Physical exercise is known to activate the sympathetic nervous system, which influences the production of saliva from salivary glands. Our examination of saliva collected from highly trained athletes before and after a number of physical competititions showed an increase in the secretion of S-type cystatins and cystatin C as a subacute response to aerobic and anaerobic exercise. The elevation in salivary cystatins was transient and the recovery time course differed from that of amylase and other salivary proteins. An in vitro assay was developed based on a cell line from a human submandibular gland (HSG) that differentiated into acinus-like structures. Treatments with the β-adrenergic agonist isoproterenol caused a shift in the intracellular distribution of S-type cystatins and cystatin C, promoting their accumulation at the outer regions of the acinus prior to release and suggesting the activation of a directional transport involving co-migration of both molecules. In another treatment using non-differentiated HSG cells, it was evident that both expression and secretion of cystatin C increased upon addition of the β-adrenergic agonist, and these effects were essentially eliminated by the antagonist propranolol. The HSG cell line appears to have potential as a model for exploring the mechanism of cystatin secretion, particularly the S-type cystatins that originate primarily in the submandibular glands.

Phytocystatins are tight-binding cysteine protease inhibitors produced by plants. The first phytocystatin described was isolated from Oryza sativa and, since then, cystatins from several plant species were reported, including from sugarcane. Sugarcane cystatins were unraveled in Sugarcane EST project database, after sequencing of cDNA libraries from various sugarcane tissues at different developmental stages and six sugarcane cystatins were cloned, expressed and characterized (CaneCPI-1 to CaneCPI-6). These recombinant proteins were produced in different expression systems and inhibited several cysteine proteases, including human cathepsins B and L, which can be involved in pathologies, such as cancer. In this review, we summarize a comprehensive history of all sugarcane cystatins, presenting an updated phylogenetic analysis; chromosomal localization, and genomic organization. We also present protein docking of CaneCPI-5 in the active site of human cathepsin B, insights about canecystatins structures; recombinant expression in different systems, comparison of their inhibitory activities against human cysteine cathepsins B, K, L, S, V, falcipains from Plasmodium falciparum and a cathepsin L-like from the sugarcane weevil Sphenophorus levis; and enlighten their potential and current applications in agriculture and health.

Cysteine proteases are implicated in proteolysis events favoring cancer cell growth, spread, and death by apoptosis. Herein, we have studied whether the net growth and survival of the leukemic cell lines Jurkat, U937, and HL-60 are affected by external addition of five proteins acting as natural cysteine protease inhibitors. None of the cystatins examined (A, C, D, and E/M) or chagasin showed consistent effects on Fas-induced apoptosis when evaluated at 1 µm. In contrast, when the intrinsic apoptosis pathway was activated by hydrogen peroxide, addition of cystatin D augmented caspase-3-like activity within all three cell lines. Flow cytometric analysis of U937 cells also showed increased numbers of annexin V-positive cells when hydrogen peroxide was used to initiate apoptosis and cells were cultured in the presence of cystatin D or C. Moreover, stimulation of hydrogen peroxide-induced apoptotic U937 cells with either cystatin C or D resulted in a dose-dependent decrease in the number of cells. Cell viability was also decreased when U937 cells were cultured in the presence of cystatin C or D (1-9 µm) only, demonstrating that these cystatins can reduce cell proliferation by themselves in addition to enhancing apoptosis induced by oxidative stress. These effects on U937 cells were paralleled by internalization of cystatins C and D, indicating these effects are caused by downregulation of intracellular proteolysis. External addition of cystatins C and D to HL-60 and Jurkat cells demonstrated similar degrees of cystatin D uptake and decreased viability as for U937 cells, indicating that these effects are general for leukemic cells.

To develop a diagnostic trial enabling the selective examination for a target cystatin in human body fluids, we attempted to prepare monoclonal antibodies against human cystatin SA1 (originally cystatin SA) and its variant form (cystatin SA2). BALB/c mice were immunized with recombinant (r-) cystatins SA1 and SA2. Two monoclonal antibodies designated Cys3F11 and Cys2E5 were selected. By ELISA analyses, the Cys2E5 was shown to react with r-cystatin SA2 but also somewhat with r-cystatin SA1 (22% cross-reactivity) and with plasma cystatin C (18% cross-reactivity), indicating a high specificity for cystatin SA2. The Cys3F11 reacted not only with r-cystatin SA1 but also with r-cystatin SA2 (89% cross-reactivity) and plasma cystatin C (47% cross-reactivity). This finding was further emphasized by immunoblotting of human submandibular-sublingual saliva samples. ELISA additivity test suggests that the two monoclonal antibodies bind to distinct epitopes. In conclusion, we have succeeded in producing two antibodies that discriminate the structural differences between salivary cystatins S and SN, which share more than 90% identity in amino acid sequence with cystatin SA.

Recently several reports have demonstrated that innate immune response and inflammation have an important role in major neurodegenerative diseases. The activation of the NF-κB family of transcription factors is a key step in the regulation of pro inflammatory cytokine expression. Microglia and other cell types in the brain can be activated in response to endogenous danger molecules as well as aggregated proteins and brain injury. During the past couple of years several studies reported the role of cystatins in neuroinflammation and neurodegeneration. In the present review, I will summarize and analyze recent findings regarding the role of cystatins in inflammation and NF-κB activation. Type I cystatin stefin B (cystatin B) is an endogenous cysteine cathepsin inhibitor localized in the cytosol, mitochondria and nucleus. Mutations in the gene of stefin B are associated with the neurodegenerative disease known as Unverricht-Lundborg disease and microglial activation plays an important role in the pathogenesis of the disease. Stefin B deficient mice have increased caspase-11 expression and secreted higher amounts of pro-inflammatory cytokines. The increased caspase-11 gene expression, was a consequence of increased NF-κB activation.

A high incidence of cholangiocarcinoma (bile duct cancer) has been observed in Thailand. This usually rare cancer has been associated with infection with the human liver fluke, Opisthorchis viverrini. Secretions of the parasite that interact with the host are thought to be a major component of its pathogenicity and proteolysis is a key biological activity of the secreted molecules. In this study, we present a molecular analysis of cysteine proteinase inhibitors (cystatins) of Opisthorchis viverrini. Six cDNA coding sequences of Opisthorchis viverrini cystatins, OvCys1-6, were cloned from the adult stage of the parasite using RT-PCR. Based on their sequences, OvCys1 and OvCys2 are classified as type 1 cystatins, while OvCys3-6 are classified as type 2 cystatins, with each containing a signal peptide and only one C-terminal disulfide bond. Their C-terminal region sequences are diverse compared with other cystatin members. Cystatins OvCys1, 3 and 4 were found in crude worm extracts and excretory-secretory (ES) products from the adult parasite using Western blot detection, while the other isoforms were not. Thus, OvCys1, 3 and 4 were selected for inhibition analysis and immune reactivity with Opisthorchis viverrini-infected hamster sera. OvCys1, 3, and 4 inhibited mammalian cathepsin L more effectively than cathepsin B. The pH range for their full activity was very wide (pH 3-9) and they were heat stable for at least 3 h. Unlike Fasciola gigantica cystatins, they showed no immune reactivity with infected hamster sera based on indirect ELISA. Our findings suggest that Opisthorchis viverrini cystatins are not major antigenic components in the ES product of this parasite and that other effects of Opisthorchis viverrini cystatins should be investigated.

Cystatins are inhibitors of cysteine proteases. The majority are only weak inhibitors of human cathepsin B, which has been associated with cancer, Alzheimer's disease and arthritis.

Phytocystatins are cysteine proteinase inhibitors from plants implicated in the endogenous regulation of protein turnover, programmed cell death, and in defense mechanisms against pathogens. To date, only few cystatin genes have been characterized in most plant species. We have previously characterized the protein Canecystatin, the first cystatin described in sugarcane. In an attempt to study novel Canecystatins, we identified two ORFs encoding cystatins (referred as CaneCPI-2 and CaneCPI-3) using the data from the Sugarcane EST genome project. These ORFs were then subcloned and expressed in Escherichia coli using pET28 expression vector. High amounts (approximately 20 mg/L) of pure recombinant proteins were obtained by affinity chromatography in a single step of purification. Polyclonal antibodies against the recombinant Canecystatins were raised, allowing the immunodetection of the endogenous proteins in the plant tissues. Moreover, the proteins were able to inhibit papain in a fluorometric assay with K(i) values of 0.2 and 0.25 microM for CaneCPI-2 and CaneCPI-3, respectively. These findings contribute to a better understanding of the activity of sugarcane cystatins and encourage future activity and structural studies of these proteins.

The evolutionary aspects of cystatins are greatly underexplored in early-emerging metazoans. Thus, we surveyed the gene organization, protein architecture, and phylogeny of cystatin homologues mined from 110 genomes and the transcriptomes of 58 basal metazoan species, encompassing free-living and parasite taxa of Porifera, Placozoa, Cnidaria (including Myxozoa), and Ctenophora. We found that the cystatin gene repertoire significantly differs among phyla, with stefins present in most of the investigated lineages but with type 2 cystatins missing in several basal metazoan groups. Similar to liver and intestinal flukes, myxozoan parasites possess atypical stefins with chimeric structure that combine motifs of classical stefins and type 2 cystatins. Other early metazoan taxa regardless of lifestyle have only the classical representation of cystatins and lack multi-domain ones. Our comprehensive phylogenetic analyses revealed that stefins and type 2 cystatins clustered into taxonomically defined clades with multiple independent paralogous groups, which probably arose due to gene duplications. The stefin clade split between the subclades of classical stefins and the atypical stefins of myxozoans and flukes. Atypical stefins represent key evolutionary innovations of the two parasite groups for which their origin might have been linked with ancestral gene chimerization, obligate parasitism, life cycle complexity, genome reduction, and host immunity.

Cystatins are thiol proteinase inhibitors (TPI), present ubiquitously in animals, plants and micro-organisms. These are not merely inhibitors rather they are at heart of many pathological conditions ranging from diabetes to renal failure. These are essential for maintenance of protein balance of the cell; once this balance gets disturbed, it may lead to cell death. Thus, cystatins cannot be merely regarded as TPI's as these have been found to play a pivotal role in tumorigenesis and neurodegenerative diseases. Many studies have reported the variation in cystatin level in incidences of different types of cancer; highlighting an important role played by these inhibitors in cancer development and progression. Cystatin C is increasingly replacing creatinine as a biomarker of glomerular filtration rate (GFR) thereby highlighting the importance of this important inhibitor. Some recent studies have also reported the interaction pattern of various anti-cancer drugs with cystatins in a bid to find how these drugs affect this important inhibitors and whether these drugs have any side effect on cystatins. Thus, in this growing disease era it can be said that cystatins are no more just inhibitors blocking the activity of thiol proteases rather they play a pivotal role in variety of pathological conditions.

Cystatins are a group of cysteine protease inhibitors responsible for physiological proteolysis regulation and present in a wide range of organisms. Studies about this class of inhibitors in parasites have contributed to clarify their roles in important physiological processes, like blood digestion and modulation of host immune response during blood feeding. Thus, cystatins are a subject of research on the development of new parasite control methods. Additionally, the characterization of proteins shared by different parasite species represents a valuable strategy to find potential targets in multi-species control methods. However, cystatin functions in ticks remain undetermined, especially in Rhipicephalus microplus and Ixodes ovatus, two species that affect livestock and human health, respectively.

Cystatins are a family of inhibitors of cysteine peptidases that comprises the salivary cystatins (D and S-type cystatins) and cystatin C. These cystatins are encoded by a multigene family (CST3, CST5, CST4, CST1 and CST2) organized in tandem in the human genome. Their presence and functional importance in human saliva has been reported, however the distribution of these proteins in other mammals is still unclear. Here, we performed a proteomic analysis of the saliva of several mammals and studied the evolution of this multigene family. The proteomic analysis detected S-type cystatins (S, SA, and SN) in human saliva and cystatin D in rat saliva. The evolutionary analysis showed that the cystatin C encoding gene is present in species of the most representative mammalian groups, i.e. Artiodactyla, Rodentia, Lagomorpha, Carnivora and Primates. On the other hand, D and S-type cystatins are mainly retrieved from Primates, and especially the evolution of S-type cystatins seems to be a dynamic process as seen in Pongo abelii genome where several copies of CST1-like gene (cystatin SN) were found. In Rodents, a group of cystatins previously identified as D and S has also evolved. Despite the high divergence of the amino acid sequence, their position in the phylogenetic tree and their genome organization suggests a common origin with those of the Primates. These results suggest that the D and S type cystatins have emerged before the mammalian radiation and were retained only in Primates and Rodents. Although the mechanisms driving the evolution of cystatins are unknown, it seems to be a dynamic process with several gene duplications evolving according to the birth-and-death model of evolution. The factors that led to the appearance of a group of saliva-specific cystatins in Primates and its rapid evolution remain undetermined, but may be associated with an adaptive advantage.

Phytocystatins constitute a multigene family that regulates the activity of endogenous and/or exogenous cysteine proteinases. Cereal crops like wheat are continuously threatened by a multitude of pathogens, therefore cystatins offer to play a pivotal role in deciding the plant response. In order to study the need of having diverse specificities and activities of various cystatins, we conducted comparative analysis of six wheat cystatins (WCs) with twelve rice, seven barley, one sorghum and ten corn cystatin sequences employing different bioinformatics tools. The obtained results identified highly conserved signature sequences in all the cystatins considered. Several other motifs were also identified, based on which the sequences could be categorized into groups in congruence with the phylogenetic clustering. Homology modeling of WCs revealed 3D structural topology so well shared by other cystatins. Protein-protein interaction of WCs with papain supported the notion that functional diversity is a con-sequence of existing differences in amino acid residues in highly conserved as well as relatively less conserved motifs. Thus there is a significant conservation at the sequential and structural levels; however, concomitant variations maintain the functional diversity in this protein family, which constantly modulates itself to reciprocate the diversity while counteracting the cysteine proteinases.

Cystatins and their putative targets, the families of cysteine proteinases C1A and C13 play key roles in plants. Comparative genomic analyses are powerful tools to obtain valuable insights into the conservation and evolution of the proteinases and their proteinaceous inhibitors, and could aid to elucidate issues concerning the function of these proteins.

Cathepsins are a group of endosomal proteases present in many cells including dendritic cells (DCs). The activity of cathepsins is regulated by their endogenous inhibitors - cystatins. Cathepsins are crucial to antigen processing during viral and bacterial infections, and as such are a prerequisite to antigen presentation in the context of major histocompatibility complex class I and II molecules. Due to the involvement of DCs in both innate and adaptive immune responses, and the quest to understand the impact of poxvirus infection on host cells, we investigated the influence of ectromelia virus (ECTV) infection on cathepsin and cystatin levels in murine conventional DCs (cDCs). ECTV is a poxvirus that has evolved many mechanisms to avoid host immune response and is able to replicate productively in DCs.

Multidrug-resistant (MDR) bacterial pneumonia can induce dysregulated pulmonary and systemic inflammation leading to morbidity and mortality. Antibiotics to treat MDR pathogens do not function to modulate the extent and intensity of inflammation and can have serious side effects. Here we evaluate the efficacy of two human cysteine proteinase inhibitors, cystatin 9 (CST9) and cystatin C (CSTC), as a novel immunotherapeutic treatment to combat MDR New Delhi metallo-beta-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae Our results showed that mice infected intranasally (i.n.) with a 90% lethal dose (LD90) challenge of NDM-1 K. pneumoniae and then treated with the combination of human recombinant CST9 (rCST9) and rCSTC (rCSTs; 50 pg of each i.n. at 1 h postinfection [p.i.] and/or 500 pg of each intraperitoneally [i.p.] at 3 days p.i.) had significantly improved survival compared to that of infected mice alone or infected mice treated with individual rCSTs (P < 0.05). Results showed that both of our optimal rCST treatment regimens modulated pulmonary and systemic proinflammatory cytokine secretion in the serum, lungs, liver, and spleen in infected mice (P < 0.05). Treatment also significantly decreased the bacterial burden (P < 0.05) while preserving lung integrity, with reduced inflammatory cell accumulation compared to that in infected mice. Further, rCST treatment regimens reduced lipid peroxidation and cell apoptosis in the lungs of infected mice. Additionally, in vitro studies showed that rCSTs (50 or 500 pg of each) directly decreased the viability of NDM-1 K. pneumoniae In conclusion, the data showed that rCST9/rCSTC worked synergistically to modulate host inflammation against MDR NDM-1 K. pneumoniae pneumonia, which significantly improved survival. Therefore, rCST9/rCSTC is a promising therapeutic candidate for the treatment of bacterial pneumonia.

Among the C1A cysteine proteases, the plant cathepsin F-like group has been poorly studied. This paper describes the molecular and functional characterization of the HvPap-1 cathepsin F-like protein from barley. This peptidase is N-glycosylated and has to be processed to become active by its own propeptide being an important modulator of the peptidase activity. The expression pattern of its mRNA and protein suggest that it is involved in different proteolytic processes in the barley plant. HvPap-1 peptidase has been purified in Escherichia coli and the recombinant protein is able to degrade different substrates, including barley grain proteins (hordeins, albumins, and globulins) stored in the barley endosperm. It has been localized in protein bodies and vesicles of the embryo and it is induced in aleurones by gibberellin treatment. These three features support the implication of HvPap-1 in storage protein mobilization during grain germination. In addition, a complex regulation exerted by the barley cystatins, which are cysteine protease inhibitors, and by its own propeptide, is also described.

The cystatin family comprises cysteine protease inhibitors distributed in 3 subfamilies (I25A-C). Family members lacking cystatin activity are currently unclassified. Little is known about the evolution of Schistosoma cystatins, their physiological roles, and expression patterns in the parasite life cycle. The present study aimed to identify cystatin homologs in the predicted proteome of three Schistosoma species and other Platyhelminthes. We analyzed the amino acid sequence diversity focused in the identification of protein signatures and to establish evolutionary relationships among Schistosoma and experimentally validated human cystatins. Gene expression patterns were obtained from different developmental stages in Schistosoma mansoni using microarray data. In Schistosoma, only I25A and I25B proteins were identified, reflecting little functional diversification. I25C and unclassified subfamily members were not identified in platyhelminth species here analyzed. The resulting phylogeny placed cystatins in different clades, reflecting their molecular diversity. Our findings suggest that Schistosoma cystatins are very divergent from their human homologs, especially regarding the I25B subfamily. Schistosoma cystatins also differ significantly from other platyhelminth homologs. Finally, transcriptome data publicly available indicated that I25A and I25B genes are constitutively expressed thus could be essential for schistosome life cycle progression. In summary, this study provides insights into the evolution, classification, and functional diversification of cystatins in Schistosoma and other Platyhelminthes, improving our understanding of parasite biology and opening new frontiers in the identification of novel therapeutic targets against helminthiases.

Irisin, a novel myokine, is secreted by the muscle following proteolytic cleavage of fibronectin type III domain containing 5 (FNDC5) and is considered a novel regulator of glucose homeostasis. Cystathionine γ-lyase (CSE) produces hydrogen sulfide (H2S) and is involved in glucose homeostasis. We examined the hypothesis that H2S deficiency leads to decreased FNDC5 and irisin secretion, and thereby alters glucose metabolism. High-fat diet-fed mice exhibited elevated blood glucose and significantly reduced levels of CSE, H2S, and PGC-1α, with decreased FNDC5/irisin levels and increased oxidative stress in the muscle compared with those of normal diet-fed mice (control). High glucose or palmitate decreases CSE/PGC-1α/FNDC5 levels and glucose uptake in myotubes. Inhibitors (propargylglycine and aminooxyacetate) of H2S producing enzymes or CSE siRNA significantly decreased levels of H2S and FNDC5 along with PGC-1α; similar H2S-deficient conditions also resulted in decreased GLUT4 and glucose uptake. The levels of H2S, PGC-1α, and FNDC5 and glucose uptake were significantly upregulated after treatment with l-cysteine or an H2S donor. Myoblast differentiation showed upregulation of PGC-1α and FNDC5, which was consistent with the increased expression of CSE/H2S. These findings suggest that the upregulation of H2S levels can have beneficial effects on glucose homeostasis via activation of the PGC-1α/FNDC5/irisin signaling pathway.

The cystatin superfamily is comprised of cysteine proteinase inhibitors and encompasses at least 3 subfamilies: stefins, cystatins and kininogens. In this study, the platyhelminth cystatin superfamily was identified and grouped into stefin and cystatin subfamilies. The conserved domain of stefins (G, QxVxG) was observed in all members of platyhelminth stefins. The three characteristics of cystatins, the cystatin-like domain (G, QxVxG, PW), a signal peptide, and one or two conserved disulfide bonds, were observed in platyhelminths, with the exception of cestodes, which lacked the conserved disulfide bond. However, it is noteworthy that cestode cystatins had two tandem repeated domains, although the second tandem repeated domain did not contain a cystatin-like domain, which has not been previously reported. Tertiary structure analysis of Taenia solium cystatin, one of the cestode cystatins, demonstrated that the N-terminus of T. solium cystatin formed a five turn α-helix, a five stranded β-pleated sheet and a hydrophobic edge, similar to the structure of chicken cystatin. Although no conserved disulfide bond was found in T. solium cystatin, the models of T. solium cystatin and chicken cystatin corresponded at the site of the first disulfide bridge of the chicken cystatin. However, the two models were not similar regarding the location of the second disulfide bridge of chicken cystatin. These results showed that T. solium cystatin and chicken cystatin had similarities and differences, suggesting that the biochemistry of T. solium cystatin could be similar to chicken cystatin in its inhibitory function and that it may have further functional roles. The same results were obtained for other cestode cystatins. Phylogenetic analysis showed that cestode cystatins constituted an independent clade and implied that cestode cystatins should be considered to have formed a new clade during evolution.