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Mei-fang Jin - One of the best experts on this subject based on the ideXlab platform.
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zinc camk ii associatEd mitophagy signaling contributEd to hippocampal mossy fibEr sprouting and cognitivE dEficits following nEonatal sEizurEs and its rEgulation by chronic lEptin trEatmEnt
Frontiers in Neurology, 2018Co-Authors: Mei-fang JinAbstract:ThE rolE of lEptin in thE pathogEnEsis of EpilEpsy is gEtting morE and morE attEntion in clinical and basic rEsEarch. Although thErE arE data indicating nEuroprotEctivE EffEcts of ElEvatEd sErum/brain lEptin lEvEls following acutE sEizurEs, no study to datE has dEalt with thE impact of chronic lEptin trEatmEnt on long-tErm brain injury following dEvElopmEntal sEizurEs. ThE aim of this study was to EvaluatE whEthEr chronic lEptin trEatmEnt may havE nEuroprotEctivE EffEcts on cognitivE and hippocampal mossy fibEr sprouting following flurothyl-inducEd rEcurrEnt nEonatal sEizurEs and whEthEr thEsE EffEcts arE mEdiatEd by thE zinc/CaMKII-associatEd mitophagy signaling pathway. Forty SpraguE-DawlEy rats (postnatal day 6, P6) wErE randomly assignEd into two groups: nEonatal sEizurE group and control group. At P13, thEy wErE furthEr dividEd into control group, sEizurE group (RS), control + lEptin (lEptin, i.p., 2 mg/kg/day for 10 days), sEizurE+lEptin group (RS+LEptin, 2mg/kg/day, i.p., for 10 consEcutivE days). Morris watEr mazE tEst was pErformEd during P27-P32. SubsEquEntly, Timm staining and WEstErn blotting wErE usEd to dEtEct thE mossy fibEr sprouting and protEin lEvEls in hippocampus. Flurothyl-inducEd sEizurEs (RS group) significantly down-rEgulatEd mitophagy markErs PINK, Drp1, PHB, and mEmory markEr CaMK II alpha whilE up-rEgulating zinc transportErs ZnT3, ZnT4, ZIP7, and autophagy ExEcution molEcular CathEpsin-E, which wErE parallElEd with hippocampal abErrant mossy fibEr sprouting and cognitivE dysfunction. HowEvEr, thEsE changEs wErE rEstorEd by chronic lEptin trEatmEnt (RS+LEptin group). ThE rEsults showEd that lEptin had nEuroprotEctivE EffEct on hippocampal pathological damagE and cognitivE dEficits inducEd by nEonatal sEizurEs and suggEstEd that Zinc/CaMK II associatEd-mitophagy signaling pathway in hippocampus may bE a nEw targEt of lEptin's nEuroprotEction, with potEntial valuE of translational mEdicinE.
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Zinc/CaMK II AssociatEd-Mitophagy Signaling ContributEd to Hippocampal Mossy FibEr Sprouting and CognitivE DEficits Following NEonatal SEizurEs and Its REgulation by Chronic LEptin TrEatmEnt
Frontiers Media S.A., 2018Co-Authors: Mei-fang JinAbstract:ThE rolE of lEptin in thE pathogEnEsis of EpilEpsy is gEtting morE and morE attEntion in clinical and basic rEsEarch. Although thErE arE data indicating nEuroprotEctivE EffEcts of ElEvatEd sErum/brain lEptin lEvEls following acutE sEizurEs, no study to datE has dEalt with thE impact of chronic lEptin trEatmEnt on long-tErm brain injury following dEvElopmEntal sEizurEs. ThE aim of this study was to EvaluatE whEthEr chronic lEptin trEatmEnt may havE nEuroprotEctivE EffEcts on cognitivE and hippocampal mossy fibEr sprouting following flurothyl-inducEd rEcurrEnt nEonatal sEizurEs and whEthEr thEsE EffEcts arE mEdiatEd by thE zinc/CaMKII-associatEd mitophagy signaling pathway. Forty SpraguE-DawlEy rats (postnatal day 6, P6) wErE randomly assignEd into two groups: nEonatal sEizurE group and control group. At P13, thEy wErE furthEr dividEd into control group, sEizurE group (RS), control + lEptin (lEptin, i.p., 2 mg/kg/day for 10 days), sEizurE+lEptin group (RS+LEptin, 2mg/kg/day, i.p., for 10 consEcutivE days). Morris watEr mazE tEst was pErformEd during P27-P32. SubsEquEntly, Timm staining and WEstErn blotting wErE usEd to dEtEct thE mossy fibEr sprouting and protEin lEvEls in hippocampus. Flurothyl-inducEd sEizurEs (RS group) significantly down-rEgulatEd mitophagy markErs PINK, Drp1, PHB, and mEmory markEr CaMK II alpha whilE up-rEgulating zinc transportErs ZnT3, ZnT4, ZIP7, and autophagy ExEcution molEcular CathEpsin-E, which wErE parallElEd with hippocampal abErrant mossy fibEr sprouting and cognitivE dysfunction. HowEvEr, thEsE changEs wErE rEstorEd by chronic lEptin trEatmEnt (RS+LEptin group). ThE rEsults showEd that lEptin had nEuroprotEctivE EffEct on hippocampal pathological damagE and cognitivE dEficits inducEd by nEonatal sEizurEs and suggEstEd that Zinc/CaMK II associatEd-mitophagy signaling pathway in hippocampus may bE a nEw targEt of lEptin's nEuroprotEction, with potEntial valuE of translational mEdicinE
Koichi Nishigaki - One of the best experts on this subject based on the ideXlab platform.
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pEptidE aptamEr modifiEd singlE wallEd carbon nanotubE basEd transistors for high pErformancE biosEnsors
Scientific Reports, 2017Co-Authors: Nguyen Thanh Tung, Koichi Nishigaki, Truong Thi Ngoc Lien, Phan Trong Tue, Yasuhide Ohno, Kenzo Maehashi, Kazuhiko Matsumoto, Manish Biyani, Yuzuru TakamuraAbstract:BiosEnsors Employing singlE-wallEd carbon nanotubE fiEld-EffEct transistors (SWCNT FETs) offEr ultimatE sEnsitivity. HowEvEr, bEsidEs thE sEnsitivity, a high sElEctivity is critically important to distinguish thE truE signal from intErfErEncE signals in a non-controllEd EnvironmEnt. This work prEsEnts thE first dEmonstration of thE succEssful intEgration of a novEl pEptidE aptamEr with a liquid-gatEd SWCNT FET to achiEvE highly sEnsitivE and spEcific dEtEction of CathEpsin E (CatE), a usEful prognostic biomarkEr for cancEr diagnosis. NovEl pEptidE aptamErs that spEcifically rEcognizE CatE arE EnginEErEd by systEmic in vitro Evolution. ThE SWCNTs wErE firstly grown using thE thErmal chEmical vapor dEposition (CVD) mEthod and thEn wErE EmployEd as a channEl to fabricatE a SWCNT FET dEvicE. NExt, thE SWCNTs wErE functionalizEd by noncovalEnt immobilization of thE pEptidE aptamEr using 1-pyrEnEbutanoic acid succinimidyl EstEr (PBASE) linkEr. ThE rEsulting FET sEnsors ExhibitEd a high sElEctivity (no rEsponsE to bovinE sErum albumin and CathEpsin K) and labEl-frEE dEtEction of CatE at unprEcEdEntEdly low concEntrations in both phosphatE-buffErEd salinE (2.3 pM) and human sErum (0.23 nM). Our rEsults highlight thE usE of pEptidE aptamEr-modifiEd SWCNT FET sEnsors as a promising platform for nEar-patiEnt tEsting and point-of-carE tEsting applications.
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sElEction by function EfficiEnt EnrichmEnt of CathEpsin E inhibitors from a dna library
Journal of Molecular Recognition, 2007Co-Authors: Mohammed Naimuddin, Kenji Yamamoto, Koichirou Kitamura, Yasunori Kinoshita, Yoko Hondatakahashi, Marina Murakami, Masato Ito, Kazunori Hanada, Yuzuru Husimi, Koichi NishigakiAbstract:A mEthod for EfficiEnt EnrichmEnt of protEasE inhibitors out of a DNA library was dEvElopEd by introducing SF-link tEchnology. A two-stEp sElEction stratEgy was dEsignEd consisting of thE initial EnrichmEnt of aptamErs basEd on binding function whilE thE sEcond EnrichmEnt stEp was basEd on thE inhibitory activity to a protEasE, CathEpsin E (CE). ThE lattEr was constructEd by covalEntly linking of a biotinylatEd pEptidE substratE to Each of thE ssDNA molEculE containEd in thE prEliminarily sElEctEd DNA library, gEnErating ‘SF-link’. Gradual EnrichmEnt of inhibitory DNAs was attainEd in thE coursE of sElEction. OnE molEculE, SFR-6-3, showEd an IC50 of around 30 nM, a Kd of around 15 nM and high sElEctivity for CE. SEquEncE and structurE analysis rEvEalEd a C-rich sEquEncE without any guaninE and possibly an i-motif structurE, which must bE novEl to bE found in in vitro-sElEctEd aptamErs. SF-link tEchnology, which is novEl as thE scrEEning tEchnology, providEd a rEmarkablE EnrichmEnt of spEcific protEasE inhibitors and has a potEntial to bE furthEr dEvElopEd. Copyright © 2006 John WilEy & Sons, Ltd.
Yuzuru Takamura - One of the best experts on this subject based on the ideXlab platform.
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pEptidE aptamEr modifiEd singlE wallEd carbon nanotubE basEd transistors for high pErformancE biosEnsors
Scientific Reports, 2017Co-Authors: Nguyen Thanh Tung, Koichi Nishigaki, Truong Thi Ngoc Lien, Phan Trong Tue, Yasuhide Ohno, Kenzo Maehashi, Kazuhiko Matsumoto, Manish Biyani, Yuzuru TakamuraAbstract:BiosEnsors Employing singlE-wallEd carbon nanotubE fiEld-EffEct transistors (SWCNT FETs) offEr ultimatE sEnsitivity. HowEvEr, bEsidEs thE sEnsitivity, a high sElEctivity is critically important to distinguish thE truE signal from intErfErEncE signals in a non-controllEd EnvironmEnt. This work prEsEnts thE first dEmonstration of thE succEssful intEgration of a novEl pEptidE aptamEr with a liquid-gatEd SWCNT FET to achiEvE highly sEnsitivE and spEcific dEtEction of CathEpsin E (CatE), a usEful prognostic biomarkEr for cancEr diagnosis. NovEl pEptidE aptamErs that spEcifically rEcognizE CatE arE EnginEErEd by systEmic in vitro Evolution. ThE SWCNTs wErE firstly grown using thE thErmal chEmical vapor dEposition (CVD) mEthod and thEn wErE EmployEd as a channEl to fabricatE a SWCNT FET dEvicE. NExt, thE SWCNTs wErE functionalizEd by noncovalEnt immobilization of thE pEptidE aptamEr using 1-pyrEnEbutanoic acid succinimidyl EstEr (PBASE) linkEr. ThE rEsulting FET sEnsors ExhibitEd a high sElEctivity (no rEsponsE to bovinE sErum albumin and CathEpsin K) and labEl-frEE dEtEction of CatE at unprEcEdEntEdly low concEntrations in both phosphatE-buffErEd salinE (2.3 pM) and human sErum (0.23 nM). Our rEsults highlight thE usE of pEptidE aptamEr-modifiEd SWCNT FET sEnsors as a promising platform for nEar-patiEnt tEsting and point-of-carE tEsting applications.
Nguyen Thanh Tung - One of the best experts on this subject based on the ideXlab platform.
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pEptidE aptamEr modifiEd singlE wallEd carbon nanotubE basEd transistors for high pErformancE biosEnsors
Scientific Reports, 2017Co-Authors: Nguyen Thanh Tung, Koichi Nishigaki, Truong Thi Ngoc Lien, Phan Trong Tue, Yasuhide Ohno, Kenzo Maehashi, Kazuhiko Matsumoto, Manish Biyani, Yuzuru TakamuraAbstract:BiosEnsors Employing singlE-wallEd carbon nanotubE fiEld-EffEct transistors (SWCNT FETs) offEr ultimatE sEnsitivity. HowEvEr, bEsidEs thE sEnsitivity, a high sElEctivity is critically important to distinguish thE truE signal from intErfErEncE signals in a non-controllEd EnvironmEnt. This work prEsEnts thE first dEmonstration of thE succEssful intEgration of a novEl pEptidE aptamEr with a liquid-gatEd SWCNT FET to achiEvE highly sEnsitivE and spEcific dEtEction of CathEpsin E (CatE), a usEful prognostic biomarkEr for cancEr diagnosis. NovEl pEptidE aptamErs that spEcifically rEcognizE CatE arE EnginEErEd by systEmic in vitro Evolution. ThE SWCNTs wErE firstly grown using thE thErmal chEmical vapor dEposition (CVD) mEthod and thEn wErE EmployEd as a channEl to fabricatE a SWCNT FET dEvicE. NExt, thE SWCNTs wErE functionalizEd by noncovalEnt immobilization of thE pEptidE aptamEr using 1-pyrEnEbutanoic acid succinimidyl EstEr (PBASE) linkEr. ThE rEsulting FET sEnsors ExhibitEd a high sElEctivity (no rEsponsE to bovinE sErum albumin and CathEpsin K) and labEl-frEE dEtEction of CatE at unprEcEdEntEdly low concEntrations in both phosphatE-buffErEd salinE (2.3 pM) and human sErum (0.23 nM). Our rEsults highlight thE usE of pEptidE aptamEr-modifiEd SWCNT FET sEnsors as a promising platform for nEar-patiEnt tEsting and point-of-carE tEsting applications.
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PEptidE aptamEr-modifiEd singlE wallEd carbon nanotubE-basEdtransistors for high-pErformancE biosEnsors
'Springer Science and Business Media LLC', 2017Co-Authors: Nguyen Thanh Tung, Phan Tue Trong, Truong Thi Ngoc Lien, Ohno Yasuhide, Maehashi Kenzo, Matsumoto Kazuhiko, Nishigaki Koichi, Biyani Manish, Takamura YuzuruAbstract:BiosEnsors Employing singlE-wallEd carbon nanotubE fiEld-EffEct transistors (SWCNT FETs) offEr ultimatE sEnsitivity. HowEvEr, bEsidEs thE sEnsitivity, a high sElEctivity is critically important to distinguish thE truE signal from intErfErEncE signals in a non-controllEd EnvironmEnt. This work prEsEnts thE first dEmonstration of thE succEssful intEgration of a novEl pEptidE aptamEr with a liquid gatEdSWCNT FET to achiEvE highly sEnsitivE and spEcific dEtEction of CathEpsin E (CatE), a usEful prognostic biomarkEr for cancEr diagnosis. NovEl pEptidE aptamErs that spEcifically rEcognizE CatE arE EnginEErEd by systEmic in vitro Evolution. ThE SWCNTs wErE firstly grown using thE thErmal chEmical vapor dEposition (CVD) mEthod and thEn wErE EmployEd as a channEl to fabricatE a SWCNT FET dEvicE.NExt, thE SWCNTs wErE functionalizEd by noncovalEnt immobilization of thE pEptidE aptamEr using1-pyrEnEbutanoic acid succinimidyl EstEr (PBASE) linkEr. ThE rEsulting FET sEnsors ExhibitEd a high sElEctivity (no rEsponsE to bovinE sErum albumin and CathEpsin K) and labEl-frEE dEtEction of CatE at unprEcEdEntEdly low concEntrations in both phosphatE-buffErEd salinE (2.3 pM) and human sErum (0.23 nM). Our rEsults highlight thE usE of pEptidE aptamEr-modifiEd SWCNT FET sEnsors as a promisingplatform for nEar-patiEnt tEsting and point-of-carE tEsting applications
Kenji Yamamoto - One of the best experts on this subject based on the ideXlab platform.
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thE critical rolE of protEolytic rElay through CathEpsins b and E in thE phEnotypic changE of microglia macrophagE
The Journal of Neuroscience, 2015Co-Authors: Christoph Peterts, Kenji Yamamoto, Hong Qing, Hiroshi NakanishiAbstract:ProtEinasE cascadEs arE part of thE basic machinEry of nEuronal dEath pathways. NEuronal CathEpsin B (CatB), a typical cystEinE lysosomal protEasE, plays a critical rolE in nEuronal dEath through lysosomal lEakagE or ExcEssivE autophagy. On thE othEr hand, much attEntion has bEEn paid to microglial CatB in nEuronal dEath. WE hErEin show thE critical rolE of protEolytic rElay through microglial CatB and CatE in thE polarization of microglia/macrophagEs in thE nEurotoxic phEnotypE, lEading to hypoxia/ischEmia (HI)-inducEd hippocampal nEuronal damagE in nEonatal micE. HI causEd ExtEnsivE brain injury in nEonatal wild-typE micE, but not in CatB(-/-) micE. FurthErmorE, HI-inducEd polarization of microglia/macrophagEs in thE nEurotoxic phEnotypE followEd by thE nEuroprotEctivE phEnotypE in wild-typE micE. On thE othEr hand, microglia/macrophagEs ExhibitEd only thE Early and transiEnt polarization in thE nEuroprotEctivE phEnotypE in CatB(-/-) micE. CA-074ME, a spEcific CatB inhibitor, significantly inhibitEd thE nEuronal dEath of primary culturEd hippocampal nEurons inducEd by thE conditionEd mEdium from culturEd microglia polarizEd in thE nEurotoxic phEnotypE. FurthErmorE, CA-074ME prEvEntEd thE activation of nuclEar factor-κB (NF-κB) in culturEd microglia by inhibiting autophagic inhibitor of κBα dEgradation following ExposurE to oxygEn-glucosE dEprivation. RathEr surprisingly, CatE incrEasEd thE CatB ExprEssion aftEr HI by thE libEration of thE tumor nEcrosis factor-rElatEd apoptosis-inducing ligand (TRAIL) from microglia through thE protEasomal pathway. A significant incrEasE in CatB and CatE lEvEls was found ExclusivEly in microglia/macrophagEs aftEr HI. Thus, a protEolytic rElay through thE Early CatE/TRAIL-dEpEndEnt protEosomal and latE CatB-dEpEndEnt autophagic pathways for NF-κB activation may play a critical rolE in thE polarization of microglia/macrophagEs in thE nEurotoxic phEnotypE. SignificancE statEmEnt: ProtEinasE cascadEs arE part of thE basic machinEry of nEuronal dEath pathways. CathEpsin B, a typical cystEinE lysosomal protEasE, plays a critical rolE in nEuronal dEath through lysosomal lEakagE or ExcEssivE autophagy in nEurons. On thE othEr hand, much attEntion has bEEn also paid to thE rolE of microglial CathEpsin B in nEuronal dEath. In this study, using in vivo and in vitro modEls of rElEvancE to brain ischEmia, wE found a critical rolE of protEolytic rElay through CathEpsin B and CathEpsin E in thE nEurotoxic polarization of microglia/macrophagEs, which is rEsponsiblE for aggravation of hypoxia/ischEmia-inducEd nEuronal injury. ThEsE findings suggEst orally activE sElEctivE inhibitors of CathEpsin B or CathEpsin E as promising pharmacological agEnts for thE trEatmEnt of ischEmic brain injury.
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dEfEctivE adiposE tissuE dEvElopmEnt associatEd with hEpatomEgaly in CathEpsin E dEficiEnt micE fEd a high fat diEt
Biochemical and Biophysical Research Communications, 2014Co-Authors: Tomoko Kadowaki, Mizuho A Kido, Junko Hatakeyama, Kuniaki Okamoto, Takayuki Tsukuba, Kenji YamamotoAbstract:CathEpsin E is an intracEllular aspartic protEinasE, which is prEdominantly distributEd in immunE-rElatEd and EpithElial cElls. HowEvEr, thE rolE of thE EnzymE in adiposE tissuEs rEmains unknown. In this study, wE invEstigatEd thE charactEristics of CathEpsin E-dEficiEnt (CatE−/−) micE fEd a high-fat diEt (HFD), as a mousE modEl of obEsity. HFD-fEd CatE−/− micE displayEd rEducEd body wEight gain and dEfEctivE dEvElopmEnt of whitE adiposE tissuE (WAT) and brown adiposE tissuE (BAT), comparEd with HFD-fEd wild-typE micE. MorEovEr, fat-inducEd CatE−/− micE showEd abnormal lipid accumulation in non-adiposE tissuEs charactErizEd by hEpatomEgaly, which is probably duE to dEfEctivE adiposE tissuE dEvElopmEnt. DEtailEd pathological and biochEmical analysEs showEd that hEpatomEgaly was accompaniEd by hEpatic stEatosis and hypErcholEstErolEmia in HFD-inducEd CatE−/− micE. In fat-inducEd CatE−/− micE, thE numbEr of macrophagEs infiltrating into WAT was significantly lowEr than in fat-inducEd wild-typE micE. Thus, thE impairEd adiposE tissuE dEvElopmEnt in HFD-inducEd CatE−/− micE was probably duE to rEducEd infiltration of macrophagEs and may lEad to hEpatomEgaly accompaniEd by hEpatic stEatosis and hypErcholEstErolEmia.
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sElEction by function EfficiEnt EnrichmEnt of CathEpsin E inhibitors from a dna library
Journal of Molecular Recognition, 2007Co-Authors: Mohammed Naimuddin, Kenji Yamamoto, Koichirou Kitamura, Yasunori Kinoshita, Yoko Hondatakahashi, Marina Murakami, Masato Ito, Kazunori Hanada, Yuzuru Husimi, Koichi NishigakiAbstract:A mEthod for EfficiEnt EnrichmEnt of protEasE inhibitors out of a DNA library was dEvElopEd by introducing SF-link tEchnology. A two-stEp sElEction stratEgy was dEsignEd consisting of thE initial EnrichmEnt of aptamErs basEd on binding function whilE thE sEcond EnrichmEnt stEp was basEd on thE inhibitory activity to a protEasE, CathEpsin E (CE). ThE lattEr was constructEd by covalEntly linking of a biotinylatEd pEptidE substratE to Each of thE ssDNA molEculE containEd in thE prEliminarily sElEctEd DNA library, gEnErating ‘SF-link’. Gradual EnrichmEnt of inhibitory DNAs was attainEd in thE coursE of sElEction. OnE molEculE, SFR-6-3, showEd an IC50 of around 30 nM, a Kd of around 15 nM and high sElEctivity for CE. SEquEncE and structurE analysis rEvEalEd a C-rich sEquEncE without any guaninE and possibly an i-motif structurE, which must bE novEl to bE found in in vitro-sElEctEd aptamErs. SF-link tEchnology, which is novEl as thE scrEEning tEchnology, providEd a rEmarkablE EnrichmEnt of spEcific protEasE inhibitors and has a potEntial to bE furthEr dEvElopEd. Copyright © 2006 John WilEy & Sons, Ltd.
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a rolE for CathEpsin E in thE procEssing of mast cEll carboxypEptidasE a
Journal of Cell Science, 2005Co-Authors: Frida Henningsson, Kenji Yamamoto, Paul Saftig, Thomas Reinheckel, Christoph Peters, Stefan D KnightAbstract:Mast-cEll carboxypEptidasE A is storEd in thE sEcrEtory granulE and is rElEasEd, togEthEr with a rangE of othEr inflammatory mEdiators, upon mast-cEll dEgranulation. CarboxypEptidasE A, likE all mast-cEll protEasEs, is storEd in thE granulE as an activE EnzymE (i.E. with its propEptidE rEmovEd). Although thE procEssing mEchanisms for thE othEr classEs of mast-cEll protEasEs (in particular thE chymasEs) havE bEEn clarifiEd to somE ExtEnt, thE procEssing of procarboxypEptidasE A is poorly charactErizEd. HErE, wE show that mast cElls from micE lacking thE aspartic protEasE CathEpsin E display an accumulation of procarboxypEptidasE A, indicating a dEfEct in carboxypEptidasE-A procEssing. By contrast, mast cElls lacking CathEpsins B, L or D havE normal carboxypEptidasE-A procEssing. FurthErmorE, rEcombinant CathEpsin E was found to procEss rEcombinant procarboxypEptidasE A in vitro, undEr conditions rEsEmbling thosE found in mast-cEll granulEs. ImmunohistochEmical analysis rEvEalEd staining for CathEpsin E in mast cElls from normal micE but not in mast cElls from micE lacking hEparin, indicating that CathEpsin E is bound to hEparin protEoglycan within mast-cEll granulEs. In accordancE with this notion, affinity chromatography showEd that rEcombinant CathEpsin E bound strongly to hEparin undEr acidic conditions (thE conditions prEvailing in mast-cEll granulEs) but not at nEutral pH. MorEovEr, mast-cEll dEgranulation rEsultEd in thE rElEasE of CathEpsin E. TakEn togEthEr, our rEsults indicatE that CathEpsin E is locatEd in mast-cEll sEcrEtory granulEs in complEx with hEparin protEoglycans, and that it has a rolE in thE procEssing of procarboxypEptidasE A into activE protEasE.
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CathEpsin d but not CathEpsin E dEgradEs dEsmosomEs during EpidErmal dEsquamation
British Journal of Dermatology, 2004Co-Authors: S Igarashi, Kenji Yamamoto, Yoshiyuki Yasuda, T Takizawa, H Uchiwa, S Hayashi, Henry Brysk, J M Robinson, Miriam M Brysk, Toshio HorikoshiAbstract:Summary Background WE prEviously rEportEd that an ambiEnt aspartic protEinasE is crucial to dEsquamation of thE stratum cornEum at pH 5. IdEntification of this aspartic protEinasE by using EnzymE inhibitors suggEstEd it to bE CathEpsin D, although wE could not ExcludE CathEpsin E. ObjEctivEs To dEtErminE thE idEntity of this aspartic protEinasE and its distribution within thE stratum cornEum. MEthods WE mEasurEd EnzymE activitiEs of CathEpsin D and CathEpsin E in thE salt and dEtErgEnt Extracts from callus stratum cornEum, using a fluorogEnic pEptidE as a substratE and comparing thE EffEct of addition of Ascaris pEpsin inhibitor (spEcific for CathEpsin E) with that of pEpstatin A (which inhibits both CathEpsin D and CathEpsin E). Both EnzymEs wErE thEn ExtractEd and purifiEd from plantar stratum cornEum samplEs and idEntifiEd by WEstErn blotting. ImmunofluorEscEncE microscopy was usEd to invEstigatE thE localization of protEinasEs within human plantar stratum cornEum samplE sEctions. REsults WE found that 20% of total aspartic protEinasE activity could bE attributEd to CathEpsin E, thE rEmaindEr to CathEpsin D. Two subunits of CathEpsin D wErE idEntifiEd, a maturE activE form at 33 kDa and an intErmEdiatE activE form at 48 kDa; CathEpsin E was also idEntifiEd at 48 kDa, although in a stainEd band 10-fold wEakEr in thE immunoblot. ImmunofluorEscEncE microscopy showEd thE antibody to CathEpsin D to bE localizEd in thE lipid EnvElopEs of thE stratum cornEum, whErEas that to CathEpsin E stainEd thE tissuE diffusEly. ThE labElling for CathEpsin D was similar to that obsErvEd for dEsmosomEs, and immunoElEctron microscopy confirmEd that CathEpsin D was prEsEnt on dEsmosomEs. On thE othEr hand, CathEpsin E occurrEd intracEllularly within thE squamEs. Conclusions WE concludE that CathEpsin D, and not CathEpsin E, causEs dEsquamation by dEgrading dEsmosomEs.