Spectroscopic study of the activation and oligomerization of the channel-forming toxin aerolysin: identification of the site of proteolytic activation.

The channel-forming protein aerolysin is secreted as a protoxin which can be activated by proteolytic removal of a C-terminal peptide. The activation and subsequent oligomerization of aerolysin were studied using a variety of spectroscopic techniques. Mass spectrometric determination of the molecular weights of proaerolysin and aerolysin permitted identification of the sites at which the protoxin is processed by trypsin and chymotrypsin. The results of far- and near-UV circular dichroism measurements indicated that processing with trypsin does not lead to major changes in secondary or tertiary structure of the protein. An increase in tryptophan fluorescence intensity and a small red shift in the maximum emission wavelength of tryptophans could be observed, suggesting that there is a change in the environment of some of the tryptophans. There was also a dramatic increase in the binding of the hydrophobic fluorescent probe 1-anilino-8-naphthalenesulfonate during activation, leading us to conclude that a hydrophobic region in the protein is exposed by trypsin treatment. Using measurements of light scattering, various parameters influencing oligomerisation of trypsin-activated aerolysin were determined. Oligomerization rates were found to increase with the concentration of aerolysin, whereas they decreased with increasing ionic strength.     

A Pore-forming Toxin Interacts with a GPI-anchored Protein and Causes Vacuolation of the Endoplasmic Reticulum

In this paper, we have investigated the effects of the pore-forming toxin aerolysin, produced by Aeromonas hydrophila, on mammalian cells. Our data indicate that the protoxin binds to an 80-kD glycosyl-phosphatidylinositol (GPI)-anchored protein on BHK cells, and that the bound toxin is associated with specialized plasma membrane domains, described as detergent-insoluble microdomains, or cholesterol-glycolipid “rafts.” We show that the protoxin is then processed to its mature form by host cell proteases. We propose that the preferential association of the toxin with rafts, through binding to GPI-anchored proteins, is likely to increase the local toxin concentration and thereby promote oligomerization, a step that it is a prerequisite for channel formation. We show that channel formation does not lead to disruption of the plasma membrane but to the selective permeabilization to small ions such as potassium, which causes plasma membrane depolarization. Next we studied the consequences of channel formation on the organization and dynamics of intracellular membranes. Strikingly, we found that the toxin causes dramatic vacuolation of the ER, but does not affect other intracellular compartments. Concomitantly we find that the COPI coat is released from biosynthetic membranes and that biosynthetic transport of newly synthesized transmembrane G protein of vesicular stomatitis virus is inhibited. Our data indicate that binding of proaerolysin to GPI-anchored proteins and processing of the toxin lead to oligomerization and channel formation in the plasma membrane, which in turn causes selective disorganization of early biosynthetic membrane dynamics.     

Activation of the hole-forming toxin aerolysin by extracellular processing.

A precursor-product relationship between aerolysin and a protein with a higher molecular weight was observed in culture supernatants of Aeromonas hydrophila. The larger protein was isolated by ammonium sulfate precipitation and ion-exchange and hydroxyapatite chromatography and compared with purified aerolysin. It was at least 250 times less hemolytic than aerolysin. Both proteins had the same amino acid sequence at the amino terminus. Cyanogen bromide fragments obtained from the two were identical except that each protein contained one unique fragment, and the fragment from the larger protein was 2,500 daltons larger than the fragment obtained from aerolysin. Treatment with trypsin or with an extracellular Aeromonas protease resulted in rapid conversion of the larger protein to a form corresponding in molecular weight and activity to aerolysin. The results indicate that aerolysin is exported to the culture supernatant as a protoxin which is later activated by proteolytic removal of a peptide from the C terminus.     

Lipids favoring inverted phase enhance the ability of aerolysin to permeabilize liposome bilayers

Channel formation by the bacterial toxin aerolysin follows oligomerization of the protein to produce heptamers that are capable of inserting into lipid bilayers. How insertion occurs is not understood, not only for aerolysin but also for other proteins that can penetrate membranes. We have studied aerolysin channel formation by measuring dye leakage from large unilamellar egg phosphatidylcholine vesicles containing varying amounts of other lipids. The rate of leakage was enhanced in a dose-dependent manner by the presence of phosphatidylethanolamine, diacylglycerol, cholesterol, or hexadecane, all of which are known to favor a lamellar-to-inverted hexagonal (L-H) phase transition. Phosphatidylethanolamine molecular species with low L-H transition temperatures had the largest effects on aerolysin activity. In contrast, the presence in the egg phosphatidylcholine liposomes of lipids that are known to stabilize the lamellar phase, such as sphingomyelin and saturated phosphatidylcholines, reduced the rate of channel formation, as did the presence of lysophosphatidylcholine, which favors positive membrane curvature. When two different lipids that favor hexagonal phase were present with egg PC in the liposomes, their stimulatory effects were additive. Phosphatidylethanolamine and lysophosphatidylcholine canceled each other's effect on channel formation.     

Site-directed mutagenesis at histidines of aerolysin from Aeromonas hydrophila: a lipid planar bilayer study

The role of histidine residues in the formation of channels by the cytolytic toxin aerolysin has been studied in planar lipid bilayers by substituting each of the six histidines in the native protein with asparagine. His341 or His186 mutants had the same channel-forming ability as native toxin, whereas the His332 and His121 mutants were less active. Mutations at His132 and His107, which interfere with the oligomerization of the toxin, drastically reduce pore formation. These findings support the conclusion that oligomerization of the toxin must precede channel formation, and that at least two of the six histidine residues are essential for this to occur. The aerolysin channel is a water-filled pore with an approximate diameter of 9.3 +/- 0.4 A.     

Structure of the stress response protein DR1199 from Deinococcus radiodurans: a member of the DJ-1 superfamily

The expression level of protein DR1199 is observed to increase considerably in the radio-resistant bacterium Deinococcus radiodurans following irradiation. This protein belongs to the DJ-1 superfamily, which includes proteins with diverse functions, such as the archaeal proteases PhpI and PfpI, the bacterial chaperone Hsp31 and hyperosmotic stress protein YhbO, and the human Parkinson's disease-related protein DJ-1. All members of the superfamily are oligomeric, and the oligomerization interface varies from protein to protein. Although for many of these proteins, their function remains obscure, most of them are involved in cellular protection against environmental stresses. We have determined the structure of DR1199 to a resolution of 2.15 A, and we have tested its function and studied its role in the response to irradiation and more generally to oxidative stress in D. radiodurans. The protein is a dimer displaying an oligomerization interface similar to that observed for the YhbO and PhpI proteins. The cysteine in the catalytic triad (Cys 115) is oxidized in our structure, similar to modifications seen in the corresponding cysteine of the DJ-1 protein. The oxidation occurs spontaneously in DR1199 crystals. In solution, no proteolytic or chaperone activity was detected. On the basis of our results, we suggest that DR1199 might work as a general stress protein involved in the detoxification of the cell from oxygen reactive species, rather than as a peptidase in D. radiodurans.     

The versatility of proteolytic enzymes

The growing realization of their physiological importance has generated renewed interest in the study of proteolytic enzymes. Modern methods of protein chemistry and molecular biology have revealed new insights into the protein and gene structure of a variety of protein precursors and their processing by limited proteolysis. Examples are given in this review for transmembrane processes and the role of signal peptidases of both eukaryotic and prokaryotic origin, the processing of prohormones and precursors of growth factors, protein components of blood coagulation, fibrinolysis, and of the complement system, and a group of granulocyte proteases, including the mast cell serine proteases. The relationship of homologous domains found in many of these proteases and their zymogens to protein evolution is a recurrent theme of this discussion.     

Substrate recognition and binding by RseP, an Escherichia coli intramembrane protease

Escherichia coli RseP belongs to the S2P family of intramembrane cleaving proteases. RseP catalyzes proteolytic cleavage of the membrane-bound anti-sigma(E) protein RseA as an essential step in transmembrane signal transduction in the sigma(E) extracytoplasmic stress response pathway. RseP cleaves transmembrane segments of membrane proteins, but the molecular mechanisms of its substrate recognition and proteolytic action remain largely unknown. Here we analyzed interaction between RseP and substrate membrane proteins. Co-immunoprecipitation assays showed that helix-destabilizing residues in a substrate transmembrane segment, which were previously shown to be required for efficient proteolysis of the substrate by RseP, stabilize the substrate-RseP interaction. Substitutions of certain amino acid residues, including those evolutionarily conserved, in the third transmembrane region (TM3) of RseP weakened the RseP-substrate interaction. Specific combinations of Cys substitutions in RseP TM3 and in the RseA transmembrane segment led to the formation of disulfide bonds upon oxidation, suggesting that TM3 of RseP directly binds the substrate. These results provide insights into the mechanism of membrane protein proteolysis by RseP.     

Hepatocyte growth factor activator inhibitor type 1 inhibits protease activity and proteolytic activation of human airway trypsin-like protease

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type transmembrane serine protease inhibitor initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA), a serine protease that converts pro-HGF to the active form. HAI-1 also has inhibitory activity against serine proteases such as matriptase, hepsin and prostasin. In this study, we examined effects of HAI-1 on the protease activity and proteolytic activation of human airway trypsin-like protease (HAT), a transmembrane serine protease that is expressed mainly in bronchial epithelial cells. A soluble form of HAI-1 inhibited the protease activity of HAT in vitro. HAT was proteolytically activated in cultured mammalian cells transfected with its expression vector, and a soluble form of active HAT was released into the conditioned medium. The proteolytic activation of HAT required its own serine protease activity. Co-expression of the transmembrane full-length HAI-1 inhibited the proteolytic activation of HAT. In addition, full-length HAI-1 associated with the transmembrane full-length HAT in co-expressing cells. Like other target proteases of HAI-1, HAT converted pro-HGF to the active form in vitro. These results suggest that HAI-1 functions as a physiological regulator of HAT by inhibiting its protease activity and proteolytic activation in airway epithelium.     

Secretion of haemolysins and proteases by Aeromonas hydrophila EO63: separation and characterization of the serine protease (caseinase) and the metalloprotease (elastase)

AIMS: To determine the haemolysins and proteases excreted by the virulent strain EO63 of Aeromonas hydrophila grown in complex media and to then fractionate and characterize them, in particular those with elastolytic activity. METHODS AND RESULTS: The amount of haemolytic and proteolytic activity in EO63 culture supernatants was dependent on the culture media used. In all media, haemolysins appeared during the phase of active growth and haemolytic activity decreased quickly thereafter, as previously described for aerolysin. In contrast, proteases were mainly released during the stationary phase. Serine protease activity in EO63 culture supernatants was four times greater than that caused by metalloproteases. Two main proteases were partially purified from EO63 culture supernatants by isoelectrophoresis: a serine protease (68 kDa) active against casein; a mixture of different protein bands (60, 44 and 31 kDa) representing a thermostable metalloprotease active against elastin and casein. This metallo-elastase was also inhibited by dithiothreitol and showed a pH optimum of 8.0. Both exoenzymes were toxic for eels at LD50 doses of 1.1 and 3.5 microg (g fish)(-1), respectively. CONCLUSIONS: A serine caseinase and a metallo-elastase may play a role in the pathogenicity of EO63 for eels. These toxins are excreted in vitro by EO63 in the ratio of 4:1 during the stationary phase of growth. Strain EO63 also produced beta-haemolysins in vitro which could correspond to aerolysin. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the purification of a metallo-elastase excreted by a wild-type A. hydrophila strain.     

Expression and properties of an aerolysin--Clostridium septicum alpha toxin hybrid protein

Aerolysin is a bilobal channel-forming toxin secreted by Aeromonas hydrophila. The alpha toxin produced by Clostridium septicum is homologous to the large lobe of aerolysin. However, it does not contain a region corresponding to the small lobe of the Aeromonas toxin, leading us to ask what the function of the small lobe is. We fused the small lobe of aerolysin to alpha toxin, producing a hybrid protein that should structurally resemble aerolysin. Unlike aerolysin, the hybrid was not secreted when expressed in Aeromonas salmonicida. The purified hybrid was activated by proteolytic processing in the same way as both parent proteins and, after activation, it formed oligomers that corresponded to the aerolysin heptamer. Like aerolysin, the hybrid was far more active than alpha toxin against human erythrocytes and mouse T lymphocytes. Both aerolysin and the hybrid bound to human glycophorin, and both were inhibited by preincubation with this erythrocyte glycoprotein, whereas alpha toxin was unaffected. We conclude that aerolysin contains two receptor binding sites, one for glycosyl-phosphatidylinositol-anchored proteins that is located in the large lobe and is also found in alpha toxin, and a second site, located in the small lobe, that binds a surface carbohydrate determinant.     

Integrin-dependent Control of Translation: Engagement of Integrin αIIbβ3 Regulates Synthesis of Proteins in Activated Human Platelets

It has been proposed that the plasma membrane of many cell types contains cholesterol-sphingolipid-rich microdomains. Here, we analyze the role of these microdomains in promoting oligomerization of the bacterial pore-forming toxin aerolysin. Aerolysin binds to cells, via glycosyl phosphatidylinositol-anchored receptors, as a hydrophilic soluble protein that must polymerize into an amphipathic ring-like complex to form a pore. We first show that oligomerization can occur at >10(5)-fold lower toxin concentration at the surface of living cells than in solution. Our observations indicate that it is not merely the number of receptors on the target cell that is important for toxin sensitivity, but their ability to associate transiently with detergent resistant microdomains. Oligomerization appears to be promoted by the fact that the toxin bound to its glycosyl phosphatidylinositol-anchored receptors, can be recruited into these microdomains, which act as concentration devices.     

Breakdown of Diazotized Proteins and Synthetic Substrates by Rumen Bacterial Proteases

Several different kinds of substrate were used to investigate the proteolytic activity of rumen bacteria and of proteases released from rumen bacteria by blending (“coat proteases”). These substrates included diazotized feed proteins and diazotized soluble and insoluble pure proteins. It was concluded that, while solubility was an important factor, the secondary and tertiary structure of a protein had a major influence on its rate of digestion. The resistance of elastin congo red to digestion indicated that similar fibrous proteins in plant material might resist proteolytic attack by rumen bacteria. Coat proteases had a broad specificity, including several exo- and endopeptidase activities, as determined by using synthetic peptide substrates.     

Channels formed by subnanomolar concentrations of the toxin aerolysin trigger apoptosis of T lymphomas

Aerolysin is a channel-forming toxin that binds to glycosylphosphatidylinositol (GPI)-anchored proteins, such as Thy-1, on target cells. Here, we show that subnanomolar concentrations of aerolysin trigger apoptosis of T lymphomas. Using inactive aerolysin variants, we determined that apoptosis was not directly triggered by binding to GPI-anchored receptors, nor was it caused by receptor clustering induced by toxin oligomerization. Apoptosis was caused by the production of a small number of channels in the cell membrane. Channel formation resulted in a rapid increase in intracellular calcium, which may have been the signal for apoptosis. Overexpression of the antiapoptotic protein bcl-2 blocked aerolysin-induced apoptosis, although this effect was overcome at higher toxin concentrations.     

Aerolysin,a hemolysin fromAeromonas hydrophila,forms voltage-gated channels in planar lipid bilayers

Summary The cytolytic toxin aerolysin was found to form ion channels which displayed slight anion selectivity in planar lipid bilayers. In voltage-clamp experiments the ion current flowing through the channels was homogeneous indicating a defined conformation and a uniform size. The channels remained open between –70 to +70 mV, but outside this range they underwent voltage-dependent inactivation which was observed as open-closed fluctuations at the single-channel level. Zinc ions not only prevented the formation of channels by inhibiting oligomerization of monomeric aerolysin but they also induced a closure of preformed channels in a voltage-dependent fashion. The results of a Hill plot indicated that 2–3 zinc ions bound to a site within the channel lumen. Proaerolysin, and a mutant of aerolysin in which histidine 132 was replaced by an asparagine, were both unable to oligomerize and neither could form channels. This is evidence that oligomerization is a necessary step in channel formation.     

Characterisation of the <Emphasis Type="Italic">Aspergillus niger dapB</Emphasis> gene,which encodes a novel fungal type IV dipeptidyl aminopeptidase

We have cloned the Aspergillus niger dapB gene. Analysis of its nucleotide sequence and the corresponding protein sequence indicates that the gene encodes a type IV dipeptidyl aminopeptidase (DPP IV). Based upon its deduced sequence we predict the presence of a transmembrane domain in the protein. Furthermore, dapB-overexpressing transformants display an increase in intracellular DPP IV activity. This is the first reported characterisation of a dipeptidyl aminopeptidase with a transmembrane domain from a filamentous fungus. Using the dapB sequence as a query, we were able to identify 14 DPP IV-encoding genes, and 12 additional DPPIV proteases in public genomic databases. Phylogenetic analysis reveals that in yeasts there are two clades of genes that encode DPP IV proteases with a transmembrane domain. In this study we demonstrate that, as in yeasts, two classes of DPP IV-encoding genes exist in filamentous fungi. However, only one of these codes for DPP IV proteases with a transmembrane domain. The second type present in filamentous fungi encodes extracellular DPP IV proteases. The dapB gene belongs to the first cluster. We propose that DapB plays a role in the proteolytic maturation of enzymes produced by A. niger.Electronic Supplementary Material Supplementary material is available for this article at     

Proteolytic action of GlpG, a rhomboid protease in the Escherichia coli cytoplasmic membrane

We characterized Escherichia coli GlpG as a membrane-embedded protease and a possible player in the regulated intramembrane proteolysis in this organism. From the sequence features, it belongs to the widely conserved rhomboid family of membrane proteases. We verified the expected topology of GlpG, and it traverses the membrane six times. A model protein having an N-terminal and periplasmically localized beta-lactamase (Bla) domain, a LacY-derived transmembrane region, and a cytosolic maltose binding protein (MBP) mature domain was found to be GlpG-dependently cleaved in vivo. This proteolytic reaction was reproduced in vitro using purified GlpG and purified model substrate protein, and the cleavage was shown to occur between Ser and Asp in a region of high local hydrophilicity, which might be located in a juxtamembrane rather than an intramembrane position. The conserved Ser and His residues of GlpG were essential for the proteolytic activities. Our results using several variant forms of the model protein suggest that GlpG recognizes features of the transmembrane regions of substrates. These results point to a detailed molecular mechanism and cellular analysis of this interesting class of membrane-embedded proteases.     

ATP-dependent Clp Proteases in Photosynthetic Organisms— A Cut Above the Rest!

Proteases are critical regulatory factors for many metabolic cellular processes as well as being vital for degrading proteins damaged during environmental stresses. Many of those responsible for targeted protein degradation require the hydrolysis of ATP, and one class that has attracted much attention recently are the Clp proteases. They are among the best characterized proteases to date, and were the first shown to rely on an ATPase regulatory subunit possessing molecular chaperone activity, which functions both within the proteolytic complex and independently. A range of Clp proteins has been identified from many different bacteria and eukaryotes, with by far the greatest number and diversity of forms in oxygenic photobionts such as cyanobacteria and higher plants. Functionally, Clp proteins have also evolved into one of the more critical proteolytic enzymes within photobionts, and it is now somewhat of a paradox that we currently know least about Clp protease functions in the photosynthetic organisms, where they have their most important roles. This discrepancy is now being addressed, with studies on Clp protein in cyanobacteria and, in an increasing number, in higher plants.     

Identification of functional domains of Clostridium septicum alpha toxin

Alpha toxin (AT) is the major virulence factor of Clostridium septicum that is a proteolytically activated pore-forming toxin that belongs to the aerolysin-like family of toxins. AT is predicted to be a three-domain molecule on the basis of its functional and sequence similarity with aerolysin, for which the crystal structure has been determined. In this study, we have substituted the entire primary structure of AT with alanine or cysteine to identify those amino acids that comprise functional domains involved in receptor binding, oligomerization, and pore formation. These studies revealed that receptor binding is restricted to domain 1 of the AT structure, whereas domains 1 and 3 are involved in oligomerization. These studies also revealed the presence of a putative functional region of AT proximal to the receptor-binding domain but distal from the pore-forming domain that is proposed to regulate the insertion of the transmembrane beta-hairpin of the prepore oligomer.