The collagenolytic proteinases, type IV collagenase and cathepsins Land H, have been implicated in tumour invasion and metastasis, by virtue of their degradative action on the extracellular matrix barriers traversed by migrating tumour cells. Type IV collagenase was isolated from human leucocytes using anti-peptide antibody immunoaffinity chromatography. The highly specific targeting of both native and denatured forms of human type IV collagenase by these anti-peptide antibodies holds much promise for immunolocalisation studies in human tumour tissue. Cathepsin L was purified in both a free; single-chain form from sheep liver, and as complexes with the endogenous cysteine proteinase inhibitor, stefin B. These complexes comprised mixtures of the usual tight-binding non-covalent, inhibitory complexes, and novel, proteolytically active, covalent cathepsin L/stefin B complexes. The latter form spontaneously in a pH-dependent manner in vitro from purified, active constituents. The primary structures of these complexing moieties from sheep liver are reported here for the first time, and showed a high degree of sequence homology with their human counterparts. Single-chain cathepsin L, both in the free, and novel, covalently complexed forms, manifested stability and increased activity at neutral pH, thus suggesting a role in extracellular tissue destruction. This potential involvement in tumour invasion was strengthened by demonstrating that the single-chain form of the enzyme, and similar covalent complexes, active under physiological conditions, could be isolated from liver tissue homogenates of higher primates, baboon (Papio ursinus) and man. A battery of versatile polyclonal anti-sheep cathepsin L and anti-human cathepsins L and H peptide antibodies were raised in chickens and rabbits. The chicken egg yolk antibodies were often of a higher titre than the corresponding rabbit serum antibodies, and additionally manifested unique immunoinhibitory properties. In the case of the polyclonal chicken anti-sheep cathepsin L antibodies, this was derived from their ability to target a peptide located in the active site of cathepsin L. The chicken anti-human cathepsins L and H peptide antibodies constitute the immunological probes of choice for immunolocalisation and in vitro tumour invasion studies to elucidate the relative contributions of these collagenolytic cathepsins to tumour invasion, and could ultimately find application in tumour immunotherapy. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1992.

No / We have previously shown that the ubiquitously expressed lysosomal cysteine protease, cathepsin L (CTSL), is essential for skin and hair follicle homeostasis. Here we examine the effect of CTSL deficiency on hair follicle development and cycling in ctsl-/- mice by light and electron microscopy, Ki67/terminal dUTP nick-end labeling, and trichohyalin immunofluorescence. Hair follicle morphogenesis in ctsl-/- mice was associated with several abnormalities. Defective terminal differentiation of keratinocytes occurred during the formation of the hair canal, resulting in disruption of hair shaft outgrowth. Both proliferation and apoptosis levels in keratinocytes and melanocytes were higher in ctsl-/- than in ctsl+/+ hair follicles. The development of the hair follicle pigmentary unit was disrupted by vacuolation of differentiating melanocytes. Hair cycling was also abnormal in ctsl-/- mice. Final stages of hair follicle morphogenesis and the induction of hair follicle cycling were retarded. Thereafter, these follicles exhibited a truncated resting phase (telogen) and a premature entry into the first growth phase. Further abnormalities of telogen development included the defective anchoring of club hairs in the skin, which resulted in their abnormal shedding. Melanocyte vacuolation was again apparent during the hair cycle-associated reconstruction of the hair pigmentary unit. A hallmark of these ctsl-/- mice was the severe disruption in the exiting of hair shafts to the skin surface. This was mostly because of a failure of the inner root sheath (keratinocyte layer next to the hair shaft) to fully desquamate. These changes resulted in a massive dilation of the hair canal and the abnormal routing of sebaceous gland products to the skin surface. In summary, this study suggests novel roles for cathepsin proteases in skin, hair, and pigment biology. Principal target tissues that may contain protein substrate(s) for this cysteine protease include the developing hair cone, inner root sheath, anchoring apparatus of the telogen club, and organelles of lysosomal origin (eg, melanosomes).

Trematode peptidases are important molecules responsible for biocatalysis in many basal biological processes and are crucial in host-parasite interactions. Therefore, these enzymes are intensively studied in order to characterize their biological functions and to use them as potential diagnostic or therapeutic targets. Lately, investigation of transcriptome and secretome revealed, that adult Fascioloides magna (giant liver fluke) expresses and secretes a variety of peptidases. Thus, this thesis focuses on three newly identified enzymes: cathepsin L (FmCL), cathepsin B (FmCB) and cathepsin D (FmCD). In other trematode species, these cathepsins are being linked mainly with the digestion of host blood. We applied quantitative PCR (qPCR) to investigate relative expression levels of the three enzymes among three developmental stages - egg, miracidium and adult. It was revealed that all cathepsins have the highest expression in adult flukes in comparison to eggs and miracidia. We also localized the place of transcription of FmCL, FmCB and FmCD in adult fluke using RNA in situ hybridization. All of the peptidases were detected in gastrodermis, and in addition, they were localized in the reproductive system. The latter surprising finding is suggesting that these enzymes might have multiple functions in adult F....

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 papaína / Cathepsin 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

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 bioinformática / After 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

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 bioinformática / After 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

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.

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.

Made available in DSpace on 2016-06-02T20:20:35Z (GMT). No. of bitstreams: 1 5217.pdf: 18042962 bytes, checksum: 6c55d259639b3b244326b14d21f608f1 (MD5) Previous issue date: 2012-02-29 / Universidade 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.

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 papaína / Cathepsin 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