Development of a homogeneous time-resolved fluorescence leukotriene B4 assay for determining the activity of leukotriene A4 hydrolase

Leukotriene A4 (LTA4) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B4 (LTB4), a potent lipid chemotactic agent and proinflammatory mediator. LTB4 has been implicated in the pathogenesis of various acute and chronic inflammatory diseases, and thus LTA4 hydrolase is regarded as an attractive therapeutic target for anti-inflammation. To facilitate identification and optimization of LTA4 hydrolase inhibitors, a specific and efficient assay to quantify LTB4 is essential. This article describes the development of a novel 384-well homogeneous time-resolved fluorescence assay for LTB4 (LTB4 HTRF assay) and its application to establish an HTRF-based LTA4 hydrolase assay for lead optimization. This LTB4 HTRF assay is based on competitive inhibition and was established by optimizing the reagent concentration, buffer composition, incubation time, and assay miniaturization. The optimized assay is sensitive, selective, and robust, with a Z' factor of 0.89 and a subnanomolar detection limit for LTB4. By coupling this LTB4 HTRF assay to the LTA4 hydrolase reaction, an HTRF-based LTA4 hydrolase assay was established and validated. Using a test set of 16 LTA4 hydrolase inhibitors, a good correlation was found between the IC50 values obtained using LTB4 HTRF with those determined using the LTB enzyme-linked immunoassay (R = 0.84). The HTRF-based LTA4 hydrolase assay was shown to be an efficient and suitable assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA4 hydrolase.     

COMMD1-mediated ubiquitination regulates CFTR trafficking

The CFTR (cystic fibrosis transmembrane conductance regulator) protein is a large polytopic protein whose biogenesis is inefficient. To better understand the regulation of CFTR processing and trafficking, we conducted a genetic screen that identified COMMD1 as a new CFTR partner. COMMD1 is a protein associated with multiple cellular pathways, including the regulation of hepatic copper excretion, sodium uptake through interaction with ENaC (epithelial sodium channel) and NF-kappaB signaling. In this study, we show that COMMD1 interacts with CFTR in cells expressing both proteins endogenously. This interaction promotes CFTR cell surface expression as assessed by biotinylation experiments in heterologously expressing cells through regulation of CFTR ubiquitination. In summary, our data demonstrate that CFTR is protected from ubiquitination by COMMD1, which sustains CFTR expression at the plasma membrane. Thus, increasing COMMD1 expression may provide an approach to simultaneously inhibit ENaC absorption and enhance CFTR trafficking, two major issues in cystic fibrosis.     

Cellulase immobilization on magnetic nanoparticles encapsulated in polymer nanospheres

Immobilization of cellulases on magnetic nanoparticles, especially magnetite nanoparticles, has been the main approach studied to make this enzyme, economically and industrially, more attractive. However, magnetite nanoparticles tend to agglomerate, are very reactive and easily oxidized in air, which has strong impact on their useful life. Thus, it is very important to provide proper surface coating to avoid the mentioned problems. This study aimed to investigate the immobilization of cellulase on magnetic nanoparticles encapsulated in polymeric nanospheres. The support was characterized in terms of morphology, average diameter, magnetic behavior and thermal decomposition analyses. The polymer nanospheres containing encapsulated magnetic nanoparticles showed superparamagnetic behavior and intensity average diameter about 150 nm. Immobilized cellulase exhibited broader temperature stability than in the free form and great reusability capacity, 69% of the initial enzyme activity was maintained after eight cycles of use. The magnetic support showed potential for cellulase immobilization and allowed fast and easy biocatalyst recovery through a single magnet.

     

PURIFIED DIPHTHERIA ANTITOXIN IN THE ULTRACENTRIFUGE AND IN THE ELECTROPHORESIS APPARATUS

Ultracentrifugation studies of diphtheria antitoxin showed that: 1. Purified antitoxin of high activity obtained from horse plasma without enzymatic treatment has exactly the same sedimentation constant as the globulin fraction obtained in a similar way from normal horse plasma s ₂₀ ^water = 6.9 x 10^–13. 2. Purified antitoxin obtained with trypsin digestion of the toxin-antitoxin complex has a sedimentation constant of s ₂₀ ^water = 5.5 ± 0.1 x 10^–13, a diffusion constant of D ₂₀ ^water = 5.7₆ x 10^–7, and a molecular weight of about 90,000. Electrophoresis experiments demonstrated that: 1. The trypsin-purified antitoxin has an isoelectric point not far from pH 7.0. 2. The reversible spreading noticed at about pH 7.3 cannot be attributed to heterogeneous preparation. 3. The large increase in the γ-globulin fraction occurring during immunization consists either of antitoxin of various degrees of activity or of some inert protein in addition to the antitoxin.     

Functional and Evolutionary Analysis of the CASPARIAN STRIP MEMBRANE DOMAIN PROTEIN Family

CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASPs) are four-membrane-span proteins that mediate the deposition of Casparian strips in the endodermis by recruiting the lignin polymerization machinery. CASPs show high stability in their membrane domain, which presents all the hallmarks of a membrane scaffold. Here, we characterized the large family of CASP-like (CASPL) proteins. CASPLs were found in all major divisions of land plants as well as in green algae; homologs outside of the plant kingdom were identified as members of the MARVEL protein family. When ectopically expressed in the endodermis, most CASPLs were able to integrate the CASP membrane domain, which suggests that CASPLs share with CASPs the propensity to form transmembrane scaffolds. Extracellular loops are not necessary for generating the scaffold, since CASP1 was still able to localize correctly when either one of the extracellular loops was deleted. The CASP first extracellular loop was found conserved in euphyllophytes but absent in plants lacking Casparian strips, an observation that may contribute to the study of Casparian strip and root evolution. In Arabidopsis (Arabidopsis thaliana), CASPL showed specific expression in a variety of cell types, such as trichomes, abscission zone cells, peripheral root cap cells, and xylem pole pericycle cells.Biological membranes are conceptually simple structures that may be generated in vitro according to simple physicochemical principles. In vivo, however, membranes are highly complex and host a plethora of proteins that mediate the transfer of molecules and communication across the membrane. Proteins may be trapped in membrane by their transmembrane domains, anchored by lipid tails, or attach to membrane-integral proteins. A further level of complexity is seen when membrane proteins are not equally distributed but occupy only a limited fraction of the available surface (i.e. when they are polarly localized or when they form small membrane subdomains in the micrometer range). The question of how membrane proteins are retained locally and prevented from diffusing freely is of high importance to cell biology. Polarly localized proteins may be retained in their respective domains by membrane fences; in such a situation, polarly localized proteins are mobile in their domains but cannot diffuse through tightly packed scaffold proteins forming a molecular fence within the membrane. Membrane fences delimiting polar domains have been described in different organisms. For example, diffusion between membrane compartments is prevented in budding yeast (Saccharomyces cerevisiae) at the level of the bud neck (Barral et al., 2000; Takizawa et al., 2000); in ciliated vertebrate cells, between ciliary and periciliary membranes (Hu et al., 2010); in epithelial cells, between apical and basolateral membranes (van Meer and Simons, 1986); in neurons, between axon and soma (Kobayashi et al., 1992; Winckler et al., 1999; Nakada et al., 2003); and in spermatozoa, at the level of the annulus (Myles et al., 1984; Nehme et al., 1993). The existence of membrane scaffolds that prevent free protein diffusion has also been described in bacteria (Baldi and Barral, 2012; Schlimpert et al., 2012). In plants, we have shown the existence of a strict membrane fence in the root endodermis, where a median domain splits the cell in two lateral halves occupied by different sets of proteins (Alassimone et al., 2010). The situation in the plant endodermis is analogous to the separation of animal epithelia into apical and basolateral domains; indeed, a parallel between epithelia and endodermal cells has been drawn, despite the different origin of multicellularity in plants and animals (Grebe, 2011).The protein complexes responsible for the formation of membrane fences have been identified. Septins are a family of proteins able to oligomerize and form filaments (Saarikangas and Barral, 2011); their role in the formation of membrane fences has been demonstrated in several organisms and cellular situations, including the yeast bud neck (Barral et al., 2000; Takizawa et al., 2000), animal cilia (Hu et al., 2010), and mammalian spermatozoa (Ihara et al., 2005; Kissel et al., 2005; Kwitny et al., 2010). At the axonal initial segment of neurons, AnkyrinG is necessary to establish and maintain a membrane scaffold where different membrane proteins are immobilized and stabilized (Hedstrom et al., 2008; Sobotzik et al., 2009). In Caulobacter crescentus, the stalk protein Stp forms a complex that prevents diffusion between the cell body and stalk and between stalk compartments. Claudins and occludin are the main components of epithelial tight junctions (Furuse et al., 1993, 1998). Occludins are four-membrane-span proteins and belong to the MARVEL protein family (Sánchez-Pulido et al., 2002), as do Tricellulin and MARVELD3, which are also tight junction-associated proteins (Furuse et al., 1993; Ikenouchi et al., 2005; Steed et al., 2009).In Arabidopsis (Arabidopsis thaliana), our group identified a family of proteins that form a membrane fence in the endodermis (Roppolo et al., 2011). These CASPARIAN STRIP MEMBRANE DOMAIN PROTEINS (CASP1 to CASP5) are four-transmembrane proteins that form a median domain referred to as the Casparian strip membrane domain (CSD). CASPs are initially targeted to the whole plasma membrane, then they are quickly removed from lateral plasma membranes and remain localized exclusively at the CSD; there, they show an extremely low turnover, although they are eventually removed (Roppolo et al., 2011). The membrane proteins NOD26-LIKE INTRINSIC PROTEIN5;1 and BORON TRANSPORTER1 are restricted from diffusing through the CSD and remain polarly localized in the outer and inner lateral membranes, respectively; a fluorescent lipophilic molecule, when integrated in the outer endodermal membrane, was blocked at the level of the CSD and could not diffuse into the inner membrane (Roppolo et al., 2011). Besides making a plasma membrane diffusion barrier, CASPs have an important role in directing the modification of the cell wall juxtaposing their membrane domain: by interacting with secreted peroxidases, they mediate the deposition of lignin and the building up of the Casparian strips (Roppolo et al., 2011; Naseer et al., 2012; Lee et al., 2013). The two CASP activities, making membrane scaffolds and directing a modification of the cell wall, can be uncoupled: indeed, (1) formation of the CASP domain is independent from the deposition of lignin, and (2) interaction between CASPs and peroxidases can take place outside the CSD when CASPs are ectopically expressed (Lee et al., 2013).As CASPs are currently the only known proteins forming membrane fences in plants and because of their essential role in directing a local cell wall modification, we were interested in characterizing the repertoire of a large number of CASP-like (CASPL) proteins in the plant kingdom. Our aim was to provide the molecular basis for the discovery of additional membrane domains in plants and for the identification of proteins involved in local cell wall modifications. We extended our phylogenetic analysis outside of the plant kingdom and found conservation between CASPLs and the MARVEL protein family. Conserved residues are located in transmembrane domains, and we provide evidence suggesting that these domains are involved in CASP localization. We explored the potential use of the CASPL module in plants by investigating CASPL expression patterns and their ability to form membrane domains in the endodermis. Moreover, we related the appearance of the Casparian strips in the plant kingdom to the emergence of a CASP-specific signature that was not found in the genomes of plants lacking Casparian strips.     

Leishmanicidal effect of Spiranthera odoratíssima (Rutaceae) and its isolated alkaloid skimmianine occurs by a nitric oxide dependent mechanism

Leishmaniasis is one of the neglected diseases. High cost, systemic toxicity, and diminished efficacy due to development of resistance by the parasites has a negative impact on the current treatment options. Thus, the search for a new, effective and safer anti-leishmanial drug becomes of paramount importance. Compounds derived from natural products may be a better and cheaper source in this regard. This study evaluated the in vitro anti-leishmanial activity of Spiranthera odoratíssima (Rutaceae) fractions and isolated compounds, using promastigote and amastigote forms of different Leishmania species. J774 A.1 macrophage was used as the parasite host cell for the in vitro assays. Evaluations of cytoxicity, nitric oxide (NO), interleukin-10 and in silico analysis were carried out. In vitro experiments showed that the fruit hexanic fraction (Fhf) and its alkaloid skimmianine (Skm) have a significant (P<0·001) effect against L. braziliensis. This anti-L. braziliensis activity of Fhf and Skm was due to increased production of NO and attenuation of IL-10 production in the macrophages at concentrations ranging from 1·6 to 40·0 μg/ml. The in silico assay demonstrated significant interaction between Skm and amino acid residues of NOS2. Skm is thus a promising drug candidate for L. braziliensis due to its potent immunomodulatory activity.     

Anti-inflammatory and redox-protective activities of citronellal

The anti-inflammatory and redox protective effects of the citronellal (CT) were evaluated using in vivo and in vitro tests. Intraperitoneal (i.p.) administration of CT (50, 100, and 200 mg/kg) inhibited (p < 0.05) the carrageenan-induced leukocyte migration to the peritoneal cavity. Additionally, the carrageenan- and arachidonic acid-induced rat hind paw edema was significantly inhibited (p < 0.05) by i.p. administration of 100 and 200 mg/kg of the compound. When the redox activity was evaluated, CT (200 mg/kg) significantly reduced hepatic lipoperoxidation (p < 0.001), as well as oxidation of plasmatic (p < 0.05) and hepatic (p < 0.01) proteins. The results of the present study support the hypothesis that CT possesses anti-inflammatory and redox protective activities. It is suggested that its effects are associated with the inhibition of the enzymes in the arachidonic acid pathway, which prevent cell migration by inhibiting leukotriene production, edema formation and the increase of reactive oxygen species in tissues. Therefore, CT is of potential benefit to manage inflammatory disorders and correlated damages caused by oxidant agents.     

Structural and Physiological Analyses of the Alkanesulphonate-Binding Protein (SsuA) of the Citrus Pathogen Xanthomonas citri

Background

The uptake of sulphur-containing compounds plays a pivotal role in the physiology of bacteria that live in aerobic soils where organosulfur compounds such as sulphonates and sulphate esters represent more than 95% of the available sulphur. Until now, no information has been available on the uptake of sulphonates by bacterial plant pathogens, particularly those of the Xanthomonas genus, which encompasses several pathogenic species. In the present study, we characterised the alkanesulphonate uptake system (Ssu) of Xanthomonas axonopodis pv. citri 306 strain (X. citri), the etiological agent of citrus canker.

Methodology/Principal Findings

A single operon-like gene cluster (ssuEDACB) that encodes both the sulphur uptake system and enzymes involved in desulphurisation was detected in the genomes of X. citri and of the closely related species. We characterised X. citri SsuA protein, a periplasmic alkanesulphonate-binding protein that, together with SsuC and SsuB, defines the alkanesulphonate uptake system. The crystal structure of SsuA bound to MOPS, MES and HEPES, which is herein described for the first time, provides evidence for the importance of a conserved dipole in sulphate group coordination, identifies specific amino acids interacting with the sulphate group and shows the presence of a rather large binding pocket that explains the rather wide range of molecules recognised by the protein. Isolation of an isogenic ssuA-knockout derivative of the X. citri 306 strain showed that disruption of alkanesulphonate uptake affects both xanthan gum production and generation of canker lesions in sweet orange leaves.

Conclusions/Significance

The present study unravels unique structural and functional features of the X. citri SsuA protein and provides the first experimental evidence that an ABC uptake system affects the virulence of this phytopathogen.