Self-assembly of influenza hemagglutinin: studies of ectodomain aggregation by in situ atomic force microscopy.
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We have used in situ tapping mode atomic force microscopy (AFM) to study the structural morphology of two fragments of the influenza hemagglutinin protein bound to supported bilayers. The two proteins that we studied are the bromelain-cleaved hemagglutinin (BHA), corresponding to the full ectodomain of the hemagglutinin protein, and FHA2, the 127 amino acid N-terminal fragment of the HA2 subunit of the hemagglutinin protein. While BHA is water soluble at neutral pH and is known to bind to membranes via specific interactions with a viral receptor, FHA2 can only be solubilized in water with an appropriate detergent. Furthermore, FHA2 is known to readily bind to membranes at neutral pH in the absence of a receptor. Our in situ AFM studies demonstrated that, when bound to supported bilayers at neutral pH, both these proteins are self-assembled as single trimeric molecules. In situ acidification resulted in further lateral association of the FHA2 without a large perturbation of the bilayer. In contrast, BHA remained largely unaffected by acidification, except in areas of exposed mica where it is aggregated. Remarkably, these results are consistent with previous observations that FHA2 promotes membrane fusion while BHA only induces liposome leakage at low pH. The results presented here are the first example of in situ imaging of the ectodomain of a viral envelope protein allowing characterization of the real-time self-assembly of a membrane fusion protein. (+info)
Proteolytic inhibition of Salmonella enterica serovar typhimurium-induced activation of the mitogen-activated protein kinases ERK and JNK in cultured human intestinal cells.
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Bromelain, a mixture of cysteine proteases from pineapple stems, blocks signaling by the mitogen-activated protein (MAP) kinases extracellular regulated kinase 1 (ERK-1) and ERK-2, inhibits inflammation, and protects against enterotoxigenic Escherichia coli infection. In this study, we examined the effect of bromelain on Salmonella enterica serovar Typhimurium infection, since an important feature of its pathogenesis is its ability to induce activation of ERK-1 and ERK-2, which leads to internalization of bacteria and induction of inflammatory responses. Our results show that bromelain dose dependently blocks serovar Typhimurium-induced ERK-1, ERK-2, and c-Jun NH(2)-terminal kinase (JNK) activation in Caco-2 cells. Bromelain also blocked signaling induced by carbachol and anisomycin, pharmacological MAP kinase agonists. Despite bromelain inhibition of serovar Typhimurium-induced MAP kinase signaling, it did not prevent subsequent invasion of the Caco-2 cells by serovar Typhimurium or alter serovar Typhimurium -induced decreases in resistance across Caco-2 monolayers. Surprisingly, bromelain also did not block serovar Typhimurium-induced interleukin-8 (IL-8) secretion but synergized with serovar Typhimurium to enhance IL-8 production. We also found that serovar Typhimurium does not induce ERK phosphorylation in Caco-2 cells in the absence of serum but that serovar Typhimurium-induced invasion and decreases in monolayer resistance are unaffected. Collectively, these data indicate that serovar Typhimurium-induced invasion of Caco-2 cells, changes in the resistance of epithelial cell monolayers, and IL-8 production can occur independently of the ERK and JNK signaling pathways. Data also confirm that bromelain is a novel inhibitor of MAP kinase signaling pathways and suggest a novel role for proteases as inhibitors of signal transduction pathways in intestinal epithelial cells. (+info)
Bromelain reversibly inhibits invasive properties of glioma cells.
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Bromelain is an aqueous extract from pineapple stem that contains proteinases and exhibits pleiotropic therapeutic effects, i.e., antiedematous, antiinflammatory, antimetastatic, antithrombotic, and fibrinolytic activities. In this study, we tested bromelain's effects on glioma cells to assess whether bromelain could be a potential contributor to new antiinvasive strategies for gliomas. Several complementary assays demonstrated that bromelain significantly and reversibly reduced glioma cell adhesion, migration, and invasion without affecting cell viability, even after treatment periods extending over several months. Immunohistochemistry and immunoblotting experiments demonstrated that alpha3 and beta1 integrin subunits and hyaluronan receptor CD44 protein levels were reduced within 24 hours of bromelain treatment. These effects were not reflected at the RNA level because RNA profiling did not show any significant effects on gene expression. Interestingly, metabolic labelling with 35-S methionine demonstrated that de novo protein synthesis was greatly attenuated by bromelain, in a reversible manner. By using a transactivating signaling assay, we found that CRE-mediated signaling processes were suppressed. These results indicate that bromelain exerts its antiinvasive effects by proteolysis, signaling cascades, and translational attenuation. (+info)
4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole as a reactivity probe for the investigation of the thiol proteinases. evidence that ficin and bromelain may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain.
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1. 4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (Nbd chloride) was used as a reactivity probe to characterize the active centres of papin (EC 3.4.22.2), ficin (EC 3.4.22.3) and bromelain (EC 3.4.22.4). 2. In the pH range 0-8 Nbd chloride probably exists mainly as a monocation, possibly with the proton located on N-1 of the oxadiazole ring. 3. Spectroscopic evidence is presented for the intermediacy of Meisenheimer-type adducts in the reaction of Nbd chloride with nucleophiles. 4. The pH-dependence of the second-order rate constants (k) of the reactions of the three enzymes with Nbd chloride was determined at 25 degrees C, I = 0.1 mol/litre in 6.7% (v/v) ethanol in the pH range 2.5-5, where, at least for papain and ficin, the reactions occur specifically with their active-centre thiol groups. The pH-k profile for the papain reaction is bell-shaped (pKaI = 3.24, pKaII = 3.44 and k = 86M(-1)-s(-1), whereas that for ficin is sigmoidal (pKa = 3.6, k = 0.36M(-1)-s(-1), the rate increasing with increasing pH. The profile for the bromelain reaction appears to resemble that for the ficin reaction, but is complicated by amino-group labelling. 5. The bell-shaped profile of the papain reaction is considered to arise from the reaction of the thiolate ion of cysteine-25, maintained in acidic media by interaction with the side chain of histidine-159, with the Nbd chloride monocation hydrogen-bonded at its nitro group to the un-ionized form of the carboxyl group of aspartic acid-158. The lack of acid catalysis in the corresponding reactions of ficin and probably of bromelain suggests that these enzymes may lack carboxyl groups conformationally equivalent to that of aspartic acid-158 of papain. The possible consequences of this for the catalytic sites of these enzymes is discussed. (+info)
Characterization of a partially folded intermediate of stem bromelain at low pH.
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Equilibrium studies on the acid included denaturation of stem bromelain (EC 3.4.22.32) were performed by CD spectroscopy, fluorescence emission spectroscopy and binding of the hydrophobic dye, 1-anilino 8-naphthalene sulfonic acid (ANS). At pH 2.0, stem bromelain lacks a well defined tertiary structure as seen by fluorescence and near-UV CD spectra. Far-UV CD spectra show retention of some native like secondary structure at pH 2.0. The mean residue ellipticities at 208 nm plotted against pH showed a transition around pH 4.5 with loss of secondary structure leading to the formation of an acid-unfolded state. With further decrease in pH, this unfolded state regains most of its secondary structure. At pH 2.0, stem bromelain exists as a partially folded intermediate containing about 42.2% of the native state secondary structure Enhanced binding of ANS was observed in this state compared to the native folded state at neutral pH or completely unfolded state in the presence of 6 m GdnHCl indicating the exposure of hydrophobic regions on the protein molecule. Acrylamide quenching of the intrinsic tryptophan residues in the protein molecule showed that at pH 2.0 the protein is in an unfolded conformation with more tryptophan residues exposed to the solvent as compared to the native conformation at neutral pH. Interestingly, stem bromelain at pH 0.8 exhibits some characteristics of a molten globule, such as an enhanced ability to bind the fluorescent probe as well as considerable retention of secondary structure. All the above data taken together suggest the existence of a partially folded intermediate state under low pH conditions. (+info)
Isolation, molecular and functional properties of the C-terminal domain of colicin A.
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Partial proteolytic digestion of colicin A with bromelain allowed the isolation of a 20-kd fragment. This fragment has been purified to homogeneity and its molecular properties have been studied. The sequence of the 54 N-terminal amino acid residues has been determined by automated Edman degradation. This sequence is identical to that of the predicted amino acid sequence of the 20-kd C-terminal part of the colicin A polypeptide deduced from the nucleotide sequence of the caa gene. This polypeptide can produce channels in phospholipid planar bilayers of the same size as those formed by colicin A. However, the voltage-dependence for opening and closing was drastically altered in the peptide fragment channels. The latter, in contrast to colicin A channels, remained open over a wide range of voltage. Large negative potentials were required to close the peptide fragment channels although opening took place in the same voltage range as for colicin A ionic pores. (+info)
Lysolipids do not inhibit influenza virus fusion by interaction with hemagglutinin.
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The interaction of a spin-labeled lysophosphatidylcholine analog with intact and bromelain-treated influenza viruses as well as with the bromelain-solubilized hemagglutinin ectodomain has been studied. The inhibition of fusion of influenza viruses with erythrocytes by the lysophosphatidylcholine analog was similar to that observed for non-labeled lysophosphatidylcholine. Only a weak interaction of the lysophosphatidylcholine analog with the hemagglutinin ectodomain was observed even upon triggering the conformational change of the ectodomain at a low pH. A significant interaction of spin-labeled lysophosphatidylcholine with the hemagglutinin ectodomain of intact viruses was observed neither at neutral nor at low pH, whereas a strong interaction of the lipid analog with the viral lipid bilayer was evident. We suggest that the high number of lipid binding sites of the virus bilayer and their affinity compete efficiently with binding sites of the hemagglutinin ectodomain. We conclude that the inhibition of influenza virus fusion by lysolipids is not mediated by binding to the hemagglutinin ectodomain, preventing its interaction with the target membrane. The results unambiguously argue for an inhibition mechanism based on the action of lysolipid inserted into the lipid bilayer. (+info)
Thermal denaturation of influenza virus and its relationship to membrane fusion.
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The X-31 strain of influenza virus was studied by differential scanning calorimetry (DSC), CD and SDS/PAGE analysis as a function of both temperature and pH. A bromelain-treated virus was also studied by these methods. The major transition observed in the intact virus was a result of the denaturation of the haemagglutinin (HA) protein. At pH 7.4, this transition was similar in the intact virus and the isolated HA, but was absent in the bromelain-treated virus. However, at pH 5 the denaturation temperature and enthalpy were both higher for HA in the virus than in the isolated protein, indicating that HA interacts with other molecular components in the intact virus. The transition observed by DSC occurs at a higher temperature than does the thermal transition observed by CD. The temperature of the CD transition coincides with the temperature at which the fusogenicity of the virus increases, and probably corresponds to the formation of an extended coiled-coil conformation. Analysis by SDS/PAGE at neutral pH under non-reducing conditions demonstrates a selective loss of the HA protein trimer, resulting in the formation of aggregates in the range of temperatures of 55 to 70 degrees C. In contrast, at acidic pH, the HA protein is largely in the monomeric form at 25 degrees C, and there is little change with temperature. There is thus a weakening of the quaternary structure of HA at acidic pH prior to heating. At the temperature at which the virus exhibits an increased fusogenicity at neutral pH, there is a loss of secondary structure and a beginning of a destabilization of the trimeric form of HA. This temperature is lower than that required for the major endothermic peak observed in DSC experiments. The results demonstrate that there is no kinetically trapped high-energy form of HA at neutral pH. (+info)