Cathepsin B-like protease from chili pepper revealed by in silico approach (original) (raw)
Related papers
Biological chemistry, 2018
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. Mut...
PLANT PHYSIOLOGY, 2012
Papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes associated with development, immunity, and senescence. Although many properties have been described for individual proteases, the distribution of these characteristics has not been studied collectively. Here, we analyzed 723 plant PLCPs and classify them into nine subfamilies that are present throughout the plant kingdom. Analysis of these subfamilies revealed previously unreported distinct subfamily-specific functional and structural characteristics. For example, the NPIR and KDEL localization signals are distinctive for subfamilies, and the carboxyl-terminal granulin domain occurs in two PLCP subfamilies, in which some individual members probably evolved by deletion of the granulin domains. We also discovered a conserved double cysteine in the catalytic site of SAG12-like proteases and two subfamily-specific disulfides in RD19A-like proteases. Protease activity profiling of representatives of the PLCP subfamilies using novel fluorescent probes revealed striking polymorphic labeling profiles and remarkably distinct pH dependency. Competition assays with peptide-epoxide scanning libraries revealed common and unique inhibitory fingerprints. Finally, we expand the detection of PLCPs by identifying common and organ-specific protease activities and identify previously undetected proteases upon labeling with cell-penetrating probes in vivo. This study provides the plant protease research community with tools for further functional annotation of plant PLCPs.
Biochimie, 2015
The tobacco-related plant species Nicotiana benthamiana has recently emerged as a versatile expression platform for the rapid generation of recombinant biopharmaceuticals, but product yield and quality frequently suffer from unintended proteolysis. Previous studies have highlighted that recombinant protein fragmentation in plants involves papain-like cysteine proteinases (PLCPs). For this reason, we have now characterized two major N. benthamiana PLCPs in detail: aleurain-like protease (NbALP) and cathepsin B (NbCathB). As typical for PLCPs, the precursor of NbCathB readily undergoes autocatalytic activation when incubated at low pH. On the contrary, maturation of NbALP requires the presence of a cathepsin L-like PLCP as processing enzyme. While the catalytic features of NbALP closely resemble those of its mammalian homologue cathepsin H, NbCathB displays remarkable differences to human cathepsin B. In particular, NbCathB appears to be a far less efficient peptidyldipeptidase (removing C-terminal dipeptides) than its human counterpart, suggesting that it functions primarily as an endopeptidase. Importantly, NbCathB was far more efficient than NbALP in processing the human anti-HIV-1 antibody 2F5 into fragments observed during its production in N. benthamiana. This suggests that targeted downregulation of NbCathB could improve the performance of this plant-based expression platform.
Biochemistry (Moscow), 2014
Proteolytic enzymes (known as proteases) catalyze the hydrolytic cleavage of peptide bonds in their target proteins, and about 2% of the genes code for these enzymes in higher organisms [1 3]. Protease inhibitors (PIs) are widely distributed in the plant kingdom and observed in storage tissues, including tubers and seeds [4]. Also, it is thought that these inhibitors play important roles in defense against herbivores that protect against digestion by proteases [5]. Previous studies revealed that the expression of many PI genes is induced by mechani cal or herbivore damage [6]. PIs have been classified into families and subfamilies based on sequence similarity with protein folds of the inhibitory domains or units. According to their inhibitor domains, PIs can be grouped into 48 families [7]. Soybean trypsin inhibitors (Kunitz), Bowman-Birk inhibitors (BBIs), and potato inhibitors I and II are widely studied classes [8]. BBIs are members of the serine protease inhibitors family and are commonly found in leguminous and cere al plants [5]. BBIs were first discovered and characterized in soybean [9, 10]. BBIs are double headed serine pro teinase inhibitors, having low molecular mass between 8 and 20 kDa and being rich the cysteine residues [11]. These cysteine residues support conservation of the con formation of the inhibitor with the disulfide link, hence allowing the inhibition of the target enzyme [12]. In dicotyledonous plants, BBIs have a single polypeptide chain with molecular mass of 8 kDa; in contrast, mono cotyledonous plants contain two groups of BBIs. The first group has a single polypeptide chain with molecular mass of about 8 kDa and a single reactive site. The second group has molecular mass of 16 kDa with two reactive sites [12, 13]. The loops (or reactive sites) contribute to inhibition of trypsin and chymotrypsin in monocotyledo nous plants and trypsin, chymotrypsin, and elastase in dicotyledonous plants. Also, the three dimensional struc ture of peanut, soybean, and barley BBIs showed that there are disulfide bridges between two conserved cysteine residues (e.g.
Protease gene families in Populus and Arabidopsis
BMC Plant …, 2006
Background: Proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. Similarities and differences between the proteases expressed in different species may give valuable insights into their physiological roles and evolution.
Activity Profiling of Papain-Like Cysteine Proteases in Plants
PLANT PHYSIOLOGY, 2004
Transcriptomic and proteomic technologies are generating a wealth of data that are frequently used by scientists to predict the function of proteins based on their expression or presence. However, activity of many proteins, such as transcription factors, kinases, and proteases, depends on posttranslational modifications that frequently are not detected by these technologies. Therefore, to monitor activity of proteases rather than their abundance, we introduce protease activity profiling in plants. This technology is based on the use of biotinylated, irreversible protease inhibitors that react with active proteases in a mechanismbased manner. Using a biotinylated derivative of the Cys protease inhibitor E-64, we display simultaneous activities of many papain-like Cys proteases in extracts from various tissues and from different plant species. Labeling is pH dependent, stimulated with reducing agents, and inhibited specifically by Cys protease inhibitors but not by inhibitors of other protease classes. Using one-step affinity capture of biotinylated proteases followed by sequencing mass spectrometry, we identified proteases that include xylem-specific XCP2, desiccation-induced RD21, and cathepsin B-and aleurain-like proteases. Together, these results demonstrate that this technology can identify differentially activated proteases and/or characterize the activity of a particular protease within complex mixtures.
Cross genome comparisons of serine proteases in Arabidopsis and rice
BMC genomics, 2006
Background: Serine proteases are one of the largest groups of proteolytic enzymes found across all kingdoms of life and are associated with several essential physiological pathways. The availability of Arabidopsis thaliana and rice (Oryza sativa) genome sequences has permitted the identification and comparison of the repertoire of serine protease-like proteins in the two plant species. Results: Despite the differences in genome sizes between Arabidopsis and rice, we identified a very similar number of serine protease-like proteins in the two plant species (206 and 222, respectively). Nearly 40% of the above sequences were identified as potential orthologues. Atypical members could be identified in the plant genomes for Deg, Clp, Lon, rhomboid proteases and species-specific members were observed for the highly populated subtilisin and serine carboxypeptidase families suggesting multiple lateral gene transfers. DegP proteases, prolyl oligopeptidases, Clp proteases and rhomboids share a significantly higher percentage orthology between the two genomes indicating substantial evolutionary divergence was set prior to speciation. Single domain architectures and paralogues for several putative subtilisins, serine carboxypeptidases and rhomboids suggest they may have been recruited for additional roles in secondary metabolism with spatial and temporal regulation. The analysis reveals some domain architectures unique to either or both of the plant species and some inactive proteases, like in rhomboids and Clp proteases, which could be involved in chaperone function. Conclusion: The systematic analysis of the serine protease-like proteins in the two plant species has provided some insight into the possible functional associations of previously uncharacterised serine protease-like proteins. Further investigation of these aspects may prove beneficial in our understanding of similar processes in commercially significant crop plant species.
The Protein Journal, 2008
A new cysteine peptidase (Granulosain I) was isolated from ripe fruits of Solanum granuloso-leprosum Dunal (Solanaceae) by means of precipitation with organic solvent and cation exchange chromatography. The enzyme showed a single band by SDS-PAGE, its molecular mass was 24,746 Da (MALDI-TOF/MS) and its isoelectric point was higher than 9.3. It showed maximum activity (more than 90%) in the pH range 7-8.6. Granulosain I was completely inhibited by E-64 and activated by the addition of cysteine or 2-mercaptoethanol, confirming its cysteinic nature. The kinetic studies carried out with PFLNA as substrate, showed an affinity (Km 0.6 mM) slightly lower than those of other known plant cysteine proteases (papain and bromelain). The N-terminal sequence of granulosain I (DRLPASVDWRGKGVLVLVKNQGQC) exhibited a close homology with other cysteine proteases belonging to the C1A family. Keywords Solanum granuloso-leprosum Á Solanaceae Á Cysteine protease Á Plant peptidases Abbreviations AMPSO N-(1,1-Dimethyl-2-hydroxyethyl)-3amino-2-hydroxypropanesulfonic acid CAPS 3-(Cyclohexylamino)-1-propanesulfonic acid E-64 trans-Epoxysuccinyl-L-leucyl-amido (4-guanidino)butane EDTA Ethylendiaminetetraacetic acid MALDI-TOF/MS Matrix assisted laser desorption ionization time of flight mass spectrometry MES 2-Morpholinoethanesulfonic acid MOPS 3-(N-Morpholino) propanesulfonic acid PFLNA pGlu-Phe-Leu p-nitroanilide PMSF Phenylmethanesulfonyl fluoride PVDF Polyvinylidene difluoride RAP Redissolved acetone precipitate TAPS N-Tris(hydroxymethyl)methyl-3aminopropanesulfonic acid
Papain-like cysteine proteases in Carica papaya: lineage-specific gene duplication and expansion
BMC genomics, 2018
Papain-like cysteine proteases (PLCPs), a large group of cysteine proteases structurally related to papain, play important roles in plant development, senescence, and defense responses. Papain, the first cysteine protease whose structure was determined by X-ray crystallography, plays a crucial role in protecting papaya from herbivorous insects. Except the four major PLCPs purified and characterized in papaya latex, the rest of the PLCPs in papaya genome are largely unknown. We identified 33 PLCP genes in papaya genome. Phylogenetic analysis clearly separated plant PLCP genes into nine subfamilies. PLCP genes are not equally distributed among the nine subfamilies and the number of PLCPs in each subfamily does not increase or decrease proportionally among the seven selected plant species. Papaya showed clear lineage-specific gene expansion in the subfamily III. Interestingly, all four major PLCPs purified from papaya latex, including papain, chymopapain, glycyl endopeptidase and caric...