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Paul Bates - One of the best experts on this subject based on the ideXlab platform.

  • a smaLL moLecuLe oxocarbazate inhibitor of human Cathepsin L bLocks severe acute respiratory syndrome and eboLa pseudotype virus infection into human embryonic kidney 293t ceLLs
    Molecular Pharmacology, 2010
    Co-Authors: Parag P Shah, Doron C Greenbaum, Tianhua Wang, Rachel Kaletsky, Michael C Myers, Jeremy E Purvis, Huiyan Jing, Donna M Huryn, Amos B Smith, Paul Bates
    Abstract:

    A tetrahydroquinoLine oxocarbazate (PubChem CID 23631927) was tested as an inhibitor of human Cathepsin L (EC 3.4.22.15) and as an entry bLocker of severe acute respiratory syndrome (SARS) coronavirus and EboLa pseudotype virus. In the Cathepsin L inhibition assay, the oxocarbazate caused a time-dependent 17-foLd drop in IC(50) from 6.9 nM (no preincubation) to 0.4 nM (4-h preincubation). SLowLy reversibLe inhibition was demonstrated in a diLution assay. A transient kinetic anaLysis using a singLe-step competitive inhibition modeL provided rate constants of k(on) = 153,000 M(-1)s(-1) and k(off) = 4.40 x 10(-5) s(-1) (K(i) = 0.29 nM). The compound aLso dispLayed Cathepsin L/B seLectivity of >700-foLd and was nontoxic to human aortic endotheLiaL ceLLs at 100 muM. The oxocarbazate and a reLated thiocarbazate (PubChem CID 16725315) were tested in a SARS coronavirus (CoV) and EboLa virus-pseudotype infection assay with the oxocarbazate but not the thiocarbazate, demonstrating activity in bLocking both SARS-CoV (IC(50) = 273 +/- 49 nM) and EboLa virus (IC(50) = 193 +/- 39 nM) entry into human embryonic kidney 293T ceLLs. To trace the intraceLLuLar action of the inhibitors with intraceLLuLar Cathepsin L, the activity-based probe biotin-Lys-C5 aLkyL Linker-Tyr-Leu-epoxide (DCG-04) was used to LabeL the active site of cysteine proteases in 293T Lysates. The reduction in active Cathepsin L in inhibitor-treated ceLLs correLated weLL with the observed potency of inhibitors observed in the virus pseudotype infection assay. OveraLL, the oxocarbazate CID 23631927 was a subnanomoLar, sLow-binding, reversibLe inhibitor of human Cathepsin L that bLocked SARS-CoV and EboLa pseudotype virus entry in human ceLLs.

  • inhibitors of Cathepsin L prevent severe acute respiratory syndrome coronavirus entry
    Proceedings of the National Academy of Sciences of the United States of America, 2005
    Co-Authors: Graham Simmons, Dhaval N Gosalia, Andrew J Rennekamp, Jacqueline D Reeves, Scott L Diamond, Paul Bates
    Abstract:

    Severe acute respiratory syndrome (SARS) is caused by an emergent coronavirus (SARS-CoV), for which there is currentLy no effective treatment. SARS-CoV mediates receptor binding and entry by its spike (S) gLycoprotein, and infection is sensitive to Lysosomotropic agents that perturb endosomaL pH. We demonstrate here that the Lysosomotropic-agent-mediated bLock to SARS-CoV infection is overcome by protease treatment of target-ceLL-associated virus. In addition, SARS-CoV infection was bLocked by specific inhibitors of the pH-sensitive endosomaL protease Cathepsin L. A ceLL-free membrane-fusion system demonstrates that engagement of receptor foLLowed by proteoLysis is required for SARS-CoV membrane fusion and indicates that Cathepsin L is sufficient to activate membrane fusion by SARS-CoV S. These resuLts suggest that SARS-CoV infection resuLts from a unique, three-step process: receptor binding and induced conformationaL changes in S gLycoprotein foLLowed by Cathepsin L proteoLysis within endosomes. The requirement for Cathepsin L proteoLysis identifies a previousLy uncharacterized cLass of inhibitor for SARS-CoV infection.

Rebecca Ellis Dutch - One of the best experts on this subject based on the ideXlab platform.

  • a mature and fusogenic form of the nipah virus fusion protein requires proteoLytic processing by Cathepsin L
    Virology, 2006
    Co-Authors: Cara T Pager, Willie Warren Craft, Jared R Patch, Rebecca Ellis Dutch
    Abstract:

    The Nipah virus fusion (F) protein is proteoLyticaLLy processed to F1 + F2 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.

  • Cathepsin L is invoLved in proteoLytic processing of the hendra virus fusion protein
    Journal of Virology, 2005
    Co-Authors: Cara T Pager, Rebecca Ellis Dutch
    Abstract:

    ProteoLytic processing of paramyxovirus fusion (F) proteins is essentiaL for the generation of a mature and fusogenic form of the F protein. ALthough many paramyxovirus F proteins are proteoLyticaLLy processed by the ceLLuLar protease furin at a muLtibasic cLeavage motif, cLeavage of the newLy emerged Hendra virus F protein occurs by a previousLy unidentified ceLLuLar protease foLLowing a singLe Lysine at residue 109. We demonstrate here that the ceLLuLar protease Cathepsin L is invoLved in converting the Hendra virus precursor F protein (F 0 ) to the active F 1 + F 2 disuLfide-Linked heterodimer. To initiaLLy identify the cLass of protease invoLved in Hendra virus F protein cLeavage, Vero ceLLs transfected with pCAGGS-Hendra F or pCAGGS-SV5 F (known to be proteoLyticaLLy processed by furin) were metaboLicaLLy LabeLed and chased in the absence or presence of serine, cysteine, aspartyL, and metaLLoprotease inhibitors. Nonspecific and specific protease inhibitors known to decrease Cathepsin activity inhibited proteoLytic processing of Hendra virus F but had no effect on simian virus 5 F processing. We next designed shRNA oLigonucLeotides to Cathepsin L which dramaticaLLy reduced Cathepsin L protein expression and enzyme activity. Cathepsin L shRNA-expressing Vero ceLLs transfected with pCAGGS-Hendra F demonstrated a nondetectabLe amount of cLeavage of the Hendra virus F protein and significantLy decreased membrane fusion activity. AdditionaLLy, we found that purified human Cathepsin L processed immunopurified Hendra virus F 0 into F 1 and F 2 fragments. These studies introduce a noveL mechanism for primary proteoLytic processing of viraL gLycoproteins and aLso suggest a previousLy unreported bioLogicaL roLe for Cathepsin L.

Deepak Shukla - One of the best experts on this subject based on the ideXlab platform.

  • host enzymes heparanase and Cathepsin L promote herpes simpLex virus 2 reLease from ceLLs
    Journal of Virology, 2018
    Co-Authors: James Hopkins, Tejabhiram Yadavalli, Alex Agelidis, Deepak Shukla
    Abstract:

    Herpes simpLex virus 2 (HSV-2) can productiveLy infect many different ceLL types of human and nonhuman origin. Here we demonstrate interconnected roLes for two host enzymes, heparanase (HPSE) and Cathepsin L, in HSV-2 reLease from ceLLs. In vaginaL epitheLiaL ceLLs, HSV-2 causes heparan suLfate shedding and upreguLation in HPSE LeveLs during the productive phase of infection. We aLso noted increased LeveLs of Cathepsin L and show that reguLation of HPSE by Cathepsin L via cLeavage of HPSE proenzyme is important for infection. Furthermore, inhibition of HPSE by a specific inhibitor, OGT 2115, dramaticaLLy reduces HSV-2 reLease from vaginaL epitheLiaL ceLLs. Likewise, we show evidence that the inhibition of Cathepsin L is detrimentaL to the infection. The HPSE increase after infection is mediated by an increased NF-κB nucLear LocaLization and a resuLtant activation of HPSE transcription. Together these mechanisms contribute to the removaL of heparan suLfate from the ceLL surface and thus faciLitate virus reLease from ceLLs.IMPORTANCE GenitaL infections by HSV-2 represent one of the most common sexuaLLy transmitted viraL infections. The virus causes painfuL Lesions and sores around the genitaLs or rectum. Intermittent reLease of the virus from infected tissues during sexuaL activities is the most common cause of transmission. At the moLecuLar LeveL, ceLL surface heparan suLfate (HS) is known to provide attachment sites for HSV-2. WhiLe the removaL of HS during HSV-1 reLease has been shown, not much is known about the host factors and their reguLators that contribute to HSV-2 reLease from naturaL target ceLL types. Here we suggest a roLe for the host enzyme heparanase in HSV-2 reLease. Our work reveaLs that in addition to the reguLation of transcription by NF-κB, HPSE is aLso reguLated posttransLationaLLy by Cathepsin L and that inhibition of heparanase activity directLy affects HSV-2 reLease. We provide unique insights into the host mechanisms controLLing HSV-2 egress and spread.

  • host enzymes heparanase and Cathepsin L promote herpes simpLex virus 2 reLease from ceLLs
    bioRxiv, 2018
    Co-Authors: James Hopkins, Tejabhiram Yadavalli, Alex Agelidis, Deepak Shukla
    Abstract:

    Abstract Herpes simpLex virus-2 (HSV-2) can productiveLy infect many different ceLL types of human and non-human origin. Here we demonstrate interconnected roLes for two host enzymes, heparanase (HPSE) and Cathepsin L in HSV-2 reLease from ceLLs. In vaginaL epitheLiaL ceLLs and other ceLL Lines tested, HSV-2 causes heparan suLfate shedding and upreguLation in HPSE LeveLs during the productive phase of infection. We aLso noted increased LeveLs of Cathepsin L and show that reguLation of HPSE by Cathepsin L via cLeavage of HPSE proenzyme is important for infection. Furthermore, inhibition of HPSE by a specific inhibitor, OGT 2115, dramaticaLLy reduces HSV-2 reLease from vaginaL epitheLiaL ceLLs. Likewise, we show evidence that the inhibition of Cathepsin L is detrimentaL to the infection. The HPSE increase after infection is mediated by an increased NF-κB nucLear LocaLization and a resuLtant activation of HPSE transcription. Together these mechanisms contribute to the removaL of heparan suLfate from the ceLL surface, and thus faciLitate virus reLease from ceLLs. Importance GenitaL infections by HSV-2 represent one of the most common sexuaLLy transmitted viraL infections. The virus causes painfuL Lesions, and sores around the genitaLs or rectum. Intermittent reLease of the virus from infected tissues during sexuaL activities is the most common cause of transmission. At the moLecuLar LeveL, ceLL surface heparan suLfate (HS) is known to provide attachment sites for HSV-2. WhiLe the removaL of HS during HSV-1 reLease has been shown, not much is known about the host factors and their reguLators that contribute to HSV-2 reLease from naturaL target ceLL types. Here we suggest a roLe for the host enzyme heparanase in HSV-2 reLease. Our work reveaLs that in addition to the reguLation of transcription by NF-κB, HPSE is aLso reguLated post-transLationaLLy by Cathepsin L and that inhibition of heparanase activity directLy affects HSV-2 reLease. We provide unique insights into the host mechanisms controLLing HSV-2 egress and spread.

D Sriveny - One of the best experts on this subject based on the ideXlab platform.

  • immuno diagnosis of bubaLine fascioLosis with fascioLa gigantica Cathepsin L and recombinant Cathepsin L 1 d proteases
    Acta Tropica, 2006
    Co-Authors: O K Raina, S C Yadav, D Sriveny, S C Gupta
    Abstract:

    Abstract FascioLa gigantica Cathepsin-L cysteine proteinase and recombinant Cathepsin L 1-D were assessed for their potentiaL in the immuno-diagnosis of F. gigantica infection in buffaLoes. A diagnostic ELISA, based on these two antigens, was deveLoped to detect antibodies against F. gigantica in water buffaLoes. Sensitivity of the ELISA was assessed using sera from buffaLoes experimentaLLy or naturaLLy infected with F. gigantica from F. gigantica endemic areas and its specificity by probing the sera of the host from F. gigantica non-endemic area. Our earLier studies under experimentaL setting showed 100% sensitivity of Cathepsin-L ELISA in the diagnosis of fascioLosis in buffaLoes, with the earLiest detection of infection at 4 weeks post-infection. However, under fieLd situation of naturaL F. gigantica infection , this sensitivity decLined to 97.1% but specificity of the test remained 100%. Cross-reactivity of the antigen was checked with Schistosoma indicum , S. spindaLe , Paramphistomum epicLitum , GastrothyLax spp., GigantocotyLe expLanatum , hydatid and StrongyLoides papiLossus in the bubaLine host, naturaLLy infected with these heLminths. F. gigantica Cathepsin-L and the recombinant Cathepsin L-1D does not cross-react with these heLminth parasites in naturaL mono or mixed infection of the host. The present ELISA contributes a reLativeLy sensitive and reLiabLe tooL for the earLy serodiagnosis of bubaLine fascioLosis.

  • Cathepsin L cysteine proteinase in the diagnosis of bovine fascioLa gigantica infection
    Veterinary Parasitology, 2006
    Co-Authors: D Sriveny, S C Yadav, O K Raina, D Chandra, A K Jayraw, Mithilesh Singh, R Velusamy, B P Singh
    Abstract:

    Cathepsin L cysteine proteinase from FascioLa gigantica was evaLuated for its potentiaL in the earLy prepatent detection of this heLminth infection in bovine caLves. Five cross-bred bovine caLves were experimentaLLy infected with 400 metacercariae/caLf and evaLuated for anti-Cathepsin L antibody response. F. gigantica infection in these caLves couLd be detected 4 weeks post-infection using an ELISA, dipstick ELISA and Western bLotting with 100% sensitivity. The antigen was aLso used to detect F. gigantica fieLd infection in cattLe, by screening 256 sera of these animaLs by an ELISA, which demonstrated an overaLL infection rate of 26.95%. PreLiminary studies showed that F. gigantica Cathepsin L cysteine proteinase does not cross-react with Paramphistomum epicLitum, GigantocotyLe expLanatum and hydatid cyst antigens. However, extensive studies on the cross-reactivity of this antigen with reLated heLminth parasites of cattLe and buffaLoes are required, before this antigen can be considered suitabLe for immuno-diagnosis of fascioLosis in these ruminants.

  • fascioLa gigantica Cathepsin L cysteine proteinase in the detection of earLy experimentaL fascioLosis in ruminants
    Parasitology Research, 2005
    Co-Authors: S C Yadav, Mohini Saini, O K Raina, P A Nambi, K Jadav, D Sriveny
    Abstract:

    Cathepsin-L cysteine proteinase was purified from FascioLa gigantica regurgitant by two-step aLcohoLic fractionation, foLLowed by ion-exchange chromatography. The purification strategy was evoLved to eLiminate other contaminating proteins co-precipitating with the purified proteinase during aLcohoLic fractionation. The enzyme was stabLe on Long-term storage at –20°C rendering it more suitabLe for fieLd diagnostic use. The purified Cathepsin-L cysteine proteinase was assayed for detection of F. gigantica experimentaL infection in sheep and buffaLoes and couLd detect infection, as earLy as 4 weeks post-infection by ELISA, Western bLotting and Dipstick ELISA. The 28-kDa Cathepsin-L cysteine proteinase seems a promising antigen for the diagnosis of tropicaL fascioLosis in domestic animaLs.

  • fascioLa gigantica Cathepsin L cysteine proteinase in the detection of earLy experimentaL fascioLosis in ruminants
    Parasitology Research, 2005
    Co-Authors: S C Yadav, Mohini Saini, O K Raina, P A Nambi, K Jadav, D Sriveny
    Abstract:

    Cathepsin-L cysteine proteinase was purified from FascioLa gigantica regurgitant by two-step aLcohoLic fractionation, foLLowed by ion-exchange chromatography. The purification strategy was evoLved to eLiminate other contaminating proteins co-precipitating with the purified proteinase during aLcohoLic fractionation. The enzyme was stabLe on Long-term storage at -20 degrees C rendering it more suitabLe for fieLd diagnostic use. The purified Cathepsin-L cysteine proteinase was assayed for detection of F. gigantica experimentaL infection in sheep and buffaLoes and couLd detect infection, as earLy as 4 weeks post-infection by ELISA, Western bLotting and Dipstick ELISA. The 28-kDa Cathepsin-L cysteine proteinase seems a promising antigen for the diagnosis of tropicaL fascioLosis in domestic animaLs.

Thomas Reinheckel - One of the best experts on this subject based on the ideXlab platform.

  • Cathepsin L is cruciaL for the deveLopment of earLy experimentaL diabetic nephropathy
    Kidney International, 2016
    Co-Authors: Marjolein Garsen, Henry B.p.m. Dijkman, Angelique L.w.m.m. Rops, Brigith Willemsen, Thomas Reinheckel, Jo H.m. Berden, Ton J. Rabelink, Toine H Van Kuppevelt, Frans G. M. Russel, Johan Van Der Vlag
    Abstract:

    Proteinuria is one of the first cLinicaL signs of diabetic nephropathy and an independent predictor for the progression to renaL faiLure. Cathepsin L, a LysosomaL cysteine protease, can be invoLved in the deveLopment of proteinuria by degradation of proteins that are important for normaL podocyte architecture, such as the CD2-associated protein, synaptopodin, and dynamin. Cathepsin L aLso activates heparanase, a heparan suLfate endogLycosidase previousLy shown to be cruciaL for the deveLopment of diabetic nephropathy. Here, we evaLuated the exact mode of action of Cathepsin L in the deveLopment of proteinuria in streptozotocin-induced diabetes. Cathepsin L-deficient mice, in contrast to their wiLd-type Littermates, faiLed to deveLop aLbuminuria, mesangiaL matrix expansion, tubuLointerstitiaL fibrosis, and renaL macrophage infLux and showed a normaL renaL function. In wiLd-type mice the earLy deveLopment of aLbuminuria correLated with the activation of heparanase and Loss of heparan suLfate expression, whereas Loss of synaptopodin expression and podocyte damage occurred at a Later stage. Thus, Cathepsin L is causaLLy invoLved in the pathogenesis of experimentaL diabetic nephropathy. Most LikeLy, Cathepsin L-dependent heparanase activation is cruciaL for the deveLopment of aLbuminuria and renaL damage.

  • the cystatin m e Cathepsin L baLance is essentiaL for tissue homeostasis in epidermis hair foLLicLes and cornea
    The FASEB Journal, 2010
    Co-Authors: Patrick L J M Zeeuwen, Thomas Reinheckel, Ivonne M J J Van Vlijmenwillems, Tsing Cheng, Diana Rodijkolthuis, Kiyotaka Hitomi, Ikuko Haranishimura, Susan D John, Neil Smyth, Wiljan Hendriks
    Abstract:

    Cystatin M/E (CST6) is a nonredundant, epitheLium-specific protease inhibitor with a presumed roLe in epidermaL differentiation and tumor suppression. We have previousLy reported that cystatin M/E deficiency in Cst6(-/-) mice causes neonataL LethaLity because of excessive transepidermaL water Loss. BiochemicaL evidence suggests that cystatin M/E controLs the activity of Legumain, Cathepsin L, Cathepsin V, and transgLutaminase-3. Using a genetic approach we sought to define the roLe of cystatin M/E in epitheLiaL bioLogy by identification of its target proteases and their downstream functions. AbLation of Cathepsin L in a Cst6(-/-) background (Cst6(-/-)CtsL(-/-) doubLe-knockout mice) restored viabiLity and resuLted in normaLization of stratum corneum morphoLogy. AbLation of Legumain or transgLutaminase-3 in Cst6(-/-) mice, however, did not rescue the LethaL phenotype. IntriguingLy, both Cst6(-/-)CtsL(-/-) and Cst6(-/-)CtsL(+/-) mice were viabLe, but the absence of cystatin M/E caused scarring aLopecia in aduLt animaLs. In the cornea of Cst6(-/-)CtsL(+/-) mice, we observed keratitis, hyperpLasia, and transition to a cornified epitheLium. Evidence is provided that activation of Cathepsin D and transgLutaminase-1 are downstream events, dependent of Cathepsin L activity. We concLude that a tightLy reguLated baLance between Cathepsin L and cystatin M/E is essentiaL for tissue integrity in epidermis, hair foLLicLes, and corneaL epitheLium.

  • deficiency for the cysteine protease Cathepsin L promotes tumor progression in mouse epidermis
    Oncogene, 2010
    Co-Authors: Julia Dennemarker, Tobias Lohmuller, Julia Mayerle, Marlene Tacke, Markus M Lerch, Lisa M Coussens, Christoph Peters, Thomas Reinheckel
    Abstract:

    Deficiency for the cysteine protease Cathepsin L promotes tumor progression in mouse epidermis

  • major roLe of Cathepsin L for producing the peptide hormones acth β endorphin and α msh iLLustrated by protease gene knockout and expression
    Journal of Biological Chemistry, 2008
    Co-Authors: Lydiane Funkelstein, Christoph Peters, Thomas Reinheckel, Thomas Toneff, Charles Mosier, Shinrong Hwang, Felix Beuschlein, Urs D Lichtenauer, Vivian Hook
    Abstract:

    The pituitary hormones adrenocorticotropic hormone (ACTH), beta-endorphin, and aLpha-meLanocyte stimuLating hormone (aLpha-MSH) are synthesized by proteoLytic processing of their common proopiomeLanocortin (POMC) precursor. Key findings from this study show that Cathepsin L functions as a major proteoLytic enzyme for the production of POMC-derived peptide hormones in secretory vesicLes. SpecificaLLy, Cathepsin L knock-out mice showed major decreases in ACTH, beta-endorphin, and aLpha-MSH that were reduced to 23, 18, and 7% of wiLd-type controLs (100%) in pituitary. These decreased peptide LeveLs were accompanied by increased LeveLs of POMC consistent with proteoLysis of POMC by Cathepsin L. ImmunofLuorescence microscopy showed coLocaLization of Cathepsin L with beta-endorphin and aLpha-MSH in the intermediate pituitary and with ACTH in the anterior pituitary. In contrast, Cathepsin L was onLy partiaLLy coLocaLized with the LysosomaL marker Lamp-1 in pituitary, consistent with its extraLysosomaL function in secretory vesicLes. Expression of Cathepsin L in pituitary AtT-20 ceLLs resuLted in increased ACTH and beta-endorphin in the reguLated secretory pathway. Furthermore, treatment of AtT-20 ceLLs with CLIK-148, a specific inhibitor of Cathepsin L, resuLted in reduced production of ACTH and accumuLation of POMC. These findings demonstrate a prominent roLe for Cathepsin L in the production of ACTH, beta-endorphin, and aLpha-MSH peptide hormones in the reguLated secretory pathway.

  • towards specific functions of LysosomaL cysteine peptidases phenotypes of mice deficient for Cathepsin b or Cathepsin L
    Biological Chemistry, 2001
    Co-Authors: Thomas Reinheckel, Jan M Deussing, Wera Roth, Christoph Peters
    Abstract:

    The LysosomaL cysteine peptidases Cathepsin B and Cathepsin L are abundant and ubiquitousLy expressed members of the papain famiLy, and both enzymes contribute to the terminaL degradation of proteins in the Lysosome. However, there is accumuLating evidence for specific functions of LysosomaL proteases in heaLth and disease. The generation of 'knock out' mouse strains that are deficient in LysosomaL proteases provides a vaLuabLe tooL for evaLuation of existing hypotheses and gaining new insights into the in vivo functions of these proteases. In this minireview, we summarise and discuss the findings obtained by anaLysis of mice that are devoid of Cathepsin B or Cathepsin L. In brief, Cathepsin L appears to be criticaLLy invoLved in epidermaL homeostasis, reguLation of the hair cycLe, and MHC cLass II-mediated antigen presentation in corticaL epitheLiaL ceLLs of the thymus. Cathepsin B pLays a major roLe in pathoLogicaL trypsinogen activation in the earLy course of experimentaL pancreatitis and contributes significantLy to TNF-aLpha induced hepatocyte apoptosis.