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 Presence of Anti-cystatin C–positive Dendritic Cells or Macrophages and Localization of Cysteine Proteases in the Apical Bud of the Enamel Organ in the Rat Incisor

2005 ◽  
Vol 53 (5) ◽  
pp. 643-651 ◽  
Author(s):  
Sumio Nishikawa
Keyword(s):  
Dendritic Cells ◽  
Cystatin C ◽  
Cysteine Protease ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Cysteine Protease Inhibitor ◽  
Enamel Organ ◽  
Apical Bud ◽  
Apical Buds ◽  
Proliferation And Differentiation

Cystatin C, a cysteine protease inhibitor, was examined in the apical buds of rat incisors by immunohistochemistry, because in transition and maturation zones most of the dendritic cells in the papillary layer are anti-cystatin C–positive. Anti-cystatin C–labeled cells were sparse and localized to the proliferation and differentiation zones, constituting the apical bud of 5-week-old rat incisors. These cells were considered macrophages or dendritic cells, based on their reactivity with OX6 and ED1, as well as their ultrastructure. Basement membrane at the periphery of apical bud was also labeled by anti-cystatin C antibody. The apical buds included a few apoptotic fragments and weak reactivity with antibody to cathepsin L, a cysteine protease. Reactivity to anti-cystatin C and anti-cathepsin ∗∗∗L antibodies was also detected in the apical bud of newborn rat incisors. These results suggest that the cystatin C–positive macrophages or dendritic cells are involved in normal incisor formation. They may be related to the clearance of apoptotic cells or protection from putative cysteine protease activity.

scholarly journals Characterisation of a cysteine protease from poultry red mites and its potential use as a vaccine for chickens

Parasite ◽  
2021 ◽  
Vol 28 ◽  
pp. 9
Author(s):  
Shiro Murata ◽  
Ayaka Taniguchi ◽  
Masayoshi Isezaki ◽  
Sotaro Fujisawa ◽  
Eishi Sakai ◽  
...  
Keyword(s):  
Cysteine Protease ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Mite Species ◽  
Economic Losses ◽  
Dermanyssus Gallinae ◽  
Physiological Processes ◽  
Poultry Houses ◽  
Poultry Red Mites ◽  
Potential Use

Poultry red mites (PRMs, Dermanyssus gallinae) are ectoparasites that negatively affect farmed chickens, leading to serious economic losses worldwide. Acaricides have been used to control PRMs in poultry houses. However, some PRMs have developed resistance to acaricides, and therefore different approaches are required to manage the problems caused by PRMs. Vaccination of chickens is one of the methods being considered to reduce the number of PRMs in poultry houses. In a previous study, a cysteine protease, Deg-CPR-1, was identified as a candidate vaccine against PRMs distributed in Europe. In this study, we investigated the characteristics of Deg-CPR-1. A phylogenetic analysis revealed that Deg-CPR-1 is closely related to the digestive cysteine proteases of other mite species, and it was classified into a cluster different from that of chicken cathepsins. Deg-CPR-1 of PRMs in Japan has an amino acid substitution compared with that of PRMs in Europe, but it showed efficacy as a vaccine, consistent with previous findings. Deg-CPR-1 exhibited cathepsin L-like enzyme activity. In addition, the Deg-CPR-1 mRNA was expressed in the midgut and in all stages of PRMs that feed on blood. These results imply that Deg-CPR-1 in the midgut may have important functions in physiological processes, and the inhibition of its expression may contribute to the efficacy of a Deg-CPR-1-based vaccine. Further research is required to fully understand the mechanisms of vaccine efficacy.

scholarly journals Structure, interdomain dynamics and pH-dependent autoactivation of pro-rhodesain, the main lysosomal cysteine protease from African trypanosomes

2020 ◽  
Author(s):  
Patrick Johé ◽  
Elmar Jaenicke ◽  
Hannes Neuweiler ◽  
Tanja Schirmeister ◽  
Christian Kersten ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cysteine Protease ◽  
Cathepsin L ◽  
Salt Bridge ◽  
Correlation Spectroscopy ◽  
African Trypanosomes ◽  
Lysosomal Cysteine Protease ◽  
Control Switch ◽  
Α Helix ◽  
Dynamics Simulations

AbstractRhodesain is the lysosomal cathepsin L-like cysteine protease of T. brucei rhodesiense, the causative agent of Human African Trypanosomiasis. The enzyme is essential for the proliferation and pathogenicity of the parasite as well as its ability to overcome the blood-brain barrier of the host. Lysosomal cathepsins are expressed as zymogens with an inactivating pro-domain that is cleaved under acidic conditions. A structure of the uncleaved maturation intermediate from a trypanosomal cathepsin L-like protease is currently not available. We thus established the heterologous expression of T. brucei rhodesiense pro-rhodesain in E. coli and determined its crystal structure. The trypanosomal pro-domain differs from non-parasitic pro-cathepsins by a unique, extended α-helix that blocks the active site and whose interactions resemble that of the antiprotozoal inhibitor K11777. Interdomain dynamics between pro- and core protease domain as observed by photoinduced electron transfer fluorescence correlation spectroscopy increase at low pH, where pro-rhodesain also undergoes autocleavage. Using the crystal structure, molecular dynamics simulations and mutagenesis, we identify a conserved interdomain salt bridge that prevents premature intramolecular cleavage at higher pH values and may thus present a control switch for the observed pH-sensitivity of pro-enzyme cleavage in (trypanosomal) CathL-like proteases.

A cysteine protease inhibitor blocks SARS-CoV-2 infection of human and monkey cells

2020 ◽  
Author(s):  
Drake M. Mellott ◽  
Chien-Te Tseng ◽  
Aleksandra Drelich ◽  
Pavla Fajtová ◽  
Bala C. Chenna ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Host Cell ◽  
Cysteine Protease ◽  
Cleavage Site ◽  
Cathepsin L ◽  
Cysteine Proteases ◽  
Protein Processing ◽  
Spike Protein ◽  
Cysteine Protease Inhibitor ◽  
Viral Infectivity

ABSTRACTK777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 μM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of ≤ 100 μM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2, differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing.SIGNIFICANCEThe virus causing COVID-19 is highly infectious and has resulted in a global pandemic. We confirm that a cysteine protease inhibitor, approved by the FDA as a clinical-stage compound, inhibits SARS-CoV-2 infection of several human and monkey cell lines with notable(nanomolar) efficacy. The mechanism of action of this inhibitor is identified as a specific inhibition of host cell cathepsin L. This in turn inhibits host cell processing of the coronaviral spike protein, a step required for cell entry. Neither of the coronaviral proteases are inhibited, and the cleavage site of spike protein processing is different from that reported in other coronaviruses. Hypotheses to explain the differential activity of the inhibitor with different cell types are discussed.

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 The Intestinal Protozoan Parasite Entamoeba histolytica Contains 20 Cysteine Protease Genes, of Which Only a Small Subset Is Expressed during In Vitro Cultivation

2003 ◽  
Vol 2 (3) ◽  
pp. 501-509 ◽  
Author(s):  
Iris Bruchhaus ◽  
Brendan J. Loftus ◽  
Neil Hall ◽  
Egbert Tannich
Keyword(s):  
Entamoeba Histolytica ◽  
Cysteine Protease ◽  
Cathepsin L ◽  
Protozoan Parasite ◽  
Cysteine Proteases ◽  
Small Subset ◽  
In Vitro Cultivation ◽  
Intestinal Protozoan ◽  
Parasite Life Cycle

ABSTRACT Cysteine proteases are known to be important pathogenicity factors of the protozoan parasite Entamoeba histolytica. So far, a total of eight genes coding for cysteine proteases have been identified in E. histolytica, two of which are absent in the closely related nonpathogenic species E. dispar. However, present knowledge is restricted to enzymes expressed during in vitro cultivation of the parasite, which might represent only a subset of the entire repertoire. Taking advantage of the current E. histolytica genome-sequencing efforts, we analyzed databases containing more than 99% of all ameba gene sequences for the presence of cysteine protease genes. A total of 20 full-length genes was identified (including all eight genes previously reported), which show 10 to 86% sequence identity. The various genes obviously originated from two separate ancestors since they form two distinct clades. Despite cathepsin B-like substrate specificities, all of the ameba polypeptides are structurally related to cathepsin L-like enzymes. None of the previously described enzymes but 7 of the 12 newly identified proteins are unique compared to cathepsins of higher eukaryotes in that they are predicted to have transmembrane or glycosylphosphatidylinositol anchor attachment domains. Southern blot analysis revealed that orthologous sequences for all of the newly identified proteases are present in E. dispar. Interestingly, the majority of the various cysteine protease genes are not expressed in E. histolytica or E. dispar trophozoites during in vitro cultivation. Therefore, it is likely that at least some of these enzymes are required for infection of the human host and/or for completion of the parasite life cycle.

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.

Cathepsin B Stability, But Not Activity, Is Affected in Cysteine:Cystine Redox Buffers

2002 ◽  
Vol 383 (7-8) ◽  
pp. 1199-1204 ◽  
Author(s):  
C. S. Pillay ◽  
C. Dennison
Keyword(s):  
Redox Potential ◽  
Cysteine Protease ◽  
Cathepsin B ◽  
Reduction Potential ◽  
Potential Range ◽  
Neutral Ph ◽  
Lysosomal Cysteine Protease ◽  
The Stability ◽  
Redox Buffers

Abstract In order to test the hypothesis that the lysosomal cysteine protease cathepsin B may be redox regulated in vivo, cathepsin B activity and stability were measured in cysteine and/or cystinecontaining buffers. Cathepsin B activity in cysteinecontaining buffers was similar at pH 6.0 and pH 7.0, over all thiol concentrations tested. In contrast, the stability of the enzyme was greater at pH 6.0 than at pH 7.0. This suggests that the enzymes operational pH in vivo may be < pH 7.0. The activity of the enzyme was depressed in glutathionecontaining buffers. When assessed in cysteine:cystine redox buffers (pH 6.0 7.0) cathepsin B was active over a broad redox potential range, suggesting that cathepsin B activity may not be redox regulated. However, at pH 7.0, the stability of cathepsin B decreased with increasing reduction potential and ambient cystine concentration. This suggests that the stability of the enzyme at neutral pH is dependent on redox potential, and on the presence of oxidising agents.

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.

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