Revealing the inhibitory potential of Yersinia enterocolitica on cysteine proteases of the papain family.

Cysteine proteases of the papain family, including mammalian cathepsins, play important physiological roles, however, their excessive activity may contribute to the development of various pathologies. Therefore, cysteine cathepsin inhibitors are being considered as promising drugs to treat cathepsin-driven diseases. Diverse saprophytic and parasitic microbes produce such inhibitors, which target the host's proteases playing pivotal roles in immune responses, thus leading to the survival of microbes within their host. Yersinia enterocolitica is a Gram-negative zoopathogenic coccobacillus, which has developed several mechanisms to evade the host's immune system. Nevertheless, the bacterium has not yet been shown to produce any cysteine protease inhibitors. Here we demonstrate that Y. enterocolitica strains of different bioserotypes and genotypes synthesize papain and human cathepsin L inhibitors, but not bovine cathepsin B inhibitors. By employing fluorimetry and zymography, the cell-surface inhibitors were shown to associate peripherally with the outer membrane, while the inhibitors present in cell-free extracts proved to: interact reversibly with their target enzymes, exhibit thermolability and stability in a range of pH values (5-9), and have high molecular weights. Batch affinity chromatography on papain-agarose resin was then undertaken to isolate putative inhibitors of cysteine proteases from the bacterial extract. The isolated 18 kDa protein was identified by LC-MS/MS as the periplasmic chaperone Skp. The Skp-containing eluate inhibited the activity of cysteine cathepsins produced by human dermal fibroblasts. The homologous Skp protein was also isolated from the extract of Escherichia coli. Our results point to a possible new biological role of the bacterial chaperone Skp.

Up-regulation of Key Glycolysis Proteins in Cancer Development.

In rapid proliferating cancer cells, there is a need for fast ATP and lactate production, therefore cancer cells turn off oxidative phosphorylation and turn on the so called "Warburg effect". This regulating the expression of genes involved in glycolysis. According to many studies, glucose transporter 1, which supplies glucose to the cell, is the most abundantly expressed transporter in cancer cells. Hexokinase 2, is one of four hexokinase isoenzymes, is also another highly expressed enzyme in cancer cells and it functions to enhance the glycolytic rate. The up-regulation of these two proteins has been established as an important factor in promoting development and metastasis in many types of cancer. Furthermore, other enzymes involved in glycolysis pathway such as phosphoglucose isomerase and glyceraldehyde 3-phosphate dehydrogenase, exhibit additional functions in promoting tumor growth in a non-glycolytic way. This review demonstrates the pivotal role of GLUT1, HK2, PGI and GAPDH in cancer development. In particular, we look at how the multifunctional proteins, PGI and GAPDH, affect cancer cell survival. We also present various clinical cancer cases in terms of the overexpression of selected proteins, which may be considered as a therapeutic target.

Different patterns of extracellular proteolytic activity in W303a and BY4742 Saccharomyces cerevisiae strains.

Protease secretion in Saccharomyces cerevisiae cultures is a complex process, important for the application of this organism in the food industry and biotechnology. Previous studies provide rather quantitative data, yielding no information about the number of enzymes involved in proteolysis and their individual biochemical properties. Here we demonstrate that W303a and BY4742 S. cerevisiae strains reveal different patterns of spontaneous and gelatin-induced extracellular proteolytic activity. We applied the gelatin zymography assay to track changes of the proteolytic profile in time, finding the protease secretion dependent on the growth phase and the presence of the protein inducer. Detected enzymes were characterized regarding their substrate specificity, pH tolerance, and susceptibility to inhibitors. In case of the W303a strain, only one type of gelatin-degrading secretory protease (presumably metalloproteinase) was observed. However, the BY4742 strain secreted different proteases of the various catalytic types, depending on the substrate availability. Our study brings the evidence that S. cerevisiae strains secrete several kinds of proteases depending on the presence and type of the substrate. Protein induction may cause not only quantitative but also qualitative changes in the extracellular proteolytic patterns.

Microbial inhibitors of cysteine proteases.

Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted.

Inhibition of cathepsin B activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent toxic isomer in the dioxin-like family. Due to its resistance to metabolic degradation, this ubiquitous environmental pollutant readily accumulates in multiple organs. Cathepsin B is a lysosomal cysteine protease playing an essential role in the intracellular protein turnover. Alterations in its expression, activity, and localization may facilitate the development of many pathologies, including cancer. TCDD, due to its extremely lipophilic nature, may diffuse through biological membranes and affect lysosomal enzymes, including cathepsins. Therefore, in this study we performed two enzymatic assays, spectrofluorimetry and gelatin zymography, in order to evaluate the effect of TCDD on purified bovine cathepsin B. We showed that the dioxin decreases the enzyme's activity in a dose-dependent manner. The reversibility of TCDD-induced inhibition of the protease was also examined, suggesting that TCDD does not bind covalently to the enzyme's active site, acting rather as a reversible inhibitor.

Innate immune properties of selected human neuropeptides against Moraxella catarrhalis and nontypeable Haemophilus influenzae.

BACKGROUND: Considerable evidence supports the concept of active communication between the nervous and immune systems. One class of such communicators are the neuropeptides (NPs). Recent reports have highlighted the antimicrobial activity of neuropeptides, placing them among the integral components of innate immune defense. This study examined the action of four human neuropeptides: calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), substance P (SP) and somatostatin (SOM), which are accessible in the upper respiratory tract, against two human-specific respiratory pathogens. We studied: (i) neuropeptide-mediated direct antibacterial activity exerted against Moraxella catarrhalis and nontypeable Haemophilus influenzae, and (ii) indirect immunomodulatory role of these neuropeptides in the neutrophil-mediated phagocytosis of indicated pathogens. RESULTS: We found that 100 micromolar concentrations of CGRP, NPY, SP, and SOM effectively permeabilized bacterial membranes and showed (except SOM) bactericidal activity against both pathogens. SOM acted only bacteriostatically. However the killing efficacy was dependent on the bactericidal assay used. The rank order of killing NP effect was: NPY ≥ CGRP > SP >> SOM and correlated with their potency to permeabilize bacterial membranes. The killing and permeabilization activity of the analyzed NPs showed significant correlation with several physicochemical properties and amino acid composition of the neuropeptides. M. catarrhalis was more sensitive to neuropeptides than nontypeable H. influenzae.The immunomodulatory bimodal effect of physiological concentrations of CGRP, NPY, and SP on the phagocytic function of human neutrophils against M. catarrhalis and H. influenzae was observed both in the ingestion (pathogen uptake) and reactive oxygen species generation stages. This effect was also dependent on the distinct type of pathogen recognition (opsonic versus nonopsonic). CONCLUSIONS: The present results indicate that neuropeptides such as CGRP, NPY, and SP can effectively participate in the direct and indirect elimination of human-specific respiratory pathogens. Because the studied NPs show both direct and indirect modulating antimicrobial potency, they seem to be important molecules involved in the innate host defense against M. catarrhalis and nontypeable H. influenzae.

[Efflux-mediated antimicrobial multidrug resistance]. / Opornosc wielolekowa zwiazana z aktywnym usuwaniem leków z komórek drobnoustrojów.

Multidrug resistance is a major problem in the treatment of infectious diseases caused by bacteria and fungi. One of the basic mechanisms of resistance is active efflux of distinct drugs from cells. Export of toxic compounds from bacterial cells is mediated by proteins of 5 distinct families: MF, SMR, ABC, RND and MATE. The substrate spectrum of efflux pumps includes antibiotics, chemotherapeutics and detergents. Genes that determine resistance can be located on chromosomes or mobile elements (plasmids, transposons, integrons). The presence of resistance genes on mobile elements enables bacteria to transfer those genes between cells and spread the multidrug resistance phenotype. There are several inhibitors of efflux pumps that are currently in the experimental phase. Proteins that mediate multidrug resistance are also present in fungal cells. They belong mainly to the ABC superfamily of transporters and PDR subfamily. These efflux pumps are widely investigated in Saccharomyces cerevisiae.

The immunogenicity of the liposome-associated outer membrane proteins (OMPs) of Moraxella catarrhalis.

The outer membrane proteins (OMPs) are the most immunogenic and attractive of the Moraxella catarrhalis vaccine antigens that may induce the protective immune response. The aim of this study was to determine the effectiveness of two types of OMP-associated phosphatidylcholine (PC) liposomal formulations (OMPs-PC, PC-OMPs) and of Zwittergent-based proteomicelles (OMPs-Z) in potentiating an anti-OMP systemic immune response in mice. The immunogenicities of the above preparations were evaluated by assessing serum anti-OMP IgG and IgA reactivity in the post-immunized mouse antisera using ELISA and Western blotting. Additionally, the cross-reactivity of the most effective anti-OMP response was determined using heterologous sera from both humans and mice. Both the proteoliposomes and the proteomicelles showed high immunogenic properties and did not elicit any distinct quantitative differences in the antibody titer or qualitative differences in the pattern of the mouse antisera. The post-immunized mouse antisera predominantly recognized a approximately 60-kDa OMP of M. catarrhalis. That protein was also found to be a highly cross-reactive antigen interacting with a panel of pooled mouse antisera produced by immunization either with whole cells or the purified OMPs of heterologous M. catarrhalis strains. Furthermore, normal sera collected from healthy children were found to be preferentially reactive with the 60-kDa OMP. The serum-specific IgG, IgA and IgM were respectively detected via immunoblotting in 90%, 85% and 30% of heterologous human sera. This similar immunogenic effectiveness of both OMP-associated liposomal formulations could contribute to the practical use of such formulations in the future in human vaccination. Moreover, the highly cross-reactive 60-kDa OMP seems to be an important antigenic marker of M. catarrhalis, and, as it is responsible for the induction of an antibody-mediated and long-lasting immune response, studying it may partially aid us in understanding the relatively low degree of pathogenicity of the bacterium in immunocompetent individuals.

Distinct roles of the last transmembrane domain in controlling Arabidopsis K+ channel activity.

The family of voltage-gated potassium channels in plants presumably evolved from a common ancestor and includes both inward-rectifying (K(in)) channels that allow plant cells to accumulate K(+) and outward-rectifying (K(out)) channels that mediate K(+) efflux. Despite their close structural similarities, the activity of K(in) channels is largely independent of K(+) and depends only on the transmembrane voltage, whereas that of K(out) channels responds to the membrane voltage and the prevailing extracellular K(+) concentration. Gating of potassium channels is achieved by structural rearrangements within the last transmembrane domain (S6). Here we investigated the functional equivalence of the S6 helices of the K(in) channel KAT1 and the K(out) channel SKOR by domain-swapping and site-directed mutagenesis. Channel mutants and chimeras were analyzed after expression in Xenopus oocytes. We identified two discrete regions that influence gating differently in both channels, demonstrating a lack of functional complementarity between KAT1 and SKOR. Our findings are supported by molecular models of KAT1 and SKOR in the open and closed states. The role of the S6 segment in gating evolved differently during specialization of the two channel subclasses, posing an obstacle for the transfer of the K(+)-sensor from K(out) to K(in) channels.

Inhibitory activity against papain, a CA1 cysteine peptidase, in Saccharomycetaceae.

A search for new biological sources of cysteine peptidase inhibitors has not only an academic aspect but is of great importance in medicine and biotechnology. The activity of CA1 peptidases can be inhibited by proteins of nine structurally different families. Although these inhibitors are widespread in nature, there is little information on them in yeast and in the kingdom of fungi overall. To gain insight into the endogenous inhibitors of CA1 cysteine peptidases in unicellular fungi, we initiated a study of the extra- and intracellular antipapain activity in yeast. We report here, for the first time, an analysis of the inhibitory activity against papain in the culture medium and the cell-free extract of 16 yeast strains belonging to the Saccharomycetaceae family. The existence of the antipapain activity, likely from protein inhibitors, in all the tested yeast strains has been demonstrated.

Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH.

Interaction of pig muscle lactate dehydrogenase (LDH) with acidic phospholipids is strongly dependent on pH and is most efficient at pH values<6.5. The interaction is ionic strength sensitive and is not observed when bilayer structures are disrupted by detergents. Bilayers made of phosphatidylcholine (PC) do not bind the enzyme. The LDH interaction with mixed composition bilayers phosphatidylserine/phosphatidylcholine (PS/PC) and cardiolipin/phosphatidylcholine (CL/PC) leads to dramatic changes in the specific activity of the enzyme above a threshold of acidic phospholipid concentration likely when a necessary surface charge density is achieved. The threshold is dependent on the kind of phospholipid. Cardiolipin (CL) is much more effective compared to phosphatidylserine, which is explained as an effect of availability of both phosphate groups in a CL molecule for interaction with the enzyme. A requirement of more than one binding point on the enzyme molecule for the modification of the specific activity is postulated and discussed. Changes in CD spectra induced by the presence of CL and PS vesicles evidence modification of the conformational state of the protein molecules. In vivo qualitative as well as quantitative phospholipid composition of membrane binding sites for LDH molecules would be crucial for the yield of the binding and its consequences for the enzyme activity in the conditions of lowered pH.

[Cystatins, thyropins and inhibitors homologous to propeptides of cysteine proteases]. / Cystatyny, tyropiny i inhibitory homologiczne do propeptydów proteaz cysteinowych.

Protein inhibitors of proteolytic enzymes play an important role in regulating the activity of endogenous proteases and in host defense mechanisms against pathogens preventing the deleterious effects of exogenous proteases. In recent years a great interest in protein inhibitors of cysteine proteases has increased due to the extensive growth of knowledge about the contribution of cysteine proteases to pathological processes associated with many human diseases, as well as due to prospects for treatment of these disorders which may arise from the thorough understanding of their inhibitory mechanisms. This paper reviews the most important aspects of three families of cysteine protease inhibitors: cystatins, thyropins and inhibitors homologous to propeptides of cysteine proteases. Special attention is given to structural bases of the interactions between the inhibitors and their target enzymes. The paper presents a general characterization of the families according to the MEROPS classification of protease inhibitors, pointing out new members.

The association of glycolytic enzymes with cellular and model membranes.

This article deals with the binding of glycolytic enzymes with membranous or protein subcellular structures. The representative papers of the last three decades dealing with this matter are reviewed. The studies evidencing the binding of some glycolytic enzymes to insoluble subcellular proteins and membranous structures are presented. It is currently generally accepted that the glycolytic enzymes work in some organisation. Such organisation undoubtedly plays a marked role, although still poorly known, in the regulation processes of glycolysis. From this review, the conclusion emerges that the regulatory ability of the binding of glycolytic enzymes to cellular membranes should be added to the list of well-known mechanisms of post-translational regulation of the glycolytic enzymes. Some of the results presented are the background for the hypothesis that planar phospholipid domains in/on the membrane surface are capable of functioning as binding sites for these enzymes. Such binding can modify the conformation state of the enzymes, which results in changes in their kinetic properties; thus, it may function as a regulator of catalytic activity

Inhibition of cathepsin B activity in human breast cancer tissue by cysteine peptidase inhibitor isolated from human placenta: immunohistochemical and biochemical studies.

Cysteine peptidases and their endogenous inhibitors (CPI) have been shown to be involved in tumor progression and metastasis. Since their activity has been found to be changed in tumor tissue and/or body fluids of cancer patients, the determination of the peptidase/inhibitor levels is considered as a procedure of diagnostic value. Determination of cathepsin B, its precursor and inhibitor activity in homogenates of tumors and control breast tissue samples of patients with invasive ductal and lobular breast carcinoma and with benign breast disease (BBD) was performed using fluorometric assay. Immunohistochemical staining of the breast tissue samples was carried out using polyclonal antibody against cysteine peptidase inhibitor isolated from human placenta. Procathepsin B and cathepsin B were found to be significantly increased and their endogenous inhibitors decreased in homogenates of tumors from patients with breast cancer. A correlation between procathepsin B or cathepsin B activities as well as cysteine peptidase inhibitor activity and the histopathological grading of the tumor was observed. All samples of the tumor tissue showed positive immunostaining with antibody raised against cysteine peptidase inhibitor, while in the control tissue samples the immunostaining was much weaker. Significant difference observed between the activities of cathepsin B and/or its precursor in malignant and benign tumors might serve as a useful clinical indicator in discrimination between benign and invasive tumors.

Activity of cysteine proteases and their inhibitors in the lymph nodes of larynx cancer patients.

BACKGROUND: Cysteine endopeptidases and their inhibitors play an important role in the process of carcinogenesis. Positive correlation has been found between tumor invasiveness, its metastatic potential and the secretion of cysteine endopeptidases. Cysteine protease inhibitory activity is also altered in malignant tumors and various body fluids of patients with cancer. MATERIAL/METHODS: Total cysteine endopeptidase activity and cysteine proteinase inhibitory activity were measured in homogenates of cervical lymph node tissue surgically obtained from the larynx of cancer patients. The tissue samples were histologically examined, and each was divided into two parts: positive (PCN), with mostly cancer cells, and negative (NCN), with no cancer cells. RESULTS: In the PCNs, the levels of the assayed enzymes and their inhibitors were significantly higher than in the NCNs. The mean values of cysteine protease activity were 2.70I2.29 and 1.59I1.28 for PCNs and NCNs, respectively (p<0.005). The mean values of cysteine protease inhibitors were 9.1I8.6 and 6.1I6.3 for PCNs and NCNs, respectively (p<0.02). An altered protease-inhibitor activity ratio was also found in PCN samples compared to NCNs. CONCLUSIONS: These data suggest increased activity of cysteine peptidases and their inhibitors in the case of secondary tumor tissue. The cancer cells metastasized to lymph node tissue produce some alteration in balance between cysteine protease activity and the endogenous inhibitors of the proteases.

The role of lipid phase structure in the interaction of lactate dehydrogenase with phosphatidylserine. Activity studies.

Lactate dehydrogenase is one of the enzymes of the glycolytic path. It has been shown to be able to bind in vitro to cellular membranes. The presence of anionic phospholipids induces changes in the catalytic properties of the enzyme similar to those found when the enzyme is bound to natural membranes. In this study, a nonionic detergent (Tween 20), at concentrations not affecting the catalytic activity of LDH, was used to study the role of the lipid supra-molecular structure in the interaction between pig skeletal muscle lactate dehydrogenase and phosphatidylserine. Tween 20 changes the equilibrium of concentrations between the lipid supra-molecular forms. The detergent at the used concentration values did not alter the activity of the enzyme when it was used on its own, but did diminish the level of inhibition induced by the studied phospholipid. The obtained results showed that the interaction is reversible and that the bilayer structure of the lipid is essential for the inhibition.

Further evidence for the importance of lipid bilayers in the interaction between lactate dehydrogenase and phosphatidylserine.

Lactate dehydrogenase (LDH) is one of the glycolytic enzymes, which have been proved to have the capability to reverse non-specific adsorption on cellular membranous structures in vitro, as well as on the structural proteins of the contractile system of muscle cells. It has been suggested that this binding may play a physiological role, as it alters the enzyme's kinetic properties. Our previous studies on this enzyme showed that its interaction with some anionic phospholipids reveals similar characteristics and similar effect on the activity of the enzyme to those which had been observed for the interaction with membranous structures. Disruption of the lipid bilayers by nonionic detergent (Tween 20) restored the enzyme activity inhibited by the presence of phosphatidylserine (PS) liposomes. In this study, we used the measurement of enzyme tryptophanyl fluorescence spectra to monitor the interaction and possible changes in the enzyme conformation. The investigation provided further evidence of the importance of the bilayer structure in this interaction. Similarly to the effect on the activity of the enzyme, the addition of Tween 20 diminishes the quenching of the LDH tryptophanyl fluorescence, and finally completely restores the fluorescence.