Cathepsin B-like Protease Research Articles

SlCathB2 as a negative regulator mediates a novel regulatory pathway upon high-temperature stress response in tomato.

High-temperature stress (HS) is a major abiotic stress that affects the yield and quality of plants. Cathepsin B-like protease 2 (CathB2) has been reported to play a role in developmental processes and stress response, but its involvement in HS response has not been identified. Here, overexpression, virus-induced gene silencing (VIGS)and RNA-sequencing analysis were performed to uncover the functional characteristics of SlCathB2-1 and SlCathB2-2 genes for HS response in tomato. The results showed that overexpression of SlCathB2-1 and SlCathB2-2 resulted in reduced heat tolerance of tomato to HS while silencing the genes resulted in enhanced heat tolerance. RNA-sequencing analysis revealed that the heat shock proteins (HSPs) exhibited higher expression in WT than in SlCathB2-1 and SlCathB2-2 overexpression lines. Furthermore, the possible molecular regulation mechanism underlying SlCathB2-1 and SlCathB2-2-mediated response to HS was investigated. We found that SlCathB2-1 and SlCathB2-2 negatively regulated antioxidant capacity by regulating a set of genes involved in antioxidant defence and reactive oxygen species (ROS) signal transduction. We also demonstrated that SlCathB2-1 and SlCathB2-2 positively regulated ER-stress-induced PCD (ERSID) by regulating unfolded protein response (UPR) gene expression. Furthermore, SlCathB2-1 and SlCathB2-2 interacting with proteasome subunit beta type-4 (PBA4) was identified in the ERSID pathway using yeast two-hybrid (Y2H) analysis and bimolecular fluorescence complementation (BiFC) screening. Overall, the study identified both SlCathB2-1 and SlCathB2-2 as new negative regulators to HS and presented a new HS response pathway. This provided the foundation for the construction of heat-tolerant molecular mechanisms and breeding strategies aiming to improve the thermotolerance of tomato plants.

Comparative transcriptome profiles of Schistosoma japonicum larval stages: Implications for parasite biology and host invasion.

Schistosoma japonicum is prevalent in Asia with a wide mammalian host range, which leads to highly harmful zoonotic parasitic diseases. Most previous transcriptomic studies have been performed on this parasite, but mainly focus on stages inside the mammalian host. Moreover, few larval transcriptomic data are available in public databases. Here we mapped the detailed transcriptome profiles of four S. japonicum larval stages including eggs, miracidia, sporocysts and cercariae, providing a comprehensive development picture outside of the mammalian host. By analyzing the stage-specific/enriched genes, we identified functional genes associated with the biological characteristic at each stage: e.g. we observed enrichment of genes necessary for DNA replication only in sporocysts, while those involved in proteolysis were upregulated in sporocysts and/or cercariae. This data indicated that miracidia might use leishmanolysin and neprilysin to penetrate the snail, while elastase (SjCE2b) and leishmanolysin might contribute to the cercariae invasion. The expression profile of stem cell markers revealed potential germinal cell conversion during larval development. Additionally, our analysis indicated that tandem duplications had driven the expansion of the papain family in S. japonicum. Notably, all the duplicated cathepsin B-like proteases were highly expressed in cercariae. Utilizing our 3rd version of S. japonicum genome, we further characterized the alternative splicing profiles throughout these four stages. Taken together, the present study provides compressive gene expression profiles of S. japonicum larval stages and identifies a set of genes that might be involved in intermediate and definitive host invasion.

Immune reactivity and host modulatory roles of two novel Haemonchus contortus cathepsin B-like proteases

BackgroundHaemonchus contortus is a blood-feeding, gastrointestinal nematode (GIN) that causes significant economic losses to the small ruminant industry worldwide. Despite extensive efforts, our understanding of the molecular mechanisms used by GIN to evade host immune responses is limited. Cathepsin B-like proteins (CBPs) are members of the cysteine protease family and are involved in parasite invasion and thus provide viable vaccine candidates.MethodsIn silico comparative analysis was used to identify conserved proteins among a subset of clade V parasitic nematodes with emphasis on blood-feeding worms, among which CBPs appeared prominently. We identified and characterized two novel CBPs designated Hc-CBP-1 and Hc-CBP-2. Rabbit anti-recombinant (r) Hc-CBP-1 and rHc-CBP-2 were used to detect the presence of native proteins in the excretory secretory products (ESP) and in worm tissues of adult H. contortus. Peptide arrays of rHc-CBP-1 and rHc-CBP-2 were screened with the homologous and heterologous anti-sera and with sera from dexamethasone-treated (Dex+) and non-treated (Dex−) H. contortus-infected animals to identify key immunogenic peptides. Gene transcription of Hc-cbp-1 and Hc-cbp-2 was also performed on H. contortus-infected animals treated with Dex+. Finally, the mature recombinant proteins were used to assess their abilities to modulate cell functions.ResultsImmunohistochemistry showed that both Hc-CBP-1 and Hc-CBP-2 are present on the brush borders of the intestine; Hc-CBP-2 was also present in the hypodermis of the body wall. Peptide displays screened with rabbit anti-rHc-CBP-1 and anti-rHc-CBP-2 revealed regions within the proteins where dominant and overlapping epitopes prevailed. ELISA results were consistent with only Hc-CBP-1 being present in H. contortus adult ESPs. H. contortus from Dex+ animals exhibited a threefold increase in Hc-cbp-2 transcript while Hc-cbp-1 expression did not change. In contrast, comparisons of immunoreactivities of rHc-CBP-1 and rHc-CBP-2 peptide arrays to sera from Dex+ and Dex− animals primarily showed changes in Hc-CBP-1 binding. Lastly, rHc-CBP-1 suppressed mRNA expression of bovine peripheral blood mononuclear cell cytokines/activation markers, including TNFα, IL-1, IL-6 and CD86.ConclusionsThese results suggest that as secreted and cryptic proteins, respectively, Hc-CBP-1 and Hc-CBP-2 influence cellular and immunological activities that have interesting dynamics during infection and may provide viable immune-related targets for attenuating H. contortus infectivity.Graphic

Transcriptomic and proteomic analysis of putative digestive proteases in the salivary gland and gut of Empoasca (Matsumurasca) onukii Matsuda

BackgroundInfestation by tea green leafhoppers (Empoasca (Matsumurasca) onukii) can cause a series of biochemical changes in tea leaves. As a typical cell-rupture feeder, E. onukii secretes proteases while using its stylet to probe the tender shoots of tea plants (Camellia sinensis). This study identified and analyzed proteases expressed specifically in the salivary gland (SG) and gut of E. onukii through enzymatic activity assays complemented with an integrated analysis of transcriptomic and proteomic data.ResultsIn total, 129 contigs representing seven types of putative proteases were identified. Transcript abundance of digestive proteases and enzymatic activity assays showed that cathepsin B-like protease, cathepsin L-like protease, and serine proteases (trypsin- and chymotrypsin-like protease) were highly abundant in the gut but moderately abundant in the SG. The abundance pattern of digestive proteases in the SG and gut of E. onukii differed from that of other hemipterans, including Nilaparvata lugens, Laodelphax striatellus, Acyrthosiphum pisum, Halyomorpha halys and Nephotettix cincticeps. Phylogenetic analysis showed that aminopeptidase N-like proteins and serine proteases abundant in the SG or gut of hemipterans formed two distinct clusters.ConclusionsAltogether, this study provides insightful information on the digestive system of E. onukii. Compared to five other hemipteran species, we observed different patterns of proteases abundant in the SG and gut of E. onukii. These results will be beneficial in understanding the interaction between tea plants and E. onukii.

Identification and expression analysis of Cathepsin B-like protease 2 genes in tomato at abiotic stresses especially at High temperature

Cathepsin B-like protease 2 (CathB2) genes have been shown to participate in programmed cell death (PCD) that is a crucial component of development and defense mechanisms in plants at biotic and abiotic stresses. However, little is known regarding CathB2 genes in tomato. In this study, we reported the analysis of two SlCathB2 genes named SlCathB2−1 and SlCathB2−2. Promoter analysis found some elements related to abiotic stress-responses in two SlCathB2 genes. Domain analysis revealed that SlCathB2 encoding proteins characterized by signal peptide, propeptide, and papain family cysteine protease domain. The phylogenetic analysis revealed their close evolutionary relationship with the CathB2 of Solanum tuberosum. Expression analysis showed that SlCathB2 differentially expressed in various tissues and SlCathB2 positively responded to high temperature, drought, salt, ABA and SA treatments with higher expression upon high-temperature stress (HS) in the cultivated tomato line ‘LA1698′ (Solanum lycopersicum) and the currant tomato line ‘LA2093′ (Solanum pimpinellifolium). Then, the expression of SlCathB2 in the leaves of tomato genotypes with different thermotolerance under moderately elevated temperature (MET, 33 °C/33 °C) and acutely elevated temperature (AET, 40 °C/40 °C) stresses were further analyzed. The results showed that SlCathB2 genes were most triggered in heat-sensitive tomato genotypes (S1 and S2) as compared to heat-tolerant genotypes (T1 and T2) and were not significantly induced in the moderate genotypes (M1 and M2) under MET and AET stresses, suggesting that they were negatively regulated by HS. Furthermore, SlCathB2 genes showed similar expression patterns in tomato genotypes with same behavior for thermotolerance under MET and AET stresses, while exhibited higher expression level under AET stress, indicating that more thermosensors and multiple HSR (high-temperature stress- responsive) pathways were activated under AET stress leading to the higher expression level of a set of HSR genes including SlCathB2−1 and SlCathB2−2 genes. This study lays a foundation for functional studies of SlCathB2 in tomato.

The two cathepsin B-like proteases of Arabidopsis thaliana are closely related enzymes with discrete endopeptidase and carboxydipeptidase activities.

The genome of the model plant Arabidopsis thaliana encodes three paralogues of the papain-like cysteine proteinase cathepsin B (AtCathB1, AtCathB2 and AtCathB3), whose individual functions are still largely unknown. Here we show that a mutated splice site causes severe truncations of the AtCathB1 polypeptide, rendering it catalytically incompetent. By contrast, AtCathB2 and AtCathB3 are effective proteases which display comparable hydrolytic properties and share most of their substrate specificities. Site-directed mutagenesis experiments demonstrated that a single amino acid substitution (Gly336→Glu) is sufficient to confer AtCathB2 with the capacity to tolerate arginine in its specificity-determining S2 subsite, which is otherwise a hallmark of AtCathB3-mediated cleavages. A degradomics approach utilizing proteome-derived peptide libraries revealed that both enzymes are capable of acting as endopeptidases and exopeptidases, releasing dipeptides from the C-termini of substrates. Mutation of the carboxydipeptidase determinant His207 also affected the activity of AtCathB2 towards non-exopeptidase substrates, highlighting mechanistic differences between plant and human cathepsin B. This was also noted in molecular modeling studies which indicate that the occluding loop defining the dual enzymatic character of cathepsin B does not obstruct the active-site cleft of AtCathB2 to the same extent as in its mammalian orthologues.

A novel antigenic cathepsin B protease induces protective immunity in Trichinella-infected mice

Trichinellosis is a foodborne disease that remains a public health hazard and an economic problem in food safety. Vaccines against the parasite can be an effective way to control this disease; however, commercial vaccines against Trichinella infection are not yet available. Trichinella cathepsin B proteins appear to be promising targets for vaccine development. Here, we reported for the first time the characterization of a novel cDNA that encodes Trichinella spiralis (T. spiralis) cathepsin B-like protease 2 gene (TsCPB2). The recombinant mature TsCPB2 protein was successfully expressed in E. coli system and purified with Ni-affinity chromatography. TsCPB2 expression was detected at all the developmental stages of T. spiralis and it was expressed as an excretory–secretory protein of T. spiralis muscle larvae. Immunization with TsCPB2 antigen induced a combination of humoral and cellular immune responses, which manifested as a mixed Th1/Th2 response, as well as remarkably elevated IgE level. Moreover, vaccination of mice with TsCPB2 that were subsequently challenged with T. spiralis larvae resulted in a 52.3% (P < .001) reduction in worm burden and a 51.2% (P < .001) reduction in muscle larval burden. Our results suggest that TsCPB2 induces protective immunity in Trichinella-infected mice and might be a novel vaccine candidate against trichinellosis.

Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination.

Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins.

Angiostrongylus cantonensis cathepsin B-like protease (Ac-cathB-1) is involved in host gut penetration.

Although the global spread of the emerging zoonosis, human angiostrongyliasis, has attracted increasing attention, understanding of specific gene function has been impeded by the inaccessibility of genetic manipulation of the pathogen nematode causing this disease, Angiostrongylus cantonensis. Many parasitic proteases play key roles in host-parasite interactions, but those of A. cantonensis are always expressed as the inactive form in prokaryotic expression systems, thereby impeding functional studies. Hence, a lentiviral system that drives secreted expression of target genes fused to a Myc-His tag was used to obtain recombinant Ac-cathB-1 with biological activity. Although this class of proteases was always reported to function in nutrition and immune evasion in parasitic nematodes, recombinant Ac-cathB-1 was capable of hydrolysis of fibronectin and laminin as well as the extracellular matrix of IEC-6 monolayer, so that the intercellular space of the IEC-6 monolayer increased 5.15 times as compared to the control, while the shape of the adherent cells partly rounded up. This suggests a probable role for this protease in intestinal epithelial penetration. The inhibition of Ac-cathB-1 enzymatic activity with antiserum partly suppressed larval penetration ability in the isolated intestine. Thus, an effective system for heterologous expression of parasite proteases is presented for studying gene function in A. cantonensis; and Ac-cathB-1 was related to larval penetration ability in the host small intestine.

The AtCathB3 gene, encoding a cathepsin B-like protease, is expressed during germination of Arabidopsis thaliana and transcriptionally repressed by the basic leucine zipper protein GBF1

Protein hydrolysis plays an important role during seed germination and post-germination seedling establishment. In Arabidopsis thaliana, cathepsin B-like proteases are encoded by a gene family of three members, but only the AtCathB3 gene is highly induced upon seed germination and at the early post-germination stage. Seeds of a homozygous T-DNA insertion mutant in the AtCathB3 gene have, besides a reduced cathepsin B activity, a slower germination than the wild type. To explore the transcriptional regulation of this gene, we used a combined phylogenetic shadowing approach together with a yeast one-hybrid screening of an arrayed library of approximately 1200 transcription factor open reading frames from Arabidopsis thaliana. We identified a conserved CathB3-element in the promoters of orthologous CathB3 genes within the Brassicaceae species analysed, and, as its DNA-interacting protein, the G-Box Binding Factor1 (GBF1). Transient overexpression of GBF1 together with a PAtCathB3::uidA (β-glucuronidase) construct in tobacco plants revealed a negative effect of GBF1 on expression driven by the AtCathB3 promoter. In stable P35S::GBF1 lines, not only was the expression of the AtCathB3 gene drastically reduced, but a significant slower germination was also observed. In the homozygous knockout mutant for the GBF1 gene, the opposite effect was found. These data indicate that GBF1 is a transcriptional repressor of the AtCathB3 gene and affects the germination kinetics of Arabidopsis thaliana seeds. As AtCathB3 is also expressed during post-germination in the cotyledons, a role for the AtCathB3-like protease in reserve mobilization is also inferred.

Analysis of a novel cathepsin B circulating antigen and its response to drug treatment in Trichinella-infected mice

In this paper, we cloned a novel full-length cDNA that encodes a Trichinella spiralis cathepsin B-like protease gene (TsCPB) using 3'-RACE PCR. The recombinant mature TsCPB protein (rTsCPB) was then expressed in an Escherichia coli expression system and purified with Ni-affinity chromatography. Real-time quantitative PCR revealed that TsCPB was expressed across all development stages of the parasite but had the highest expression level during the adult stage. Furthermore, rTsCPB was detected in Trichinella excretory-secretory products with anti-rTsCPB rabbit polyclonal antibodies. Interestingly, rTsCPB was strongly recognized by the T. spiralis-infected sera in Western blotting, implying that TsCPB protein appeared in the peripheral blood of Trichinella-infected mice as circulating antigens (CAg). We then analyzed the dynamic levels of TsCPB CAg and its antibodies in T. spiralis-infected sera by using an improved double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) and indirect ELISA, respectively. The results showed that TsCPB CAg can be detected much earlier compared to antibody detection in Trichinella-infected mice. In addition, we monitored the effects of albendazole drug therapy (a dosage of 370mg/kg body weight, twice a day) on T. spiralis-infected mice by detecting the levels of TsCPB CAg and its antibody in the sera of drug-treated mice. The results showed that the levels of CAg dramatically decreased after successful drug treatment, while the antibody level remained unchanged. Overall, the novel Trichinella antigen TsCPB could be a promising novel circulating antigen molecule for the detection of Trichinella infection and for monitoring the efficacy of drug treatment of trichinellosis.

Nicotinamide Inhibits the Lysosomal Cathepsin b-like Protease and Kills African Trypanosomes

Nicotinamide, a soluble compound of the vitamin B3 group, has antimicrobial activity against several microorganisms ranging from viruses to parasite protozoans. However, the mode of action of this antimicrobial activity is unknown. Here, we investigate the trypanocidal activity of nicotinamide on Trypanosoma brucei, the causative agent of African trypanosomiasis. Incubation of trypanosomes with nicotinamide causes deleterious defects in endocytic traffic, disruption of the lysosome, failure of cytokinesis, and, ultimately, cell death. At the same concentrations there was no effect on a cultured mammalian cell line. The effects on endocytosis and vesicle traffic were visible within 3 h and can be attributed to inhibition of lysosomal cathepsin b-like protease activity. The inhibitory effect of nicotinamide was confirmed by a direct activity assay of recombinant cathepsin b-like protein. Taken together, these data demonstrate that inhibition of the lysosomal protease cathepsin b-like blocks endocytosis, causing cell death. In addition, these results demonstrate for the first time the inhibitory effect of nicotinamide on a protease.

Eimeripain, a Cathepsin B-Like Cysteine Protease, Expressed throughout Sporulation of the Apicomplexan Parasite Eimeria tenella

The invasion and replication of Eimeria tenella in the chicken intestine is responsible for avian coccidiosis, a disease that has major economic impacts on poultry industries worldwide. E. tenella is transmitted to naïve animals via shed unsporulated oocysts that need contact with air and humidity to form the infectious sporulated oocysts, which contain the first invasive form of the parasite, the sporozoite. Cysteine proteases (CPs) are major virulence factors expressed by protozoa. In this study, we show that E. tenella expresses five transcriptionally regulated genes encoding one cathepsin L, one cathepsin B and three cathepsin Cs. Biot-LC-LVG-CHN2, a cystatin derived probe, tagged eight polypeptides in unsporulated oocysts but only one in sporulated oocysts. CP-dependant activities were found against the fluorescent substrates, Z-FR-AMC and Z-LR-AMC, throughout the sporulation process. These activities corresponded to a cathepsin B-like enzyme since they were inhibited by CA-074, a specific cathepsin B inhibitor. A 3D model of the catalytic domain of the cathepsin B-like protease, based on its sequence homology with human cathepsin B, further confirmed its classification as a papain-like protease with similar characteristics to toxopain-1 from the related apicomplexan parasite, Toxoplasma gondii; we have, therefore, named the E. tenella cathepsin B, eimeripain. Following stable transfection of E. tenella sporozoites with a plasmid allowing the expression of eimeripain fused to the fluorescent protein mCherry, we demonstrated that eimeripain is detected throughout sporulation and has a punctate distribution in the bodies of extra- and intracellular parasites. Furthermore, CA-074 Me, the membrane-permeable derivative of CA-074, impairs invasion of epithelial MDBK cells by E. tenella sporozoites. This study represents the first characterization of CPs expressed by a parasite from the Eimeria genus. Moreover, it emphasizes the role of CPs in transmission and dissemination of exogenous stages of apicomplexan parasites.

Cloning and characterization of a novel cathepsin B-like cysteine proteinase from Angiostrongylus cantonensis

Cysteine protease plays a key role in host-parasite interactions. In this study, we identified a novel gene encoding a cathepsin B-like cysteine protease (AcCBL1) from the cDNA library of Angiostrongysus cantonensis fourth-stage larvae (L4) and characterized its biological role in the parasite. Sequence and phylogeny analysis showed that AcCBL1 is related to other cathepsin B family members with the conserved catalytic triad (Cys, His, Asn) and diagnostic occluding loop. In addition, the sequence contains a specific "hemoglobinase motif" and might have a hemoglobinase (Hb)-degrading function. The recombinant AcCBL1 (rAcCBL1) exhibited the protease activity by gelation SDS/PAGE assay; rAcCBL1 can cleave the fluorogenic substrate Z-Arg-Arg-AMC, and the optimum pH was 5.5. The enzyme can hydrolyse several host proteins including Hb and human IgG in acidic pH, but low levels of hydrolysis were observed in neutral pH. Reverse transcription polymerase chain reaction revealed that AcCBL1 expression was detected throughout various developmental stages, L3, L4, adult male and female worms. Western blotting analysis indicated that AcCBL1 was an excretory/secretory product of L4 in mature form of protease. Immunolocalization demonstrated that AcCBL1 was mainly localized in the intestine of L4. These results suggest that rAcCBL1 may play an important role in the parasite nutrition uptake.

Structural Basis for the Regulation of Cysteine-Protease Activity by a New Class of Protease Inhibitors in Plasmodium

Plasmodium cysteine proteases are essential for host-cell invasion and egress, hemoglobin degradation, and intracellular development of the parasite. The temporal, site-specific regulation of cysteine-protease activity is a prerequisite for survival and propagation of Plasmodium. Recently, a new family of inhibitors of cysteine proteases (ICPs) with homologs in at least eight Plasmodium species has been identified. Here, we report the 2.6Å X-ray crystal structure of the C-terminal, inhibitory domain of ICP from P. berghei (PbICP-C) in a 1:1 complex with falcipain-2, an important hemoglobinase of Plasmodium. The structure establishes Plasmodium ICP as a member of the I42 class of chagasin-like protease inhibitors but with large insertions and differences in the binding mode relative to other family members. Furthermore, the PbICP-C structure explains why host-cell cathepsin B-like proteases and, most likely, also the protease-like domain of Plasmodium SERA5 (serine-repeat antigen 5) are no targets for ICP.

Immunodominant antigens in Naegleria fowleri excretory–secretory proteins were potential pathogenic factors

Naegleria fowleri, a ubiquitous pathogenic free-living amoeba, is the most virulent species and causes primary amoebic meningoencephalitis in laboratory animals and humans. The parasite secretes various inducing molecules as biological responses, which are thought to be involved in pathophysiological and immunological events during infection. To investigate what molecules of N. fowleri excretory-secretory proteins (ESPs) are related with amoebic pathogenicity, N. fowleri ESPs fractionated by two-dimensional electrophoresis were reacted with N. fowleri infection or immune sera. To identify immunodominant ESPs, six major protein spots were selected and analyzed by N-terminal sequencing. Finally, six proteins, 58, 40, 24, 21, 18, and 16 kDa of molecular weight, were partially cloned and matched with reference proteins as follow: 58 kDa of exendin-3 precursor, 40 kDa of secretory lipase, 24 kDa of cathepsin B-like proteases and cysteine protease, 21 kDa of cathepsin B, 18 kDa of peroxiredoxin, and 16 kDa of thrombin receptor, respectively. These results suggest that N. fowleri ESPs contained important proteins, which may play an important role in the pathogenicity of N. fowleri.

Molecular Cloning and Expression of a Caenorhabditis elegans Cathepsin B-Like Protease

Molecular Cloning and Expression of a Caenorhabditis elegans Cathepsin B-Like Protease

A Parasite Cysteine Protease Is Key to Host Protein Degradation and Iron Acquisition

Cysteine proteases of the Clan CA (papain) family are the predominant protease group in primitive invertebrates. Cysteine protease inhibitors arrest infection by the protozoan parasite, Trypanosoma brucei. RNA interference studies implicated a cathepsin B-like protease, tbcatB, as a key inhibitor target. Utilizing parasites in which one of the two alleles of tbcatb has been deleted, the key role of this protease in degradation of endocytosed host proteins is delineated. TbcatB deficiency results in a decreased growth rate and dysmorphism of the flagellar pocket and the subjacent endocytic compartment. Western blot and microscopic analysis indicate that deficiency in tbcatB results in accumulation of both host and parasite proteins, including the lysosomal marker p67. A critical function for parasitism is the degradation of host transferrin, which is necessary for iron acquisition. Substrate specificity analysis of recombinant tbcatB revealed the optimal peptide cleavage sequences for the enzyme and these were confirmed experimentally using FRET-based substrates. Degradation of transferrin was validated by SDS-PAGE and the specific cleavage sites identified by N-terminal sequencing. Because even a modest deficiency in tbcatB is lethal for the parasite, tbcatB is a logical target for the development of new anti-trypanosomal chemotherapy.

Construction of the recombinant baculovirus AcMNPV with cathepsin B-like proteinase and its insecticidal activity against Helicoverpa armigera

The Helicoverpa armigera cathepsin B-like proteinase (HCB) has been shown to have a wide spectrum of substrates. It has been involved in the degradation of yolk protein during embryonic development and also in the decomposition of the adult fat body. To study the possibility of using HCB to improve the insecticidal activity of bioinsecticides, it was inserted into the pFASTBACDUAL-green fluorescent protein (GFP) donor plasmid under the strong polyhedrin promoter, and the polyhedrin gene was retained behind the HCB gene so that the virus could orally infect the host and survive in the natural environment. After the recombinant plasmid transfected the Sf21 cells, the recombinant baculovirus Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV)-GFP-HCB-Polh + was produced. A 37-kDa recombinant procathepsin B was expressed in AcMNPV-GFP-HCB-Polh +-infected Sf21 cells and processed into 29/25-kDa mature forms. Gelatin zymography revealed that the proteolytic activities of the recombinant HCB and other proteases were activated or enhanced by HCB in AcMNPV-GFP-HCB-Polh +-infected larvae. Green fluorescence was observed earlier and was more intense in AcMNPV-GFP-HCB-Polh +-infected larvae than in HCB-free AcMNPV-GFP-Polh +-infected ones that were infected at the same time; this indicated that the AcMNPV-GFP-HCB-Polh + virus spreaded faster in larvae than the AcMNPV-GFP-Polh + virus. A bioassay revealed that infection with the AcMNPV-GFP-HCB-Polh + virus shortened the median survival time of H. armigera larvae by 12 h as compared with those infected with the AcMNPV-GFP-Polh + or the wild-type AcMNPV virus. These results suggest that HCB may possibly play a role in the recombinant virus in improving the rate of killing larvae.

Molecular and Biochemical Characterization of a Cathepsin B-Like Protease Family Unique toTrypanosoma congolense

Cysteine proteases have been shown to be essential virulence factors and drug targets in trypanosomatids and an attractive antidisease vaccine candidate for Trypanosoma congolense. Here, we describe an important amplification of genes encoding cathepsin B-like proteases unique to T. congolense. More than 13 different genes were identified, whereas only one or two highly homologous genes have been identified in other trypanosomatids. These proteases grouped into three evolutionary clusters: TcoCBc1 to TcoCBc5 and TcoCBc6, which possess the classical catalytic triad (Cys, His, and Asn), and TcoCBs7 to TcoCBs13, which contains an unusual catalytic site (Ser, Xaa, and Asn). Expression profiles showed that members of the TcoCBc1 to TcoCBc5 and the TcoCBs7 to TcoCBs13 groups are expressed mainly in bloodstream forms and localize in the lysosomal compartment. The expression of recombinant representatives of each group (TcoCB1, TcoCB6, and TcoCB12) as proenzymes showed that TcoCBc1 and TcoCBc6 are able to autocatalyze their maturation 21 and 31 residues, respectively, upstream of the predicted start of the catalytic domain. Both displayed a carboxydipeptidase function, while only TcoCBc1 behaved as an endopeptidase. TcoCBc1 exhibited biochemical differences regarding inhibitor sensitivity compared to that of other cathepsin B-like proteases. Recombinant pro-TcoCBs12 did not automature in vitro, and the pepsin-matured enzyme was inactive in tests with cathepsin B fluorogenic substrates. In vivo inhibition studies using CA074Me (a cell-permeable cathepsin B-specific inhibitor) demonstrated that TcoCB are involved in lysosomal protein degradation essential for survival in bloodstream form. Furthermore, TcoCBc1 elicited an important immune response in experimentally infected cattle. We propose this family of proteins as a potential therapeutic target and as a plausible antigen for T. congolense diagnosis.