Papers by Mauro Dalla Serra
Journal of Natural Products, 1997
From the marine sponge Reniera sarai 3-alkylpyridinium oligomers and polymers have been isolated.... more From the marine sponge Reniera sarai 3-alkylpyridinium oligomers and polymers have been isolated. 3-Alkylpyridinium polymers are potent anticholinesterase agents; in addition, they show hemolytic and cytotoxic activities. Oligomers with a molecular weight lower than 3000 Da do not possess any significant activity. We report structural characterization of 3-alkylpyridinium polymers and their behavior in aqueous solutions. We found that biologically active polymers are composed of head-to-tail 3-alkylpyridinium units. According to MALDI-TOF spectrometry two species of polymers exist, the smaller with a molecular weight of 5520 Da and the larger with a molecular weight of 18 900 Da. Both polymers are soluble only in water, while low molecular oligomers are readily soluble in organic solvents. Polymers form large water-dissolved supramolecular structures with an average hydrodynamic radius of 23 ( 2 nm and, therefore, cannot be separated with size-exclusion chromatography.
Scientific reports, 2015
Pore formation of cellular membranes is an ancient mechanism of bacterial pathogenesis that allow... more Pore formation of cellular membranes is an ancient mechanism of bacterial pathogenesis that allows efficient damaging of target cells. Several mechanisms have been described, however, relatively little is known about the assembly and properties of pores. Listeriolysin O (LLO) is a pH-regulated cholesterol-dependent cytolysin from the intracellular pathogen Listeria monocytogenes, which forms transmembrane β-barrel pores. Here we report that the assembly of LLO pores is rapid and efficient irrespective of pH. While pore diameters at the membrane surface are comparable at either pH 5.5 or 7.4, the distribution of pore conductances is significantly pH-dependent. This is directed by the unique residue H311, which is also important for the conformational stability of the LLO monomer and the rate of pore formation. The functional pores exhibit variations in height profiles and can reconfigure significantly by merging to other full pores or arcs. Our results indicate significant plasticity...
Biophysical Chemistry, 2013
Pore-forming toxins are an important group of natural molecules that damage cellular membranes by... more Pore-forming toxins are an important group of natural molecules that damage cellular membranes by forming transmembrane pores. They are used by many organisms for attack or defense and similar proteins are employed in the immune system of vertebrates. Various biophysical approaches have been used to understand how these proteins act at the molecular level. One of the most useful, in terms of monitoring pore formation in real time, is a method that employs planar lipid membranes and involves ionic current measurements. Here we highlight the advantages and possibilities that this approach offers and show how it can advance understanding of the pore-forming mechanism and pore properties for one of the most important families of natural toxins, the cholesterol-dependent cytolysins.
European Journal of Biochemistry, 1999
Equinatoxin II is a cysteineless pore-forming protein from the sea anemone Actinia equina. It rea... more Equinatoxin II is a cysteineless pore-forming protein from the sea anemone Actinia equina. It readily creates pores in membranes containing sphingomyelin. Its topology when bound in lipid membranes has been studied using cysteine-scanning mutagenesis. At approximately every tenth residue, a cysteine was introduced. Nineteen single cysteine mutants were produced in Escherichia coli and purified. The accessibility of the thiol groups in lipid-embedded cysteine mutants was studied by reaction with biotin maleimide. Most of the mutants were modified, except those with cysteines at positions 105 and 114. Mutants R144C and S160C were modified only at high concentrations of the probe. Similar results were obtained if membrane-bound biotinylated mutants were tested for avidin binding, but in this case three more mutants gave a negative result: S1C, S13C and K43C. Furthermore, mutants S1C, S13C, K20C, K43C and S95C reacted with biotin only after insertion into the lipid, suggesting that they were involved in major conformational changes occurring upon membrane binding. These results were further confirmed by labeling the mutants with acrylodan, a polarity-sensitive fluorescent probe. When labeled mutants were combined with vesicles, the following mutants exhibited blue-shifts, indicating the transfer of acrylodan into a hydrophobic environment: S13C, K20C, S105C, S114C, R120C, R144C and S160C. The overall results suggest that at least two regions are embedded within the lipid membrane: the N-terminal 13±20 region, probably forming an amphiphilic helix, and the tryptophan-rich 105±120 region. Arg144, Ser160 and residues nearby could be involved in making contacts with lipid headgroups. The association with the membrane appears to be unique and different from that of bacterial pore-forming proteins and therefore equinatoxin II may serve as a model for eukaryotic channel-forming toxins.
Plant Pathology, 2013
Ppdfn1 is a defensin gene previously identified in peach (Prunus persica). The biological role of... more Ppdfn1 is a defensin gene previously identified in peach (Prunus persica). The biological role of Ppdfn1 was investigated by analysing its expression profile in leaves, flowers and fruits, either inoculated with the Monilinia laxa fungal pathogen or mock-inoculated. Ppdfn1 expression was highest in flowers and, in fruits, did not vary upon M. laxa inoculation. To characterize the PpDFN1 antifungal activity, the recombinant mature peptide was expressed in Escherichia coli and purified; recombinant PpDFN1 displays antifungal activity against Botrytis cinerea, M. laxa and Penicillium expansum, with IC 50 values of 15AE1, 9AE9 and 1AE1 lg mL )1 , respectively. Treatment of fungal hyphae with FITC-labelled PpDFN1 indicated that the peptide is not internalized by fungal hyphae, but localizes on their external cell surface. At this site, PpDFN1 is capable of membrane destabilization and permeabilization, as demonstrated by SYTOX Green fluorescence uptake by the treated mycelia. Using artificial lipid monolayers, it was shown that PpDFN1 interacts with sphingolipid-containing membranes; however the strongest interaction occurs with monolayers composed of lipids extracted from sensitive fungi, such as P. expansum. These data suggest that the lipid composition of fungal membranes is of key relevance for defensin specificity.
Biophysical chemistry, 2013
Pore-forming toxins are an important group of natural molecules that damage cellular membranes by... more Pore-forming toxins are an important group of natural molecules that damage cellular membranes by forming transmembrane pores. They are used by many organisms for attack or defense and similar proteins are employed in the immune system of vertebrates. Various biophysical approaches have been used to understand how these proteins act at the molecular level. One of the most useful, in terms of monitoring pore formation in real time, is a method that employs planar lipid membranes and involves ionic current measurements. Here we highlight the advantages and possibilities that this approach offers and show how it can advance understanding of the pore-forming mechanism and pore properties for one of the most important families of natural toxins, the cholesterol-dependent cytolysins.
Nature Communications, 2014
Previous efforts to control cellular behaviour have largely relied upon various forms of genetic ... more Previous efforts to control cellular behaviour have largely relied upon various forms of genetic engineering. Once the genetic content of a living cell is modified, the behaviour of that cell typically changes as well. However, other methods of cellular control are possible. All cells sense and respond to their environment. Therefore, artificial, non-living cellular mimics could be engineered to activate or repress already existing natural sensory pathways of living cells through chemical communication. Here we describe the construction of such a system. The artificial cells expand the senses of Escherichia coli by translating a chemical message that E. coli cannot sense on its own to a molecule that activates a natural cellular response. This methodology could open new opportunities in engineering cellular behaviour without exploiting genetically modified organisms.
Cardioscience, 1991
Sarcolemmal membrane vesicles isolated from bovine ventricular tissue accumulate Ca2+ through the... more Sarcolemmal membrane vesicles isolated from bovine ventricular tissue accumulate Ca2+ through the Na+/Ca2+ exchanger when exposed to an outwardly directed Na+ gradient. This Ca2+ is then released by the same mechanism if the vesicles are transferred to a Ca(2+)-depleted Na+ buffer. Using the Ca+ indicator, arsenazo III, and a stopped-flow spectrophotometer, we can directly follow the kinetics of Ca2+ extrusion. We can thus measure the activity of the Na+/Ca2+ exchanger by the initial rate of Ca2+ release. We found that it depends upon the external Na+ concentration in a cooperative way, with a Hill coefficient of 2. By studying the temperature dependence of Na+/Ca2+ exchange, we found that it can be described by a single activation energy: Ea = 8.3 +/- 0.4 Kcal/mol. When the Na+/Ca2+ exchanger is reconstituted into lipid vesicles of defined composition, we observe a higher activity if cholesterol is among the lipids. The activation energy becomes 6.1 +/- 0.1 Kcal/mol in this system,...
Biophysical Chemistry, 2015
The interfacing of artificial devices with biological systems is a challenging field that crosses... more The interfacing of artificial devices with biological systems is a challenging field that crosses several disciplines ranging from fundamental research (biophysical chemistry, neurobiology, material and surface science) to frontier technological application (nanotechnology, bioelectronics). The memristor is the fourth fundamental circuit element, whose electrical properties favor applications in signal processing, neural networks, and brain-computer interactions and it represents a new frontier for technological applications in many fields including the nanotechnologies, bioelectronics and the biosensors. Using multidisciplinary approaches, covering surface science, cell biology and electrophysiology, we successfully implemented a living bio-hybrid system constituted by cells adhering to films of poly(aniline) (PANI), a semiconductor polymer having memristive properties assembled with polyelectrolytes. Here we tested whether the PANI devices could support survivor, adhesion and differentiation of several cell lines, including the neuron-like SHSY5Y cells. Moreover, we performed electrophysiology on these cells showing that the biophysical properties are retained with differences occurring in the recorded ion currents. Taken together, the cell viability here reported is the key requirement to design and develop a reliable functional memristor-based bio-hybrid able to mimic neuronal activity and plasticity.
Biophysical Journal, 2005
Bax and Bid are proapoptotic proteins of the Bcl-2 family that regulate the release of apoptogeni... more Bax and Bid are proapoptotic proteins of the Bcl-2 family that regulate the release of apoptogenic factors from mitochondria. Although they localize constitutively in the cytoplasm, their apoptotic function is exerted at the mitochondrial outer membrane, and is related to their ability to form transbilayer pores. Here we report the poration activity of fragments from these two proteins, containing the first a-helix of a colicinlike hydrophobic hairpin (a-helix 5 of Bax and a-helix 6 of Bid). Both peptides readily bind to synthetic lipid vesicles, where they adopt predominantly a-helical structures and induce the release of entrapped calcein. In planar lipid membranes they form ion conducting channels, which in the case of the Bax-derived peptide are characterized by a two-stage pattern, a large conductivity and lipid-charge-dependent ionic selectivity. These features, together with the influence of intrinsic lipid curvature on the poration activity and the existence of two helical stretches of different orientations for the membrane-bound peptide, suggest that it forms mixed lipidic/peptidic pores of toroidal structure. In contrast, the assayed Bid fragment shows a markedly different behavior, characterized by the formation of discrete, steplike channels in planar lipid bilayers, as expected for a peptidic pore lined by a bundle of helices.
FEBS Journal, Jan 2012
Listeriolysin O (LLO) is the major factor implicated in the escape of Listeria monocytogenes from... more Listeriolysin O (LLO) is the major factor implicated in the escape of Listeria monocytogenes from the phagolysosome. It is the only representative of cholesterol-dependent cytolysins that exhibits pH-dependent activity. Despite intense studies of LLO pH-dependence, this feature of the toxin still remains incompletely explained. Here we used fluorescence and CD spectroscopy to show that the structure of LLO is not detectably affected by pH at room temperature. We observed slightly altered haemolytic and permeabilizing activities at different pH values, which we relate to reduced binding of LLO to the lipid membranes. However, alkaline pH and elevated temperatures caused rapid denaturation of LLO. Aggregates of the toxin were able to bind Congo red and Thioflavin T dyes and were visible under transmission electron microscopy as large, amorphous, micrometer-sized assemblies. The aggregates had the biophysical properties of amyloid. Analytical ultracentrifugation indicated dimerization of the protein in acidic conditions, which protects the protein against premature denaturation in the phagolysosome, where toxin activity takes place. We therefore suggest that LLO spontaneously aggregates at the neutral pH found in the host cell cytosol and that this is a major mechanism of LLO inactivation.
Nature Communications, 2014
Previous efforts to control cellular behaviour have largely relied upon various forms of genetic ... more Previous efforts to control cellular behaviour have largely relied upon various forms of genetic engineering. Once the genetic content of a living cell is modified, the behaviour of that cell typically changes as well. However, other methods of cellular control are possible. All cells sense and respond to their environment. Therefore, artificial, non-living cellular mimics could be engineered to activate or repress already existing natural sensory pathways of living cells through chemical communication. Here we describe the construction of such a system. The artificial cells expand the senses of Escherichia coli by translating a chemical message that E. coli cannot sense on its own to a molecule that activates a natural cellular response. This methodology could open new opportunities in engineering cellular behaviour without exploiting genetically modified organisms.
Pore formation of cellular membranes is an ancient mechanism of bacterial pathogenesis that allow... more Pore formation of cellular membranes is an ancient mechanism of bacterial pathogenesis that allows efficient damaging of target cells. Several mechanisms have been described, however, relatively little is known about the assembly and properties of pores. Listeriolysin O (LLO) is a pH-regulated cholesterol-dependent cytolysin from the intracellular pathogen Listeria monocytogenes, which forms transmembrane β-barrel pores. Here we report that the assembly of LLO pores is rapid and efficient irrespective of pH. While pore diameters at the membrane surface are comparable at either pH 5.5 or 7.4, the distribution of pore conductances is significantly pH-dependent. This is directed by the unique residue H311, which is also important for the conformational stability of the LLO monomer and the rate of pore formation. The functional pores exhibit variations in height profiles and can reconfigure significantly by merging to other full pores or arcs. Our results indicate significant plasticity of large β-barrel pores, controlled by environmental cues like pH.
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Papers by Mauro Dalla Serra