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Cystatins are the thiol Proteinase inhibitors, present ubiquitously in mammalian body. They prevent unwanted proteolysis and play important role in several diseases. Regulation of cysteine Proteinase and their inhibitors is of utmost importance in neurodegenerative diseases like Alzheimer, amyloid angiopathy and in many other diseases. The action of these cysteine proteases is biologically controlled by proteinase inhibitors namely cystatins(cys) they constitute a superfamily of homologous proteins. The major role of cystatins is to protect the organism against endogenous proteases released from lysosomes, invading microorganisms and parasites that use cysteine proteases to enter the body. An enormous progress has been made in understanding of protein degradation process under normal and pathological conditions; in fact proteases are now clearly viewed as important drug targets. Some studies have suggested that cystatin C is a target for intervention in neurological disorders because its expression increases in response to human neurological disorders and in animal models of neurodegenerative states. Although, these studies did not clarify whether CysC up-regulation is a pathogenic factor in neurodegenerative disorders or whether it represents a neuroprotective compensatory response of the organisms aimed to prevent progression of the disease. However, for other diseases in some cases cystatins other than cys C are up regulated and in some it is down regulated.Cystatins have been implicated in the processes of neuronal degeneration and repair of the nervous system. Both CysC and CysB are potent, reversible inhibitors of most of the currently known cathepsins. The extent of proteolytic activity at any given time and location is the result of a balance between active proteases and physiological inhibitors. Uncontrolled proteolysis as a result of imbalance between active proteases and their endogenous inhibitors has been associated with neuronal cell death in different neuronal diseases, including brain tumors, stroke, some forms of epilepsy, Alzheimer's disease, and neurological autoimmune diseases.An antidepressant is a psychiatric medication used to alleviate mood disorders, major depression and other brain diseases. Drugs including the monoamine oxidase inhibitors (MAOIs), tricyclic antidepressants (TCAs), and serotonin-norepinephrine reuptake inhibitors (SNRIs) are most commonly used antidepressant. They are also used to treat other conditions, such as anxiety disorders, obsessive compulsive disorder, eating disorders, and chronic pain. Although the mechanisms of the action of these antidepressants are not precisely understood, their principal target of action is at the monoamine transporter proteins located at nerve endings. Monoamine neurotransmitter transporters act to terminate synaptic neurotransmission. Selective serotonin reuptake inhibitors or SSRIs are also most widely used class of antidepressants. They work by increasing the level of serotonin in the brain. SSRIs have fewer and milder side effects, fewer drug interactions, and are much less likely to be associated with suicide than TCAs.These antidepressants shows binding when incubated with cystatin, presenting the involvement of these antidepressant in cascade of disease, as leaving no cystatin to inhibit the cathepsin showing the myriad side effect after the administration of antidepressant. This might be one of the reason in the mechanism of action of antidepressant.So this review expound about the role of cystatins in neurological diseases which is considered to be highly significant as it pave the way for commanding tool in the drug design.Communicated by Ramaswamy H. Sarma.

Recently, a salivary gland transcriptome study demonstrated that the transcripts of a putative cystatin gene (SeqID AAEL013287; Aacystatins) from Aedes aegypti were increased in DENV2-infected mosquitoes and that silencing of the Aacystatin gene resulted in an increase in DENV titres. In this work, Aacystatin was biochemically characterized; the purified recombinant inhibitor was able to inhibit typical cysteine proteases with a Ki in the nM range. Pulldown assays using Aag2 cell extracts identified a cathepsin L-like peptidase (AaCatL) as a possible target of Aacystatin. Purified recombinant AaCatL had an optimal pH of 5.0 and displayed a preference for Leu, Val and Phe residues at P2, which is common for other cathepsin L-like peptidases. Transcription analysis of Aacystatin and AaCatL in the salivary glands and midgut of DENV2-infected mosquitoes revealed a negative correlation between DENV2 titres and levels of the inhibitor and peptidase, suggesting their involvement in DENV2-mosquito interactions. Considering that apoptosis may play an important role during viral infections, the possible involvement of Aacystatin in staurosporine-induced apoptosis in Aag2 cells was investigated; the results showed higher expression of the inhibitor in treated cells; moreover, pre incubation with rAacystatin was able to increase Aag2 cell viability., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Schistosomula (the post-infective stages) of the neurotropic schistosome Trichobilharzia regenti possess multiple isoforms of cathepsin B1 peptidase (TrCB1.1-TrCB1.6) with involvement in nutrient digestion. The comparison of substrate preferences of TrCB1.1 and TrCB1.4 showed that TrCB1.4 had a very narrow substrate specificity and after processing it was less effective toward protein substrates when compared to TrCB1.1. Self-processing of both isoforms could be facilitated by sulfated polysaccharides due to a specific binding motif in the pro-sequence. Trans-activation by heterologous enzymes was also successfully employed. Expression profiling revealed a high level of transcription of genes encoding the enzymatically inactive paralogs TrCB1.5 and TrCB1.6. The transcription level of TrCB1.6 was comparable with that of TrCB1.1 and TrCB1.2, the most abundant active isoforms. Recombinant TrCB1.6wt, a wild type paralog with a Cys 29 -to-Gly substitution in the active site that renders the enzyme inactive, was processed by the active TrCB1 forms and by an asparaginyl endopeptidase. Although TrCB1.6wt lacked hydrolytic activity, endopeptidase, but not dipeptidase, activity could be restored by mutating Gly 29 to Cys 29 . The lack of exopeptidase activity may be due to other mutations, such as His 110 -to-Asn in the occluding loop and Asp 224 -to-Gly in the main body of the mature TrCB1.6, which do not occur in the active isoforms TrCB1.1 and TrCB1.4 with exopeptidase activity. The catalytically active enzymes and the inactive TrCB1.6 paralog formed complexes with chicken cystatin, thus supporting experimentally the hypothesis that inactive paralogs could potentially regulate the activity of the active forms or protect them from being inhibited by host inhibitors. The effect on cell viability and nitric oxide production by selected immune cells observed for TrCB1.1 was not confirmed for TrCB1.6. We show here that the active isoforms of TrCB1 have different affinities for peptide substrates thereby facilitating diversity in protein-derived nutrition for the parasite. The inactive paralogs are unexpectedly highly expressed and one of them retains the ability to bind cystatins, likely due to specific mutations in the occluding loop and the enzyme body. This suggests a role in sequestration of inhibitors and protection of active cysteine peptidases., (Copyright © 2020 Dvořáková, Leontovyč, Macháček, O'Donoghue, Šedo, Zdráhal, Craik, Caffrey, Horák and Mikeš.)

Ticks must durably suppress vertebrate host responses (hemostasis, inflammation, immunity) to avoid rejection and act as vectors of many pathogenic microorganisms that cause disease in humans and animals. Transcriptomics and proteomics studies have been used to study tick-host-pathogen interactions and have facilitated the systematic characterization of salivary composition and molecular dynamics throughout tick feeding. Tick saliva contains a complement of protease inhibitors that are differentially produced during feeding, many of which inhibit blood coagulation, platelet aggregation, vasodilation, and immunity. Here we focus on two major groups of protease inhibitors, the small molecular weight Kunitz inhibitors and cystatins. We discuss their role in tick-host-pathogen interactions, how they mediate the interaction between ticks and their hosts, and how they might be exploited both by pathogens to invade hosts and as candidates for the treatment of various human pathologies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Protease inhibitors of the cystatin protein superfamily show potential in plant protection for the control of herbivorous pests. Here, we describe a cystatin activity-based profiling procedure for the selection of potent cystatin candidates, using single functional variants of tomato cystatin SlCYS8 and digestive Cys proteases of the herbivore insect Colorado potato beetle as a case study. The procedure involves the capture of target Cys proteases with biotinylated versions of the cystatins, followed by the identification and quantitation of captured proteases by mass spectrometry. An example is given to illustrate usefulness of the approach as an alternative to current procedures for recombinant inhibitor selection based on in vitro assays with synthetic peptide substrates. A second example is given showing its usefulness as a tool to compare the affinity spectra of inhibitor variants toward different subsets of target protease complements.

Plant cystatins, also called phytocystatins constitute a family of specific cysteine protease inhibitors found in several monocots and dicots. In soybean, phytocystatins regulate several endogenous processes contributing immensely to this crop's tolerance to abiotic stress factors. Soybeans offer numerous nutritional, pharmaceutical and industrial benefits; however, their growth and yields is hampered by drought, which causes more than 10% yield losses recorded every harvest period worldwide. This review analyses the role of papain-like cysteine proteases and their inhibitors in soybean plant growth and development under drought stress. It also describes their localisation, regulation, target organs and tissues, and the overall impact of cystatins on generating drought tolerance soybean plants. These proteins have many functions that remain poorly characterized, particularly under abiotic stress. Although much information is available on the utilisation of proteases for industrial applications, very few reports have focused on the impact of proteases on plant stress responses. The exploitation of cystatins in plant engineering, as competitive proteases inhibitors is one of the means that will guarantee the continued utilisation of soybeans as an important oilseed crop., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Infection with helminth parasites or the administration of their antigens can prevent or attenuate autoimmune diseases. To date, the specific molecules that prime the amelioration are only limited. In this study, recombinant Schistosoma japonicum cystatin (rSjcystatin) and fructose-1,6-bisphosphate aldolase (rSjFBPA) were administered to female NOD mice via intraperitoneal (i.p.) injection to characterize the immunological response by the recombinant proteins. We have shown that the administration of rSjcystatin or rSjFBPA significantly reduced the diabetes incidence and ameliorated the severity of type 1 diabetes mellitus (T1DM). Disease attenuation was associated with suppressed interferon-gamma (IFN-γ) production in autoreactive T cells and with a switch to the production of Th2 cytokines. Following rSjcystatin or rSjFBPA injection, regulatory T cells (Tregs) were remarkably increased, which was accompanied by increased expression of interleukin-10 (IL-10) and transforming growth factor beta (TGF-β). Our study suggests that helminth-derived proteins may be useful in strategies to limit pathology by promoting the Th2 response and upregulating Tregs during the inflammatory tissue-damage process in T1DM.

Background: Acetamiprid (ACMP) is a member of the neonicotinoid group of insecticides. It is extensively used worldwide. The misuse of ACMP creates danger hazards to human and animal., Methods: ACMP induced renal damage evidenced by an increase in kidney injury biomarkers. So the goal of this work is to clarify the reno protective effect of Quercetin (Qrctn) and/or Nano-glutathione (N-Gluta) solely or in combination to counterbalance the danger effect of ACMP. All treatments with the previous agents were coadministered orally with ACMP for one month., Results: ACMP ingestion caused a significant rise in serum creatinin, urea, and uric acid, TNF α along with renal cystatin C, lipid peroxidation and nitric oxide with the concomitant decline in the levels of reduced glutathione and IL-10 levels. Protein expression of ICAM was upregulated as well as mRNA expression of NF-κB while mRNA expression of Nrf2 was down-regulated. Immune histochemistry of TLR 4 revealed strong immune reaction. The administration of Qrctn or N-Gluta either individually or together modulated all the preceding aforementioned parameters., Conclusion: Fascinatingly Qrctn and N-Gluta combination was the most powerful regimen to frustrate ACMP reno-toxicity and may be deliberate as a hopeful applicant for renal therapy., (Copyright © 2019 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.)

Stefin B (cystatin B) is an intracellular inhibitor of cysteine cathepsins and mutations in the stefin B gene, resulting in the development of Unverricht-Lundborg disease, which is a form of myoclonic epilepsy. It was suggested that a key mechanism behind stefin B-mediated disease progression was impaired redox homeostasis. Stefin B-deficient mice were found more sensitive to lipopolysaccharide (LPS)-induced sepsis as a consequence of increased expression of caspase-11 and Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing (NLRP nflammasome activation and higher levels of mitochondrial reactive oxygen species (ROS). In the present study, we investigated if LPS-triggered oxidative stress affected the protein levels and redox status of redox sensitive proteins-thioredoxin, peroxiredoxins, and superoxide dismutases in macrophages and spleens of LPS-injected mice. LPS challenge was found to result in a marked elevation in mitochondrial peroxiredoxin 3 (Prx3), sulfiredoxin, and superoxide dismutase 2 (Sod2) in stefin B-deficient macrophages and spleens. We determined that sulfiredoxin is targeted to mitochondria after LPS challenge. In conclusion, the upregulation of mitochondrial redox-sensitive proteins Prx3 and Sod2 in stefin B-deficient cells implies a protective role of stefin B in mitochondrial function., Competing Interests: The authors declare no conflict of interest.

Cystatins are endogenous and reversible inhibitors of cysteine peptidases that are important players in cancer progression. Besides their primary role as regulators of cysteine peptidase activity, cystatins are involved in cancer development and progression through proteolysis-independent mechanisms. Mechanistic studies of cystatin function revealed that they affect all stages of cancer progression including tumor growth, apoptosis, invasion, metastasis and angiogenesis. Recently, the involvement of cystatins in the antitumor immune responses was reported. In this review, we discuss molecular mechanisms and clinical aspects of cystatins in cancer. Altered expression of cystatins in cancer resulting in harmful excessive cysteine peptidase activity has been a subject of several studies in order to find correlations with clinical outcome and therapy response. However, involvement in anti-tumor immune response and signaling cascades leading to cancer progression designates cystatins as possible targets for development of new anti-tumor drugs., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Here, we aim at exploring the effect of CST5 on bone resorption and activation of osteoclasts in osteoporosis (OP) rats through the NF-κB pathway. Microarray analysis was used to screen the OP-related differentially expressed genes. Osteoporosis was induced in rats by intragastric retinoic acid administration. The serum levels of tartrate-resistant acid phosphatase (TRAP), bone alkaline phosphatase (BALP) and osteocalcin (OC) and the expression of CD61 on the surface of osteoclasts were examined. The number of osteoclasts and the number and area of resorption pits were detected. Besides, the pathological changes and bone mineral density in bone tissues of rats were assessed. Also, the relationship between CST5 and the NF-κB pathway was identified through determining the expression of CST5, RANKL, RANK, OPG, p65 and IKB. Poorly expressed CST5 was indicated to affect the OP. CST5 elevation and inhibition of the NF-κB pathway decreased serum levels of TRAP, BALP and OC and expression of CD61 in vivo and in vitro. In OP rats, CST5 overexpression increased trabecular bones and bone mineral density of bone tissues, but decreased trabecular separation, fat within the bone marrow cavities and the number of osteoclasts through inhibiting the NF-κB pathway. In vivo experiments showed that CST5 elevation inhibited growth in number and area of osteoclastic resorption pits and restrained osteoclastic bone absorption by inhibiting the NF-κB pathway. In summary, overexpression of CST5 suppresses the activation and bone resorption of osteoclasts by inhibiting the activation of the NF-κB pathway., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Phytocystatins are cysteine proteinase inhibitors ubiquitously present in plants and animals. They are known to carry out various significant physiological functions and also maintain the balance of protease-antiprotease activity. In the present disquisition, a phytocystatin after preliminary treatment has been isolated and purified to homogeneity from soybean (Glycine max) by a simple two-step stratagem using ammonium sulfate fractionation and gel filtration chromatography performed on Sephacryl S-100-HR. Soybean phytocystatin (SBPC) was purified with a fold purification of 635 and percent yield of 77.6%. A single band was observed on native gel electrophoresis confirming the homogeneity of the purified SBPC. The molecular weight of SBPC was found to be 19.05 kDa as determined by SDS-PAGE. The SBPC was found to be devoid of carbohydrate moieties and sulfhydryl group content. The binding stoichiometry of SBPC-papain interaction was determined by isothermal calorimetry suggesting 1:1 complex, and the value of binding constant (K) was found to be 2.78 × 10 5  M -1 The affinity of binding (K d ) value obtained through ITC was 3.59 × 10 -6  M. The purified SBPC was found to be stable in the pH range of 3 to 7 and is thermostable up to 50°C. The UV-visible and fluorescence studies showed significant changes in the conformation upon the formation of the SBPC-papain complex. Furthermore, fluorescence spectroscopy, ANS binding, and caseinolytic activity assay were conducted out to explore the effect of metal ions on SBPC which showed that there was a loss in the inhibitory activity along with conformational changes of SBPC upon complex formation with Cd +2 and Ni +2 ., (© 2019 John Wiley & Sons, Ltd.)

Abiotic stress induced by heavy metals retards the growth and development of plants. Therefore, it is essential to have an insight into the potential toxic effects of heavy metals. The present article investigates the effect of zinc and cadmium on the structure and function of garlic phytocystatin (GPhyCys). The cysteine proteinase inhibitory assay showed a reduction in the inhibitory activity upon binding with zinc and cadmium. UV-vis absorption spectroscopy revealed the complex formation of zinc and cadmium with garlic phytocystatin. Fluorescence quenching experiment confirmed the quenching of fluorophores upon binding of zinc and cadmium. Synchronous and 3-dimensional fluorescence spectroscopy suggest the alteration in the microenvironment around aromatic residues of garlic phytocystatin upon binding with the above metals. Circular dichroism showed a reduction in the alpha-helical content of native garlic phytocystatin. Scanning electron micrographs showed the morphological changes in the native garlic phytocystatin upon addition of zinc and cadmium. The observations confirmed the alteration in structure and conformation of garlic phytocystatin upon interaction with zinc and cadmium. It can be safely concluded that the high concentration of zinc and cadmium can alter the functioning of cysteine proteinase present in garlic and affects the growth and development of plants., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, and early diagnosis and assessment may enhance the quality of life and survival of patients. The prognostic value of key family 2 cystatins subunit in PDAC patients remains unknown. The potential molecular roles of family 2 cystatins and related pathways were investigated using bioinformatics analysis. The relationship of family 2 cystatin expression levels and clinical outcomes of 112 patients with early‑stage PDAC were evaluated via univariate and combined survival analysis. A prognostic nomogram model was also constructed and gene set enrichment analysis was performed to investigate potential pathways in PDAC. The pathways, interaction networks, and Gene Ontology term analysis of the cystatin gene family were analyzed in the present study. Cystatin F (CST7) was identified as the key subunit of family 2 cystatins in survival analysis. PDAC patients who harbored a higher expression level of CST7 had a lower risk in overall survival (adjusted HROS=0.44, 95% CI=0.25‑0.77, P=0.004) and a longer survival time in various subgroups. The prognostic nomogram indicated that the CST7 expression model effectively predicted the outcomes of patients with early‑stage PDAC (predictive ability >0.75). In the gene set enrichment analysis, it was revealed that CST7 expression may be involved in immune regulation and be associated with cell adhesion. CST7 could be a useful biomarker for the prognostic prediction of early‑stage PDAC after pancreaticoduodenectomy.

Cystatins are the inhibitors of thiol proteinases and are ubiquitously present in mammalian system. In brain, they put off unwanted proteolysis and are also involved in several neurodegenerative diseases. In the present study, it was demonstrated that photo-activated HOCl-induced modifications in brain cystatin leading to its inactivation and degradation due to hydroxyl radicals. It has been shown that oxidation of cystatin by ROS in vivo leads to oxidative modification which may direct the damage of this significant protein, as it is so well pronounced in vitro. The interplay between free radicals, antioxidants and co-factors is important in maintaining health, aging and age-related diseases. Body's endogenous antioxidant systems stabilize free radical-induced oxidative stress by the ingestion of exogenous antioxidants. If the generation of free radicals goes beyond the protective effect of antioxidants, this can cause oxidative damage which accumulates during the life cycle and has been implicated in aging and age-related diseases such as cardiovascular disease, cancer, neurodegenerative disorders and other chronic conditions. Activation of neutrophils in certain diseases (e.g., inflammatory conditions and atherosclerosis) results in the production of highly reactive species, such as OH • and the release of the enzyme myeloperoxidase. Stimulated monocytes and neutrophils generate hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. Hypochlorous acid (HOCl) is a potent oxidant formed by myeloperoxidase that causes aggregation of many proteins and damage of proteins by reaction with amino-acid side-chains or backbone cleavage. Communicated by Ramaswamy H. Sarma.

Infection with canine heartworm ( Dirofilaria immitis ), spread via mosquito vectors, causes coughing, asthma, pneumonia, and bronchitis in humans and other animals. The disease is especially severe and often fatal in dogs and represents a serious threat to public health worldwide. Cysteine protease inhibitors (CPIs), also known as cystatins, are major immunomodulators of the host immune response during nematode infections. Herein, we cloned and expressed the cystatin Di -CPI from D. immitis . Sequence analysis revealed two specific cystatin-like domains, a Q-x-V-x-G motif, and a SND motif. Phylogenetic analysis indicates that Di -CPI is a member of the second subgroup of nematode type II cystatins. Probing of D. immitis total proteins with anti-r Di -CPI polyclonal antibody revealed a weak signal, and immunofluorescence-based histochemical analysis showed that native Di -CPI is mainly localized in the cuticle of male and female worms and the gut of male worms. Treatment of canine peripheral blood mononuclear cells (PMBCs) with recombinant Di -CPI induced a Th2-type immune response characterized by high expression of the anti-inflammatory factor interleukin-10. Proliferation assays showed that Di -CPI inhibits the proliferation of canine PMBCs by 15%. Together, the results indicate that Di -CPI might be related to cellular hyporesponsiveness in dirofilariasis and may help D. immitis to evade the host immune system.

Background: Treatment with apremilast has recently demonstrated clinically meaningful improvement in moderate hidradenitis suppurativa (HS)., Objective: To evaluate the change in expression of inflammatory markers in lesional skin of HS patients receiving apremilast 30 mg twice daily (n = 15) for 16 weeks compared with placebo (n = 5)., Methods: At baseline, 5-mm punch biopsies were obtained from an index lesion (HSL) and non-lesional (HSN) skin in the same anatomical area. Subsequent HSL samples were taken as close as possible to the previously biopsied site at week 4 and week 16. After sampling, biopsies were split; one half was processed for in vivo mRNA analysis using real-time quantitative PCR; the other half was cultured for ex vivo protein analysis using a proximity extension assay (Olink). Linear mixed effects models were calculated to compare the levels of inflammatory markers in HSL skin between apremilast and placebo over time., Results: At baseline, 17 proteins with a fold change >2 in HSL vs. HSN skin were identified in 20 patients. The top five were IL-17A (5), S100A12, CST5, IL-12/23p40, CD6 (1) with fold changes ranging from 6.6 to 1638, respectively (FDR <0.044). Linear mixed effects models for 75 assays were calculated. Protein levels of S100A12 decreased during treatment in the apremilast group compared with the placebo group (p = 0.014, FDR = 0.186). None of the 14 genes exhibited significant changes in expression over time. However, an evident downward trend in relative mRNA expression of IL-17A and IL-17F was demonstrated in patients receiving apremilast., Conclusion: We did not detect statistically significant changes in inflammatory markers in HSL skin of HS patients receiving apremilast compared with placebo, despite clinical improvement in the apremilast group. Nonetheless, S100A12 and IL-17A were significantly elevated in HSL skin and showed a decrease in response to apremilast. The translational model in clinical trials involving HS clearly needs further improvement., (© 2018 The Authors. Journal of the European Academy of Dermatology and Venereology published by John Wiley & Sons Ltd on behalf of European Academy of Dermatology and Venereology.)

Background Cystatin F is a protein inhibitor of cysteine peptidases, expressed predominantly in immune cells and localised in endosomal/lysosomal compartments. In cytotoxic immune cells cystatin F inhibits both the major pro-granzyme convertases, cathepsins C and H that activate granzymes, and cathepsin L, that acts as perforin activator. Since perforin and granzymes are crucial molecules for target cell killing by cytotoxic lymphocytes, defects in the activation of either granzymes or perforin can affect their cytotoxic potential. Materials and methods Levels of cystatin F were assessed by western blot and interactions of cystatin F with cathepsins C, H and L were analysed by immunoprecipitation and confocal microscopy. In TALL-104 cells specific activities of the cathepsins and granzyme B were determined using peptide substrates. Results Two models of reduced T cell cytotoxicity of TALL-104 cell line were established, either by treatment by ionomycin or by immunosuppressive transforming growth factor beta. Reduced cytotoxicity correlated with increased levels of cystatin F and with attenuated activities of cathepsins C, H and L and of granzyme B. Co-localisation of cystatin F and cathepsins C, H and L and interactions between cystatin F and cathepsins C and H were demonstrated. Conclusions Cystatin F is designated as a possible regulator of T cell cytotoxicity, similar to its role in natural killer cells.

Multiple sclerosis (MS) is an autoimmune disease characterized by immune-mediated inflammation, which attacks the myelin sheath. MS pursues a relapsing and remitting course with varying intervals between symptoms. The main clinical pathological features include inflammation, myelin sheath destruction and plaque formation in the central nervous system (CNS). We previously reported that cystatin F (CysF) expression is induced in demyelinating lesions that are accompanied by active remyelination (referred to as shadow plaques) but is down-regulated in chronic demyelinated lesions (plaques) in the spinal cord of MS patients and in several murine models of demyelinating disease. CysF is a cathepsin protease inhibitor whose major target is cathepsin C (CatC), which is co-expressed in demyelinating regions in Plp 4e/- mice, a model of chronic demyelination. Here, we report the time course of CatC and CysF expression and describe the symptoms in a mouse experimental autoimmune encephalomyelitis (EAE) model using CatC knockdown (KD) and CatC over-expression (OE) mice. In myelin oligodendrocyte glycoprotein (MOG)-EAE, CatC positive cells were found to infiltrate the CNS at an early stage prior to any clinical signs, in comparison to WT mice. CysF expression was not observed at this early stage, but appeared later within shadow plaques. CatC expression was found in chronic demyelinated lesions but was not associated with CysF expression, and CatCKD EAE mouse showed delayed demyelination. Whereas, CatCOE in microglia significantly increased severity of demyelination in the MOG-EAE model. Thus, these results demonstrate that CatC plays a major role in MOG-EAE., (© 2018 International Society for Neurochemistry.)

Cysteine cathepsins are lysosomal peptidases involved in the regulation of innate and adaptive immune responses. Among the diverse processes, regulation of granule-dependent cytotoxicity of cytotoxic T-lymphocytes (CTLs) and natural killer (NK) cells during cancer progression has recently gained significant attention. The function of cysteine cathepsins is regulated by endogenous cysteine protease inhibitors-cystatins. Whereas other cystatins are generally cytosolic or extracellular proteins, cystatin F is present in endosomes and lysosomes and is thus able to regulate the activity of its target directly. It is delivered to endosomal/lysosomal vesicles as an inactive, disulphide-linked dimer. Proteolytic cleavage of its N-terminal part leads to the monomer, the only form that is a potent inhibitor of cathepsins C, H and L, involved in the activation of granzymes and perforin. In NK cells and CTLs the levels of active cathepsin C and of granzyme B are dependent on the concentration of monomeric, active cystatin F. In tumour microenvironment, inactive dimeric cystatin F can be secreted from tumour cells or immune cells and further taken up by the cytotoxic cells. Subsequent monomerization and inhibition of cysteine cathepsins within the endosomal/lysosomal vesicles impairs granzyme and perforin activation, and provokes cell anergy. Further, the glycosylation pattern has been shown to be important in controlling secretion of cystatin F from target cells, as well as internalization by cytotoxic cells and trafficking to endosomal/lysosomal vesicles. Cystatin F is therefore an important mediator used by bystander cells to reduce NK and T-cell cytotoxicity.

Intrinsic and extrinsic factors are responsible for the transition of soluble proteins into aggregated form. Trifluoroethanol is among such potent extrinsic factor which facilitates the formation of aggregated structure. It disrupts the interactive forces and destabilizes the native structure of the protein. The present study investigates the effect of trifluoroethanol (TFE) on garlic cystatin. Garlic cystatin was incubated with increasing concentration of TFE (0-90% v/v) for 4 h. Incubation of GPC with TFE induces structural changes thereby resulting in the formation of aggregates. Inactivation of garlic phytocystatin was confirmed by cysteine proteinase inhibitory activity. Garlic cystatin at 30% TFE exhibits native-like secondary structure and high ANS fluorescence, thus suggesting the presence of molten globule state. Circular dichroism and FTIR confirmed the transition of the native alpha-helical structure of garlic cystatin to the beta-sheet structure at 60% TFE. Furthermore, increased ThT fluorescence and redshift in Congo red absorbance assay confirmed the presence of aggregates. Rayleigh and turbidity assay was also performed to validate the aggregation results. Scanning electron microscopy was followed to analyze the morphological changes which confirm the presence of sheath-like structure at 60% TFE. The study sheds light on the conformational behavior of a plant protein when kept under stress condition induced by an extrinsic factor., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cystatins are thiol proteinase inhibitors present ubiquitously in mammalian system. They prevent unwanted proteolysis and are involved in several neurodegenerative diseases. Hydrogen peroxide, hydroxyl radical and superoxide radical were produced during the autoxidation of various cytotoxic agents namely 6-hydroxydopamine, 6-aminodopamine, 6,7-dihydroxytryptamine, and dialuric acid. Reactive oxygen species (ROS) such as singlet oxygen [O 2 - ] and hydroxyl radicals result in nonspecific reactivity, thus involved in several pathological conditions such as inflammation and reperfusion injury leading to damage of different biological molecules due to the production of enzyme myeloperoxidase because of activation of neutrophils in these diseases. As Myeloperoxidase [MPO] is released extracellularly by activated monocytes and is highly basic protein that damages the localized sites. The onset and progression of several diseases is a complex process in which oxidation is believed to play significant role. Oxidized lipids and proteins that are detected in early, intermediate and advanced stage of human neurodegenerative diseases (plaques) have revealed considerably elevated levels of reactive oxygen species showing their involvement in brain diseases. In the present study, it has been demonstrated that H 2 O 2 induced modifications in brain cystatin leads to its inactivation and degradation. It was also found that the damage from the oxidants occurred mainly because of the (ROS) hydroxyl radicals [OH] produced by H 2 O 2 which in turn changes the conformation of the Cystatin, a major factor for the damage of this significant protein. It is one of the cause of protease-antiprotease imbalance in mammals leading to progression of several diseases., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Several mammalian proteins form pathological deposits under nonphysiological conditions that are associated with many degenerative diseases. Protein aggregation is associated with aging, as well as a variety of diseases, including cystic fibrosis, amyotrophic lateral sclerosis (ALS), and hypertrophic cardiomyopathy. There is a lack of any potential anti-amyloidogenic agents and therapeutics till date. Polyphenols have been accredited with myriad biological effects. An analysis of the effects of natural agents like baicalin (BC) and gallocatechin (GC) on aggregation process can open new avenues for the treatment of protein misfolding diseases. Thus, investigation of the effects of these flavonoids on Buffalo Heart Cystatin (BHC) aggregation induced by a reactive metabolic dialdehyde, glyoxal (GO), was taken up. Results have shown that elevated concentration of GO forms aggregates of BHC, which was characterized by an increase in the ANS fluorescence intensity, an increase in ThT fluorescence intensity, red shift in Congo red absorbance, negative ellipticity peak at 217 nm in the far-UVCD and BHC aggregates displaying by TEM. Using fluorescence spectroscopic analysis with Thioflavin T, CD and electron microscopic studies, anti-aggregation effects of polyphenols, BC and GC were analyzed. The study showed that BC and GC produced concentration-dependent anti-aggregation effects with GC producing a more pronounced effect than BC. The study proposed a mechanistic approach assuming structural constraints and specific aromatic interactions of polyphenols with sheets of BHC aggregates.

Triple-negative breast cancer (TNBC) accounts for a disproportionately high number of deaths due to a lack of targeted therapies and an increased likelihood of distant recurrence. Estrogen receptor beta (ERβ), a well-characterized tumor suppressor, is expressed in 30% of TNBCs, and its expression is associated with improved patient outcomes. We demonstrate that therapeutic activation of ERβ elicits potent anticancer effects in TNBC through the induction of a family of secreted proteins known as the cystatins, which function to inhibit canonical TGFβ signaling and suppress metastatic phenotypes both in vitro and in vivo. These data reveal the involvement of cystatins in suppressing breast cancer progression and highlight the value of ERβ-targeted therapies for the treatment of TNBC patients., Competing Interests: The authors declare no conflict of interest.

Cystatin F is an inhibitor of cysteine peptidases expressed solely in immune cells. It is the only type II cystatin able to enter endosomal/lysosomal vesicles and to regulate directly the activity of intracellular cysteine cathepsins. Its expression in promonocytic U937 and promyeloblastic HL-60 cells is highly upregulated but, after differentiation with phorbol 12-myristate 13-acetate - PMA, its levels drop significantly. In contrast, the activities of intracellular cysteine cathepsins C, L and S are higher in differentiated cells than in non-differentiated ones due, presumably, to the lower inhibitory capacity of cystatin F. Using immunofluorescence confocal microscopy, proximity ligation assay and co-immunoprecipitation, cathepsins C, L and S were confirmed to be the main interacting partners of cystatin F in U937 and HL-60 cells. The promoter region of the cystatin F gene, CST7, contains a unique binding site for transcription factor C/EBP α, one of the main myeloid differentiation instructors. Using the chromatin immunoprecipitation assay, C/EBP α was shown to bind to CST7 gene in U937 cells. Following cell differentiation with PMA, the binding of C/EBP α was decreased significantly. The protein level of C/EBP α was also significantly lower in differentiated than in non-differentiated cells. It was shown that, during monocyte to macrophage differentiation, the endosomal/lysosomal proteolytic activity can be regulated by cystatin F whose expression is under the control of transcriptional factor C/EBP α., (Copyright © 2018. Published by Elsevier GmbH.)

The equilibrium between protein synthesis and degradation is key to maintaining efficiency in different physiological processes. The proteinase inhibitor cystatin regulates protease activities in different developmental and physiological contexts. Here we describe for the first time the identification and the biological function of the cysteine protease inhibitor cystatin of Fragaria chiloensis, FchCYS1. Based on primary sequence and 3D-structural homology modelling, FchCYS1 is a type II phytocystatin with high identity to other cystatins of the Fragaria genus. Both the papain-like and the legumain-like protease inhibitory domains are indeed functional, based on in vitro assays performed with Escherichia coli protein extracts containing recombinant FchCYS1. FchCYS1 is differentially-expressed in achenes of F. chiloensis fruits, with highest expression as the fruit reaches the ripened stage, suggesting a role in preventing degradation of storage proteins that will nourish the embryo during seed germination. Furthermore, FchCYS1 responds transcriptionally to the application of salicylic acid and to mechanical injury, strongly suggesting that FchCYS1 could be involved in the response against pathogen attack. Overall these results point to a role for FchCYS1 in diverse physiological processes in F. chiloensis., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Protein breakdown and mobilization are some of the major metabolic features associated with abiotic stresses, essential for nutrient recycling and plant survival. Genetic manipulation of protease and/or protease inhibitors may contribute to modulate proteolytic processes and plant responses. The expression analysis of the whole cystatin family, inhibitors of C1A cysteine proteases, after water deprivation in barley leaves highlighted the involvement of Icy-2 and Icy-4 cystatin genes. Artificial microRNA lines independently silencing the two drought-induced cystatins were generated to assess their function in planta. Phenotype alterations at the final stages of the plant life cycle are represented by the stay-green phenotype of silenced cystatin 2 lines. Besides, the enhanced tolerance to drought and differential responses to water deprivation at the initial growing stages are observed. The mutual compensating expression of Icy-2 and Icy-4 genes in the silencing lines pointed to their cooperative role. Proteolytic patterns by silencing these cystatins were concomitant with modifications in the expression of potential target proteases, in particular, HvPap-1, HvPap-12, and HvPap-16 C1A proteases. Metabolomics analysis lines also revealed specific modifications in the accumulation of several metabolites. These findings support the use of plants with altered proteolytic regulation in crop improvement in the face of climate change., (© 2018 John Wiley & Sons Ltd.)

Reactive dicarbonyl species such as methylglyoxal (MGO) and glyoxal (GO) have recently received extensive attention due to their high reactivity and ability to modify biological substances such as proteins, phospholipids, and DNA. In case of proteins these reactive species mainly react with lysine and arginine residues to form AGEs, oxidative products, and aggregates. Chickpea cystatin (CPC) was incubated with varying concentrations of glyoxal and methylglyoxal which caused, along with altered secondary and tertiary structures, glycation, functional inactivation, altered redox state, cross-linking and high-molecular-mass aggregation. All these processes were examined and characterized by UV-Vis, fluorescence, and CD spectroscopies. Further characterization of CPC modified by reactive dicarbonyls was done by polyacrylamide gel electrophoresis which also showed alterations in the CPC molecules. Thus, in addition to describing the effects of GO and MGO on structure, conformation and function of CPC, this study also shows the relatively superior modifying effect of methylglyoxal for CPC in terms of glycation, oxidation and aggregation. This model system could shed some more light on the role of the reactive dicarbonyls in the specific alterations of proteins with different biological consequences having implications to ageing and disease such as diabetes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Pesticides are being used globally to improve agricultural production. They are applied specifically to combat with pathogens that are a major threat for reduced optimum yield of crops. This study was carried out to see the effect of commercially used pesticides on a specific plant protein viz. phytocystatin isolated from yellow mustard seeds (YMP). Phytocystatin is a thiol proteinase inhibitor, which regulates endogenous and exogenous cysteine proteinases and plays a vital physiological role in plants. Different classes of pesticides like fungicide (iprodione) of dicarboximide class and an insecticide (malathion) of class organophosphate are retorted for our study. In the presence of these pesticides, biophysical and biochemical changes were observed in phytocystatin. These changes were evaluated making use of caseinolytic activity assay, UV-vis spectroscopy, fluorescence spectroscopy, FTIR, and circular dichroism. Isothermal titration calorimetry was employed to see interaction pattern of these pesticides with phytocystatin. The results obtained clearly depict that the pesticides bind with the phytocystatin thereby changing its native conformation and reducing its intrinsic property of inhibition on cysteine proteinase as evident by reduced anti-papain inhibition in the presence of pesticides. Furthermore, CD and FTIR spectroscopy results clearly show a decrease in α-helical content upon interaction with malathion and iprodione. Among the two pesticides, iprodione has far more pronounced effect on YMP evident from striking changes in UV, Fluorescence, CD and FTIR spectroscopy. 2,4-dinitrophenylhydrazine spectrophotometric assay was also carried out to check production of ROS, generation of ROS was observed in the presence of these pesticides thus implying that ROS might be responsible for changes in native structure of phytocystatin induced by pesticides., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ticks, blood-feeding arthropods, and secrete immunosuppressive molecules that inhibit host immune responses and provide survival advantages to pathogens. In this study, we characterized the immunosuppressive function of a novel tick salivary protein, DsCystatin, from Dermacentor silvarum of China. DsCystatin directly interacted with human Cathepsins L and B and inhibited their enzymatic activities. DsCystatin impaired the expression of inflammatory cytokines such as IL1β, IFNγ, TNFα, and IL6 from mouse bone marrow-derived macrophages (BMDMs) that had been stimulated with LPS or Borrelia burgdorferi . Consistently, DsCystatin inhibited the activation of mouse BMDMs and bone marrow-derived dendritic cells by downregulating the surface expression of CD80 and CD86. Mechanically, DsCystatin inhibited LPS- or B. burgdorferi -induced NFκB activation. For the first time, we identified that DsCystatin-attenuated TLR4 signaling by targeting TRAF6. DsCystatin enhanced LPS-induced autophagy, mediated TRAF6 degradation via an autophagy dependent manner, thereby impeded the downstream phosphorylation of IκBα and the nuclear transport of NFκB. Finally, DsCystatin relieved the joint inflammation in B. burgdorferi or complete Freund's adjuvant induced mouse arthritis models. These data suggested that DsCystatin is a novel immunosuppressive protein and can potentially be used in the treatment of inflammatory diseases.

Strigolactones (SL) fulfil important roles in plant development and stress tolerance. Here, we characterized the role of SL in the dark chilling tolerance of pea and Arabidopsis by analysis of mutants that are defective in either SL synthesis or signalling. Pea mutants (rms3, rms4, and rms5) had significantly greater shoot branching with higher leaf chlorophyll a/b ratios and carotenoid contents than the wild type. Exposure to dark chilling significantly decreased shoot fresh weights but increased leaf numbers in all lines. Moreover, dark chilling treatments decreased biomass (dry weight) accumulation only in rms3 and rms5 shoots. Unlike the wild type plants, chilling-induced inhibition of photosynthetic carbon assimilation was observed in the rms lines and also in the Arabidopsis max3-9, max4-1, and max2-1 mutants that are defective in SL synthesis or signalling. When grown on agar plates, the max mutant rosettes accumulated less biomass than the wild type. The synthetic SL, GR24, decreased leaf area in the wild type, max3-9, and max4-1 mutants but not in max2-1 in the absence of stress. In addition, a chilling-induced decrease in leaf area was observed in all the lines in the presence of GR24. We conclude that SL plays an important role in the control of dark chilling tolerance., (© 2018 John Wiley & Sons Ltd.)

Key Message: Expression of the TaMDC1 in transgenic tomato plants confer resistance to bacterial and fungal pathogens, as well as an insect pest and thus prove in planta function of the wheat cystatin. Cystatins are the polypeptides with cysteine proteinase inhibitory activities. Plant cystatins or phytocystatins are known to contribute to plant resistance against insect pests. Recently, increasing data proved that some of the phytocystatins also have antifungal activities in vitro. Here, we functionally characterized a wheat multidomain cystatin, TaMDC1, using in planta assays. Expression of TaMDC1 in wheat seedlings is up-regulated in response to methyl jasmonate and salicylic acid, indicating that TaMDC1 is involved in biotic stress responses mediated by these plant hormones. The TaMDC1 cDNA was integrated in tomato genome and expressed under cauliflower mosaic virus 35S promoter. Four transgenic plants that show high level of the transgene expression were selected by RNA gel blot and immunoblot analysis and utilized to assess biotic stress resistance against the bacterial pathogen Pseudomonas syringae, the fungal pathogens Botrytis cinerea and Alternaria alternata, and the insect pest Colorado potato beetle (CPB, Leptinotarsa decemlineata). Detached leaf inoculation assays revealed that the tomato plants expressing TaMDC1 showed high levels of resistance against P. syringae and A. alternata, and elevated tolerance against B. cinerea. Sustenance of L. decemlineata larvae to the transgenic plants demonstrated inhibition of CPB larvae growth. Inhibitory activity of TaMDC1 against selected pathogens was also demonstrated by in vitro assays with total protein extracted from transgenic tomato plants. Taken together, the presented data suggest that TaMDC1 is involved in a broad spectrum biotic stress resistance in planta.

Our previous studies have shown adenosine A 2A R activation markedly promotes the expression of cystatin F (CF) and exacerbates the white matter lesions induced by hypoxic brain injuries. Thus, we hypothesized that CF was probably involved in neuroinflammation of activated microglia induced by A 2A R activation. We transfected the BV2 cells with a CF shRNA vector and examined the production of pro-inflammatory cytokines in hypoxic-BV2 cells in which A 2A R was activated or inactivated to confirm this hypothesis. Additionally, we also investigated the probable signaling pathways involved in modulation of A 2A R activation on CF expression in hypoxia-activated BV2 cells. Activation of A 2A R promoted CF expression, which was significantly increased after the low glucose and hypoxia treatments in BV2 cells. CF gene knockdown markedly inhibited the increase in the expression of pro-inflammatory cytokines induced by A 2A R activation in hypoxic-BV2 cells. Furthermore, the increased expression of the CF induced by A 2A R activation was remarkably inhibited in hypoxic-BV2 cells administrated with the PKA inhibitor H-89 and the PKC inhibitor staurosporine. Hence, these results indicate that hypoxia BV2 cells highly express CF, which is involved in A 2A R activation-mediated neuroinflammation via the PKA/CREB and PKC/CREB or ERK1/2 signaling pathways.

Rodents use the vomeronasal olfactory system to acquire both inter- and intra-specific information from the external environment and take appropriate actions. For example, urinary proteins from predator species elicit avoidance in mice, while those from male mice attract female mice. In addition to urinary proteins, recent studies have highlighted the importance of lacrimal proteins for intra-specific communications in mice. However, whether the tear fluid of other species also mediates social signals remains unknown. Here, we show that a lacrimal protein in rats (predators of mice), called cystatin-related protein 1 (ratCRP1), activates the vomeronasal system of mice. This protein is specifically produced by adult male rats in a steroid hormone-dependent manner, activates the vomeronasal system of female rats, and enhances stopping behavior. When detected by mice, ratCRP1 activates the medial hypothalamic defensive circuit, resulting in decreased locomotion coupled with lowered body temperature and heart rate. Notably, ratCRP1 is recognized by multiple murine type 2 vomeronasal receptors, including Vmn2r28. CRISPR/Cas9-mediated deletion of vmn2r28 impaired both ratCRP1-induced neural activation of the hypothalamic center and decrease of locomotor activity in mice. Taken together, these data reveal the neural and molecular basis by which a tear fluid compound in rats affects the behavior of mice. Furthermore, our study reveals a case in which a single compound that mediates an intra-specific signal in a predator species also functions as an inter-specific signal in the prey species., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The presence and localization of cystatin, a cysteine protease inhibitor involved in barrier formation in human and mice epidermis, has been studied in the epidermis of piscine and terrestrial vertebrates using a mouse monoclonal antibody. Cystatin has been localized by Immunostaining in the pre-corneous and corneous layers of monotreme, marsupial and placental mammals, and sparsely in the thin corneous layer of birds. Cystatin-immunolabeling is present in the pre-corneous and corneous layer of crocodilian and turtle epidermis, in the alpha-corneous layer and likely also in the beta-corneous layer of the epidermis in lizards, snakes and the tuatara. In keratinocytes of the pre-corneous (transitional) layers the protein initially shows a peripheral distribution that becomes compacted in mature corneocytes. The protein is not detected using the antibody in the epidermis of cyclostome, teleosts, sarcopterigian fish, and in amphibians. The study concludes that while in fish and amphibians cystatin is absent or however uncertainly localized in the epidermis, the protein instead appears present in the more external pre-corneous and corneous layers of amniotes. This special regionalization suggests a specific role of cystatin in the formation of the corneous epidermal barrier and regulation of desquamation originally evolved in the terrestrial environment., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Asthma arises from the complex interplay of inflammatory pathways in diverse cell types and tissues. We sought to undertake a comprehensive transcriptomic assessment of the epithelium and airway T cells that remain understudied in asthma and investigate interactions between multiple cells and tissues. Epithelial brushings and flow-sorted CD3 + T cells from sputum and BAL were obtained from healthy subjects (n = 19) and patients with asthma (mild, moderate, and severe asthma; n = 46). Gene expression was assessed using Affymetrix HT HG-U133 + PM GeneChips, and results were validated by real-time quantitative PCR. In the epithelium, IL-13 response genes (POSTN, SERPINB2, and CLCA1), mast cell mediators (CPA3 and TPSAB1), inducible nitric oxide synthase, and cystatins (CST1, CST2, and CST4) were upregulated in mild asthma, but, except for cystatins, were suppressed by corticosteroids in moderate asthma. In severe asthma-with predominantly neutrophilic phenotype-several distinct processes were upregulated, including neutrophilia (TCN1 and MMP9), mucins, and oxidative stress responses. The majority of the disease signature was evident in sputum T cells in severe asthma, where 267 genes were differentially regulated compared with health, highlighting compartmentalization of inflammation. This signature included IL-17-inducible chemokines (CXCL1, CXCL2, CXCL3, IL8, and CSF3) and chemoattractants for neutrophils (IL8, CCL3, and LGALS3), T cells, and monocytes. A protein interaction network in severe asthma highlighted signatures of responses to bacterial infections across tissues (CEACAM5, CD14, and TLR2), including Toll-like receptor signaling. In conclusion, the activation of innate immune pathways in the airways suggests that activated T cells may be driving neutrophilic inflammation and steroid-insensitive IL-17 response in severe asthma.

Parasite inhibitors of cysteine peptidases are known to influence a vast range of processes linked to a degradation of either the parasites' own proteins or proteins native to their hosts. We characterise a novel type I cystatin (stefin) found in a sanguinivorous fish parasite Eudiplozoon nipponicum (Platyhelminthes: Monogenea). We have identified a transcript of its coding gene in the transcriptome of adult worms. Its amino acid sequence is similar to other stefins except for containing a legumain-binding domain, which is in this type of cystatins rather unusual. As expected, the recombinant form of E. nipponicum stefin (rEnStef) produced in Escherichia coli inhibits clan CA peptidases - cathepsins L and B of the worm - via the standard papain-binding domain. It also blocks haemoglobinolysis by cysteine peptidases in the worm's excretory-secretory products and soluble extracts. Furthermore, we had confirmed its ability to inhibit clan CD asparaginyl endopeptidase (legumain). The presence of a native EnStef in the excretory-secretory products of adult worms, detected by mass spectrometry, suggests that this protein has an important biological function at the host-parasite interface. We discuss the inhibitor's possible role in the regulation of blood digestion, modulation of antigen presentation, and in the regeneration of host tissues.

Cystatins and crystallins are both neuroprotective proteins. Except for their function as cysteine cathepsins inhibitors, cystatins are Aβ binding proteins and presumably protect neurons from intracellular Aβ and extracellular Aβ plaques. Pathological mutations of cystatin C cause amyloid angiopathy. Crystallins, known as small heat shock proteins, bind not only Aβ peptide but also other crystallins in the eye lens and prevent their aggregation. Mutations in crystallins cause cataracts and myopathies. Cross-interactions between amyloidogenic proteins, intrinsically disordered and folded proteins, can also occur. I term the nonspecific binding between amyloidogenic proteins and peptides "co-chaperoning." A wide range of other Aβ binding proteins exist, such as catalase, lysozyme, β-lactoglobulin and some other abundant proteins found in plasma.

Phytocystatins have been ascribed several protective roles against abiotic and biotic stress conditions. It was, therefore, thought worthwhile to document the effect of heavy metal stress on the endogenous plant cystatin. The mustard cystatin, purified from Brassica juncea (B. juncea) seeds retained its functional property of cysteine proteinase inhibition, despite exposure to high concentrations of metal ions, Cd 2+ and Ni 2+ . An increase in inhibitory activity, ∼26% for Ni 2+ and ∼16% for Cd 2+ was observed, suggesting changes in protein conformation upon metal ion interaction. Isothermal calorimetric (ITC) studies show formation of a 1:1 binary complex on interaction with both metal ions but suggest a higher affinity for Ni 2+ . Fluorescence quenching data suggest a static quenching mechanism of interaction. Various spectroscopic analyses, namely, synchronous fluorescence, ANS fluorescence, far UV CD and ATR-FTIR spectroscopy show that the native mustard cystatin acquires a more ordered conformation upon interaction with metal ions. Differential Scanning Calorimetry indicates that the thermo-stability of the Ni 2+ bound protein (T m =109.4°C) is greater than both, the Cd 2+ bound (T m =104.5°C) and the native (T m =99.5°C) forms. The B. juncea seed cystatin, is thus, identified as a potent and resilient member of the phytocystatin family with considerable inhibitory capacity despite exposure to heavy metal stress., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cystatins are small, phylogenetically conserved proteins that are tight-binding inhibitors of cysteine proteinases. The liver fluke Fasciola hepatica uses a diverse set of cysteine proteinases of the papain superfamily for host invasion, immune evasion and nutrition, but little is known about the regulation of these enzymes. The aim of this work is to characterize the cystatin repertoire of F. hepatica. For this purpose, we first surveyed the available sequence databases, identifying three different F. hepatica single-domain cystatins. In agreement with the in silico predictions, at least three small proteins with cysteine proteinase binding activity were identified. Phylogenetic analyses showed that the three cystatins (named FhStf-1, -2 and -3) are members of the I25A subfamily (stefins). Whereas FhStf-1 grouped with classical stefins, FhStf-2 and 3 fell in a divergent stefin subgroup unusually featuring signal peptides. Recombinant rFhStf-1, -2 and -3 had potent inhibitory activity against F. hepatica cathepsin L cysteine proteinases but differed in their capacity to inhibit mammalian cathepsin B, L and C. FhStf-1 was localized in the F. hepatica reproductive organs (testes and ovary), and at the surface lamella of the adult gut, where it may regulate cysteine proteinases related with reproduction and digestion, respectively. FhStf-1 was also detected among F. hepatica excretion-secretion (E/S) products of adult flukes. This suggests that it is secreted by non-classical secretory pathway and that it may interact with host lysosomal cysteine proteinases.

Phytocystatins are a group of proteins with significant potential to regulate activities of cysteine proteinases of native and pest/pathogen origins. The two-domain triticale (x Triticosecale Wittm.) phytocystatin TrcC-8 was characterized in this study. This protein belongs to the second group of phytocystatins and contains all the conserved sequences and motifs as well as both N-terminal (CY) and C-terminal (CY-L) domains that are characteristic of phytocystatins with the C-terminal extension. We demonstrated that TrcC-8 forms stable dimers with a significantly reduced inhibitory activity against papain compared to the activity of monomers, indicating the regulatory nature of the oligomerization. Moreover, according to our research, only the N-terminal domain possesses the ability to form dimers, indicating that this part of TrcC-8 is involved in the dimerization of the full-length protein. Homology modelling of TrcC-8 strongly suggests distinct specificities for the CY and CY-L domains, confirmed in experiments with inhibition of the papain. Our results suggest that the CY domain of TrcC-8 may, although markedly weakly and suboptimally, interact with papain in an analogous mode to tarocystatin, while the CY-L domain of TrcC-8 has distinct specificity than tarocystatin., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Colorado potato beetle (CPB; Leptinotarsa decemlineata Say, Coleoptera: Chrysomelidae) has shown a remarkable adaptability to a variety of control measures. Although oryzacystatin I and II (OCI and OCII) have potential in controlling pests that use cysteine proteinases for food digestion, expression of a single OC gene in potato exhibited a minimal or no effect on CPB fitness traits. The aim of this study was to examine the effect of coexpressed OCI and OCII in potato (Solanum tuberosum L.) cultivars Desiree, Dragačevka and Jelica on CPB larvae. Growth parameters, consumption rates and food utilization, as well as activity of proteases of CPB larvae were assayed. Second and third instar larvae fed on transformed leaves molted earlier and had higher relative growth and consumption rates than larvae fed on nontransformed leaves, while efficiency of food utilization was unaffected. In contrast, fourth instar maximum weight gain and amount of leaves consumed were about 20% lower for the larvae fed on transgenic potato. Analysis of total protease activity of third instar larvae revealed reduction in overall proteolytic activity measured by azocasein hydrolysis, accompanied with inhibition of cysteine proteinase activity 24 h after ingestion of potato leaves expressing OCI and OCII. However, after long-term feeding on transformed leaves proteolytic activities of larvae became similar to the controls. Although feeding on OCI/OCII leaves did not affect larval survival, coexpression of OC genes reduced the development time and thus significantly decreased plant damage caused by CPB larvae., (© 2016 Institute of Zoology, Chinese Academy of Sciences.)

Objective: This study evaluated changes in protein profile of the acquired enamel pellicle (AEP) formed in vivo, after application of gels containing chlorhexidine or EGCG and further challenge with citric acid., Design: AEP was formed in 9 volunteers for 2h and then treated with one of the following gels: placebo, 400μM EGCG or 0.012% chlorhexidine. A thin layer of gel was applied and after 1min the excess was removed. One hour after gel application, the AEP was collected from the buccal surface (upper and lower jaw) of one of the sides with filter paper dipped in 3% citric acid. On the other side, erosive challenge was performed through gentle application of 1% citric acid (pH 2.5) for 20s (using a pipette) followed by washing with deionized water. The AEP was collected as mentioned before. Proteomic analysis was performed through liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The MS/MS spectra obtained were compared with human protein databases (SWISS-PROT). Label-free quantitation was done using the PLGS software., Results: In total, 223 proteins were identified. After treatment with EGCG and CHX gels, proteins with potential functions to protect against caries and erosion such as PRPs, calcium-bind proteins and Statherin were increased. When EGCG and CHX-treated AEPs were challenged with citric acid, there was increase in cystatins and Profilin-1., Conclusion: CHX- and EGCG-treated AEPs, submitted to challenge with citric acid or not, had remarkable changes in their proteomic profiles., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Haemonchosis is a disease of the small ruminant caused by a nematode parasite Haemonchus contortus, and it is most important and alarming challenges to the small ruminant's production. The infection of the H. contortus could cause high economic losses worldwide. H. contortus is a blood feeding parasite which penetrates into the abomasal mucosa to feed the blood of the host and causing the anemia and decreased total plasma protein. Modulation and suppression of the immune response of the host by nematode parasites have been reported extensively, and the cysteine protease inhibitor (cystatin) is identified as one of the major immunomodulators., Methods: The recombinant protein of HCcyst-3 was expressed in a histidine-tagged fusion soluble form in Escherichia coli, and its inhibitory activity against cathepsin L, B, as well as papain, were identified by fluorogenic substrate analysis. Native HCcyst-3 protein was localized by an Immunohistochemical test. The immunomodulatory effects of HCcyst-3 on cytokine secretion, MHC molecule expression, NO production and phagocytosis were observed by co-incubation of rHCcyst-3 with goat monocytes., Results: We cloned and produced recombinant cystatin protein from H. contortus (rHCcyst-3) and investigated its immunomodulatory effects on goat monocyte. The rHCcyst-3 protein is biologically functional as shown by its ability to inhibit the protease activity of cathepsin L, cathepsin B, and papain. The immunohistochemical test demonstrated that the native HCcyst-3 protein was predominantly localized at the body surface and internal surface of the worm's gut. We demonstrated that rHCcyst-3 could be distinguished by antisera from goat experimentally infected with H. contortus and could uptake by goat monocytes. The results showed that the engagement of rHCcyst-3 decreased the production of TNF-α, IL-1β and IL-12p40. However, it significantly increased the secretion of IL-10 and TGF-β1 in goat monocytes. After rHCcyst-3 exposure, the expression of MHC-II on goat monocytes was restricted. Moreover, rHCcyst-3 could upregulate LPS induced NO production of goat monocytes. Phagocytotic assay by FITC-dextran internalization showed that rHCcyst-3 inhibited the phagocytosis of goat monocytes., Conclusions: Our results suggested that the recombinant cystatin from H. contortus (rHCcyst-3) significantly modulated goat monocyte function in multiple aspects.

The ratio between proteases and their inhibitors is unbalanced in cancer. The cysteine protease inhibitor cystatin C is internalized by some cancer cells, which affects cellular properties. Here we aimed to investigate if uptake of cystatin C and the related inhibitor cystatin E/M occur in melanoma cell lines and to evaluate to what extent the uptake affects the legumain activity that is typically increased in melanoma. First we studied the basic expression, secretion, and intracellular content of all type 2 cystatins as well as expression and activity of their possible target enzymes legumain and cathepsin B in MDA-MB-435S, A375, and C8161 melanoma cells. Legumain activity was measureable in all cell lines, and of the potential legumain inhibitors, cystatin C, E/M, and F, cystatin C was the one mainly produced. All cells internalized cystatin C added to culture media, leading to increased intracellular cystatin C levels by 120-200%. Cystatin E/M was internalized as well but at a modest rate. The effects on intracellular legumain activity were nevertheless pronounced, probably because the cells lacked this inhibitor, and its affinity for legumain is 100-fold higher than that of cystatin C. Likewise, the low-degree uptake resulted in reduced migration and invasion of A375 cells in Matrigel to an extent comparable with the W106F variant of cystatin C with optimal uptake properties and resulting in much higher intracellular levels. Thus, cystatin E/M appears to be a good candidate to efficiently down-regulate the increased legumain activity, possibly important for the malignant phenotype of melanoma cells., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

To ensure successful feeding tick saliva contains a number of inhibitory proteins that interfere with the host immune response and help to create a permissive environment for pathogen transmission. Among the potential targets of the salivary cystatins are two host cysteine proteases, cathepsin S, which is essential for antigen- and invariant chain-processing, and cathepsin C (dipeptidyl peptidase 1, DPP1), which plays a critical role in processing and activation of the granule serine proteases. Here, the effect of salivary cystatin OmC2 from Ornithodoros moubata was studied using differentiated MUTZ-3 cells as a model of immature dendritic cells of the host skin. Following internalization, cystatin OmC2 was initially found to inhibit the activity of several cysteine cathepsins, as indicated by the decreased rates of degradation of fluorogenic peptide substrates. To identify targets, affinity chromatography was used to isolate His-tagged cystatin OmC2 together with the bound proteins from MUTZ-3 cells. Cathepsins S and C were identified in these complexes by mass spectrometry and confirmed by immunoblotting. Furthermore, reduced increase in the surface expression of MHC II and CD86, which are associated with the maturation of dendritic cells, was observed. In contrast, human inhibitor cystatin C, which is normally expressed and secreted by dendritic cells, did not affect the expression of CD86. It is proposed that internalization of salivary cystatin OmC2 by the host dendritic cells targets cathepsins S and C, thereby affecting their maturation.

Phytocystatins are a well-characterized class of naturally occurring protease inhibitors that prevent the catalysis of papain-like cysteine proteases. The action of cystatins in stress tolerance has been studied intensively, but relatively little is known about their functions in plants during leaf senescence. Here, we examined the potential roles of the apple cystatin, MpCYS4, in leaf photosynthesis as well as the concentrations and composition of leaf proteins when plants encounter natural or stress-induced senescence. Overexpression of this gene in apple rootstock M26 effectively slowed the senescence-related declines in photosynthetic activity and chlorophyll concentrations and prevented the action of cysteine proteinases during the process of degrading proteins (e.g., Rubisco) in senescing leaves. Moreover, MpCYS4 alleviated the associated oxidative damage and enhanced the capacity of plants to eliminate reactive oxygen species by activating antioxidant enzymes such as ascorbate peroxidase, peroxidase, and catalase. Consequently, plant cells were protected against damage from free radicals during leaf senescence. Based on these results, we conclude that MpCYS4 functions in delaying natural and stress-induced senescence of apple leaves., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

In demyelinating diseases such as multiple sclerosis (MS), an imbalance between the demyelination and remyelination rates underlies the degenerative processes. Microglial activation is observed in demyelinating lesions; however, the molecular mechanism responsible for the homeostatic/environmental change remains elusive. We previously found that cystatin F (CysF), a cysteine protease inhibitor, is selectively expressed in microglia only in actively demyelinating/remyelinating lesions but ceases expression in chronic lesions, suggesting its role in remyelination. Here, we report the effects of manipulating the expression of CysF and cathepsin C (CatC), a key target of CysF, in a murine model of transgenic demyelinating disease, Plp 4e/- . During the active remyelinating phase, both CysF knockdown (CysFKD) and microglial-selective CatC overexpression (CatCOE) showed a worsening of the demyelination in Plp 4e/- transgenic mice. Conversely, during the chronic demyelinating phase, CatC knockdown (CatCKD) ameliorated the demyelination. Our results suggest that the balance between CatC and CysF expression controls the demyelination and remyelination process., (© 2017 Wiley Periodicals, Inc.)

Cystatins, known for their ubiquitous presence in mammalian system are thiol protease inhibitors serving important physiological functions. Here, we present a variant of cystatin isolated from brain of Capra hircus (goat) which is glycosylated but lacks disulphide bonds. Caprine brain cystatin (CBC) was isolated using alkaline treatment, ammonium sulphate fractionation (40-60%) and gel filtration chromatography on Sephacryl S-100HR column with an overall yield of 26.29% and 322-fold purification. The inhibitor gave a molecular mass of ~44 kDa as determined by SDS-PAGE and gel filtration behaviour. The Stokes radius and diffusion coefficient of CBC were 27.14 Å and 8.18 × 10 -7  cm 2  s -1 , respectively. Kinetic data revealed that CBC inhibited thiol proteases reversibly and competitively, with the highest inhibition towards papain (K i  = 4.10 nM) followed by ficin and bromelain. CBC possessed 34.7% α-helical content as observed by CD spectroscopy. UV, fluorescence, CD and FTIR spectroscopy revealed significant conformational change upon CBC-papain complex formation. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic parameters - ΔH, ΔS, ΔG along with N (binding stoichiometry) for CBC-papain complex formation. Binding stoichiometry (N = .97 ± .07 sites) for the CBC-papain complex indicates that cystatin is surrounded by nearly one papain molecule. Negative ΔH (-5.78 kcal mol -1 ) and positive ΔS (11.01 cal mol -1  deg -1 ) values suggest that the interaction between CBC and papain is enthalpically as well as entropically favoured process. The overall negative ΔG (-9.19 kcal mol -1 ) value implies a spontaneous CBC-papain interaction.