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1
Herms, Max. "Genetische Analyse des Cathepsin L bei chronischer Pankreatitis." Doctoral thesis, Universitätsbibliothek Leipzig, 2012. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-90473.
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Die chronische Pankreatitis (CP) ist eine wiederkehrende, entzündliche Erkrankung des Pankreas. In den letzten Jahren wurden mehrere Kandidatengene, die zur Entstehung einer CP prädisponieren, identifiziert. Zu diesen Genen gehören PRSS1, PRSS2, SPINK1, CFTR und CTRC. Der Pathogenese der genetisch bedingten CP scheint dabei eine frühzeitige, intrapankreatische Aktivierung von Trypsin zugrunde zu liegen.
Cathepsin B (CTSB), eine in Lysosomen vorkommenden Protease, ist in der Lage Trypsinogen zu aktivieren. Genetisch zeigte sich eine Assoziation der p.L26V Variante bei tropisch-kalzifizierender CP, welche bei idiopathischer CP nicht bestätigt wurde. Neben CTSB ist CTSL die am zweithäufigsten vorkommende lysosomale Protease. Funktionelle Untersuchungen zeigten, dass CTSL ein inaktives Trypsin freisetzt. Im Mausmodell zeigten sich bei Ctsl-/- Tieren bei experimentell induzierter Pankreatitis zwei Effekte. Zum einen war die Trypsinaktivität erhöht, zum anderen verlief die Pankreatitis milder, da vermehrt Apoptose anstelle von Nekrose der Azinuszellen auftrat.
In dieser Studie wurde mittels uni-direktionaler DNA-Sequenzierung das gesamte CTSL1 untersucht. Dabei fanden wir insgesamt drei seltene nicht-synonyme Varianten. Die Variante c.5A>C (p.N2T, rs112682750) fanden wir bei einem Patienten, wobei diese Variante bereits bei Kontrollen beschrieben wurde. Die Varianten c.126+1G>A und c.915A>C (p.E305D) lagen bei jeweils einer Kontrolle vor. Sowohl seltene als auch häufige Varianten und die berechneten Haplotypen zeigten keinen signifikanten Verteilungsunterschied zwischen Patienten und Kontrollen. Demnach besteht keine Assoziation von Varianten des CTSL1 und CP.
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Wilcox, Donna. "The role of cathepsin L in elastin degradation." Thesis, Open University, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.277118.
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Dong, Qian. "Functional Characterization of a Cathepsin L in Drosophila Melanogaster." TopSCHOLAR®, 2015. http://digitalcommons.wku.edu/theses/1525.
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The Drosophila dorsal Air Sac Primordium (ASP) is a tracheal tube that invasively grows toward and into the wing imaginal disc. The unfolding of Drosophila wing is a process following eclosion with a cuticular bilayer replacing epithelial cells originally packing the wing. We reasoned that protease functions might be needed for the invasion of ASP into the wing imaginal disc as well as the rearrangement of epithelia cells during wing unfolding. Our study is particularly focused on understanding the role of a Cathepsin L like cysteine protease (CP1) in the development of dorsal ASP and wing development of Drosophila melanogaster. To analyze the function of CP1, we overexpressed and knocked down CP1, respectively, using UAS-GAL4 system in combination with RNA interference technology. We found that both the knockdown and overexpression of CP1 in ASP resulted in perturbed growth, migration and weakened invasion of ASPs. We further explored the mechanism by which CP1 regulates ASP development and found that CP1 is capable of degrading collagen IV, a component of extracellular matrix. For wing development, we observed that both the knockdown and overexpression of CP1 in wing imaginal discs interrupted with normal wing development. In summary, our study demonstrated that CP1 facilitates the normal development of ASPs by degrading extracellular matrix and regulates wing development via a complex network of signaling pathways and protein interactions. Knowledge gained from this study has the potential to help us better understand the invasion of tumor cells through the extracellular matrix in humans.
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Arispe, Angulo Wara Milenka Trawick Mary Lynn. "Inhibitors of human cathepsin L and cruzain as therapeutic agents." Waco, Tex. : Baylor University, 2008. http://hdl.handle.net/2104/5290.
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Burton, LizaJoy. "Snail-Cathepsin L Signaling in Human Breast and Prostate Cancers." DigitalCommons@Robert W. Woodruff Library, Atlanta University Center, 2017. http://digitalcommons.auctr.edu/cauetds/60.
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Prostate and breast cancer are the leading causes of cancer-related death in men and women, respectively, and metastasis is the primary factor underlying the high mortality rates.1 Snail transcription factor is an important molecule that drives prostate and breast cancer metastasis through the process of epithelial mesenchymal transition (EMT). Proteolytic enzymes that promote invasion and metastasis such as the lysosomal cysteine protease cathepsin L (Cat L) have been shown to degrade E-cadherin, promoting the epithelial mesenchymal transition (EMT).2 It has also been shown that silencing Cat L can inhibit transforming growth factor-beta (TGF-β)-mediated EMT by suppressing Snail transcription factor.3 Several recent studies have highlighted an additional unexpected localization and site of action for Cat L within the nucleus in breast, colon and prostate cancer.4 Natural products have been shown to be efficacious in prevention and possible treatment of cancer.5 Specifically, we have been studying Muscadine Grape Skin Extract (MSKE) as a possible candidate to inhibit Snail signaling. MSKE has previously been shown to promote prostate cancer apoptosis.6 We hypothesized that Snail promotes nuclear localization of Cat L, which promotes EMT associated with increased migration and invasion, and that antagonizing Snail-Cat L signaling would lead to mesenchymal epithelial transition (MET). We showed for the first time that MSKE promotes apoptosis through induction of endoplasmic reticulum stress response and autophagy. Additionally, MSKE could inhibit Snail-mediated EMT via scavenging reactive oxygen species. Moreover, Snail could promote nuclear localization of Cat L, which then promoted cleavage of CDP/Cux, increased Snail transcription and decreased E-cadherin transcription by direct promoter binding of cleaved CDP/Cux, leading to EMT associated with increased migration and invasion. Interestingly, Z-FY-CHO, a small molecule specific inhibitor of Cat L, as well as MSKE could antagonize this signaling by promoting nuclear to cytoplasmic re-localization of Cat L. Therefore, we have dissected novel mechanisms of action of Snail and how it can be antagonized by MSKE natural product.
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Patel, Amita. "Transcriptional regulation of cathepsin L during mouse mammary gland involution a test of STAT3 involvement /." Click here for download, 2006. http://wwwlib.umi.com/cr/villanova/fullcit?p1432835.
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Norbury, Luke James, and s9806495@student rmit edu au. "Structure, Function and Evolutionary Studies of Fasciola Cathepsin L-like Proteases." RMIT University. Applied Science, 2008. http://adt.lib.rmit.edu.au/adt/public/adt-VIT20081204.160915.
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Fasciola cause considerable monetary loss in the agriculture industry, while parasitism of humans is an emerging disease. Fasciola cathepsin L-like proteases are believed to aid parasite invasion and survival through a range of functions including feeding, immune evasion and modulation, tissue migration, egg production and excystment. As such these proteases are considered good targets for chemotherapies and vaccine development. Fasciola cathepsins are evolutionarily divided into clades that reflect function and life stage of expression. Analysis of F. gigantica genomic DNA and mRNA identified novel cathepsin L-like sequences which are incorporated into a phylogenetic analysis of the complete Fasciola cathepsin L-like protease family. Analysis of mRNA transcripts isolated in this study also points to trans-splicing occurring amongst cathepsin transcripts, the first time this has been identified in Fasciola species. S2 subsite specificity is important in determining substrate interactions with cathepsin L-like proteases. Previous work has shown that amino acid substitutions at this site can dramatically influence substrate specificity. A number of substitutions, specifically those that have been observed, or predicted to occur during the evolution of Fasciola cathepsins L-like proteases, were introduced into the S2 subsite of FhCatL5 at aa69 to determine their influence. The introduction of L69C and L69S substitutions resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants amongst fluke. The L69F variant showed an increase in the ability to cleave substrates with P2 proline, indicating F69 variants expressed by fluke are also likely to have this ability, similar to that shown with L69Y and FhCatL2. The introduction of a L69W substitution leads to increased cleavage of substrates with P2 proline, along with a decrease in cleavage of substrates with P2 phenylalanine. FgCatL1G transcripts were isolated from F. gigantica metacercariae. This contrasts with FhCatL5 and FhCatL2 which have been isolated in adult F. hepatica. These cathepsins differ at aa69, possessing tryptophan, leucine and tyrosine respectively. The processing and substrate specificities of each recombinant enzyme was analysed and compared. While FhCatL5 and FhCatL2 process in vitro in a manner similar to that reported for FhCatL1, FgCatL1G requires different processing conditions, including neutral pH. Combined with FgCatL1G possessing increased stability at acidic pH, this reflects the different environment into which FgCatL1G is expressed by immature compared to the adult flukes. The substrate specificity of FgCatL1G also differed from previously reported cathepsins, with a preference for P2 proline and low activity against substrates with P2 phenylalanine. This is the first time recombinant expression and purification of a cathepsin L-like protease specific to the immature life stages of Fasciola has been undertaken and had enzyme specificity analysed. This work has expanded knowledge of the repertoire of cathepsin proteases expressed at various life-stages of the liver fluke. Vaccination and/or drug inhibition studies may in the future be targeted towards cathepsins that are expressed in either the adult or immature stage, or perhaps both in a multi-targeted approach. The knowledge gained in this study may allow such targets to be chosen.
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Spira, Daniel. "Genetische Analyse zellspezifischer Funktionen der lysosomalen Cysteinprotease Cathepsin L im Mausherz." [S.l. : s.n.], 2006. http://nbn-resolving.de/urn:nbn:de:bsz:25-opus-61727.
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Stairiker, Patricia A. "The role of cathepsin L in involution and the termination of lactation in the mouse mammary gland." Click here for download, 2006. http://wwwlib.umi.com/cr/villanova/fullcit?p1432836.
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Gewies, Andreas. "Investigation of the ubiquitin-specific protease UBP41 and of the lysosomal cysteine proteases cathepsin-L and cathepsin-B as potential mediators of proapoptotic signalling." Diss., lmu, 2004. http://nbn-resolving.de/urn:nbn:de:bvb:19-16836.
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McCarroll, Douglas. "The effects of Trypanosoma brucei and mammalian-derived extracellular cathepsin-L on myocardial function." Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/5170/.
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African trypanosomiasis is a neglected tropical disease affecting both animals and humans in sub-Saharan Africa. The disease is caused bythe protozoan parasite Trypanosoma brucei, which is transmitted by the tsetse fly (Glossina sp.) vector. In animals, infection leads to severe muscle atrophy and anaemia resulting in significant production and economic losses. In humans, infection leads to both neurological and cardiac dysfunction and can be fatal if untreated. While the neurological-related pathogenesis is well studied, and indeed is responsible for the colloquial name “Sleeping Sickness”, the cardiac pathogenesis remains unknown. Previous studies interpreted cardiac dysfunction as being due to immune/inflammatory responses. However, recent work examining the parasite’s interaction with the blood brain barrier, the traversal of which is important for development of neurological signs, has identified direct immune/inflammatory independent mechanisms involving calcium (Ca2+) signalling. The current study exposed isolated ventricular cardiomyocytes and adult rat hearts to T. brucei to test whether trypanosomes can alter Ca2+ signalling and cardiac function independent of a systemic immune/inflammatory response. Using a high-throughput method of observing spontaneous contractile activity in isolated cardiomyocytes, we were able to determine that the presence of T. b. brucei parasites resulted in more cardiomyocytes exhibiting spontaneous contractile events. Moreover, when the parasites were removed by careful centrifugation, the culture supernatant had the same effect. Confocal Ca2+ imaging identified an increase in the frequency of arrhythmogenic spontaneous diastolic sarcoplasmic reticulum (SR)-mediated Ca2+ release (Ca2+ waves). Studies utilising specific inhibitors, recombinant protein and RNA interference all demonstrated that this altered SR function was due to cathepsin-L; a cysteine protease produced by T. brucei (TbCatL). Experiments utilising a Langendorff perfusion method revealed that trypanosome culture supernatant could induce ventricular premature contractions in 50% of a cohort of ex vivo whole rat hearts. Mechanistic experiments were performed on single isolated cardiomyocytes stimulated at 1.0 Hz and perfused first with control media followed by trypanosome culture supernatant. The protocol utilised triple caffeine applications: (i) prior to stimulation to empty the SR of Ca2+, (ii) after perfusion with control media and after supernatant to determine the SR Ca2+ content and sarcolemmal extrusion of Ca2+ following each solution. Results were normalised to a parallel set of cardiomyocytes perfused with control media only as time controls. These experiments revealed a 10-15% increase in SR Ca2+ reuptake by the SR Ca2+ ATPase (SERCA) but a reduced SR Ca2+ content suggesting a concomitant increase in SR-mediated Ca2+ leak. This conclusion was supported by the data demonstrating that TbCatL increased Ca2+ wave frequency. These effects were abolished by autocamtide-2-related inhibitory peptide (AIP), highlighting a role for Ca2+/calmodulin kinase II (CaMKII) in the TbCatL action on SR function. When cytosolic diastolic Ca2+ was measured in cardiomyocytes with SR function inhibited by ryanodine and thapsigargin, trypanosome supernatant prevented a decline in cytosolic diastolic Ca2+ that was observed in control media. AIP did not abolish this effect suggesting that TbCatL may raise diastolic Ca2+ that could activate CaMKII leading to the observed effects. These data demonstrated for the first time that African trypanosomes alter cardiac function independent of a systemic immune response via a mechanism involving extracellular cathepsin-L-mediated changes in SR function. Utilising the same (culture adapted and monomorphic) strain of T. brucei as the in vitro experiments, Lister 427, in a rat model of infection we found no significant increase in the arrhythmia frequency as measured by a 15 min electrocardiogram (ECG). However, when hearts were removed and Langendorff perfused with the addition of isoproterenol the arrhythmia frequency was increased. When the pleomorphic strain T. b. brucei TREU 927 was used in rats with continuous ECG recording from biopotential telemetry there was a significant increase in arrhythmia frequency in the infected rats. When hearts were removed and Langendorff perfused with isoproterenol there was a similar increase in arrhythmia frequency as observed with the 427 infected hearts. This suggests that a cardiac dysfunction phenotype is present during trypanosome infections in an animal model providing the basis for future therapeutic work. The relationship between arrhythmogenic SR-mediated Ca2+ release and TbCatL has parallels with endogenous extracellular cathepsin-L (CatL). It has been demonstrated that a basal level of CatL is necessary for normal cardiac function. However, in coronary heart disease (CHD) CatL levels are increased in the serum of patients correlating with the severity of disease. The effects of raised CatL on cardiac function remain unknown. Work in our lab has identified that ex vivo Langendorff perfused hearts that have undergone a 30 min period of ischaemia followed by 90 min reperfusion show greater CatL activity in coronary effluent than hearts perfused without ischaemia. In addition, preliminary data collected in this thesis suggest that human patients that have suffered a myocardial infarction and have undergone reperfusion via percutaneous coronary intervention (PCI) showed higher CatL levels in post-reperfusion serum samples compared to pre-reperfusion serum. When severity of heart function in patients (measured as left ventricular volume at systole and diastole, ejection fraction, infarct size and area at risk) was assessed by magnetic resonance imaging (MRI) in a preliminary study, there was a positive correlation with serum CatL levels. Using recombinant CatL on isolated rat ventricular cardiomyocytes it was found that the SR Ca2+ content and the stimulated Ca2+ transient were significantly reduced in a concentration dependent manner. This suggests a CatL dependent SR dysfunction. This conclusion was supported by an increase in Ca2+ wave frequency measured by confocal Ca2+ imaging in isolated cardiomyocytes. The work in this thesis demonstrates a role for both mammalian-derived and exogenous extracellular cathepsin-L proteases in arrhythmogenic SR-mediated Ca2+ release.
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胡可進 and Kejin Hu. "Molecular cloning and characterization of the cathepsin L gene from the marine shrimp metapenaeus ensis." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2003. http://hub.hku.hk/bib/B3124421X.
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Behn, Claas-Olsen [Verfasser]. "Die Rolle von Sequenzvarianten im Cathepsin L als Risikofaktoren der chronischen Pankreatitis / Claas-Olsen Behn." Greifswald : Universitätsbibliothek Greifswald, 2016. http://d-nb.info/1122310722/34.
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Lamore, Sarah Diane. "Identification of Cathepsin B and L as Novel Uva Targets Upstream of Cutaneous Lysosomal-Autophagic Dysregulation." Diss., The University of Arizona, 2012. http://hdl.handle.net/10150/228173.
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Chronic exposure to solar UVA plays a causative role in skin photoaging and photocarcinogenesis. Guided by exploratory difference-in-gel-electrophoresis (DIGE)-proteomics, we identified the thiol-dependent cysteine-proteases cathepsin B and cathepsin L as novel UVA-targets undergoing photo-oxidative inactivation upstream of autophagic-lysosomal dysfunction. In human skin fibroblasts, exposure to noncytotoxic doses of chronic UVA (9.9 J/cm ², twice a week, 3 weeks) caused pronounced photooxidative impairment of cathepsin B and L enzymatic activity suppressed by antioxidant intervention. Western blot analysis revealed extensive 4-hydroxy-2-trans-nonenal (4-HNE) modification of cathepsin B in UVA-exposed fibroblasts. Consistent with lysosomal impairment, accumulation of cellular autofluorescent material colocalizing with lysosomes was observed by confocal fluorescence microscopy, and extensive deposition of lipofuscin was detectable by transmission electron microscopy (TEM). Lysosomal expansion was further evidenced by increased immunodetection of lysosomal associated membrane protein-1 (Lamp-1) and Lysotracker-based flow cytometric analysis. While lysosomal membrane integrity remained intact, autophagic blockade was suggested by accumulation of cellular protein levels of LC3-II and p62 (sequestosome 1) in UVA-exposed fibroblasts. Furthermore, UVA-exposure modulated transcriptional levels of p62 (sequestosome 1, SQSTM1), α-synuclein (SNCA), and transglutaminase-2 (TGM2). Strikingly, pharmacological cathepsin B/L inhibition using CA074Me mimicked UVA-induced accumulation of lipofuscin and autophagic-lysosomal proteins (Lamp-1, LC3-II, and p62), as well as changes at the transcriptional levels. In order to determine if UVA-induced lysosomal impairment requires single or dual inactivation of cathepsin B and/or L, we used a genetic approach (siRNA) to selectively downregulate enzymatic activity of these target cathepsins. Monitoring protein levels of Lamp-1, LC3-II, and p62, we observed that only dual genetic antagonism (targeting both CTSB and CTSL expression) could mimic UVA-induced autophagic-lysosomal alterations, whereas single knockdown (targeting CTSB or CTSL only) did not reproduce the UVA-induced phenotype. Similarly, TEM revealed massive accumulation of lipofuscin-containing lysosomal vesicles in fibroblasts only after CTSB/CTSL-double knockdown. Taken together, our data indicate for the first time that UVA impairs lysosomal function causing autophagic-lysosomal alterations downstream of cathepsin B/L enzymatic inactivation. This work provides evidence for a heretofore unrecognized 'double-hit' mechanism of UVA skin photodamage where primary photo-oxidative insult occurs simultaneously with impaired clearance of damaged molecules and organelles downstream of dual inactivation of cathepsin B and L.
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Beton, Daniela. "Estrutura e função das cisteína proteinases intestinais do besouro Tenebrio molitor." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-28042010-141010/.
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A catepsina L é uma cisteína proteinase da família da papaína (clã CA, família C1), sendo esta família a mais conhecida entre as cisteína proteinase. A catepsina L, como outras proteinases da família C1, é sintetizada como uma pró-enzima inativa que é ativada através da remoção do pró-peptídeo. Os pró-peptídeos das catepsinas da subfamília catepsina L apresentam um motivo consenso, denominado motivo ERFNIN. A catepsina L corresponde a principal proteinase digestiva em Tenebrio molitor. No nosso laboratório 3 pró-catepsinas L (pCALs) foram clonadas e seqüenciadas a partir de uma biblioteca de cDNA de intestino médio de larvas de T. molitor: pCAL1 (CAL lisossomal), pCAL2 e pCAL3 (enzimas digestivas). Estas proteinases apresentam o motivo ERFNIN e os resíduos envolvidos na catálise: Cys25, His169, e Asn175 com Gln19 (numeração da papaína). Neste trabalho descrevemos a clonagem em vetores de expressão e a expressão em bactérias das sequências codificadoras de pCAL1, pCAL2 e pCAL3. As pró-catepsinas L recombinantes foram purificadas por cromatografia de afinidade e a incubação em pH ácido resultou na formação das enzimas maduras CAL1, CAL2 e CAL3 com atividade sobre o substrato Z-FR-MCA. O anticorpo policlonal anti-pCAL2 foi produzido em coelho e reconheceu pCAL2 e CAL2 em immunoblots. Experimentos de immunoblots com diferentes tecidos de T. molitor mostraram que o anticorpo policlonal anti-pCAL3 reconheceu pCAL3 e CAL3 nos dois terços anteriores do intestino médio de larvas de T. molitor. Estudos de imunocitolocalização indicam que a catepsina L 3 ocorre em vesículas no intestino médio anterior e em microvilosidades no intestino médio posterior. Para os experimentos de cristalização, nós expressamos pCAL1, pCAL2 e pCAL3 como mutantes Cys25→Ser inativos. pCAL3Cys26Ser foi cristalizada por difusão de vapor (gota sentada) contra 0,1-1,6M de dihidrogênio fosfato de amônio. Os cristais são monoclínicos com grupo espacial C2 e parâmetros de célula: a=57,634 Å, b=89,322 Å, c=70,076 Å, α=γ=90°, β=92,502° e uma molécula na unidade assimétrica. A e strutura foi determinada por substituição molecular usando a estrutura de Fasciola hepatica (42% de identidade) como modelo. O modelo foi refinado a 2,1 Å com fator R final de 16,19% (Rfree=20,5%). pCAL2Cys25Ser foi cristalizada por difusão de vapor (gota sentada) contra acetato de sódio 0,2M, cacodilato de sódio 0,1M pH6,6-6,7 e 20% de PEG 8000. Os cristais são triclínicos com grupo espacial P1 e parâmetros de célula: a=51,669 Å, b=52,37 Å, c=59,716 Å, α= 91,278°, γ=109,586°, β=91,547° e duas moléculas na unidade assimétrica. A estrutura foi determinada por substituição molecular usando a estrutura da pCAL3 (44% de identidade) como modelo. O modelo foi refinado a 2,0 Å com fator R final de 17,61% (Rfree=22,48%). A estrutura terciária da pró-catepsinas L digestivas é muito similar as estruturas de cisteína proteinases da família da papaínaCathepsin L is a cysteine proteinase of the papain family (clan CA, family C1), which is the most known among the cysteine proteinases. Cathepsin L, like other proteinases of family C1, is synthesized as an inactive proenzyme that is activated by propeptide removal. The propeptide of cathepsin L-like subfamily contain a highly conserved motif, the so called ERFNIN motif. Cathepsin L corresponds to the major digestive proteinase in Tenebrio molitor. In our laboratory, 3 procathepsins L (pCALs) were cloned and sequenced from a cDNA library prepared from T. molitor larval midguts: pCAL1 (lysosomal CAL), pCAL2 and pCAL3 (digestive enzymes). These proteinases have ERFNIN motif and 3 residues directly involved in catalysis: Cys25, His169, Asn175 with Gln19 (papain numbering). In this work we report the cloning into the expression vector and bacterial expression of the sequences coding pCAL1, pCAL2 and pCAL3. The recombinant procathepsins L were purified by affinity chromatography and activation of these enzymes occurs under acidic conditions. The cathepsins L (CAL1, CAL2 and CAL3) were able to hydrolyse Z-FR-MCA. The polyclonal antibody anti-pCAL2 was produced in rabbit and recognized pCAL2 and CAL2 on immunoblots. Immunoblot analyses of different T. molitor larval tissues demonstrated that the polyclonal antibody anti-pCAL3 recognised pCAL3 and CAL3 in the anterior two-thirds of midgut tissue of T. molitor larvae. Immunolocalization studies indicate that cathepsin L 3 occurs in vesicles in the anterior midgut and microvilli in posterior midgut. To crystallographic studies we expressed pCAL1, pCAL2 and pCAL3 as inactive Cys25→Ser mutants. pCAL3Cys26Ser was crystallized by vapor diffusion in sitting drops against 0.1-1.6 M mono-ammonium dihydrogen phosphate. The crystals are monoclinic, belonging to space group C2, with cell parameters: a = 57.634 Å, b = 89.322 Å, c = 70.076 Å, α = γ =90°, β = 92.502° and contain one molecule in the asymmetric unit. The structure was determined by molecular replacement using the structure of Fasciola hepatica procathepsin L (42.5% identity) as a model. The model was refined at 2.1 Å resolution with an R factor of 16.19% (Rfree = 20.5%). pCAL2Cys25Ser was crystallized by vapor diffusion in sitting drops against 0.2M sodium acetate, 0.1M sodium cacodylate pH 6.6-6.7 and 20% polyethylene glycol 8,000. The crystals are triclinic, belonging to space group P1, with cell parameters: a = 51.669 Å, b = 52.37 Å, c = 59.716 Å, α = 91.278° γ = 109.586°, β = 91.547° and contain two molecules in the asymmetric unit. The structure was determined by molecular replacement using the structure of procathepsin L 3 (44 % identity) as a model. The model was refined at 2.0 Å resolution with an R factor of 17.61% (Rfree = 22.48%). The tertiary structure ofdigestive procathepsins L is very similar to papain-like cysteine proteinases structures
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Bryce, Steven David. "Identification and characterisation of a novel family of human genomic sequences closely related to the Cathepsin L gene." Thesis, University of Newcastle Upon Tyne, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.309763.
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He, Weihong. "Investigation of Runx1 and Cathepsin-L as potential therapeutic targets for myocardial infarction and cardiac ischaemia-reperfusion injury." Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8201/.
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Myocardial infarction (MI) is a leading cause of death worldwide. The fundamental treatment for MI is reperfusion of the ischaemic myocardium by primary percutaneous intervention (PPCI). Advances in PPCI techniques in recent years has led to an increased survival rate in patients undergoing MI. A larger proportion of patients surviving post- MI following PPCI face a later risk of developing heart failure due to the substantial left ventricular (LV) injury caused by MI. Post MI heart failure has become a frequent complication of MI and is associated with an extremely high mortality. To develop novel therapies for MI, two phases of the disease can be targeted. One is the period following PPCI in which ischaemia-reperfusion (IR) injury contributes to cardiac damage. At this stage, reducing IR injury can reduce cardiac damage and thereby improve cardiac function and survival. The other is the period of developing heart failure post MI, where adverse cardiac remodelling contributes to worsening cardiac function and progression of heart failure. Therefore, therapies targeting the remodelling process following MI may prevent deterioration of cardiac function and protect against heart failure. Development of novel therapy for MI and IR injury requires experimental models which model the human disease. The clinical relevance of the animal model is associated with successful translation of novel therapies. Mouse models have become commonly used in translational research due to their cost efficiency and the ease of developing genetically-modified mice. However, inducing MI and IR injury in mice is a complex technique due to their small size and thus application of mouse models of MI and IR injury has been limited. The work in this thesis, has focussed on the MI and IR injury models in mice. MI and IR injury were successfully induced by performing left anterior descending coronary artery ligation in open-chest microsurgery and cardiac function following MI and IR injury was characterized using echocardiography and intra-left ventricular pressure-volume (PV) loops. Using these models and phenotyping techniques, two potential therapeutic targets were evaluated, one is cathepsin-L inhibition by a pharmacological inhibitor CAA0225, while the other was Runx1 deficiency which was assessed in a cardiomyocyte-specific Runx1-deficient mouse. Cathespin-L is a cysteine protease typically localized in lysosomes. In patients with coronary heart diseases (CHD), cathepsin-L has been found at increased levels in the serum and plasma, and the elevation of cathepsin-L is correlated with disease severity. In this study, using a cathepsin-L inhibitor CAA0225, we tested the effects of cathepsin-L inhibition during IR injury in both Langendorff perfused ex vivo isolated rat hearts and an in vivo mouse model. In the Langendorff isolated heart model of IR injury, applying CAA0225 significantly improved systolic and diastolic cardiac function during IR injury and this improvement was paralleled by reduced infarct size. An inhibitor (CA074Me) of a separate member of the cathepsin family (cathepsin-B) was also tested in the Langendorff perfused hearts and although it improved the speed at which systolic function recovered post I/R injury, no other changes where noted in cardiac function. The beneficial effect of CAA0225 was further tested in in vivo mouse models of MI and IR injury. Intravenous injection of CAA0225 during the ischaemic period significantly improved cardiac systolic function following MI and IR injury at both 2 and 4 weeks, respectively. In vivo administration of CAA0225 reduced infarct size, as measured by a double-dye staining technique and may relate to normalization of adverse calcium handling in cardiomyocytes by CAA0225 during IR injury. The Runx1 gene is known to be related to lineage differentiation and function within the hematopoietic system. RUNX1 has been reported to be activated in border zone cardiomyocytes in humans and mice post MI. This finding indicated that the Runx1 gene may play a role in cardiac remodelling post MI. To investigate the role of Runx1, MI and IR injury was induced in a tamoxifen-inducible cardiomyocyte-specific Runx1-deficient mouse model. Runx1 deficiency significantly preserved cardiac function 8 weeks post MI and post IR injury injury and prevented LV dilation post MI. The underlying mechanism may relate to improved cardiomyocyte calcium handling. In conclusion, the work in this thesis has assessed two separate targets, cathepsin-L and Runx1 for their therapeutic potential in MI and IR injury. This work has significantly enhanced our knowledge on these novel targets and will inform the development of translational strategies for treatment of patients with MI.
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Florence, William C. "Increased stability of class II MHC-peptide complexes in macrophages infected with Mycobacterium avium and the examination of a novel role for Cathepsin L in the innate immune response to Francisella Novicida infection." The Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=osu1173298339.
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Kürpick, Birte Verena [Verfasser]. "Seroepidemiologische Studie zur Verbreitung von Fasciola hepatica in Deutschland und Evaluierung eines rekombinanten Cathepsin L-ELISAs / Birte Verena Kürpick." Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2013. http://d-nb.info/1037856473/34.
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Nepal, Rajeev Mani. "Class II MHC function in macrophages and mice infected with mycobacterium." The Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=osu1141761508.
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Vöckler, Steffi [Verfasser]. "Die Bedeutung von Cysteinproteasen (Cathepsin B, H, L und S) für die Pathogenese der AA- und AL Amyloidose / Steffi Vöckler." Magdeburg : Universitätsbibliothek, 2012. http://d-nb.info/1051501679/34.
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Herms, Max [Verfasser], Jonas [Akademischer Betreuer] Rosendahl, Joachim [Akademischer Betreuer] Mössner, Markus M. [Gutachter] Lerch, and Thomas [Gutachter] Berg. "Genetische Analyse des Cathepsin L bei chronischer Pankreatitis / Max Herms ; Gutachter: Markus M. Lerch, Thomas Berg ; Jonas Rosendahl, Joachim Mössner." Leipzig : Universitätsbibliothek Leipzig, 2012. http://d-nb.info/1238077242/34.
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Paula, Fernando Fonseca Pereira de. "Biologia molecular aplicada à identificação de alvos para o controle do bicudo da cana‐de‐açúcar, Sphenophorus levis." Universidade Federal de São Carlos, 2012. https://repositorio.ufscar.br/handle/ufscar/5410.
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Previous issue date: 2012-02-29Universidade Federal de Minas Gerais
The sugarcane weevil, Sphenophorus levis, is an insect that feeds on the rhizome of sugarcane in its larval stage, boring channels that cause damage and death to the plant. Conventional methods of insect control have not been efficient. The aim of the present study was to generate knowledge on the biology of this insect on the molecular level using a transcriptomic approach to determine potential targets genes for the engineering of insect-resistant plants. After sequence processing and assembly using the dCAS program, 3804 sequences were grouped into 201 contigs and 1363 singlets, which were manually annotated. Several plant cell wall degrading enzymes were identified, including pectinases and cellulases. An invertasecontaining contig was identified for the first time in a coleopteran. Total probable digestive enzymes accounted for 19.3% and unknown genes accounted for 28.8% of the total number of expressed sequence tags. Considering the predominance of cathepsin L enzymes among the digestive enzymes found in the transcriptome (54.3%) and their importance to the breakdown of proteins in the insect digestive process, a cDNA clone encoding a cathepsin L enzyme, denominated Sl-CathL, was chosen for recombinant expression in Pichia pastoris cells, characterization and in vitro inhibition by the sugarcane cystatin CaneCPI-4 (Ki = 0.196 nM). Immunolocalization assays demonstrated the production of Sl-CathL in the midgut epithelium and secretion into the gut lumen from vesicles containing the enzyme. S. levis demonstrated sensitivity in triggering RNA interference machinery induced by dsRNA injections in the body cavity and gene-specific knockdown was confirmed by qRT-PCR. Larvae injected with V-ATPase E dsRNA died within three weeks after injection and serpin 1-silenced larvae either exhibited delayed development, arresting in the pupal stage, or died as pharate adults. In conclusion, transcriptome analyses, together with the inhibition of the main digestive enzyme by Cane-CPI-4 and gene silencing using RNAi, are promising procedures for the development of transgenic sugarcane plants to enhance resistance to Sphenophorus levis.
O bicudo da cana-de-açúcar, Sphenophorus levis, é um inseto que na fase larval se alimenta do rizoma da cana-de-açúcar cavando galerias que causam danos e levam à morte das plantas. Os métodos convencionais de controle disponíveis não tem sido eficientes contra esse inseto. O objetivo desse estudo foi gerar conhecimento sobre a biologia desse inseto em nível molecular utilizando uma abordagem transcriptômica para a identificação de possíveis genes alvo, visando futuros estudos para desenvolvimento de plantas transgênicas resistentes ao ataque deste inseto. Após o processamento e montagem das sequências utilizando o programa dCAS, 3804 sequências foram agrupadas em 201 contigs e 1363 singlets, os quais foram manualmente anotados. Foram identificados diversos genes de degradação de parede celular, incluindo pectinases e celulases. Pela primeira vez um contig contendo uma sequência que codifica uma invertase foi identificado em um coleóptero. As prováveis enzimas digestivas totalizam 19,3% e os genes desconhecidos representam 28,8% do total de sequências expressas. Considerando a predominância de catepsinas L dentre as enzimas digestivas identificadas no transcriptoma (54,3%) e a sua importância para a hidrólise de proteínas nos processos digestivos, um clone de cDNA codificando uma catepsina L, denominado Sl-CathL, foi escolhido para a expressão recombinante em células de Pichia pastoris, caracterização e inibição in vitro utilizando a cistatina da cana-de-açúcar CaneCPI-4 (Ki = 0,196 nM). Os ensaios de imunolocalização evidenciaram a produção da Sl-CathL no epitélio do intestino médio e a secreção de vesículas, contendo a enzima, no lúmen do intestino. Larvas do inseto S. levis também apresentaram sensibilidade para disparar a maquinaria de RNA de interferência induzida por injeções de dsRNA na cavidade corpórea e o silenciamento gênico específico foi confirmado por qRT-PCR. As larvas que receberam injeções com dsRNA da V-ATPase E morreram dentro de três semanas, enquanto as larvas que tiveram a serpina 1 silenciada exibiram atraso no desenvolvimento, aprisionamento na fase pupal, ou morreram como adultos farados. Desse modo, concluímos neste trabalho iniciado a partir da análise do transcriptoma, que a inibição da principal enzima digestiva pela Cane-CPI-4 e o silenciamento gênico via RNAi são alternativas promissoras para o estabelecimento de plantas resistentes ao inseto e podem ser aplicadas no desenvolvimento de plantas de cana-de-açúcar transgênicas com o objetivo de aumentar sua resistência ao Sphenophorus levis.
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Bifano, Thaís Duarte. "Fisiologia molecular intestinal de Dysdercus Peruvianus (Hemiptera)." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-27112008-093426/.
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A partir da identificação de catepsinas L em ensaios in vitro e em zimogramas partimos para purificação desta enzima no inseto. A região V2 foi selecionada como fonte de obtenção da cisteína proteinase já que dentre os três ventrículos o segundo apresentou maior atividade específica. Após diversas tentativas de isolar esta proteinase, foi estabelecida uma marcha de purificação que envolvia em todas as etapas a participação de metil metanosulfonato (MMTS), o que inativa a proteinase evitando assim autólise ao longo do processo de purificação. A marcha consistiu de três passos cromatográficos (troca-iônica, filtração em gel e afinidade, nesta ordem) onde foi observada a presença de duas cisteína proteinases, cada uma apresentando respectivamente as seguintes massas moleculares 32 e 45 kDa (SDSPAGE). As duas cisteína proteinases possuem o mesmo pH ótimo igual a 6,3. Além disso, estas enzimas foram termicamente inativadas a 40 ºC segundo uma cinética de primeira ordem aparente, sugerindo a existência de apenas uma espécie molecular de cada enzima na preparação com meia vida de 5 minutos para cis 1 e 4,8 minutos para cis 2. Foi determinada a constante de dissociação entre enzimainibidor, onde foi observado os valores de 17,3 nM para cis 1 e 7,11 nM para cis 2 através da titulação por E-64. A eficiência de catálise cis 1 e cis 2 é maior para o substrato sintético Z-FR-MCA do que para Z-RR-MCA, indicando que tratava-se de catepsinas L. Com o intuito de descrever os mecanismos moleculares por trás dos fenômenos fisiológicos no intestino médio do Hemiptera Dysdercus peruvianus foi construída uma biblioteca de cDNA a partir de mRNA deste tecido. Utilizamos ESTs provenientes desta biblioteca com o objetivo de identificar genes transcritos relacionados com proteínas de transporte de glicose além de enzimas digestivas. Após o processamento das leituras, surgiram 1053 ESTs úteis. Montando estes ESTs por alinhamento de bases, foram produzidos 62 contíguos e 841 singletos, o que totaliza 903 seqüências únicas. Entre as seqüências homólogas encontradas as mais relevantes para o nosso estudo foram: β-glicosidase (marcadora de membranas microvilares), α-glicosidase (marcadora de membranas 8 perimicrovilares), aminopeptidase (espaço perimicrovilar), catepsina L (conteúdo de vesículas secretoras) e proteína transportadora de açúcar do tipo GLUT. Estas seqüências encontradas tiveram a sua transcrição específica (ou preferencial) averiguada por RT-PCR semiquantitativo nos diferentes tecidos do inseto estudado (intestino médio, túbulo de Malpighi, corpo gorduroso, glândula salivar, ventrículo 1, ventrículo 2 e ventrículo 3 do intestino médio). O transporte de glicose e água in vivo foi estudado. Para isso, os insetos foram alimentados com uma solução contendo glicose e corante não absorvível seguido de dissecação e análise do conteúdo luminal. O transporte de água e glicose foi inibido por floretina 0,2 mM (inibidor do uniportador GLUT) e por florizina 0,1 mM (inibidor do simportador SGLT) e ativado por K2SO4 50 mM. Isto sugere a presença do transportador do tipo uniportador (GLUT), e do simportador K+-glicose (SGLT), ambos co-transportando água. O transcriptoma revelou proteína homóloga a transportador GLUT cuja seqüência está parcialmente completa e foi analisada com ferramentas de bioinformáticaAfter identification of cathepsins L in vitro assays and in zimograms we began to purify this enzyme in insect midgut. The region V2 was selected as a source of material for purifying a cysteine proteinase because it contains most of the activity of that proteinase. After several attempts to purify this proteinase, an effective process was developed that avoid autolysis with methyl methanethiosulfonate (MMTS), a sulfhydryl-reactive and reversible sulfonating reagent for thiol-containing molecules. The purify process was made by three chromatographic steps (anion-exchange column, gel filtration column and affinity column in this order), where two cysteine proteinase were purified, cys 1 and cys 2 with 32 and 45 kDa (SDS-PAGE). The two cysteine proteinases have the same pH optimum of 6.3. Besides that, these enzymes were thermicaly inactivated following apparent first-order kinetics with a half-life of 5 min (cys1) and 4.8 min (cys2) at 40 ºC. Both Cys are inhibited by E-64 with a KD of 17.3 nM (Cys 1) and 7.11 nM (Cys2). Both Cys are more active on Z-FR-MCA than on Z-RR-MCA, suggesting they are cathepsins-L. With purpose of describe the molecular mechanisms underlying physiological phenomena in midgut of the Hemiptera Dysdercus peruvianus a cDNA library was prepared from midgut mRNA. We used ESTs from this library to identify transcripts genes related with glucose transport proteins besides digestive enzymes. Analysis of 1053 high-quality expressed sequence tags (ESTs) yielded 903 unique sequences comprised of 62 contigs and 841 singlets. Among the homologous sequences found the following are more relevant to our aim: β-glucosidase (microvillar membrane marker), α-glucosidase (perimicrovillar membrane marker), aminopeptidase (perimicrovillar space marker), cathepsin L (vesicles content) and sugar transporter protein, GLUT. These sequences had its specific transcription (or preferential) verified by semi-quantitative RT-PCR on different insect tissues (malpighian tubules, salivary gland, fat body, midgut, midgut first ventriculus, second ventriculus and third ventriculus). The glucose and water absorption across the first ventriculus of the midgut of the Hemiptera Dysdercus peruvianus were determined. The insects were fed with a 10 glucose-non-absorbable dye solution, followed by periodical dissection of insects and analysis of ventriculus contents. The transport of water and glucose can be inhibited by 0.2 mM phloretin (GLUT inhibitor) and by 0.1 mM phlorizin (SGLT inhibitor) and is activated by 50 mM K2SO4 The results suggest that D. peruvianus has a transporter uniporter like (GLUT) and K+-glucose symporter like SGLT, both co-transporting water. The transcriptome showed a GLUT homologous protein which sequence is almost complete and was analyzed by bioinformatics tools
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Pimenta, Marcela Valente. "Avaliação da resistência de formas mutantes da enzima L-asparaginase a proteases séricas humanas." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/9/9134/tde-10092018-173712/.
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O tratamento para a Leucemia Linfoblástica Aguda LLA utiliza, entre outros fármacos, a enzima L-asparaginase (ASNase) proveniente da bactéria Escherichia coli. Reações imunológicas estão entre os problemas do tratamento com ASNase, e a formação de anticorpos contra essa proteína pode impedir o sucesso no tratamento. Duas cisteíno proteases lisossomais estão relacionadas com a degradação de ASNase nos seres humanos, a Catepsina B (CTSB) e Asparagina Endopeptidase (AEP). Em estudos prévios do nosso grupo obteve-se mutantes de ASNase resistentes a degradação por CTSB e/ou AEP in vitro. Nesse trabalho avaliamos essas mutantes quanto a sua citotoxicidade em linhagens celulares de leucemia e conduzimos estudos in vivo, aplicando as proteoformas de ASNases em camundongos Balb C para avaliar a atividade asparaginase sérica das enzimas ao longo do tempo, bem como obter informações sobre a formação de anticorpos contra essas proteoformas. Nos ensaios de citotoxicidade, duas das proteoformas testadas tiveram efeito citotóxico semelhante a forma selvagem, enquanto uma outra proteoforma tem a citotoxicidade sensivelmente reduzida. Já nos ensaios in vivo, uma proteoforma demonstrou meia vida sérica maior da atividade asparaginásica, e duas proteoformas causaram reduzida formação de anticorpos. Juntos, esses resultados colaboram para a obtenção de uma nova geração de ASNases com melhor biodisponibilidade, e efeitos adversos reduzidos, gerando a possibilidade de menores doses e frequência de aplicações.The Treatment for Acute Lymphoblastic Leukemia (ALL) includes the biopharmaceutical L-asparaginase (ASNase) from Escherichia coli. Immunological reactions are among the problems of treatment using ASNase, and the antibodies formation protein may prevent success in treatment. Lysosomal cysteine proteases are related to ASNase degradation, Cathepsin B (CTSB) and Asparagine Endopeptidase (AEP). In previous studies, ASNase mutants resistant to CTSB and / or AEP degradation in vitro were obtained. In this work, mutants were evaluated in cytotoxicity in ALL cell lines and, in vivo studies, applying doses of the wild and mutant ASNases in Balb C mice to evaluate serum asparaginase activity of the enzymes over time, as well as to obtain information on the formation of antibodies against these proteoforms. Regarding to the cytotoxicity, two proteoforms among the tested had similar cytotoxicity than the wild-type. While another proteoform had the cytotoxicity severely reduced. One proteoform have demonstrated greater serum half-life of asparaginase activity, while two other mutants caused reduced antibody formation. Together, these results collaborate to obtain a new generation of ASNases with increased bioavailability and reduced side effects, generating the possibility of lower doses and frequency of applications.
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Dufour, Éric. "Contribution a la connaissance de la structure d'une cysteine proteinase : purification, sequence en acides amines et structure secondaire de la cathepsine l de foie de poulet." Clermont-Ferrand 2, 1988. http://www.theses.fr/1988CLF21109.
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Vargas, Paola Andrea Ortiz. "Genes de cisteíno proteases (Catepsina L-like) de Trypanosoma rangeli: polimorfismo, relações filogenéticas e alvos para diagnóstico e genotipagem." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-16072009-154538/.
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Nós isolamos e seqüenciamos genes que codificam Catepsina L-like em diversos isolados de T.rangeli de humano, mamíferos silvestres e Rhodnius spp., do centro e sul da América. Análises filogenéticas de seqüências que codificam a proteína madura de T. rangeli, outras espécies de Trypanosoma e Leishmania e duas espécies de bodonídeos, posicionaram T.rangeli próximo a T.cruzi de acordo com a ordem de divergência determinada em filogenias baseadas em SSUrDNA. Uma análise de 17 seqüências do domínio catalítico de CatL-like de isolados representativos da diversidade filogenética e distribuição geográfica de T. rangeli, apoiaram as mesmas linhagens filogenéticas previamente definidas. Seqüências do gene CatL-like também foram usados para padronizar ensaios de PCR para diagnóstico de T. cruzi e T. rangeli. Além disso, um método de genotipagem por PCR multiplex segregou os isolados de T. rangeli nas principais linhagens previamente estabelecidas. Este é o primeiro estudo usando um gene codificador de proteína para comparar isolados de T. rangeli de linhagens distintas.We have isolated and sequenced genes encoding cathepsin L-like (CatL-like) cysteine proteases from isolates of T. rangeli from human, wild mammals and Rhodnius spp., from Central and South America. Phylogenetic analysis of sequences encoding the mature CatL-like enzymes from T. rangeli (Rangelipain), other Trypanosoma and Leishmania species, and two species of bodonids, positioned T. rangeli closest to T. cruzi corroborating the same order of divergence showed in phylogenies based on SSU rDNA. Analysis of 17 sequences of the catalytic domains of CatL-like genes isolates representative of the phylogenetic diversity and geographical range of T.rangeli supported previously defined phylogenetic lineages. Sequences of CatL-like genes were used to standardize PCR assays for the diagnosis of T. rangeli and T. cruzi, and a genotyping method of multiplex-PCR distributed of isolates of T. rangeli in the major phylogenetic lineages previously established. This is the first study using protein-encoding genes to compare isolates from T. rangeli of distinct lineages.
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Florence, William Clinton. "Increased stability of class II MHC-peptide complexes in macrophages infected with mycobacterium avium and the examination of a novel role for cathepsin L in the innate immune response to Francisella Novicida infection." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1173298339.
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Damasceno, Ticiane Fraga. "Função de subsítios de uma catepsina digestiva de Tenebrio molitor." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-20012015-143853/.
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A catepsina L, uma cisteína proteinase da família da papaína, é a principal proteinase digestiva do besouro Tenebrio molitor. Estudos anteriores do nosso grupo mostraram que existem três catepsinas L no intestino médio do T. molitor, uma delas é lisossômica (CAL 1) e as outras duas são digestivas (CAL 2 e CAL 3). As estruturas 3D das enzimas digestivas foram recentemente elucidadas. Com o objetivo de estudar em detalhes as propriedades das enzimas digestivas, CAL 3 foi expressa como um zimógeno em E. coli, purificada por cromatografia de afinidade e autoativada em meio ácido. Foram realizados ensaios de atividade com 63 peptídeos FRET derivados da sequência Abz-KLRSSKQ-EDDnp em um espectrofluorímetro termostatizado a 30 ºC, monitorando-se continuamente a variação de fluorescência em 320 nm (λex) e 420 nm (λem). Os parâmetros kcat e KM obtidos foram utilizados na determinação da hidrofobicidade dos subsítios (H) e da função de cada subsítio através da razão das energias livres de ativação do complexo enzima-substrato (ΔG‡T) e de ligação da enzima com o substrato (ΔGs). Os resultados mostram que o subsítio S2 está envolvido prioritariamente em catálise e é bastante seletivo para substratos com resíduos hidrofóbicos em P2. Esse subsítio é o mais hidrofóbico dentre os analisados, encontrando-se num bolsão localizado no interior da enzima. O subsítio S\'2, por outro lado, é o que apresentou a menor especificidade dentre os analisados. Este subsítio está envolvido prioritariamente na ligação com o substrato e se localiza na superfície da enzima, o que pode facilitar a acomodação de diferentes cadeias laterais em P\'2 do substrato, não oferecendo muitas restrições espaciais. O subsítio S1, hidrofílico, não é muito seletivo, o que pode ser consequência de sua localização na superfície da enzima. Esse subsítio está prioritariamente envolvido na ligação com o substrato. O subsítio S\'1, assim como S1, está localizado na superfície da enzima, é hidrofílico e não muito seletivo. No entanto, esse subsítio tem papel na catálise além de atuar na ligação do substrato. Numa análise inicial da estrutura 3D deste subsítio, sua função catalítica foi atribuída à presença de parte da cavidade oxiânica. Uma enzima com mutação no resíduo W187, pertencente à cavidade oxiânica e a S\'1, foi produzida e purificada, no entanto essa enzima não apresentou atividade. Uma análise mais aprofundada mostrou que a falta de atividade pode ser atribuída ao fato do resíduo de aminoácido mutado fazer parte de um cluster aromático essencial à estabilização da tríade catalítica. Os dados obtidos na caracterização de S\'1 e S\'2 permitem inferir que a acilação é o passo limitante da reação da CAL 3. Além disso, os resultados deste trabalho mostram que o conceito de hidrofobicidade de subsítios proposto anteriormente pelo grupo parece ser aplicável a subsítios que apresentem especificidades mais restritas.Cathepsin L, a cysteine proteinase of the papain family, is the major digestive proteinase in the beetle Tenebrio molitor. Previous studies of our group showed that there are three cathepsins L in T. molitor midgut, one is lysosomal (CAL1) and two are digestive (CAL2 and CAL3). The 3D structures of the digestive enzymes were recently elucidated. With the aim to study in details the digestive enzymes specificities, CAL3 was expressed in E. coli as a zymogen, purified by affinity chromatography and autoactivated in acid conditions. Activity assays were performed in a thermostated spectrofluorometer at 30 ºC with 63 FRET peptides derived from the lead sequence Abz-KLRSSKQ-EDDnp, continuously monitoring the fluorescence changes at 320 nm (λex) and 420 nm (λem). The parameters kcat and KM were used in the determination of subsite hydrophobicity (H) and subsite role based on the ratio of complex enzyme-substrate activation energy (ΔG‡T) and free energy of substrate binding (ΔGs). The data obtained suggest that the S2 is mainly involved in catalysis and is very selective to substrates with hydrophobic residues in P2. This subsite is the most hydrophobic among the analyzed and is located in a pocket in the enzyme interior. S\'2, on the other hand, is the less selective subsite and is mainly involved in substrate binding and is located on the enzyme surface, what can ease the accommodation of different side chains located in P\'2 by not imposing many spatial restrictions. S1, is hydrophilic and not very selective, what may be a consequence of its location on the enzyme surface. This subsite is mainly involved in substrate binding. S\'1, just like S1, is located on the enzyme surface, is hydrophilic and not very selective. However this subsite has a role in catalysis besides the role in substrate binding. In an initial 3D structure analysis its catalytic function was attributed to the presence of a part of the oxyanion hole. An enzyme with mutation in the residue W187, which apparently belonged both to the oxyanion hole and S\'1, was produced and purified, but this enzyme was inactive. A better analysis showed that the lack of activity can be attributed to the fact that the mutated residue belongs to an aromatic cluster that is essential to the catalytic triad stabilization. The data obtained in S\'1 and S\'2 characterization suggest that acylation is the limiting step in CAL 3 reaction. The results presented in this work support the concept of subsite hydrophobicity previously proposed by our group, which seems to be true to subsites with more restrict specificities
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Vargas, Paola Andrea Ortiz. "Genes de cisteíno-proteases de Trypanosoma spp. de mamíferos: polimorfismo e relações filogenéticas." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-22092014-175527/.
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Tripanossomas de mamíferos constituem um dos grupos mais complexos da família Trypanosomatidae, abrangendo parasitas com ciclos de vida e estruturas populacionais heterogêneos. De acordo com a diversidade, filogenias baseadas em genes SSUrDNA e gGAPDH segregaram estes parasitas em 4 Clados principais: T. brucei, T. cruzi, T. theileri e T. lewisi. Catepsinas L e B (CATL e CATB), as principais atividades proteolíticas dos tripanossomas, participam não apenas na degradação de proteínas como também em eventos biológicos como diferenciação, invasão celular, virulência e evasão do sistema imune. Comparamos os perfis proteolíticos de enzimas CATL em tripanossomas patogênicos e não patogênicos e também isolamos e sequenciamos os domínios catalíticos dos genes CATL e CATB em diversas espécies dos principais clados. Os resultados provaram a utilidade destes marcadores no diagnóstico e genotipagem de T. cruzi, T. rangeli, T. theileri e T. congolense, assim como na construção de filogenias robustas da família Trypanosomatidae, congruentes com os marcadores tradicionais.Trypanosomes of mammals comprise one of the most complex groups of the family Trypanosomatidae, including parasites with heterogeneous life cycles and population structures. According to such diversity, phylogenetic analyzes based on SSUrDNA and gGAPDH genes segregate these parasites in 4 major clades: T. brucei, T. cruzi, T. lewisi and T. theileri. Cathepsins L and B (CATL and CATB), the main proteolytic activities of trypanosomes, are not only involved in protein degradation but also in biological events such as cell differentiation, cell invasion, virulence, and evasion from the immune system. We comparatively analysed the CATL proteolytic profiles in pathogenic and non-pathogenic trypanosomes, and isolated and sequenced the catalytic domains of CATB and CATL genes in several species of the major clades. Our results demonstrated the usefulness of both markers in the diagnosis and genotyping of T. cruzi, T. rangeli, T. congolense and T. theileri as well as in the construction of robust phylogenies of the family Trypanosomatidae, congruent with traditional markers.
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Rodrigues, Mariane Augusta Domingues. "Avaliação da atividade e resistência à clivagem proteolítica de L-asparaginases recombinantes obtidas por reação em cadeia da polimerase propensa a erro." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/9/9134/tde-24082016-163433/.
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A L-Asparaginase II de Escherichia coli (EcA II) é uma enzima amplamente utilizada no tratamento da Leucemia Linfoblástica Aguda (LLA), atuando na depleção do aminoácido L-asparagina, o qual é fundamental para a multiplicação das células cancerosas. Contudo, o tratamento com a EcA II está associado a altos índices de hipersensibilidade, devido à formação de anticorpos anti-L-asparaginase e à clivagem da enzima pelas proteases sanguíneas asparagina endopeptidase (AEP) e catepsina B (CTSB). Também ocorre neurotoxicidade associada ao efeito L-glutaminase da enzima. O principal objetivo do presente trabalho é a obtenção de mutantes da EcA II (gene ansB) com equivalente eficiência catalítica, maior resistência à clivagem proteolítica e menor atividade glutaminase. Para este propósito, através da reação em cadeia da polimerase propensa a erro (epPCR) do gene ansB, foi construída uma biblioteca de 1128 clones expressos no vetor pET15b em BL21(DE3). Nenhum mutante com atividade asparaginásica equivalente à EcA II selvagem apresentou atividade glutaminásica inferior à esta. Dentre os clones triados obtivemos um mutante (T161I) resistente à clivagem proteolítica pela CTSB e dois mutantes (Q190L e P40S/S206C) resistentes à clivagem proteolítica por ambas AEP e CTSB. Estes três mutantes apresentaram atividade asparaginásica e glutaminásica equivalentes a EcA II selvagem. Nossos resultados mostram promissoras possibilidades de EcA II mutantes com maior estabilidade frente às proteases sanguíneas humanas e possivelmente menos imunogênicas.Escherichia coli L-asparaginase (EcA II) is an enzyme widely used in the treatment of acute lymphoblastic leukemia (ALL), acting in the depletion of the amino acid L-asparagine, which is essential for cancer cells proliferation. However, treatment with L-asparaginase is associated with a high rate of hypersensitivity, due to formation of anti-L-asparaginase antibody and the enzyme cleavage by the serum proteases asparagine endopeptidase (AEP) and cathepsin B (CTSB). Furthermore, the neurotoxicity is associated with the effect of the enzyme L-glutaminase activity. The main aim of the current work is to obtain variants of EcA II (gene ansB) with an equivalent catalytic efficiency, greater resistance to proteolytic cleavage and a reduced glutaminase activity. For such purpose, through error-prone polymerase chain reaction (epPCR) of gene ansB, a library of 1128 clones was constructed in pET15b vector and expressed in BL21(DE3). None mutant with an asparaginase activity equivalent to EcA II wild type showed a reduced glutaminase activity. Among the screened clones, one mutant (T161I) was resistant to CTSB proteolytic cleavage and two mutants (Q190L e P40S/S206C) were resistant to both CTSB and AEP proteolytic cleavages. These three mutants were EcA II wild type equivalents in asparaginase and glutaminase activities. Our data show promising new possibilities of mutant EcA II presenting higher stability against human serum proteolytic cleavage and maybe lower immunogenicity.
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Wolters, Brit Kristin [Verfasser]. "Cathepsins L and V in human keratinocytes / Brit Kristin Wolters." Bremen : IRC-Library, Information Resource Center der Jacobs University Bremen, 2008. http://d-nb.info/1034895729/34.
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Friedrich, Beate. "Cathepsine B, H, L und ihre Inhibitoren im Gewebe und in Zellkulturen der Prostata." Doctoral thesis, [S.l.] : [s.n.], 1999. http://deposit.ddb.de/cgi-bin/dokserv?idn=957675186.
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TOURNU, CECILE. "Controle nutritionnel de l'expression des cathepsines dans des fibroblastes transformes par l'oncogene k-ras : role du glucose." Clermont-Ferrand 1, 1998. http://www.theses.fr/1998CLF1MM04.
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Gläser, Kerstin Elisabeth. "Endochondral ossification : new insights into the involvement of cathepsins L and B." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.616277.
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Corry, Jacqueline D. "Prevention of Respiratory Syncytial Virus Attachment Protein Cleavage in Vero Cells Rescues Infectivity of Progeny Virions for Primary Human Airway Cultures." The Ohio State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=osu1449193388.
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Qu, Hong [Verfasser]. "Significance of cathepsins B, D and L for homeostasis of the small intestine / Hong Qu." Bremen : IRC-Library, Information Resource Center der Jacobs University Bremen, 2008. http://d-nb.info/1034891707/34.
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LE, BOULAY CHRISTINE. "Caracterisation des adnc et des genes codant pour les cathepsines l de crustaces decapodes." Rennes 1, 1997. http://www.theses.fr/1997REN10041.
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L'activite des cysteine proteinases apparentees aux cathepsines l a ete recherchee dans la glande digestive de crustaces decapodes. La teneur de cette enzyme varie en fonction de l'espece etudiee (crevette, langoustine, homard) et chez une meme espece en fonction des differents stades de la mue. Une etude immunocytochimique a permis de localiser cette proteine dans les granules de secretion des cellules f de l'hepatopancreas. Le criblage des banques d'adnc extraits des pedoncules oculaires et de l'estomac de la langoustine nephrops norvegicus a permis d'isoler deux clones de 1168 (ncp1) et 1094 (ncp2) paires de bases. Les proteines matures sont composees de 217 et 215 residus. Chez la crevette penaeus vannamei, le criblage effectue sur la banque d'adnc d'hepatopancreas a permis d'obtenir deux clones (pcp1 et pcp2) de 1084 et 1172 nucleotides. Pcp1 et pcp2 codent respectivement pour des preprocathepsines l de 326 et 322 acides amines. Un site unique de glycosylation existe dans la region c-terminale des deux proteines matures. Une analyse des homologies de sequences par la methode de parcimonie montre que les cathepsines l de la langoustine et de la crevette sont beaucoup plus proches de la cathepsine l de rat (61 - 65%) que des cathepsines h et b. D'autre part, une grande similitude est constatee avec les cysteine proteinases du homard h. Americanus (82%). Le gene codant pour la cathepsine l de la crevette p. Vannamei comprend 1793 paires de bases reparties en six exons et cinq introns. L'un d'entre eux est situe en aval du residu catalytique cys#2#5. L'architecture de ce gene est comparable a celle de son homologue chez le rat. Par contre, aucune identite n'est observee avec le gene codant pour la cathepsine l de la drosophile drosophila melanogaster.
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Naour, Nadia. "Implication physiopathologique des cathepsines S, K, L et de leur inhibiteur endogène, la cystatine C, dans l'obésité et ses complications." Paris 6, 2009. http://www.theses.fr/2009PA066525.
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Parmi les adipokines sécrétées par le tissu adipeux figurent des protéases à cystéine de la famille des cathepsines. L’enjeu de mon travail de thèse a été d’analyser l’importance relative de trois cathepsines S, L et K et de leur inhibiteur endogène, la cystatine C, dans la physiopathologie de l’obésité. Nos résultats montrent que le tissu adipeux humain est une source de cystatine C, produite en quantité plus élevée chez l’obèse. Ainsi, la cystatine C peut être considérée comme une adipokine dont la production est dérégulée dans l’obésité. Nous avons comparé l’expression des cathepsines S, L et K dans le tissu adipeux d’un même sujet. Nous montrons que seule la cathepsine S est surexprimée dans le tissu adipeux du sujet obèse et diminuée après perte de poids, avec des modifications parallèles des concentrations circulantes. Enfin, nous montrons que l’absence de cathepsine S chez la souris provoque une amélioration de l’homéostasie glucidique. En conclusion, ces travaux suggèrent que la cathepsine S est plus modulable que les cathepsines K et L par les variations de la balance énergétique chez l’homme. L’augmentation concomitante de la cystatine C représente potentiellement un mécanisme compensatoire visant à réduire l’impact de la cathepsine S chez le sujet obèse. Cette protéase reste néanmoins une cible potentielle dont l’inhibition pourrait améliorer l’homéostasie glucidique chez la personne obèse.
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Sauerbrei, Ramona Katharina Elisabeth. "Untersuchungen zur Modulation der Aktivitäten der Cathepsine B, L und S in endosomalen und lysosomalen Fraktionen menschlicher Monozyten-Makrophagen." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=970704119.
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Lefrançais, Emma. "L' interleukine-33, cytokine de la famille IL-1 : régulation par les protéases inflammatoires." Toulouse 3, 2012. http://thesesups.ups-tlse.fr/1771/.
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L'interleukine-33 (IL-33) est le dernier membre identifié de la famille des cytokines IL-1. L'intérêt qui lui est porté est grandissant, notamment depuis la découverte de ses cellules cibles majeures, les cellules lymphoïdes innées de type 2 (ILC2), impliquées dans la pathogénie des infections parasitaires et des maladies allergiques comme l'asthme. L'IL-33 se lie au récepteur ST2 via son domaine C-terminal et induit des réponses inflammatoires et de type 2, caractérisées par la sécrétion de l'IL-6, l'IL-5, et l'IL-13. L'IL-33 est une cytokine atypique puisqu'on la retrouve associée à la chromatine via sa partie N-terminale dans le noyau des cellules endothéliales et épithéliales de tissus exposés à l'environnement. A l'inverse des autres membres de la famille IL-1, comme l'IL-1beta ou l'IL-18, l'IL-33 n'a pas besoin d'être clivée par la caspase 1 pour être activée et libérée dans le milieu extracellulaire. Au contraire, l'IL-33 est inactivée par les caspases au cours d'une mort cellulaire programmée par apoptose. En fait, l'IL-33 est libérée sous sa forme de pleine taille et active, par les cellules nécrotiques lors de dommages cellulaires, agissant ainsi comme un signal d'alarme ou alarmine. Toutefois, il n'était pas exclu que d'autres protéases puissent la cliver et réguler son activité. En effet, nos travaux montrent que l'IL-33 peut être clivée et suractivée par les protéases du microenvironnement inflammatoire, issues des neutrophiles (la cathepsine G et l'élastase) ou des mastocytes (la chymase, la tryptase et le granzyme B). Nous avons identifié précisément les sites de clivage par ces diverses protéases et montré que les fragments ainsi générés, contenant le domaine C-terminal de type IL-1, ont une activité biologique augmentée d'un facteur 10 par rapport à la forme de pleine taille. Enfin, nous avons pu mettre en évidence l'existence de telles formes in vivo, dans un modèle murin de lésion pulmonaire aïgue. Ces nouvelles formes physiologiques de l'IL-33 induisent de profonds changements morphologiques in vivo, associés à une forte sécrétion de cytokines pro-inflammatoires et de type 2. Cette découverte apporte de nouveaux éléments quant à l'importance du microenvironnement inflammatoire dans la régulation de l'activité de l'IL-33. Le recrutement et l'activation de neutrophiles et/ou de mastocytes sur le site du dommage entraîneraient une amplification du signal d'alerte par la génération de fragments "super-actifs" de l'IL-33 et pourraient jouer un rôle particulièrement important dans les pathologies infectieuses et inflammatoires dans lesquelles la voie IL-33/ST2 est impliquéeInterleukin-33 (IL-33) is the latest member of the IL-1 family which has attracted much attention since the recent discovery of its major target cells, the Innate lymphoid Cells type 2 (ILC2), involved in the initiation of inflammatory and type 2 immune responses, characterised by secretion of IL-6, IL-5 and IL13, during parasitic infection and allergic diseases such as asthma. IL-33 is a chromatin associated cytokine which possesses an N-terminal chromatin binding motif and is constitutively expressed in the nuclei of endothelial and epithelial cells of tissues exposed to the environment. Extracellularly, IL-33 binds to its receptor ST2 through the C-terminal part of the protein to induce inflammatory and type 2 responses. It has been shown that the full length form of IL-33 is released upon cell injury, acting as an endogenous alarm signal or alarmin. It was initially believed that IL-33, like IL-1ß and IL-18, requires processing by caspase-1 for biological activity. On the contrary, it has been shown that full length IL-33 is biologically active, and that processing by apoptotic caspases results in IL-33 inactivation. However, it was as yet unclear whether other proteases could be involved in the regulation of IL-33. We demonstrate here that the bioactivity of IL-33 can be increased around 10 fold, due to the cleavage by inflammatory proteases secreted in the microenvironment. Immune cells recruited to the site of injury, neutrophils and mastocytes, can regulate IL-33 by releasing elastase and cathepsin G, or chymase, tryptase and granzyme B, respectively. We precisely mapped the different cleavage sites and we show that each of these proteases generate "superactive" forms of IL-33, containing the IL-1 domain. We also demonstrate that these forms do exist in vivo, using a murine model of acute lung injury. We speculate that resident mastocytes and/or recruited neutrophils activated on the site of tissue injury can amplify the IL-33/ST2 pathway and the disease-associated function of the alarmin IL-33
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Nascimento, Michelle Nauara Gomes do. "Estudo químico de erythroxylum suberosum (erythroxylaceae) frente às catepsinas K, L e V." Universidade Federal de Goiás, 2014. http://repositorio.bc.ufg.br/tede/handle/tede/3970.
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This work describes the chemical study of E. suberosum, aiming to elucidate structures of secondary metabolites in the leaves, stem and roots. Cathepsins assays were used to evaluate in vitro activity of extracts, fractions and isolated compounds, using fluorogenic substrate ZFR-MCA. These enzymes, also known as lysossomal cysteine peptidases are involved in many physiological processes, and are associated with many pathological conditions. Cathepsins K, L and V are involved in the development of diseases such as osteoporosis, skin cancer and atherosclerosis, respectively. Based on pathological processes in which cathepsins are involved, the search for specific inhibitors can be a new approach for the treatment these diseases. The study of ethanolic extract from leaves led to the identification of four flavonoids belonging to the flavonol class: quercetin and its derivatives 3-O-monoglycosides hyperin and isoquercitrin, and 3-O-diglycosides, ombuin-3-rutinoside, all previously reported for this species. The mixture of flavanols, catechin and epicatechin were isolated from ethanolic extract from roots, besides the fatty ester of sitosterol. The mixture of steroids campesterol, stigmasterol and β-sitosterol were identified from the stem extract. This study also suggests the presence of tropane alkaloids, tropacocaine and nortropacocaine, in the ethanolic extract from leaves of E. suberosum, reported for the first time in this species. In the evaluation for cathepsins K, L and V, the extracts (concentration of 50 mg/mL), showed significant inhibition of all enzymes with percentage inhibition values above 60%. The fractions showed significant activity against cathepsin V and L when evaluated at concentrations of 5 and 50 mg/mL. The quercetin flavonol showed IC50 value of 2.2 ± 0.2 μM against cathepsin V and low affinity for cathepsins K and L (100 μM). This is an important result since literature reports for flavonoids as inhibitors of cathepsin V are quite limited.
O presente trabalho descreve o estudo químico de E. suberosum, no intuito de elucidar estruturas de metabólitos secundários presentes nas folhas, caule e raiz, além de avaliar, por meio de teste in vitro, a atividade dos extratos, frações e, quando possível das substâncias isoladas, em busca de inibidores de catepsinas utilizando como ferramenta o substrato fluorogênico ZFR-MCA. Estas enzimas, também conhecidas como cisteíno peptidases lisossomais, estão envolvidas em diversos processos fisiológicos, além de estarem associadas a muitas condições patológicas, estando as catepsinas K, L e V, envolvidas no desenvolvimento de doenças como osteoporose, câncer de pele e aterosclerose, respectivamente. Baseado nos processos patológicos em que estas catepsinas estão envolvidas, a busca de inibidores específicos pode ser uma nova abordagem para o tratamento destas doenças. O estudo do extrato etanólico das folhas levou a identificação de quatro flavonoides pertencentes à classe dos flavonois, sendo estes a quercetina e seus derivados 3-Omonoglicosídeos hiperina e isoquercitrina e 3-O-diglicosídeo, ombuina-3- rutinosídeo, todos já relatados para esta espécie. Do extrato etanólico da raiz, foi isolada a mistura dos flavanois catequina e epicatequina, além do éster graxo do β-sitosterol. No caule foram identificados a mistura dos esteroides campesterol, estigmasterol e β-sitosterol. Este estudo também sugere a presença dos alcaloides tropanos, tropacocaína e nortropacocaína no extrato etanólico das folhas, sendo estes relatados pela primeira vez na espécie. Nos ensaios frente às catepsinas K, L e V, todos os extratos apresentaram inibição superior a 60% quando avaliados na concentração de 50 μg/mL. Já as frações oriundas da partição líquido-líquido, apresentaram inibição mais expressiva frente às catepsinas V e L, quando avaliadas nas concentrações de 5 e 50 μg/mL. O flavonol quercetina apresentou um valor de IC50 de 2,2 ± 0,2 μM para a catepsina V e baixa afinidade para as catepsinas K e L quando avaliado na concentração de 100 μM. Este é um resultado importante, uma vez que os relatos na literatura para flavonoides como inibidores de catepsinas são bastante restritos.
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GUINEC, NATHALIE. "Les proteases lysosomiales humaines : action des cathepsines b, h, l normales et b tumorale sur une matrice extracellulaire intacte." Paris 6, 1993. http://www.theses.fr/1993PA066378.
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Les cellules cancereuses ont la particularite de secreter des proteases normalement lysosomiales. Ces proteases pourraient etre responsables de la lyse de la matrice extracellulaire et des membranes basales observee dans l'environnement tumoral. Parmi ces enzymes, les cysteine-proteases de la super-famille de la papaine ont ete decrites. Cette these etudie la digestion d'une membrane basale intacte, la capsule de cristallin de buf par les cathepsines b, b tumorale, h et l humaines. Dans un premier chapitre, la capacite de digestion est mise en evidence et les produits de digestion sont identifies, caracterises et quantifies par des methodes immunochimiques. Le deuxieme chapitre etudie les activites proteolytiques associees aux polypeptides issus de la digestion de la fibronectine. Ce sont des activites dirigees contre les composants des membranes basales et qui apparaissent sous l'action d'une protease exogene, elles s'integrent dans une cascade proteolytique qui contribue a l'efficacite de la lyse. Un autre facteur explique l'importance de ce phenomene, c'est la fixation des cysteine-proteases sur/dans la structure de la membrane basale. L'etude de ses parametres fait l'objet du troisieme chapitre. En complement, la presence de polypeptides issus des composants des membranes basales a ete recherchee dans le plasma de malades souffrant de differents types de cancer
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Friedrich, Beate [Verfasser], A. [Gutachter] Sokolowski, H. [Gutachter] Seiter, and K. [Gutachter] Jung. "Cathepsine B, H, L und ihre Inhibitoren im Gewebe und in Zellkulturen der Prostata / Beate Friedrich ; Gutachter: A. Sokolowski, H. Seiter, K. Jung." Berlin : Humboldt-Universität zu Berlin, 1999. http://d-nb.info/1207671819/34.
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Bohne, Silvia [Verfasser]. "In vitro und in vivo Untersuchungen zur funktionellen Bedeutung der Cathepsine B, K und L bei der systemischen AA- und AL-Amyloidose / Silvia Bohne." Magdeburg : Universitätsbibliothek, 2012. http://d-nb.info/1053227523/34.
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Dufour, Eric. "Contribution à la connaissance de la structure d'une cystéine protéinase purification, séquence en acides aminés et structure secondaire de la cathepsine L de poulet /." Grenoble 2 : ANRT, 1988. http://catalogue.bnf.fr/ark:/12148/cb376132465.
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Lussier, Carine. "Rôles de la cathepsine L et des facteurs de transcription Hnf4[alpha] et Hnf1[alpha] dans la différenciation fonctionnelle de l'épithélium de l'intestin grêle." Thèse, Université de Sherbrooke, 2009. http://savoirs.usherbrooke.ca/handle/11143/4282.
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L'épithélium de l'intestin grêle est en constant renouvellement.L'équilibre entre la prolifération et la différenciation cellulaire y est finement régulé par de nombreuses interactions entre l'épithélium et son environnement. Les interactions épithéliomésenchymateuses engendrent une signalisation qui affecte l'expression génique essentielle au développement et au maintien d'une muqueuse fonctionnelle. Plusieurs facteurs de transcription endodermiques ont été identifiés comme régulateurs de l'épithélium intestinal. Cdx2 en est un exemple, son expression dans les cellules épithéliales intestinales indifférenciées est suffisante à l'induction de leur différenciation en culture. Manuscrit 1: Afin de mieux comprendre les voies moléculaires impliquées dans la différenciation de l'épithélium intestinal, les gènes dont l'expression est modulée suite à l'induction de Cdx2 dans les cellules épithéliales intestinales ont été analysés. La cathepsine L est induite avec la polarisation et la différenciation des cellules épithéliales intestinales et l'inhibition de son activité interfère avec ces processus. De plus, la perte de l'activité de cette protéase conduit à une augmentation de la multiplicité tumorale dans l'intestin des souris APC[indice supérieur Min]. Ces résultats montrent une fonction intracellulaire non suspectée pour la cathepsine L dans l'épithélium intestinal au cours de la polarisation et de l'initiation tumorale. Manuscrit 2 : Pour identifier de nouveaux régulateurs de la différenciation épithéliale intestinale, un modèle de co-culture cellulaire permettant de récapituler la différenciation de cet épithélium a été établi et caractérisé génétiquement. Hnf-4[alpha] et Hnf-1[alpha] sont fortement induits au cours de cette différenciation.L'expression forcée de Hnf-4[alpha] dans les cellules indifférenciées entraîne l'activation de Hnf-1[alpha] et de plusieurs marqueurs des fonctions digestives, ainsi que l'établissement d'une barrière fonctionnelle en co-culture. Ces observations suggèrent que Hnf-4[alpha] est un joueur important dans la régulation de la différenciation des cellules épithéliales intestinales. Manuscrit 3 : Afin de démontrer les fonctions de Hnf-1[alpha] dans l'homéostasie de l'épithélium intestinal in vivo, le phénotype intestinal des souris invalidées pour Hnf-1[alpha] a été analysé. Ces souris présentent une intestinalomégalie caractérisée par une augmentation de la prolifération cellulaire et cryptale. Ces phénomènes étant associés à une augmentation de l'activité de mTOR. De plus, les souris invalidées pour Hnf-1[alpha] présentent une modulation de l'expression de plusieurs marqueurs de cellules entéroendocrines. Ces observations suggèrent un rôle insoupçonné pour Hnf-1[alpha] dans le maintien de la croissance et le devenir des cellules entéroendocrines de l'intestin grêle. Section 4 : Afin de confirmer la collaboration entre les facteurs Hnf-4[alpha] et Hnf-1[alpha] dans l'homéostasie de l'épithélium intestinal in vivo, des souris invalidées pour Hnf-1[alpha] de façon classique et pour Hnf-4[alpha] uniquement dans l'épithélium intestinal (doublement invalidées), au cours du développement embryonnaire (villine-Cre) ainsi que chez l'adulte (CYP1A1-Cre), ont été produites. Les souris doublement invalidées au cours du développement meurent dans les premières 36 heures suivant leur naissance et les souris doublement invalidées à l'âge adulte meurent au cours du traitement qui permet l'invalidation de Hnf-4[alpha]. Ces résultats indiquent que la double invalidation de Hnf-1[alpha] et de Hnf-4[alpha] au niveau de la muqueuse de l'intestin grêle est incompatible avec la survie des souris.
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Gonzalez-Leal, Iris Janet [Verfasser], Heidrun [Gutachter] Moll, Manfred [Gutachter] Lutz, and Tanja [Gutachter] Schirmeister. "Roles of cathepsins B and L in the Th1/Th2 polarization by dendritic cells / Iris Janet Gonzalez-Leal. Gutachter: Heidrun Moll ; Manfred Lutz ; Tanja Schirmeister." Würzburg : Universität Würzburg, 2015. http://d-nb.info/1109750447/34.
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Herms, Max. "Genetische Analyse des Cathepsin L bei chronischer Pankreatitis." Doctoral thesis, 2011. https://ul.qucosa.de/id/qucosa%3A11462.
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Die chronische Pankreatitis (CP) ist eine wiederkehrende, entzündliche Erkrankung des Pankreas. In den letzten Jahren wurden mehrere Kandidatengene, die zur Entstehung einer CP prädisponieren, identifiziert. Zu diesen Genen gehören PRSS1, PRSS2, SPINK1, CFTR und CTRC. Der Pathogenese der genetisch bedingten CP scheint dabei eine frühzeitige, intrapankreatische Aktivierung von Trypsin zugrunde zu liegen.
Cathepsin B (CTSB), eine in Lysosomen vorkommenden Protease, ist in der Lage Trypsinogen zu aktivieren. Genetisch zeigte sich eine Assoziation der p.L26V Variante bei tropisch-kalzifizierender CP, welche bei idiopathischer CP nicht bestätigt wurde. Neben CTSB ist CTSL die am zweithäufigsten vorkommende lysosomale Protease. Funktionelle Untersuchungen zeigten, dass CTSL ein inaktives Trypsin freisetzt. Im Mausmodell zeigten sich bei Ctsl-/- Tieren bei experimentell induzierter Pankreatitis zwei Effekte. Zum einen war die Trypsinaktivität erhöht, zum anderen verlief die Pankreatitis milder, da vermehrt Apoptose anstelle von Nekrose der Azinuszellen auftrat.
In dieser Studie wurde mittels uni-direktionaler DNA-Sequenzierung das gesamte CTSL1 untersucht. Dabei fanden wir insgesamt drei seltene nicht-synonyme Varianten. Die Variante c.5A>C (p.N2T, rs112682750) fanden wir bei einem Patienten, wobei diese Variante bereits bei Kontrollen beschrieben wurde. Die Varianten c.126+1G>A und c.915A>C (p.E305D) lagen bei jeweils einer Kontrolle vor. Sowohl seltene als auch häufige Varianten und die berechneten Haplotypen zeigten keinen signifikanten Verteilungsunterschied zwischen Patienten und Kontrollen. Demnach besteht keine Assoziation von Varianten des CTSL1 und CP.
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seboka, Mpoti. "Cathepsin L and dynamin-biomarkers of proteinuric renal disease." Thesis, 2016. http://hdl.handle.net/10539/21279.
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A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, in partial fulfilment for the degree of Master of Medicine in
Internal Medicine
Johannesburg 2016Background Chronic kidney disease (CKD) is a major public health problem. It is important to be able to identify those individuals at high risk of CKD progression in order to implement strategies to delay progression to end stage renal disease. Hence, early more sensitive biomarkers are required. Recently, promising new biomarkers have been identified for monitoring CKD progression. Objectives To determine whether Dynamin and Cathepsin L can be used as biomarkers for proteinuric chronic kidney disease (CKD). To compare the levels of Dynamin and Cathepsin L in serum and urine of participants with proteinuric kidney disease to those of normal controls. To determine if the levels of Cathepsin L and Dynamin correlates with the degree of proteinuria. Methods A prospective study of 37 patients with proteinuric kidney disease versus a healthy control group of 40 individuals, where the serum and urine levels of Cathepsin L and Dynamin were determined using an Enzyme Linked immunosorbent assay and the levels compared between the two groups. Data Analysis v The sample size was determined from previous similar studies, with assistance of a statistician. Sample size was calculated by comparing the means of the groups where the average value for sample 1= 1.0 (standard deviation=0.5); average value sample 2= 1.5 (standard deviation=0.5; alpha= 5% and beta= 20%. A sample size of 20 was initially selected for the kidney disease group and 20 for the Control group (to give a 1:1 ratio). The numbers were there after doubled to increase sample size in order to improve the statistics. An independent sample t-test was used to assess whether the mean serum Dynamin, urine Dynamin, serum Cathepsin L and urine Cathepsin L differed for the control group compared with kidney disease group. Pearson’s correlation analysis was used to measure the strength of the relationship between variables. Statistical significance was p<0.05. Results There was a significant increase in the level of urine Cathepsin L in the renal disease group 10.44±11.47 pg/ml compared with the control group 2.91±2.88 pg/ml; p= 0.000. There was no difference in the levels of serum Cathepsin L between the renal disease and the control groups (p= 0.23). There were no significant differences in the levels of Dynamin in the serum and urine of patients with proteinuric renal disease and controls (p-values 0.11 and 0.13 respectively). Although serum Cathepsin L (r = -0.22, p-value = 0.19), urine Cathepsin (r = -0.07, p-value = 0.68), and urine Dynamin (r = -0.04, p-value = 0.83) are negatively related to the degree of proteinuria, the correlation is not significant; all the p-values were greater than 0.05. Serum Dynamin (r = 0.12, p-value = 0.49) had a positive correlation to the degree of proteinuria but vi the correlation was not significant at the 5% significance level. Thus, there is no correlation between Cathepsin L and Dynamin levels with the degree of proteinuria. Discussion Podocyte dysfunction is a key element in understanding the progression of CKD resulting in proteinuria. In this study, levels of Cathepsin L and Dynamin were determined in participants with proteinuric renal disease and compared with healthy controls. Cathepsin L levels were elevated in the urine of the renal disease group, in keeping with the notion that Cathepsin L proteolysis plays a critical role in the various forms of proteinuria. There was negative correlation between the levels of proteinuria and Dynamin in the serum; however the correlation was not significant statistically. Conclusion Cathepsin L could potentially serve as a biomarker of proteinuric kidney disease.
MT2016