scholarly journals Cysteine Proteases and Cell Differentiation: Excystment of the Ciliated Protist Sterkiella histriomuscorum

2003 ◽  
Vol 2 (6) ◽  
pp. 1234-1245 ◽  
Author(s):  
Eduardo Villalobo ◽  
Clara Moch ◽  
Ghislaine Fryd-Versavel ◽  
Anne Fleury-Aubusson ◽  
Loïc Morin
Keyword(s):  
Cyst Wall ◽  
Protein Sequence ◽  
Calcium Binding ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Signal Transduction Pathway ◽  
Cysteine Proteases ◽  
Reverse Transcription Pcr ◽  
Phylogenetic Cluster ◽  
Extracellular Matrix Components

ABSTRACT The process of excystment of Sterkiella histriomuscorum (Ciliophora, Oxytrichidae) leads in a few hours, through a massive influx of water and the resorption of the cyst wall, from an undifferentiated resting cyst to a highly differentiated and dividing vegetative cell. While studying the nature of the genes involved in this process, we isolated three different cysteine proteases genes, namely, a cathepsin B gene, a cathepsin L-like gene, and a calpain-like gene. Excystation was selectively inhibited at a precise differentiating stage by cysteine proteases inhibitors, suggesting that these proteins are specifically required during the excystment process. Reverse transcription-PCR experiments showed that both genes display differential expression between the cyst and the vegetative cells. A phylogenetic analysis showed for the first time that the cathepsin B tree is paraphyletic and that the diverging S. histriomuscorum cathepsin B is closely related to its Giardia homologues, which take part in the cyst wall breakdown process. The deduced cathepsin L-like protein sequence displays the structural signatures and phylogenetic relationships of cathepsin H, a protein that is known only in plants and animals and that is involved in the degradation of extracellular matrix components in cancer diseases. The deduced calpain-like protein sequence does not display the calcium-binding domain of conventional calpains; it belongs to a diverging phylogenetic cluster that includes Aspergillus palB, a protein which is involved in a signal transduction pathway that is sensitive to ambient pH.

scholarly journals Expression and alteration of the S2 subsite of the Leishmania major cathepsin B-like cysteine protease

1999 ◽  
Vol 340 (1) ◽  
pp. 113-117 ◽  
Author(s):  
Victor J. CHAN ◽  
Paul M. SELZER ◽  
James H. McKERROW ◽  
Judy A. SAKANARI
Keyword(s):  
Cathepsin B ◽  
Leishmania Major ◽  
Cathepsin L ◽  
Expression System ◽  
Specific Inhibitor ◽  
Structural Similarity ◽  
Cysteine Proteases ◽  
Recombinant Enzyme ◽  
Yeast Expression System ◽  
S2 Subsite

The mature form of the cathepsin B-like protease of Leishmania major (LmajcatB) is a 243 amino acid protein belonging to the papain family of cysteine proteases and is 54% identical to human-liver cathepsin B. Despite the high identity and structural similarity with cathepsin B, LmajcatB does not readily hydrolyse benzyloxycarbonyl-Arg-Arg-7-amino-4-methyl coumarin (Z-Arg-Arg-AMC), which is cleaved by cathepsin B enzymes. It does, however, hydrolyse Z-Phe-Arg-AMC, a substrate typically cleaved by cathepsin L and B enzymes. Based upon computer generated protein models of LmajcatB and mammalian cathepsin B, it was predicted that this variation in substrate specificity was attributed to Gly234 at the S2 subsite of LmajcatB, which forms a larger, more hydrophobic pocket compared with mammalian cathepsin B. To test this hypothesis, recombinant LmajcatB was expressed in the Pichia pastoris yeast expression system. The quality of the recombinant enzyme was confirmed by kinetic characterization, N-terminal sequencing, and Western blot analysis. Alteration of Gly234 to Glu, which is found at the corresponding site in mammalian cathepsin B, increased recombinant LmajcatB (rLmajcatB) activity toward Z-Arg-Arg-AMC 8-fold over the wild-type recombinant enzyme (kcat/Km = 3740±413 M-1·s-1 versus 472±72.4 M-1·s-1). The results of inhibition assays of rLmajcatB with an inhibitor of cathepsin L enzymes, K11002 (morpholine urea-Phe-homoPhe-vinylsulphonylphenyl, kinact/Ki = 208200±36000 M-1·s-1), and a cathepsin B specific inhibitor, CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-ʟ-isoleucyl-L-proline, kinact/Ki = 199200±32900 M-1·s-1], support the findings that this protozoan protease has the P2 specificity of cathepsin L-like enzymes while retaining structural homology to mammalian cathepsin B.

scholarly journals Cathepsin Cs Are Key for the Intracellular Survival of the Protozoan Parasite, Toxoplasma gondii

2006 ◽  
Vol 282 (7) ◽  
pp. 4994-5003 ◽  
Author(s):  
Xuchu Que ◽  
Juan C. Engel ◽  
David Ferguson ◽  
Annette Wunderlich ◽  
Stanislas Tomavo ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Cathepsin B ◽  
Parasitophorous Vacuole ◽  
Cathepsin L ◽  
Protozoan Parasite ◽  
Cysteine Proteases ◽  
Intracellular Survival ◽  
Chemotherapeutic Agents ◽  
Cathepsin C

Cysteine proteases play key roles in apicomplexan invasion, organellar biogenesis, and intracellular survival. We have now characterized five genes encoding papain family cathepsins from Toxoplasma gondii, including three cathepsin Cs, one cathepsin B, and one cathepsin L. Unlike endopeptidases cathepsin B and L, T. gondii cathepsin Cs are exopeptidases and remove dipeptides from unblocked N-terminal substrates of proteins or peptides. TgCPC1 was the most highly expressed cathepsin mRNA in tachyzoites (by real-time PCR), but three cathepsins, TgCPC1, TgCPC2, and TgCPB, were undetectable in in vivo bradyzoites. The specific cathepsin C inhibitor, Gly-Phe-dimethylketone, selectively inhibited the TgCPCs activity, reducing parasite intracellular growth and proliferation. The targeted disruption of TgCPC1 does not affect the invasion and growth of tachyzoites as TgCPC2 is then up-regulated and may substitute for TgCPC1. TgCPC1 and TgCPC2 localize to constitutive secretory vesicles of tachyzoites, the dense granules. T. gondii cathepsin Cs are required for peptide degradation in the parasitophorous vacuole as the degradation of the marker protein, Escherichia coli β-lactamase, secreted into the parasitophorous vacuole of transgenic tachyzoites was completely inhibited by the cathepsin C inhibitor. Cathepsin C inhibitors also limited the in vivo infection of T. gondii in the chick embryo model of toxoplasmosis. Thus, cathepsin Cs are critical to T. gondii growth and differentiation, and their unique specificities could be exploited to develop novel chemotherapeutic agents.

scholarly journals Bioinformatics analysis and characterization of a secretory cystatin from Thelohanellus kitauei

2020 ◽  
Author(s):  
Fengli Zhang ◽  
Yalin Yang ◽  
Chenchen Gao ◽  
Yuanyuan Yao ◽  
Rui Xia ◽  
...  
Keyword(s):  
Binding Energy ◽  
Inhibitory Activity ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Economic Losses ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Multiple Sequence ◽  
Cystatin Gene

Abstract Thelohanellus kitauei , is a group of obligate parasitic Myxozoans, which causes intestinal giant-cystic disease of common carp ( Cyprinus carpio) and has resulted in significant economic losses in carp farms. Cystatin secreted by parasites can regulate the immune response of host to facilitate parasite’s survival. In this study, the secretory TK-cystatin gene, encoding a protein of 120 amino acid residues (13.65 kDa), was cloned from T. kitauei genome. Phylogenetic analysis showed that TK-cystatin gene is closely related to the cystatin-A from Hydra vulgaris . Multiple sequence alignment revealed that TK-cystatin had three conserved motifs: N-terminal G 19 G 20 , Q 73 VVAG 77 , and C-terminal L 102 P 103 . Molecular docking between TK-cystatin and three cysteine proteases showed a lower binding energy (-13 kal/mol) with cathepsin L whereas a higher binding energy (-8.6 kal/mol) with cathepsin B. TK-cystatin gene was expressed in Escherichia coli . Activity assays revealed that TK-cystatin has stronger inhibitory activity on endopeptidases (papain and cathepsin L) and weaker inhibitory activity on exopeptidase (cathepsin B). TK-cystatin was stable under the condition of acidity or alkalinity or below 57 °C. This study laid a foundation for the design and development of the anti-TK-cystatin vaccine in carp culture in the future.

Prognostic Impact of Cysteine Proteases Cathepsin B and Cathepsin L in Pancreatic Adenocarcinoma

Pancreas ◽  
2004 ◽  
Vol 29 (3) ◽  
pp. 204-211 ◽  
Author(s):  
Marco Niedergethmann ◽  
Birgit Wostbrock ◽  
J??rg W. Sturm ◽  
Frank Willeke ◽  
Stefan Post ◽  
...  
Keyword(s):  
Pancreatic Adenocarcinoma ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Prognostic Impact

scholarly journals Human cathepsin L rescues the neurodegeneration and lethality in cathepsin B/L double-deficient mice

2006 ◽  
Vol 387 (7) ◽  
pp. 885-891 ◽  
Author(s):  
Lisa Sevenich ◽  
Len A. Pennacchio ◽  
Christoph Peters ◽  
Thomas Reinheckel
Keyword(s):  
Cerebral Cortex ◽  
Purkinje Cells ◽  
Cathepsin B ◽  
Single Gene ◽  
Cathepsin L ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Cysteine Proteases ◽  
Deficient Mice

Abstract Cathepsin B (CTSB) and cathepsin L (CTSL) are two widely expressed cysteine proteases thought to predominantly reside within lysosomes. Functional analysis of CTSL in humans is complicated by the existence of two CTSL-like homologs (CTSL and CTSL2), in contrast to mice, which possess only one CTSL enzyme. Thus, transgenic expression of human CTSL in CTSL-deficient mice provides an opportunity to study the in vivo functions of this human protease without interference by its highly related homolog. While mice with single-gene deficiencies for murine CTSB or CTSL survive without apparent neuromuscular impairment, murine CTSB/CTSL double-deficient mice display degeneration of cerebellar Purkinje cells and neurons of the cerebral cortex, resulting in severe hypotrophy, motility defects, and lethality during their third to fourth week of life. Here we show that expression of human CTSL through a genomic transgene results in widespread expression of human CTSL in the mouse that is capable of rescuing the lethality found in CTSB/CTSL double-deficient animals. Human CTSL is expressed in the brain of these compound mutants, predominantly in neurons of the cerebral cortex and in Purkinje cells of the cerebellum, where it appears to prevent neuronal cell death.

Expression and regulation of three lysosomal cysteine protease activities during growth of a differentiating L6 rat myoblast cell line and its nonfusing variant

1994 ◽  
Vol 72 (7-8) ◽  
pp. 267-274 ◽  
Author(s):  
Derek T. Jane ◽  
Michael J. Dufresne
Keyword(s):  
Cysteine Protease ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Inhibitor Activity ◽  
Stages Of Growth ◽  
Lysosomal Cysteine Protease ◽  
Myoblast Cell Line ◽  
Myoblast Cell ◽  
Related Increase

The expression of three lysosomal cysteine protease activities, cathepsins B, H, and L, was examined during differentiation of L6 rat myoblasts. Analyses of intracellular levels of these proteases in unfractionated homogenates prepared from cells at different stages of growth and in parallel HPLC-fractionated samples demonstrated a fusion-related increase in all three cathepsins. Analyses of total levels of endogenous inhibitor activity against purified cathepsin B demonstrated a threefold increase in the ratio of protease to inhibitor during myoblast-myotube formation; however, levels of inhibitor activity remained constant. Extracellular levels of cathepsin B, H, and L activities were also examined in the serum-free defined media of differentiating L6 cells. These studies demonstrated a fusion-related increase in extracellular levels of acid/pepsin-activated (i.e., latent) cathepsin L. While increases in intracellular and extracellular levels of cathepsin activities were temporally related to the fusion process, fusion may not be a prerequisite for increased expression, since the nonfusing L6 variant L6-D3 demonstrated high levels of intracellular cathepsins B and L and extracellular latent cathepsin L activities throughout growth. Taken together, these results support the hypotheses that fusion or fusion-related processes play an important role in the controlled expression of cathepsins in L6 myoblasts and that cathepsins, in turn, play an important role in myoblast-myotube differentiation.Key words: L6 myoblasts, differentiation, lysosomal cysteine proteases.

scholarly journals Bioinformatics analysis and characterization of a secretory cystatin from Thelohanellus kitauei

2020 ◽  
Author(s):  
Fengli Zhang ◽  
Yalin Yang ◽  
Chenchen Gao ◽  
Yuanyuan Yao ◽  
Rui Xia ◽  
...  
Keyword(s):  
Binding Energy ◽  
Inhibitory Activity ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Economic Losses ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
Multiple Sequence ◽  
Cystatin Gene

Abstract Thelohanellus kitauei, is a group of obligate parasitic Myxozoans, which causes intestinal giant-cystic disease of common carp (Cyprinus carpio) and has resulted in significant economic losses in carp farms. Cystatin secreted by parasites can regulate the immune response of host to facilitate parasite’s survival. In this study, the secretory TK-cystatin gene, encoding a protein of 120 amino acid residues (13.65 kDa), was cloned from T. kitauei genome. Phylogenetic analysis showed that TK-cystatin gene is closely related to the cystatin-A from Hydra vulgaris. Multiple sequence alignment revealed that TK-cystatin had three conserved motifs: N-terminal G19G20, Q73VVAG77, and C-terminal L102P103. Molecular docking between TK-cystatin and three cysteine proteases showed a lower binding energy (-13 kal/mol) with cathepsin L whereas a higher binding energy (-8.6 kal/mol) with cathepsin B. TK-cystatin gene was expressed in Escherichia coli. Activity assays revealed that TK-cystatin has stronger inhibitory activity on endopeptidases (papain and cathepsin L) and weaker inhibitory activity on exopeptidase (cathepsin B). TK-cystatin was stable under the condition of acidity or alkalinity or below 57 °C. This study laid a foundation for the design and development of the anti-TK-cystatin vaccine in carp culture in the future.

The cysteine proteinases of Schistosoma mansoni cercariae

Parasitology ◽  
1997 ◽  
Vol 114 (2) ◽  
pp. 105-112 ◽  
Author(s):  
J. P. DALTON ◽  
K. A. CLOUGH ◽  
M. K. JONES ◽  
P. J. BRINDLEY
Keyword(s):  
Schistosoma Mansoni ◽  
Cathepsin B ◽  
Cathepsin L ◽  
Serine Proteinase ◽  
Skin Penetration ◽  
Scanning Electron Micrographs ◽  
Cysteine Proteinases ◽  
Similar Function ◽  
Substrate Preferences ◽  
Serine Proteinase Activity

Based on substrate preferences, cercariae of Schistosoma mansoni were seen to express both cathepsin L and cathepsin B cysteine proteinases, although the former activity was many -fold greater. Two cathepsin L activities identified in cercarial extracts by zymography co-migrated with activities in extracts of 3 h and 24 h schisotosomula and in extracts of adult worms. Since these enzymes have been implicated in haemoglob in digestion by adult worms, they may perform a similar function in schistosomula. Immunolocalization using scanning electron micrographs showed that cathepsin L and cathepsin B proteinases were present in the cercarial post-acetabular glands. In addition, cercarial serine proteinase activities considered to facilitate skin penetration efficiently cleaved the substrates Z-Gly-Pro-Arg-NHMec and Z-Gly-Pro-Lys-NHMec. Cercariae release most of this serine proteinase activity when induced to secrete the contents of their acetabular glands. In contrast, newly transformed 3 h and 24 h schistosomula did not express this activity.

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