Arabidopsis thaliana Phytocystatin 6 Forms Functional Oligomer and Amyloid Fibril States.

Cystatins encode a high functional variability not only because of their ability to inhibit different classes of proteases but also because of their propensity to form oligomers and amyloid fibrils. Phytocystatins, essential regulators of protease activity in plants, specifically inhibit papain-like cysteine proteases (PLCPs) and legumains through two distinct cystatin domains. Mammalian cystatins can form amyloid fibrils; however, the potential for amyloid fibril formation of phytocystatins remains unknown. In this study, we demonstrate that Arabidopsis thaliana phytocystatin 6 (AtCYT6) exists as a mixture of monomeric, dimeric, and oligomeric forms in solution. Noncovalent oligomerization was facilitated by the N-terminal cystatin domain, while covalent dimerization occurred through disulfide bond formation in the interdomain linker. The noncovalent dimeric form of AtCYT6 retained activity against its target proteases, papain and legumain, albeit with reduced inhibitory potency. Additionally, we observed the formation of amyloid fibrils by AtCYT6 under acidic pH conditions and upon heating. The amyloidogenic potential could be attributed to the AtCYT6's N-terminal domain (AtCYT6-NTD). Importantly, AtCYT6 amyloid fibrils harbored inhibitory activities against both papain and legumain. These findings shed light on the oligomerization and amyloidogenic behavior of AtCYT6, expanding our understanding of phytocystatin biology and its potential functional implications for plant protease regulation.

Protease-bound structure of Ricistatin provides insights into the mechanism of action of tick salivary cystatins in the vertebrate host.

Tick saliva injected into the vertebrate host contains bioactive anti-proteolytic proteins from the cystatin family; however, the molecular basis of their unusual biochemical and physiological properties, distinct from those of host homologs, is unknown. Here, we present Ricistatin, a novel secreted cystatin identified in the salivary gland transcriptome of Ixodes ricinus ticks. Recombinant Ricistatin inhibited host-derived cysteine cathepsins and preferentially targeted endopeptidases, while having only limited impact on proteolysis driven by exopeptidases. Determination of the crystal structure of Ricistatin in complex with a cysteine cathepsin together with characterization of structural determinants in the Ricistatin binding site explained its restricted specificity. Furthermore, Ricistatin was potently immunosuppressive and anti-inflammatory, reducing levels of pro-inflammatory cytokines IL-6, IL-1ß, and TNF-α and nitric oxide in macrophages; IL-2 and IL-9 levels in Th9 cells; and OVA antigen-induced CD4+ T cell proliferation and neutrophil migration. This work highlights the immunotherapeutic potential of Ricistatin and, for the first time, provides structural insights into the unique narrow selectivity of tick salivary cystatins determining their bioactivity.

Immunomodulatory in vitro effects of Trichinella cystatin-like protein on mouse splenocytes.

Trichinella parasites have developed specific mechanisms allowing successful completion of their life cycle. These mechanisms are in a great part involved in immunomodulation and studying them may provide a valuable insight into the functioning of the immune system. Trichinella products may be also used as potential therapeutic agents to treat immune diseases. This study investigates the immunomodulatory potential of recombinant multi cystatin-like protein (CLP) derived from T. britovi to determine whether CLP has anti-inflammatory properties in vitro. CLP is a highly antigenic glycoprotein present in Trichinella excetory-secretory (ES) products. AlphaFold structure prediction confirms that it consists of three type-two cystatin-like domains. Mouse splenocytes were stimulated in vitro with lipopolysaccharide (LPS) and co-stimulated with recombinant CLP. The culture supernatants were collected and tested for secreted cytokine levels using ELISA. CLP was found to reduce LPS-induced secretion of inflammatory cytokines TNFα and IL-6. On the contrary, in some experimental groups, co-stimulation with CLP resulted in increased secretion of the regulatory cytokine IL-10. The obtained results indicate that CLP has anti-inflammatory properties and future research on its function is advisable, specifically in the context of the therapy of inflammatory disorders.

Recombinant sugarcane cystatin CaneCPI-5 promotes osteogenic differentiation.

Cysteine proteases orchestrate bone remodeling, and are inhibited by cystatins. In reinforcing our hypothesis that exogenous and naturally obtained inhibitors of cysteine proteases (cystatins) act on bone remodeling, we decided to challenge osteoblasts with sugarcane-derived cystatin (CaneCPI-5) for up to 7 days. To this end, we investigated molecular issues related to the decisive, preliminary stages of osteoblast biology, such as adhesion, migration, proliferation, and differentiation. Our data showed that CaneCPI-5 negatively modulates both cofilin phosphorylation at Ser03, and the increase in cytoskeleton remodeling during the adhesion mechanism, possibly as a prerequisite to controlling cell proliferation and migration. This is mainly because CaneCPI-5 also caused the overexpression of the CDK2 gene, and greater migration of osteoblasts. Extracellular matrix remodeling was also evaluated in this study by investigating matrix metalloproteinase (MMP) activities. Our data showed that CaneCPI-5 overstimulates both MMP-2 and MMP-9 activities, and suggested that this cellular event could be related to osteoblast differentiation. Additionally, differentiation mechanisms were better evaluated by investigating Osterix and alkaline phosphatase (ALP) genes, and bone morphogenetic protein (BMP) signaling members. Altogether, our data showed that CaneCPI-5 can trigger biological mechanisms related to osteoblast differentiation, and broaden the perspectives for better exploring biotechnological approaches for bone disorders.

An undefined cystatin CsCPI1 from tea plant Camellia sinensis harbors antithrombotic activity.

Tea consumption has been linked to a decreased risk of cardiovascular disease (CVD) mortality, which imposes a heavy burden on the healthcare system; however, which components in tea cause this beneficial effect is not fully understood. Here we uncovered a cystatin (namely CsCPI1), which is a cysteine proteinase inhibitor (CPI) of the tea plant (Camellia sinensis) that promotes antithrombotic activity. Since thrombosis is a common pathogenesis of fatal CVDs, we investigated the effects of CsCPI1, which showed good therapeutic effects in mouse models of thrombotic disease and ischemic stroke. CsCPI1 significantly increases endothelial cell production of nitric oxide (NO) and inhibits platelet aggregation. Notably, CsCPI1 exhibited no cytotoxicity or resistance to pH and temperature changes, which indicates that CsCPI1 might be a potent antithrombotic agent that contributes to the therapeutic effects of tea consumption against CVD. Specifically, the antithrombotic effects of CsCPI1 are distinct from the classical function of plant cystatins against herbivorous insects. Therefore, our study proposes a new potential role of cystatins in CVD prevention and treatment, which requires further study.

An active domain SA-2 derived from cystatin-SA, and its antifungal activity.

Infections induced by fungi, especially the drug-resistant fungi, are difficult clinical problems. Conventional antifungal treatment is effective but due to resistance, treatment failure, and treatment-related toxicity, there is a need for new antifungal drugs. In this study, SA-2 (YYRRLLRVLRRRW) was derived from Cystatin-SA, a saliva protein with a molecular weight of 14 kDa. Meanwhile, the structure-activity of SA-2 and its mutants was also studied. We detected the antimicrobial activity and cytotoxicity of SA-2 and found that SA-2 had a low cytotoxicity toward mammalian cells but a good inhibitory effect on Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans), with MIC values of 16-64 µg/mL and 8-32 µg/mL, respectively. Interestingly, SA-2 effectively killed fluconazole-resistant C. neoformans and C. albicans within 12 h. This antifungal activity against fluconazole-resistant fungi was comparable to that of amphotericin B. In addition, the C. neoformans-infected mice model was established to evaluate the anti-infective activity of SA-2 in vivo. Results showed that SA-2 significantly reduced the counts of fungi in lung and brain tissues to protect fluconazole-resistant C. neoformans-infected mice from death without changing mice body weights. Moreover, the dramatically increased pro-inflammatory cytokines TNF-α, IL-6 and IL-1ß induced by intranasal infection of C. neoformans could be obviously declined due to the treatment of SA-2, which may be attributed to the elimination of C. neoformans in time in the infected tissue. For the mode of actions underlying SA-2 against C. neoformans, we found that the cationic peptide SA-2 could adhere to the negatively charged fungal cell membrane to increase the surface potential of C. neoformans in a dose-dependent manner, and finally disrupted the integrity of fungal cell membrane, reflecting as a 60% positive rate of propidium iodide uptake of C. neoformans cells after SA-2 (4 × MIC) treatment. Our study indicated that SA-2 has the potential to develop as a new therapeutic agent against infection induced by drug-resistant fungi.

Hypouricemic effect of gallic acid, a bioactive compound from Sonneratia apetala leaves and branches, on hyperuricemic mice.

As a tropical medicinal plant, Sonneratia apetala is mainly distributed in the southeast coastal areas of China. Recently, the hypouricemic effect of Sonneratia apetala leaves and branches (SAL) has been reported, but the active compound and its mechanism are unclear. Thus, this study aims to explore the effective fraction of SAL and the mechanism of its active compound on uric acid formation and excretion. SAL was extracted with ethyl acetate and concentrated to obtain solvent-free extracts (SAL-EA). The remains fraction (SAL-E) and the supernatant fraction (SAL-S) of SAL resulting from water extraction and alcohol precipitation were collected and dried. The effects of different fractions were explored on hyperuricemic mice. SAL-S showed excellent activities in decreasing the levels of uric acid (UA), blood urea nitrogen (BUN), and creatinine (CRE) in serum and in attenuating kidney damage. Then, the active compound gallic acid (GA) identified by HPLC was assayed for its mechanism of regulating uric acid metabolism in hyperuricemic mice. The hypouricemic effect of GA was probably associated with the downregulation of URAT1 and GLUT9, upregulation of ABCG2 and decreased activities of adenosine deaminase (ADA) and xanthine oxidase (XOD). Moreover, GA suppressed the level of MDA, IL-6, IL-1ß, TNF-α, TGF-ß1, COX-2 and cystatin-C (Cys-C), and enhanced the activities of SOD, GSH-Px, CAT, and Na+-K+-ATPase (NKA) in the kidneys. These results indicated that GA protects against hyperuricemia-induced kidney injury via suppressing oxidative stress and inflammation as well as decreasing the serum levels of UA by regulating urate transporters.

New insights into the anti-erosive property of a sugarcane-derived cystatin: different vehicle of application and potential mechanism of action.

OBJECTIVE: A new sugarcane-derived cystatin (CaneCPI-5) showed anti-erosive properties when included in solutions and strong binding force to enamel, but the performance of this protein when added to gel formulations and its effect on surface free energy (SFE) requires further studies. 1) to evaluate the protective effect of gels containing different concentrations of CaneCPI-5 against initial enamel erosion (Experiment 1); and 2) to analyze the SFE (γS) after treating the enamel surface with CaneCPI-5 solution (Experiment 2). METHODOLOGY: In Experiment 1, 75 bovine enamel specimens were divided into five groups according to the gel treatments: placebo (negative control); 0.27%mucin+0.5%casein (positive control); 0.1 mg/mL CaneCPI-5; 1.0 mg/mL CaneCPI-5; or 2.0 mg/mL CaneCPI-5. Specimens were treated with the gels for 1 min, the AP was formed (human saliva) for 2 h and the specimens were incubated in 0.65% citric acid (pH=3.4) for 1 min. The percentage of surface hardness change (%SHC) was estimated. In Experiment 2, measurements were performed by an automatic goniometer using three probing liquids: diiodomethane, water and ethylene glycol. Specimens (n=10/group) remained untreated (control) or were treated with solution containing 0.1 mg/mL CaneCPI-5, air-dried for 45 min, and 0.5 µL of each liquid was dispensed on the surface to measure contact angles. RESULTS: Gels containing 0.1 and 1.0 mg/mL CaneCPI-5 significantly reduced %SHC compared to the other treatments (p<0.05). Treated enamel showed significantly lower γS than control, without changes in the apolar component (γSLW), but the polar component (γSAB=Lewis acid-base) became more negative (p<0.01). Moreover, CaneCPI-5 treatment showed higher γS - (electron-donor) values compared to control (p<0.01). CONCLUSIONS: Gels containing 0.1 mg/mL or 1.0 mg/mL CaneCPI-5 protected enamel against initial dental erosion. CaneCPI-5 increased the number of electron donor sites on the enamel surface, which may affect AP formation and could be a potential mechanism of action to protect from erosion.

Phytocystatin CsinCPI-2 Reduces Osteoclastogenesis and Alveolar Bone Loss.

Periodontal disease (PD) is a polymicrobial chronic inflammatory condition of the supporting tissues around the teeth, leading to the destruction of surrounding connective tissue. During the progression of PD, osteoclasts play a crucial role in the resorption of alveolar bone that eventually leads to the loss of teeth if the PD is left untreated. Therefore, the development of antiresorptive therapies targeting bone-resorbing cells will significantly benefit the treatment of PD. Here, we demonstrate the inhibitory effect of CsinCPI-2, a novel cysteine peptidase inhibitor from the orange tree, on periodontitis-induced inflammation, alveolar bone loss, and osteoclast differentiation. Using the ligature-induced periodontitis model in mice, we show that treatment with CsinCPI-2 (0.8 µg/g of body weight) significantly reduced inflammatory cell infiltrate in the connective tissue and prevented the loss of alveolar bone mass (BV/TV) caused by PD, effects associated with diminished numbers of TRAP-positive multinucleated cells. Furthermore, CsinCPI-2 significantly downregulated the numbers of inflammatory cells expressing CD3, CD45, MAC387, and IL-1ß. In vitro, CsinCPI-2 inhibited RANKL-induced TRAP+ multinucleated osteoclast formation in mouse bone marrow macrophage cultures in a concentration-dependent manner. This effect was not due to cytotoxicity, as demonstrated by the MTT assay. CsinCPI-2 inhibited RANKL-induced mRNA expression of Acp5, Calcr, and Ctsk, as well as the RANKL-induced upregulation of Nfatc1, a crucial transcription factor for osteoclast differentiation. Based on our findings, CsinCPI-2 prevents bone loss induced by PD by controlling the inflammatory process and acting directly on osteoclastogenesis, suggesting an interesting potential for CsinCPI-2 in the strategy for PD treatment.

Harnessing the functional diversity of plant cystatins to design inhibitor variants highly active against herbivorous arthropod digestive proteases.

Protein engineering approaches have been proposed to improve the inhibitory properties of plant cystatins against herbivorous arthropod digestive proteases, generally involving the site-directed mutagenesis of functionally relevant amino acids or the selection of improved inhibitor variants by phage display approaches. Here, we propose a novel approach where the function-related structural elements of a cystatin are substituted by the corresponding elements of an alternative cystatin. Inhibitory assays were first performed with 20 representative plant cystatins and model Cys proteases, including arthropod proteases, to appreciate the extent of functional variability among the plant cystatin family. The most, and less, potent of these cystatins were then used as 'donors' of structural elements to create hybrids of tomato cystatin SlCYS8 used as a model 'recipient' inhibitor. In brief, inhibitory activities against Cys proteases strongly differed from one plant cystatin to another, with Ki (papain) values diverging by more than 30-fold and inhibitory rates against arthropod proteases varying by up to 50-fold depending on the enzymes assessed. In line with theoretical assumptions from docking models generated for different Cys protease-cystatin combinations, structural element substitutions had a strong impact on the activity of recipient cystatin SlCYS8, positive or negative depending on the basic inhibitory potency of the donor cystatin. Our data confirm the wide variety of cystatin inhibitory profiles among plant taxa. They also demonstrate the usefulness of these proteins as a pool of discrete structural elements for the design of cystatin variants with improved potency against herbivorous pest digestive Cys proteases.

Discovery and prioritization of variants and genes for kidney function in >1.2 million individuals.

Genes underneath signals from genome-wide association studies (GWAS) for kidney function are promising targets for functional studies, but prioritizing variants and genes is challenging. By GWAS meta-analysis for creatinine-based estimated glomerular filtration rate (eGFR) from the Chronic Kidney Disease Genetics Consortium and UK Biobank (n = 1,201,909), we expand the number of eGFRcrea loci (424 loci, 201 novel; 9.8% eGFRcrea variance explained by 634 independent signal variants). Our increased sample size in fine-mapping (n = 1,004,040, European) more than doubles the number of signals with resolved fine-mapping (99% credible sets down to 1 variant for 44 signals, ≤5 variants for 138 signals). Cystatin-based eGFR and/or blood urea nitrogen association support 348 loci (n = 460,826 and 852,678, respectively). Our customizable tool for Gene PrioritiSation reveals 23 compelling genes including mechanistic insights and enables navigation through genes and variants likely relevant for kidney function in human to help select targets for experimental follow-up.

Effect of a sugarcane cystatin on the profile and viability of microcosm biofilm and on dentin demineralization.

To analyze the effect of a sugarcane cystatin (CaneCPI-5) on the microbial profile and viability, as well as on the prevention of dentin demineralization using a microcosm biofilm model. Ninety bovine dentine specimens were divided into five experimental groups according with the solution they were treated for 60 s: (1) PBS (negative control), (2) 0.12% chlorhexidine (positive control), (3) Fluoride (500 ppm F, as NaF), (4) 0.025 mg/ml CaneCPI-5, and (5) 0.05 mg/ml CaneCPI-5. Specimens were incubated with inoculum (McBain's saliva plus human saliva) in the first 8 h, and from then on, they were exposed to McBain saliva containing sucrose and daily treated (60 s) with the solutions for 5 days. Resazurin and colony-forming unit counting assays were performed. Dentin demineralization was measured by transverse micro-radiography (TMR). 0.12% chlorhexidine significantly reduced the metabolic activity of the microcosm biofilm in relation to the negative control and treated groups (p < 0.01). CHX and F significantly reduced the counts of total microorganisms, mutans group streptococci, and lactobacilli when compared with the negative control. None of the treatments was able to significantly reduce dentin demineralization in comparison with the negative control. In the model evaluated, CaneCPI-5 neither altered the microcosm biofilm profile and viability nor protected dentin against demineralization.

Recombinant sugarcane cystatin CaneCPI-5 down regulates inflammation and promotes angiogenesis and collagen deposition in a mouse subcutaneous sponge model.

Cystatins are natural inhibitors of cysteine peptidases that are found practically in all living organisms. CaneCPI-5 is a sugarcane cystatin with inhibitory activity against human cathepsins B, K and L, which are cysteine proteases highly expressed in a variety of pathological conditions, usually marked by persistent inflammation and processing of the extracellular matrix. This work evaluated the effects of daily administration of the recombinant cystatin CaneCPI-5 [0.01, 0.1 or 1.0 µg in 10 µL of Phosphate-Buffered Saline (PBS)] on the inflammatory, angiogenic and fibrogenic components during chronic inflammatory response induced by subcutaneous sponge implants. The anti-inflammatory effect of treatment with CaneCPI-5 was confirmed by reduction of the levels of the pro-inflammatory mediators TNF-α, CXCL1 and CCL2/JE/MCP-1, as well as the activity of the myeloperoxidase and n-acetyl-ß-D-glucosaminidase. Treatment with CaneCPI-5 promoted angiogenesis in the implants, increasing the production of cytokines VEGF and FGF and the formation of new blood vessels. Finally, the administration of the recombinant cystatin favored the production of the pro-fibrogenic cytokine TGF-ß1 and collagen deposition next to the implants. Together, these results show the potential therapeutic application of CaneCPI-5 as an anti-inflammatory agent, capable of favoring angiogenesis and fibrogenesis processes, necessary for tissue repair.

In vitro biochemical characterization and genotoxicity assessment of Sapindus saponaria seed extract.

ETHNOPHARMACOLOGICAL RELEVANCE: Sapindus saponaria, also popularly known as soapberry, has been used in folk medicinal values because of its therapeutic properties and several compounds in its composition, which represent a target in potential for drug discovery. However, few data about its potential toxicity has been reported. AIM OF THE STUDY: Plant proteins can perform essential roles in survival, acting as defense mechanism, as well functioning as important molecular reserves for its natural metabolism. The aim of the current study was to investigate the in vitro toxicity profile of protein extract of S. saponaria and detect protein potentially involved in biological effects such as collagen hydrolysis and inhibition of viral proteases. MATERIALS AND METHODS: Protein extract of soapberry seeds was investigated for its cytotoxic and genotoxic action using the Ames test. The protein extract was also subjected to a partial purification process of a protease and a protease inhibitor by gel chromatography filtration techniques and the partially isolated proteins were characterized biochemically. RESULTS: Seed proteins extract of S. saponaria was evaluated until 100 µg/mL concentration, presenting cytotoxicity and mutagenicity in bacterial model mostly when exposed to exogenous metabolic system and causing cytotoxic and genotoxic effects in HepG2 cells. The purification and partial characterization of a serine protease (43 kDa) and a cysteine protease inhibitor (32.8 kDa) from protein extract of S. Saponaria, corroborate the idea of ​​the biological use of the plant as an insecticide and larvicide. Although it shows cytotoxic, mutagenic and genotoxic effects. CONCLUSION: The overall results of the present study provide supportive data on the potential use of proteins produced in S. saponaria seeds as pharmacological and biotechnological agents that can be further explored for the development of new drugs.

Trichinella spiralis cystatin, TsCstN, modulates STAT4/IL-12 to specifically suppress IFN-γ production.

We have previously identified a cystatin, TsCstN, derived from the L1 stage of Trichinella spiralis and have shown that this protein is internalised in macrophages. Here we sought to address if this macrophage-TsCstN interaction could alter downstream T-cell priming. Using LPS-primed macrophages to stimulate T-cells in a co-culture system with or without TsCstN we assessed the resultant T-cell outcomes. IFN-γ, both protein and mRNA, but not IL-17A was negatively regulated by inclusion of TsCstN during macrophage priming. We identified a cell-cell contact independent change in the levels of IL-12 that led to altered phosphorylated STAT4 levels and translocation. TsCstN also negatively regulated the autonomous response in the myotubule cell line, C2C12. This work identifies a potential pathyway for L1 larvae to evade protective Th1 based immune responses and establish muscle-stage T. spiralis infection.

Schistosoma japonicum cystatin suppresses osteoclastogenesis via manipulating the NF­κB signaling pathway.

Abnormal osteoclastic activation and secretion of cysteine proteinases result in excessive bone resorption, which is one of the primary factors in the development of bone metabolic disorders, such as rheumatoid arthritis and osteoporosis. Mammalian cystatins have been demonstrated to restrain osteoclastic bone resorption and to alleviate severe osteolytic destruction via blocking the activity of cysteine proteinases. However, the specific effects of parasite cystatins on the formation and function of osteoclasts remain unclear. The purpose of the current study was to explore the effects of cystatins from Schistosoma japonicum (Sj­Cys) on macrophage colony­stimulating factor (M­CSF) and receptor activator of NF­κB ligand (RANKL)­induced osteoclast differentiation, as well as the underlying molecular mechanisms. Recombinant Sj­Cys (rSj­Cys) dose­dependently restrained osteoclast formation, with a half­maximal inhibitory concentration (IC50) value of 0.3 µM, and suppressed osteoclastic bone resorptive capability in vitro. The findings were based on tartrate resistant acid phosphatase (TRAP) staining and bone resorption assays, respectively. However, the cell viability assay showed that the repression of rSj­Cys on osteoclast formation did not depend on effects on cell viability or apoptosis. Based on the results of reverse transcription­quantitative PCR and western blot analysis, it was found that rSj­Cys downregulated the expression levels of osteoclastogenesis­related genes and proteins, by interfering with M­CSF and RANKL­induced NF­κB signaling and downstream transcription factors during early­phase osteoclastogenesis. Overall, the results of the present study revealed that rSj­Cys exerted an inhibitory role in osteoclast differentiation and could be a prospective biotherapeutic candidate for the treatment and prevention of bone metabolic disorders.

Mechanisms Applied by Protein Inhibitors to Inhibit Cysteine Proteases.

Protein inhibitors of proteases are an important tool of nature to regulate and control proteolysis in living organisms under physiological and pathological conditions. In this review, we analyzed the mechanisms of inhibition of cysteine proteases on the basis of structural information and compiled kinetic data. The gathered structural data indicate that the protein fold is not a major obstacle for the evolution of a protease inhibitor. It appears that nature can convert almost any starting fold into an inhibitor of a protease. In addition, there appears to be no general rule governing the inhibitory mechanism. The structural data make it clear that the "lock and key" mechanism is a historical concept with limited validity. However, the analysis suggests that the shape of the active site cleft of proteases imposes some restraints. When the S1 binding site is shaped as a pocket buried in the structure of protease, inhibitors can apply substrate-like binding mechanisms. In contrast, when the S1 binding site is in part exposed to solvent, the substrate-like inhibition cannot be employed. It appears that all proteases, with the exception of papain-like proteases, belong to the first group of proteases. Finally, we show a number of examples and provide hints on how to engineer protein inhibitors.

Sugarcane cystatins: From discovery to biotechnological applications.

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.

Secreted cystatins decrease proliferation and enhance apoptosis of human leukemic cells.

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.

A new Piper nigrum cysteine proteinase inhibitor, PnCPI, with antifungal activity: molecular cloning, recombinant expression, functional analyses and molecular modeling.

MAIN CONCLUSION: A new Piper nigrum cysteine proteinase inhibitor, PnCPI, belonging to group I of phytocystatins, with inhibitory activity against papain and growth of Fusarium solani f. sp. piperis, was isolated and characterized. Previous studies (de Souza et al. 2011) have identified a partial cDNA sequence of putative cysteine proteinase inhibitor differentially expressed in roots of black pepper (P. nigrum L.) infected by F. solani f. sp. piperis. Here, we aimed to isolate the full-length cDNA and genomic sequences of the P. nigrum cysteine proteinase inhibitor gene, named PnCPI. Sequence analyses showed that the PnCPI gene encodes a deduced protein of 108 amino acid residues with a predicted molecular mass of 12.3 kDa and isoelectric point of 6.51. Besides the LARFAV-like sequence, common to all phytocystatins, PnCPI contains three conserved motifs of the superfamily cystatin: a glycine residue at the N-terminal region, the QxVxG reactive site more centrally positioned, and one tryptophan in the C-terminal region. PnCPI, belonging to group I of phytocystatins, showed high identity with cystatins isolated from several plant species. Sequence analyses also revealed no putative signal peptide at the N-terminal of PnCPI, as well as no introns within the genomic sequence corresponding to the PnCPI coding region. Molecular modeling showed the ability of PnCPI to interact with papain, while its inhibitory activity against this protease was confirmed after heterologous expression in Escherichia coli. The effects of heat treatments on the inhibitory activity of recombinant PnCPI, rPnCPI, were evaluated. In addition, rPnCPI exhibited in vitro activity against F. solani f. sp. piperis, revealing a new cystatin with the potential antifungal application. The identification of PnCPI as a functional cystatin able to inhibit the in vitro growth of F. solani f. sp. piperis indicates other factors contributing to in vivo susceptibility of black pepper to root rot disease.