursor ion concentration from relatively high charge states followed by ion isolation and subsequent generation of precursor ions within the optimal m/z range via a second proton transfer reaction step. It is shown that the majority of product ion abundance is concentrated into cleavages C-terminal to aspartic acid residues and N-terminal to proline residues for ions generated by this process. Implementation of a scoring system that weights both ion fragment type and ion fragment area demonstrated identification of standard proteins, ranging in mass from 8.5 to 29.0 kDa. [Figure not available: see fulltext.

  7. 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.

  8. Plasmodium falciparum: VAR2CSA expressed during pregnancy-associated malaria is partially resistant to proteolytic cleavage by trypsin

    DEFF Research Database (Denmark)

    Nielsen, Morten A; Resende, Mafalda; Alifrangis, Michael

    2007-01-01

    In areas of high Plasmodium falciparum transmission, immunity to malaria is acquired during childhood, so that adults in general are clinically immune. One exception is that first-time pregnant women are susceptible to pregnancy-associated malaria caused by accumulation of parasites in the placen...

  9. Quantitative kinetics of proteolytic enzymes determined by a surface concentration-based assay using peptide arrays.

    Science.gov (United States)

    Jung, Se-Hui; Kong, Deok-Hoon; Park, Seoung-Woo; Kim, Young-Myeong; Ha, Kwon-Soo

    2012-08-21

    Peptide arrays have emerged as a key technology for drug discovery, diagnosis, and cell biology. Despite the promise of these arrays, applications of peptide arrays to quantitative analysis of enzyme kinetics have been limited due to the difficulty in obtaining quantitative information of enzymatic reaction products. In this study, we developed a new approach for the quantitative kinetics analysis of proteases using fluorescence-conjugated peptide arrays, a surface concentration-based assay with solid-phase peptide standards using dry-off measurements, and compared it with an applied concentration-based assay. For fabrication of the peptide arrays, substrate peptides of cMMP-3, caspase-3, caspase-9, and calpain-1 were functionalized with TAMRA and cysteine, and were immobilized onto amine-functionalized arrays using a heterobifunctional linker, N-[γ-maleimidobutyloxy]succinimide ester. The proteolytic activities of the four enzymes were quantitatively analyzed by calculating changes induced by enzymatic reactions in the concentrations of peptides bound to array surfaces. In addition, this assay was successfully applied for calculating the Michaelis constant (K(m,surf)) for the four enzymes. Thus, this new assay has a strong potential for use in the quantitative evaluation of proteases, and for drug discovery through kinetics studies including the determination of K(m) and V(max).

  10. Biological and Proteolytic Variation in the Venom of Crotalus scutulatus scutulatus from Mexico

    Directory of Open Access Journals (Sweden)

    Miguel Borja

    2018-01-01

    Full Text Available Rattlesnake venoms may be classified according to the presence/absence and relative abundance of the neurotoxic phospholipases A 2 s (PLA 2 s, such as Mojave toxin, and snake venom metalloproteinases (SVMPs. In Mexico, studies to determine venom variation in Mojave Rattlesnakes (Crotalus scutulatus scutulatus are limited and little is known about the biological and proteolytic activities in this species. Tissue (34 and venom (29 samples were obtained from C. s. scutulatus from different locations within their distribution in Mexico. Mojave toxin detection was carried out at the genomic (by PCR and protein (by ELISA levels for all tissue and venom samples. Biological activity was tested on representative venoms by measuring LD 50 and hemorrhagic activity. To determine the approximate amount of SVMPs, 15 venoms were separated by RP-HPLC and variation in protein profile and proteolytic activity was evaluated by SDS-PAGE (n = 28 and Hide Powder Azure proteolytic analysis (n = 27. Three types of venom were identified in Mexico which is comparable to the intraspecific venom diversity observed in the Sonoran Desert of Arizona, USA: Venom Type A (∼Type II, with Mojave toxin, highly toxic, lacking hemorrhagic activity, and with scarce proteolytic activity; Type B (∼Type I, without Mojave toxin, less toxic than Type A, highly hemorrhagic and proteolytic; and Type A + B, containing Mojave toxin, as toxic as venom Type A, variable in hemorrhagic activity and with intermediate proteolytic activity. We also detected a positive correlation between SVMP abundance and hemorrhagic and proteolytic activities. Although more sampling is necessary, our results suggest that venoms containing Mojave toxin and venom lacking this toxin are distributed in the northwest and southeast portions of the distribution in Mexico, respectively, while an intergradation in the middle of both zones is present.

  11. Nuclear import of influenza B virus nucleoprotein: Involvement of an N-terminal nuclear localization signal and a cleavage-protection motif

    International Nuclear Information System (INIS)

    Wanitchang, Asawin; Narkpuk, Jaraspim; Jongkaewwattana, Anan

    2013-01-01

    The nucleoprotein of influenza B virus (BNP) shares several characteristics with its influenza A virus counterpart (ANP), including localization in the host's nucleus. However, while the nuclear localization signal(s) (NLS) of ANP are well characterized, little is known about those of BNP. In this study, we showed that the fusion protein bearing the BNP N-terminus fused with GFP (N70–GFP) is exclusively nuclear, and identified a highly conserved KRXR motif spanning residues 44–47 as a putative NLS. In addition, we demonstrated that residues 3–15 of BNP, though not an NLS, are also crucial for nuclear import. Results from mutational analyses of N70–GFP and the full-length BNP suggest that this region may be required for protection of the N-terminus from proteolytic cleavage. Altogether, we propose that the N-terminal region of BNP contains the NLS and cleavage-protection motif, which together drive its nuclear localization. - Highlights: • The N-terminal region of BNP is required for nuclear accumulation. • The conserved motif at position 44–47 is a putative nuclear localization signal. • The first 15 amino acids of BNP may function as a cleavage-protection motif. • BNP may get access to the nucleus via a mechanism distinct from ANP

  12. Possible cleavage sites of glutelin partial degradation confirmed by immunological analysis in globulin-less mutants of rice (Oryza sativa L.).

    Science.gov (United States)

    Khan, Nadar; Yamaguchi, Satoru; Katsube-Tanaka, Tomoyuki

    2017-10-01

    Proteolytic cleavage or partial degradation of proteins is one of the important post-translational modifications for various biological processes, but it is difficult to analyze. Previously, we demonstrated that some subunits of the major rice (Oryza sativa L.) seed storage protein glutelin are partially degraded to produce newly identified polypeptides X1-X5 in mutants in which another major seed storage protein globulin is absent. In this study, the new polypeptides X3 and X4/X5 were immunologically confirmed to be derived from GluA3 and GluA1/GluA2 subunits, respectively. Additionally, the new polypeptides X1 and X2 were at least in part the α polypeptides of the GluB4 subunit partially degraded at the C-terminus. Simulated 2D-PAGE migration patterns of intact and partially degraded α polypeptides based on the calculation of their MWs and pIs enabled us to narrow or predict the possible locations of cleavage sites. The predicted cleavage sites were also verified by the comparison of 2D-PAGE patterns between seed-extracted and E. coli-expressed proteins of the intact and truncated α polypeptides. The results and methodologies demonstrated here would be useful for analyses of partial degradation of proteins and the structure-function relationships of rice seed protein bodies. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Nuclear import of influenza B virus nucleoprotein: Involvement of an N-terminal nuclear localization signal and a cleavage-protection motif

    Energy Technology Data Exchange (ETDEWEB)

    Wanitchang, Asawin; Narkpuk, Jaraspim; Jongkaewwattana, Anan, E-mail: anan.jon@biotec.or.th

    2013-08-15

    The nucleoprotein of influenza B virus (BNP) shares several characteristics with its influenza A virus counterpart (ANP), including localization in the host's nucleus. However, while the nuclear localization signal(s) (NLS) of ANP are well characterized, little is known about those of BNP. In this study, we showed that the fusion protein bearing the BNP N-terminus fused with GFP (N70–GFP) is exclusively nuclear, and identified a highly conserved KRXR motif spanning residues 44–47 as a putative NLS. In addition, we demonstrated that residues 3–15 of BNP, though not an NLS, are also crucial for nuclear import. Results from mutational analyses of N70–GFP and the full-length BNP suggest that this region may be required for protection of the N-terminus from proteolytic cleavage. Altogether, we propose that the N-terminal region of BNP contains the NLS and cleavage-protection motif, which together drive its nuclear localization. - Highlights: • The N-terminal region of BNP is required for nuclear accumulation. • The conserved motif at position 44–47 is a putative nuclear localization signal. • The first 15 amino acids of BNP may function as a cleavage-protection motif. • BNP may get access to the nucleus via a mechanism distinct from ANP.

  14. Quantification of DNA cleavage specificity in Hi-C experiments.

    Science.gov (United States)

    Meluzzi, Dario; Arya, Gaurav

    2016-01-08

    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. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  15. Analysis by the reductive-cleavage method of linkage positions in a polysaccharide containing 4-linked D-glucopyranosyluronic residues.

    Science.gov (United States)

    Vodonik, S A; Gray, G R

    1988-04-01

    The fate of 4-linked D-glucopyranosyluronic residues under reductive-cleavage conditions was investigated by using the Klebsiella aerogenes type 54 strain A3 capsular polysaccharide. Treatment of the fully methylated polysaccharide with triethylsilane and trimethylsilyl trifluoromethanesulfonate in dichloromethane, followed by in situ acetylation, yielded 1,5-anhydro-2,3,4,6-tetra-O-methyl-D-glucitol, 3,4-di-O-acetyl-1,5-anhydro-2,6-di-O-methyl-D-glucitol, and 3-O-acetyl-1,5-anhydro-2,4-di-O-methyl-L-fucitol, as expected, but the expected product of reductive cleavage of the 4-linked D-glucopyranosyluronic residue, namely, methyl 3-O-acetyl-2,6-anhydro-4,5-di-O-methyl-L-gulonate, was not observed. Instead, methyl 2-O-acetyl-3,6-anhydro-4,5-di-O-methyl-L-gulonate (6) was identified as the sole product of reductive cleavage of the 4-linked D-glucopyranosyluronic residue. That compound 6 arose as a result of rearrangement during reductive cleavage rather than by reductive cleavage of a 5-linked D-glucofuranosyluronic residue, was established by reductive cleavage of the fully methylated polysaccharide following reduction of its ester groups with either lithium aluminum hydride or lithium aluminum deuteride. The products of the latter reductive cleavage were the same as before, except for the absence of 6 and the presence of 4,6-di-O-acetyl-1,5-anhydro-2,3-di-O-methyl-D-glucitol, or its 6,6-dideuterio isomer. Although the reductive-cleavage technique is suitable for the direct analysis of polysaccharides containing 4-linked D-glucopyranosyluronic residues, it does not establish whether the uronic residue is a 4-linked pyranoside or a 5-linked furanoside. The expected product is, however, derived from the 4-linked D-glucopyranosyluronic residue after sequential methylation, reduction of its ester group and reductive cleavage.

  16. The potato carotenoid cleavage dioxygenase 4 catalyzes a single cleavage of β-ionone ring-containing carotenes and non-epoxidated xanthophylls

    KAUST Repository

    Bruno, Mark

    2015-04-01

    Down-regulation of the potato carotenoid cleavage dioxygenase 4 (StCCD4) transcript level led to tubers with altered morphology and sprouting activity, which also accumulated higher levels of violaxanthin and lutein leading to elevated carotenoid amounts. This phenotype indicates a role of this enzyme in tuber development, which may be exerted by a cleavage product. In this work, we investigated the enzymatic activity of StCCD4, by expressing the corresponding cDNA in carotenoid accumulating Escherichia coli strains and by performing in vitro assays with heterologously expressed enzyme. StCCD4 catalyzed the cleavage of all-. trans-β-carotene at the C9\\'-C10\\' double bond, leading to β-ionone and all-. trans-β-apo-10\\'-carotenal, both in vivo and in vitro. The enzyme also cleaved β,β-cryptoxanthin, zeaxanthin and lutein either at the C9\\'-C10\\' or the C9-C10 double bond in vitro. In contrast, we did not observe any conversion of violaxanthin and only traces of activity with 9-. cis-β-carotene, which led to 9-. cis-β-apo-10\\'-carotenal. Our data indicate that all-. trans-β-carotene is the likely substrate of StCCD4 in planta, and that this carotene may be precursor of an unknown compound involved in tuber development.

  17. The potato carotenoid cleavage dioxygenase 4 catalyzes a single cleavage of β-ionone ring-containing carotenes and non-epoxidated xanthophylls.

    Science.gov (United States)

    Bruno, Mark; Beyer, Peter; Al-Babili, Salim

    2015-04-15

    Down-regulation of the potato carotenoid cleavage dioxygenase 4 (StCCD4) transcript level led to tubers with altered morphology and sprouting activity, which also accumulated higher levels of violaxanthin and lutein leading to elevated carotenoid amounts. This phenotype indicates a role of this enzyme in tuber development, which may be exerted by a cleavage product. In this work, we investigated the enzymatic activity of StCCD4, by expressing the corresponding cDNA in carotenoid accumulating Escherichia coli strains and by performing in vitro assays with heterologously expressed enzyme. StCCD4 catalyzed the cleavage of all-trans-β-carotene at the C9'-C10' double bond, leading to β-ionone and all-trans-β-apo-10'-carotenal, both in vivo and in vitro. The enzyme also cleaved β,β-cryptoxanthin, zeaxanthin and lutein either at the C9'-C10' or the C9-C10 double bond in vitro. In contrast, we did not observe any conversion of violaxanthin and only traces of activity with 9-cis-β-carotene, which led to 9-cis-β-apo-10'-carotenal. Our data indicate that all-trans-β-carotene is the likely substrate of StCCD4 in planta, and that this carotene may be precursor of an unknown compound involved in tuber development. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Tau truncation is a productive posttranslational modification of neurofibrillary degeneration in Alzheimer's disease.

    Science.gov (United States)

    Kovacech, B; Novak, M

    2010-12-01

    Deposits of the misfolded neuronal protein tau are major hallmarks of neurodegeneration in Alzheimer's disease (AD) and other tauopathies. The etiology of the transformation process of the intrinsically disordered soluble protein tau into the insoluble misordered aggregate has attracted much attention. Tau undergoes multiple modifications in AD, most notably hyperphosphorylation and truncation. Hyperphosphorylation is widely regarded as the hottest candidate for the inducer of the neurofibrillary pathology. However, the true nature of the impetus that initiates the whole process in the human brains remains unknown. In AD, several site-specific tau cleavages were identified and became connected to the progression of the disease. In addition, western blot analyses of tau species in AD brains reveal multitudes of various truncated forms. In this review we summarize evidence showing that tau truncation alone is sufficient to induce the complete cascade of neurofibrillary pathology, including hyperphosphorylation and accumulation of misfolded insoluble forms of tau. Therefore, proteolytical abnormalities in the stressed neurons and production of aberrant tau cleavage products deserve closer attention and should be considered as early therapeutic targets for Alzheimer's disease.

  19. The proteolytic profile of human cancer procoagulant suggests that it promotes cancer metastasis at the level of activation rather than degradation.

    Science.gov (United States)

    Kee, Nalise Low Ah; Krause, Jason; Blatch, Gregory L; Muramoto, Koji; Sakka, Kazuo; Sakka, Makiko; Naudé, Ryno J; Wagner, Leona; Wolf, Raik; Rahfeld, Jens-Ulrich; Demuth, Hans-Ulrich; Mielicki, Wojciech P; Frost, Carminita L

    2015-10-01

    Proteases are essential for tumour progression and many are over-expressed during this time. The main focus of research was the role of these proteases in degradation of the basement membrane and extracellular matrix (ECM), thereby enabling metastasis to occur. Cancer procoagulant (CP), a protease present in malignant tumours, but not normal tissue, is a known activator of coagulation factor X (FX). The present study investigated the function of CP in cancer progression by focussing on its enzymatic specificity. FX cleavage was confirmed using SDS-PAGE and MALDI-TOF MS and compared to the proteolytic action of CP on ECM proteins, including collagen type IV, laminin and fibronectin. Contrary to previous reports, CP cleaved FX at the conventional activation site (between Arg-52 and Ile-53). Additionally, degradation of FX by CP occurred at a much slower rate than degradation by conventional activators. Complete degradation of the heavy chain of FX was only visible after 24 h, while degradation by RVV was complete after 30 min, supporting postulations that the procoagulant function of CP may be of secondary importance to its role in cancer progression. Of the ECM proteins tested, only fibronectin was cleaved. The substrate specificity of CP was further investigated by screening synthetic peptide substrates using a novel direct CP assay. The results indicate that CP is not essential for either cancer-associated blood coagulation or the degradation of ECM proteins. Rather, they suggest that this protease may be required for the proteolytic activation of membrane receptors.

  20. Detection of nucleic acid sequences by invader-directed cleavage

    Science.gov (United States)

    Brow, Mary Ann D.; Hall, Jeff Steven Grotelueschen; Lyamichev, Victor; Olive, David Michael; Prudent, James Robert

    1999-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The 5' nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based by charge.

  1. Photooxidative cleavage of 4(1H)-quinolinones to 2-acylaminobenzoic acids and derivatives

    Energy Technology Data Exchange (ETDEWEB)

    Staskun, B. (University of the Witwatersrand, Johannesburg (South Africa). Dept. of Chemistry); Foote, C.S. (California Univ., Los Angeles (USA). Dept. of Chemistry)

    1984-12-01

    4(1H)-Quinolinones undergo oxidative cleavage to afford the corresponding 2-acylaminobenzoic acids when subjected to dye-sensitized photooxygenation in methanol-aqueous sodium hydroxide solution. The derived 2-aminobenzoic acid was the predominant product in certain instances. The reaction, with singlet oxygen suggested as the active species, provides an alternative methodology for access to nuclear- substituted anthranilic acids and derivatives.

  2. Influence of different proteolytic strains of Streptococcus thermophilus in co-culture with Lactobacillus delbrueckii subsp. bulgaricus on the metabolite profile of set-yoghurt.

    Science.gov (United States)

    Settachaimongkon, Sarn; Nout, M J Robert; Antunes Fernandes, Elsa C; Hettinga, Kasper A; Vervoort, Jacques M; van Hooijdonk, Toon C M; Zwietering, Marcel H; Smid, Eddy J; van Valenberg, Hein J F

    2014-05-02

    Proto-cooperation between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is one of the key factors that determine the fermentation process and final quality of yoghurt. In this study, the interaction between different proteolytic strains of S. thermophilus and L. delbrueckii subsp. bulgaricus was investigated in terms of microbial growth, acidification and changes in the biochemical composition of milk during set-yoghurt fermentation. A complementary metabolomics approach was applied for global characterization of volatile and non-volatile polar metabolite profiles of yoghurt associated with proteolytic activity of the individual strains in the starter cultures. The results demonstrated that only non-proteolytic S. thermophilus (Prt-) strain performed proto-cooperation with L. delbrueckii subsp. bulgaricus. The proto-cooperation resulted in significant higher populations of the two species, faster milk acidification, significant abundance of aroma volatiles and non-volatile metabolites desirable for a good organoleptic quality of yoghurt. Headspace SPME-GC/MS and (1)H NMR resulted in the identification of 35 volatiles and 43 non-volatile polar metabolites, respectively. Furthermore, multivariate statistical analysis allows discriminating set-yoghurts fermented by different types of starter cultures according to their metabolite profiles. Our finding underlines that selection of suitable strain combinations in yoghurt starters is important for achieving the best technological performance regarding the quality of product. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Cell growth and proteolytic activity of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus in milk as affected by supplementation with peptide fractions.

    Science.gov (United States)

    Gandhi, Akanksha; Shah, Nagendra P

    2014-12-01

    The present investigation examined the effects of supplementation of milk peptide fractions produced by enzymatic hydrolysis on the fermentation of reconstituted skim milk (RSM). Changes in pH, cell growth, proteolytic activity, and angiotensin-converting enzyme (ACE)-inhibitory activity were monitored during fermentation of RSM by pure cultures of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus. The study showed that supplementation with peptide fractions of different molecular weights did not significantly affect the bacterial growth in RSM. All bacteria showed an increased proteolytic activity in RSM supplemented with large peptides (>10 kDa), and L. helveticus in general exhibited the highest proteolytic activity among the bacteria studied. The ACE-inhibitory activity was observed to be the maximum in RSM supplemented with larger peptides (>10 kDa) for all bacteria. The results suggest that proteolysis by bacteria leads to increased production of ACE-inhibitory peptides compared to the supplemented peptides produced by enzymatic hydrolysis.

  4. Fangchinoline inhibits human immunodeficiency virus type 1 replication by interfering with gp160 proteolytic processing.

    Directory of Open Access Journals (Sweden)

    Zhitao Wan

    Full Text Available The introduction of highly active antiretroviral therapy has led to a significant reduction in the morbidity and mortality of acquired immunodeficiency syndrome patients. However, the emergence of drug resistance has resulted in the failure of treatments in large numbers of patients and thus necessitates the development of new classes of anti-HIV drugs. In this study, more than 200 plant-derived small-molecule compounds were evaluated in a cell-based HIV-1 antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (fangchinoline. Fangchinoline, a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae, exhibited antiviral activity against HIV-1 laboratory strains NL4-3, LAI and BaL in MT-4 and PM1 cells with a 50% effective concentration ranging from 0.8 to 1.7 µM. Mechanism-of-action studies showed that fangchinoline did not exhibit measurable antiviral activity in TZM-b1 cells but did inhibit the production of infectious virions in HIV-1 cDNA transfected 293T cells, which suggests that the compound targets a late event in infection cycle. Furthermore, the antiviral effect of fangchinoline seems to be HIV-1 envelope-dependent, as the production of infectious HIV-1 particles packaged with a heterologous envelope, the vesicular stomatitis virus G glycoprotein, was unaffected by fangchinoline. Western blot analysis of HIV envelope proteins expressed in transfected 293T cells and in isolated virions showed that fangchinoline inhibited HIV-1 gp160 processing, resulting in reduced envelope glycoprotein incorporation into nascent virions. Collectively, our results demonstrate that fangchinoline inhibits HIV-1 replication by interfering with gp160 proteolytic processing. Fangchinoline may serve as a starting point for developing a new HIV-1 therapeutic approach.

  5. The Role of Trop2 Cleavage Products in Prostate Tumorigenesis

    Science.gov (United States)

    2014-10-01

    Ranganathan et al. 2011). A recent study revealed that EpCAM, an adhesion protein that shares 50% homology with Trop2, functions in cellular...epithelial cancers. PLoS ONE 5: e14130. doi: 10.1371/journal.pone.0014130. Ranganathan P, Weaver KL, Capobianco AJ. 2011. Notch signal- ling in solid tumours

  6. Hydrolytic cleavage of ammonia-borane complex for hydrogen production

    Energy Technology Data Exchange (ETDEWEB)

    Mohajeri, Nahid; T-Raissi, Ali; Adebiyi, Olawale [Hydrogen R and D Division, Florida Solar Energy Center, University of Central Florida 1679 Clearlake Rd., Cocoa, FL 32955 (United States)

    2007-05-15

    A new process for generating hydrogen via near room temperature hydrolysis of AB complex using small amounts of platinum group metal catalyst has been studied. Using in situ {sup 11}B NMR spectroscopy, the overall rate of K{sub 2}Cl{sub 6}Pt catalyzed hydrolysis of AB complex was calculated to be third-order. The pre-exponential factor (A) and the activation energy (E{sub a}) of Arrhenius equation, ln k = ln A - E{sub a}/RT, were determined to be: A = 1.6 x 10{sup 11} L mol{sup -1} s{sup -1} and E{sub a} = 86.6 kJ mol{sup -1} for temperature range of (25-35 C). X-ray photoelectron spectroscopy of the residue suggested that the platinum salt was reduced from Pt{sup 4+} to Pt{sup 0} within the course of the reaction and X-ray diffraction analysis pattern for the residue showed crystallized single-phase boric acid. (author)

  7. 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...

  8. Macrophage-mediated proteolytic remodeling of the extracellular matrix in atherosclerosis results in neoepitopes

    DEFF Research Database (Denmark)

    Skjøt-Arkil, Helene; Barascuk, Natasha; Register, Thomas

    2010-01-01

    in almost all stages of atherosclerosis, by both initiating atherosclerotic plaques and degrading them through the secretion of proteolytic enzymes leading to rupture. This review summarizes the literature on the role of macrophages and their proteolytic activity on proteins in the extracellular matrix (ECM......) of the atherosclerotic plaque with a view to suggest a novel approach for identification of vulnerable plaques and turnover by the use of a new type of biomarker. The PubMed database was searched using the terms macrophages, foam cells, atherosclerosis, CVD, ECM remodeling, biomarker, neoepitope, matrix...... of the constituents of the ECM of the atherosclerotic plaque. At present it is not clear which proteases play pivotal roles at distinct stages of pathogenesis, rather that the combined proteolytic potential with some proteases at early stages and other at later stages may result in plaque rupture. This macrophage...

  9. Glycoside bond cleavage in the radiolysis of aqueous solutions of methylglycosides and disaccharides

    International Nuclear Information System (INIS)

    Shadyro, O.I.; Kisel', R.M.

    2007-01-01

    The kinetics of formation of methylglycoside and disaccharide radiolysis products resulting from the O-glycoside bond cleavage under the action of 137 Cs γ-radiation (0-2.5 kGy radiation doses, 0.28 Gy/s dose rate) was studied, and the yields of these products were determined. It was found that oxygen inhibits these processes. The findings suggest that the fragmentation reaction of C' 2 radicals plays an important role in the formation of carbohydrate degradation products in the radiolysis of aqueous carbohydrate solutions [ru

  10. 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.

  11. 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.

  12. Enzymatic carotenoid cleavage in star fruit (Averrhoa carambola).

    Science.gov (United States)

    Fleischmann, Peter; Watanabe, Naoharu; Winterhalter, Peter

    2003-05-01

    This paper presents the first description of an enzyme fraction exhibiting carotenoid cleavage activity isolated from fruit skin of Averrhoa carambola. Partial purification of the enzyme could be achieved by acetone precipitation, ultrafiltration (300 kDa, 50 kDa), isoelectric focusing (pH 3-10) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (7.5%). In this way, an enzymatically active protein fraction was obtained, consisting of four proteins in the molecular weight range of between 12 and 90 kDa. Using beta-carotene as substrate, the enzyme activity was detected spectrophotometrically at 505 nm. The main reaction product, detected by GC analysis, was beta-ionone. This proves that the isolated enzymes are closely related to aroma metabolism and release of star fruit. The time constant of the reaction was 16.6 min, the Michaelis Constant K(m)=3.6 micromol 1(-1) and the maximum velocity V(max)=10.5 x 10(-3) micromol l(-1) s(-1) mg((Protein))(-1). The optimum temperature was 45 degrees C.

  13. Characterisation of a novel proteolytic enzyme localised to goblet cells in rat and man

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier

    1984-01-01

    A proteolytic enzyme, ingobsin , purified from rat duodenal extracts is shown to be localised to intestinal goblet cells of both man and rat. Enzyme positive cells decrease in number from duodenum to colon. The enzyme is a 33 000 Mr protein with an isoelectric point of 5.1. The pH optimum...... for enzymatic activity is 7.4-8.0. Based on substrate specificity for arg-x, lys-x and to a lesser degree tyr-x, on the effect of diisopropylphosphorofluoride , Trasylol and phenylmethylsulfonylfluoride and on proteolytic activity towards intact proteins, ingobsin is classified as a serine proteinase...

  14. A novel branched chain amino acids responsive transcriptional regulator, BCARR, negatively acts on the proteolytic system in Lactobacillus helveticus.

    Directory of Open Access Journals (Sweden)

    Taketo Wakai

    Full Text Available Transcriptional negative regulation of the proteolytic system of Lactobacillus helveticus CM4 in response to amino acids seems to be very important for the control of antihypertensive peptide production; however, it remains poorly understood. A 26-kDa protein with N-terminal cystathionine β-synthase domains (CBS domain protein, which seems to be involved in the regulatory system, was purified by using a DNA-sepharose bound 300-bp DNA fragment corresponding to the upstream regions of the six proteolytic genes that are down-regulated by amino acids. The CBS domain protein bound to a DNA fragment corresponding to the region upstream of the pepV gene in response to branched chain amino acids (BCAAs. The expression of the pepV gene in Escherichia coli grown in BCAA-enriched medium was repressed when the CBS domain protein was co-expressed. These results reveal that the CBS domain protein acts as a novel type of BCAA-responsive transcriptional regulator (BCARR in L. helveticus. From comparative analysis of the promoter regions of the six proteolysis genes, a palindromic AT-rich motif, 5'-AAAAANNCTWTTATT-3', was predicted as the consensus DNA motif for the BCARR protein binding. Footprint analysis using the pepV promotor region and gel shift analyses with the corresponding short DNA fragments strongly suggested that the BCARR protein binds adjacent to the pepV promoter region and affects the transcription level of the pepV gene in the presence of BCAAs. Homology search analysis of the C-terminal region of the BCARR protein suggested the existence of a unique βαββαβ fold structure that has been reported in a variety of ACT (aspartate kinase-chorismate mutase-tyrA domain proteins for sensing amino acids. These results also suggest that the sensing of BCAAs by the ACT domain might promote the binding of the BCARR to DNA sequences upstream of proteolysis genes, which affects the gene expression of the proteolytic system in L. helveticus.

  15. Oxidative stress and nitrosative stress are involved in different stages of proteolytic pulmonary emphysema.

    Science.gov (United States)

    Lanzetti, Manuella; da Costa, Cristiane Aguiar; Nesi, Renata Tiscoski; Barroso, Marina Valente; Martins, Vanessa; Victoni, Tatiana; Lagente, Vincent; Pires, Karla Maria Pereira; e Silva, Patrícia Machado Rodrigues; Resende, Angela Castro; Porto, Luis Cristóvão; Benjamim, Cláudia Farias; Valença, Samuel Santos

    2012-12-01

    Our aim was to investigate the role of oxidative stress in elastase-induced pulmonary emphysema. C57BL/6 mice were subjected to pancreatic porcine elastase (PPE) instillation (0.05 or 0.5 U per mouse, i.t.) to induce pulmonary emphysema. Lungs were collected on days 7, 14, and 21 after PPE instillation. The control group was sham injected. Also, mice treated with 1% aminoguanidine (AMG) and inducible NO synthase (iNOS) knockout mice received 0.5 U PPE (i.t.), and lungs were analyzed 21 days after. We performed bronchoalveolar lavage, biochemical analyses of oxidative stress, and lung stereology and morphometry assays. Emphysema was observed histologically at 21 days after 0.5 U PPE treatment; tissues from these mice exhibited increased alveolar linear intercept and air-space volume density in comparison with the control group. TNF-α was elevated at 7 and 14 days after 0.5 U PPE treatment, concomitant with a reduction in the IL-10 levels at the same time points. Myeloperoxidase was elevated in all groups treated with 0.5 U PPE. Oxidative stress was observed during early stages of emphysema, with increased nitrite levels and malondialdehyde and superoxide dismutase activity at 7 days after 0.5 U PPE treatment. Glutathione peroxidase activity was increased in all groups treated with 0.5 U PPE. The emphysema was attenuated when iNOS was inhibited using 1% AMG and in iNOS knockout mice. Furthermore, proteolytic stimulation by PPE enhanced the expression of nitrotyrosine and iNOS, whereas the PPE+AMG group showed low expression of iNOS and nitrotyrosine. PPE stimulus also induced endothelial (e) NOS expression, whereas AMG reduced eNOS. Our results suggest that the oxidative and nitrosative stress pathways are triggered by nitric oxide production via iNOS expression in pulmonary emphysema. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Metaproteomics of cellulose methanisation under thermophilic conditions reveals a surprisingly high proteolytic activity.

    Science.gov (United States)

    Lü, Fan; Bize, Ariane; Guillot, Alain; Monnet, Véronique; Madigou, Céline; Chapleur, Olivier; Mazéas, Laurent; He, Pinjing; Bouchez, Théodore

    2014-01-01

    Cellulose is the most abundant biopolymer on Earth. Optimising energy recovery from this renewable but recalcitrant material is a key issue. The metaproteome expressed by thermophilic communities during cellulose anaerobic digestion was investigated in microcosms. By multiplying the analytical replicates (65 protein fractions analysed by MS/MS) and relying solely on public protein databases, more than 500 non-redundant protein functions were identified. The taxonomic community structure as inferred from the metaproteomic data set was in good overall agreement with 16S rRNA gene tag pyrosequencing and fluorescent in situ hybridisation analyses. Numerous functions related to cellulose and hemicellulose hydrolysis and fermentation catalysed by bacteria related to Caldicellulosiruptor spp. and Clostridium thermocellum were retrieved, indicating their key role in the cellulose-degradation process and also suggesting their complementary action. Despite the abundance of acetate as a major fermentation product, key methanogenesis enzymes from the acetoclastic pathway were not detected. In contrast, enzymes from the hydrogenotrophic pathway affiliated to Methanothermobacter were almost exclusively identified for methanogenesis, suggesting a syntrophic acetate oxidation process coupled to hydrogenotrophic methanogenesis. Isotopic analyses confirmed the high dominance of the hydrogenotrophic methanogenesis. Very surprising was the identification of an abundant proteolytic activity from Coprothermobacter proteolyticus strains, probably acting as scavenger and/or predator performing proteolysis and fermentation. Metaproteomics thus appeared as an efficient tool to unravel and characterise metabolic networks as well as ecological interactions during methanisation bioprocesses. More generally, metaproteomics provides direct functional insights at a limited cost, and its attractiveness should increase in the future as sequence databases are growing exponentially.

  17. Translation of Polioviral mRNA Is Inhibited by Cleavage of Polypyrimidine Tract-Binding Proteins Executed by Polioviral 3Cpro

    Science.gov (United States)

    Back, Sung Hoon; Kim, Yoon Ki; Kim, Woo Jae; Cho, Sungchan; Oh, Hoe Rang; Kim, Jung-Eun; Jang, Sung Key

    2002-01-01

    The translation of polioviral mRNA occurs through an internal ribosomal entry site (IRES). Several RNA-binding proteins, such as polypyrimidine tract-binding protein (PTB) and poly(rC)-binding protein (PCBP), are required for the poliovirus IRES-dependent translation. Here we report that a poliovirus protein, 3Cpro (and/or 3CDpro), cleaves PTB isoforms (PTB1, PTB2, and PTB4). Three 3Cpro target sites (one major target site and two minor target sites) exist in PTBs. PTB fragments generated by poliovirus infection are redistributed to the cytoplasm from the nucleus, where most of the intact PTBs are localized. Moreover, these PTB fragments inhibit polioviral IRES-dependent translation in a cell-based assay system. We speculate that the proteolytic cleavage of PTBs may contribute to the molecular switching from translation to replication of polioviral RNA. PMID:11836431

  18. The N-terminal cleavage of chondromodulin-I in growth-plate cartilage at the hypertrophic and calcified zones during bone development.

    Directory of Open Access Journals (Sweden)

    Shigenori Miura

    Full Text Available Chondromodulin-I (ChM-I is a 20-25 kDa anti-angiogenic glycoprotein in cartilage matrix. In the present study, we identified a novel 14-kDa species of ChM-I by immunoblotting, and purified it by immunoprecipitation with a newly raised monoclonal antibody against ChM-I. The N-terminal amino acid sequencing indicated that it was an N-terminal truncated form of ChM-I generated by the proteolytic cleavage at Asp37-Asp38. This 14-kDa ChM-I was shown by the modified Boyden chamber assay to have very little inhibitory activity on the VEGF-A-induced migration of vascular endothelial cells in contrast to the intact 20-25 kDa form of ChM-I (ID50 = 8 nM. Immunohistochemistry suggested that 20-25 kDa ChM-I was exclusively localized in the avascular zones, i.e. the resting, proliferating, and prehypertrophic zones, of the cartilaginous molds of developing long bone, whereas the 14-kDa form of ChM-I was found in hypertrophic and calcified zones. Immunoblotting demonstrated that mature growth-plate chondrocytes isolated from rat costal cartilage actively secrete ChM-I almost exclusively as the intact 20-25 kDa form into the medium in primary culture. Taken together, our results suggest that intact 20-25 kDa ChM-I is stored as a component of extracellular matrix in the avascular cartilage zones, but it is inactivated by a single N-terminal proteolytic cleavage in the hypertrophic zone of growth-plate cartilage.

  19. Proteolytic activity of recombinant DegP from Chromohalobacter salexigens BKL5

    Directory of Open Access Journals (Sweden)

    Dewi Fitriani

    2017-09-01

    Conclusions: Recombinant DegP from C. salexigens BKL5 showed proteolytic activity when β-casein was used as a substrate. In silico analysis indicated that recombinant DegP had characteristics similar to those of halophilic proteins depending on its amino acid composition.

  20. Glucocorticoids activate the ATP-ubiquitin-dependent proteolytic system in skeletal muscle during fasting

    Science.gov (United States)

    Wing, S. S.; Goldberg, A. L.; Goldberger, A. L. (Principal Investigator)

    1993-01-01

    Glucocorticoids are essential for the increase in protein breakdown in skeletal muscle normally seen during fasting. To determine which proteolytic pathway(s) are activated upon fasting, leg muscles from fed and fasted normal rats were incubated under conditions that block or activate different proteolytic systems. After food deprivation (1 day), the nonlysosomal ATP-dependent process increased by 250%, as shown in experiments involving depletion of muscle ATP. Also, the maximal capacity of the lysosomal process increased 60-100%, but no changes occurred in the Ca(2+)-dependent or the residual energy-independent proteolytic processes. In muscles from fasted normal and adrenalectomized (ADX) rats, the protein breakdown sensitive to inhibitors of the lysosomal or Ca(2+)-dependent pathways did not differ. However, the ATP-dependent process was 30% slower in muscles from fasted ADX rats. Administering dexamethasone to these animals or incubating their muscles with dexamethasone reversed this defect. During fasting, when the ATP-dependent process rises, muscles show a two- to threefold increase in levels of ubiquitin (Ub) mRNA. However, muscles of ADX animals failed to show this response. Injecting dexamethasone into the fasted ADX animals increased muscle Ub mRNA within 6 h. Thus glucocorticoids activate the ATP-Ub-dependent proteolytic pathway in fasting apparently by enhancing the expression of components of this system such as Ub.

  1. Proteolytic activity of IgGs from blood serum of wistar rats at experimental rheumatoid arthritis

    Directory of Open Access Journals (Sweden)

    Yu. Ya. Kit

    2014-10-01

    Full Text Available The aim of this work was to study the proteolytic activity of IgGs purified from blood serum of Wistar rats at experimental rheumatoid arthritis (ERA induced by an injection of bovine collagen of type II. Twenty rats were immunized with a preparation of bovine collagen II (Sigma-Aldrich, USA in the presence of complete Freund’s adjuvant. ERA development was determined by inflammation in limbs of treated animals. IgG preparations were isolated from blood serum of immunized and non-immunized animals by precipitation of antibodies with 33% ammonium sulfate followed by chromatography on the Protein G-Sepharose column. Human histone H1, bovine collagen II, calf thymus histones, myelin basic protein (MBP, bovine serum albumin (BSA, and bovine casein were used as substrates of the proteolytic activity of IgGs. It was found that IgG preparations from blood serum of rats with ERA were capable of cleaving histone H1 and MBP, however, they were catalytically inactive towards collagen II, casein, BSA, and core histones. IgGs from blood serum of non-immunized rats were proteolytically inactive towards all used protein substrates. Thus, we demonstrated that immunization of rats with bovine collagen II induced IgG-antibodies possessing the proteolytic activity towards histone H1 and MBP. This activity might be associated with the development of inflammatory processes in the immunized rats.

  2. Proteolytic Enzymes in Detergents: Evidence of Their Presence through Activity Measurements Based on Electrophoresis

    Science.gov (United States)

    Saperas, Nuria; Fonfria-Subiros, Elsa

    2011-01-01

    This laboratory exercise uses a problem-based approach to expose students to some basic concepts relating to proteins and enzymes. One of the main applications of enzymes at the industrial level is their use in the detergent market. The students examine a detergent sample to ascertain whether proteolytic enzymes are a component and, if so, which…

  3. Proteolytic stability in colloidal systems : interaction of proteins with the solid-water interface

    NARCIS (Netherlands)

    Maste, M.C.L.

    1996-01-01


    Proteolytic enzymes in liquid detergents suffer from lack of stability in the sense that activity diminishes with time. Although the phenomenon could be attributed to several factors, the influence of colloidal surfaces on the enzymatic stability was investigated. Besides the types of

  4. Proteolytic system of blood-feeding ticks: An update on protein structures

    Czech Academy of Sciences Publication Activity Database

    Kovářová, Zuzana; Hobizalová, Radka; Žebrakovská, Iva; Brynda, Jiří; Řezáčová, Pavlína; Horn, Martin; Mareš, Michael

    2015-01-01

    Roč. 22, č. 1 (2015), s. 43-44 ISSN 1211-5894. [Discussions in Structural Molecular Biology. Annual Meeting of the Czech Society for Structural Biology /13./. 19.03.2015-21.03.2015, Nové Hrady] Institutional support: RVO:61388963 ; RVO:68378050 Keywords : blood-feeding ticks * protein structure * proteolytic system Subject RIV: CE - Biochemistry

  5. Genetics of Proteolytic Enzymes of Lactococci and Their Role in Cheese Flavor Development

    NARCIS (Netherlands)

    Kok, Jan

    In recent years, knowledge of the genetics and biochemistry of the enzymes that constitute the proteolytic system of starter lactococci has increased tremendously. This paper summarizes the data obtained largely in the last 5 yr of intensive research by various research groups operative in this

  6. Stimulation of NADH-dependent microsomal DNA strand cleavage by rifamycin SV.

    Science.gov (United States)

    Kukiełka, E; Cederbaum, A I

    1995-04-15

    Rifamycin SV is an antibiotic anti-bacterial agent used in the treatment of tuberculosis. This drug can autoxidize, especially in the presence of metals, and generate reactive oxygen species. A previous study indicated that rifamycin SV can increase NADH-dependent microsomal production of reactive oxygen species. The current study evaluated the ability of rifamycin SV to interact with iron and increase microsomal production of hydroxyl radical, as detected by conversion of supercoiled plasmid DNA into the relaxed open circular state. The plasmid used was pBluescript II KS(-), and the forms of DNA were separated by agarose-gel electrophoresis. Incubation of rat liver microsomes with plasmid plus NADH plus ferric-ATP caused DNA strand cleavage. The addition of rifamycin SV produced a time- and concentration-dependent increase in DNA-strand cleavage. No stimulation by rifamycin SV occurred in the absence of microsomes, NADH or ferric-ATP. Stimulation occurred with other ferric complexes besides ferric-ATP, e.g. ferric-histidine, ferric-citrate, ferric-EDTA, and ferric-(NH4)2SO4. Rifamycin SV did not significantly increase the high rates of DNA strand cleavage found with NADPH as the microsomal reductant. The stimulation of NADH-dependent microsomal DNA strand cleavage was completely blocked by catalase, superoxide dismutase, GSH and a variety of hydroxyl-radical-scavenging agents, but not by anti-oxidants that prevent microsomal lipid peroxidation. Redox cycling agents, such as menadione and paraquat, in contrast with rifamycin SV, stimulated the NADPH-dependent reaction; menadione and rifamycin SV were superior to paraquat in stimulating the NADH-dependent reaction. These results indicate that rifamycin SV can, in the presence of an iron catalyst, increase microsomal production of reactive oxygen species which can cause DNA-strand cleavage. In contrast with other redox cycling agents, the stimulation by rifamycin SV is more pronounced with NADH than with NADPH as the

  7. Planar integrated optical waveguide used as a transducer to yield chemical information: detection of the activity of proteolytic enzymes e.g. serine-proteases

    Science.gov (United States)

    Zhylyak, Gleb; Ramoz-Perez, Victor; Linnhoff, Michael; Hug, Thomas; Citterio, Daniel; Spichiger-Keller, Ursula E.

    2005-03-01

    The paper shows the very first results of a feasibility study where the activity of proteolytic enzymes towards dye-labelled artificial substrates immobilized on the surface of planar optical Ta2O5 waveguide was investigated. Within this project, a chromophore label was developed, synthesized and attached to the carboxy-terminus of specific tripeptides. The goal was to develop a highly sensitive optical assay in order to monitor the activity of serine-proteases by cleavage of the amide bond between peptide and chromophore. On the one hand, a strategy was developed to immobilize the labeled tripeptide unto integrated planar waveguides. On the other hand, an instrument, the so-called "chip-reader" was developed to detect the biological process on the surface of the integrated planar optical waveguide. Surface characteristics were analyzed by XPS, TOF-SIMS and contact angle measurements. A comparison between the effectivity of ATR-photometry on chip using TE0 mode and photometry in transmission mode is discussed.

  8. Secondary isotope effects on alpha-cleavage reactions

    International Nuclear Information System (INIS)

    Ingemann, S.; Hammerum, S.

    1980-01-01

    Kinetic deuterium isotope effects on mass spectral reactions have in several instances been utilized to provide structural information and to answer mechanistic questions. Typically, the influence of the deuterium label on the rate of one of a number of competing reactions has been studied. Secondary isotope effects have usually been assumed to be relatively insignificant in comparison with the observed kinetic effects, even though various workers have shown that secondary isotope effects may indeed exert a considerable influence on the rates of competing simple cleavages. Recent studies have provided quantitative data to show that the mere presence of deuterium atoms up to six bonds away may influence the rate of a simple cleavage reaction. In relation to an investigation of rearrangements accompanying simple cleavage reactions, a semi-quantitative measure was needed of the variation of the secondary isotope effect with the number of bonds between the deuterium label and the point of rupture. The influence has therefore been examined of the presence of remote deuterium atoms on a typical simple cleavage reaction, the α-cleavage of aliphatic amines. As a model compound, N-methyldipentylamine was chosen, systematically labelled with deuterium. (author)

  9. 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

    2014-01-01

    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. PMID:25548410

  10. Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells' Migration.

    Directory of Open Access Journals (Sweden)

    Monica Salamone

    Full Text Available In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a "resting" phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4 and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs or Serine Integral Membrane Peptidases (SIMPs caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process.

  11. Optical Imaging of Matrix Metalloproteinase-7 Activity in Vivo Using a Proteolytic Nanobeacon

    Directory of Open Access Journals (Sweden)

    Randy L. Scherer

    2008-05-01

    Full Text Available Matrix metalloproteinases (MMPs are extracellular proteolytic enzymes involved in tumor progression. We present the in vivo detection and quantitation of MMP7 activity using a specific near-infrared polymer-based proteolytic beacon, PB-M7NIR. PB-M7NIR is a pegylated polyamidoamine PAMAM-Generation 4 dendrimer core covalently coupled to a Cy5.5-labeled peptide representing a selective substrate that monitors MMP7 activity (sensor and AF750 as an internal reference to monitor relative substrate concentration (reference. In vivo imaging of tumors expressing MMP7 had a median sensor to reference ratio 2.2-fold higher than a that of a bilateral control tumor. Ex vivo imaging of intestines of multiple intestinal neoplasia (APCMin mice injected systemically with PB-M7NIR revealed a sixfold increase in the sensor to reference ratio in the adenomas of APCMin mice compared with control intestinal tissue or adenomas from MMP7-null Min mice. PB-M7NIR detected tumor sizes as small as 0.01 cm2, and the sensor to reference ratio was independent of tumor size. Histologic sectioning of xenograft tumors localized the proteolytic signal to the extracellular matrix; MMP7-overexpressing tumors displayed an approximately 300-fold enhancement in the sensor to reference ratio compared with nonexpressing tumor cells. In APCMin adenomas, the proteolytic signal colocalized with the endogenously expressed MMP7 protein, with sensor to reference ratios approximately sixfold greater than that of normal intestinal epithelium. PB-M7NIR provides a useful reagent for the in vivo and ex vivo quantitation and localization of MMP-selective proteolytic activity.

  12. Proteolytic Digestion and TiO2 Phosphopeptide Enrichment Microreactor for Fast MS Identification of Proteins

    Science.gov (United States)

    Deng, Jingren; Lazar, Iulia M.

    2016-04-01

    The characterization of phosphorylation state(s) of a protein is best accomplished by using isolated or enriched phosphoprotein samples or their corresponding phosphopeptides. The process is typically time-consuming as, often, a combination of analytical approaches must be used. To facilitate throughput in the study of phosphoproteins, a microreactor that enables a novel strategy for performing fast proteolytic digestion and selective phosphopeptide enrichment was developed. The microreactor was fabricated using 100 μm i.d. fused-silica capillaries packed with 1-2 mm beds of C18 and/or TiO2 particles. Proteolytic digestion-only, phosphopeptide enrichment-only, and sequential proteolytic digestion/phosphopeptide enrichment microreactors were developed and tested with standard protein mixtures. The protein samples were adsorbed on the C18 particles, quickly digested with a proteolytic enzyme infused over the adsorbed proteins, and further eluted onto the TiO2 microreactor for enrichment in phosphopeptides. A number of parameters were optimized to speed up the digestion and enrichments processes, including microreactor dimensions, sample concentrations, digestion time, flow rates, buffer compositions, and pH. The effective time for the steps of proteolytic digestion and enrichment was less than 5 min. For simple samples, such as standard protein mixtures, this approach provided equivalent or better results than conventional bench-top methods, in terms of both enzymatic digestion and selectivity. Analysis times and reagent costs were reduced ~10- to 15-fold. Preliminary analysis of cell extracts and recombinant proteins indicated the feasibility of integration of these microreactors in more advanced workflows amenable for handling real-world biological samples.

  13. Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells' Migration.

    Science.gov (United States)

    Salamone, Monica; Carfì Pavia, Francesco; Ghersi, Giulio

    2016-01-01

    In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a "resting" phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process.

  14. Proteolytic Enzymes Clustered in Specialized Plasma-Membrane Domains Drive Endothelial Cells’ Migration

    Science.gov (United States)

    Salamone, Monica; Carfì Pavia, Francesco

    2016-01-01

    In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a “resting” phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process. PMID:27152413

  15. A method for the detection of proteolytic enzymes in electropherograms

    NARCIS (Netherlands)

    Venrooij, W.J.W. van

    As has been described earlier, it is possible to localize amylase on agar electropherograms exactly and with great sensitivity with the help of a "sandwich method". This method is generally applicable to enzymes, the substrates of which can be differentiated from their degradation products by

  16. 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.

  17. 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.

  18. Short RNA guides cleavage by eukaryotic RNase III.

    Directory of Open Access Journals (Sweden)

    Bruno Lamontagne

    Full Text Available In eukaryotes, short RNAs guide a variety of enzymatic activities that range from RNA editing to translation repression. It is hypothesized that pre-existing proteins evolved to bind and use guide RNA during evolution. However, the capacity of modern proteins to adopt new RNA guides has never been demonstrated. Here we show that Rnt1p, the yeast orthologue of the bacterial dsRNA-specific RNase III, can bind short RNA transcripts and use them as guides for sequence-specific cleavage. Target cleavage occurred at a constant distance from the Rnt1p binding site, leaving the guide RNA intact for subsequent cleavage. Our results indicate that RNase III may trigger sequence-specific RNA degradation independent of the RNAi machinery, and they open the road for a new generation of precise RNA silencing tools that do not trigger a dsRNA-mediated immune response.

  19. Prediction of proteasome cleavage motifs by neural networks

    DEFF Research Database (Denmark)

    Kesimir, C.; Nussbaum, A.K.; Schild, H.

    2002-01-01

    physiological conditions. Our algorithm has been trained not only on in vitro data, but also on MHC Class I ligand data, which reflect a combination of immunoproteasome and constitutive proteasome specificity. This feature, together with the use of neural networks, a non-linear classification technique, make...... the prediction of MHC Class I ligand boundaries more accurate: 65% of the cleavage sites and 85% of the non-cleavage sites are correctly determined. Moreover, we show that the neural networks trained on the constitutive proteasome data learns a specificity that differs from that of the networks trained on MHC...

  20. 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...... reduced compared to the conventional chemical cleavage method (CCM), and even by using a uniformly labelled probe, the exact position and nature of the mutation can be revealed. The SpCCM is suitable for automatization using a workstation to carry out the reactions and a fluorescent detection-based DNA...

  1. Distinctive proteolytic activity of cell envelope proteinase of Lactobacillus helveticus isolated from airag, a traditional Mongolian fermented mare's milk.

    Science.gov (United States)

    Miyamoto, Mari; Ueno, Hiroshi M; Watanabe, Masayuki; Tatsuma, Yumi; Seto, Yasuyuki; Miyamoto, Taku; Nakajima, Hadjime

    2015-03-16

    Airag is a traditional fermented milk of Mongolia that is usually made from raw mare's milk. Lactobacillus helveticus is one of the lactic acid bacteria most frequently isolated from airag. In this study, we investigated the genetic and physiological characteristics of L. helveticus strains isolated from airag and clarified their significance in airag by comparing them with strains from different sources. Six strains of L. helveticus were isolated from five home-made airag samples collected from different regions of Mongolia. The optimal temperature for acidification in skim milk was 30 to 35°C for all the Mongolian strains, which is lower than those for the reference strains (JCM 1554 and JCM 1120(T)) isolated from European cheeses. All of the strains had a prtH1-like gene encoding a variant type of cell envelope proteinase (CEP). The CEP amino acid sequence in Snow Brand Typeculture (SBT) 11087 isolated from airag shared 71% identity with PrtH of L. helveticus CNRZ32 (AAD50643.1) but 98% identity with PrtH of Lactobacillus kefiranofaciens ZW3 (AEG40278.1) isolated from a traditional fermented milk in Tibet. The proteolytic activities of the CEP from SBT11087 on artificial substrate (N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) and pure casein were measured using an intact-cell degradation assay. The activity of the CEP from SBT11087 was observed to be weak and exhibited a lower optimal temperature (40°C) than those from the reference strains (45-50°C). The specificity of the SBT11087 CEP for αS1-casein was typical of the CEPs previously reported in L. helveticus, as determined through the degradation profiles obtained through gel electrophoresis and mass spectrometry analyses. In contrast, the degradation profile of β-casein revealed that the CEP of SBT11087 primarily hydrolyzes its C-terminal domain and hydrolyzed nine of the 16 cleavage sites shared among the CEPs of other L. helveticus strains. Thus, the CEP of SBT11087 is distinct from those from

  2. Abnormal early cleavage events predict early embryo demise: sperm oxidative stress and early abnormal cleavage.

    Science.gov (United States)

    Burruel, Victoria; Klooster, Katie; Barker, Christopher M; Pera, Renee Reijo; Meyers, Stuart

    2014-10-13

    Human embryos resulting from abnormal early cleavage can result in aneuploidy and failure to develop normally to the blastocyst stage. The nature of paternal influence on early embryo development has not been directly demonstrated although many studies have suggested effects from spermatozoal chromatin packaging, DNA damage, centriolar and mitotic spindle integrity, and plasma membrane integrity. The goal of this study was to determine whether early developmental events were affected by oxidative damage to the fertilizing sperm. Survival analysis was used to compare patterns of blastocyst formation based on P2 duration. Kaplan-Meier survival curves demonstrate that relatively few embryos with short (P2 times reached blastocysts, and the two curves diverged beginning on day 4, with nearly all of the embryos with longer P2 times reaching blastocysts by day 6 (p < .01). We determined that duration of the 2nd to 3rd mitoses were sensitive periods in the presence of spermatozoal oxidative stress. Embryos that displayed either too long or too short cytokineses demonstrated an increased failure to reach blastocyst stage and therefore survive for further development. Although paternal-derived gene expression occurs later in development, this study suggests a specific role in early mitosis that is highly influenced by paternal factors.

  3. Mechanisms of Bond Cleavage during Manganese Oxide and UV Degradation of Glyphosate: Results from Phosphate Oxygen Isotopes and Molecular Simulations.

    Science.gov (United States)

    Jaisi, Deb P; Li, Hui; Wallace, Adam F; Paudel, Prajwal; Sun, Mingjing; Balakrishna, Avula; Lerch, Robert N

    2016-11-16

    Degradation of glyphosate in the presence of manganese oxide and UV light was analyzed using phosphate oxygen isotope ratios and density function theory (DFT). The preference of C-P or C-N bond cleavage was found to vary with changing glyphosate/manganese oxide ratios, indicating the potential role of sorption-induced conformational changes on the composition of intermediate degradation products. Isotope data confirmed that one oxygen atom derived solely from water was incorporated into the released phosphate during glyphosate degradation, and this might suggest similar nucleophilic substitution at P centers and C-P bond cleavage both in manganese oxide- and UV light-mediated degradation. The DFT results reveal that the C-P bond could be cleaved by water, OH - or • OH, with the energy barrier opposing bond dissociation being lowest in the presence of the radical species, and that C-N bond cleavage is favored by the formation of both nitrogen- and carbon-centered radicals. Overall, these results highlight the factors controlling the dominance of C-P or C-N bond cleavage that determines the composition of intermediate/final products and ultimately the degradation pathway.

  4. Proteolytic activities in fillets of selected underutilized Australian fish species.

    Science.gov (United States)

    Ahmed, Z; Donkor, O; Street, W A; Vasiljevic, T

    2013-09-01

    The hydrolytic activity of major endogenous proteases, responsible for proteolysis of myofibrillar proteins during post-mortem storage, may be an indicator of the textural quality of fish which influences consumer purchasing behaviour and thus market value of the final product. Furthermore, it may also influence the type and bioactive properties of the peptides released during post-mortem proteolysis of myofibrillar proteins. This study compared the activities of cathepsins B, B+L, D, H and calpain-like enzymes in crude muscle extracted from 16 Australian underutilized fish species. Fish species had a significant effect on the activity of these enzymes with barracouta showing the highest cathepsins B, B+L, D and H activities. Activities of cathepsins B and B+L were higher than cathepsin H for all studied species. The more commercially important rock ling and tiger flathead demonstrated higher cathepsin B+L activity, whereas gemfish and eastern school whiting showed higher activity towards cathepsin B. Underutilized fish species showing higher endogenous protease activities may be suitable for fish sauce production, whereas those with lower protease activities for surimi processing. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Affect of Presenilin Mutations on APP Cleavage; Insights into the Pathogenesis of FAD

    Directory of Open Access Journals (Sweden)

    Nuomin eLi

    2016-03-01

    Full Text Available Alzheimer disease (AD is characterized by progressive memory loss, reduction in cognitive functions, and damage to the brain. The β-amyloid precursor protein can be sequentially cleaved by β- secretase and γ-secretase. Mutations in the presenilin1(PS1) are the most common cause of Familial Alzheimer’s disease ( FAD. PS1 mutations can alter the activity of γ-secretase on the cleavage of the β-amyloid precursor protein, causing increased Aβ production. Previous studies show that the βAPP-C-terminal fragment is first cleaved by β-scretase, primarily generating long fragments of Aβ48 and Aβ49, followed by the stepwise cleavage of every three amino acid residues at the C terminus, resulting in Aβ48-, 45-, 42 line and Aβ49-, 46-, 43-, 40 line. Here, we used LC-MS/MS to analyze unique peptides IAT, VVIA, ITL,TVI,IVI through sequential cleavage, combined with ELISA to test the level of Aβ42 and Aβ40 for validation. The results show that most FAD mutant PS1 can alter the level of Aβ42 and Aβ40 monitored by the Aβ42/Aβ40 ratio. Among them, 6 mutants (I143T, H163P, S170F, Q223R, M233V and G384A affect the Aβ42/40 ratio through both Aβ49-40 and Aβ48-38 lines; L166P through decreasing the Aβ49-40 line, 6 mutants (I143V, M146V, G217A, E280A, L381V and L392V through increasing the Aβ48-42 line. More importantly, we found some mutations can affect the γ-secretase cleavage preference of α-CTF and β-CTF. In conclusion, we found that the FAD PS1 mutations mainly increase the generation of Aβ42 by decreasing the cleavage of Aβ42-Aβ38 and Aβ43-Aβ40.

  6. 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 ass...

  7. DNA Cleavage Activity of Diazonium Salts: Chemical Nucleases

    OpenAIRE

    KIZIL, Murat

    2014-01-01

    4-Fenoldiazonium tetrafluoroborate and 4-benzoicaciddiazonium tetrafluoroborate was prepared and was shown to be an effective DNA cleavage agent in the presence of the 1-electron donor copper(II) chloride. Its mechanism involves the generation of the aryl radical cleaving DNA by hydrogen atom abstraction from deoxyribose sugar.

  8. Cleavage sites within the poliovirus capsid protein precursors

    International Nuclear Information System (INIS)

    Larsen, G.R.; Anderson, C.W.; Dorner, A.J.; Semler, B.L.; Wimmer, E.

    1982-01-01

    Partial amino-terminal sequence analysis was performed on radiolabeled poliovirus capsid proteins VP1, VP2, and VP3. A computer-assisted comparison of the amino acid sequences obtained with that predicted by the nucleotide sequence of the poliovirus genome allows assignment of the amino terminus of each capsid protein to a unique position within the virus polyprotein. Sequence analysis of trypsin-digested VP4, which has a blocked amino terminus, demonstrates that VP4 is encoded at or very near to the amino terminus of the polyprotein. The gene order of the capsid proteins is VP4-VP2-VP3-VP1. Cleavage of VP0 to VP4 and VP2 is shown to occur between asparagine and serine, whereas the cleavages that separate VP2/VP3 and VP3/VP1 occur between glutamine and glycine residues. This finding supports the hypothesis that the cleavage of VP0, which occurs during virion morphogenesis, is distinct from the cleavages that separate functional regions of the polyprotein

  9. 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 mo...

  10. 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...

  11. Cell-surface acceleration of urokinase-catalyzed receptor cleavage

    DEFF Research Database (Denmark)

    Høyer-Hansen, G; Ploug, M; Behrendt, N

    1997-01-01

    by a prior incubation of the cells with uPA inactivated by diisopropyl fluorophosphate, demonstrating a requirement for specific receptor binding of the active uPA to obtain the high-efficiency cleavage of cell-bound uPAR. Furthermore, amino-terminal sequence analysis revealed that uPAR(2+3), purified from U...

  12. Effect of Presenilin Mutations on APP Cleavage; Insights into the Pathogenesis of FAD

    OpenAIRE

    Li, Nuomin; Liu, Kefu; Qiu, Yunjie; Ren, Zehui; Dai, Rongji; Deng, Yulin; Qing, Hong

    2016-01-01

    Alzheimer disease (AD) is characterized by progressive memory loss, reduction in cognitive functions, and damage to the brain. The β-amyloid precursor protein can be sequentially cleaved by β- secretase and γ-secretase. Mutations in the presenilin1(PS1) are the most common cause of Familial Alzheimer’s disease (FAD). PS1 mutations can alter the activity of γ-secretase on the cleavage of the β-amyloid precursor protein, causing increased Aβ production. Previous studies show that the βAPP-C-ter...

  13. Furin proteolytically processes the heparin-binding region of extracellular superoxide dismutase

    DEFF Research Database (Denmark)

    Bowler, Russell P; Nicks, Mike; Olsen, Dorte Aa

    2002-01-01

    Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that attenuates brain and lung injury from oxidative stress. A polybasic region in the carboxyl terminus distinguishes EC-SOD from other superoxide dismutases and determines EC-SOD's tissue half-life and affinity for heparin....... There are two types of EC-SOD that differ based on the presence or absence of this heparin-binding region. It has recently been shown that proteolytic removal of the heparin-binding region is an intracellular event (Enghild, J. J., Thogersen, I. B., Oury, T. D., Valnickova, Z., Hojrup, P., and Crapo, J. D...... of intracellular proteases implicate furin as a processing protease. In vitro experiments using furin and purified EC-SOD suggest that furin proteolytically cleaves EC-SOD in the middle of the polybasic region and then requires an additional carboxypeptidase to remove the remaining lysines and arginines...

  14. [Treatment of surface burns with proteolytic enzymes: mathematic description of lysis kinetics].

    Science.gov (United States)

    Domogatskaia, A S; Domogatskiĭ, S P; Ruuge, E K

    2003-01-01

    The lysis of necrotic tissue by a proteolytic enzyme applied to the surface of a burn wound was studied. A mathematical model was proposed, which describes changes in the thickness of necrotic tissue as a function of the proteolytic activity of the enzyme. The model takes into account the inward-directed diffusion of the enzyme, the counterflow of interstitial fluid (exudates) containing specific inhibitors, and the extracellular matrix proteolysis. It was shown in terms of the quasi-stationary approach that the thickness of the necrotic tissue layer decreases exponentially with time; i.e., the lysis slows down as the thickness of the necrotic tissue layer decreases. The dependence of the characteristic time of this decrease on enzyme concentration was obtained. It was shown that, at high enzyme concentrations (more than 5 mg/ml), the entire time of lysis (after the establishment of quasi-stationary equilibrium) is inversely proportional to the concentration of the enzyme.

  15. Heterogeneous production of proteases from Brazilian clinical isolates of Pseudomonas aeruginosa.

    Science.gov (United States)

    Galdino, Anna Clara M; Viganor, Lívia; Ziccardi, Mariangela; Nunes, Ana Paula F; Dos Santos, Kátia R N; Branquinha, Marta H; Santos, André L S

    2017-12-01

    Pseudomonas aeruginosa is an important human pathogen that causes severe infections in a wide range of immunosuppressed patients. Herein, we evaluated the proteolytic profiles of 96 Brazilian clinical isolates of P. aeruginosa recovered from diverse anatomical sites. Cell-associated and extracellular proteases were evidenced by gelatin-SDS-PAGE and by the cleavage of soluble gelatin. Elastase was measured by using the peptide substrate N-succinyl-Ala-Ala-Ala-p-nitroanilide. The prevalence of elastase genes (lasA and lasB) was evaluated by PCR. Bacterial extracts were initially applied on gelatin-SDS-PAGE and the results revealed four distinct zymographic profiles as follows: profile I (composed by bands of 145, 118 and 50kDa), profile II (118 and 50kDa), profile III (145kDa) and profile IV (118kDa). All the proteolytic enzymes were inhibited by EDTA, identifying them as metalloproteases. The profile I was the most detected in both cellular (79.2%) and extracellular (84.4%) extracts. Overall, gelatinase and elastase activities measured in the spent culture media were significantly higher (around 2-fold) compared to the cellular extracts and the production level varied according to the site of bacterial isolation. For instance, tracheal secretion isolates produced elevated amount of gelatinase and elastase measured in both cellular and extracellular extracts. The prevalence of elastase genes revealed that 100% isolates were lasB-positive and 85.42% lasA-positive. Some positive/negative correlations were showed concerning the production of gelatinase, elastase, isolation site and antimicrobial susceptibility. The protease production was highly heterogeneous in Brazilian clinical isolates of P. aeruginosa, which corroborates the genomic/metabolic versatility of this pathogen. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

  16. Mass spectrometry analysis of proteome-wide proteolytic post-translational degradation of proteins

    OpenAIRE

    Shen, Yufeng; Hixson, Kim K.; Tolić, Nikola; Camp, David G.; Purvine, Samuel O.; Moore, Ronald J.; Smith, Richard D.

    2008-01-01

    Protein proteolytic degradation is an essential component to proper cell function and its life cycle. Here, we study the protein degradation in yeast Saccharomyces cerevisiae cells on a proteome-wide scale by detection of the intermediate peptides produced from the intracellular degradation of proteins using sequencing-based tandem mass spectrometry. By tracing the detected ~1,100 peptides and their ~200 protein substrate origins we obtain evidence for new insights into the proteome-wide prot...

  17. Proteolytic activity and cooperative hemolytic effect of dermatophytes with different species of bacteria

    Science.gov (United States)

    Pakshir, K; Mohamadi, T; Khodadadi, H; Motamedifar, M; Zomorodian, K; Alipour, S; Motamedi, M

    2016-01-01

    Background and Purpose: Globally, dermatophytes are the most common filamentous group of fungi causing cutaneous mycoses. Dermatophytes were shown to secrete a multitude of enzymes that play a role in their pathogenesis. There is limited data on co-hemolytic (CAMP-like) effect of different bacterial species on dermatophyte species. In this study, we sought to the evaluate exoenzyme activity and co-hemolytic effect of four bacteria on clinical dermatophytes isolated from patients in Shiraz, Iran. Materials and Methods: A total of 84 clinical dermatophyte species were isolated from patients suffering dermatophytosis and identified by conventional methods. Hemolytic activity was evaluated with Columbia 5% sheep blood agar. Proteolytic activity was determined by plate clearance assay method, using gelatin 8% agar. CAMP-like factor was evaluated with four bacteria, namely, S. areus, S. saprophyticus, S. pyogenes, and S. agalactiae. Fisher's exact test was run for statistical analysis. Results: T. mentagrophytes was the most predominant agent (27 [32.1%]) followed by T. verrucosum(20 [23.8%]), T. tonsurans (10 [11.9%]), Microsporum canis (7 [8.3%]), T. rubrum (6 [7.1%]), E. floccosum (6 [7.1%]), M. gypseum (5 [6%]), and T. violaceum (3[3.6%]). The most common clinical area of dermatophytosis was the skin. All the isolates expressed the zone of incomplete alpha hemolysis. All the isolates had CAMP- positive reaction with S. aureus and the other bacteria were CAMP-negative. All the isolates expressed proteolytic activity and no significant differences were noted among diverse genera of dermatophytes and severities of proteolytic activity. Conclusion: This study indicated that hemolysin and proteolytic enzymes potentially play a role in dermatophyte pathogenesis and S. aureus could be considered as a main bacterium for creation of co-hemolytic effect in association with dermatophyte species. PMID:28959790

  18. Fibrin Clots Are Equilibrium Polymers That Can Be Remodeled Without Proteolytic Digestion

    OpenAIRE

    Chernysh, Irina N.; Nagaswami, Chandrasekaran; Purohit, Prashant K.; Weisel, John W.

    2012-01-01

    Fibrin polymerization is a necessary part of hemostasis but clots can obstruct blood vessels and cause heart attacks and strokes. The polymerization reactions are specific and controlled, involving strong knob-into-hole interactions to convert soluble fibrinogen into insoluble fibrin. It has long been assumed that clots and thrombi are stable structures until proteolytic digestion. On the contrary, using the technique of fluorescence recovery after photobleaching, we demonstrate here that the...

  19. Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum.

    Science.gov (United States)

    Carter, Andrew T; Paul, Catherine J; Mason, David R; Twine, Susan M; Alston, Mark J; Logan, Susan M; Austin, John W; Peck, Michael W

    2009-03-19

    Proteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray. Whole genome microarray analysis revealed that 63% of the coding sequences (CDSs) present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes), and the flagellar glycosylation island (FGI). These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5) has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI. Proteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism outbreaks.

  20. Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum

    Directory of Open Access Journals (Sweden)

    Twine Susan M

    2009-03-01

    Full Text Available Abstract Background Proteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray. Results Whole genome microarray analysis revealed that 63% of the coding sequences (CDSs present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes, and the flagellar glycosylation island (FGI. These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5 has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI. Conclusion Proteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism

  1. The role of lysosomal proteolytic enzymes in invasion and dissemination of malignant melanoma

    International Nuclear Information System (INIS)

    Bassalyk, L.S.; Tsanev, P.E.; Parshikova, S.M.; Demidov, L.V.

    1992-01-01

    Preoperative chemo- and radiation therapy was followed by a decrease in lysosomal cathepsins activity in metastatic lymph nodes which, however, did not reach the level established for intact lymph nodes. The pathogenetic role of proteolytic endopeptidases in invasion and sissemination of malignant melanoma is discussed as well as the value of their level measurement for assessing metastatic potential of tumor and prognosis of disease of disease on the basis of tumor site, degree of invasion regional lymph node status

  2. Flanking signal and mature peptide residues influence signal peptide cleavage

    Directory of Open Access Journals (Sweden)

    Ranganathan Shoba

    2008-12-01

    Full Text Available Abstract Background Signal peptides (SPs mediate the targeting of secretory precursor proteins to the correct subcellular compartments in prokaryotes and eukaryotes. Identifying these transient peptides is crucial to the medical, food and beverage and biotechnology industries yet our understanding of these peptides remains limited. This paper examines the most common type of signal peptides cleavable by the endoprotease signal peptidase I (SPase I, and the residues flanking the cleavage sites of three groups of signal peptide sequences, namely (i eukaryotes (Euk (ii Gram-positive (Gram+ bacteria, and (iii Gram-negative (Gram- bacteria. Results In this study, 2352 secretory peptide sequences from a variety of organisms with amino-terminal SPs are extracted from the manually curated SPdb database for analysis based on physicochemical properties such as pI, aliphatic index, GRAVY score, hydrophobicity, net charge and position-specific residue preferences. Our findings show that the three groups share several similarities in general, but they display distinctive features upon examination in terms of their amino acid compositions and frequencies, and various physico-chemical properties. Thus, analysis or prediction of their sequences should be separated and treated as distinct groups. Conclusion We conclude that the peptide segment recognized by SPase I extends to the start of the mature protein to a limited extent, upon our survey of the amino acid residues surrounding the cleavage processing site. These flanking residues possibly influence the cleavage processing and contribute to non-canonical cleavage sites. Our findings are applicable in defining more accurate prediction tools for recognition and identification of cleavage site of SPs.

  3. Proteolytic signatures define unique thrombin-derived peptides present in human wound fluid in vivo.

    Science.gov (United States)

    Saravanan, Rathi; Adav, Sunil S; Choong, Yeu Khai; van der Plas, Mariena J A; Petrlova, Jitka; Kjellström, Sven; Sze, Siu Kwan; Schmidtchen, Artur

    2017-10-13

    The disease burden of failing skin repair and non-healing ulcers is extensive. There is an unmet need for new diagnostic approaches to better predict healing activity and wound infection. Uncontrolled and excessive protease activity, of endogenous or bacterial origin, has been described as a major contributor to wound healing impairments. Proteolytic peptide patterns could therefore correlate and "report" healing activity and infection. This work describes a proof of principle delineating a strategy by which peptides from a selected protein, human thrombin, are detected and attributed to proteolytic actions. With a particular focus on thrombin-derived C-terminal peptides (TCP), we show that distinct peptide patterns are generated in vitro by the human S1 peptidases human neutrophil elastase and cathepsin G, and the bacterial M4 peptidases Pseudomonas aeruginosa elastase and Staphylococcus aureus aureolysin, respectively. Corresponding peptide sequences were identified in wound fluids from acute and non-healing ulcers, and notably, one peptide, FYT21 (FYTHVFRLKKWIQKVIDQFGE), was only present in wound fluid from non-healing ulcers colonized by P. aeruginosa and S. aureus. Our result is a proof of principle pointing at the possibility of defining peptide biomarkers reporting distinct proteolytic activities, of potential implication for improved diagnosis of wound healing and infection.

  4. 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

    2012-01-01

    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. PMID:22357960

  5. Disruption of TLR3 signaling due to cleavage of TRIF by the hepatitis A virus protease-polymerase processing intermediate, 3CD.

    Directory of Open Access Journals (Sweden)

    Lin Qu

    2011-09-01

    Full Text Available Toll-like receptor 3 (TLR3 and cytosolic RIG-I-like helicases (RIG-I and MDA5 sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-β (TRIF, and mitochondrial antiviral signaling protein (MAVS, respectively. Previously, we demonstrated that hepatitis A virus (HAV, a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3C(pro, that is derived by auto-processing of the P3 (3ABCD segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C-stimulated dimerization of IFN regulatory factor 3 (IRF-3, IRF-3 translocation to the nucleus, and IFN-β promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3C(pro protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3C(pro and downstream 3D(pol sequence, but not 3D(pol polymerase activity. Cleavage occurs at two non-canonical 3C(pro recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3D(pol sequence modulates the substrate specificity of the upstream 3C(pro protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3C(pro. HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate.

  6. AKTIVITAS PROTEOLITIK BAKTERI ASAM LAKTAT DALAM FERMENTASI SUSU KEDELAI [Proteolytic Activities of Lactic Acid Bacteria in Fermentation of Soymilk

    OpenAIRE

    Yusmarini1,2)*; R. Indrati1); T. Utami1); Y. Marsono1)

    2010-01-01

    Some lactic acid bacteria (LAB) strains had been isolated from spontaneously fermented soymilk which have proteolytic system. The purpose of this research was to study ability of isolates in fermentation of soymilk. The changes in bacterial growth, pH, titrable acidity, and proteolytic activities during fermentation were examined. Isolates of Lactobacillus plantarum 1 R.1.3.2; L. plantarum 1 R.11.1.2 and L. acidophilus FNCC 0051 (as a control) were capable growing in soymilk. The results indi...

  7. Alkali metal control over N-N cleavage in iron complexes.

    Science.gov (United States)

    Grubel, Katarzyna; Brennessel, William W; Mercado, Brandon Q; Holland, Patrick L

    2014-12-03

    Though N2 cleavage on K-promoted Fe surfaces is important in the large-scale Haber-Bosch process, there is still ambiguity about the number of Fe atoms involved during the N-N cleaving step and the interactions responsible for the promoting ability of K. This work explores a molecular Fe system for N2 reduction, particularly focusing on the differences in the results obtained using different alkali metals as reductants (Na, K, Rb, Cs). The products of these reactions feature new types of Fe-N2 and Fe-nitride cores. Surprisingly, adding more equivalents of reductant to the system gives a product in which the N-N bond is not cleaved, indicating that the reducing power is not the most important factor that determines the extent of N2 activation. On the other hand, the results suggest that the size of the alkali metal cation can control the number of Fe atoms that can approach N2, which in turn controls the ability to achieve N2 cleavage. The accumulated results indicate that cleaving the triple N-N bond to nitrides is facilitated by simultaneous approach of least three low-valent Fe atoms to a single molecule of N2.

  8. Sequence specificity of DNA cleavage by Micrococcus luteus γ endonuclease

    International Nuclear Information System (INIS)

    Hentosh, P.; Henner, W.D.; Reynolds, R.J.

    1985-01-01

    DNA fragments of defined sequence have been used to determine the sites of cleavage by γ-endonuclease activity in extracts prepared from Micrococcus luteus. End-labeled DNA restriction fragments of pBR322 DNA that had been irradiated under nitrogen in the presence of potassium iodide or t-butanol were treated with M. luteus γ endonuclease and analyzed on irradiated DNA preferentially at the positions of cytosines and thymines. DNA cleavage occurred immediately to the 3' side of pyrimidines in irradiated DNA and resulted in fragments that terminate in a 5'-phosphoryl group. These studies indicate that both altered cytosines and thymines may be important DNA lesions requiring repair after exposure to γ radiation

  9. Relationship between synthesis and cleavage of poliovirus-specific proteins.

    OpenAIRE

    Thomas, A A; Voorma, H O; Boeye, A

    1983-01-01

    Poliovirus proteinase was studied in vitro in lysates from poliovirus-infected HeLa cells. Preincubation of these lysates caused (i) a reduction in poliovirus proteinase activity and (ii) a partial dependence on exogenous mRNA for optimal translation. Proteins translated from endogenous poliovirus RNA in preincubated extracts from virus-infected HeLa cells are poorly cleaved. This cleavage deficiency is alleviated by adding fresh poliovirus RNA to the translation system, thus, allowing re-ini...

  10. 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.

  11. Effects of Cysteamine on Sheep Embryo Cleavage Rates

    Directory of Open Access Journals (Sweden)

    Sinem Ö. ENGİNLER

    2015-01-01

    Full Text Available Oxidative stress during in vitro culture leads to defects in development of gametes and embryos. Several antioxidants such as cysteamine, L-ascorbic acid, beta mercaptoethanol, cysteine, glutathione, proteins, vitamins have been used to supplement culture media to counter the oxidative stress. This study was conducted to detect the effect of adding cysteamine to the maturation medium to subsequent cleavage rates of sheep embryos. Totally 604 ovaries were obtained by ten replica and 2060 oocytes were collected. The cumulus oocyte complexes were recovered by the slicing method. A total of 1818 selected oocytes were divided into two groups and used for maturation (88.25%. The first group was created as supplemented with cysteamine (Group A and second group (Group B, control without cysteamine in TCM-199. The two groups were incubated for 24 h at 38.8 °C in an atmosphere of 5% CO2 in humidified air for in vitro maturation (IVM. After IVM, oocytes were fertilized with 50 x 107 / mL fresh ram semen in BSOF medium for 18 h. After fertilization, maturation groups were divided into two subgroups with different culture media: Group AI-SOF (Synthetic Oviduct Fluid medium, Group AII-CR1aa (Charles Rosencrans medium, Group BI-SOF and Group BII-CR1aa were achieved. Cleavage rates were evaluated at day 2. post insemination. The rates of cleavage were detected as 59.54% (184/309, 55.44% (173/312, 65.34% (215/329, 59.34% (200/337 respectively, with showing no statistically significant difference between the groups at the level of P>0.05. In conclusion, supplementing cysteamine to maturation media in TCM-199 did not affect the cleavage rates of sheep embryos in SOF and CR1aa culture media.

  12. Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis

    KAUST Repository

    Frusciante, Sarah; Diretto, Gianfranco; Bruno, Mark; Ferrante, Paola; Pietrella, Marco; Prado-Cabrero, Alfonso; Rubio-Moraga, Á ngela L.; Beyer, Peter D.; Gó mez-Gó mez, Lourdes; Al-Babili, Salim; Giuliano, Giovanni

    2014-01-01

    Crocus sativus stigmas are the source of the saffron spice and accumulate the apocarotenoids crocetin, crocins, picrocrocin, and safranal, responsible for its color, taste, and aroma. Through deep transcriptome sequencing, we identified a novel dioxygenase, carotenoid cleavage dioxygenase 2 (CCD2), expressed early during stigma development and closely related to, but distinct from, the CCD1 dioxygenase family. CCD2 is the only identified member of a novel CCD clade, presents the structural features of a bona fide CCD, and is able to cleave zeaxanthin, the presumed precursor of saffron apocarotenoids, both in Escherichia coli and in maize endosperm. The cleavage products, identified through high-resolution mass spectrometry and comigration with authentic standards, are crocetin dialdehyde and crocetin, respectively. In vitro assays show that CCD2 cleaves sequentially the 7,8 and 7′,8′ double bonds adjacent to a 3-OH-β-ionone ring and that the conversion of zeaxanthin to crocetin dialdehyde proceeds via the C30 intermediate 3-OH-β-apo-8′-carotenal. In contrast, zeaxanthin cleavage dioxygenase (ZCD), an enzyme previously claimed to mediate crocetin formation, did not cleave zeaxanthin or 3-OH-β-apo-8′-carotenal in the test systems used. Sequence comparison and structure prediction suggest that ZCD is an N-truncated CCD4 form, lacking one blade of the β-propeller structure conserved in all CCDs. These results constitute strong evidence that CCD2 catalyzes the first dedicated step in crocin biosynthesis. Similar to CCD1, CCD2 has a cytoplasmic localization, suggesting that it may cleave carotenoids localized in the chromoplast outer envelope.

  13. Novel carotenoid cleavage dioxygenase catalyzes the first dedicated step in saffron crocin biosynthesis

    KAUST Repository

    Frusciante, Sarah

    2014-08-05

    Crocus sativus stigmas are the source of the saffron spice and accumulate the apocarotenoids crocetin, crocins, picrocrocin, and safranal, responsible for its color, taste, and aroma. Through deep transcriptome sequencing, we identified a novel dioxygenase, carotenoid cleavage dioxygenase 2 (CCD2), expressed early during stigma development and closely related to, but distinct from, the CCD1 dioxygenase family. CCD2 is the only identified member of a novel CCD clade, presents the structural features of a bona fide CCD, and is able to cleave zeaxanthin, the presumed precursor of saffron apocarotenoids, both in Escherichia coli and in maize endosperm. The cleavage products, identified through high-resolution mass spectrometry and comigration with authentic standards, are crocetin dialdehyde and crocetin, respectively. In vitro assays show that CCD2 cleaves sequentially the 7,8 and 7′,8′ double bonds adjacent to a 3-OH-β-ionone ring and that the conversion of zeaxanthin to crocetin dialdehyde proceeds via the C30 intermediate 3-OH-β-apo-8′-carotenal. In contrast, zeaxanthin cleavage dioxygenase (ZCD), an enzyme previously claimed to mediate crocetin formation, did not cleave zeaxanthin or 3-OH-β-apo-8′-carotenal in the test systems used. Sequence comparison and structure prediction suggest that ZCD is an N-truncated CCD4 form, lacking one blade of the β-propeller structure conserved in all CCDs. These results constitute strong evidence that CCD2 catalyzes the first dedicated step in crocin biosynthesis. Similar to CCD1, CCD2 has a cytoplasmic localization, suggesting that it may cleave carotenoids localized in the chromoplast outer envelope.

  14. A novel carotenoid cleavage activity involved in the biosynthesis of Citrus fruit-specific apocarotenoid pigments

    KAUST Repository

    Rodrigo, María J.

    2013-09-04

    Citrus is the first tree crop in terms of fruit production. The colour of Citrus fruit is one of the main quality attributes, caused by the accumulation of carotenoids and their derivative C30 apocarotenoids, mainly ?-citraurin (3-hydroxy-?-apo-8?-carotenal), which provide an attractive orange-reddish tint to the peel of oranges and mandarins. Though carotenoid biosynthesis and its regulation have been extensively studied in Citrus fruits, little is known about the formation of C30 apocarotenoids. The aim of this study was to the identify carotenoid cleavage enzyme(s) [CCD(s)] involved in the peel-specific C30 apocarotenoids. In silico data mining revealed a new family of five CCD4-type genes in Citrus. One gene of this family, CCD4b1, was expressed in reproductive and vegetative tissues of different Citrus species in a pattern correlating with the accumulation of C30 apocarotenoids. Moreover, developmental processes and treatments which alter Citrus fruit peel pigmentation led to changes of ?-citraurin content and CCD4b1 transcript levels. These results point to the involvement of CCD4b1 in ?-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and ?-cryptoxanthin. Functional analysis of CCD4b1 by in vitro assays unequivocally demonstrated the asymmetric cleavage activity at the 7?,8? double bond in zeaxanthin and ?-cryptoxanthin, confrming its role in C30 apocarotenoid biosynthesis. Thus, a novel plant carotenoid cleavage activity targeting the 7?,8? double bond of cyclic C40 carotenoids has been identified. These results suggest that the presented enzyme is responsible for the biosynthesis of C30 apocarotenoids in Citrus which are key pigments in fruit coloration. The Author 2013.

  15. A novel carotenoid cleavage activity involved in the biosynthesis of Citrus fruit-specific apocarotenoid pigments

    KAUST Repository

    Rodrigo, Marí a J.; Alqué zar, Berta; Aló s, Enriqueta; Medina, Ví ctor; Carmona, Lourdes; Bruno, Mark; Al-Babili, Salim; Zacarí as, Lorenzo

    2013-01-01

    Citrus is the first tree crop in terms of fruit production. The colour of Citrus fruit is one of the main quality attributes, caused by the accumulation of carotenoids and their derivative C30 apocarotenoids, mainly ?-citraurin (3-hydroxy-?-apo-8?-carotenal), which provide an attractive orange-reddish tint to the peel of oranges and mandarins. Though carotenoid biosynthesis and its regulation have been extensively studied in Citrus fruits, little is known about the formation of C30 apocarotenoids. The aim of this study was to the identify carotenoid cleavage enzyme(s) [CCD(s)] involved in the peel-specific C30 apocarotenoids. In silico data mining revealed a new family of five CCD4-type genes in Citrus. One gene of this family, CCD4b1, was expressed in reproductive and vegetative tissues of different Citrus species in a pattern correlating with the accumulation of C30 apocarotenoids. Moreover, developmental processes and treatments which alter Citrus fruit peel pigmentation led to changes of ?-citraurin content and CCD4b1 transcript levels. These results point to the involvement of CCD4b1 in ?-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and ?-cryptoxanthin. Functional analysis of CCD4b1 by in vitro assays unequivocally demonstrated the asymmetric cleavage activity at the 7?,8? double bond in zeaxanthin and ?-cryptoxanthin, confrming its role in C30 apocarotenoid biosynthesis. Thus, a novel plant carotenoid cleavage activity targeting the 7?,8? double bond of cyclic C40 carotenoids has been identified. These results suggest that the presented enzyme is responsible for the biosynthesis of C30 apocarotenoids in Citrus which are key pigments in fruit coloration. The Author 2013.

  16. Uncoupling of Protease trans-Cleavage and Helicase Activities in Pestivirus NS3.

    Science.gov (United States)

    Zheng, Fengwei; Lu, Guoliang; Li, Ling; Gong, Peng; Pan, Zishu

    2017-11-01

    The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å 2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A cis -cleavage event. Although this conformation is incompatible with protease trans -cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein. IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis -cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different

  17. Numerical modeling of ductile tearing effects on cleavage fracture toughness

    International Nuclear Information System (INIS)

    Dodds, R.H. Jr.; Tang, M.; Anderson, T.L.

    1994-05-01

    Experimental studies demonstrate a significant effect of specimen size, a/W ratio and prior ductile tearing on cleavage fracture toughness values (J c ) measured in the ductile-to-brittle transition region of ferritic materials. In the lower-transition region, cleavage fracture often occurs under conditions of large-scale yielding but without prior ductile crack extension. The increased toughness develops when plastic zones formed at the crack tip interact with nearby specimen surfaces which relaxes crack-tip constraint (stress triaxiality). In the mid-to-upper transition region, small amounts of ductile crack extension (often c -values. Previous work by the authors described a micromechanics fracture model to correct measured J c -values for the mechanistic effects of large-scale yielding. This new work extends the model to also include the influence of ductile crack extension prior to cleavage. The paper explores development of the new model, provides necessary graphs and procedures for its application and demonstrates the effects of the model on fracture data sets for two pressure vessel steels (A533B and A515)

  18. Cleavage mechanoluminescence in elemental and III-V semiconductors

    International Nuclear Information System (INIS)

    Chandra, B.P.; Patel, R.P.; Gour, Anubha S.; Chandra, V.K.; Gupta, R.K.

    2003-01-01

    The present paper reports the theory of mechanoluminescence (ML) produced during cleavage of elemental and III-V semiconductors. It seems that the formation of crack-induced localized states is responsible for the ML excitation produced during the cleavage of elemental and III-V semiconductors. According to this mechanism, as the atoms are drawn away from each other in an advancing crack tip, the decreasing wave function overlap across the crack may result in localized states which is associated with increasing electron energy. If the energy of these localized states approach that of the conduction band, transition to the conduction band via tunnelling would be possible, creating minority carriers, and consequently the electron-hole recombination may give rise to mechanoluminescence. When an elemental or III-V semiconductor is cleaved, initially the ML intensity increases with time, attains a peak value I m at the time t m corresponding to completion of the cleavage of the semiconductor, and then it decreases following power law decay. Expressions are derived for the ML intensity I m corresponding to the peak of the ML intensity versus time curve and for the total ML intensity I T . It is shown that both I m and I T should increase directly with the area of the newly created surfaces of the crystals. From the measurements of the ML intensity, the velocity of crack propagation in material can be determined by using the relation v=H/t m

  19. An analysis of the proteolytic system in Aspergillus in order to improve protein production

    NARCIS (Netherlands)

    Hombergh, van den J.P.T.W.

    1996-01-01


    The filamentous fungus Aspergillus niger is capable of producing and secreting large amounts of homologous proteins (2040 g/l; Montenecourt and Eveleigh, 1985; Nevalainen et al., 1991). Because of this very efficient secretion large effort is being made to use

  20. Proteolytic processing of the primary translation products of cowpea mosaic virus RNAs

    NARCIS (Netherlands)

    Franssen, H.

    1984-01-01

    Cowpea mosaic virus (CPMV) is the type member of a group of plant viruses, the comoviruses, with a genome consisting of two single stranded RNA molecules separately encapsidated in icosahedral particles. A characteristic feature of the two genome RNAs is that they are both polyadenylated at their

  1. [Recent knowledge about intestinal absorption and cleavage of carotenoids].

    Science.gov (United States)

    Borel, P; Drai, J; Faure, H; Fayol, V; Galabert, C; Laromiguière, M; Le Moël, G

    2005-01-01

    Our knowledge about intestinal absorption and cleavage of carotenoids has rapidly grown during the last years. New facts about carotenoid absorption have emerged while some controversies about cleavage are close to end. The knowledge of the absorption and conversion processes is indispensable to understand and interpret the perturbations that can occur in the metabolism of carotenoids and vitamin A. Recently, it has been shown that the absorption of certain carotenoids is not passive - as believed for a long time - but is a facilitated process that requires, at least for lutein, the class B-type 1 scavenger receptor (SR-B1). Various epidemiological and clinical studies have shown wide variations in carotenoid absorption from one subject to another, such differences are now explained by the structure of the concerned carotenoid, by the nature of the food that is absorbed with the carotenoid, by diverse exogenous factors like the intake of medicines or interfering components, by diet factors, by genetic factors, and by the nutritional status of the subject. Recently, the precise mechanism of beta-carotene cleavage by betabeta-carotene 15,15' monooxygenase (EC 1.14.99.36) - formerly called beta-carotene 15,15' dioxygenase (ex EC 1.13.11.21) - has been discovered, and a second enzyme which cleaves asymmetrically the beta-carotene molecule has been found. beta-carotene 15,15' monooxygenase only acts on the 15,15' bond, thus forming two molecules of retinal from one molecule of beta-carotene by central cleavage. Even though the betabeta-carotene 15,15' monooxygenase is much more active on the beta-carotene molecule, a study has shown that it can act on all carotenoids. Searchers now agree that other enzymes that can catalyse an eccentric cleavage of carotenoids probably exist, but under physiological conditions the betabeta-carotene 15,15' monooxygenase is by far the most active, and it is mainly effective in the small bowel mucosa and in the liver. However the

  2. Interaction of Leptospira interrogans with Human Proteolytic Systems Enhances Dissemination through Endothelial Cells and Protease Levels

    Science.gov (United States)

    Vieira, Monica L.; Alvarez-Flores, Miryam P.; Kirchgatter, Karin; Romero, Eliete C.; Alves, Ivy J.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Chudzinski-Tavassi, Ana M.

    2013-01-01

    We have recently reported the ability of Leptospira to capture plasminogen (PLG) and generate plasmin (PLA) bound on the microbial surface in the presence of exogenous activators. In this work, we examined the effects of leptospiral PLG binding for active penetration through the endothelial cell barrier and activation. The results indicate that leptospires with PLG association or PLA activation have enhanced migration activity through human umbilical vein endothelial cell (HUVEC) monolayers compared with untreated bacteria. Leptospira cells coated with PLG were capable of stimulating the expression of PLG activators by HUVECs. Moreover, leptospires endowed with PLG or PLA promoted transcriptional upregulation matrix metalloprotease 9 (MMP-9). Serum samples from patients with confirmed leptospirosis showed higher levels of PLG activators and total MMP-9 than serum samples from normal (healthy) subjects. The highest level of PLG activators and total MMP-9 was detected with microscopic agglutination test (MAT)-negative serum samples, suggesting that this proteolytic activity stimulation occurs at the early stage of the disease. Furthermore, a gelatin zymography profile obtained for MMPs with serum samples from patients with leptospirosis appears to be specific to leptospiral infection because serum samples from patients with unrelated infectious diseases produced no similar degradation bands. Altogether, the data suggest that the Leptospira-associated PLG or PLA might represent a mechanism that contributes to bacterial penetration of endothelial cells through an activation cascade of events that enhances the proteolytic capability of the organism. To our knowledge, this is the first proteolytic activity associated with leptospiral pathogenesis described to date. PMID:23478319

  3. Proteolytic processing of connective tissue growth factor in normal ocular tissues and during corneal wound healing.

    Science.gov (United States)

    Robinson, Paulette M; Smith, Tyler S; Patel, Dilan; Dave, Meera; Lewin, Alfred S; Pi, Liya; Scott, Edward W; Tuli, Sonal S; Schultz, Gregory S

    2012-12-13

    Connective tissue growth factor (CTGF) is a fibrogenic cytokine that is up-regulated by TGF-β and mediates most key fibrotic actions of TGF-β, including stimulation of synthesis of extracellular matrix and differentiation of fibroblasts into myofibroblasts. This study addresses the role of proteolytic processing of CTGF in human corneal fibroblasts (HCF) stimulated with TGF-β, normal ocular tissues and wounded corneas. Proteolytic processing of CTGF in HCF cultures, normal animal eyes, and excimer laser wounded rat corneas were examined by Western blot. The identity of a 21-kDa band was determined by tandem mass spectrometry, and possible alternative splice variants of CTGF were assessed by 5' Rapid Amplification of cDNA Ends (RACE). HCF stimulated by TGF-β contained full length 38-kDa CTGF and fragments of 25, 21, 18, and 13 kDa, while conditioned medium contained full length 38- and a 21-kDa fragment of CTGF that contained the middle "hinge" region of CTGF. Fragmentation of recombinant CTGF incubated in HCF extracts was blocked by the aspartate protease inhibitor, pepstatin. Normal mouse, rat, and rabbit whole eyes and rabbit ocular tissues contained abundant amounts of C-terminal 25- and 21-kDa fragments and trace amounts of 38-kDa CTGF, although no alternative transcripts were detected. All forms of CTGF (38, 25, and 21 kDa) were detected during healing of excimer ablated rat corneas, peaking on day 11. Proteolytic processing of 38-kDa CTGF occurs during corneal wound healing, which may have important implications in regulation of corneal scar formation.

  4. Sensitive microplate assay for the detection of proteolytic enzymes using radiolabeled gelatin

    International Nuclear Information System (INIS)

    Robertson, B.D.; Kwan-Lim, G.E.; Maizels, R.M.

    1988-01-01

    A sensitive, microplate assay is described for the detection of a wide range of proteolytic enzymes, using radio-iodine-labeled gelatin as substrate. The technique uses the Bolton-Hunter reagent to label the substrate, which is then coated onto the wells of polyvinyl chloride microtiter plates. By measuring the radioactivity released the assay is able to detect elastase, trypsin, and collagenase in concentrations of 1 ng/ml or less, while the microtiter format permits multiple sample handling and minimizes sample volumes required for analysis

  5. A radiolabel-release microwell assay for proteolytic enzymes present in cell culture media

    International Nuclear Information System (INIS)

    Rucklidge, G.J.; Milne, G.

    1990-01-01

    A modified method for the measurement of proteolytic enzyme activity in cell culture-conditioned media has been developed. Using the release of 3H-labeled peptides from 3H-labeled gelatin the method is performed in microwell plates. The substrate is insolubilized and attached to the wells by glutaraldehyde treatment, thus eliminating the need for a precipitation step at the end of the assay. The assay is sensitive, reproducible, and convenient for small sample volumes. The effect of different protease inhibitors on activity can be assessed rapidly allowing an early characterization of the enzyme. It can also be adapted to microplate spectrophotometric analysis by staining residual substrate with Coomassie blue

  6. The Oxygenase CAO-1 of Neurospora crassa Is a Resveratrol Cleavage Enzyme

    KAUST Repository

    Diaz-Sanchez, V.; F. Estrada, A.; Limon, M. C.; Al-Babili, Salim; Avalos, J.

    2013-01-01

    The genome of the ascomycete Neurospora crassa encodes CAO-1 and CAO-2, two members of the carotenoid cleavage oxygenase family that target double bonds in different substrates. Previous studies demonstrated the role of CAO-2 in cleaving the C40 carotene torulene, a key step in the synthesis of the C35 apocarotenoid pigment neurosporaxanthin. In this work, we investigated the activity of CAO-1, assuming that it may provide retinal, the chromophore of the NOP-1 rhodopsin, by cleaving β-carotene. For this purpose, we tested CAO-1 activity with carotenoid substrates that were, however, not converted. In contrast and consistent with its sequence similarity to family members that act on stilbenes, CAO-1 cleaved the interphenyl Cα-Cβ double bond of resveratrol and its derivative piceatannol. CAO-1 did not convert five other similar stilbenes, indicating a requirement for a minimal number of unmodified hydroxyl groups in the stilbene background. Confirming its biological function in converting stilbenes, adding resveratrol led to a pronounced increase in cao-1 mRNA levels, while light, a key regulator of carotenoid metabolism, did not alter them. Targeted Δcao-1 mutants were not impaired by the presence of resveratrol, a phytoalexin active against different fungi, which did not significantly affect the growth and development of wild-type Neurospora. However, under partial sorbose toxicity, the Δcao-1 colonies exhibited faster radial growth than control strains in the presence of resveratrol, suggesting a moderate toxic effect of resveratrol cleavage products.

  7. Mechanisms of catalytic cleavage of benzyl phenyl ether in aqueous and apolar phases

    Energy Technology Data Exchange (ETDEWEB)

    He, Jiayue; Lu, Lu; Zhao, Chen; Mei, Donghai; Lercher, Johannes A.

    2014-03-01

    Catalytic pathways for the cleavage of ether bonds in benzyl phenyl ether (BPE) in liquid phase using Ni- and zeolite-based catalysts are explored. In the absence of catalysts, the C-O bond is selectively cleaved in water by hydrolysis, forming phenol and benzyl alcohol as intermediates, followed by alkylation. The hydronium ions catalyzing the reactions are provided by the dissociation of water at 523 K. Upon addition of HZSM-5, rates of hydrolysis and alkylation are markedly increased in relation to proton concentrations. In the presence of Ni/SiO2, the selective hydrogenolysis dominates for cleaving the Caliphatic-O bond. Catalyzed by the dual-functional Ni/HZSM-5, hydrogenolysis occurs as the major route rather than hydrolysis (minor route). In apolar undecane, the non-catalytic thermal pyrolysis route dominates. Hydrogenolysis of BPE appears to be the major reaction pathway in undecane in the presence of Ni/SiO2 or Ni/HZSM-5, almost completely suppressing radical reactions. Density functional theory (DFT) calculations strongly support the proposed C-O bond cleavage mechanisms on BPE in aqueous and apolar phases. These calculations show that BPE is initially protonated and subsequently hydrolyzed in the aqueous phase. Finally, DFT calculations suggest that the radical reactions in non-polar solvents lead to primary benzyl and phenoxy radicals in undecane, which leads to heavier condensation products as long as metals are absent for providing dissociated hydrogen.

  8. Fast cleavage of phycocyanobilin from phycocyanin for use in food colouring.

    Science.gov (United States)

    Roda-Serrat, Maria Cinta; Christensen, Knud Villy; El-Houri, Rime Bahij; Fretté, Xavier; Christensen, Lars Porskjær

    2018-02-01

    Phycocyanins from cyanobacteria are possible sources for new natural blue colourants. Their chromophore, phycocyanobilin (PCB), was cleaved from the apoprotein by solvolysis in alcohols and alcoholic aqueous solutions. In all cases two PCB isomers were obtained, while different solvent adducts were formed upon the use of different reagents. The reaction is believed to take place via two competing pathways, a concerted E2 elimination and a S N 2 nucleophilic substitution. Three cleavage methods were compared in terms of yield and purity: conventional reflux, sealed vessel heated in an oil bath, and microwave assisted reaction. The sealed vessel method is a new approach for fast cleavage of PCB from phycocyanin and gave at 120°C the same yield within 30min compared to 16h by the conventional reflux method (P<0.05). In addition the sealed vessel method resulted in improved purity compared to the other methods. Microwave irradiation increased product degradation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. The Oxygenase CAO-1 of Neurospora crassa Is a Resveratrol Cleavage Enzyme

    KAUST Repository

    Diaz-Sanchez, V.

    2013-07-26

    The genome of the ascomycete Neurospora crassa encodes CAO-1 and CAO-2, two members of the carotenoid cleavage oxygenase family that target double bonds in different substrates. Previous studies demonstrated the role of CAO-2 in cleaving the C40 carotene torulene, a key step in the synthesis of the C35 apocarotenoid pigment neurosporaxanthin. In this work, we investigated the activity of CAO-1, assuming that it may provide retinal, the chromophore of the NOP-1 rhodopsin, by cleaving β-carotene. For this purpose, we tested CAO-1 activity with carotenoid substrates that were, however, not converted. In contrast and consistent with its sequence similarity to family members that act on stilbenes, CAO-1 cleaved the interphenyl Cα-Cβ double bond of resveratrol and its derivative piceatannol. CAO-1 did not convert five other similar stilbenes, indicating a requirement for a minimal number of unmodified hydroxyl groups in the stilbene background. Confirming its biological function in converting stilbenes, adding resveratrol led to a pronounced increase in cao-1 mRNA levels, while light, a key regulator of carotenoid metabolism, did not alter them. Targeted Δcao-1 mutants were not impaired by the presence of resveratrol, a phytoalexin active against different fungi, which did not significantly affect the growth and development of wild-type Neurospora. However, under partial sorbose toxicity, the Δcao-1 colonies exhibited faster radial growth than control strains in the presence of resveratrol, suggesting a moderate toxic effect of resveratrol cleavage products.

  10. Does transgenic Cry1Ac + CpTI cotton pollen affect hypopharyngeal gland development and midgut proteolytic enzyme activity in the honey bee Apis mellifera L. (Hymenoptera, Apidae)?

    Science.gov (United States)

    Han, Peng; Niu, Chang-Ying; Biondi, Antonio; Desneux, Nicolas

    2012-11-01

    The transgenic Cry1Ac (Bt toxin) + CpTI (Cowpea Trypsin Inhibitor) cotton cultivar CCRI41 is increasingly used in China and potential side effects on the honey bee Apis mellifera L. have been documented recently. Two studies have assessed potential lethal and sublethal effects in young bees fed with CCRI41 cotton pollen but no effect was observed on learning capacities, although lower feeding activity in exposed honey bees was noted (antifeedant effect). The present study aimed at providing further insights into potential side effects of CCRI41 cotton on honey bees. Emerging honey bees were exposed to different pollen diets using no-choice feeding protocols (chronic exposure) in controlled laboratory conditions and we aimed at documenting potential mechanisms underneath the CCRI41 antifeedant effect previously reported. Activity of midgut proteolytic enzyme of young adult honey bees fed on CCRI41 cotton pollen were not significantly affected, i.e. previously observed antifeedant effect was not linked to disturbed activity of the proteolytic enzymes in bees' midgut. Hypopharyngeal gland development was assessed by quantifying total extractable proteins from the glands. Results suggested that CCRI41 cotton pollen carries no risk to hypopharyngeal gland development of young adult honey bees. In the two bioassays, honey bees exposed to 1 % soybean trypsin inhibitor were used as positive controls for both midgut proteolytic enzymes and hypopharyngeal gland proteins quantification, and bees exposed to 48 ppb (part per billion) (i.e. 48 ng g(-1)) imidacloprid were used as controls for exposure to a sublethal concentration of toxic product. The results show that the previously reported antifeedant effect of CCRI41 cotton pollen on honey bees is not linked to effects on their midgut proteolytic enzymes or on the development of their hypopharyngeal glands. The results of the study are discussed in the framework of risk assessment of transgenic crops on honey bees.

  11. Biochemical and Functional Characterization of Parawixia bistriata Spider Venom with Potential Proteolytic and Larvicidal Activities

    Science.gov (United States)

    Gimenez, Gizeli S.; Coutinho-Neto, Antonio; Kayano, Anderson M.; Simões-Silva, Rodrigo; Trindade, Frances; de Almeida e Silva, Alexandre; Marcussi, Silvana; da Silva, Saulo L.; Fernandes, Carla F. C.; Zuliani, Juliana P.; Calderon, Leonardo A.; Soares, Andreimar M.; Stábeli, Rodrigo G.

    2014-01-01

    Toxins purified from the venom of spiders have high potential to be studied pharmacologically and biochemically. These biomolecules may have biotechnological and therapeutic applications. This study aimed to evaluate the protein content of Parawixia bistriata venom and functionally characterize its proteins that have potential for biotechnological applications. The crude venom showed no phospholipase, hemorrhagic, or anti-Leishmania activities attesting to low genotoxicity and discrete antifungal activity for C. albicans. However the following activities were observed: anticoagulation, edema, myotoxicity and proteolysis on casein, azo-collagen, and fibrinogen. The chromatographic and electrophoretic profiles of the proteins revealed a predominance of acidic, neutral, and polar proteins, highlighting the presence of proteins with high molecular masses. Five fractions were collected using cation exchange chromatography, with the P4 fraction standing out as that of the highest purity. All fractions showed proteolytic activity. The crude venom and fractions P1, P2, and P3 showed larvicidal effects on A. aegypti. Fraction P4 showed the presence of a possible metalloprotease (60 kDa) that has high proteolytic activity on azo-collagen and was inhibited by EDTA. The results presented in this study demonstrate the presence of proteins in the venom of P. bistriata with potential for biotechnological applications. PMID:24895632

  12. Smart biomaterials: Surfaces functionalized with proteolytically stable osteoblast-adhesive peptides

    Directory of Open Access Journals (Sweden)

    Annj Zamuner

    2017-09-01

    Full Text Available Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion, growth, proliferation and differentiation. Recently, covalent and selective functionalization of glass and titanium surfaces with an adhesive peptide (HVP mapped on [351–359] sequence of human Vitronectin allowed to selectively increase osteoblast attachment and adhesion strength in in vitro assays, and to promote osseointegration in in vivo studies. For the first time to our knowledge, in this study we investigated the resistance of adhesion sequences to proteolytic digestion: HVP was completely cleaved after 5 h. In order to overcome the enzymatic degradation of the native peptide under physiological conditions we synthetized three analogues of HVP sequence. A retro-inverted peptide D-2HVP, composed of D amino acids, was completely stable in serum-containing medium. In addition, glass surfaces functionalized with D-2HVP increased human osteoblast adhesion as compared to the native peptide and maintained deposition of calcium. Interestingly, D-2HVP increased expression of IBSP, VTN and SPP1 genes as compared to HVP functionalized surfaces. Total internal reflection fluorescence microscope analysis showed cells with numerous filopodia spread on D-2HVP-functionalized surfaces. Therefore, the D-2HVP sequence is proposed as new osteoblast adhesive peptide with increased bioactivity and high proteolytic resistance.

  13. Smart biomaterials: Surfaces functionalized with proteolytically stable osteoblast-adhesive peptides.

    Science.gov (United States)

    Zamuner, Annj; Brun, Paola; Scorzeto, Michele; Sica, Giuseppe; Castagliuolo, Ignazio; Dettin, Monica

    2017-09-01

    Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion, growth, proliferation and differentiation. Recently, covalent and selective functionalization of glass and titanium surfaces with an adhesive peptide (HVP) mapped on [351-359] sequence of human Vitronectin allowed to selectively increase osteoblast attachment and adhesion strength in in vitro assays, and to promote osseointegration in in vivo studies. For the first time to our knowledge, in this study we investigated the resistance of adhesion sequences to proteolytic digestion: HVP was completely cleaved after 5 h. In order to overcome the enzymatic degradation of the native peptide under physiological conditions we synthetized three analogues of HVP sequence. A retro-inverted peptide D-2HVP, composed of D amino acids, was completely stable in serum-containing medium. In addition, glass surfaces functionalized with D-2HVP increased human osteoblast adhesion as compared to the native peptide and maintained deposition of calcium. Interestingly, D-2HVP increased expression of IBSP, VTN and SPP1 genes as compared to HVP functionalized surfaces. Total internal reflection fluorescence microscope analysis showed cells with numerous filopodia spread on D-2HVP-functionalized surfaces. Therefore, the D-2HVP sequence is proposed as new osteoblast adhesive peptide with increased bioactivity and high proteolytic resistance.

  14. Comparing Proteolytic Fingerprints of Antigen-Presenting Cells during Allergen Processing.

    Science.gov (United States)

    Hofer, Heidi; Weidinger, Tamara; Briza, Peter; Asam, Claudia; Wolf, Martin; Twaroch, Teresa E; Stolz, Frank; Neubauer, Angela; Dall, Elfriede; Hammerl, Peter; Jacquet, Alain; Wallner, Michael

    2017-06-08

    Endolysosomal processing has a critical influence on immunogenicity as well as immune polarization of protein antigens. In industrialized countries, allergies affect around 25% of the population. For the rational design of protein-based allergy therapeutics for immunotherapy, a good knowledge of T cell-reactive regions on allergens is required. Thus, we sought to analyze endolysosomal degradation patterns of inhalant allergens. Four major allergens from ragweed, birch, as well as house dust mites were produced as recombinant proteins. Endolysosomal proteases were purified by differential centrifugation from dendritic cells, macrophages, and B cells, and combined with allergens for proteolytic processing. Thereafter, endolysosomal proteolysis was monitored by protein gel electrophoresis and mass spectrometry. We found that the overall proteolytic activity of specific endolysosomal fractions differed substantially, whereas the degradation patterns of the four model allergens obtained with the different proteases were extremely similar. Moreover, previously identified T cell epitopes were assigned to endolysosomal peptides and indeed showed a good overlap with known T cell epitopes for all four candidate allergens. Thus, we propose that the degradome assay can be used as a predictor to determine antigenic peptides as potential T cell epitopes, which will help in the rational design of protein-based allergy vaccine candidates.

  15. Investigation of plant latices of Asteraceae and Campanulaceae regarding proteolytic activity.

    Science.gov (United States)

    Sytwala, Sonja; Domsalla, André; Melzig, Matthias F

    2015-12-01

    Occurrence of plant latices is widespread, there are more than 40 families of plants characterized to establish lactiferous structures. The appearance of hydrolytic active proteins, incorporated in latices is already characterized, and hydrolytic active proteins are considerable, and for several plant families, the occurrence of hydrolytic active proteins is already specified e.g. Apocynaceae Juss., Caricaceae Dumort, Euphorbiaceae Juss., Moraceae Gaudich and Papaveraceae Juss. In our investigation, focused on latex bearing plants of order Asterales, Asteraceae and Campanulaceae in particular. The present outcomes represent a comprehensive study, relating to the occurrence of proteolytic active enzymes of order Asterales for the first time. 131 different species of Asteraceae and Campanulaceae were tested, and the appearance of plant latex proteases were determined in different quantities. Proteolytic activity was investigated by inhibitory studies and determination of residual activity in the following, enable us to characterize the proteases. Most of the considered species exhibit a serine protease activity and a multiplicity of species exhibited two or more subclasses of proteases. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  16. Proteolytic action of the crude extract ofDuddingtonia flagrans on cyathostomins (Nematoda: Cyathostominae in coprocultures

    Directory of Open Access Journals (Sweden)

    Fabio Ribeiro Braga

    Full Text Available The objective of this study was to examine the action of the crude extract of Duddingtonia flagrans (isolates AC001 and CG722 on infective larvae (L3 of cyathostomins in coprocultures and to confirm its proteolytic activity by means of a zymogram. The following groups were formed in coprocultures: Group 1: 10 mL of crude extract of D. flagrans (AC001; group 2: 10 mL of crude extract of AC001 with 10 mM of Ca2+; group 3: 10 mL of crude extract of D. flagrans (CG722; group 4: 10 mL of crude extract of CG722 with 10 mM of Ca2+; and group 5: control group (distilled water. The third-stage larvae (L3 were obtained after eight days. The crude extract of D. flagrans was effective in reducing the number of L3, with the following percentage reductions: group 1, 49.5%; group 2, 52.5%; group 3, 36.8%; and group 4, 57.7%; in relation to the control group (p > 0.05. The proteolytic activity of the crude extract was confirmed through the zymogram. The results from this study confirmed that the crude extract of the fungusD. flagrans could be used for controlling cyathostomin L3, and suggested that at least one protease of approximately 38 kDa was present.

  17. Copper-Catalyzed Oxidative Reaction of β-Keto Sulfones with Alcohols via C-S Bond Cleavage: Reaction Development and Mechanism Study.

    Science.gov (United States)

    Du, Bingnan; Wang, Wenmin; Wang, Yang; Qi, Zhenghang; Tian, Jiaqi; Zhou, Jie; Wang, Xiaochen; Han, Jianlin; Ma, Jing; Pan, Yi

    2018-02-16

    A Cu-catalyzed cascade oxidative radical process of β-keto sulfones with alcohols has been achieved by using oxygen as an oxidant. In this reaction, β-keto sulfones were converted into sulfinate esters under the oxidative conditions via cleavage of C-S bond. Experimental and computational studies demonstrate that a new pathway is involved in this reaction, which proceeds through the formation of the key four-coordinated Cu II intermediate, O-O bond homolysis induced C-S bond cleavage and Cu-catalyzed esterification to form the final products. This reaction provides a new strategy to sulfonate esters and enriches the research content of C-S bond cleavage and transformations. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Specific in vitro cleavage of Mason-Pfizer monkey virus capsid protein: evidence for a potential role of retroviral protease in early stages of infection

    International Nuclear Information System (INIS)

    Rumlova, Michaela; Ruml, Tomas; Pohl, Jan; Pichova, Iva

    2003-01-01

    Processing of Gag polyproteins by viral protease (PR) leads to reorganization of immature retroviral particles and formation of a ribonucleoprotein core. In some retroviruses, such as HIV and RSV, cleavage of a spacer peptide separating capsid and nucleocapsid proteins is essential for the core formation. We show here that no similar spacer peptide is present in the capsid-nucleocapsid (CA-NC) region of Mason-Pfizer monkey virus (M-PMV) and that the CA protein is cleaved in vitro by the PR within the major homology region (MHR) and the NC protein in several sites at the N-terminus. The CA cleavage product was also identified shortly after penetration of M-PMV into COS cells, suggesting that the protease-catalyzed cleavage is involved in core disintegration

  19. Programmable RNA recognition and cleavage by CRISPR/Cas9.

    Science.gov (United States)

    O'Connell, Mitchell R; Oakes, Benjamin L; Sternberg, Samuel H; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A

    2014-12-11

    The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

  20. AKTIVITAS PROTEOLITIK BAKTERI ASAM LAKTAT DALAM FERMENTASI SUSU KEDELAI [Proteolytic Activities of Lactic Acid Bacteria in Fermentation of Soymilk

    Directory of Open Access Journals (Sweden)

    Yusmarini1,2*

    2010-12-01

    Full Text Available Some lactic acid bacteria (LAB strains had been isolated from spontaneously fermented soymilk which have proteolytic system. The purpose of this research was to study ability of isolates in fermentation of soymilk. The changes in bacterial growth, pH, titrable acidity, and proteolytic activities during fermentation were examined. Isolates of Lactobacillus plantarum 1 R.1.3.2; L. plantarum 1 R.11.1.2 and L. acidophilus FNCC 0051 (as a control were capable growing in soymilk. The results indicated that initial pH of soymilk was 6,6 and decreased to 4,6 after fermentation and titrable acidity of 0.11 increased to 0.34 after fermentation. The proteolytic activities were 0.352 U/ml – 0.468 U/ml. The electrophoretic pattern of the proteins showed changes during fermentation of soymilk.

  1. Mixed-substrate (glycerol tributyrate and fibrin) zymography for simultaneous detection of lipolytic and proteolytic enzymes on a single gel.

    Science.gov (United States)

    Choi, Nack-Shick; Choi, Jong Hyun; Kim, Bo-Hye; Han, Yun-Jon; Kim, Joong Su; Lee, Seung-Goo; Song, Jae Jun

    2009-06-01

    A new zymography method for simultaneous detection of two different enzymatic activities (lipolytic and proteolytic) using a single SDS-containing or native-conformation gel and a mixed-substrate (glycerol tributyrate and fibrin) (MS)(1) gel was developed. After routine electrophoresis, SDS in the gel was removed by treatment with Triton X-100. Gel proteins were electrotransferred to the MS gel. To visualize lipolytic activity, the MS gel was incubated at 37 degrees C (for 6 or 24 h) until clear bands against an opaque background were observed. To detect proteolytic activity, the same MS gel was stained with Coomassie brilliant blue. Using this method, we show that six lipolytic enzymes from Staphylococcus pasteuri NJ-1 and four proteolytic enzymes from two Bacillus strains, B. licheniformis DJ-2 and B. licheniformis NJ-5, isolated from soil, can be simultaneously detected.

  2. What are the proteolytic enzymes of honey and what they do tell us? A fingerprint analysis by 2-D zymography of unifloral honeys.

    Science.gov (United States)

    Rossano, Rocco; Larocca, Marilena; Polito, Teresa; Perna, Anna Maria; Padula, Maria Carmela; Martelli, Giuseppe; Riccio, Paolo

    2012-01-01

    Honey is a sweet and healthy food produced by honeybees (Apis mellifera L.) from flower nectars. Using bidimensional zymography, we have detected the, until now unrevealed, proteolytic activities present in row honey samples. The resulting zymograms were specific for each type of the four unifloral honey under study, and enzymes were identified as serine proteases by the use of specific inhibitors. Further, using bidimensional electrophoresis, we have shown that honey proteases are able to degrade the major Royal Jelly proteins and in particular MRPJ-1, the protein that promotes queen differentiation in honeybees. Our findings open new perspectives for the better understanding of honeybee development, social behaviour and role in honey production. The now discovered honey proteases may influence honey properties and quality, and bidimensional zymograms might be useful to distinguish between different honey types, establish their age and floral origin, and allow honey certification.

  3. What are the proteolytic enzymes of honey and what they do tell us? A fingerprint analysis by 2-D zymography of unifloral honeys.

    Directory of Open Access Journals (Sweden)

    Rocco Rossano

    Full Text Available Honey is a sweet and healthy food produced by honeybees (Apis mellifera L. from flower nectars. Using bidimensional zymography, we have detected the, until now unrevealed, proteolytic activities present in row honey samples. The resulting zymograms were specific for each type of the four unifloral honey under study, and enzymes were identified as serine proteases by the use of specific inhibitors. Further, using bidimensional electrophoresis, we have shown that honey proteases are able to degrade the major Royal Jelly proteins and in particular MRPJ-1, the protein that promotes queen differentiation in honeybees. Our findings open new perspectives for the better understanding of honeybee development, social behaviour and role in honey production. The now discovered honey proteases may influence honey properties and quality, and bidimensional zymograms might be useful to distinguish between different honey types, establish their age and floral origin, and allow honey certification.

  4. Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris.

    Science.gov (United States)

    Sreenivas, Suma; Krishnaiah, Sateesh M; Govindappa, Nagaraja; Basavaraju, Yogesh; Kanojia, Komal; Mallikarjun, Niveditha; Natarajan, Jayaprakash; Chatterjee, Amarnath; Sastry, Kedarnath N

    2015-01-01

    Glargine is an analog of Insulin currently being produced by recombinant DNA technology using two different hosts namely Escherichia coli and Pichia pastoris. Production from E. coli involves the steps of extraction of inclusion bodies by cell lysis, refolding, proteolytic cleavage and purification. In P. pastoris, a single-chain precursor with appropriate disulfide bonding is secreted to the medium. Downstream processing currently involves use of trypsin which converts the precursor into two-chain final product. The use of trypsin in the process generates additional impurities due to presence of Lys and Arg residues in the Glargine molecule. In this study, we describe an alternate approach involving over-expression of endogenous Kex2 proprotein convertase, taking advantage of dibasic amino acid sequence (Arg-Arg) at the end of B-chain of Glargine. KEX2 gene over-expression in Pichia was accomplished by using promoters of varying strengths to ensure production of greater levels of fully functional two-chain Glargine product, confirmed by HPLC and mass analysis. In conclusion, this new production process involving Kex2 protease over-expression improves the downstream process efficiency, reduces the levels of impurities generated and decreases the use of raw materials.

  5. Active site mutations change the cleavage specificity of neprilysin.

    Directory of Open Access Journals (Sweden)

    Travis Sexton

    Full Text Available Neprilysin (NEP, a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe(563 and Ser(546. Among the mutants studied in detail we observed changes in their activity towards leucine(5-enkephalin, insulin B chain, and amyloid β(1-40. For example, NEP(F563I displayed an increase in preference towards cleaving leucine(5-enkephalin relative to insulin B chain, while mutant NEP(S546E was less discriminating than neprilysin. Mutants NEP(F563L and NEP(S546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß(1-40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential.

  6. RecA-mediated cleavage activates UmuD for mutagenesis: Mechanistic relationship between transcriptional derepression and posttranslational activation

    International Nuclear Information System (INIS)

    Nohmi, Takehiko; Battista, J.R.; Dodson, L.A.; Walker, G.C.

    1988-01-01

    The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli. It has been shown that the UmuD protein shares homology with LexA, the repressor of the SOS genes. In this paper the authors describe a series of genetic experiments that indicate that the purpose of RecA-mediated cleavage of UmuD at its bond between Cys-24 and Gly-25 is to activate UmuD for its role in mutagenesis and that the COOH-terminal fragment of UmuD is necessary and sufficient for the role of UmuD in UV mutagenesis. Other genetic experiments are presented that (i) support the hypothesis that the primary role of Ser-60 in UmuD function is to act as a nucleophile in the RecA-mediated cleavage reaction and (ii) raise the possibility that RecA has a third role in UV mutagenesis besides mediating the cleavage of LexA and UmuD

  7. Insights into the Reaction Mechanism of Aromatic Ring Cleavage by Homogentisate Dioxygenase: A Quantum Mechanical/Molecular Mechanical Study.

    Science.gov (United States)

    Qi, Yue; Lu, Jiarui; Lai, Wenzhen

    2016-05-26

    To elucidate the reaction mechanism of the ring cleavage of homogentisate by homogentisate dioxygenase, quantum mechanical/molecular mechanical (QM/MM) calculations were carried out by using two systems in different protonation states of the substrate C2 hydroxyl group. When the substrate C2 hydroxyl group is ionized (the ionized pathway), the superoxo attack on the substrate is the rate-limiting step in the catalytic cycle, with a barrier of 15.9 kcal/mol. Glu396 was found to play an important role in stabilizing the bridge species and its O-O cleavage product by donating a proton via a hydrogen-bonded water molecule. When the substrate C2 hydroxyl group is not ionized (the nonionized pathway), the O-O bond cleavage of the bridge species is the rate-limiting step, with a barrier of 15.3 kcal/mol. The QM/MM-optimized geometries for the dioxygen and alkylperoxo complexes using the nonionized model (for the C2 hydroxyl group) are in agreement with the experimental crystal structures, suggesting that the C2 hydroxyl group is more likely to be nonionized.

  8. Targeted cleavage of hepatitis E virus 3' end RNA mediated by hammerhead ribozymes inhibits viral RNA replication

    International Nuclear Information System (INIS)

    Sriram, Bandi; Thakral, Deepshi; Panda, Subrat Kumar

    2003-01-01

    The 3' end of hepatitis E virus (HEV) contains cis-acting regulatory element, which plays an important role in viral replication. To develop specific replication inhibitor at the molecular level, mono- and di-hammerhead ribozymes (Rz) were designed and synthesized against the conserved 3' end sequences of HEV, which cleave at nucleotide positions 7125 and 7112/7125, respectively. Di-hammerhead ribozyme with two catalytic motifs in tandem was designed to cleave simultaneously at two sites spaced 13 nucleotides apart, which increases the overall cleavage efficiency and prevents the development of escape mutants. Specific cleavage products were obtained with both the ribozymes in vitro at physiological conditions. The inactive control ribozymes showed no cleavage. The ribozymes showed specific inhibition of HEV 3' end fused-luciferase reporter gene expression by ∼37 and ∼60%, respectively in HepG2 cells. These results demonstrate a feasible approach to inhibit the HEV replication to a limited extent by targeting the cis-acting 3' end of HEV with hammerhead ribozymes

  9. Activities of indigenous proteolytic enzymes in caprine milk of different somatic cell counts.

    Science.gov (United States)

    Albenzio, M; Santillo, A; Kelly, A L; Caroprese, M; Marino, R; Sevi, A

    2015-11-01

    Individual caprine milk with different somatic cell counts (SCC) were studied with the aim of investigating the percentage distribution of leukocyte cell types and the activities of indigenous proteolytic enzymes; proteolysis of casein was also studied in relation to cell type following recovery from milk. The experiment was conducted on 5 intensively managed dairy flocks of Garganica goats; on the basis of SCC, the experimental groups were denoted low (L-SCC; 1,501,000 cells/mL) SCC. Leukocyte distribution differed between groups; polymorphonuclear neutrophilic leukocytes were higher in M-SCC and H-SCC milk samples, the percentage macrophages was the highest in H-SCC, and levels of nonviable cells significantly decreased with increasing SCC. Activities of all the main proteolytic enzymes were affected by SCC; plasmin activity was the highest in H-SCC milk and the lowest in L-SCC, and elastase and cathepsin D activities were the highest in M-SCC. Somatic cell count influenced casein hydrolysis patterns, with less intact α- and β-casein in H-SCC milk. Higher levels of low electrophoretic mobility peptides were detected in sodium caseinate incubated with leukocytes isolated from L-SCC milk, independent of cell type, whereas among cells recovered from M-SCC milk, macrophages yielded the highest levels of low electrophoretic mobility peptides from sodium caseinate. The level of high electrophoretic mobility peptides was higher in sodium caseinate incubated with polymorphonuclear neutrophilic leukocytes and macrophages isolated from M-SCC, whereas the same fraction of peptides was always the highest, independent of leukocyte type, for cells recovered from H-SCC milk. In caprine milk, a level of 700,000 cells/mL represented the threshold for changes in leukocyte distribution, which is presumably related to the immune status of the mammary gland. Differences in the profile of indigenous lysosomal proteolytic enzymes in caprine milk may influence the integrity of casein

  10. Altered cleavage patterns in human tripronuclear embryos and their association to fertilization method

    DEFF Research Database (Denmark)

    Joergensen, Mette Warming; Agerholm, Inge; Hindkjaer, Johnny

    2014-01-01

    PURPOSE: To analyze the cleavage patterns in dipronuclear (2PN) and tripronuclear (3PN) embryos in relation to fertilization method. METHOD: Time-lapse analysis. RESULTS: Compared to 2PN, more 3PN IVF embryos displayed early cleavage into 3 cells (p ... stage (p embryos, the 2nd and 3rd cleavage cycles were completed within the expected time frame. However, timing of the cell divisions within the cleavage cycles differed between the two groups. In contrast......, the completion of the 1st, 2nd, and 3rd cleavage cycle was delayed, but with a similar division pattern for 3PN ICSI compared with the 2PN ICSI embryos. 3PN, more often than 2PN ICSI embryos, displayed early cleavage into 3 cells (p = 0.03) and arrested development from the compaction stage and onwards (p = 0...

  11. Oxidative C-C bond cleavage of 1,2-diols by silver(II)

    International Nuclear Information System (INIS)

    Kumar, A.

    1981-01-01

    Oxidation of ethylene glycol and related compounds by Ag(II) has been investigated. Complexation of these substrates by Ag(II) precedes their oxidation. Oxidation occurs through electron transfer from an OH group to the Ag(II) within the complex resulting in the formation of alkoxyl-type radicals. The radicals thus formed undergo β-scission to give cleavage products. For ethylene glycol a complexation rate 1.3 x 10 6 M -1 s -1 and oxidation rate approx. 3 x 10 3 s -1 were observed. A general trend for the type of the substrates which would undergo C-C bond scission by Ag(II) is discussed

  12. Fluorescence-based high-throughput screening of dicer cleavage activity.

    Science.gov (United States)

    Podolska, Katerina; Sedlak, David; Bartunek, Petr; Svoboda, Petr

    2014-03-01

    Production of small RNAs by ribonuclease III Dicer is a key step in microRNA and RNA interference pathways, which employ Dicer-produced small RNAs as sequence-specific silencing guides. Further studies and manipulations of microRNA and RNA interference pathways would benefit from identification of small-molecule modulators. Here, we report a study of a fluorescence-based in vitro Dicer cleavage assay, which was adapted for high-throughput screening. The kinetic assay can be performed under single-turnover conditions (35 nM substrate and 70 nM Dicer) in a small volume (5 µL), which makes it suitable for high-throughput screening in a 1536-well format. As a proof of principle, a small library of bioactive compounds was analyzed, demonstrating potential of the assay.

  13. Cleavage of nicotinamide adenine dinucleotide by the ribosome-inactivating protein from Momordica charantia.

    Science.gov (United States)

    Vinkovic, M; Dunn, G; Wood, G E; Husain, J; Wood, S P; Gill, R

    2015-09-01

    The interaction of momordin, a type 1 ribosome-inactivating protein from Momordica charantia, with NADP(+) and NADPH has been investigated by X-ray diffraction analysis of complexes generated by co-crystallization and crystal soaking. It is known that the proteins of this family readily cleave the adenine-ribose bond of adenosine and related nucleotides in the crystal, leaving the product, adenine, bound to the enzyme active site. Surprisingly, the nicotinamide-ribose bond of oxidized NADP(+) is cleaved, leaving nicotinamide bound in the active site in the same position but in a slightly different orientation to that of the five-membered ring of adenine. No binding or cleavage of NADPH was observed at pH 7.4 in these experiments. These observations are in accord with current views of the enzyme mechanism and may contribute to ongoing searches for effective inhibitors.

  14. Cleavage preference distinguishes the two-component NS2B-NS3 serine proteinases of Dengue and West Nile viruses.

    Science.gov (United States)

    Shiryaev, Sergey A; Kozlov, Igor A; Ratnikov, Boris I; Smith, Jeffrey W; Lebl, Michal; Strongin, Alex Y

    2007-02-01

    Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.

  15. Disruption of KEX1 gene reduces the proteolytic degradation of secreted two-chain Insulin glargine in Pichia pastoris.

    Science.gov (United States)

    Sreenivas, Suma; Krishnaiah, Sateesh M; Shyam Mohan, Anil H; Mallikarjun, Niveditha; Govindappa, Nagaraja; Chatterjee, Amarnath; Sastry, Kedarnath N

    2016-02-01

    Insulin glargine is a slow acting analog of insulin used in diabetes therapy. It is produced by recombinant DNA technology in different hosts namely E. coli and Pichia pastoris. In our previous study, we have described the secretion of fully folded two-chain Insulin glargine into the medium by over-expression of Kex2 protease. The enhanced levels of the Kex2 protease was responsible for the processing of the glargine precursor with in the host. Apart from the two-chain glargine product we observed a small proportion of arginine clipped species. This might be due to the clipping of arginine present at the C-terminus of the B-chain as it is exposed upon Kex2 cleavage. The carboxypeptidase precursor Kex1 is known to be responsible for clipping of C-terminal lysine or arginine of the proteins or peptides. In order to address this issue we created a Kex1 knock out in the host using Cre/loxP mechanism of targeted gene deletion. When two-chain glargine was expressed in the Kex1 knock out host of P. pastoris GS115 the C-terminal clipped species reduced by ∼80%. This modification further improved the process by reducing the levels of product related impurities. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Peptidomic analysis reveals proteolytic activity of kefir microorganisms on bovine milk proteins

    Science.gov (United States)

    Dallas, David C.; Citerne, Florine; Tian, Tian; Silva, Vitor L. M.; Kalanetra, Karen M.; Frese, Steven A.; Robinson, Randall C.; Mills, David A.; Barile, Daniela

    2015-01-01

    Scope The microorganisms that make up kefir grains are well known for lactose fermentation, but the extent to which they hydrolyze and consume milk proteins remains poorly understood. Peptidomics technologies were used to examine the proteolytic activity of kefir grains on bovine milk proteins. Methods and results Gel electrophoresis revealed substantial digestion of milk proteins by kefir grains, with mass spectrometric analysis showing the release of 609 protein fragments and alteration of the abundance of >1,500 peptides that derived from 27 milk proteins. Kefir contained 25 peptides identified from the literature as having biological activity, including those with antihypertensive, antimicrobial, immunomodulatory, opioid and anti-oxidative functions. 16S rRNA and shotgun metagenomic sequencing identified the principle taxa in the culture as Lactobacillus species. Conclusion The model kefir sample contained thousands of protein fragments released in part by kefir microorganisms and in part by native milk proteases. PMID:26616950

  17. Peptidomic analysis reveals proteolytic activity of kefir microorganisms on bovine milk proteins.

    Science.gov (United States)

    Dallas, David C; Citerne, Florine; Tian, Tian; Silva, Vitor L M; Kalanetra, Karen M; Frese, Steven A; Robinson, Randall C; Mills, David A; Barile, Daniela

    2016-04-15

    The microorganisms that make up kefir grains are well known for lactose fermentation, but the extent to which they hydrolyze and consume milk proteins remains poorly understood. Peptidomics technologies were used to examine the proteolytic activity of kefir grains on bovine milk proteins. Gel electrophoresis revealed substantial digestion of milk proteins by kefir grains, with mass spectrometric analysis showing the release of 609 protein fragments and alteration of the abundance of >1500 peptides that derived from 27 milk proteins. Kefir contained 25 peptides identified from the literature as having biological activity, including those with antihypertensive, antimicrobial, immunomodulatory, opioid and anti-oxidative functions. 16S rRNA and shotgun metagenomic sequencing identified the principle taxa in the culture as Lactobacillus species. The model kefir sample contained thousands of protein fragments released in part by kefir microorganisms and in part by native milk proteases. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Microbiological and biochemical response of certain proteolytic bacterial isolates to varying levels of gamma irradiation

    International Nuclear Information System (INIS)

    El-Hifnawi, H.M.N.E.A.

    1997-01-01

    Amniotic membrane allo - and xeno grafts prepared from human foetal placenta, and their potential replacement of skin autotransplant, would significantly contribute to the success of clinical treatment of skin burns. Allo-and xenografts of human amniotic membrane should be ensured for their sterility, bio-mechanics and tissue antigenicity. The present study has been focused on sterilization and sterility assurance of the membrane grafts. Physico-chemical properties and antigenicity of the grafts await investigation. In the present study the isolation and identification of the bacteria contaminating the amniotic membrane allo-and xenografts prepared from human foetal placenta and the effect of gamma irradiation on it has been investigated. The proteolytic activity of these bacteria and the role of gamma irradiation in the control of bacterial activity were similarly investigated

  19. The proteolytic system of pineapple stems revisited: Purification and characterization of multiple catalytically active forms.

    Science.gov (United States)

    Matagne, André; Bolle, Laetitia; El Mahyaoui, Rachida; Baeyens-Volant, Danielle; Azarkan, Mohamed

    2017-06-01

    Crude pineapple proteases extract (aka stem bromelain; EC 3.4.22.4) is an important proteolytic mixture that contains enzymes belonging to the cysteine proteases of the papain family. Numerous studies have been reported aiming at the fractionation and characterization of the many molecular species present in the extract, but more efforts are still required to obtain sufficient quantities of the various purified protease forms for detailed physicochemical, enzymatic and structural characterization. In this work, we describe an efficient strategy towards the purification of at least eight enzymatic forms. Thus, following rapid fractionation on a SP-Sepharose FF column, two sub-populations with proteolytic activity were obtained: the unbound (termed acidic) and bound (termed basic) bromelain fractions. Following reversible modification with monomethoxypolyethylene glycol (mPEG), both fractions were further separated on Q-Sepharose FF and SP-Sepharose FF, respectively. This procedure yielded highly purified molecular species, all titrating ca. 1 mol of thiol group per mole of enzyme, with distinct biochemical properties. N-terminal sequencing allowed identifying at least eight forms with proteolytic activity. The basic fraction contained previously identified species, i.e. basic bromelain forms 1 and 2, ananain forms 1 and 2, and comosain (MEROPS identifier: C01.027). Furthermore, a new proteolytic species, showing similarities with basic bomelain forms 1 and 2, was discovered and termed bromelain form 3. The two remaining species were found in the acidic bromelain fraction and were arbitrarily named acidic bromelain forms 1 and 2. Both, acidic bromelain forms 1, 2 and basic bromelain forms 1, 2 and 3 are glycosylated, while ananain forms 1 and 2, and comosain are not. The eight protease forms display different amidase activities against the various substrates tested, namely small synthetic chromogenic compounds (DL-BAPNA and Boc-Ala-Ala-Gly-pNA), fluorogenic compounds

  20. Effects of proteolytic enzymes and neuraminidase on the I and i erythrocyte antigen sites

    International Nuclear Information System (INIS)

    Doinel, C.; Ropars, C.; Salmon, C.

    1978-01-01

    Homogeneous cold agglutinins, purified and labelled with 125 I, have been used in a study of the effects of neuraminidase and proteolytic enzymes on the I and i reactivities of human adult erythrocytes. Measurements were made of antigen site numbers, equilibrium constants and thermodynamic parameters. There was enhanced reactivity after enzyme treatment as well as after the release of N-acetylneuraminic acid. Steric factors were shown to be of primary importance in the accessibility of the I and i antigenic determinant. After enzyme treatment, the antigenic structures became more homogeneous in their reaction with antibodies. The heterogeneity of binding constants observed with antigenic determinants of non-treated erythrocytes is probably due to the wide range of spatial distribution of these receptors within the membrane. (author)

  1. Reversible Heterolytic Cleavage of the H-H Bond by Molybdenum Complexes: Controlling the Dynamics of Exchange Between Proton and Hydride

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Shaoguang; Appel, Aaron M.; Bullock, R. Morris

    2017-05-18

    Controlling the heterolytic cleavage of the H-H bond of dihydrogen is critically important in catalytic hydrogenations and in the catalytic oxidation of H2. We show how the rate of reversible heterolytic cleavage of H2 can be controlled over nearly four orders of magnitude at 25 °C, from 2.1 × 103 s-1 to ≥107 s-1. Bifunctional Mo complexes, [CpMo(CO)(κ3-P2N2)]+ (P2N2 = 1,5-diaza-3,7-diphosphacyclooctane with alkyl/aryl groups on N and P), have been developed for heterolytic cleavage of H2 into a proton and a hydride, akin to Frustrated Lewis Pairs. The H-H bond cleavage is enabled by the basic amine in the second coordination sphere. The products of heterolytic cleavage of H2, Mo hydride complexes bearing protonated amines, [CpMo(H)(CO)(P2N2H)]+, were characterized by spectroscopic studies and by X-ray crystallography. Variable temperature 1H, 15N and 2-D 1H-1H ROESY NMR spectra indicated rapid exchange of the proton and hydride. The exchange rates are in the order [CpMo(H)(CO)(PPh2NPh2H)]+ > [CpMo(H)(CO)(PtBu2NPh2H)]+ > [CpMo(H)(CO)(PPh2NBn2H)]+ > [CpMo(H)(CO)(PtBu2NBn2H)]+ > [CpMo(H)(CO)(PtBu2NtBu2H)]+. The pKa values determined in acetonitrile range from 9.3 to 17.7, and show a linear correlation with the logarithm of the exchange rates. Thus the exchange dynamics are controlled through the relative acidity of the [CpMo(H)(CO)(P2N2H)]+ and [CpMo(H2)(CO)(P2N2)]+ isomers, providing a design principle for controlling heterolytic cleavage of H2.

  2. A novel synthetic quinolinone inhibitor presents proteolytic and hemorrhagic inhibitory activities against snake venom metalloproteases.

    Science.gov (United States)

    Baraldi, Patrícia T; Magro, Angelo J; Matioli, Fábio F; Marcussi, Silvana; Lemke, Ney; Calderon, Leonardo A; Stábeli, Rodrigo G; Soares, Andreimar M; Correa, Arlene G; Fontes, Marcos R M

    2016-02-01

    Metalloproteases play a fundamental role in snake venom envenomation inducing hemorrhagic, fibrigen(ogen)olytic and myotoxic effects in their victims. Several snake venoms, such as those from the Bothrops genus, present important local effects which are not efficiently neutralized by conventional serum therapy. Consequently, these accidents may result in permanent sequelae and disability, creating economic and social problems, especially in developing countries, leading the attention of the World Health Organization that considered ophidic envenomations a neglected tropical disease. Aiming to produce an efficient inhibitor against bothropic venoms, we synthesized different molecules classified as quinolinones - a group of low-toxic chemical compounds widely used as antibacterial and antimycobacterial drugs - and tested their inhibitory properties against hemorrhage caused by bothropic venoms. The results from this initial screening indicated the molecule 2-hydroxymethyl-6-methoxy-1,4-dihydro-4-quinolinone (Q8) was the most effective antihemorrhagic compound among all of the assayed synthetic quinolinones. Other in vitro and in vivo experiments showed this novel compound was able to inhibit significantly the hemorrhagic and/or proteolytic activities of bothropic crude venoms and isolated snake venom metalloproteases (SVMPs) even at lower concentrations. Docking and molecular dynamic simulations were also performed to get insights into the structural basis of Q8 inhibitory mechanism against proteolytic and hemorrhagic SVMPs. These structural studies demonstrated that Q8 may form a stable complex with SVMPs, impairing the access of substrates to the active sites of these toxins. Therefore, both experimental and structural data indicate that Q8 compound is an interesting candidate for antiophidic therapy, particularly for the treatment of the hemorrhagic and necrotic effects induced by bothropic venoms. Copyright © 2015 Elsevier B.V. and Société Française de

  3. Development of a solid-phase assay for measurement of proteolytic enzyme activity

    International Nuclear Information System (INIS)

    Varani, J.; Johnson, K.; Kaplan, J.

    1980-01-01

    A solid-phase, plate assay was developed for the measurement of proteolytic enzyme activity. In this assay procedure, radiolabeled substrates were dried onto the surface of microtiter wells. Following drying, the wells were washed two times with saline to remove the nonadherent substrate. When proteolytic enzymes were added to the wells, protein hydrolysis occurred, releasing radioactivity into the supernatant fluid. The amount of protein hydrolysis that occurred was reflected by the amount of radioactivity in the supernatant fluid. When 125 I-hemoglobin was used as the substrate, it was as susceptible to hydrolysis by trypsin in the solid-phase assay as it was in solution in a standard assay procedure. Protease activity from a variety of sources (including from viable cells as well as from extracellular sources) were also able to hydrolyze the hemoglobin on the plate. 125 I-Labeled serum albumen, fibrinogen, and rat pulmonary basement membrane were also susceptible to hydrolysis by trypsin in the solid phase. When [ 14 C]elastin was dried onto the plate, it behaved in a similar manner to elastin in solution. It was resistant to hydrolysis by nonspecific proteases such as trypsin and chymotrypsin but was highly susceptible to hydrolysis by elastase. The solid-phase plate assay has several features which recommended it for routine use. It is as sensitive as standard tube assays (and much more sensitive than routinely used colormetric assays). It is quick and convenient; there are no precipitation, centrifugation, or filtration steps. In addition, very small volumes of radioactive wastes are generated. Another advantage of the solid-phase plate assay is the resistance of the dried substrates to spontaneous breakdown and to microbial contamination. Finally, this assay is suitable for use with viable cells as well as for extracellular proteases

  4. Multiplexed homogeneous assays of proteolytic activity using a smartphone and quantum dots.

    Science.gov (United States)

    Petryayeva, Eleonora; Algar, W Russ

    2014-03-18

    Semiconductor quantum dot (QD) bioconjugates, with their unique and highly advantageous physicochemical and optical properties, have been extensively utilized as probes for bioanalysis and continue to generate widespread interest for these applications. An important consideration for expanding the utility of QDs and making their use routine is to make assays with QDs more accessible for laboratories that do not specialize in nanomaterials. Here, we show that digital color imaging of QD photoluminescence (PL) with a smartphone camera is a viable, easily accessible readout platform for quantitative, multiplexed, and real-time bioanalyses. Red-, green-, and blue-emitting CdSeS/ZnS QDs were conjugated with peptides that were labeled with a deep-red fluorescent dye, Alexa Fluor 647, and the dark quenchers, QSY9 and QSY35, respectively, to generate Förster resonance energy transfer (FRET) pairs sensitive to proteolytic activity. Changes in QD PL caused by the activity of picomolar to nanomolar concentrations of protease were detected as changes in the red-green-blue (RGB) channel intensities in digital color images. Importantly, measurements of replicate samples made with smartphone imaging and a sophisticated fluorescence plate reader yielded the same quantitative results, including initial proteolytic rates and specificity constants. Homogeneous two-plex and three-plex assays for the activity of trypsin, chymotrypsin, and enterokinase were demonstrated with RGB imaging. Given the ubiquity of smartphones, this work largely removes any instrumental impediments to the adoption of QDs as routine tools for bioanalysis in research laboratories and is a critical step toward the use of QDs for point-of-care diagnostics. This work also adds to the growing utility of smartphones in analytical methods by enabling multiplexed fluorimetric assays within a single sample volume and across multiple samples in parallel.

  5. Embryo apoptosis identification: Oocyte grade or cleavage stage?

    Science.gov (United States)

    Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

    2015-01-01

    Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced. PMID:26858565

  6. The effects of Capn1 gene inactivation on skeletal muscle growth, development, and atrophy, and the compensatory role of other proteolytic systems.

    Science.gov (United States)

    Kemp, C M; Oliver, W T; Wheeler, T L; Chishti, A H; Koohmaraie, M

    2013-07-01

    Myofibrillar protein turnover is a key component of muscle growth and degeneration, requiring proteolytic enzymes to degrade the skeletal muscle proteins. The objective of this study was to investigate the role of the calpain proteolytic system in muscle growth development using μ-calpain knockout (KO) mice in comparison with control wild-type (WT) mice, and evaluate the subsequent effects of silencing this gene on other proteolytic systems. No differences in muscle development between genotypes were observed during the early stages of growth due to the up regulation of other proteolytic systems. The KO mice showed significantly greater m-calpain protein abundance (P proteolytic systems to ensure muscle protein homeostasis in vivo. Furthermore, these data contribute to the existing evidence of the importance of the calpain system's involvement in muscle growth, development, and atrophy. Collectively, these data suggest that there are opportunities to target the calpain system to promote the growth and/or restoration of skeletal muscle mass.

  7. Proteolytic degradation of regulator of G protein signaling 2 facilitates temporal regulation of Gq/11 signaling and vascular contraction.

    Science.gov (United States)

    Kanai, Stanley M; Edwards, Alethia J; Rurik, Joel G; Osei-Owusu, Patrick; Blumer, Kendall J

    2017-11-24

    Regulator of G protein signaling 2 (RGS2) controls signaling by receptors coupled to the G q/11 class heterotrimeric G proteins. RGS2 deficiency causes several phenotypes in mice and occurs in several diseases, including hypertension in which a proteolytically unstable RGS2 mutant has been reported. However, the mechanisms and functions of RGS2 proteolysis remain poorly understood. Here we addressed these questions by identifying degradation signals in RGS2, and studying dynamic regulation of G q/11 -evoked Ca 2+ signaling and vascular contraction. We identified a novel bipartite degradation signal in the N-terminal domain of RGS2. Mutations disrupting this signal blunted proteolytic degradation downstream of E3 ubiquitin ligase binding to RGS2. Analysis of RGS2 mutants proteolyzed at various rates and the effects of proteasome inhibition indicated that proteolytic degradation controls agonist efficacy by setting RGS2 protein expression levels, and affecting the rate at which cells regain agonist responsiveness as synthesis of RGS2 stops. Analyzing contraction of mesenteric resistance arteries supported the biological relevance of this mechanism. Because RGS2 mRNA expression often is strikingly and transiently up-regulated and then down-regulated upon cell stimulation, our findings indicate that proteolytic degradation tightly couples RGS2 transcription, protein levels, and function. Together these mechanisms provide tight temporal control of G q/11 -coupled receptor signaling in the cardiovascular, immune, and nervous systems. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Interactions between Lactobacillus sakei and CNC (Staphylococcus xylosus and Kocuria varians) and their influence on proteolytic activity.

    Science.gov (United States)

    Tremonte, P; Reale, A; Di Renzo, T; Tipaldi, L; Di Luccia, A; Coppola, R; Sorrentino, E; Succi, M

    2010-11-01

    To evaluate interactions between Lactobacillus sakei and coagulase negative cocci (CNC) (Staphylococcus xylosus and Kocuria varians) and to investigate the influence of these interactions on their own proteolytic activity. Interactions occurring between strains of Lact. sakei and CNC were assessed by spectrophotometric analysis. The growth of 35 strains of Lact. sakei, used as indicators, was compared to that obtained combining the same strains with growing cells or cell-free supernatants of 20 CNC (18 Staph. xylosus and 2 K. varians). The proteolytic activity expressed by single strains or by their combinations was assessed on sarcoplasmic protein extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results evidenced that interactions are able to affect not only the growth but also the in vitro proteolytic activity of Lact. sakei and CNC used in combination. A relationship between the presence of interactions among useful strains and the strength of technological characteristics, such as proteolysis, was defined. The study highlighted that CNC are able to stimulate the growth of some Lact. sakei strains. At the same time, this interaction positively influences the proteolytic activity of strains used in combination. Given the importance of proteolysis during the ripening of fermented meats, this phenomenon should be taken into account to select meat starter cultures. © 2010 The Authors. © 2010 The Society for Applied Microbiology.

  9. Antibody-Mediated Neutralization of uPA Proteolytic Function Reduces Disease Progression in Mouse Arthritis Models

    DEFF Research Database (Denmark)

    Almholt, Kasper; Hebsgaard, Josephine B; Nansen, Anneline

    2018-01-01

    the potential in mice of an Ab that blocks the proteolytic capacity of uPA in the CIA model and the delayed-type hypersensitivity arthritis model. A second aim was to determine the cellular origins of uPA and the uPA receptor (uPAR) in joint tissue from patients with rheumatoid arthritis. A mAb that neutralizes...

  10. Single-Molecule Analysis for RISC Assembly and Target Cleavage.

    Science.gov (United States)

    Sasaki, Hiroshi M; Tadakuma, Hisashi; Tomari, Yukihide

    2018-01-01

    RNA-induced silencing complex (RISC) is a small RNA-protein complex that mediates silencing of complementary target RNAs. Biochemistry has been successfully used to characterize the molecular mechanism of RISC assembly and function for nearly two decades. However, further dissection of intermediate states during the reactions has been warranted to fill in the gaps in our understanding of RNA silencing mechanisms. Single-molecule analysis with total internal reflection fluorescence (TIRF) microscopy is a powerful imaging-based approach to interrogate complex formation and dynamics at the individual molecule level with high sensitivity. Combining this technique with our recently established in vitro reconstitution system of fly Ago2-RISC, we have developed a single-molecule observation system for RISC assembly. In this chapter, we summarize the detailed protocol for single-molecule analysis of chaperone-assisted assembly of fly Ago2-RISC as well as its target cleavage reaction.

  11. Modelling of ductile and cleavage fracture by local approach

    International Nuclear Information System (INIS)

    Samal, M.K.; Dutta, B.K.; Kushwaha, H.S.

    2000-08-01

    This report describes the modelling of ductile and cleavage fracture processes by local approach. It is now well known that the conventional fracture mechanics method based on single parameter criteria is not adequate to model the fracture processes. It is because of the existence of effect of size and geometry of flaw, loading type and rate on the fracture resistance behaviour of any structure. Hence, it is questionable to use same fracture resistance curves as determined from standard tests in the analysis of real life components because of existence of all the above effects. So, there is need to have a method in which the parameters used for the analysis will be true material properties, i.e. independent of geometry and size. One of the solutions to the above problem is the use of local approaches. These approaches have been extensively studied and applied to different materials (including SA33 Gr.6) in this report. Each method has been studied and reported in a separate section. This report has been divided into five sections. Section-I gives a brief review of the fundamentals of fracture process. Section-II deals with modelling of ductile fracture by locally uncoupled type of models. In this section, the critical cavity growth parameters of the different models have been determined for the primary heat transport (PHT) piping material of Indian pressurised heavy water reactor (PHWR). A comparative study has been done among different models. The dependency of the critical parameters on stress triaxiality factor has also been studied. It is observed that Rice and Tracey's model is the most suitable one. But, its parameters are not fully independent of triaxiality factor. For this purpose, a modification to Rice and Tracery's model is suggested in Section-III. Section-IV deals with modelling of ductile fracture process by locally coupled type of models. Section-V deals with the modelling of cleavage fracture process by Beremins model, which is based on Weibulls

  12. Micromechanisms and toughness for cleavage fracture of steel

    International Nuclear Information System (INIS)

    Rosenfield, A.R.; Majumdar, B.S.

    1987-01-01

    A complete understanding of the fracture mechanisms of steel in the ductile/brittle transition region requires analysis not only of crack initiation, but also of crack propagation. This paper reviews micrographic and fractographic experiments that give insight into both phenomena, and suggests a frame-work through which both may be related. Unstable cleavage crack initiation can occur after some blunting of the original fatigue precrack or after some stable crack growth. In either event, instability appears to be triggered by the fracture of a brittle micro-constituent ahead of the precrack. The large scatter in reported K IC values within the transition region reflects the size distribution and relative scarcity of these 'trigger' particles. While a large number of models have attempted to correlate toughness in the ductile/brittle transition regime to events occurring ahead of the crack tip, surprisingly little attention has been paid to events occurring behind the crack front. Fractographic evidence as well as metallographic sectioning of arrested cracks show that the mechanism of rapid crack propagation by cleavage is affected strongly by partial crack-plane deflection which leaves unbroken ligaments in its wake. The tearing of these ligaments by dimple-rupture is the dominant energy-absorbing mechanism. Etch-pit experiments using an Fe-Si alloy show that the crack-tip stress intensity based on plastic zone size is extremely low. It is suggested that the mechanism of crack arrest should be modeled using a sharp crack which is restrained by a distribution of discrete pinching forces along its faces. The same model is applied to crack initiation. (orig.)

  13. Cleavage of sp3 C-O bonds via oxidative addition of C-H bonds.

    Science.gov (United States)

    Choi, Jongwook; Choliy, Yuriy; Zhang, Xiawei; Emge, Thomas J; Krogh-Jespersen, Karsten; Goldman, Alan S

    2009-11-04

    (PCP)Ir (PCP = kappa(3)-C(6)H(3)-2,6-[CH(2)P(t-Bu)(2)](2)) is found to undergo oxidative addition of the methyl-oxygen bond of electron-poor methyl aryl ethers, including methoxy-3,5-bis(trifluoromethyl)benzene and methoxypentafluorobenzene, to give the corresponding aryloxide complexes (PCP)Ir(CH(3))(OAr). Although the net reaction is insertion of the Ir center into the C-O bond, density functional theory (DFT) calculations and a significant kinetic isotope effect [k(CH(3))(OAr)/k(CD(3))(OAr) = 4.3(3)] strongly argue against a simple insertion mechanism and in favor of a pathway involving C-H addition and alpha-migration of the OAr group to give a methylene complex followed by hydride-to-methylene migration to give the observed product. Ethoxy aryl ethers, including ethoxybenzene, also undergo C-O bond cleavage by (PCP)Ir, but the net reaction in this case is 1,2-elimination of ArO-H to give (PCP)Ir(H)(OAr) and ethylene. DFT calculations point to a low-barrier pathway for this reaction that proceeds through C-H addition of the ethoxy methyl group followed by beta-aryl oxide elimination and loss of ethylene. Thus, both of these distinct C-O cleavage reactions proceed via initial addition of a C(sp(3))-H bond, despite the fact that such bonds are typically considered inert and are much stronger than C-O bonds.

  14. The mitochondrion-like organelle of Trimastix pyriformis contains the complete glycine cleavage system.

    Directory of Open Access Journals (Sweden)

    Zuzana Zubáčová

    Full Text Available All eukaryotic organisms contain mitochondria or organelles that evolved from the same endosymbiotic event like classical mitochondria. Organisms inhabiting low oxygen environments often contain mitochondrial derivates known as hydrogenosomes, mitosomes or neutrally as mitochondrion-like organelles. The detailed investigation has shown unexpected evolutionary plasticity in the biochemistry and protein composition of these organelles in various protists. We investigated the mitochondrion-like organelle in Trimastix pyriformis, a free-living member of one of the three lineages of anaerobic group Metamonada. Using 454 sequencing we have obtained 7 037 contigs from its transcriptome and on the basis of sequence homology and presence of N-terminal extensions we have selected contigs coding for proteins that putatively function in the organelle. Together with the results of a previous transcriptome survey, the list now consists of 23 proteins - mostly enzymes involved in amino acid metabolism, transporters and maturases of proteins and transporters of metabolites. We have no evidence of the production of ATP in the mitochondrion-like organelle of Trimastix but we have obtained experimental evidence for the presence of enzymes of the glycine cleavage system (GCS, which is part of amino acid metabolism. Using homologous antibody we have shown that H-protein of GCS localizes into vesicles in the cell of Trimastix. When overexpressed in yeast, H- and P-protein of GCS and cpn60 were transported into mitochondrion. In case of H-protein we have demonstrated that the first 16 amino acids are necessary for this transport. Glycine cleavage system is at the moment the only experimentally localized pathway in the mitochondrial derivate of Trimastix pyriformis.

  15. Comparative and phylogenetic perspectives of the cleavage process in tailed amphibians.

    Science.gov (United States)

    Desnitskiy, Alexey G; Litvinchuk, Spartak N

    2015-10-01

    The order Caudata includes about 660 species and displays a variety of important developmental traits such as cleavage pattern and egg size. However, the cleavage process of tailed amphibians has never been analyzed within a phylogenetic framework. We use published data on the embryos of 36 species concerning the character of the third cleavage furrow (latitudinal, longitudinal or variable) and the magnitude of synchronous cleavage period (up to 3-4 synchronous cell divisions in the animal hemisphere or a considerably longer series of synchronous divisions followed by midblastula transition). Several species from basal caudate families Cryptobranchidae (Andrias davidianus and Cryptobranchus alleganiensis) and Hynobiidae (Onychodactylus japonicus) as well as several representatives from derived families Plethodontidae (Desmognathus fuscus and Ensatina eschscholtzii) and Proteidae (Necturus maculosus) are characterized by longitudinal furrows of the third cleavage and the loss of synchrony as early as the 8-cell stage. By contrast, many representatives of derived families Ambystomatidae and Salamandridae have latitudinal furrows of the third cleavage and extensive period of synchronous divisions. Our analysis of these ontogenetic characters mapped onto a phylogenetic tree shows that the cleavage pattern of large, yolky eggs with short series of synchronous divisions is an ancestral trait for the tailed amphibians, while the data on the orientation of third cleavage furrows seem to be ambiguous with respect to phylogeny. Nevertheless, the midblastula transition, which is characteristic of the model species Ambystoma mexicanum (Caudata) and Xenopus laevis (Anura), might have evolved convergently in these two amphibian orders.

  16. 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.

  17. RecA-mediated cleavage reaction of Lambda repressor and DNA ...

    African Journals Online (AJOL)

    PRECIOUS

    2010-01-11

    Jan 11, 2010 ... hydrolyze ATP at all, but fulfills RecA functions such as cleavage of Lambda repressor and strand .... DNA binding properties of RecA and may result in an in- .... AMP-PNP there is no cleavage of Lambda repressor (Figure.

  18. Photochemically promoted bond-cleavage and -capture in a diazomethane derivative of a triamidoamine uranium(IV) complex

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, Benedict M.; Patel, Dipti; Lewis, William; Blake, Alexander J.; Liddle, Stephen T. [School of Chemistry, University of Nottingham (United Kingdom)

    2011-10-24

    Photolysis of [U(tren{sup TMS})(μ-N(SiMe{sub 3})NC)]{sub 2} results in multiple bond cleavage and capture to give a well-defined product [U{N(CH_2CH_2NSiMe_3)_2(μ-CH_2CH_2N-C≡N)}{N(SiMe_3)_2}]{sub 2}. This transformation has no precedent in diazoalkane chemistry and is not thermally accessible. (Copyright copyright 2011 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  19. Comparison of the cleavage of pyrimidine dimers by the bacteriophage T4 and Micrococcus luteus uv-specific endonucleases

    International Nuclear Information System (INIS)

    Gordon, L.K.; Haseltine, W.A.

    1980-01-01

    A comparison was made of the activity of the uv-specific endonucleases of bacteriophage T4 (T4 endonuclease V) and of Micrococcus luteus on ultraviolet light-irradiated DNA substrates of defined sequence. The two enzyms cleave DNA at the site of pyrimidine dimers with the same frequency. The products of the cleavage reaction are the same. The pyrimidine dimer DNA-glycosylase activity of both enzymes is more active on double-stranded DNA than it is on single-stranded DNA

  20. Phosphorylation of iRhom2 Controls Stimulated Proteolytic Shedding by the Metalloprotease ADAM17/TACE

    Directory of Open Access Journals (Sweden)

    Miguel Cavadas

    2017-10-01

    Full Text Available Cell surface metalloproteases coordinate signaling during development, tissue homeostasis, and disease. TACE (TNF-α-converting enzyme, is responsible for cleavage (“shedding” of membrane-tethered signaling molecules, including the cytokine TNF, and activating ligands of the EGFR. The trafficking of TACE within the secretory pathway requires its binding to iRhom2, which mediates the exit of TACE from the endoplasmic reticulum. An important, but mechanistically unclear, feature of TACE biology is its ability to be stimulated rapidly on the cell surface by numerous inflammatory and growth-promoting agents. Here, we report a role for iRhom2 in TACE stimulation on the cell surface. TACE shedding stimuli trigger MAP kinase-dependent phosphorylation of iRhom2 N-terminal cytoplasmic tail. This recruits 14-3-3 proteins, enforcing the dissociation of TACE from complexes with iRhom2, promoting the cleavage of TACE substrates. Our data reveal that iRhom2 controls multiple aspects of TACE biology, including stimulated shedding on the cell surface.

  1. Proteolytic Processing and Assembly of gag and gag-pol Proteins of TED, a Baculovirus-Associated Retrotransposon of the Gypsy Family

    Science.gov (United States)

    Hajek, Kathryn L.; Friesen, Paul D.

    1998-01-01

    TED (transposable element D) is an env-containing member of the gypsy family of retrotransposons that represents a possible retrovirus of invertebrates. This lepidopteran (moth) retroelement contains gag and pol genes that encode proteins capable of forming viruslike particles (VLP) with reverse transcriptase. Since VLP are likely intermediates in TED transposition, we investigated the roles of gag and pol in TED capsid assembly and maturation. By using constructed baculovirus vectors and TED Gag-specific antiserum, we show that the principal translation product of gag (Pr55gag) is cleaved to produce a single VLP structural protein, p37gag. Replacement of Asp436 within the retrovirus-like active site of the pol-encoded protease (PR) abolished Pr55gag cleavage and demonstrated the requirement for PR in capsid processing. As shown by expression of an in-frame fusion of TED gag and pol, PR is derived from the Gag-Pol polyprotein Pr195gag-pol. The PR cleavage site within Pr55gag was mapped to a position near the junction of a basic, nucleocapsid-like domain and a C-terminal acidic domain. Once released by cleavage, the C-terminal fragment was not detected. This acidic fragment was dispensable for VLP assembly, as demonstrated by the formation of VLP by C-terminal Pr55gag truncation proteins and replacement of the acidic domain with a heterologous protein. In contrast, C-terminal deletions that extended into the adjacent nucleocapsid-like domain of Pr55gag abolished VLP recovery and demonstrated that this central region contributes to VLP assembly or stability, or both. Collectively, these data suggest that the single TED protein p37gag provides both capsid and nucleocapsid functions. TED may therefore use a simple processing strategy for VLP assembly and genome packaging. PMID:9765414

  2. Amino-terminal domain of the v-fms oncogene product includes a functional signal peptide that directs synthesis of a transforming glycoprotein in the absence of feline leukemia virus gag sequences

    International Nuclear Information System (INIS)

    Wheeler, E.F.; Roussel, M.F.; Hampe, A.; Walker, M.H.; Fried, V.A.; Look, A.T.; Rettenmier, C.W.; Sherr, C.J.

    1986-01-01

    The nucleotide sequence of a 5' segment of the human genomic c-fms proto-oncogene suggested that recombination between feline leukemia virus and feline c-fms sequences might have occurred in a region encoding the 5' untranslated portion of c-fms mRNA. The polyprotein precursor gP180/sup gag-fms/ encoded by the McDonough strain of feline sarcoma virus was therefore predicted to contain 34 v-fms-coded amino acids derived from sequences of the c-fms gene that are not ordinarily translated from the proto-oncogene mRNA. The (gP180/sup gag-fms/) polyprotein was cotranslationally cleaved near the gag-fms junction to remove its gag gene-coded portion. Determination of the amino-terminal sequence of the resulting v-fms-coded glycoprotein, gp120/sup v-fms/, showed that the site of proteolysis corresponded to a predicted signal peptidase cleavage site within the c-fms gene product. Together, these analyses suggested that the linked gag sequences may not be necessary for expression of a biologically active v-fms gene product. The gag-fms sequences of feline sarcoma virus strain McDonough and the v-fms sequences alone were inserted into a murine retroviral vector containing a neomycin resistance gene. The authors conclude that a cryptic hydrophobic signal peptide sequence in v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms gene product within membranous organelles. It seems likely that the proteolytic cleavage of gP180/gag-fms/ is mediated by signal peptidase and that the amino termini of gp140/sup v-fms/ and the c-fms gene product are identical

  3. Amino-terminal domain of the v-fms oncogene product includes a functional signal peptide that directs synthesis of a transforming glycoprotein in the absence of feline leukemia virus gag sequences

    Energy Technology Data Exchange (ETDEWEB)

    Wheeler, E.F.; Roussel, M.F.; Hampe, A.; Walker, M.H.; Fried, V.A.; Look, A.T.; Rettenmier, C.W.; Sherr, C.J.

    1986-08-01

    The nucleotide sequence of a 5' segment of the human genomic c-fms proto-oncogene suggested that recombination between feline leukemia virus and feline c-fms sequences might have occurred in a region encoding the 5' untranslated portion of c-fms mRNA. The polyprotein precursor gP180/sup gag-fms/ encoded by the McDonough strain of feline sarcoma virus was therefore predicted to contain 34 v-fms-coded amino acids derived from sequences of the c-fms gene that are not ordinarily translated from the proto-oncogene mRNA. The (gP180/sup gag-fms/) polyprotein was cotranslationally cleaved near the gag-fms junction to remove its gag gene-coded portion. Determination of the amino-terminal sequence of the resulting v-fms-coded glycoprotein, gp120/sup v-fms/, showed that the site of proteolysis corresponded to a predicted signal peptidase cleavage site within the c-fms gene product. Together, these analyses suggested that the linked gag sequences may not be necessary for expression of a biologically active v-fms gene product. The gag-fms sequences of feline sarcoma virus strain McDonough and the v-fms sequences alone were inserted into a murine retroviral vector containing a neomycin resistance gene. The authors conclude that a cryptic hydrophobic signal peptide sequence in v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms was unmasked by gag deletion, thereby allowing the correct orientation and transport of the v-fms gene product within membranous organelles. It seems likely that the proteolytic cleavage of gP180/gag-fms/ is mediated by signal peptidase and that the amino termini of gp140/sup v-fms/ and the c-fms gene product are identical.

  4. Prion protein cleavage fragments regulate adult neural stem cell quiescence through redox modulation of mitochondrial fission and SOD2 expression.

    Science.gov (United States)

    Collins, Steven J; Tumpach, Carolin; Groveman, Bradley R; Drew, Simon C; Haigh, Cathryn L

    2018-03-24

    Neurogenesis continues in the post-developmental brain throughout life. The ability to stimulate the production of new neurones requires both quiescent and actively proliferating pools of neural stem cells (NSCs). Actively proliferating NSCs ensure that neurogenic demand can be met, whilst the quiescent pool makes certain NSC reserves do not become depleted. The processes preserving the NSC quiescent pool are only just beginning to be defined. Herein, we identify a switch between NSC proliferation and quiescence through changing intracellular redox signalling. We show that N-terminal post-translational cleavage products of the prion protein (PrP) induce a quiescent state, halting NSC cellular growth, migration, and neurite outgrowth. Quiescence is initiated by the PrP cleavage products through reducing intracellular levels of reactive oxygen species. First, inhibition of redox signalling results in increased mitochondrial fission, which rapidly signals quiescence. Thereafter, quiescence is maintained through downstream increases in the expression and activity of superoxide dismutase-2 that reduces mitochondrial superoxide. We further observe that PrP is predominantly cleaved in quiescent NSCs indicating a homeostatic role for this cascade. Our findings provide new insight into the regulation of NSC quiescence, which potentially could influence brain health throughout adult life.

  5. Alanine substitutions in the GXXXG motif alter C99 cleavage by γ-secretase but not its dimerization.

    Science.gov (United States)

    Higashide, Hidekazu; Ishihara, Seiko; Nobuhara, Mika; Ihara, Yasuo; Funamoto, Satoru

    2017-03-01

    The amyloid β (Aβ) protein is a major component of senile plaques, one of the neuropathological hallmarks of Alzheimer's disease. Amyloidogenic processing of amyloid precursor protein (APP) by β- and γ-secretases leads to production of Aβ. APP contains tandem triple repeats of the GXXXG motif in its extracellular juxtamembrane and transmembrane regions. It is reported that the GXXXG motif is related to protein-protein interactions, but it remains controversial whether the GXXXG motif in APP is involved in substrate dimerization and whether dimerization affects γ-secretase-dependent cleavage. Therefore, the relationship between the GXXXG motifs, substrate dimerization, and γ-secretase-dependent cleavage sites remains unclear. Here, we applied blue native poly acrylamide gel electrophoresis to examine the effect of alanine substitutions within the GXXXG motifs of APP carboxyl terminal fragment (C99) on its dimerization and Aβ production. Surprisingly, alanine substitutions in the motif failed to alter C99 dimerization in detergent soluble state. Cell-based and solubilized γ-secretase assays demonstrated that increasing alanine substitutions in the motif tended to decrease long Aβ species such as Aβ42 and Aβ43 and to increase in short Aβ species concomitantly. Our data suggest that the GXXXG motif is crucial for Aβ production, but not for C99 dimerization. © 2016 International Society for Neurochemistry.

  6. Cleavage of olefinic double bonds by mediated anodic oxidation

    International Nuclear Information System (INIS)

    Baeumer, U.-St.; Schaefer, H.J.

    2003-01-01

    Seven alkenes, e.g. 1-decene, methyl oleate, cyclododecene, norbornene, are cleaved by indirect anodic oxidation with IO 4 - /RuCl 3 as mediator to carboxylic acids. The best performance was achieved with two alternative ex cell-methods. Periodate is regenerated from iodate in a divided cell at a PbO 2 /Ti-anode. In the chemical reactor alkene and the produced carboxylic acid are immobilized in a chromatography column on Chromosorb W and oxidized with IO 4 - /RuO 4 in CH 3 CN/water. In the alternative version the alkene is oxidized in an emulsion generated by sonication and the organic phase is retained in the reactor by a separator. Acids and diacids are obtained in 61-91% chemical yield and good current yields. The amount of consumed periodate can be reduced to less than 5% of the amount needed for the chemical oxidation. The mediated anodic cleavage of alkenes is altogether an interesting alternative to ozonolysis

  7. Diaphanous gene mutation affects spiral cleavage and chirality in snails

    Science.gov (United States)

    Kuroda, Reiko; Fujikura, Kohei; Abe, Masanori; Hosoiri, Yuji; Asakawa, Shuichi; Shimizu, Miho; Umeda, Shin; Ichikawa, Futaba; Takahashi, Hiromi

    2016-01-01

    L-R (left and right) symmetry breaking during embryogenesis and the establishment of asymmetric body plan are key issues in developmental biology, but the onset including the handedness-determining gene locus still remains unknown. Using pure dextral (DD) and sinistral (dd) strains of the pond snail Lymnaea stagnalis as well as its F2 through to F10 backcrossed lines, the single handedness-determining-gene locus was mapped by genetic linkage analysis, BAC cloning and chromosome walking. We have identified the actin-related diaphanous gene Lsdia1 as the strongest candidate. Although the cDNA and derived amino acid sequences of the tandemly duplicated Lsdia1 and Lsdia2 genes are very similar, we could discriminate the two genes/proteins in our molecular biology experiments. The Lsdia1 gene of the sinistral strain carries a frameshift mutation that abrogates full-length LsDia1 protein expression. In the dextral strain, it is already translated prior to oviposition. Expression of Lsdia1 (only in the dextral strain) and Lsdia2 (in both chirality) decreases after the 1-cell stage, with no asymmetric localization throughout. The evolutionary relationships among body handedness, SD/SI (spiral deformation/spindle inclination) at the third cleavage, and expression of diaphanous proteins are discussed in comparison with three other pond snails (L. peregra, Physa acuta and Indoplanorbis exustus). PMID:27708420

  8. Sox11 Reduces Caspase-6 Cleavage and Activity.

    Directory of Open Access Journals (Sweden)

    Elaine Waldron-Roby

    Full Text Available The apoptotic cascade is an orchestrated event, whose final stages are mediated by effector caspases. Regulatory binding proteins have been identified for caspases such as caspase-3, -7, -8, and -9. Many of these proteins belong to the inhibitor of apoptosis (IAP family. By contrast, caspase-6 is not believed to be influenced by IAPs, and little is known about its regulation. We therefore performed a yeast-two-hybrid screen using a constitutively inactive form of caspase-6 for bait in order to identify novel regulators of caspase-6 activity. Sox11 was identified as a potential caspase-6 interacting protein. Sox11 was capable of dramatically reducing caspase-6 activity, as well as preventing caspase-6 self- cleavage. Several regions, including amino acids 117-214 and 362-395 within sox11 as well as a nuclear localization signal (NLS all contributed to the reduction in caspase-6 activity. Furthermore, sox11 was also capable of decreasing other effector caspase activity but not initiator caspases -8 and -9. The ability of sox11 to reduce effector caspase activity was also reflected in its capacity to reduce cell death following toxic insult. Interestingly, other sox proteins also had the ability to reduce caspase-6 activity but to a lesser extent than sox11.

  9. A plant Bcl-2-associated athanogene is proteolytically activated to confer fungal resistance

    Directory of Open Access Journals (Sweden)

    Mehdi Kabbage

    2016-04-01

    Full Text Available The Bcl-2-associated athanogene (BAG family is a multifunctional group of proteins involved in numerous cellular functions ranging from apoptosis to tumorigenesis. These proteins are evolutionarily conserved and encode a characteristic region known as the BAG domain. BAGs function as adapter proteins forming complexes with signaling molecules and molecular chaperones. In humans, a role for BAG proteins has been suggested in tumor growth, HIV infection, and neurodegenerative diseases; as a result, the BAGs are attractive targets for therapeutic interventions, and their expression in cells may serve as a predictive tool for disease development. The Arabidopsis genome contains seven homologs of BAG family proteins (Figure 1, including four with a domain organization similar to animal BAGs (BAG1-4. The remaining three members (BAG5-7 contain a predicted calmodulin-binding motif near the BAG domain, a feature unique to plant BAG proteins that possibly reflects divergent mechanisms associated with plant-specific functions. As reported for animal BAGs, plant BAGs also regulate several stress and developmental processes (Figure 2. The recent article by Li et al. focuses on the role of BAG6 in plant innate immunity. This study shows that BAG6 plays a key role in basal plant defense against fungal pathogens. Importantly, this work further shows that BAG6 is proteolytically activated to induce autophagic cell death and resistance in plants. This finding underscores the importance of proteases in the execution of plant cell death, yet little is known about proteases and their substrates in plants.

  10. Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis) 1

    Science.gov (United States)

    Samac, Deborah; Storey, Richard

    1981-01-01

    Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling. Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination. PMID:16662104

  11. Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis).

    Science.gov (United States)

    Samac, D; Storey, R

    1981-12-01

    Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination.

  12. Effect of wine inhibitors on the proteolytic activity of papain from Carica papaya L. latex.

    Science.gov (United States)

    Benucci, Ilaria; Esti, Marco; Liburdi, Katia

    2015-01-01

    The influence of potential inhibitors naturally present in wine on the proteolytic activity of papain from Carica papaya latex was investigated to evaluate its applicability in white wine protein haze stabilization. Enzymatic activity was tested against a synthetic tripeptide chromogenic substrate in wine-like acidic medium that consisted of tartaric buffer (pH 3.2) supplemented with ethanol, free sulfur dioxide (SO2 ), grape skin and seed tannins within the average ranges of concentrations that are typical in wine. The diagnosis of inhibition type, performed with the graphical method, demonstrated that all of tested wine constituents were reversible inhibitors of papain. The strongest inhibition was exerted by free SO2 , which acted as a mixed-type inhibitor, similar to grape skin and seed tannins. Finally, when tested in table white wines, the catalytic activity of papain, even when if it was ascribable to the hyperbolic behavior of Michaelis-Menten equation, was determined to be strongly affected by free SO2 and total phenol level. © 2014 American Institute of Chemical Engineers.

  13. Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development

    Science.gov (United States)

    Uchiyama, Kazuhiko; Sakiyama, Toshio; Hasebe, Takumu; Musch, Mark W.; Miyoshi, Hiroyuki; Nakagawa, Yasushi; He, Tong-Chuan; Lichtenstein, Lev; Naito, Yuji; Itoh, Yoshito; Yoshikawa, Toshikazu; Jabri, Bana; Stappenbeck, Thaddeus; Chang, Eugene B.

    2016-01-01

    Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation. PMID:27561676

  14. Lipidperoxidative and proteolytic processes in fungi under the influence of xenobiotica. [Botrytis cinerea; Phytophora infestans

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, H M; Edlich, W; Lyr, H

    1986-01-01

    As a model system for the investigation of peroxidative effects in the phytopathogenic fungi Botrytis cinerea in submers culture was used. The influence was investigated of hydrogenperoxide with biochemical and cytological methods. The hyphae showed significant changes of the ultrastructure: swelling and vacuolization of mitochondria, loss of ribosomes, vesiculation of endoplasmic reticulum, degradation and lysis of the cell ribosomes, and deposition of lipid droplets on the membranes and in the cytoplasm, dependent on the concentration of the peroxide. Same effects with higher injuries of the membrane system are found in Phytophthorea infestans. A parallel investigation on fibroblasts showed similar effects: pathological changes of mitochondria, but also pycnosis in the nucleus. Comparative ultrastructure investigations were performed in cultures of Phytophthora infestans with tetrachlorcarbon and chloroneb, a specific fungicide substance with a new mode of action. Scavengers like ..cap alpha..-tocopherol and piperonyl butoxide inhibit the toxic effect of the investigated substances in various degrees. Results are discussed in regard to the effect of a lipid peroxidative and proteolytic attack in pesticides of unknown mechanism of action.

  15. Mitochondrial AAA proteases--towards a molecular understanding of membrane-bound proteolytic machines.

    Science.gov (United States)

    Gerdes, Florian; Tatsuta, Takashi; Langer, Thomas

    2012-01-01

    Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Fermentation of sugar to ethyl alcohol in the presence of proteolytic enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Coates, E W; Conde Julio, C

    1963-06-11

    Sugar is fermented to EtOH by yeasts capable of elaborating zymase and proteolytic enzymes, the zymase component comprising exceptionally large amounts of phosphatase. Saccharomyces ellipsoideus was acclimated to 20% EtOH by growing on fresh pineapple juice in a medium consisting of malt sirup 15, sugar sirup 3, and pineapple juice 82%. An aqueous solution of 2000 gallons of sugar cane molasses in H/sub 2/O to give a Brix of 16/sup 0/ was placed in a 48,000-gallon fermentor. S. ellipsoideus with a cell constant of 1 x 10/sup 9/ cells/ml was added, with sufficient H/sub 2/SO/sub 4/ to adjust the pH to approximately 4.5. Fermentation was carried out at 35/sup 0/ until the Brix dropped to 8/sup 0/, after which it was brought back to 16/sup 0/ by adding 6000 gallons of sirup containing nutrients in H/sub 2/O. This process was repeated with another 6000 and then 2000 gallons of sirup. The total fermentation required 48 h and the EtOH content was 15.25% by volume. Te EtOH was recovered in the usual manner by removal of solids and fractional distillation.

  17. Enzymatic surface hydrolysis of polyamide 6,6 with mixtures of proteolytic and lipolytic enzymes.

    Science.gov (United States)

    Parvinzadeh Gashti, Mazeyar; Assefipour, Reza; Kiumarsi, Amir; Parvinzadeh Gashti, Mahyar

    2013-01-01

    This study investigated the changes induced on nylon 6,6 fabric by a mixture of proteolytic and lipolytic enzymes. Technical measurements were studied including those of Fourier-transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), weight loss (WL), bending lengths (BL), scanning electron microscopy (SEM), moisture absorbency (MA), and reflectance spectroscopy (RS). For this purpose, nylon 6,6 fabrics were treated separately with different concentrations of protease and lipase mixtures in solution. The dyeing process was then carried out on the treated fabrics with two reactive and acid dyes. The intensity of major peaks in the FTIR spectra of the protease-treated samples is in favor of chemical changes the polypeptide functional groups in the fabrics. Thermal studies also show a significant decrease in the thermal degradation temperature of the treated polymer at temperatures higher than 400°C. The protease and lipase mixtures decreased the sample weight, while lipase intensified the weight loss comparing with protease. It was observed that the concentration of lipase enzyme had a direct influence on the darkness of dyed samples.

  18. Studies on the membrane-associated proteolytic activities of Escherichia coli

    International Nuclear Information System (INIS)

    Palmer, S.M.

    1986-01-01

    The membrane fraction contains three proteolytic activities which can be resolved from whole membrane detergent extracts by DEAE-cellulose chromatography. The first two eluting activities have been previously reported as protease V and protease IV. These two enzymes were further purified by gel permeation HPLC. Protease V has a M. W. of 31,000 in SDS-PAGE gels. Protease IV has a M. W. of 62,000 and exists in two distinct isoforms of pl ≅ 6.7 and 6.9. The third enzyme eluting from the DEAE-cellulose column was further purified by affinity chromatography on Benzamidine-Sepharose 6B. This enzyme, referred to herein as protease VI, is a 43 kdal protein which has not been previously characterized. Protease VI was sensitive to inhibition by the serine protease inhibitors phenylmethylsulfony fluoride (PMSF), diisopropylfluorophosphate (DFP) and p-amino-benzamidine (PAB). Additionally, this enzyme exhibited maximal activity at pH ≅ 8.0. Incubation of whole membrane preparations with [ 3 H] DFP resulted in 11 specific proteins acquiring the radioactive label, included in this group of proteins were proteases IV, V, and VI. Several of the DFP-reactive proteins were also shown to bind [ 125 I] ampicillin

  19. Membrane-associated proteolytic activity in Escherichia coli that is stimulated by ATP

    International Nuclear Information System (INIS)

    Klemes, Y.; Voellmy, R.W.; Goldberg, A.L.

    1986-01-01

    The degradation of proteins in bacteria requires metabolism energy. One important enzyme in this process is protease La, a soluble ATP-dependent protease encoded by the lon gene. However, lon mutants that lack a functional protease La still show some ATP-dependent protein breakdown. The authors have reported an ATP-stimulated endoproteolytic activity associated with the inner membrane of E. coli. This ATP-stimulated activity is found in normal levels in membranes derived from lon mutants, including strains carrying insertions in the lon gene. The membrane-bound activity hydrolyzes 14 C-methylglobin at a linear rate for up to 3 hours. These fractions also contain appreciable proteolytic activity that is not affected by ATP. The stimulation by ATP requires the presence of Mg 2+ . Nonhydrolyzable ATP analogs (e.g. AMPPNP or ATP-γ-S) and ADP do not enhance proteolysis. Unlike protease La, the membrane-associated enzyme does not degrade the fluorometric substrate, Glt-Ala-Ala-Phe-MNA, in an ATP-stimulated fashion, and its level is not influenced by high temperature of by the gene which regulates the heat-shock response. The enzyme is inhibited by dichloroisocoumarin and certain peptide chloromethyl ketones. They conclude that E. coli contain at least two ATP-dependent proteases with distinct specificities: one is soluble and the other is membrane-associated

  20. Serine proteases as candidates for proteolytic processing of angiotensin-I converting enzyme.

    Science.gov (United States)

    Aragão, Danielle S; de Andrade, Maria Claudina C; Ebihara, Fabiana; Watanabe, Ingrid K M; Magalhães, Dayane C B P; Juliano, Maria Aparecida; Hirata, Izaura Yoshico; Casarini, Dulce Elena

    2015-01-01

    Somatic angiotensin-I converting enzyme (sACE) is a broadly distributed peptidase which plays a role in blood pressure and electrolyte homeostasis by the conversion of angiotensin I into angiotensin II. N-domain isoforms (nACE) with 65 and 90 kDa have been described in body fluids, tissues and mesangial cells (MC), and a 90 kDa nACE has been described only in spontaneously hypertensive rats. The aim of this study was to investigate the existence of proteolytic enzymes that may act in the hydrolysis of sACE generating nACEs in MC. After the confirmation of the presence of ACE sheddases in Immortalized MC (IMC), we purified and characterized these enzymes using fluorogenic substrates specifically designed for ACE sheddases. Purified enzyme identified as a serine protease by N-terminal sequence was able to generate nACE. In the present study, we described for the first time the presence of ACE sheddases in IMC, identified as serine proteases able to hydrolyze sACE in vitro. Further investigations are necessary to elucidate the mechanisms responsible for the expression and regulation of ACE sheddases in MC and their roles in the generation of nACEs, especially the 90 kDa form possibly related to hypertension. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. PAPP-A proteolytic activity enhances IGF bioactivity in ascites from women with ovarian carcinoma

    DEFF Research Database (Denmark)

    Thomsen, Jacob; Hjortebjerg, Rikke; Espelund, Ulrick

    2015-01-01

    Pregnancy-associated plasma protein-A (PAPP-A) stimulates insulin-like growth factor (IGF) action through proteolysis of IGF-binding protein (IGFBP)-4. In experimental animals, PAPP-A accelerates ovarian tumor growth by this mechanism. To investigate the effect of PAPP-A in humans, we compared.......03). Ascites was more potent than serum in activating the IGF-I receptor (IGF-IR) in vitro (+31%, P IGF-I, and lower levels of IGF-II (P ... of IGF-IR in all but one tumor, whereas all tumors expressed PAPP-A, IGFBP-4, IGF-I and IGF-II. Addition of recombinant PAPP-A to ascites increased the cleavage of IGFBP-4 and enhanced IGF-IR activation (P IGFs and these proteins...

  2. Downstream element determines RNase Y cleavage of the saePQRS operon in Staphylococcus aureus.

    Science.gov (United States)

    Marincola, Gabriella; Wolz, Christiane

    2017-06-02

    In gram-positive bacteria, RNase J1, RNase J2 and RNase Y are thought to be major contributors to mRNA degradation and maturation. In Staphylococcus aureus, RNase Y activity is restricted to regulating the mRNA decay of only certain transcripts. Here the saePQRS operon was used as a model to analyze RNase Y specificity in living cells. A RNase Y cleavage site is located in an intergenic region between saeP and saeQ. This cleavage resulted in rapid degradation of the upstream fragment and stabilization of the downstream fragment. Thereby, the expression ratio of the different components of the operon was shifted towards saeRS, emphasizing the regulatory role of RNase Y activity. To assess cleavage specificity different regions surrounding the sae CS were cloned upstream of truncated gfp, and processing was analyzed in vivo using probes up- and downstream of CS. RNase Y cleavage was not determined by the cleavage site sequence. Instead a 24-bp double-stranded recognition structure was identified that was required to initiate cleavage 6 nt upstream. The results indicate that RNase Y activity is determined by secondary structure recognition determinants, which guide cleavage from a distance. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Deep Sequencing Insights in Therapeutic shRNA Processing and siRNA Target Cleavage Precision.

    Science.gov (United States)

    Denise, Hubert; Moschos, Sterghios A; Sidders, Benjamin; Burden, Frances; Perkins, Hannah; Carter, Nikki; Stroud, Tim; Kennedy, Michael; Fancy, Sally-Ann; Lapthorn, Cris; Lavender, Helen; Kinloch, Ross; Suhy, David; Corbau, Romu

    2014-02-04

    TT-034 (PF-05095808) is a recombinant adeno-associated virus serotype 8 (AAV8) agent expressing three short hairpin RNA (shRNA) pro-drugs that target the hepatitis C virus (HCV) RNA genome. The cytosolic enzyme Dicer cleaves each shRNA into multiple, potentially active small interfering RNA (siRNA) drugs. Using next-generation sequencing (NGS) to identify and characterize active shRNAs maturation products, we observed that each TT-034-encoded shRNA could be processed into as many as 95 separate siRNA strands. Few of these appeared active as determined by Sanger 5' RNA Ligase-Mediated Rapid Amplification of cDNA Ends (5-RACE) and through synthetic shRNA and siRNA analogue studies. Moreover, NGS scrutiny applied on 5-RACE products (RACE-seq) suggested that synthetic siRNAs could direct cleavage in not one, but up to five separate positions on targeted RNA, in a sequence-dependent manner. These data support an on-target mechanism of action for TT-034 without cytotoxicity and question the accepted precision of substrate processing by the key RNA interference (RNAi) enzymes Dicer and siRNA-induced silencing complex (siRISC).Molecular Therapy-Nucleic Acids (2014) 3, e145; doi:10.1038/mtna.2013.73; published online 4 February 2014.

  4. Differential spatio-temporal expression of carotenoid cleavage dioxygenases regulates apocarotenoid fluxes during AM symbiosis.

    Science.gov (United States)

    López-Ráez, Juan A; Fernández, Iván; García, Juan M; Berrio, Estefanía; Bonfante, Paola; Walter, Michael H; Pozo, María J

    2015-01-01

    Apocarotenoids are a class of compounds that play important roles in nature. In recent years, a prominent role for these compounds in arbuscular mycorrhizal (AM) symbiosis has been shown. They are derived from carotenoids by the action of the carotenoid cleavage dioxygenase (CCD) enzyme family. In the present study, using tomato as a model, the spatio-temporal expression pattern of the CCD genes during AM symbiosis establishment and functioning was investigated. In addition, the levels of the apocarotenoids strigolactones (SLs), C13 α-ionol and C14 mycorradicin (C13/C14) derivatives were analyzed. The results suggest an increase in SLs promoted by the presence of the AM fungus at the early stages of the interaction, which correlated with an induction of the SL biosynthesis gene SlCCD7. At later stages, induction of SlCCD7 and SlCCD1 expression in arbusculated cells promoted the production of C13/C14 apocarotenoid derivatives. We show here that the biosynthesis of apocarotenoids during AM symbiosis is finely regulated throughout the entire process at the gene expression level, and that CCD7 constitutes a key player in this regulation. Once the symbiosis is established, apocarotenoid flux would be turned towards the production of C13/C14 derivatives, thus reducing SL biosynthesis and maintaining a functional symbiosis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  5. Functional analysis of coordinated cleavage in V(D)J recombination.

    Science.gov (United States)

    Kim, D R; Oettinger, M A

    1998-08-01

    V(D)J recombination in vivo requires a pair of signals with distinct spacer elements of 12 and 23 bp that separate conserved heptamer and nonamer motifs. Cleavage in vitro by the RAG1 and RAG2 proteins can occur at individual signals when the reaction buffer contains Mn2+, but cleavage is restricted to substrates containing two signals when Mg2+ is the divalent cation. By using a novel V(D)J cleavage substrate, we show that while the RAG proteins alone establish a moderate preference for a 12/23 pair versus a 12/12 pair, a much stricter dependence of cleavage on the 12/23 signal pair is produced by the inclusion of HMG1 and competitor double-stranded DNA. The competitor DNA serves to inhibit the cleavage of substrates carrying a 12/12 or 23/23 pair, as well as the cutting at individual signals in 12/23 substrates. We show that a 23/33 pair is more efficiently recombined than a 12/33 pair, suggesting that the 12/23 rule can be generalized to a requirement for spacers that differ from each other by a single helical turn. Furthermore, we suggest that a fixed spatial orientation of signals is required for cleavage. In general, the same signal variants that can be cleaved singly can function under conditions in which a signal pair is required. However, a chemically modified substrate with one noncleavable signal enables us to show that formation of a functional cleavage complex is mechanistically separable from the cleavage reaction itself and that although cleavage requires a pair of signals, cutting does not have to occur simultaneously at both. The implications of these results are discussed with respect to the mechanism of V(D)J recombination and the generation of chromosomal translocations.

  6. Evidence for the presence of proteolytically active secreted aspartic proteinase 1 of Candida parapsilosis in the cell wall

    Czech Academy of Sciences Publication Activity Database

    Vinterová, Zuzana; Šanda, Miloslav; Dostál, Jiří; Hrušková-Heidingsfeldová, Olga; Pichová, Iva

    2011-01-01

    Roč. 20, č. 12 (2011), s. 2004-2012 ISSN 0961-8368 R&D Projects: GA MŠk(CZ) LC531; GA ČR GA310/09/1945 Institutional research plan: CEZ:AV0Z40550506 Keywords : Candida parapsilosis * secreted aspartic proteinases * Sapp1p * cell wall * biotin * proteolytic activity Subject RIV: CE - Biochemistry Impact factor: 2.798, year: 2011

  7. Analysis of Proteolytic and Fibrinolytic Activity of the Blood Plasma in Patients with Bronchial Asthma Combined with Obesity

    Directory of Open Access Journals (Sweden)

    Fediv A.I. Fediv A.I.

    2016-09-01

    Full Text Available Aim of the study — to conduct the analysis of the performance of proteolytic and fibrinolytic activity of the blood plasma in patients with bronchial asthma (BA combined with obesity. Materials and methods. The study included 40 patients divided into groups. The basic group consisted of 20 patients with BA associated with obesity (ІI group, two comparison groups — of 10 patients with BA and normal body weight (I group and 10 patients with obesity and without pathology of the bronchopulmonary system (ІІІ group. Control group included 10 apparently healthy persons. In patients with BA, there were investigated the indexes of proteolytic and fibrinolytic activity of the blood plasma. Results. Analysis of the obtained data in patients with BA showed an increase of activity of plasma coagulation factors: total fibrinolytic activity increased in comparison to the control group, and in patients with BA it was 1.46 ± 0.13 Е440/ml/h, while in apparently healthy persons — 1.23 ± 0.16 Е440/ml/h (р < 0.05. In addition, an increase was marked and in the group of patients with the isolated obesity (by 38.5 % as compared to the group of apparently healthy persons, that can be explained by the growth of activity of inflammatory process due to the immune changes related to biological activity of fatty tissue as an additional source of proinflammatory cytokines. Conclusions. In combination of bronchial asthma and obesity, there is an activation of fibrinolytic and proteolytic systems of the blood that leads to microcirculatory and hemostatic disorders, enhancement of inflammatory process. In obesity as the state of systemic inflammatory response, the activation of fibrinolytic and proteolytic blood systems is more significant, than in bronchial asthma.

  8. Pathogenic Assay of Probiotic Bacteria Producing Proteolytic Enzymes as Bioremediation Bacteria Against Vannamei Shrimp Larvae (Litopenaeus vannamei)

    OpenAIRE

    Wilis Ari Setyati; Muhammad Zainuddin; Person Pesona Renta

    2017-01-01

    Application of bacteria in bioremediation of shrimp culture ponds is one of the methods used to clean internal pollutants. This study aimed to evaluate the pathogenicity of extracellular proteolytic enzyme produced by the probiotic bacteria as bioremediation bacteria on vannamei shrimp larvae culture. There were five probiotic bacteria, which were successfully isolated from the sediments served as substrate in mangrove area. The isolated bacteria were coded in number as 13, 19, 30, 33, and 36...

  9. Remarkable weakness against cleavage stress for YBCO-coated conductors and its effect on the YBCO coil performance

    International Nuclear Information System (INIS)

    Yanagisawa, Y.; Nakagome, H.; Takematsu, T.; Takao, T.; Sato, N.; Takahashi, M.; Maeda, H.

    2011-01-01

    Cleavage strength for YBCO-coated conductor is extremely low, typically 0.5 MPa. The remarkable weakness is due to cracks on the slit edge of the conductor. The cleavage stress appears on YBCO double pancake coils impregnated with epoxy. The cleavage stress should be avoided in the coil winding. Cleavage strength for an YBCO-coated conductor at 77 K was investigated with a model experiment. The nominal cleavage strength for an YBCO-coated conductor is extremely low, typically 0.5 MPa. This low nominal cleavage strength is due to stress concentration on a small part of the YBCO-coated conductor in cleavage fracture. Debonding by the cleavage stress occurs at the interface between the buffer layer and the Hastelloy substrate. The nominal cleavage strength for a slit edge of the conductor is 2.5-times lower than that for the original edge of the conductor; cracks and micro-peel existing over the slit edge reduce the cleavage strength for the slit edge. Cleavage stress and peel stress should be avoided in coil winding, as they easily delaminate the YBCO-coated conductor, resulting in substantial degradation of coil performance. These problems are especially important for epoxy impregnated YBCO-coated conductor coils. It appears that effect of cleavage stress and peel stress are mostly negligible for paraffin impregnated YBCO-coated conductor coils or dry wound YBCO-coated conductor coils.

  10. Endocytic down-regulation of ErbB2 is stimulated by cleavage of its C-terminus

    DEFF Research Database (Denmark)

    Lerdrup, Mads; Bruun, Silas; Grandal, Michael Vibo

    2007-01-01

    inhibition of HSP90 with geldanamycin this cleavage is accompanied by proteasome-dependent endocytosis of ErbB2. However, it is unknown whether C-terminal cleavage is linked to endocytosis. To study ErbB2 cleavage and endocytic trafficking, we fused yellow fluorescent protein (YFP) and cyan fluorescent...

  11. Cleavage and synthesis function of high and low redox potential laccases towards 4-morpholinoaniline and aminated as well as chlorinated phenols.

    Science.gov (United States)

    Hahn, Veronika; Mikolasch, Annett; Schauer, Frieder

    2014-02-01

    Laccases are able to mediate both cleavage and synthesis processes. The basis for this dual reaction capability lies in the property of the enzyme laccase to oxidize phenolic, and to some extent non-phenolic substances, to reactive radicals which can undergo on the one hand separations of small substitutents or large molecule parts from the parent compound and on the other hand coupling reactions with other radicals or molecules which are not themselves oxidizable by laccase. The cleavage of the non-phenolic compound 4-morpholinoaniline as well as the deamination of 4-aminophenol and the dechlorination of 4-chlorophenol resulted in the formation of 1,4-hydroquinone which is immediately oxidized by laccase to 1,4-benzoquinone. The formation of the 1,4-hydroquinone/1,4-benzoquinone is the rate limiting step for the synthesis of the heteromolecular dimers and trimers composed of 1,4-benzoquinone and one or two molecules of morpholine. In addition to the synthesis of new compounds from the cleavage products, 4-morpholinoaniline polymerized probably via azo groups and C-N bonds to a homomolecular dimer and trimer. Similarities and differences in cleavage and synthesis reactions catalyzed by the low redox potential laccase of Myceliophthora thermophila (0.46 V) and the high redox potential laccase of Pycnoporus cinnabarinus (0.79 V) were determined. In addition, the dependency of the cleavage and synthesis efficiencies on the (a) structure and redox potential of the laccase, (b) structure and redox potential of the substrate, (c) pH value of the buffer used, (d) incubation temperature, (e) solvent concentration, and (f) laccase activity is discussed in general.

  12. An evaluation of the proteolytic and lipolytic potential of Penicillium spp. isolated from traditional Greek sausages in submerged fermentation.

    Science.gov (United States)

    Papagianni, Maria

    2014-01-01

    A number of novel Penicillium strains belonging to Penicillium nalgiovense, Penicillium solitum, Penicillium commune, Penicillium olsonii, and Penicillium oxalicum species, isolated from the surface of traditional Greek sausages, were evaluated for their proteolytic and lipolytic potential in a solid substrate first and next in submerged fermentations, using complex media. Extracellular proteolytic activity was assessed at acid, neutral, and alkaline pH, while the lipolytic activity was assessed using olive oil, the short-chain triacylglycerol tributyrin, and the long-chain triolein, as substrates. The study revealed that although closely related, the tested strains produce enzymes of distinct specificities. P. nalgiovense PNA9 produced the highest alkaline proteolytic activity (13.2 unit (U)/ml) and the highest lipolytic activity with tributyrin (92 U/ml). Comparisons with known sources show that proteases and/or lipases can be secreted effectively by some Penicillia (P. nalgiovense PNA4, PNA7, and PNA9 and P. solitum PSO1), and further investigations on their properties and characteristics would be promising.

  13. Secreted Aspartic Protease Cleavage of Candida albicans Msb2 Activates Cek1 MAPK Signaling Affecting Biofilm Formation and Oropharyngeal Candidiasis

    Science.gov (United States)

    Chadha, Sonia; Tati, Swetha; Conti, Heather R.; Hube, Bernhard; Cullen, Paul J.; Edgerton, Mira

    2012-01-01

    Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK) pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1) phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA), a selective inhibitor of aspartic proteases (Saps). Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ) or Sap8 (sap8Δ/Δ) resulted in higher C. albicans surface β-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence. PMID:23139737

  14. Positioning the 5'-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage

    KAUST Repository

    Song, Bo

    2018-02-09

    Flap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair and recombination, and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca2+ have suggested that base unpairing at the 5\\'-flap:template junction is an important step in the reaction, but the new structural data suggest otherwise. To clarify the role of DNA changes in the kinetic mechanism, we measured a series of transient steps - from substrate binding to product release - during the hFEN1-catalyzed reaction in the presence of Mg2+. We found that while hFEN1 binds and bends DNA at a fast, diffusion-limited rate, much slower Mg2+-dependent conformational changes in DNA around the active site are subsequently necessary and rate-limiting for 5\\'-flap cleavage. These changes are reported overall by fluorescence of 2-aminopurine at the 5\\'-flap:template junction, indicating that local DNA distortion (e.g., disruption of base stacking observed in structures), associated with positioning the 5\\'-flap scissile phosphodiester bond in the hFEN1 active site, controls catalysis. hFEN1 residues with distinct roles in the catalytic mechanism, including those binding metal ions (Asp-34, Asp-181), steering the 5\\'-flap through the active site and binding the scissile phosphate (Lys-93, Arg-100), and stacking against the base 5\\' to the scissile phosphate (Tyr-40), all contribute to these rate-limiting conformational changes, ensuring efficient and specific cleavage of 5\\'-flaps.

  15. Positioning the 5'-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage

    KAUST Repository

    Song, Bo; Hamdan, Samir; Hingorani, Manju M

    2018-01-01

    Flap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair and recombination, and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca2+ have suggested that base unpairing at the 5'-flap:template junction is an important step in the reaction, but the new structural data suggest otherwise. To clarify the role of DNA changes in the kinetic mechanism, we measured a series of transient steps - from substrate binding to product release - during the hFEN1-catalyzed reaction in the presence of Mg2+. We found that while hFEN1 binds and bends DNA at a fast, diffusion-limited rate, much slower Mg2+-dependent conformational changes in DNA around the active site are subsequently necessary and rate-limiting for 5'-flap cleavage. These changes are reported overall by fluorescence of 2-aminopurine at the 5'-flap:template junction, indicating that local DNA distortion (e.g., disruption of base stacking observed in structures), associated with positioning the 5'-flap scissile phosphodiester bond in the hFEN1 active site, controls catalysis. hFEN1 residues with distinct roles in the catalytic mechanism, including those binding metal ions (Asp-34, Asp-181), steering the 5'-flap through the active site and binding the scissile phosphate (Lys-93, Arg-100), and stacking against the base 5' to the scissile phosphate (Tyr-40), all contribute to these rate-limiting conformational changes, ensuring efficient and specific cleavage of 5'-flaps.

  16. Efficient debridement of necrotic wounds using proteolytic enzymes derived from Antarctic krill: a double-blind, placebo-controlled study in a standardized animal wound model

    NARCIS (Netherlands)

    Mekkes, J. R.; Le Poole, I. C.; Das, P. K.; Bos, J. D.; Westerhof, W.

    1998-01-01

    Wound healing can be accelerated by removing necrotic tissue. Various methods of wound debridement have been developed, including enzymatic debridement. Recently potent proteolytic enzymes were isolated from the intestine of Euphausia superba (Antarctic krill) that might be useful for degrading

  17. Assessment of proteolytic degradation of the basement membrane: a fragment of type IV collagen as a biochemical marker for liver fibrosis

    DEFF Research Database (Denmark)

    Veidal, Sanne S.; Karsdal, Morten A.; Nawrocki, Arkadiusz

    2011-01-01

    Collagen deposition and an altered matrix metalloproteinase (MMP) expression profile are hallmarks of fibrosis. Type IV collagen is the most abundant structural basement membrane component of tissue, which increases 14-fold during fibrogenesis in the liver. Proteolytic degradation of collagens...

  18. Dynamic propagation and cleavage crack arrest in bainitic steel

    International Nuclear Information System (INIS)

    Hajjaj, M.

    2006-06-01

    In complement of the studies of harmfulness of defects, generally realized in term of initiation, the concept of crack arrest could be used as complementary analyses to the studies of safety. The stop occurs when the stress intensity factor becomes lower than crack arrest toughness (KIa) calculated in elasto-statics (KI ≤ KIa). The aim of this thesis is to understand and predict the stop of a crack propagating at high speed in a 18MND5 steel used in the pressure water reactor (PWR). The test chosen to study crack arrest is the disc thermal shock test. The observations under the scanning electron microscope of the fracture surface showed that the crack arrest always occurs in cleavage mode and that the critical microstructural entity with respect to the propagation and crack arrest corresponds to at least the size of the prior austenitic grain. The numerical analyses in elasto-statics confirm the conservatism of the codified curve of the RCC-M with respect to the values of KIa. The dynamic numerical analyses show that the deceleration of the crack measured at the end of the propagation is related to the global dynamic of the structure (vibrations). The transferability to components of crack arrest toughness obtained from tests analysed in static is thus not assured. The disc thermal shock tests were also modelled by considering a criterion of propagation and arrest of the type 'RKR' characterized by a critical stress sc which depends on the temperature. The results obtained account well for the crack jump measured in experiments as well as the shape of the crack arrest front. (author)

  19. Mechanism of Excretion of a Bacterial Proteinase: Demonstration of Two Proteolytic Enzymes Produced by a Sarcina Strain (Coccus P)

    Energy Technology Data Exchange (ETDEWEB)

    SARNER, NITZA Z; BISSELL, MINA J; GIROLAMO, MARIO Di; GORINI, LUIGI

    1970-06-29

    A Sarcina strain (Coccus P) produces two proteolytic enzymes. One is found only extracellularly, is far more prevalent, and is actively excreted during exponential growth. It is the enzyme responsible for the known strong proteolytic activity of the cultures of this strain. A second protease is, however, produced which remains associated with the intact cells but is released by the protoplasts. The two enzymes appear unrelated in their derivation. Calcium ions play an essential role in preventing autodigestion of the excreted enzyme. Bacterial proteins are found outside the cell boundary as a consequence either of passive processes such as leakage or lysis or of active excretion. Under conditions in which leakage and lysis do not occur, as during exponential growth, the cell boundary is a barrier causing a complete separation of the bulk of the intracellular proteins from the one or very few extracellular proteins, with no trace of either type being detectable on the wrong side of the boundary. Since in bacteria there is no evidence of protein being produced other than internally, the separation into intraand extracellular proteins should occur after peptide chain formation. The question arises as to whether the structure of the cell boundary or that of the excreted proteins themselves determines this separation. Coccus P, a Sarcina closely related to Micrococcus lysodeikticus (3), produces an extracellular proteinase during the exponential phase of growth so that the process appears to be active excretion. The organism grows exponentially in a defined synthetic medium (12) to relatively high cell density (10{sup 9} cells/ml); therefore the mechanism of excretion can be studied over an extended period of time without the difficulties of changing growth rates. Coagulation of reconstituted skim milk provides a simple and sensitive assay for enzyme activity (I 1). The extracellular proteinase has also been purified and partially characterized (6-8). It has been shown

  20. Identifying and quantifying proteolytic events and the natural N terminome by terminal amine isotopic labeling of substrates.

    Science.gov (United States)

    Kleifeld, Oded; Doucet, Alain; Prudova, Anna; auf dem Keller, Ulrich; Gioia, Magda; Kizhakkedathu, Jayachandran N; Overall, Christopher M

    2011-09-22

    Analysis of the sequence and nature of protein N termini has many applications. Defining the termini of proteins for proteome annotation in the Human Proteome Project is of increasing importance. Terminomics analysis of protease cleavage sites in degradomics for substrate discovery is a key new application. Here we describe the step-by-step procedures for performing terminal amine isotopic labeling of substrates (TAILS), a 2- to 3-d (depending on method of labeling) high-throughput method to identify and distinguish protease-generated neo-N termini from mature protein N termini with all natural modifications with high confidence. TAILS uses negative selection to enrich for all N-terminal peptides and uses primary amine labeling-based quantification as the discriminating factor. Labeling is versatile and suited to many applications, including biochemical and cell culture analyses in vitro; in vivo analyses using tissue samples from animal and human sources can also be readily performed. At the protein level, N-terminal and lysine amines are blocked by dimethylation (formaldehyde/sodium cyanoborohydride) and isotopically labeled by incorporating heavy and light dimethylation reagents or stable isotope labeling with amino acids in cell culture labels. Alternatively, easy multiplex sample analysis can be achieved using amine blocking and labeling with isobaric tags for relative and absolute quantification, also known as iTRAQ. After tryptic digestion, N-terminal peptide separation is achieved using a high-molecular-weight dendritic polyglycerol aldehyde polymer that binds internal tryptic and C-terminal peptides that now have N-terminal alpha amines. The unbound naturally blocked (acetylation, cyclization, methylation and so on) or labeled mature N-terminal and neo-N-terminal peptides are recovered by ultrafiltration and analyzed by tandem mass spectrometry (MS/MS). Hierarchical substrate winnowing discriminates substrates from the background proteolysis products and

  1. Selection of proteolytic bacteria with ability to inhibit Vibrio harveyi during white shrimp (Litopenaeus vannamei cultivation

    Directory of Open Access Journals (Sweden)

    Suntinanalert, P.

    2007-03-01

    Full Text Available Five isolates of bacteria with high proteolytic activity, isolated from water samples of intensive shrimp ponds in southern Thailand, were selected to test for the ability to control the shrimp pathogen Vibrioharveyi. 70 μl of each culture broth were investigated for their ability to inhibit V. harveyi using an agar well diffusion test but only one isolate W3 gave a reasonable sized inhibition zone of 21.62 mm. This zone wassimilar to that of oxolinic acid (2 μg and sulfamethoxazole (25 μg. The W3 isolate was identified as Pseudomonas sp. Shrimp cultivation in aquaria was conducted to investigate the inhibition of V. harveyi bythe isolate W3. The experiment consisted of a treatment of the shrimp culture with an inoculum of the isolate W3 and V. harveyi (biocontrol set, a positive control set (only inoculation of V. harveyi and a negativecontrol set as without inoculation. No mortality was found in the negative control. Shrimp mortality in the biocontrol set (33% was lower than that in the positive control set (40%; however, it showed no significantdifference (p>0.05. The average numbers of V. harveyi over 12 days of the biocontrol set were lower than those in the positive control set by about 1 log cycle although the numbers were not significantly different(p>0.05. The shrimp growth rate at day 32 of cultivation was in order of the biocontrol treatment (10.17% > the negative control treatment (9.44% > the positive control set (9.28%, but no significant difference (p>0.05 was observed among treatments.

  2. Dynamics of digestive proteolytic system during blood feeding of the hard tick Ixodes ricinus

    Directory of Open Access Journals (Sweden)

    Sojka Daniel

    2010-12-01

    Full Text Available Abstract Background Ticks are vectors of a wide variety of pathogens causing severe diseases in humans and domestic animals. Intestinal digestion of the host blood is an essential process of tick physiology and also a limiting factor for pathogen transmission since the tick gut represents the primary site for pathogen infection and proliferation. Using the model tick Ixodes ricinus, the European Lyme disease vector, we have previously demonstrated by genetic and biochemical analyses that host blood is degraded in the tick gut by a network of acidic peptidases of the aspartic and cysteine classes. Results This study reveals the digestive machinery of the I. ricinus during the course of blood-feeding on the host. The dynamic profiling of concentrations, activities and mRNA expressions of the major digestive enzymes demonstrates that the de novo synthesis of peptidases triggers the dramatic increase of the hemoglobinolytic activity along the feeding period. Overall hemoglobinolysis, as well as the activity of digestive peptidases are negligible at the early stage of feeding, but increase dramatically towards the end of the slow feeding period, reaching maxima in fully fed ticks. This finding contradicts the established opinion that blood digestion is reduced at the end of engorgement. Furthermore, we show that the digestive proteolysis is localized intracellularly throughout the whole duration of feeding. Conclusions Results suggest that the egressing proteolytic system in the early stage of feeding and digestion is a potential target for efficient impairment, most likely by blocking its components via antibodies present in the host blood. Therefore, digestive enzymes are promising candidates for development of novel 'anti-tick' vaccines capable of tick control and even transmission of tick-borne pathogens.

  3. Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.

    Science.gov (United States)

    Nakatsukasa, Kunio; Okumura, Fumihiko; Kamura, Takumi

    2015-01-01

    Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes.

  4. On the formation and nature of quasi-cleavage fracture surfaces in hydrogen embrittled steels

    Energy Technology Data Exchange (ETDEWEB)

    Martin, May L.; Fenske, Jamey A.; Liu, Grace S.; Sofronis, Petros [University of Illinois, Dept. of Materials Science and Engineering, 1304 W. Green St., Urbana, IL 61801 (United States); Robertson, Ian M., E-mail: ianr@illinois.edu [University of Illinois, Dept. of Materials Science and Engineering, 1304 W. Green St., Urbana, IL 61801 (United States)

    2011-02-15

    Quasi-cleavage, a common feature of hydrogen-induced fracture surfaces, is generally taken as being cleavage-like but not along a known cleavage plane. Despite the frequency with which this surface is observed, the relationship to the underlying microstructure remains unknown. Through a combination of topographical reconstruction of secondary electron microscope fractographs and a transmission electron microscopy study of the microstructure from site-specific locations, it will be shown that the features on quasi-cleavage surfaces are ridges that can be correlated with sub-surface intense and highly localized deformation bands. It will be demonstrated that the fracture surface arises from the growth and coalescence of voids that initiate at and extend along slip band intersections. This mechanism and process is fully consistent with hydrogen enhancing and localizing plastic processes.

  5. Changes in Contact Area in Meniscus Horizontal Cleavage Tears Subjected to Repair and Resection.

    Science.gov (United States)

    Beamer, Brandon S; Walley, Kempland C; Okajima, Stephen; Manoukian, Ohan S; Perez-Viloria, Miguel; DeAngelis, Joseph P; Ramappa, Arun J; Nazarian, Ara

    2017-03-01

    To assess the changes in tibiofemoral contact pressure and contact area in human knees with a horizontal cleavage tear before and after treatment. Ten human cadaveric knees were tested. Pressure sensors were placed under the medial meniscus and the knees were loaded at twice the body weight for 20 cycles at 0°, 10°, and 20° of flexion. Contact area and pressure were recorded for the intact meniscus, the meniscus with a horizontal cleavage tear, after meniscal repair, after partial meniscectomy (single leaflet), and after subtotal meniscectomy (double leaflet). The presence of a horizontal cleavage tear significantly increased average peak contact pressure and reduced effective average tibiofemoral contact area at all flexion angles tested compared with the intact state (P contact pressure after creation of the horizontal cleavage tear. Repairing the horizontal cleavage tear restored peak contact pressures and areas to within 15% of baseline, statistically similar to the intact state at all angles tested (P contact pressure and reduced average contact area at all degrees of flexion compared with the intact state (P contact area and a significant elevation in contact pressure. These changes may accelerate joint degeneration. A suture-based repair of these horizontal cleavage tears returns the contact area and contact pressure to nearly normal, whereas both partial and subtotal meniscectomy lead to significant reductions in contact area and significant elevations in contact pressure within the knee. Repairing horizontal cleavage tears may lead to improved clinical outcomes by preserving meniscal tissue and the meniscal function. Understanding contact area and peak contact pressure resulting from differing strategies for treating horizontal cleavage tears will allow the surgeon to evaluate the best strategy for treating his or her patients who present with this meniscal pathology. Copyright © 2016 Arthroscopy Association of North America. Published by Elsevier

  6. Control of extracellular cleavage of ProBDNF by high frequency neuronal activity

    OpenAIRE

    Nagappan, Guhan; Zaitsev, Eugene; Senatorov, Vladimir V.; Yang, Jianmin; Hempstead, Barbara L.; Lu, Bai

    2009-01-01

    Pro- and mature neurotrophins often elicit opposing biological effects. For example, mature brain-derived neurotrophic factor (mBDNF) is critical for long-term potentiation induced by high-frequency stimulation, whereas proBDNF facilitate long-term depression induced by low-frequency stimulation. Because mBDNF is derived from proBDNF by endoproteolytic cleavage, mechanisms regulating the cleavage of proBDNF may control the direction of BDNF regulation. Using methods that selectively detect pr...

  7. Condensed tannins: A novel rearrangement of procyanidins and prodelphinidins in thiolytic cleavage

    Science.gov (United States)

    G. Wayne McGraw; Jan P. Steynberg; Richard W. Hemingway

    1993-01-01

    Conditions commonly used for the thiolytic cleavage of interflavanoid bonds of condensed tannins also result in cleavage of the C4 to C10 bond of flavan units. Subsequenet lectrophilic attack of the C4 carbocation on the C2' or C6' of the B-ring, and loss of phloroglucinol (the A-ring), result in the formation of a mixture of 1,3-dithiobenzyl-2,4,s,6-...

  8. Visualization of proteolytic activity associated with the apoptotic response in cancer cells

    Science.gov (United States)

    Tice, Brian George

    Caspases execute programmed cell death, where low levels of caspase activity are linked to cancer. Chemotherapies utilize induction of apoptosis as a key mechanism for cancer treatment, where caspase-3 is a major player involved in dismantling these aberrant cells. The ability to sensitively measure the initial caspase-3 cleavage events during apoptosis is important for understanding the initiation of this complex cellular process, however, current ensemble methods are not sensitive enough to measure single cleavage events in cells. By utilizing the optical properties of plasmon coupling, peptide-linked gold nanoparticles were developed to enable single molecule imaging of caspase-3 activity in two different cancer systems. Au crown nanoparticles were assembled in a multimeric fashion to overcome the high and heterogeneous background scattering of live cells. In a colon cancer (SW620) cell line challenged with tumor necrosis factor-alpha (TNF-alpha), single molecule trajectories show early stage caspase-3 activation within minutes, which was not detectable by ensemble assays until 23 hours. Variability in caspase-3 activation among the population of cells was identified and likely a result of each cell's specific resistance to death receptor-induced apoptosis. Following these studies, improvements by way of sensitivity and selectivity were tailored into an improved nanosensor construct. Au nanoshell dimers were prepared as a comparably bright construct with 1) reduced heterogeneity compared to the synthesis of the crown nanoparticles and 2) a peptide sequence highly selective for caspase-3. Chronic myeloid leukemia (CML) K562 cells were assessed for their early apoptotic response upon treatment with dasatinib, a clinically approved tyrosine kinase inhibitor that specifically targets BCR-ABL. It has been demonstrated that inhibition of BCR-ABL by dasatinib commits K562 cells to apoptosis. Single molecule experiments with Au nanoshell dimers show caspase-3 activation

  9. Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein.

    Science.gov (United States)

    Welch, Brett D; Liu, Yuanyuan; Kors, Christopher A; Leser, George P; Jardetzky, Theodore S; Lamb, Robert A

    2012-10-09

    The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein.

  10. On the temperature independence of statistical model parameters for cleavage fracture in ferritic steels

    Science.gov (United States)

    Qian, Guian; Lei, Wei-Sheng; Niffenegger, M.; González-Albuixech, V. F.

    2018-04-01

    The work relates to the effect of temperature on the model parameters in local approaches (LAs) to cleavage fracture. According to a recently developed LA model, the physical consensus of plastic deformation being a prerequisite to cleavage fracture enforces any LA model of cleavage fracture to observe initial yielding of a volume element as its threshold stress state to incur cleavage fracture in addition to the conventional practice of confining the fracture process zone within the plastic deformation zone. The physical consistency of the new LA model to the basic LA methodology and the differences between the new LA model and other existing models are interpreted. Then this new LA model is adopted to investigate the temperature dependence of LA model parameters using circumferentially notched round tensile specimens. With the published strength data as input, finite element (FE) calculation is conducted for elastic-perfectly plastic deformation and the realistic elastic-plastic hardening, respectively, to provide stress distributions for model calibration. The calibration results in temperature independent model parameters. This leads to the establishment of a 'master curve' characteristic to synchronise the correlation between the nominal strength and the corresponding cleavage fracture probability at different temperatures. This 'master curve' behaviour is verified by strength data from three different steels, providing a new path to calculate cleavage fracture probability with significantly reduced FE efforts.

  11. Synthesis and DNA cleavage activity of Bis-3-chloropiperidines as alkylating agents.

    Science.gov (United States)

    Zuravka, Ivonne; Roesmann, Rolf; Sosic, Alice; Wende, Wolfgang; Pingoud, Alfred; Gatto, Barbara; Göttlich, Richard

    2014-09-01

    Nitrogen mustards are an important class of bifunctional alkylating agents routinely used in chemotherapy. They react with DNA as electrophiles through the formation of highly reactive aziridinium ion intermediates. The antibiotic 593A, with potential antitumor activity, can be considered a naturally occurring piperidine mustard containing a unique 3-chloropiperidine ring. However, the total synthesis of this antibiotic proved to be rather challenging. With the aim of designing simplified analogues of this natural product, we developed an efficient bidirectional synthetic route to bis-3-chloropiperidines joined by flexible, conformationally restricted, or rigid diamine linkers. The key step involves an iodide-catalyzed double cyclization of unsaturated bis-N-chloroamines to simultaneously generate both piperidine rings. Herein we describe the synthesis and subsequent evaluation of a series of novel nitrogen-bridged bis-3-chloropiperidines, enabling the study of the impact of the linker structure on DNA alkylation properties. Our studies reveal that the synthesized compounds possess DNA alkylating abilities and induce strand cleavage, with a strong preference for guanine residues. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Caspase cleavage of viral proteins, another way for viruses to make the best of apoptosis.

    Science.gov (United States)

    Richard, A; Tulasne, D

    2012-03-08

    Viral infection constitutes an unwanted intrusion that needs to be eradicated by host cells. On one hand, one of the first protective barriers set up to prevent viral replication, spread or persistence involves the induction of apoptotic cell death that aims to limit the availability of the cellular components for viral amplification. On the other hand, while they completely depend on the host molecular machinery, viruses also need to evade the cellular responses that are meant to destroy them. The existence of numerous antiapoptotic products within the viral kingdom proves that apoptosis constitutes a major threat that should better be bypassed. Among the different strategies developed to deal with apoptosis, one is based on what viruses do best: backfiring the cell on itself. Several unrelated viruses have been described to take advantage of apoptosis induction by expressing proteins targeted by caspases, the key effectors of apoptotic cell death. Caspase cleavage of these proteins results in various consequences, from logical apoptosis inhibition to more surprising enhancement or attenuation of viral replication. The present review aims at discussing the characterization and relevance of this post-translational modification that adds a new complexity in the already intricate host-apoptosis-virus triangle.

  13. Cleavage of rRNA ensures translational cessation in sperm at fertilization

    Science.gov (United States)

    Johnson, G.D.; Sendler, E.; Lalancette, C.; Hauser, R.; Diamond, M.P.; Krawetz, S.A.

    2011-01-01

    Intact ribosomal RNAs (rRNAs) comprise the majority of somatic transcripts, yet appear conspicuously absent in spermatozoa, perhaps reflecting cytoplasmic expulsion during spermatogenesis. To discern their fate, total RNA retained in mature spermatozoa from three fertile donors was characterized by Next Generation Sequencing. In all samples, >75% of total sequence reads aligned to rRNAs. The distribution of reads along the length of these transcripts exhibited a high degree of non-uniformity that was reiterated between donors. The coverage of sequencing reads was inversely correlated with guanine-cytosine (GC)-richness such that sequences greater than ∼70% GC were virtually absent in all sperm RNA samples. To confirm the loss of sequence, the relative abundance of specific regions of the 28S transcripts in sperm was established by 7-Deaza-2′-deoxy-guanosine-5′-triphosphate RT–PCR. The inability to amplify specific regions of the 28S sequence from sperm despite the abundant representation of this transcript in the sequencing libraries demonstrates that approximately three-quarters of RNA retained in the mature male gamete are products of rRNA fragmentation. Hence, cleavage (not expulsion of the RNA component of the translational machinery) is responsible for preventing spurious translation following spermiogenesis. These results highlight the potential importance of those transcripts, including many mRNAs, which evade fragmentation and remain intact when sperm are delivered at fertilization. Sequencing data are deposited in GEO as: GSE29160. PMID:21831882

  14. Anodic carbon-boron bond cleavage through the intermediacy of electrogenerated bromonium ion

    International Nuclear Information System (INIS)

    Shi Deqing; Gitkis, Anna; Becker, James Y.

    2007-01-01

    The electrochemical properties of a series of cyclic arylboronic esters, XC 6 H 4 B(OR) 2 [RR = CH 2 CH 2 ; X = H (1a); p-Me (1b); p-OMe (1c); p-Cl (1d); p-Ph (1e); m-Cl (1f); m-OMe (1g); CF 3 (1h); OMe (1i); 2,6-dimethyl (1j); 1b with RR = (CH 2 ) 3 , (1k); 1b with RR = CMe 2 CMe 2 , (1m)] has been studied in acetonitrile by cyclic voltammetry (CV) and controlled-potential electrolysis (CPE). The CV of representative examples of aryl borates with different substituents show one irreversible oxidation wave on a Pt cathode, at 1.8-1.9 V (vs. Ag/AgCl), with a negligible substituent effect. The cathodic CPE process led to small amounts of biaryls only, whereas the direct anodic CPE could not be carried out practically due to low currents. However, in the presence of electrogenerated bromonium (or iodonium) ions a C-B bond cleavage does take place to yield the corresponding bromoaryls, brominated phenols, and arylboronic acids as the major products

  15. Anodic carbon-boron bond cleavage through the intermediacy of electrogenerated bromonium ion

    Energy Technology Data Exchange (ETDEWEB)

    Shi Deqing; Gitkis, Anna [Department of Chemistry, Ben-Gurion University of the Negev, Beer Sheva 84105 (Israel); Becker, James Y. [Department of Chemistry, Ben-Gurion University of the Negev, Beer Sheva 84105 (Israel)], E-mail: becker@bgu.ac.il

    2007-12-31

    The electrochemical properties of a series of cyclic arylboronic esters, XC{sub 6}H{sub 4}B(OR){sub 2} [RR = CH{sub 2}CH{sub 2}; X = H (1a); p-Me (1b); p-OMe (1c); p-Cl (1d); p-Ph (1e); m-Cl (1f); m-OMe (1g); CF{sub 3} (1h); OMe (1i); 2,6-dimethyl (1j); 1b with RR = (CH{sub 2}){sub 3}, (1k); 1b with RR = CMe{sub 2}CMe{sub 2}, (1m)] has been studied in acetonitrile by cyclic voltammetry (CV) and controlled-potential electrolysis (CPE). The CV of representative examples of aryl borates with different substituents show one irreversible oxidation wave on a Pt cathode, at 1.8-1.9 V (vs. Ag/AgCl), with a negligible substituent effect. The cathodic CPE process led to small amounts of biaryls only, whereas the direct anodic CPE could not be carried out practically due to low currents. However, in the presence of electrogenerated bromonium (or iodonium) ions a C-B bond cleavage does take place to yield the corresponding bromoaryls, brominated phenols, and arylboronic acids as the major products.

  16. Social economy partnerships and the public/private cleavages

    Directory of Open Access Journals (Sweden)

    Joxerramon Bengoetxea

    2012-06-01

    Full Text Available Public/Private Partnerships can be seen as one particular topos where the divide between the public domain, all levels of the Public Administration and the private initiative and private property is turned into a joint venture rather than a confrontation or a cleavage. Some of the possible combinations of public and private and where public/private partnerships might fit are displayed analytically. The importance of political theory or ideology in conceiving the relationships between ‘public’ and ‘private’, and the conceptions of a market economy as opposed to a social market economy cannot be exaggerated enough, but equally important are the legal or regulatory framework and the underlying dominant legal culture and legal principles, and of course the economic and financial situation. Public/private partnerships thrive in some conditions, but seem to wane in others, and the current predicament is not favourable, taking into account that only the regulatory framework is supportive of these ventures. Los partenariados público-privados se pueden entender como un espacio particular, en el que el sector público, todos los niveles de la administración pública, y la iniciativa privada y la propiedad privada, abordan una empresa conjunta, en lugar mantener posturas contrapuestas. Se muestran algunas de las posibles combinaciones del sector público y privado, en las que tendrían cabida los partenariados público/privados. Es patente la importancia de la teoría o la ideología política para entender las relaciones entre lo público y lo privado, y las concepciones de una economía de mercado frente a una economía social, pero tampoco se puede negar la importancia del marco legal o reglamentario y la cultura jurídica dominante subyacente, y los principios jurídicos, sin olvidar la situación económica y financiera. Los partenariados público-privados prosperan en algunas condiciones, pero no lo hacen siempre, y la situación econ

  17. RISC-interacting clearing 3'- 5' exoribonucleases (RICEs) degrade uridylated cleavage fragments to maintain functional RISC in Arabidopsis thaliana.

    Science.gov (United States)

    Zhang, Zhonghui; Hu, Fuqu; Sung, Min Woo; Shu, Chang; Castillo-González, Claudia; Koiwa, Hisashi; Tang, Guiliang; Dickman, Martin; Li, Pingwei; Zhang, Xiuren

    2017-05-02

    RNA-induced silencing complex (RISC) is composed of miRNAs and AGO proteins. AGOs use miRNAs as guides to slice target mRNAs to produce truncated 5' and 3' RNA fragments. The 5' cleaved RNA fragments are marked with uridylation for degradation. Here, we identified novel cofactors of Arabidopsis AGOs, named RICE1 and RICE2. RICE proteins specifically degraded single-strand (ss) RNAs in vitro; but neither miRNAs nor miRNA*s in vivo. RICE1 exhibited a DnaQ-like exonuclease fold and formed a homohexamer with the active sites located at the interfaces between RICE1 subunits. Notably, ectopic expression of catalytically-inactive RICE1 not only significantly reduced miRNA levels; but also increased 5' cleavage RISC fragments with extended uridine tails. We conclude that RICEs act to degrade uridylated 5' products of AGO cleavage to maintain functional RISC. Our study also suggests a possible link between decay of cleaved target mRNAs and miRNA stability in RISC.

  18. Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells.

    Science.gov (United States)

    Mandal, Manoj K; Fischer, Rainer; Schillberg, Stefan; Schiermeyer, Andreas

    2014-08-01

    Recombinant proteins produced in plant suspension cultures are often degraded by endogenous plant proteases when secreted into the medium, resulting in low yields. To generate protease-deficient tobacco BY-2 cell lines and to retrieve the sequence information, we cloned four different protease cDNAs from tobacco BY-2 cells (NtAP, NtCP, NtMMP1, and NtSP), which represent the major catalytic classes. The simultaneous expression of antisense RNAs against these endogenous proteases led to the establishment of cell lines with reduced levels of endogenous protease expression and activity at late stages of the cultivation cycle. One of the cell lines showing reduced proteolytic activity in the culture medium was selected for the expression of the recombinant full-length IgG1(κ) antibody 2F5, recognizing the gp41 surface protein of HIV-1. This cell line showed significantly reduced degradation of the 2F5 heavy chain, resulting in four-fold higher accumulation of the intact antibody heavy chain when compared to transformed wild type cells expressing the same antibody. N-terminal sequencing data revealed that the antibody has two cleavage sites within the CDR-H3 and one site at the end of the H4-framework region. These cleavage sites are found to be vulnerable to serine proteases. The data provide a basis for further improvement of plant cells for the production of recombinant proteins in plant cell suspension cultures. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Molecular mechanism of the intramembrane cleavage of the β-carboxyl terminal fragment of amyloid precursor protein by γ-secretase

    Directory of Open Access Journals (Sweden)

    Maho eMorishima-Kawashima

    2014-11-01

    Full Text Available Amyloid β-protein (Aβ plays a central role in the pathogenesis of Alzheimer’s disease, the most common age-associated neurodegenerative disorder. Aβ is generated through intramembrane proteolysis of the β-carboxyl terminal fragment (βCTF of β-amyloid precursor protein (APP by γ-secretase. The initial cleavage by γ-secretase occurs in the membrane/cytoplasm boundary of the βCTF, liberating the APP intracellular domain (AICD. The remaining βCTFs, which are truncated at the C-terminus (longer Aβs, are then cropped sequentially in a stepwise manner, predominantly at three residue intervals, to generate Aβ. There are two major Aβ product lines which generate Aβ40 and Aβ42 with concomitant release of three and two tripeptides, respectively. Additionally, many alternative cleavages occur, releasing peptides with three to six residues. These modulate the Aβ product lines and define the species and quantity of Aβ generated. Here, we review our current understanding of the intramembrane cleavage of the βCTF by γ-secretase, which may contribute to the future goal of developing an efficient therapeutic strategy for Alzheimer’s disease.

  20. Tomato carotenoid cleavage dioxygenases 1A and 1B: Relaxed double bond specificity leads to a plenitude of dialdehydes, mono-apocarotenoids and isoprenoid volatiles

    KAUST Repository

    Ilg, Andrea; Bruno, Mark; Beyer, Peter; Al-Babili, Salim

    2014-01-01

    The biosynthetic processes leading to many of the isoprenoid volatiles released by tomato fruits are still unknown, though previous reports suggested a clear correlation with the carotenoids contained within the fruit. In this study, we investigated the activity of the tomato (Solanum lycopersicum) carotenoid cleavage dioxygenase (SlCCD1B), which is highly expressed in fruits, and of its homolog SlCCD1A. Using in vitro assays performed with purified recombinant enzymes and by analyzing products formed by the two enzymes in carotene-accumulating Escherichia coli strains, we demonstrate that SlCCD1A and, to a larger extent, SlCCD1B, have a very relaxed specificity for both substrate and cleavage site, mediating the oxidative cleavage of cis- and all-. trans-carotenoids as well as of different apocarotenoids at many more double bonds than previously reported. This activity gives rise to a plenitude of volatiles, mono-apocarotenoids and dialdehyde products, including cis-pseudoionone, neral, geranial, and farnesylacetone. Our results provide a direct evidence for a carotenoid origin of these compounds and point to CCD1s as the enzymes catalyzing the formation of the vast majority of tomato isoprenoid volatiles, many of which are aroma constituents. © 2014 The Authors.

  1. Tomato carotenoid cleavage dioxygenases 1A and 1B: Relaxed double bond specificity leads to a plenitude of dialdehydes, mono-apocarotenoids and isoprenoid volatiles

    KAUST Repository

    Ilg, Andrea

    2014-06-25

    The biosynthetic processes leading to many of the isoprenoid volatiles released by tomato fruits are still unknown, though previous reports suggested a clear correlation with the carotenoids contained within the fruit. In this study, we investigated the activity of the tomato (Solanum lycopersicum) carotenoid cleavage dioxygenase (SlCCD1B), which is highly expressed in fruits, and of its homolog SlCCD1A. Using in vitro assays performed with purified recombinant enzymes and by analyzing products formed by the two enzymes in carotene-accumulating Escherichia coli strains, we demonstrate that SlCCD1A and, to a larger extent, SlCCD1B, have a very relaxed specificity for both substrate and cleavage site, mediating the oxidative cleavage of cis- and all-. trans-carotenoids as well as of different apocarotenoids at many more double bonds than previously reported. This activity gives rise to a plenitude of volatiles, mono-apocarotenoids and dialdehyde products, including cis-pseudoionone, neral, geranial, and farnesylacetone. Our results provide a direct evidence for a carotenoid origin of these compounds and point to CCD1s as the enzymes catalyzing the formation of the vast majority of tomato isoprenoid volatiles, many of which are aroma constituents. © 2014 The Authors.

  2. Tomato carotenoid cleavage dioxygenases 1A and 1B: Relaxed double bond specificity leads to a plenitude of dialdehydes, mono-apocarotenoids and isoprenoid volatiles

    Directory of Open Access Journals (Sweden)

    Andrea Ilg

    2014-01-01

    Full Text Available The biosynthetic processes leading to many of the isoprenoid volatiles released by tomato fruits are still unknown, though previous reports suggested a clear correlation with the carotenoids contained within the fruit. In this study, we investigated the activity of the tomato (Solanum lycopersicum carotenoid cleavage dioxygenase (SlCCD1B, which is highly expressed in fruits, and of its homolog SlCCD1A. Using in vitro assays performed with purified recombinant enzymes and by analyzing products formed by the two enzymes in carotene-accumulating Escherichia coli strains, we demonstrate that SlCCD1A and, to a larger extent, SlCCD1B, have a very relaxed specificity for both substrate and cleavage site, mediating the oxidative cleavage of cis- and all-trans-carotenoids as well as of different apocarotenoids at many more double bonds than previously reported. This activity gives rise to a plenitude of volatiles, mono-apocarotenoids and dialdehyde products, including cis-pseudoionone, neral, geranial, and farnesylacetone. Our results provide a direct evidence for a carotenoid origin of these compounds and point to CCD1s as the enzymes catalyzing the formation of the vast majority of tomato isoprenoid volatiles, many of which are aroma constituents.

  3. Quantitative characterization of cleavage and hydrogen-assisted quasi-cleavage fracture surfaces with the use of confocal laser scanning microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Merson, E. [Institute of Advanced Technologies, Togliatti State University, 445667 (Russian Federation); Kudrya, A.V.; Trachenko, V.A. [Department of Physical Metallurgy and the Physics of Strength, NUST MISiS, Moscow 119490 (Russian Federation); Merson, D. [Institute of Advanced Technologies, Togliatti State University, 445667 (Russian Federation); Laboratory for Advanced Materials, Kazan Federal University, Naberezhnye Chelny 423812, Republic of Tatarstan (Russian Federation); Danilov, V. [Institute of Advanced Technologies, Togliatti State University, 445667 (Russian Federation); Vinogradov, A. [Institute of Advanced Technologies, Togliatti State University, 445667 (Russian Federation); Department of Engineering Design and Materials, Norwegian University of Science and Technology – NTNU, N-7491 Trondheim (Norway)

    2016-05-17

    “True” cleavage (TC) and quasi-cleavage (QC) fracture surfaces of low-carbon steel specimens tested in liquid nitrogen and after hydrogen charging respectively were investigated by quantitative confocal laser scanning microscopy (CLSM) and conventional scanning electron microscopy (SEM) with electron-backscattered diffraction (EBSD). Topological and crystallographic features of the TC fracture surface are found in good agreement with the generally accepted cleavage mechanism: TC facets diameters correspond to those of grains; the crack path strictly follows the crystallographic orientation of grains and the most of the cleavage cracks are parallel to {100} planes. On the 2D SEM images, the QC facets appeared resembling the TC ones in terms of river line patterns, shapes and sizes. However, the substantial differences between the topography of these two kinds of fracture surfaces were revealed by 3D CLSM: the average misorientation angle between QC facets and the roughness of the QC fracture surface were much lower than those measured for TC. It is demonstrated that all these features are attributed to the specific fracture mechanism operating during hydrogen-assisted cracking.

  4. Quantitative characterization of cleavage and hydrogen-assisted quasi-cleavage fracture surfaces with the use of confocal laser scanning microscopy

    International Nuclear Information System (INIS)

    Merson, E.; Kudrya, A.V.; Trachenko, V.A.; Merson, D.; Danilov, V.; Vinogradov, A.

    2016-01-01

    “True” cleavage (TC) and quasi-cleavage (QC) fracture surfaces of low-carbon steel specimens tested in liquid nitrogen and after hydrogen charging respectively were investigated by quantitative confocal laser scanning microscopy (CLSM) and conventional scanning electron microscopy (SEM) with electron-backscattered diffraction (EBSD). Topological and crystallographic features of the TC fracture surface are found in good agreement with the generally accepted cleavage mechanism: TC facets diameters correspond to those of grains; the crack path strictly follows the crystallographic orientation of grains and the most of the cleavage cracks are parallel to {100} planes. On the 2D SEM images, the QC facets appeared resembling the TC ones in terms of river line patterns, shapes and sizes. However, the substantial differences between the topography of these two kinds of fracture surfaces were revealed by 3D CLSM: the average misorientation angle between QC facets and the roughness of the QC fracture surface were much lower than those measured for TC. It is demonstrated that all these features are attributed to the specific fracture mechanism operating during hydrogen-assisted cracking.

  5. Phenylpropanoid 2,3-dioxygenase involved in the cleavage of the ferulic acid side chain to form vanillin and glyoxylic acid in Vanilla planifolia.

    Science.gov (United States)

    Negishi, Osamu; Negishi, Yukiko

    2017-09-01

    Enzyme catalyzing the cleavage of the phenylpropanoid side chain was partially purified by ion exchange and gel filtration column chromatography after (NH 4 ) 2 SO 4 precipitation. Enzyme activities were dependent on the concentration of dithiothreitol (DTT) or glutathione (GSH) and activated by addition of 0.5 mM Fe 2+ . Enzyme activity for ferulic acid was as high as for 4-coumaric acid in the presence of GSH, suggesting that GSH acts as an endogenous reductant in vanillin biosynthesis. Analyses of the enzymatic reaction products with quantitative NMR (qNMR) indicated that an amount of glyoxylic acid (GA) proportional to vanillin was released from ferulic acid by the enzymatic reaction. These results suggest that phenylpropanoid 2,3-dioxygenase is involved in the cleavage of the ferulic acid side chain to form vanillin and GA in Vanilla planifolia.

  6. Pyrexia's effect on the CBG-cortisol thermocouple, rather than CBG cleavage, elevates the acute free cortisol response to TNF-α in humans

    DEFF Research Database (Denmark)

    Nenke, Marni Anne; Nielsen, Signe Tellerup; Lehrskov, Louise Lang

    2017-01-01

    an inflammatory stimulus is unknown. Hence our aim was to determine the immediate effect of the key pro-inflammatory cytokine TNF-α on CBG levels and cleavage. We performed a crossover study of 12 healthy males receiving a TNF-α versus saline infusion, measuring total CBG, haCBG, laCBG and free and total cortisol...... hourly for 6 h. There was no change in total CBG or haCBG levels in the first 6 h of inflammation between the groups, suggesting that CBG cleavage is not activated nor is hepatic CBG production affected by TNF-α in this time frame. There was an early increase in the ratio of free:total cortisol...

  7. HIV-1 incorporates and proteolytically processes human NDR1 and NDR2 serine-threonine kinases

    International Nuclear Information System (INIS)

    Devroe, Eric; Silver, Pamela A.; Engelman, Alan

    2005-01-01

    Mammalian genomes encode two related serine-threonine kinases, nuclear Dbf2 related (NDR)1 and NDR2, which are homologous to the Saccharomyces cerevisiae Dbf2 kinase. Recently, a yeast genetic screen implicated the Dbf2 kinase in Ty1 retrotransposition. Since several virion-incorporated kinases regulate the infectivity of human immunodeficiency virus type 1 (HIV-1), we speculated that the human NDR1 and NDR2 kinases might play a role in the HIV-1 life cycle. Here we show that the NDR1 and NDR2 kinases were incorporated into HIV-1 particles. Furthermore, NDR1 and NDR2 were cleaved by the HIV-1 protease (PR), both within virions and within producer cells. Truncation at the PR cleavage site altered NDR2 subcellular localization and inhibited NDR1 and NDR2 enzymatic activity. These studies identify two new virion-associated host cell enzymes and suggest a novel mechanism by which HIV-1 alters the intracellular environment of human cells

  8. PAPP-A proteolytic activity enhances IGF bioactivity in ascites from women with ovarian carcinoma

    Science.gov (United States)

    Thomsen, Jacob; Hjortebjerg, Rikke; Espelund, Ulrick; Ørtoft, Gitte; Vestergaard, Poul; Magnusson, Nils E.; Conover, Cheryl A.; Tramm, Trine; Hager, Henrik; Høgdall, Claus; Høgdall, Estrid; Oxvig, Claus; Frystyk, Jan

    2015-01-01

    Pregnancy-associated plasma protein-A (PAPP-A) stimulates insulin-like growth factor (IGF) action through proteolysis of IGF-binding protein (IGFBP)-4. In experimental animals, PAPP-A accelerates ovarian tumor growth by this mechanism. To investigate the effect of PAPP-A in humans, we compared serum and ascites from 22 women with ovarian carcinoma. We found that ascites contained 46-fold higher PAPP-A levels as compared to serum (P IGF-I receptor (IGF-IR) in vitro (+31%, P IGF-I, and lower levels of IGF-II (P IGF-IR in all but one tumor, whereas all tumors expressed PAPP-A, IGFBP-4, IGF-I and IGF-II. Addition of recombinant PAPP-A to ascites increased the cleavage of IGFBP-4 and enhanced IGF-IR activation (P IGFs and these proteins are also present in ascites. We suggest that both soluble PAPP-A in ascites and tissue-associated PAPP-A serve to increase IGF bioactivity and, thereby, to stimulate IGF-IR-mediated tumor growth. PMID:26336825

  9. Characterization of Goat Milk Hydrolyzed by Cell Envelope Proteinases from Lactobacillus plantarum LP69: Proteolytic System Optimization, Bioactivity, and Storage Stability Evaluation

    Directory of Open Access Journals (Sweden)

    Guowei Shu

    2018-05-01

    Full Text Available Despite the widespread application of lactic acid bacterium in dairy production through its contribution to acidification, development of sensorial properties, and health-promoting effects, relatively little information is available on the cell envelope proteinases (CEPs of Lactobacillus plantarum, especially on the proteolytic system and the production of bioactivity peptides. In this study, CEPs from a novel L. plantarum LP69 were involved in goat milk hydrolysis and generated a product with high activity that showed a degree of hydrolysis of 15.68 ± 0.74%, Angiotensin I-Converting Enzyme (ACE-inhibitory rate of 83.25 ± 1.05%, 2,2-diphenyl-1-picrylhydrazyl (DPPH radical scavenging rate of 64.91 ± 1.27%, and hydroxyl radical scavenging rate of 89.17 ± 1.13%. The optimized hydrolysis conditions were time of 4.5 h, temperature of 41 °C, initial pH of 8.5, and enzyme to substrate ratio (E/S of 12% (w/w by orthogonal experiments. Application of a stabilizer greatly promoted milk stability. A well-designed stabilizer consists of 0.05% carrageenan, 0.15% gellan gum, and 0.15% sucrose esters, which significantly raised the milk stability coefficient, R, from 70.67% to 98.57%. The storage stability of milk was evaluated during 84 days at room temperature or 4 °C. Our study depicts the contribution of CEPs from L. plantarum LP69 in goat milk, exploring a new way for the development of a functional milk product.

  10. Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells

    International Nuclear Information System (INIS)

    Amacher, S.L.; Goodman, L.J.; Bravo, D.A.; Wong, K.Y.; Goldfine, I.D.; Hawley, D.M.; Firestone, G.L.

    1989-01-01

    Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by [125I] insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways

  11. Coronavirus and influenza virus proteolytic priming takes place in tetraspanin-enriched membrane microdomains.

    Science.gov (United States)

    Earnest, James T; Hantak, Michael P; Park, Jung-Eun; Gallagher, Tom

    2015-06-01

    Coronaviruses (CoVs) and low-pathogenicity influenza A viruses (LP IAVs) depend on target cell proteases to cleave their viral glycoproteins and prime them for virus-cell membrane fusion. Several proteases cluster into tetraspanin-enriched microdomains (TEMs), suggesting that TEMs are preferred virus entry portals. Here we found that several CoV receptors and virus-priming proteases were indeed present in TEMs. Isolated TEMs, when mixed with CoV and LP IAV pseudoparticles, cleaved viral fusion proteins to fusion-primed fragments and potentiated viral transductions. That entering viruses utilize TEMs as a protease source was further confirmed using tetraspanin antibodies and tetraspanin short hairpin RNAs (shRNAs). Tetraspanin antibodies inhibited CoV and LP IAV infections, but their virus-blocking activities were overcome by expressing excess TEM-associated proteases. Similarly, cells with reduced levels of the tetraspanin CD9 resisted CoV pseudoparticle transductions but were made susceptible by overproducing TEM-associated proteases. These findings indicated that antibodies and CD9 depletions interfere with viral proteolytic priming in ways that are overcome by surplus proteases. TEMs appear to be exploited by some CoVs and LP IAVs for appropriate coengagement with cell receptors and proteases. Enveloped viruses use their surface glycoproteins to catalyze membrane fusion, an essential cell entry step. Host cell components prime these viral surface glycoproteins to catalyze membrane fusion at specific times and places during virus cell entry. Among these priming components are proteases, which cleave viral surface glycoproteins, unleashing them to refold in ways that catalyze virus-cell membrane fusions. For some enveloped viruses, these proteases are known to reside on target cell surfaces. This research focuses on coronavirus and influenza A virus cell entry and identifies TEMs as sites of viral proteolysis, thereby defining subcellular locations of virus

  12. Yeast ribonuclease III uses a network of multiple hydrogen bonds for RNA binding and cleavage.

    Science.gov (United States)

    Lavoie, Mathieu; Abou Elela, Sherif

    2008-08-19

    Members of the bacterial RNase III family recognize a variety of short structured RNAs with few common features. It is not clear how this group of enzymes supports high cleavage fidelity while maintaining a broad base of substrates. Here we show that the yeast orthologue of RNase III (Rnt1p) uses a network of 2'-OH-dependent interactions to recognize substrates with different structures. We designed a series of bipartite substrates permitting the distinction between binding and cleavage defects. Each substrate was engineered to carry a single or multiple 2'- O-methyl or 2'-fluoro ribonucleotide substitutions to prevent the formation of hydrogen bonds with a specific nucleotide or group of nucleotides. Interestingly, introduction of 2'- O-methyl ribonucleotides near the cleavage site increased the rate of catalysis, indicating that 2'-OH are not required for cleavage. Substitution of nucleotides in known Rnt1p binding site with 2'- O-methyl ribonucleotides inhibited cleavage while single 2'-fluoro ribonucleotide substitutions did not. This indicates that while no single 2'-OH is essential for Rnt1p cleavage, small changes in the substrate structure are not tolerated. Strikingly, several nucleotide substitutions greatly increased the substrate dissociation constant with little or no effect on the Michaelis-Menten constant or rate of catalysis. Together, the results indicate that Rnt1p uses a network of nucleotide interactions to identify its substrate and support two distinct modes of binding. One mode is primarily mediated by the dsRNA binding domain and leads to the formation of stable RNA/protein complex, while the other requires the presence of the nuclease and N-terminal domains and leads to RNA cleavage.

  13. Controllable synthesis of silver and silver sulfide nanocrystals via selective cleavage of chemical bonds

    International Nuclear Information System (INIS)

    Tang Aiwei; Wang Yu; Ye Haihang; Zhou Chao; Yang Chunhe; Li Xu; Peng Hongshang; Zhang Fujun; Hou Yanbing; Teng Feng

    2013-01-01

    A one-step colloidal process has been adopted to prepare silver (Ag) and silver sulfide (Ag 2 S) nanocrystals, thus avoiding presynthesis of an organometallic precursor and the injection of a toxic phosphine agent. During the reaction, a layered intermediate compound is first formed, which then acts as a precursor, decomposing into the nanocrystals. The composition of the as-obtained products can be controlled by selective cleavage of S–C bonds or Ag–S bonds. Pure Ag 2 S nanocrystals can be obtained by directly heating silver acetate (Ag(OAc)) and n-dodecanethiol (DDT) at 200 ° C without any surfactant, and pure Ag nanocrystals can be synthesized successfully if the reaction temperature is reduced to 190 ° C and the amount of DDT is decreased to 1 ml in the presence of a non-coordinating organic solvent (1-octadecene, ODE). Otherwise, the mixture of Ag and Ag 2 S is obtained by directly heating Ag(OAc) in DDT by increasing the reaction temperature or in a mixture of DDT and ODE at 200 ° C. The formation mechanism has been discussed in detail in terms of selective S–C and Ag–S bond dissociation due to the nucleophilic attack of DDT and the lower bonding energy of Ag–S. Interestingly, some products can easily self-assemble into two- or three-dimensional (2D or 3D) highly ordered superlattice structures on a copper grid without any additional steps. The excess DDT plays a key role in the superlattice structure due to the bundling and interdigitation of the thiolate molecules adsorbed on the as-obtained nanocrystals. (paper)

  14. Triphenylphosphonium Moiety Modulates Proteolytic Stability and Potentiates Neuroprotective Activity of Antioxidant Tetrapeptides in Vitro

    Directory of Open Access Journals (Sweden)

    Rezeda A. Akhmadishina

    2018-02-01

    Full Text Available Although delocalized lipophilic cations have been identified as effective cellular and mitochondrial carriers for a range of natural and synthetic drug molecules, little is known about their effects on pharmacological properties of peptides. The effect of triphenylphosphonium (TPP cation on bioactivity of antioxidant tetrapeptides based on the model opioid YRFK motif was studied. Two tetrapeptide variants with L-arginine (YRFK and D-arginine (YrFK were synthesized and coupled with carboxyethyl-TPP (TPP-3 and carboxypentyl-TPP (TPP-6 units. The TPP moiety noticeably promoted YRFK cleavage by trypsin, but effectively prevented digestion of more resistant YrFK attributed, respectively, to structure-organizing and shielding effects of the TPP cation on conformational variants of the tetrapeptide motif. The TPP moiety enhanced radical scavenging activity of the modified YRFK in a model Fenton-like reaction, whereas decreased reactivity was revealed for both YrFK and its TPP derivative. The starting motifs and modified oligopeptides, especially the TPP-6 derivatives, suppressed acute oxidative stress in neuronal PC-12 cells during a brief exposure similarly with glutathione. The effect of oligopeptides was compared upon culturing of PC-12 cells with CoCl2, L-glutamic acid, or menadione to mimic physiologically relevant oxidative states. The cytoprotective activity of oligopeptides significantly depended on the type of oxidative factor, order of treatment and peptide structure. Pronounced cell-protective effect was established for the TPP-modified oligopeptides, which surpassed that of the unmodified motifs. The protease-resistant TPP-modified YrFK showed the highest activity when administered 24 h prior to the cell damage. Our results suggest that the TPP cation can be used as a modifier for small therapeutic peptides to improve their pharmacokinetic and pharmacological properties.

  15. Protein cleavage strategies for an improved analysis of the membrane proteome

    Directory of Open Access Journals (Sweden)

    Poetsch Ansgar

    2006-03-01

    Full Text Available Abstract Background Membrane proteins still remain elusive in proteomic studies. This is in part due to the distribution of the amino acids lysine and arginine, which are less frequent in integral membrane proteins and almost absent in transmembrane helices. As these amino acids are cleavage targets for the commonly used protease trypsin, alternative cleavage conditions, which should improve membrane protein analysis, were tested by in silico digestion for the three organisms Saccharomyces cerevisiae, Halobacterium sp. NRC-1, and Corynebacterium glutamicum as hallmarks for eukaryotes, archea and eubacteria. Results For the membrane proteomes from all three analyzed organisms, we identified cleavage conditions that achieve better sequence and proteome coverage than trypsin. Greater improvement was obtained for bacteria than for yeast, which was attributed to differences in protein size and GRAVY. It was demonstrated for bacteriorhodopsin that the in silico predictions agree well with the experimental observations. Conclusion For all three examined organisms, it was found that a combination of chymotrypsin and staphylococcal peptidase I gave significantly better results than trypsin. As some of the improved cleavage conditions are not more elaborate than trypsin digestion and have been proven useful in practice, we suppose that the cleavage at both hydrophilic and hydrophobic amino acids should facilitate in general the analysis of membrane proteins for all organisms.

  16. Cleavage and Cell Adhesion Properties of Human Epithelial Cell Adhesion Molecule (HEPCAM)*

    Science.gov (United States)

    Tsaktanis, Thanos; Kremling, Heidi; Pavšič, Miha; von Stackelberg, Ricarda; Mack, Brigitte; Fukumori, Akio; Steiner, Harald; Vielmuth, Franziska; Spindler, Volker; Huang, Zhe; Jakubowski, Jasmine; Stoecklein, Nikolas H.; Luxenburger, Elke; Lauber, Kirsten; Lenarčič, Brigita; Gires, Olivier

    2015-01-01

    Human epithelial cell adhesion molecule (HEPCAM) is a tumor-associated antigen frequently expressed in carcinomas, which promotes proliferation after regulated intramembrane proteolysis. Here, we describe extracellular shedding of HEPCAM at two α-sites through a disintegrin and metalloprotease (ADAM) and at one β-site through BACE1. Transmembrane cleavage by γ-secretase occurs at three γ-sites to generate extracellular Aβ-like fragments and at two ϵ-sites to release human EPCAM intracellular domain HEPICD, which is efficiently degraded by the proteasome. Mapping of cleavage sites onto three-dimensional structures of HEPEX cis-dimer predicted conditional availability of α- and β-sites. Endocytosis of HEPCAM warrants acidification in cytoplasmic vesicles to dissociate protein cis-dimers required for cleavage by BACE1 at low pH values. Intramembrane cleavage sites are accessible and not part of the structurally important transmembrane helix dimer crossing region. Surprisingly, neither chemical inhibition of cleavage nor cellular knock-out of HEPCAM using CRISPR-Cas9 technology impacted the adhesion of carcinoma cell lines. Hence, a direct function of HEPCAM as an adhesion molecule in carcinoma cells is not supported and appears to be questionable. PMID:26292218

  17. Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis*

    Science.gov (United States)

    Leroy, Catherine; Belkina, Natalya V.; Long, Thavy; Deruy, Emeric; Dissous, Colette; Shaw, Stephen; Tulasne, David

    2016-01-01

    The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK−/− and LOK+/− lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments. PMID:26945071

  18. Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis.

    Science.gov (United States)

    Leroy, Catherine; Belkina, Natalya V; Long, Thavy; Deruy, Emeric; Dissous, Colette; Shaw, Stephen; Tulasne, David

    2016-05-06

    The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK(-/-) and LOK(+/-) lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Fetal hemoglobin is much less prone to DNA cleavage compared to the adult protein

    Directory of Open Access Journals (Sweden)

    Sandeep Chakane

    2017-08-01

    Full Text Available Hemoglobin (Hb is well protected inside the red blood cells (RBCs. Upon hemolysis and when free in circulation, Hb can be involved in a range of radical generating reactions and may thereby attack several different biomolecules. In this study, we have examined the potential damaging effects of cell-free Hb on plasmid DNA (pDNA. Hb induced cleavage of supercoiled pDNA (sc pDNA which was proportional to the concentration of Hb applied. Almost 70% of sc pDNA was converted to open circular or linear DNA using 10 µM of Hb in 12 h. Hb can be present in several different forms. The oxy (HbO2 and met forms are most reactive, while the carboxy-protein shows only low hydrolytic activity. Hemoglobin A (HbA could easily induce complete pDNA cleavage while fetal hemoglobin (HbF was three-fold less reactive. By inserting, a redox active cysteine residue on the surface of the alpha chain of HbF by site-directed mutagenesis, the DNA cleavage reaction was enhanced by 82%. Reactive oxygen species were not directly involved in the reaction since addition of superoxide dismutase and catalase did not prevent pDNA cleavage. The reactivity of Hb with pDNA can rather be associated with the formation of protein based radicals. Keywords: Adult hemoglobin, Fetal hemoglobin, Supercoiled plasmid DNA, DNA cleavage, Cysteine, Protein radicals

  20. The use of Fluorescence Resonance Energy Transfer (FRET peptidesfor measurement of clinically important proteolytic enzymes

    Directory of Open Access Journals (Sweden)

    Adriana K. Carmona

    2009-09-01

    Full Text Available Proteolytic enzymes have a fundamental role in many biological processes and are associated with multiple pathological conditions. Therefore, targeting these enzymes may be important for a better understanding of their function and development of therapeutic inhibitors. Fluorescence Resonance Energy Transfer (FRET peptides are convenient tools for the study of peptidases specificity as they allow monitoring of the reaction on a continuous basis, providing a rapid method for the determination of enzymatic activity. Hydrolysis of a peptide bond between the donor/acceptor pair generates fluorescence that permits the measurement of the activity of nanomolar concentrations of the enzyme. The assays can be performed directly in a cuvette of the fluorimeter or adapted for determinations in a 96-well fluorescence plate reader. The synthesis of FRET peptides containing ortho-aminobenzoic acid (Abz as fluorescent group and 2, 4-dinitrophenyl (Dnp or N-(2, 4-dinitrophenylethylenediamine (EDDnp as quencher was optimized by our group and became an important line of research at the Department of Biophysics of the Federal University of São Paulo. Recently, Abz/Dnp FRET peptide libraries were developed allowing high-throughput screening of peptidases substrate specificity. This review presents the consolidation of our research activities undertaken between 1993 and 2008 on the synthesis of peptides and study of peptidases specificities.As enzimas proteolíticas têm um papel fundamental em muitos processos biológicos e estão associadas a vários estados patológicos. Por isso, o estudo da especificidade das peptidases pode ser importante para uma melhor compreensão da função destas enzimas e para o desenvolvimento de inibidores. Os substratos com supressão intramolecular de fluorescência constituem uma excelente ferramenta, pois permitem o monitoramento da reação de forma contínua, proporcionando um método prático e rápido para a determinação da

  1. PHYSICOCHEMICAL PROPERTIES OF THE PROTEOLYTIC ENZYME FROM THE LATEX OF THE MILKWEED, ASCLEPIAS SPECIOSA TORR. SOME COMPARISONS WITH OTHER PROTEASES

    Science.gov (United States)

    Winnick, Theodore; Davis, Alva R.; Greenberg, David M.

    1940-01-01

    1. A study has been made of the properties of a hitherto unreported proteolytic enzyme from the latex of the milkweed, Asclepias speciosa. The new protease has been named asclepain by the authors. 2. The results of chemical, diffusion, and denaturation tests indicate that asclepain is a protein. 3. Like papain, asclepain dots milk and digests most proteins, particularly if they are dissolved in concentrated urea solution. Unlike papain, asclepain did not clot blood. 4. The activation and inhibition phenomena of asclepain resemble those of papain, and seem best explained on the assumption that free sulfhydryl in the enzyme is necessary for proteolytic activity. The sulfhydryl of asclepain appears more labile than that of papain. 5. The measurement of pH-activity curves of asclepain on casein, ovalbumin, hemoglobin, edestin, and ovovitellin showed no definite digestion maxima for most of the undenatured proteins, while in urea solution there were well defined maxima near pH 7.0. Native hemoglobin and ovovitellin were especially undigestible, while native casein was rapidly attacked. 6. Temperature-activity curves were determined for asclepain on hemoglobin, casein, and milk solutions. The optimum temperature was shown to increase with decreasing time of digestion. PMID:19873154

  2. Effects of black-eyed pea trypsin/chymotrypsin inhibitor on proteolytic activity and on development of Anthonomus grandis.

    Science.gov (United States)

    Franco, Octávio L; dos Santos, Roseane C; Batista, João A N; Mendes, Ana Cristina M; de Araújo, Marcus Aurélio M; Monnerat, Rose G; Grossi-de-Sá, Maria Fátima; de Freitas, Sonia M

    2003-06-01

    The cotton boll weevil Anthonomus grandis (Boheman) is one of the major pests of cotton (Gossypium hirsutum L.) in tropical and sub-tropical areas of the New World. This feeds on cotton floral fruits and buds causing severe crop losses. Digestion in the boll weevil is facilitated by high levels of serine proteinases, which are responsible for the almost all proteolytic activity. Aiming to reduce the proteolytic activity, the inhibitory effects of black-eyed pea trypsin/chymotrypsin inhibitor (BTCI), towards trypsin and chymotrypsin from bovine pancreas and from midguts of A. grandis larvae and adult insects were analyzed. BTCI, purified from Vigna unguiculata (L.) seeds, was highly active against different trypsin-like proteinases studied and moderately active against the digestive chymotrypsin of adult insects. Nevertheless, no inhibitory activity was observed against chymotrypsin from A. grandis larval guts. To test the BTCI efficiency in vivo, neonate larvae were reared on artificial diet containing BTCI at 10, 50 and 100 microM. A reduction of larval weight of up to approximately 54% at the highest BTCI concentration was observed. At this concentration, the insect mortality was 65%. This work constitutes the first observation of a Bowman-Birk type inhibitor active in vitro and in vivo toward the cotton boll weevil A. grandis. The results of bioassays strongly suggest that BTCI may have potential as a transgene protein for use in engineered crop plants modified for heightened resistance to the cotton boll weevil.

  3. Effect of culturing conditions on the expression of key enzymes in the proteolytic system of Lactobacillus bulgaricus *

    Science.gov (United States)

    Hou, Jun-cai; Liu, Fei; Ren, Da-xi; Han, Wei-wei; Du, Yue-ou

    2015-01-01

    The proteolytic system of Lactobacillus bulgaricus breaks down milk proteins into peptides and amino acids, which are essential for the growth of the bacteria. The aim of this study was to determine the expressions of seven key genes in the proteolytic system under different culturing conditions (different phases, initial pH values, temperatures, and nitrogen sources) using real-time polymerase chain reaction (RT-PCR). The transcriptions of the seven genes were reduced by 30-fold on average in the stationary phase compared with the exponential growth phase. The transcriptions of the seven genes were reduced by 62.5-, 15.0-, and 59.0-fold in the strains KLDS 08006, KLDS 08007, and KLDS 08012, respectively, indicating that the expressions of the seven genes were significantly different among strains. In addition, the expressions of the seven genes were repressed in the MRS medium containing casein peptone. The effect of peptone supply on PepX transcription was the weakest compared with the other six genes, and the impact on OppD transcription was the strongest. Moreover, the expressions of the seven genes were significantly different among different strains (PLactobacillus bulgaricus at the transcription level. PMID:25845365

  4. Nano-zymography Using Laser-Scanning Confocal Microscopy Unmasks Proteolytic Activity of Cell-Derived Microparticles.

    Science.gov (United States)

    Briens, Aurélien; Gauberti, Maxime; Parcq, Jérôme; Montaner, Joan; Vivien, Denis; Martinez de Lizarrondo, Sara

    2016-01-01

    Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs.

  5. Effect of culturing conditions on the expression of key enzymes in the proteolytic system of Lactobacillus bulgaricus.

    Science.gov (United States)

    Hou, Jun-cai; Liu, Fei; Ren, Da-xi; Han, Wei-wei; Du, Yue-ou

    2015-04-01

    The proteolytic system of Lactobacillus bulgaricus breaks down milk proteins into peptides and amino acids, which are essential for the growth of the bacteria. The aim of this study was to determine the expressions of seven key genes in the proteolytic system under different culturing conditions (different phases, initial pH values, temperatures, and nitrogen sources) using real-time polymerase chain reaction (RT-PCR). The transcriptions of the seven genes were reduced by 30-fold on average in the stationary phase compared with the exponential growth phase. The transcriptions of the seven genes were reduced by 62.5-, 15.0-, and 59.0-fold in the strains KLDS 08006, KLDS 08007, and KLDS 08012, respectively, indicating that the expressions of the seven genes were significantly different among strains. In addition, the expressions of the seven genes were repressed in the MRS medium containing casein peptone. The effect of peptone supply on PepX transcription was the weakest compared with the other six genes, and the impact on OppD transcription was the strongest. Moreover, the expressions of the seven genes were significantly different among different strains (Pproteolytic system genes in Lactobacillus bulgaricus at the transcription level.

  6. Force-Induced Calpain Cleavage of Talin Is Critical for Growth, Adhesion Development, and Rigidity Sensing.

    Science.gov (United States)

    Saxena, Mayur; Changede, Rishita; Hone, James; Wolfenson, Haguy; Sheetz, Michael P

    2017-12-13

    Cell growth depends upon formation of cell-matrix adhesions, but mechanisms detailing the transmission of signals from adhesions to control proliferation are still lacking. Here, we find that the scaffold protein talin undergoes force-induced cleavage in early adhesions to produce the talin rod fragment that is needed for cell cycle progression. Expression of noncleavable talin blocks cell growth, adhesion maturation, proper mechanosensing, and the related property of EGF activation of motility. Further, the expression of talin rod in the presence of noncleavable full-length talin rescues cell growth and other functions. The cleavage of talin is found in early adhesions where there is also rapid turnover of talin that depends upon calpain and TRPM4 activity as well as the generation of force on talin. Thus, we suggest that an important function of talin is its control over cell cycle progression through its cleavage in early adhesions.

  7. Pressure modulates the self-cleavage step of the hairpin ribozyme

    Science.gov (United States)

    Schuabb, Caroline; Kumar, Narendra; Pataraia, Salome; Marx, Dominik; Winter, Roland

    2017-03-01

    The ability of certain RNAs, denoted as ribozymes, to not only store genetic information but also catalyse chemical reactions gave support to the RNA world hypothesis as a putative step in the development of early life on Earth. This, however, might have evolved under extreme environmental conditions, including the deep sea with pressures in the kbar regime. Here we study pressure-induced effects on the self-cleavage of hairpin ribozyme by following structural changes in real-time. Our results suggest that compression of the ribozyme leads to an accelerated transesterification reaction, being the self-cleavage step, although the overall process is retarded in the high-pressure regime. The results reveal that favourable interactions between the reaction site and neighbouring nucleobases are strengthened under pressure, resulting therefore in an accelerated self-cleavage step upon compression. These results suggest that properly engineered ribozymes may also act as piezophilic biocatalysts in addition to their hitherto known properties.

  8. Cell elongation is an adaptive response for clearing long chromatid arms from the cleavage plane

    Science.gov (United States)

    Kotadia, Shaila; Montembault, Emilie; Sullivan, William

    2012-01-01

    Chromosome segregation must be coordinated with cell cleavage to ensure correct transmission of the genome to daughter cells. Here we identify a novel mechanism by which Drosophila melanogaster neuronal stem cells coordinate sister chromatid segregation with cleavage furrow ingression. Cells adapted to a dramatic increase in chromatid arm length by transiently elongating during anaphase/telophase. The degree of cell elongation correlated with the length of the trailing chromatid arms and was concomitant with a slight increase in spindle length and an enlargement of the zone of cortical myosin distribution. Rho guanine-nucleotide exchange factor (Pebble)–depleted cells failed to elongate during segregation of long chromatids. As a result, Pebble-depleted adult flies exhibited morphological defects likely caused by cell death during development. These studies reveal a novel pathway linking trailing chromatid arms and cortical myosin that ensures the clearance of chromatids from the cleavage plane at the appropriate time during cytokinesis, thus preserving genome integrity. PMID:23185030

  9. Electrostatic instability of some jellium model lattices of high symmetry to their plane cleavage

    International Nuclear Information System (INIS)

    Kholopov, Eugene V; Kalashnikova, Vita V

    2007-01-01

    Jellium model structures composed of regular lattices of equal point charges immersed in a neutralizing uniform background are considered. The symmetric description eliminating the effect of potentials without transverse structural modulation is extended to the events specified by alternating distances between point-charge planes. Based on modulated potentials typical of plane-wise lattice summation, the energy of interaction between two semi-infinite hemi-crystals divided by a plane is obtained for cubic and hexagonal crystals, where all the characteristic orientations of the cleavage plane are taken into account. We found that simple cubic and hexagonal lattices, as well as cubic and hexagonal diamond structures, turn out to be unstable for certain cleavage planes. The most favourable cleavage planes for the bcc, fcc and hcp structures are also emphasized

  10. Effect of microstructure on the cleavage fracture strength of low carbon Mn-Ni-Mo bainitic steels

    International Nuclear Information System (INIS)

    Im, Young-Roc; Lee, Byeong-Joo; Oh, Yong Jun; Hong, Jun Hwa; Lee, Hu-Chul

    2004-01-01

    The effects of the microstructure on the cleavage fracture strength of low carbon Mn-Ni-Mo bainitic steels were examined. A four-point bend test and double-notched bend specimens were used to measure the cleavage fracture strength of the alloys and identify the cleavage initiating micro-cracks, respectively. The cleavage fracture strength and DBTT of Mn-Ni-Mo bainitic steels were strongly affected by the alloy carbon content. The decrease in the alloy carbon content resulted in a decrease in the inter-lath cementite-crowded layers and higher cleavage fracture strength. Micro-cracks that formed across the inter-lath cementite-crowded layers were observed to initiate cleavage fracture. The width of these inter-lath cementite-crowded layers was accepted as a cleavage initiating micro-crack size in the micro-mechanical modeling of the cleavage fracture, and the measured cleavage strength values of the bainitic Mn-Ni-Mo steels were well represented by the modified Griffith relationship

  11. Cleavage strain in the Variscan fold belt, County Cork, Ireland, estimated from stretched arsenopyrite rosettes

    Science.gov (United States)

    Ford, M.; Ferguson, C.C.

    1985-01-01

    In south-west Ireland, hydrothermally formed arsenopyrite crystals in a Devonian mudstone have responded to Variscan deformation by brittle extension fracture and fragment separation. The interfragment gaps and terminal extension zones of each crystal are infilled with fibrous quartz. Stretches within the cleavage plane have been calculated by the various methods available, most of which can be modified to incorporate terminal extension zones. The Strain Reversal Method is the most accurate currently available but still gives a minimum estimate of the overall strain. The more direct Hossain method, which gives only slightly lower estimates with this data, is more practical for field use. A strain ellipse can be estimated from each crystal rosette composed of three laths (assuming the original interlimb angles were all 60??) and, because actual rather than relative stretches are estimated, this provides a lower bound to the area increase in the plane of cleavage. Based on the average of our calculated strain ellipses this area increase is at least 114% and implies an average shortening across the cleavage of at least 53%. However, several lines of evidence suggest that the cleavage deformation was more intense and more oblate than that calculated, and we argue that a 300% area increase in the cleavage plane and 75% shortening across the cleavage are more realistic estimates of the true strain. Furthermore, the along-strike elongation indicated is at least 80%, which may be regionally significant. Estimates of orogenic contraction derived from balanced section construction should therefore take into account the possibility of a substantial strike elongation, and tectonic models that can accommodate such elongations need to be developed. ?? 1985.

  12. Atomistic Details of the Associative Phosphodiester Cleavage in Human Ribonuclease H

    International Nuclear Information System (INIS)

    Elsasser, Brigitta M.; Fels, Gregor

    2010-01-01

    During translation of the genetic information of DNA into proteins, mRNA is synthesized by RNA polymerase and after the transcription process degraded by RNase H. The endoribonuclease RNase H is a member of the nucleotidyl-transferase (NT) superfamily and is known to hydrolyze the phosphodiester bonds of RNA which is hybridized to DNA. Retroviral RNase H is part of the viral reverse transcriptase enzyme that is indispensable for the proliferation of retroviruses, such as HIV. Inhibitors of this enzyme could therefore provide new drugs against diseases like AIDS. In our study we investigated the molecular mechanism of RNA cleavage by human RNase H using a comprehensive high level DFT/B3LYP QM/MM theoretical method for the calculation of the stationary points and nudged elastic band (NEB) and free energy calculations to identify the transition state structures, the rate limiting step and the reaction barrier. Our calculations reveal that the catalytic mechanism proceeds in two steps and that the nature of the nucleophile is a water molecule. In the first step, the water attack on the scissile phosphorous is followed by a proton transfer from the water to the O2P oxygen and a trigonal bipyramidal pentacoordinated phosphorane is formed. Subsequently, in the second step the proton is shuttled to the O30 oxygen to generate the product state. During the reaction mechanism two Mg2+ ions support the formation of a stable associated in-line SN2-type phosphorane intermediate. Our calculated energy barrier of 19.3 kcal mol*1 is in excellent agreement with experimental findings (20.5 kcal mol*1). These results may contribute to the clarification and understanding of the RNase H reaction mechanism and of further enzymes from the RNase family.

  13. Production of proteolytic enzymes in mast cells, fibroblasts, vascular smooth muscle and endothelial cells cultivated under normoxic or hypoxic conditions

    Czech Academy of Sciences Publication Activity Database

    Maxová, H.; Bačáková, Lucie; Lisá, Věra; Novotná, J.; Tomášová, H.; Vízek, M.; Herget, J.

    2010-01-01

    Roč. 59, č. 5 (2010), s. 711-719 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GA305/08/0108; GA MŠk(CZ) 1M0510 Institutional research plan: CEZ:AV0Z50110509 Keywords : hypoxia * metalloproteinases * tryptase * chymase * immunofluorescence Subject RIV: EI - Biotechnology ; Bionics Impact factor: 1.646, year: 2010

  14. Characterization of cleavage events in the multifunctional cilium adhesin Mhp684 (P146) reveals a mechanism by which Mycoplasma hyopneumoniae regulates surface topography.

    Science.gov (United States)

    Bogema, Daniel R; Deutscher, Ania T; Woolley, Lauren K; Seymour, Lisa M; Raymond, Benjamin B A; Tacchi, Jessica L; Padula, Matthew P; Dixon, Nicholas E; Minion, F Chris; Jenkins, Cheryl; Walker, Mark J; Djordjevic, Steven P

    2012-01-01

    Mycoplasma hyopneumoniae causes enormous economic losses to swine production worldwide by colonizing the ciliated epithelium in the porcine respiratory tract, resulting in widespread damage to the mucociliary escalator, prolonged inflammation, reduced weight gain, and secondary infections. Protein Mhp684 (P146) comprises 1,317 amino acids, and while the N-terminal 400 residues display significant sequence identity to the archetype cilium adhesin P97, the remainder of the molecule is novel and displays unusual motifs. Proteome analysis shows that P146 preprotein is endogenously cleaved into three major fragments identified here as P50(P146), P40(P146), and P85(P146) that reside on the cell surface. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) identified a semitryptic peptide that delineated a major cleavage site in Mhp684. Cleavage occurred at the phenylalanine residue within sequence (672)ATEF↓QQ(677), consistent with a cleavage motif resembling S/T-X-F↓X-D/E recently identified in Mhp683 and other P97/P102 family members. Biotinylated surface proteins recovered by avidin chromatography and separated by two-dimensional gel electrophoresis (2-D GE) showed that more-extensive endoproteolytic cleavage of P146 occurs. Recombinant fragments F1(P146)-F3(P146) that mimic P50(P146), P40(P146), and P85(P146) were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3(P146) generated from M. hyopneumoniae strain J and 232 sequences strongly bind porcine plasminogen, and the removal of their respective C-terminal lysine and arginine residues significantly reduces this interaction. These data reveal that P146 is an extensively processed, multifunctional adhesin of M. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by which M. hyopneumoniae regulates protein topography. Vaccines used to control Mycoplasma

  15. Change in radiosensitivity on the development of sea urchin eggs during the early cleavage stage, 2

    International Nuclear Information System (INIS)

    Nakamura, Izumi

    1975-01-01

    The effect of cysteamine on the fluctuation of X-ray sensitivity during early cleavage stage of sea urchin eggs expressed by pluteus formation rate was examined. Sea urchin eggs were very resistant to radiation immediately after insemination or after S phase of the cleavage. When irradiation was given just prior to S phase or in the phase of cytokinesis, the eggs were very sensitive. In these sensitive stages, existence of cysteamine during X-irradiation apparently protected eggs against radiation effect. Dose modifying factor increased linearly from 1.8 to 3.4 with increasing dose of cysteamine (25mM-75mM) added. (auth.)

  16. Change in radiosensitivity of sea-urchin eggs during early cleavage stages

    International Nuclear Information System (INIS)

    Nakamura, I.

    1977-01-01

    When sea-urchin eggs were irradiated with 137 Cs γ-rays, their radiosensitivity, expressed by the percentage which formed pluteus larvae, fluctuated during the early cleavage cycle. Split-dose irradiations were made both in the sensitive and resistant phases. For eggs in the sensitive phase, the effect of the first exposure of 500 rad was not diminished during the interval before the second exposure. Eggs irradiated in the resistant phase were only slightly damaged. Results implied that fluctuations in radiosensitivity of sea-urchin eggs were caused mainly by different degrees of non-repairable damage in each phase of cleavage rather than by different recovery abilities. (author)

  17. On the mechanism of action of ribonucleases: dinucleotide cleavage catalyzed by imidazole and Zn2+.

    OpenAIRE

    Breslow, R; Huang, D L; Anslyn, E

    1989-01-01

    Cyclization/cleavage of the 2-(p-nitrophenyl) phosphate ester of propylene glycol is catalyzed by imidazole and, much more effectively, by Zn2+ with imidazole. In the latter case, the mechanism involves simultaneous Lewis acid/base catalysis. Similar Zn2+ and imidazole catalysis of cyclization/cleavage is seen with the dinucleotide 3',5'-UpU (uridylyluridine). Again, the zinc system is much more effective than is catalysis by imidazole alone, and in this case simultaneous Lewis acid/base cata...

  18. [Cleavage of DNA fragments induced by UV nanosecond laser excitation at 193 nm].

    Science.gov (United States)

    Vtiurina, N N; Grokhovskiĭ, S L; Filimonov, I V; Medvedkov, O I; Nechipurenko, D Iu; Vasil'ev, S A; Nechipurenko, Iu D

    2011-01-01

    The cleavage of dsDNA fragments in aqueous solution after irradiation with UV laser pulses at 193 nm has been studied. Samples were investigated using polyacrylamide gel electrophoresis. The intensity of damage of particular phosphodiester bond after hot alkali treatment was shown to depend on the base pair sequence. It was established that the probability of cleavage is twice higher for sites of DNA containing two or more successively running guanine residues. A possible mechanism of damage to the DNA molecule connected with the migration of holes along the helix is discussed.

  19. The proteolytic activity of pregnancy-associated plasma protein-A is potentially regulated by stanniocalcin-1 and -2 during human ovarian follicle development

    DEFF Research Database (Denmark)

    Jepsen, Malene R.; Kløverpris, Søren; Bøtkjær, Jane A.

    2016-01-01

    STUDY QUESTION: Is the proteolytic activity of pregnancy-associated plasma protein-A (PAPP-A) regulated by the stanniocalcins (STC1 and STC2) during human follicle maturation? SUMMARY ANSWER: The STCs and PAPP-A show similar expression by immunohistochemistry in developing follicles, and regulation...... of PAPP-A proteolytic activity is suggested by the identification of inhibited protein complexes between PAPP-A and STC1 or STC2 in human follicular fluid (FF). WHAT IS KNOWN ALREADY: The insulin-like growth factor (IGF)-regulating proteinase PAPP-A is secreted by the granulosa cells of estrogen...

  20. Pregnancy-associated plasma protein-A (PAPP-A) modulates early developmental rate in zebrafish independent of its proteolytic activity

    DEFF Research Database (Denmark)

    Kjær-Sørensen, Kasper; Engholm, Ditte Høyer; Kamei, Hiroyasu

    2013-01-01

    the developmental rate beginning during gastrulation without affecting the normal patterning of the embryo. This phenotype is different from those resulting from deficiency of Igf receptor or ligand in zebrafish, suggesting a function of Papp-a outside the Igf system. Biochemical analysis of recombinant zebrafish...... Papp-a demonstrates conservation of proteolytic activity, specificity, and intrinsic regulatory mechanism. However, in vitro transcribed mRNA, which encodes a proteolytically inactive Papp-a mutant, recues the papp-a knockdown phenotype as efficient as wild-type Papp-a. Thus, the developmental...

  1. Characterization of a Non-Canonical Signal Peptidase Cleavage Site in a Replication Protein from Tomato Ringspot Virus.

    Directory of Open Access Journals (Sweden)

    Ting Wei

    Full Text Available The NTB-VPg polyprotein from tomato ringspot virus is an integral membrane replication protein associated with endoplasmic reticulum membranes. A signal peptidase (SPase cleavage was previously detected in the C-terminal region of NTB-VPg downstream of a 14 amino acid (aa-long hydrophobic region (termed TM2. However, the exact location of the cleavage site was not determined. Using in vitro translation assays, we show that the SPase cleavage site is conserved in the NTB-VPg protein from various ToRSV isolates, although the rate of cleavage varies from one isolate to another. Systematic site-directed mutagenesis of the NTB-VPg SPase cleavage sites of two ToRSV isolates allowed the identification of sequences that affect cleavage efficiency. We also present evidence that SPase cleavage in the ToRSV-Rasp2 isolate occurs within a GAAGG sequence likely after the AAG (GAAG/G. Mutation of a downstream MAAV sequence to AAAV resulted in SPase cleavage at both the natural GAAG/G and the mutated AAA/V sequences. Given that there is a distance of seven aa between the two cleavage sites, this indicates that there is flexibility in the positioning of the cleavage sites relative to the inner surface of the membrane and the SPase active site. SPase cleavage sites are typically located 3-7 aa downstream of the hydrophobic region. However, the NTB-VPg GAAG/G cleavage site is located 17 aa downstream of the TM2 hydrophobic region, highlighting unusual features of the NTB-VPg SPase cleavage site. A putative 11 aa-long amphipathic helix was identified immediately downstream of the TM2 region and five aa upstream of the GAAG/G cleavage site. Based on these results, we present an updated topology model in which the hydrophobic and amphipathic domains form a long tilted helix or a bent helix in the membrane lipid bilayer, with the downstream cleavage site(s oriented parallel to the membrane inner surface.

  2. Integrated Bottom-Up and Top-Down Liquid Chromatography-Mass Spectrometry for Characterization of Recombinant Human Growth Hormone Degradation Products.

    Science.gov (United States)

    Wang, Yu Annie; Wu, Di; Auclair, Jared R; Salisbury, Joseph P; Sarin, Richa; Tang, Yang; Mozdzierz, Nicholas J; Shah, Kartik; Zhang, Anna Fan; Wu, Shiaw-Lin; Agar, Jeffery N; Love, J Christopher; Love, Kerry R; Hancock, William S

    2017-12-05

    With the advent of biosimilars to the U.S. market, it is important to have better analytical tools to ensure product quality from batch to batch. In addition, the recent popularity of using a continuous process for production of biopharmaceuticals, the traditional bottom-up method, alone for product characterization and quality analysis is no longer sufficient. Bottom-up method requires large amounts of material for analysis and is labor-intensive and time-consuming. Additionally, in this analysis, digestion of the protein with enzymes such as trypsin could induce artifacts and modifications which would increase the complexity of the analysis. On the other hand, a top-down method requires a minimum amount of sample and allows for analysis of the intact protein mass and sequence generated from fragmentation within the instrument. However, fragmentation usually occurs at the N-terminal and C-terminal ends of the protein with less internal fragmentation. Herein, we combine the use of the complementary techniques, a top-down and bottom-up method, for the characterization of human growth hormone degradation products. Notably, our approach required small amounts of sample, which is a requirement due to the sample constraints of small scale manufacturing. Using this approach, we were able to characterize various protein variants, including post-translational modifications such as oxidation and deamidation, residual leader sequence, and proteolytic cleavage. Thus, we were able to highlight the complementarity of top-down and bottom-up approaches, which achieved the characterization of a wide range of product variants in samples of human growth hormone secreted from Pichia pastoris.

  3. Structural and Biochemical Characterization of Organotin and Organolead Compounds Binding to the Organomercurial Lyase MerB Provide New Insights into Its Mechanism of Carbon–Metal Bond Cleavage

    Energy Technology Data Exchange (ETDEWEB)

    Wahba, Haytham M. [Département; Faculty; Stevenson, Michael J. [Department; Mansour, Ahmed [Département; Sygusch, Jurgen [Département; Wilcox, Dean E. [Department; Omichinski, James G. [Département

    2017-01-03

    The organomercurial lyase MerB has the unique ability to cleave carbon–Hg bonds, and structural studies indicate that three residues in the active site (C96, D99, and C159 in E. coli MerB) play important roles in the carbon–Hg bond cleavage. However, the role of each residue in carbon–metal bond cleavage has not been well-defined. To do so, we have structurally and biophysically characterized the interaction of MerB with a series of organotin and organolead compounds. Studies with two known inhibitors of MerB, dimethyltin (DMT) and triethyltin (TET), reveal that they inhibit by different mechanisms. In both cases the initial binding is to D99, but DMT subsequently binds to C96, which induces a conformation change in the active site. In contrast, diethyltin (DET) is a substrate for MerB and the SnIV product remains bound in the active site in a coordination similar to that of HgII following cleavage of organomercurial compounds. The results with analogous organolead compounds are similar in that trimethyllead (TML) is not cleaved and binds only to D99, whereas diethyllead (DEL) is a substrate and the PbIV product remains bound in the active site. Binding and cleavage is an exothermic reaction, while binding to D99 has negligible net heat flow. These results show that initial binding of organometallic compounds to MerB occurs at D99 followed, in some cases, by cleavage and loss of the organic moieties and binding of the metal ion product to C96, D99, and C159. Th