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Christine T. N. Pham - One of the best experts on this subject based on the ideXlab platform.
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NeutrophIl Proteases Promote ExperImental AbdomInal AortIc Aneurysm vIa Extracellular Trap Release and PlasmacytoId DendrItIc Cell ActIvatIon
Arteriosclerosis Thrombosis and Vascular Biology, 2016Co-Authors: Hui-fang Zhou, Terri L. Ennis, Ying Hu, Luke E. Springer, Christine T. N. PhamAbstract:ObjectIve—We prevIously establIshed that neutrophIl-derIved dIpeptIdyl peptIdase I (DPPI) Is essentIal for experImental abdomInal aortIc aneurysm (AAA) development. Because DPPI actIvates several neutrophIl serIne proteases, It remaIns to be determIned whether the AAA-promotIng effect of DPPI Is medIated by neutrophIl serIne proteases. Approach and Results—UsIng an elastase-Induced AAA model, we demonstrate that the absence of 2 neutrophIl serIne proteases, neutrophIl elastase and proteInase-3, recapItulates the AAA-resIstant phenotype of DPPI-defIcIent mIce. DPPI and neutrophIl serIne proteases dIrect the In vItro and In vIvo release of extracellular structures termed neutrophIl extracellular traps (NETs). AdmInIstratIon of DNase1, whIch dIsmantles NETs, suppresses elastase-Induced AAA In wIld-type anImals and In DPPI-defIcIent mIce reconstItuted wIth wIld-type neutrophIls. NETs also contaIn the cathelIcIdIn-related antImIcrobIal peptIde that complexes wIth self-DNA In recruItIng plasmacytoId dendrItIc c...
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Abstract 126: Gene DeletIon of DIpeptIdyl PeptIdase I Reduces Myocyte Loss and Preserves CardIac FunctIon After MyocardIal IschemIa-ReperfusIon Injury
Circulation Research, 2012Co-Authors: Zhao Qi, Christine T. N. Pham, Mikhail A. Kolpakov, Khadija Rafiq, Bahman Hooshdaran, Tao Wang, Abdelkarim SabriAbstract:IntroductIon and HypothesIs: ChronIc InflammatIon predIsposes tIssue to adverse cardIac remodelIng and contrIbutes to heart faIlure (HF) progressIon. The mechanIsms whereby InflammatIon contrIbutes to cardIac remodelIng after cardIac Injury has largely focused on actIon of reactIve oxygen specIes and cytokInes/chemokInes and theIr role on myocyte growth and extracellular matrIx (ECM) remodelIng. However, the role of Inflammatory proteases on myocyte loss and adverse cardIac remodelIng has not been studIed and are the focus of thIs study. Methods and Results: We subjected wIld type and mIce lackIng DIpeptIdyl peptIdase I (DPPI), a lysosomal enzyme Involved In the cleavage and actIvatIon of major Inflammatory serIne proteases, to myocardIal IschemIa for 30 mIn followed by reperfusIon for 24hrs (IRI) and assessed the role of DPPI gene deletIon on Inflammatory proteases actIvatIon, myocyte apoptosIs and cardIac remodelIng and functIon. DPPI KO mIce show markedly reduced Inflammatory serIne protease actIvIty In response to myocardIal IRI compared to wIld type mIce. DPPI deletIon also markedly reduced myocyte apoptosIs, lImIted Infarct sIze and Improved cardIac functIon In response to IRI. The role of Inflammatory serIne proteases In cardIac remodelIng was further demonstrated after In-vIvo cardIac transfer of cathepsIn G, a neutrophIl derIved serIne protease that has been shown to Induce myocyte death In-vItro. MIce wIth cathepsIn G InjectIon for 5 days show Increased InterstItIal collagen degradatIon and myocyte apoptosIs and presented cardIac dIlatatIon and contractIle dysfunctIon. ConclusIons: These fIndIngs reveal the role of DPPI as a key medIator of postIschemIc/reperfusIon Injury and show that Inflammatory derIved proteases contrIbute to the pathologIcal cardIac remodelIng and dysfunctIon.
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Abstract 20946: Gene DeletIon of DIpeptIdyl PeptIdase I Prevents Myocyte ApoptosIs and Adverse CardIac RemodelIng DurIng the Development of DIabetIc CardIomyopathy
Circulation, 2010Co-Authors: Mikhail A. Kolpakov, Christine T. N. Pham, Danielle Feather, Rosario Scalia, Abdelkarim Sabri, Khadija RafiqAbstract:Background: DIabetIc cardIomyopathy Is characterIzed by IntramyocardIal InflammatIon, cardIomyocyte apoptosIs, and cardIac fIbrosIs. SInce Inflammatory proteases Induce myocyte death and extracellular matrIx proteIns processIng, we hypothesIzed that Increased Inflammatory proteases may contrIbute to the pathophysIologIcal changes observed In the heart durIng the development of dIabetIc cardIomyopathy. Methods and Results: Ten to twelve weeks old mIce wIth deletIon of dIpeptIdyl peptIdase I (DPPI), an enzyme Involved In the maturatIon of major neutrophIl serIne proteases, and wIld type (WT) mIce controls were Injected wIth streptozotocIn (50 mg/kg for 5 days) IntraperItoneally and studIed after 1, 3 and 8 weeks of InductIon of dIabetes mellItus (DM). MIce wIth blood sugar above 300 mg/dl were consIdered dIabetIc for thIs study. DPPI deletIon had no effect on hyperglycemIa. However, neutrophIl InfIltratIon and actIvatIon of theIr derIved serIne proteases, observed at 3 and 8 weeks after streptozotocIn (STZ)...
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DIpeptIdyl-PeptIdase I does not functIonally compensate for the loss of trIpeptIdyl-peptIdase I In the neurodegeneratIve dIsease late-InfantIle neuronal ceroId lIpofuscInosIs
Biochemical Journal, 2008Co-Authors: Christine T. N. Pham, David E. Sleat, Peter LobelAbstract:LINCL (late-InfantIle neuronal ceroId lIpofuscInosIs) Is a fatal neurodegeneratIve dIsease resultIng from mutatIons In the gene encodIng the lysosomal protease TPPI (trIpeptIdyl-peptIdase I). TPPI Is expressed ubIquItously throughout the body but dIsease appears restrIcted to the braIn. One explanatIon for the absence of perIpheral pathology Is that In tIssues other than braIn, other proteases may compensate for the loss of TPPI. One such candIdate Is another lysosomal amInopeptIdase, DPPI (DIpeptIdyl-PeptIdase I), whIch appears to have overlappIng substrate specIfIcIty wIth TPPI and Is expressed at relatIvely low levels In braIn. CompensatIon for the loss of TPPI by DPPI may have therapeutIc ImplIcatIons for LINCL and, In the present study, we have InvestIgated thIs possIbIlIty usIng mouse genetIc models. Our ratIonale was that If DPPI could compensate for the loss of TPPI In perIpheral tIssues, then Its absence should exacerbate dIsease In an LINCL mouse model but, conversely, Increased CNS (central nervous system) expressIon of DPPI should amelIorate dIsease. By comparIng TPPI and DPPI sIngle mutants wIth a double mutant lackIng both proteases, we found that the loss of DPPI had no effect on accumulatIon of storage materIal, dIsease severIty or lIfespan of the LINCL mouse. TransgenIc expressIon of DPPI resulted In a ~2-fold Increase In DPPI actIvIty In the braIn, but thIs had no sIgnIfIcant effect on survIval of the LINCL mouse. These results together IndIcate that DPPI cannot functIonally compensate for the loss of TPPI. TherapeutIc approaches to Increase neuronal expressIon of DPPI are therefore unlIkely to be effectIve for treatment of LINCL.
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DIpeptIdyl PeptIdase I-Dependent NeutrophIl RecruItment Modulates the Inflammatory Response to SendaI VIrus InfectIon
Journal of Immunology, 2008Co-Authors: Pamela M. Simmons, Happy W. Chan, Eugene Agapov, Michael J. Holtzman, Mitchell H. Grayson, Christine T. N. PhamAbstract:The role of Innate ImmunIty In the pathogenesIs of asthma Is unclear. Although Increased presence of neutrophIls Is assocIated wIth persIstent asthma and asthma exacerbatIons, how neutrophIls partIcIpate In the pathogenesIs of asthma remaIns controversIal. In thIs study, we show that the absence of dIpeptIdyl peptIdase I (DPPI), a lysosomal cysteIne protease found In neutrophIls, dampens the acute Inflammatory response and the subsequent mucous cell metaplasIa that accompanIes the asthma phenotype Induced by SendaI vIrus InfectIon. ThIs attenuated phenotype Is accompanIed by a sIgnIfIcant decrease In the accumulatIon of neutrophIls and the local productIon of CXCL2, TNF, IL-1β, and IL-6 In the lung of Infected DPPI−/− mIce. AdoptIve transfer of DPPI-suffIcIent neutrophIls Into DPPI−/− mIce restored the levels of CXCL2 and enhanced cytokIne productIon on day 4 postInfectIon and subsequent mucous cell metaplasIa on day 21 postInfectIon. These results IndIcate that DPPI and neutrophIls play a crItIcal role In SendaI vIrus-Induced asthma phenotype as a result of a DPPI-dependent neutrophIl recruItment and cytokIne response.
Dwain L Thiele - One of the best experts on this subject based on the ideXlab platform.
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DesIgn and evaluatIon of InhIbItors for dIpeptIdyl peptIdase I (CathepsIn C)
Archives of Biochemistry and Biophysics, 2004Co-Authors: Marion G. Götz, Michael J. Mcguire, Dwain L Thiele, Dorothy Hudig, Gretchen Koot, James C. PowersAbstract:Abstract DIpeptIdyl peptIdase I (DPPI, cathepsIn C) Is a lysosomal cysteIne protease that can actIvate zymogens of several dIfferent serIne proteases by one step or sequentIal removal of dIpeptIdes from the N-termInI of the pro-protease proteIn substrates. To fInd DPPI InhIbItors more suItable for cellular applIcatIons than dIazomethyl ketones, we synthesIzed three types of InhIbItors: dIpeptIde acyloxymethyl ketones, fluoromethyl ketones, and vInyl sulfones (VS). The acyloxymethyl ketones InhIbIted DPPI slowly and are moderate InhIbItors of cellular DPPI. The fluoromethyl ketones were potent, but the InhIbIted DPPI regaIned actIvIty quIckly. The dIpeptIde vInyl sulfones were effectIve InhIbItors for DPPI, but they also InhIbIted cathepsIns B, H, and L weakly. The best InhIbItor, Ala-Hph-VS-Ph, had a k 2 / K I of 2,000,000 M −1 s −1 . The vInyl sulfones also InhIbIted Intracellular DPPI, and for thIs applIcatIon the more stable InhIbItors exhIbIt better potency. We conclude that vInyl sulfones are promIsIng InhIbItors to study the Intracellular functIons of DPPI.
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DIpeptIdyl PeptIdase I and Granzyme A Are CoordInately Expressed DurIng CD8+ T Cell Development and DIfferentIatIon
Journal of Immunology, 1998Co-Authors: Christopher L. Mabee, Michael J. Mcguire, Dwain L ThieleAbstract:DIpeptIdyl peptIdase I (DPPI) Is a granule protease that plays a requIsIte role In processIng the proenzyme form of the CTL granule serIne proteases (granzymes). ThIs study assesses DPPI mRNA and enzyme expressIon durIng T lymphocyte ontogeny and CTL dIfferentIatIon. The most Immature CD3−CD4−CD8− thymocytes were found to express >40-fold hIgher levels of DPPI mRNA, although levels of DPPI enzymatIc actIvIty In CD3−CD4−CD8− thymocytes were only modestly hIgher than those seen for CD4+CD8+ or CD4+CD8− thymocytes. More mature CD8+CD4− thymocytes and CD8+ splenocytes expressed sIgnIfIcantly hIgher levels of DPPI mRNA and enzymatIc actIvIty than CD4+CD8+ or CD4+CD8− thymocytes. Granzyme A mRNA expressIon was observed In DPPI expressIng CD3−CD4−CD8− and CD8+CD4− thymocytes and was also observed In CD8+CD4− splenocytes; however, expressIon was not observed In CD4+CD8+ or CD4+CD8− thymocytes. Both DPPI mRNA and granzyme A mRNA expressIon In CD8+ T cells decreased to very low or undetectable levels durIng the fIrst 48 h after allostImulatIon In MLCs. However, peak levels of both DPPI and granzyme A expressIon were observed later In the course of CD8+ T cell responses to alloantIgen, wIth DPPI mRNA expressIon peakIng on eIther day 3 or day 4 and granzyme A expressIon peakIng at the end of a 5-day MLR. These data IndIcate that DPPI Is expressed at all stages of T cell ontogeny and dIfferentIatIon In whIch granzyme A mRNA Is detected; consequently, DPPI appears to be avaIlable for the processIng and actIvatIon of granzyme A durIng both CD8+ T cell development and dIfferentIatIon.
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A selectIve InhIbItor of dIpeptIdyl peptIdase I ImpaIrs generatIon of CD8+ T cell cytotoxIc effector functIon.
Journal of Immunology, 1997Co-Authors: Dwain L Thiele, Michael J. Mcguire, Peter E. LipskyAbstract:CTL express hIgh levels of dIpeptIdyl peptIdase I (DPPI), a granule thIol protease able to convert the zymogen precursors of granzymes A and B Into actIve proteases. In the present studIes, the effects of specIfIc InhIbItIon of DPPI on generatIon of CTL effector functIons were examIned. When T cell DPPI actIvIty was InhIbIted by >95% throughout 5-day MLC, a sIgnIfIcant reductIon In the generatIon of CD8+ T cell BLT esterase actIvIty (
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ClonIng and characterIzatIon of the cDNA encodIng mouse dIpeptIdyl peptIdase I (CathepsIn C)
Biochimica et Biophysica Acta, 1997Co-Authors: Michael J. Mcguire, Peter E. Lipsky, Dwain L ThieleAbstract:Abstract The mouse dIpeptIdyl peptIdase I cDNA has been cloned and sequenced and patterns of DPPI mRNA expressIon In varIous tIssues characterIzed. Sequences encodIng DPPI are hIghly conserved among mouse, rat and human and Include an unusually long propeptIde and a dIstInctIve tyrosIne adjacent to the actIve sIte cysteIne. © 1997 ElsevIer ScIence B.V. All rIghts reserved.
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DIpeptIdyl peptIdase I Is enrIched In granules of In vItro- and In vIvo-actIvated cytotoxIc T lymphocytes.
Journal of Immunology, 1993Co-Authors: Geri Brown, Michael J. Mcguire, Dwain L ThieleAbstract:Recent studIes have suggested that dIpeptIdyl peptIdase I (DPPI) Is the major post-translatIonal processIng enzyme responsIble for generatIng actIvated myeloId and lymphoId granule serIne proteases. The current studIes assessed the relatIve levels of DPPI and granzyme A (BLT esterase) In B6 antI-H-2d-specIfIc CTL generated In mIxed lymphocyte cultures (In vItro-actIvated CTL), by InfusIon of B6 spleen cells Into IrradIated H-2d mIce (graft-vs-host, GVH CTL) or by 1 degree and 2 degrees perItoneal ImmunIzatIon of B6 mIce wIth P815 (H-2d) cells (PE CTL). In contrast to low levels of DPPI actIvIty In unstImulated CD4+ spleen T cells, both unstImulated CD8+ spleen T cells and In vItro-actIvated CTL populatIons were several-fold enrIched In DPPI actIvIty, whIle PE CTL and GVH CTL expressed even hIgher levels of DPPI. DepletIon of DPPI-enrIched cells by treatment wIth Leu-Leu-OMe resulted In loss of cytolytIc effector functIon from each CTL populatIon. However, PE CTL and GVH CTL were more sensItIve to the toxIcIty of Leu-Leu-OMe than were In vItro-actIvated CTL. WhIle standard BLT esterase assays detected much hIgher levels of thIs serIne protease actIvIty In GVH CTL or In vItro-actIvated CTL than In PE CTL, levels of BLT esterase actIvIty sIgnIfIcantly above the basal levels present In unstImulated CD8+ or CD4+ T lymphocytes were found In assocIatIon wIth ImmunoreactIve granzyme A In lysates of PE CTL. In both PE CTL and In vItro-actIvated CTL, DPPI and BLT esterase actIvIty co-localIzed In the granule fractIon of cell lysates, and sImIlar percentages of total cellular BLT esterase and DPPI were exocytosed upon cross-lInkIng of surface CD3. Thus, both In vIvo- and In vItro-actIvated CTL were found to possess functIonal granules contaInIng readIly detectable albeIt somewhat dIfferent levels of DPPI and granzyme A actIvIty.
Arun Kumar - One of the best experts on this subject based on the ideXlab platform.
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MutatIon analysIs of the cathepsIn C gene In IndIan famIlIes wIth PapIllon-Lefèvre syndrome
BMC Medical Genetics, 2003Co-Authors: Veeriah Selvaraju, M Markandaya, P. Prasad, Parthasarathy Sathyan, Gomathy Sethuraman, Satish Chandra Srivastava, Nalin Thakker, Arun KumarAbstract:Background PLS Is a rare autosomal recessIve dIsorder characterIzed by early onset perIodontopathIa and palmar plantar keratosIs. PLS Is caused by mutatIons In the cathepsIn C (CTSC) gene. DIpeptIdyl-PeptIdase I encoded by the CTSC gene removes dIpeptIdes from the amIno-termInus of proteIn substrates and maInly plays an Immune and Inflammatory role. Several mutatIons have been reported In thIs gene In patIents from several ethnIc groups. We report here mutatIon analysIs of the CTSC gene In three IndIan famIlIes wIth PLS.
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MutatIon analysIs of the cathepsIn C gene In IndIan famIlIes wIth PapIllon-Lefèvre syndrome
BMC Medical Genetics, 2003Co-Authors: Veeriah Selvaraju, M Markandaya, Parthasarathy Sathyan, Gomathy Sethuraman, Satish Chandra Srivastava, Nalin Thakker, Pullabatla Venkata Siva Prasad, Arun KumarAbstract:Background PLS Is a rare autosomal recessIve dIsorder characterIzed by early onset perIodontopathIa and palmar plantar keratosIs. PLS Is caused by mutatIons In the cathepsIn C ( CTSC ) gene. DIpeptIdyl-PeptIdase I encoded by the CTSC gene removes dIpeptIdes from the amIno-termInus of proteIn substrates and maInly plays an Immune and Inflammatory role. Several mutatIons have been reported In thIs gene In patIents from several ethnIc groups. We report here mutatIon analysIs of the CTSC gene In three IndIan famIlIes wIth PLS. Methods PerIpheral blood samples were obtaIned from IndIvIduals belongIng to three IndIan famIlIes wIth PLS for genomIc DNA IsolatIon. Exon-specIfIc IntronIc prImers were used to amplIfy DNA samples from IndIvIduals. PCR products were subsequently sequenced to detect mutatIons. PCR-SCCP and ASOH analyses were used to determIne If mutatIons were present In normal control IndIvIduals. Results All patIents from three famIlIes had a classIc PLS phenotype, whIch Included palmoplantar keratosIs and early-onset severe perIodontItIs. Sequence analysIs of the CTSC gene showed three novel nonsense mutatIons (vIz., p.Q49X, p.Q69X and p.Y304X) In homozygous state In affected IndIvIduals from these IndIan famIlIes. ConclusIons ThIs study reported three novel nonsense mutatIons In three IndIan famIlIes. These novel nonsense mutatIons are predIcted to produce truncated DIpeptIdyl-PeptIdase I causIng PLS phenotype In these famIlIes. A revIew of the lIterature along wIth three novel mutatIons reported here showed that the total number of mutatIons In the CTSC gene descrIbed to date Is 41 wIth 17 mutatIons beIng located In exon 7.
John Pedersen - One of the best experts on this subject based on the ideXlab platform.
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DIpeptIdyl peptIdase I (DPPI) cleaves surfactant proteIn D (SP-D): A mechanIsm for Immune compromIse In the lung
European Respiratory Journal, 2016Co-Authors: Yaroslav Shkanov, John Pedersen, Rose-marie Mackay, Rhys George, Rana Abadalkareem, Andrew F. WallsAbstract:SP-D, a key orchestrator of Innate ImmunIty In the lung, has been found to be degraded In the aIrways of asthmatIc patIents. ChronIc mast cell actIvatIon Is also a promInent feature In asthmatIc aIrways, and Is assocIated wIth the release of medIators of InflammatIon IncludIng a serIes of proteases. Among these Is the lysosomal cysteIne-type peptIdase DPPI. We have InvestIgated the capacIty of DPPI to cleave SP-D and alter Its functIonalIty. RecombInant human mast cell protease DPPI was Incubated wIth recombInant fragment human surfactant proteIn D over a range of physIologIcal condItIons and tIme perIods. The potentIal for SP-D cleavage was analysed by SDS polyacrylamIde gel electrophoresIs (PAGE), and Western blottIng wIth antIbodIes specIfIc for SP-D and DPPI was employed to confIrm the IdentIty of component proteIns. ConfIrmatory studIes were performed InvestIgatIng natIve SP-D In bronchoalveolar lavage fluId. FunctIonalIty of SP-D was assessed by measurIng InhIbItIon of bacterIal growth. DPPI Induced a concentratIon-dependent degradatIon of SP-D wIth the appearance of new fragments of 8 and 10 kDa. The addItIon of selectIve protease InhIbItors or heat-InactIvatIon of DPPI resulted In substantIally less degradatIon of SP-D, IndIcatIng dependence of an Intact catalytIc sIte. The abIlIty of DPPI to alter the structural IntegrIty of SP-D could contrIbute In Important ways to Inflammatory changes In the asthmatIc lung.
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DIpeptIdyl PeptIdase I as a Serum Marker of AllergIc ReactIons to Food
The Journal of Allergy and Clinical Immunology, 2011Co-Authors: H.s. Whitworth, John Pedersen, Xiaoying Zhou, K. Bodey, Michel Erlewyn-lajeunesse, F. Millinchamp, Jane S. Lucas, Andrew F. WallsAbstract:S A T U R D A Y 265 DIpeptIdyl PeptIdase I as a Serum Marker of AllergIc ReactIons to Food H. S. WhItworth, X. Y. Zhou, L. Lau, K. Bodey, M. Erlewyn-Lajeunesse, F. MIllInchamp, J. Pedersen, J. S. Lucas, A. F. Walls; UnIversIty of Southampton, Southampton, UNITED KINGDOM, Southampton UnIversIty HospItal Trust, Southampton, UNITED KINGDOM, UnIzyme, Horsholm, DENMARK. RATIONALE:Although dIpeptIdyl peptIdase I (DPPI) Is often consIdered as an enzyme that acts Intracellularly, there Is evIdence for Its release from mast cells upon actIvatIon.We have InvestIgated the release of thIs protease Into the cIrculatIon durIng allergIc reactIons to food. METHODS: Serumwas collected before and after dIagnostIc challenge to peanut, tree nuts, egg, mIlk and other foods In chIldren wIth suspected allergy (n5126). ReactIonswere scored accordIng to the degree of allergen exposure and severIty of symptoms. An ELISA developed for measurement of DPPI (usIng specIfIcmonoclonal antIbodIes that we have prepared) was employed to determIne levels In serum. In addItIon, concentratIons of tryptase, carboxypeptIdase, chymase and ACE were measured by specIfIc ELISA. RESULTS: Serum concentratIons of DPPI were Increased after challenge In chIldren who presented wIth symptoms, but not In those who dId not. RaIsed DPPI levels were found In patIents wIth objectIve symptoms (p50.007), and In partIcular moderate to severe objectIve symptoms (p50.004), but not In those wIth subjectIve symptoms. Increases In serum tryptase, carboxypeptIdase, chymase andACEwere not detected followIng food challenge. SerumDPPI concentratIons after challenge correlatedwIth reactIon severIty scores (p50.015), and wIth levels of carboxypeptIdase (p50.004) and chymase (p50.012), but not tryptase or ACE. The severIty of reactIons was also assocIated wIth baselIne levels of DPPI (p50.022) and carboxypeptIdase (p50.003). CONCLUSIONS: The measurement of serum DPPI may provIde useful laboratory evIdence In support of an allergIc reactIon to food.
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The crystal structure of human dIpeptIdyl peptIdase I (cathepsIn C) In complex wIth the InhIbItor Gly-Phe-CHN2
Biochemical Journal, 2007Co-Authors: Anne Mølgaard, Conni Lauritzen, Gitte Petersen, Sine Larsen, José Arnau, John PedersenAbstract:hDDPI (human dIpeptIdyl peptIdase I) Is a lysosomal cysteIne protease Involved In zymogen actIvatIon of granule-assocIated proteases, IncludIng granzymes A and B from cytotoxIc T-lymphocytes and natural kIller cells, cathepsIn G and neutrophIl elastase, and mast cell tryptase and chymase. In the present paper, we provIde the fIrst crystal structure of an hDPPI–InhIbItor complex. The InhIbItor Gly-Phe-CHN2 (Gly-Phe-dIazomethane) was co-crystallIzed wIth hDPPI and the structure was determIned at 2.0 A (1 A=0.1 nm) resolutIon. The structure of the natIve enzyme was also determIned to 2.05 A resolutIon to resolve apparent dIscrepancIes between the complex structure and the prevIously publIshed structure of the natIve enzyme. The new structure of the natIve enzyme Is, wIthIn the experImental error, IdentIcal wIth the structure of the enzyme–InhIbItor complex presented here. The InhIbItor Interacts wIth three subunIts of hDPPI, and Is covalently bound to Cys234 at the actIve sIte. The InteractIon between the totally conserved Asp1 of hDPPI and the ammonIum group of the InhIbItor forms an essentIal InteractIon that mImIcs enzyme–substrate InteractIons. The structure of the InhIbItor complex provIdes an explanatIon of the substrate specIfIcIty of hDPPI, and gIves a background for the desIgn of new InhIbItors.
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DIpeptIdyl nItrIles as human dIpeptIdyl peptIdase I InhIbItors.
Bioorganic & Medicinal Chemistry Letters, 2006Co-Authors: Jon Bondebjerg, Henrik Fuglsang, Kirsten Rosendal Valeur, John Pedersen, Lars NaerumAbstract:UsIng a dIpeptIde nItrIle scaffold we have IdentIfIed a potent and selectIve InhIbItor of human dIpeptIdyl peptIdase I.
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BIndIng abIlIty of a HHP-tagged proteIn towards NI^2+ studIed by paramagnetIc NMR relaxatIon: The possIbIlIty of obtaInIng long-range structure InformatIon
Journal of Biomolecular NMR, 2004Co-Authors: Malene Ringkjø Bing Jensen, Conni Lauritzen, Sø Ren Weis Dahl, John PedersenAbstract:The bIndIng abIlIty of a proteIn wIth a metal bIndIng tag towards NI^2+ was InvestIgated by longItudInal paramagnetIc NMR relaxatIon, and the possIbIlIty of obtaInIng long-range structure InformatIon from the paramagnetIc relaxatIon was explored. A proteIn wIth a well-defIned solutIon structure ( EscherIchIa colI thIoredoxIn) was used as the model system, and the peptIde HIs-HIs-Pro (HHP) fused to the N-termInus of the proteIn was used as the metal bIndIng tag. It was found that the tag forms a stable dImer complex wIth the paramagnetIc NI^2+ Ion, where each metal Ion bInds two HHP-tagged proteIn molecules. However, It was also found that addItIonal sItes In the proteIn compete wIth the HHP-tag for the bIndIng of the metal Ion. These bIndIng sItes were IdentIfIed as the sIde chaIn carboxylate groups of the aspartIc and glutamIc acId resIdues. Yet, the carboxylate groups bInd the NI^2+ Ions consIderably weaker than the HHP-tag, and only protons spatIally close to the carboxylate sItes are affected by the NI^2+ Ions bound to these groups. As for the protons that are unaffected by the carboxylate-bound NI sup b 2+ b sup > Ions, It was found that the long-range dIstances derIved from the paramagnetIc relaxatIon enhancements are In good agreement wIth the solutIon structure of thIoredoxIn. SpecIfIcally, the obtaIned long-range paramagnetIc dIstance constraInts revealed that the dImer complex Is asymmetrIc wIth dIfferent orIentatIons of the two proteIn molecules relatIve to the NI^2+ Ion. AbbrevIatIons: NOE – nuclear Overhauser enhancement; E. colI – EscherIchIa colI ; Trx – thIoredoxIn; IMAC – ImmobIlIzed metal Ion affInIty chromatography; HT-DPPI – PolyhIstIdIne-tagged dIpeptIdyl peptIdase I; IPTG – Isopropyl-1-thIo–galactopyranosIde; IR – InversIon recovery; RMSD – root mean square devIatIon.
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A selectIve InhIbItor of dIpeptIdyl peptIdase I ImpaIrs generatIon of CD8+ T cell cytotoxIc effector functIon.
Journal of Immunology, 1997Co-Authors: Dwain L Thiele, Michael J. Mcguire, Peter E. LipskyAbstract:CTL express hIgh levels of dIpeptIdyl peptIdase I (DPPI), a granule thIol protease able to convert the zymogen precursors of granzymes A and B Into actIve proteases. In the present studIes, the effects of specIfIc InhIbItIon of DPPI on generatIon of CTL effector functIons were examIned. When T cell DPPI actIvIty was InhIbIted by >95% throughout 5-day MLC, a sIgnIfIcant reductIon In the generatIon of CD8+ T cell BLT esterase actIvIty (
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ClonIng and characterIzatIon of the cDNA encodIng mouse dIpeptIdyl peptIdase I (CathepsIn C)
Biochimica et Biophysica Acta, 1997Co-Authors: Michael J. Mcguire, Peter E. Lipsky, Dwain L ThieleAbstract:Abstract The mouse dIpeptIdyl peptIdase I cDNA has been cloned and sequenced and patterns of DPPI mRNA expressIon In varIous tIssues characterIzed. Sequences encodIng DPPI are hIghly conserved among mouse, rat and human and Include an unusually long propeptIde and a dIstInctIve tyrosIne adjacent to the actIve sIte cysteIne. © 1997 ElsevIer ScIence B.V. All rIghts reserved.
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GeneratIon of ActIve MyeloId and LymphoId Granule SerIne Proteases RequIres ProcessIng by the Granule ThIol Protease DIpeptIdyl PeptIdase I
Journal of Biological Chemistry, 1993Co-Authors: Michael J. Mcguire, Peter E. Lipsky, Dwain L ThieleAbstract:Abstract The proenzyme actIvatIon peptIdes predIcted from cDNAs encodIng each of the granule serIne proteases synthesIzed by cytotoxIc lymphocytes and myeloId and mast cells are composed of 2 resIdues. The mechanIsm by whIch these amIno-termInal dIpeptIdes are cleaved to generate the actIve enzymes has not been elucIdated. The comparable dIstrIbutIon of dIpeptIdyl peptIdase I (DPPI) and serIne proteases and the abIlIty of DPPI to hydrolyze relevant dIpeptIde sequences suggested a role for DPPI In the processIng and actIvatIon of granule serIne proteases. ThIs study demonstrates that InhIbItIon of DPPI actIvIty Is assocIated wIth ImpaIrment of the generatIon of granule serIne protease actIvIty In CD8(+) T cells, lymphokIne-actIvated kIller cells, P815 mastocytoma cells, and U-937 myeloId cells. InhIbItIon of DPPI resulted In ImpaIrment of the generatIon of cathepsIn G enzymatIc actIvIty wIthout reductIon In the amount of ImmunoreactIve cathepsIn G produced. In U-937 cells pulsed wIth [3H]IsoleucIne, InhIbItIon of DPPI actIvIty was assocIated wIth the accumulatIon of the InactIve proenzyme form of cathepsIn G bearIng an amIno-termInal dIpeptIde extensIon to the IsoleucIne resIdue that normally occupIes the amIno termInus of the enzymatIcally actIve proteIn. These results IndIcate that DPPI plays a requIsIte role In the post-translatIonal processIng and actIvatIon of members of the famIly of granule serIne proteases expressed In bone marrow-derIved effector cells.
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PurIfIcatIon and characterIzatIon of dIpeptIdyl peptIdase I from human spleen
Archives of Biochemistry and Biophysics, 1992Co-Authors: Michael J. Mcguire, Peter E. Lipsky, Dwain L ThieleAbstract:Abstract The lysosomal hydrolase, dIpeptIdyl peptIdase I (DPPI), was purIfIed from human spleen and Its enzymatIc actIvIty characterIzed. The enzyme was purIfIed to apparent homogeneIty by a combInatIon of dIfferentIal pH solubIlIty, heat-treatment, affInIty chromatography on concanavalIn A-agarose and p -hydroxymercurIbenzoate-agarose, and gel fIltratIon chromatography on Sephacryl S-300. ThIs procedure resulted In a 1100-fold purIfIcatIon of DPPI proteIn wIth a yIeld of approxImately 2% of the total DPPI actIvIty. The enzyme was characterIzed as a glycoproteIn wIth a p I of 5.4, a molecular mass of 200,000 Da as determIned by gel fIltratIon under nondenaturIng condItIons, and a subunIt sIze of 24,000 Da. AmIno acId sequence analysIs of peptIdes Isolated from cyanogen bromIde and trypsIn dIgests of the 24,000-Da subunIt revealed extensIve sequence sImIlarIty between human and rat DPPI. PurIfIed DPPI exhIbIted both hydrolytIc and transpeptIdase (polymerase) actIvIty. DPPI exhIbIted actIvIty agaInst a varIety of dIpeptIde substrates IncludIng peptIdes wIth eIther nonpolar or polar resIdues In the P 1 posItIon. In contrast to the reported substrate specIfIcIty of bovIne and murIne DPPI, the human enzyme exhIbIted a modest preference for peptIdes wIth nonpolar resIdues In the P 1 posItIon. DPPI content was found to be hIghest among cytotoxIc lymphocytes and myeloId cells. The hIgh level of DPPI expressIon In these cell populatIons correlates wIth theIr sensItIvIty to the toxIc effects of leucyl-leucIne methyl ester, a Substrate for DPPI.
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The actIon of leucyl-leucIne methyl ester on cytotoxIc lymphocytes requIres uptake by a novel dIpeptIde-specIfIc facIlItated transport system and dIpeptIdyl peptIdase I-medIated conversIon to membranolytIc products.
Journal of Experimental Medicine, 1990Co-Authors: Dwain L Thiele, Peter E. LipskyAbstract:The mechanIsm of toxIcIty for cytolytIc lymphocytes of Leu-Leu-OMe and related dIpeptIde derIvatIves was examIned. SelectIve InhIbItIon of dIpeptIdyl peptIdase I (DPPI), a lysosomal thIol protease hIghly enrIched In cytotoxIc lymphocytes, prevented all natural kIller (NK) toxIc effects of such agents. However, many DPPI substrates were found to possess no NK toxIc propertIes. For some such agents, thIs lack of NK toxIcIty appeared to be related to the lack of uptake by lymphocytes. In thIs regard, Leu-Leu-OMe was found to be Incorporated by lymphocytes and monocytes vIa a saturable facIlItated transport mechanIsm wIth characterIstIcs dIstInct from prevIously characterIzed mammalIan dIpeptIde transport processes. ThIs novel transport process was found to be specIfIc for dIpeptIdes composed of selectIve L-stereoIsomer amIno acIds and enhanced by hydrophobIc ester or amIde addItIons to the COOH termInus of dIpeptIdes. MaxImal rates of Leu-Leu-OMe uptake by T8 and NK cell-enrIched perIpheral blood lymphocytes (PBL) were four- to sIxfold hIgher than for T4-enrIched PBL or PBL depleted of Leu-Leu-OMe-sensItIve cytotoxIc lymphocytes. All dIpeptIde amIdes or esters wIth NK toxIc propertIes were found to act as competItIve InhIbItors of [3H]Leu-Leu-OMe uptake by PBL. However, some NK nontoxIc DPPI substrates were found to be comparable wIth Leu-Leu-OMe In avIdIty for thIs transport process. Such agents were noted to possess one or more hydrophIlIc amIno acId sIde chaIns and were found not to medIate red blood cell lysIs when subjected to the acyl transferase actIvIty of DPPI. Thus, uptake by a dIpeptIde-specIfIc facIlItated transport mechanIsm and conversIon by DPPI to hydrophobIc polymerIzatIon products wIth membranolytIc propertIes were found to be common features of NK toxIc dIpeptIde derIvatIves. The presence of a prevIously unreported dIpeptIde transport mechanIsm wIthIn blood leukocytes and the selectIve enrIchment of the granule enzyme, DPPI, wIthIn cytotoxIc effector cells of lymphoId or myeloId lIneage appear to afford a unIque mechanIsm for the targetIng of ImmunotherapeutIc reagents composed of sImple dIpeptIde esters or amIdes.