Endogenous Cysteine Protease Inhibitor Research Articles

Chagasin from Trypanosoma cruzi as a molecular scaffold to express epitopes of TSA-1 as soluble recombinant chimeras

Trypanosoma cruzi is the causative agent of Chagas disease, a global public health problem. New therapeutic drugs and biologics are needed. The TSA-1 recombinant protein of T. cruzi is one such promising antigen for developing a therapeutic vaccine. However, it is overexpressed in E. coli as inclusion bodies, requiring an additional refolding step. As an alternative, in this study, we propose the endogenous cysteine protease inhibitor chagasin as a molecular scaffold to generate chimeric proteins. These proteins will contain combinations of two of the five conserved epitopes (E1 to E5) of TSA-1 in the L4 and L6 chagasin loops. Twenty chimeras (Q1-Q20) were designed, and their solubility was predicted using bioinformatics tools. Nine chimeras with different degrees of solubility were selected and expressed in E. coli BL21 (DE3). Western blot assays with anti-6x-His and anti-chagasin antibodies confirmed the expression of soluble recombinant chimeras. Both theoretically and experimentally, the Q12 (E5-E3) chimera was the most soluble, and the Q20 (E4-E5) the most insoluble protein. Q4 (E5-E1) and Q8 (E5-E2) chimeras were classified as proteins with medium solubility that exhibited the highest yield in the soluble fraction. Notably, Q4 has a yield of 239 mg/L, well above the yield of recombinant chagasin (16.5 mg/L) expressed in a soluble form. The expression of the Q4 chimera was scaled up to a 7 L fermenter obtaining a yield of 490 mg/L. These data show that chagasin can serve as a molecular scaffold for the expression of TSA-1 epitopes in the form of soluble chimeras.

Role of cystatin C in urogenital malignancy.

Urogenital malignancy accounts for one of the major causes of cancer-related deaths globally. Numerous studies have investigated novel molecular markers in the blood circulation, tumor tissue, or urine in order to assist in the clinical identification of tumors at early stages, predict the response of therapeutic strategies, and give accurate prognosis assessment. As an endogenous inhibitor of lysosomal cysteine proteinases, cystatin C plays an integral role in diverse processes. A substantial number of studies have indicated that it may be such a potential promising biomarker. Therefore, this review was intended to provide a detailed overview of the role of cystatin C in urogenital malignancy.

SERPINB3 (SCCA1) inhibits cathepsin L and lysoptosis, protecting cervical cancer cells from chemoradiation

The endogenous lysosomal cysteine protease inhibitor SERPINB3 (squamous cell carcinoma antigen 1, SCCA1) is elevated in patients with cervical cancer and other malignancies. High serum SERPINB3 is prognostic for recurrence and death following chemoradiation therapy. Cervical cancer cells genetically lacking SERPINB3 are more sensitive to ionizing radiation (IR), suggesting this protease inhibitor plays a role in therapeutic response. Here we demonstrate that SERPINB3-deficient cells have enhanced sensitivity to IR-induced cell death. Knock out of SERPINB3 sensitizes cells to a greater extent than cisplatin, the current standard of care. IR in SERPINB3 deficient cervical carcinoma cells induces predominantly necrotic cell death, with biochemical and cellular features of lysoptosis. Rescue with wild-type SERPINB3 or a reactive site loop mutant indicates that protease inhibitory activity is required to protect cervical tumor cells from radiation-induced death. Transcriptomics analysis of primary cervix tumor samples and genetic knock out demonstrates a role for the lysosomal protease cathepsin L in radiation-induced cell death in SERPINB3 knock-out cells. These data support targeting of SERPINB3 and lysoptosis to treat radioresistant cervical cancers.

Aggregation of Cystatin C Changes Its Inhibitory Functions on Protease Activities and Amyloid β Fibril Formation.

Cystatin C (CST3) is an endogenous cysteine protease inhibitor, which is implicated in cerebral amyloid angiopathy (CAA). In CAA, CST3 is found to be aggregated. The purpose of this study is to investigate whether this aggregation could alter the activity of the protein relevant to the molecular pathology of CAA. A system of CST3 protein aggregation was established, and the aggregated protein was characterized. The results showed that CST3 aggregated both at 80 °C without agitation, and at 37 °C with agitation in a time-dependent manner. However, the levels of aggregation were high and appeared earlier at 80 °C. Dot-blot immunoassay for oligomers revealed that CST3 could make oligomeric aggregates at the 37 °C condition. Electron microscopy showed that CST3 could make short fibrillary aggregates at 37 °C. Cathepsin B activity assay demonstrated that aggregated CST3 inhibited the enzyme activity less efficiently at pH 5.5. At 7.4 pH, it lost the inhibitory properties almost completely. In addition, aggregated CST3 did not inhibit Aβ1-40 fibril formation, rather, it slightly increased it. CST3 immunocytochemistry showed that the protein was positive both in monomeric and aggregated CST3-treated neuronal culture. However, His6 immunocytochemistry revealed that the internalization of exogenous recombinant CST3 by an astrocytoma cell culture was higher when the protein was aggregated compared to its monomeric form. Finally, MTT cell viability assay showed that the aggregated form of CST3 was more toxic than the monomeric form. Thus, our results suggest that aggregation may result in a loss-of-function phenotype of CST3, which is toxic and responsible for cellular degeneration.

Cystatin-like protein of sweet orange (CsinCPI-2) modulates pre-osteoblast differentiation via \u03b2-Catenin involvement

Phytocystatins are endogenous cysteine-protease inhibitors present in plants. They are involved in initial germination rates and in plant defense mechanisms against phytopathogens. Recently, a new phytocystatin derived from sweet orange, CsinCPI-2, has been shown to inhibit the enzymatic activity of human cathepsins, presenting anti-inflammatory potential and pro-osteogenic effect in human dental pulp cells. The osteogenic potential of the CsinCPI-2 protein represents a new insight into plants cysteine proteases inhibitors and this effect needs to be better addressed. The aim of this study was to investigate the performance of pre-osteoblasts in response to CsinCPI-2, mainly focusing on cell adhesion, proliferation and differentiation mechanisms. Together our data show that in the first hours of treatment, protein in CsinCPI-2 promotes an increase in the expression of adhesion markers, which decrease after 24 h, leading to the activation of Kinase-dependent cyclines (CDKs) modulating the transition from G1 to S phases cell cycle. In addition, we saw that the increase in ERK may be associated with activation of the differentiation profile, also observed with an increase in the B-Catenin pathway and an increase in the expression of Runx2 in the group that received the treatment with CsinCPI-2.

Profiling selectivity of chagasin mutants towards cysteine proteases cruzain or cathepsin L through molecular dynamics simulations

Chagasin, an endogenous cysteine protease inhibitor from Trypanosoma cruzi, can control the activity of the parasitic cruzain and its homologous human cathepsin L. While chagasin inhibits both enzymes with similar potency, mutations have different effects on binding to these enzymes. Mutants T31A and T31A/T32A bind well to cathepsin L, but their affinity for cruzain drops ∼40 to 140–fold. On the other hand, the mutant W93A binds well to cruzain, but it loses potency against cathepsin L. Here, we employed molecular dynamics simulations to understand the selectivity in inhibition of cruzain or cathepsin L by chagasin mutants W93A, T31A, and T31A/T32A. Our results allowed profiling the nonbonded interactions in the interfaces of each mutant with these cysteine proteases. Additionally, we observed differences in the binding conformation of the chagasin loops L2 and L6 of the W93A mutant, favoring interactions with cruzain and reducing interactions with cathepsin L. These differences are associated with a partial dissociation of the W93A-cathepsin L complex, providing a likely cause for the selectivity of the mutant W93A towards cruzain. Communicated by Ramaswamy H. Sarma.

Fowlerstefin, a cysteine protease inhibitor of Naegleria fowleri, induces inflammatory responses in BV-2 microglial cells in vitro

BackgroundNaegleria fowleri is a free-living amoeba that causes an opportunistic fatal infection known as primary amoebic meningoencephalitis (PAM) in humans. Cysteine proteases produced by the amoeba may play critical roles in the pathogenesis of infection. In this study, a novel cysteine protease inhibitor of N. fowleri (fowlerstefin) was characterized to elucidate its biological function as an endogenous cysteine protease inhibitor of the parasite as well as a pathogenic molecule that induces immune responses in microglial cells.MethodsRecombinant fowlerstefin was expressed in Escherichia coli. The inhibitory activity of fowlerstefin against several cysteine proteases, including human cathepsins B and L, papain and NfCPB-L, was analyzed. Fowlerstefin-induced pro-inflammatory response in BV-2 microglial cells was anayzed by cytokine array assay, reverse transcription polymerase chain reaction, and enzyme-linked immunosorbent assay.ResultsFowlerstefin is a cysteine protease inhibitor with a monomeric structure, and belongs to the stefin family. Recombinant fowlerstefin effectively inhibited diverse cysteine proteases including cathepsin B-like cysteine proteases of N. fowleri (NfCPB-L), human cathepsins B and L, and papain. Expression of fowlerstefin in the amoeba was optimal during the trophozoite stage and gradually decreased in cysts. Fowlerstefin induced an inflammatory response in BV-2 microglial cells. Fowlerstefin induced the expression of several pro-inflammatory cytokines and chemokines including IL-6 and TNF in BV-2 microglial cells. Fowlerstefin-induced expression of IL-6 and TNF in BV-2 microglial cells was regulated by mitogen-activated protein kinase (MAPKs). The inflammatory response induced by fowlerstefin in BV-2 microglial cells was downregulated via inhibition of NF-κB and AP-1.ConclusionsFowlerstefin is a pathogenic molecule that stimulates BV-2 microglial cells to produce pro-inflammatory cytokines through NF-κB- and AP-1-dependent MAPK signaling pathways. Fowlerstefin-induced inflammatory cytokines exacerbate the inflammatory response in N. fowleri-infected areas and contribute to the pathogenesis of PAM.

Functional characterization of Cystatin C in orange-spotted grouper, Epinephelus coioides

Cystatin C is an endogenous inhibitor of cysteine proteases and widely exist in organisms. Several studies in mammals have showed that Cystatin C plays critical role in the immune defense against microorganisms. It is also well known that some fish Cystatin C have important immune regulation functions in inflammatory responses. However, the function of fish Cystatin C in virus infection as well as its underlying molecular mechanisms remain to be elucidated. In the present study, a Cystatin C gene termed Ec-CysC was identified from orange-spotted grouper, Epinephelus coioides. The full-length of Ec-CysC cDNA was 817 bp with a 387 bp open reading frame (ORF) that encoded a 129-amino acid (aa) protein, including 18-aa signal peptide and 111-aa mature polypeptide. The deduced amino acid of Ec-CysC shared three conserved domains containing Glycine at the N-terminus region, QVVAG motif in the middle and PW motif near the C-terminus region. Transcription analysis of the Ec-CysC gene showed its expression in all twelve examined tissues including liver, spleen, kidney, brain, intestine, heart, skin, muscle, fin, stomach, gill and head kidney. Its expression following stimulation with Singapore grouper iridovirus (SGIV) was further tested in spleen, the relative expression of Ec-CysC was significantly up-regulated at 12 h post-infection. The subcellular localization experiment revealed that Ec-CysC was mainly distributed in the cytoplasm in Grouper Spleen (GS) cells. In vitro, Overexpression of Ec-CysC in GS cells significantly reduced the expression of viral genes, namely, ORF162, ORF049 and ORF072. Meanwhile, we found that overexpression of Ec-CysC resulted in upward trend of expression of inflammatory cytokines TNF-a, IL-1β and IL8 during SGIV infection. Further, SGIV-inducible apoptosis and Caspase-3 activity were also weakened by overexpression Ec-CysC in fathead minnow (FHM) cells. These results indicated that Ec-CysC might have a deeper involvement in fish immune defense, and played important roles in inflammation and apoptosis induced by SGIV.

Oxidation of cystatin imparted by riboflavin generated free radicals: Spectral analysis

Thiol Protease inhibitors (cystatins) are endogenous natural inhibitors of cysteine proteases. They are present in all mammalians cells and body fluids. Cystatin are allocated into three major families. Family –I stefins, family –II cystatins and family –III kininogens, according to their amino acid sequence, molecular weight, carbohydrate content and disulphide bonds. It has been investigated that thiol proteases (cathepsin) and their endogenous inhibitor, cystatins have been closely associated with diseases like Alzheimer's, Prions, neurodegenerative diseases, cancer and diabetes. Photodynamic effect of various sensitizers' have long been applied to delineate structural and functional properties of biologically active proteins. Flavins are well known to photo oxidize amino acids which effects conformation of proteins. Riboflavin (Vit B2) with a recommended daily requirement of approximately 2–3 mg is a yellow pigment, It is widely distributed in human tissues and blood, in both free and conjugated forms.In the present Study it has been shown that cystatin purified from buffalo brain (BC) is susceptible to reactive oxygen species generated by photo activation of riboflavin. It was observed that Photo activated riboflavin leads to inactivation of BC. Major Loss of tryptophan intensity was observed in the presence of purified thiol protease inhibitor upon incubation with 50 μM of riboflavin.In order to inspect the type of reactive oxygen species involved in inactivation of the inhibitor, different scavenger's were used namely glucose, potassium Iodide, sodium azide, manitol, thiourea, sodium benzoate, curcumin, quercetin, ascorbic acid and uric acid. It was found that Glucose, Potassium Iodide and sodium azide, have preventive effect on photo inactivation of the purified cystatin whilst other scavengers illustrated diminutive defensive effect.

Human cystatin SN is an endogenous protease inhibitor that prevents allergic rhinitis

Protease allergens disrupt epithelial barriers to exert their allergenicity. Cystatin SN (encoded by CST1) is an endogenous cysteine protease inhibitor upregulated in nasal epithelia in patients with allergic rhinitis (AR). We sought to investigate the protective effect of human cystatin SN on AR symptoms using pollen-induced AR mouse models. We performed an invitro protease activity assay to evaluate the effect of recombinant human cystatin SN (rhCystatin SN) on Japanese cedar (JC) or ragweed proteases. Ahuman nasal epithelial cell line, RPMI 2650, was used to examine tight junction (TJ) disruption invitro. Mice were sensitized and nasally challenged with JC or ragweed pollens with or without rhCystatin SN to examine the effect of rhCystatin SN on AR symptoms and the epithelial barrier invivo. Because mice lack CST1, we generated transgenic (Tg) mice expressing human CST1 under control of its genomic control region (hCST1-Tg mice) to examine the role of cystatin SN in physiologically expressed conditions. rhCystatin SN inhibited JC but not ragweed protease activities and prevented JC-induced but not ragweed-induced TJ disruption invitro. Exogenous administration of rhCystatin SN ameliorated JC-induced but not ragweed-induced sneezing and nasal TJ disruption invivo. Furthermore, hCST1-Tg mice showed decreased JC-induced but not ragweed-induced sneezing symptoms and nasal TJ disruption compared with wild-type mice. Human cystatin SN suppresses AR symptoms through inhibiting allergen protease activities and protecting the nasal TJ barrier in an allergen-specific manner. We propose that upregulation of nasal endogenous protease inhibitors, including cystatin SN, is a novel therapeutic strategy for protease allergen-induced AR.

Characterization of a novel endogenous cysteine proteinase inhibitor, trichocystatin-3 (TC-3), localized on the surface of Trichomonas vaginalis.

Trichomonas vaginalis is a flagellated protist responsible for human trichomoniasis. T. vaginalis has three genes encoding for endogenous cysteine proteinase (CP) inhibitors, known as trichocystatin-1 through trichocystatin-3 (TC-1, TC-2, and TC-3). These inhibitors belong to the cystatin family. In this study, we characterized trichocystatin-3 (TC-3), an endogenous cysteine proteinase (CP) inhibitor of T. vaginalis. TC-3 possesses a signal peptide in the N-terminus and two putative glycosylation sites (typical of family 2, cystatins) but lacks the PW motif and cysteine residues (typical of family 1, stefins). Native TC-3 was recognized as an ∼18 kDa protein band in a T. vaginalis protein extract. By confocal microscopy, endogenous TC-3 was found in the Golgi complex, cytoplasm, large vesicles, and the plasma membrane. These localizations are consistent with an in silico prediction. In addition, the purified recombinant protein (TC-3r) functions as an inhibitor of cathepsin L CPs, such as human liver cathepsin L and trichomonad CPs, present in a proteinase-resistant extract (PRE). Via a pull-down assay using TC-3r as bait and PRE, we identified several trichomonad CPs targeted by TC-3, primarily TvCP3. These CP-TC-3 interactions occur in vesicles, in the cytoplasm, and on the parasite surface. In addition, TC-3r showed a protective effect on HeLa cell monolayers by inhibiting trichomonad surface CPs involved in cellular damage. Our results show that the endogenous inhibitor TC-3 plays a key role in the regulation of endogenous CP proteolytic activity.

Aggregation mechanism of cystatin c in neuronal cell and its implication in neurodegeneration

Background: CystatinC (CysC) is an endogenous cysteine protease inhibitor, and expressed in all nucleated cells. This protein is mainly localized in lysosome, but is also secreted in body fluids. L68Q mutation of CysC increases its amyloid aggregation property, leading to Hereditary CysC Cerebral Amyloid Angiopathy. Moreover, CysC is known as a major component of Bunina bodies, an ALS-specific neuronal inclusion body, suggesting its relationship with neurodegeneration.

Cystatin C as a potential therapeutic mediator against Parkinson\u2019s disease via VEGF-induced angiogenesis and enhanced neuronal autophagy in neurovascular units

Cystatin C (CYS C, Cst3) is an endogenous cysteine protease inhibitor that plays neuroprotective roles in neurodegenerative diseases. We aimed to explore the association of CYS C with Parkinson’s disease (PD) models and investigate its involvement in the role of neurovascular units (NVUs) in PD neuro-pathogenesis. We used A53T α-synuclein (SNCA) transgenic mice and 6-hydroxydopamine-lesioned DAergic PC12 cells as experimental PD models to investigate the mechanisms behind this association. The injections of CYS C were administered to the right substantia nigra (SN) of A53T SNCA transgenic mice to measure the effects of CYS C in transgenic A53T SNCA mice. To explore the angiogenesis in vivo and in vitro, we used the chick embryo chorioallantoic membrane (CAM) assay and tube formation (TF) assay. We found that CYS C has a neuroprotective effect in this in vivo PD model. We observed increased VEGF, NURR1 and autophagy markers LC3B and decreased SNCA and apoptosis marker cleaved CASP3 in different brain regions of CYS C-treated A53T SNCA transgenic mice. In vitro, we observed that CYS C-induced VEGF, a secreted protein, attenuated 6-OHDA-lesioned DAergic PC12 cell degeneration by regulating p-PKC-α/p-ERK1/2-Nurr1 signaling and inducing autophagy. VEGF-mediated angiogenesis was markedly enhanced in the conditioned media of 6-OHDA-lesioned PC12 cells with CYS C-overexpression, whereas blockage of autophagy in CYS C-overexpressing PC12 cells significantly downregulated VEGF expression and the associated angiogenesis. Our data indicate that CYS C displays dual neuronal–vascular functions, promoting PC12 cell survival and angiogenesis via regulating the level of secreted VEGF in NVUs. Our study provides evidence that may aid in the development of an alternative approach for the treatment of PD through modulation of CYS C-mediated neuronal-vascular pathways.

Protease activity and phytocystatin expression in Arabidopsis thaliana upon Heterodera schachtii infection

The activity of plant proteases is important for amino acids recycling, removal of damaged proteins as well as defence responses. The second-stage juvenile of the beet cyst nematode Heterodera schachtii penetrates host roots and induces the feeding site called a syncytium. To determine whether infection by H. schachtii affects proteolysis, the protease activity was studied in Arabidopsis roots and shoots at the day of inoculation and 3, 7 and 15 days post inoculation (dpi). Nematode infection caused a decrease of protease activities in infected roots over the entire examination period at all studied pH values. In contrast, the activities of the low molecular mass as well as Ca2+-dependent cysteine proteases were found to be stimulated. In shoots of infected plants, the protease activity was diminished only at 15 dpi at all tested pH values. It was accompanied by changes in total soluble protein content, a higher protein carbonylation and a total polyphenol content. To go deeper into proteolysis regulation, the expression of phytocystatin genes, endogenous inhibitors of cysteine proteases, was examined in syncytia, roots and shoots. Expression of AtCYS1, AtCYS5 and AtCYS6 genes was enhanced upon cyst nematode infection. Our results suggest that changes in protease activities in roots and shoots and altered cystatin expression patterns in syncytia, roots and shoots are important for protein metabolism during cyst nematode infection.

The association of plasma cystatin C proteoforms with diabetic chronic kidney disease.

BackgroundCystatin C (CysC) is an endogenous cysteine protease inhibitor that can be used to assess the progression of kidney function. Recent studies demonstrate that CysC is a more specific indicator of glomerular filtration rate (GFR) than creatinine. CysC in plasma exists in multiple proteoforms. The goal of this study was to clarify the association of native CysC, CysC missing N-terminal Serine (CysC des-S), and CysC without three N-terminal residues (CysC des-SSP) with diabetic chronic kidney disease (CKD).ResultsUsing mass spectrometric immunoassay, the plasma concentrations of native CysC and the two CysC truncation proteoforms were examined in 111 individuals from three groups: 33 non-diabetic controls, 34 participants with type 2 diabetes (DM) and without CKD and 44 participants with diabetic CKD. Native CysC concentrations were 1.4 fold greater in CKD compared to DM group (p = 0.02) and 1.5 fold greater in CKD compared to the control group (p = 0.001). CysC des-S concentrations were 1.55 fold greater in CKD compared to the DM group (p = 0.002) and 1.9 fold greater in CKD compared to the control group (p = 0.0002). CysC des-SSP concentrations were 1.8 fold greater in CKD compared to the DM group (p = 0.008) and 1.52 fold greater in CKD compared to the control group (p = 0.002). In addition, the concentrations of CysC proteoforms were greater in the setting of albuminuria. The truncated CysC proteoform concentrations were associated with estimated GFR independent of native CysC concentrations.ConclusionOur findings demonstrate a greater amount of CysC proteoforms in diabetic CKD. We therefore suggest assessing the role of cystatin C proteoforms in the progression of CKD.Electronic supplementary materialThe online version of this article (doi:10.1186/s12953-016-0096-7) contains supplementary material, which is available to authorized users.

Cystatin C is a Better Marker of Renal Dysfunction in Hypertensive Pregnancies

ABSTRACT Background Glomerular endotheliosis is an essential component in the pathophysiology of gestational hypertension (GH) and preeclampsia (PE) which results in renal dysfunction. This is not always detected by routine renal function tests, such as serum creatinine, urea, and uric acid. Cystatin C, an endogenous cysteine protease inhibitor, is completely absorbed by renal tubules and has been shown to be an ideal marker of glomerular filtration rate (GFR), which needs to be evaluated in assessing renal dysfunction occurring in GH and PE. Aims The present study is designed to evaluate serum cystatin C levels in normal pregnancy, GH, and PE and compare its efficacy with traditional renal function tests. Materials and methods In this prospective cross-sectional study, 75 subjects enrolled, comprised of 25 subjects each of normal pregnancy, GH, and PE. Serum cystatin C, blood urea, serum creatinine, serum uric acid, and urinary protein/creatinine ratio were estimated in all subjects prior to delivery. Results All renal parameters including cystatin C were significantly raised in GH and PE compared with control group. However, only serum cystatin C level (and no other renal parameters) was significantly higher in PE group compared with GH group. Area under the curve for cystatin C was maximum (0.917) compared with other parameters. Cystatin C had a higher sensitivity and specificity than other conventional markers. Conclusion Serum cystatin C is a better marker of renal dysfunction in hypertensive pregnancies. How to cite this article Singh A, Gupta M, Ranjan R, Saini V, Gupta SK. Cystatin C is a Better Marker of Renal Dysfunction in Hypertensive Pregnancies. Indian J Med Biochem 2016; 20(1):21-27.

A91: Endogenous inhibitors of cysteine proteases and preform of cathepsin B in cancer of reproductive system

Background The research for procathepsin B and endogenous inhibitors of cysteine proteases in tumor markers of human reproductive system is important for early diagnostics of cancer. Preform of cathepsin B and cystatins B and C also are universally involved into development of different tumors. Tumor cells as well as tumor-associated macrophages have been shown to secrete active forms of proteases and their inhibitors; however, their roles, especially those of proenzymes as markers of malignancy, are still under investigation. Aim to evaluate procathepsin B and endogenous inhibitors of cysteine proteases cystatins B and C in tumors of reproductive system. Materials and methods Serum and ascites fluid of 38 patients with ovarian cancer (among them 15 patients after treatment) and benign ovarian tumors ( n = 9), endometrial cancer ( n = 31, before treatment 14), mammalian cancer ( n = 29, before treatment 18) of stages II–IV for all groups, from Department of Gynecology of Regional Oncology Center, Novosibirsk, were under investigation. Serum of practically healthy women aged 18–80 ( n = 82) from Regional Diagnostic Center, Novosibirsk, was used as a control group. Serum of women with tumors of the reproductive system and ascites fluids of women with ovarian tumors (aged 18–80 years), before operation were used for assay of procathepsin B, cysteine protease inhibitors cystatins B and C. Serum procathepsin B concentration was measured by ELISA commercial kit for human (RD cystatin C using BioVendor commercial kits (Czechia), cystatin B – with help of ELISA kits for human (USCN Life Science Inc., Wuhan, China). Common biomarker of ovarian cancer, CA-125, was assayed by using a commercial kit (Vector, Koltsovo, Novosibirsk Region, Russia). Statistical analysis performed by one a way ANOVA Statistic 12, program with help of Kruskall–Wallis test and the Mann–Whitney U test used to assess differences in procathepsin B or cystatins B and C levels between patient groups. Statistical analysis was performed with the soft package Statistics 12, with the level of significance being set at Results In serum of patients with endometrial cancer, ovarian cancer, mammalian cancer – significant increases in serum procathepsin B ( p p p p = 0.005 vs. healthy controls) was shown without any changes in serum level of procathepsin B, cystatins B and C. Concentrations procathepsin B and Cystatin B in serum and ascites of patients with ovarian tumor was used to assess differences in procathepsin B or cystatins B and C levels between groups. Serum procathepsin B( p p p p p > 0.1). Conclusion One can conclude that serum procathepsin B and endogenous inhibitors of cysteine proteases cystatin B are prospective tumor biomarkers in reproductive system tumors. Procathepsin B and cystatin B seem to be important in differential diagnosis of ovarian cancer and benign ovarian tumors. Procathepsin B and cystatin B are involved in breast and endometrial cancer that warrants further investigation of their role in cancer.

P43: Endogenous inhibitors of cysteine proteases cystatin C and cystatin SN in biological fluids of patients with intraocular melanoma as possible biomarkers and therapy targets

Cystatins are natural endogenous inhibitors of cysteine proteases universally involved into development of different tumors. Tumor growth and metastazing include increased consumption of these inhibitors, followed by decreased level of cystatins and dysregulation of proteases/protease inhibitors system. However, the biological role of individual cystatins is still not clear. The most known cystatin C was shown to relate to some types of tumor development. Cystatins include a group of intracellular cystatins (belonging to type 1) and extracellular cystatins (type 2), among them cystatins C, D, E/M, F, G, S, SN and SA, which functions were not studied enough. It was suggested that cystatin SN was responsible in regulation of tumor growth locally. To investigate cystatin C and cystatin SN concentrations in biological fluids of patients with intraocular melanoma as tumor biomarkers and possible therapy targets. The patients with melanoma chorioidea (57 patients; among them woman 36, men 21; aged from 28 to 80, of middle age of 56.6 ± 2.4 years) were under investigation. In all cases the pathological process involved one eye. The control group consisted of 37 healthy persons (volunteers), medical personnel in clinic and students, aged from 20 to 49 years; the middle age 31 ± 4.1 years); 7 patients with age-related cataract aged from 57 to 80 (middle age 71 ± 2.6 years; man 3, woman 4). The biological fluids studied: tears, intraocular fluid (obtained during operation) and blood serum. In all cases investigation was made according to informed agreement of patients and control group members. Cystatins concentration was measured by ELISA kits: for Cystatin C (BioVendor, Chechia) and for cystatin SN with help of Human Cystatin SN (CST1) Elisa Kit Cusabio, China. Statistical analysis was made by non-parametric statistic test of Kruskal–Wallis, for correlations – Spearman test. The difference between groups studied was considered significant p < 0.05. Increased serum level of cystatin C was revealed in patients with melanoma chorioides (1023.5 ± 78.9 ng/ml, p = 0.019) compared with the control (809.9 ± 146.8 ng/ml). In tears of patients with melanoma chorioidea, cystatin C concentration (441.7 ± 14.5 ng/ml) had a tendency to increase as compared to the data obtained in tears of the control group (287.5 ± 20.01 ng/ml) as well as the cystatin C level of intraocular fluid of these patients vs the control group (p < 0.1). Cystatin SN concentration in serum of patients with melanoma chorioidea (1.45 ± 0.30 ng/ml) was lower vs the control group (3.12 ± 0.32 ng/ml, p = 0.0038), as well as Cystatin SN level in intraocular fluid (1.43 ± 0.10 ng/ml) vs the control (2.60 ± 0.60 ng/ml, p < 0.05). There was no difference in cystatin SN concentration in tears of patients and control group. In serum of control (healthy) group, cystatin C concentration is significantly higher than cystatin SN level in serum, tears and intraocular fluid. The reverse correlation was revealed between the level of these inhibitors in serum, that is suggested their possible interaction. In melanoma patients the reciprocal changes in cystatin C and cystatin SN were shown: increased cystatin C and decreased cystatin SN level in all biological fluids studied. On the basis of cystatins distribution in biological fluids of patients one can suggest their involvement in pathological process as system reaction of organism on tumor development.

Efficient expression, purification and characterization of native human cystatin C in Escherichia coli periplasm

Human cystatin C (HCC), encoded by cystatin 3 gene, is a 13.3kDa endogenous cysteine proteinase inhibitor and an important biomarker of renal function. However, expressing recombinant cystatin C is difficult because of low yield and inclusion bodies in Escherichia coli (E. coli). In this study, we cloned HCC gene into pET-22b vector containing PelB leader signal sequence, which could direct the protein to the bacterial periplasm. Large amounts of soluble HCC could be efficiently expressed in the bacterial periplasm at 16°C with 0.1mM IPTG induction. The recombinant HCC was isolated in high purity by cation exchange chromatography and gel filtration chromatography. Furthermore, the HCC was characterized by circular dichroism (CD) and dynamic light scattering (DLS), and displayed biological activity against papain. Here, we provide a method to produce large amounts of soluble mature HCC in E. coli.

Modulation of endogenous Cysteine Protease Inhibitor (ICP) 1 expression in Entamoeba histolytica affects amoebic adhesion to Extracellular Matrix proteins

Entamoeba histolytica is an enteric tissue-invading protozoan parasite that causes amoebic colitis and occasionally liver abscess in humans. During tissue invasion, amoebic adhesion to host components is an important event for host cell death leading to successful invasion and infection. Among amoebic virulence factors, Gal/GalNAc lectin is known to be major adhesion factor to host cells. In this study, we investigated the role of amoebic secreted CP (Cysteine Proteases) in amoebic adhesion to extracellular matrix (ECM) protein using CP inhibitor and E. histolytica strains in which the endogenous inhibitor of cysteine protease (ICP) 1 gene was overexpressed (ICP1+) or repressed by antisense small RNA-mediated gene silencing (ICP1−). We found that pretreatment of wild-type amoebae with CP inhibitor E64, or thiol-group modifiers such as diamide and N-Ethylmaleimide resulted in a significant decrease in adhesion to laminin and collagen ECM proteins. Furthermore, ICP1+ strain, with a reduction of secreted CP activity, exhibited reduced ability by 40% to adhere to laminin. In contrast, ICP1− strain, with a 1.9-fold increase of secreted CP activity, showed a two-fold increase in amoebic adherence to laminin compared to the control strain. In addition, total amount of secreted CP5 was decreased in ICP1+ amoeba. Conversely, total amount of secreted CP1 and mature-form CP5 were increased in ICP1− amoeba. We also found that ICP1 was secreted into extracellular milieu. These results suggest that secreted CP activity by E. histolytica may be an important factor affecting adhesion to host proteins, and regulation of CP secretion by ICP plays a major role in pathogenesis. This study provides insight into the CP-mediated tissue pathogenesis in amoeba-invaded lesions during human amoebiasis.