Biomedical subjects

Bruno Maggio

Publications and source records attributed to Bruno Maggio.

At least 19 recordsLinked to original sources

PubMed2006

Biophysics of sphingolipids II. Glycosphingolipids: an assortment of multiple structural information transducers at the membrane surface.

Glycosphingolipids are ubiquitous components of animal cell membranes. They are constituted by the basic structure of ceramide with its hydroxyl group linked to single carbohydrates or oligosaccharide chains of different complexity. The combination of the properties of their hydrocarbon moiety with those derived from the variety and complexity of their hydrophilic polar head groups confers to these lipids an extraordinary capacity for molecular-to-supramolecular transduction across the lateral/transverse planes in biomembranes and beyond. In our opinion, most of the advances made over the last decade on the biophysical behavior of glycosphingolipids can be organized into three related aspects of increasing structural complexity: (1) intrinsic codes: local molecular interactions of glycosphingolipids translated into structural self-organization. (2) Surface topography: projection of molecular shape and miscibility of glycosphingolipids into formation of coexisting membrane domains. (3) Beyond the membrane interface: glycosphingolipid as modulators of structural topology, bilayer recombination and surface biocatalysis.

Biophysical Phenomena↗

PubMed2006

Effect of externally applied electrostatic fields on the surface topography of ceramide-enriched domains in mixed monolayers with sphingomyelin.

Lipid and protein molecules anisotropically oriented at a hydrocarbon-aqueous interface configure a dynamic array of self-organized molecular dipoles. Electrostatic fields applied to lipid monolayers have been shown to induce in-plane migration of domains or phase separation in a homogeneous system. In this work, we have investigated the effect of externally applied electrostatic fields on the distribution of the condensed ceramide-enriched domains in mixed monolayers with sphingomyelin. In these monolayers, the lipids segregate in different phases at all pressures. This allows analyzing by epifluorescence microscopy the effect of the electrostatic field at all lateral pressure because coexistence of lipid domains in condensed state are always present. Our observations indicate that a positive potential applied to an electrode placed over the monolayer promotes a repulsion of the ceramide-enriched domains which is rather insensitive to the film composition, depends inversely on the lateral pressure and exhibits threshold dependence on the in-plane elasticity.

Ceramides↗

PubMed2006

Surface thermodynamics reveals selective structural information storage capacity of c-Fos-phospholipid interactions.

The surface properties of c-Fos, a regulator of normal and pathologic cell growth and a modulator of phospholipid metabolism, suggest that it has the potential to transduce information through molecular reorganization, placing the nature of its interaction with phospholipids at the basis of its possible effects at the membrane level. Previous studies established that c-Fos induces condensation and depolarization of PIP2 films and expansion and hyperpolarization of PC. We have now explored more in depth the thermodynamic aspects of these lipid-protein interactions, finding that the mixtures have associated hysteresis. The analysis of the excess thermodynamic functions provides evidence of entropic-enthalpic compensations that result in a favorable enthalpic contribution derived from the interaction of c-Fos with PIP2, which exceeds the unfavorable configurational entropy. On the contrary, favorable entropy terms dominate the interaction of c-Fos with PC over the unfavorable enthalpy. The free energy of hysteresis is stored as excess free energy. A shift in molecular packing-dependent surface reorganization, compared to that of ideally mixed films, indicates a gain in information content at the lipid-protein interface in mixed films of c-Fos with PIP2 but not with PC. It is postulated that the free energy stored in these mixtures could act as a bidirectional structural information transducer for dynamic compression-expansion processes occurring on the membrane surface.

Animals↗

PubMed2006

Effects of a short-chain ceramide on bilayer domain formation, thickness, and chain mobililty: DMPC and asymmetric ceramide mixtures.

An important part of natural ceramides contain asymmetric hydrocarbon chains. We have used calorimetry, atomic force microscopy, and electron paramagnetic resonance to study the effect of ceramide chain asymmetry in mixtures of C8Cer with DMPC as a model system of hydrocarbon chain disparity. A phase diagram is provided along with information on the thickness of the membrane and the mobility of the chains at different temperatures both below and above the phase transition temperature of the mixtures. The results indicate a partial interdigitation of C8Cer chains in the gel phase, producing a correlation between the organization of both hemilayers. Our data suggest that the effects of ceramides on biomembranes may be bimodal and similar to those of cholesterol.

Biophysics↗

PubMed2005

c-Jun interacts with phospholipids and c-Fos at the interface.

We describe c-Jun, a widely studied transcription factor that participates in cell proliferation, differentiation, and tumorigenesis, as amphitropic. We show that c-Jun forms stable monolayers and interacts favorably, although in a nonselective manner, with phospholipids at the interface. The surface activity of c-Jun, together with that of c-Fos, its common partner in AP-1 transcription heterodimers, drives interfacial complex formation. We show that AP-1 is very stable at the air-water interface and suggest that AP-1 may not be substantially formed in solution as a stable equimolar association of both proteins.

Binding, Competitive↗

PubMed2005

Penetration and interactions of the antimicrobial peptide, microcin J25, into uncharged phospholipid monolayers.

Microcin J25 forms stable monolayers at the air-water interface showing a collapse at a surface pressure of 5 mN/m, 220 mV of surface potential, and 6 fV per squared centimeter of surface potential per unit of molecular surface density. The adsorption of microcin J25 from the subphase at clean interfaces leads to a rise of 10 mN/m in surface pressure and a surface potential of 220 mV. From these data microcin appears to be a poor surfactant per se. Nevertheless, the interaction with the lipid monolayer further increase the stability of the peptide at the interface depending on the mode in which the monolayer is formed. Spreading with egg PC leads to nonideal mixing up to 7 mN/m, with hyperpolarization and expansion of components at the interface, with a small excess free energy of mixing caused by favorable contributions to entropy due to molecular area expansion compensating for the unfavorable enthalpy changes arising from repulsive dipolar interactions. Above 7 mN/m microcin is squeezed out, leaving a film of pure phospholipid. Nevertheless, the presence of lipid at 10 and 20 mN/m stabilize further microcin at the interface and adsorption from the subphase proceeds up to 30 mN/m, equivalent to surface pressure in bilayers.

Adsorption↗

PubMed2005

Effect of molecular surface packing on the enzymatic activity modulation of an anchored protein on phospholipid Langmuir monolayers.

The catalytic activity of a glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase has been studied in Langmuir phospholipid monolayers at different surface pressures. The enzyme substrate, p-nitrophenyl phosphate, was injected into the subphase of mixed enzyme/lipid Langmuir monolayers. Its hydrolysis product was followed by monitoring the absorbance at 410 nm in situ in the monolayer subphase of the Langmuir trough. Several surface pressures, corresponding to different molecular surface densities, were attained by lateral compression of the monolayers. The morphology of the monolayers, observed by fluorescence microscopy, showed three different types of domains owing to the heterogeneous partition of the enzyme within the mixed enzyme/lipid film. The catalytic activity was modulated by the enzyme surface density, and it increased until a pressure of 18 mN/m was reached, but it decreased significantly when the equilibrium in-plane elasticity (surface compressional modulus) increased more noticeably, resulting in alterations in the interface morphology. A model for the modulation of the enzyme orientation and catalytic activity by lipid/enzyme surface morphology and enzyme surface packing at the air/liquid interface is proposed. The results might have an important impact on the comprehension of the enzymatic activity regulation of GPI-anchored proteins in biomembranes.

Air↗

PubMed2005

Na,K-ATPase reconstituted in liposomes: effects of lipid composition on hydrolytic activity and enzyme orientation.

In this paper, the reconstitution of Na,K-ATPase in liposomes (formed by single or mixed phospholipids and cholesterol) was investigated and the enzyme orientation was determined on kinetic basis using only specific inhibitors of ATP hydrolysis. A condition of foremost importance for enzyme reconstitution is the achievement of complete solubilization of the lipid in the initial stage of the cosolubilization process for the subsequent formation of the liposomes and/or proteoliposomes. PC-liposomes showed that increasing the fatty acid chain length increases the percentage of Na,K-ATPase incorporated. The average diameter of the proteoliposomes also increases in proportion, reaching a maximum with phospholipids with 16 carbon chains, resulting in 75.1% protein reconstitution and 319.4 nm diameter size, respectively. Binary lipid systems with PC and PE were efficient for incorporation of Na,K-ATPase, depending on the lipid:protein ratio used, varying from 15 to 80% recovery of total ATPase activity. The best results for Na,K-ATPase reconstitution using PC and PE mixture were obtained using a lipid:lipid ratio 1:1 (w/w) and lipid:protein 1:3 (w/w). Integrity studies using calcein release mediated by detergent or alamethicin, in association with inhibition of ATPase activity (ouabain and vanadate) showed that the enzyme is oriented inside-out in DPPC:DPPE proteoliposomes. In these vesicular systems, the enzyme is reconstituted with about 78.9% ATPase activity recovery and 89% protein incorporation, with an average diameter of 140 nm. Systems constituted by DPPC:DPPE, DPPC:DLOPE or DLOPC:DLOPE showed approximately 80, 71 and 70% of recovery of total ATPase activity, but no homogeneity in the distribution of Na,K-ATPase orientation. Reconstitution of Na,K-ATPase in DPPC:DPPE:cholesterol or DPPC:DLOPE:cholesterol systems (55% of cholesterol) showed recovery of about 86 and 82%, respectively, of its total ATPase activity. The results point to an important effect of the lipid acyl chain length and lipid-protein ratio in relation to the composition of the lipid matrix to finely tune the structural asymmetry and the amount of enzyme that can be incorporated a lipid bilayer vesicle while preserving membrane permeability.

Animals↗

PubMed2005

c-Fos and phosphatidylinositol-4,5-bisphosphate reciprocally reorganize in mixed monolayers.

The transcription factor c-Fos has surface thermodynamic properties that allow it to differentially interact with phospholipids, especially PIP2. It regulates phospholipid metabolism both in vivo and in vitro, and modulates degradation of phospholipid monolayers by phospholipases in a way that depends on the membrane intermolecular packing (i.e., surface lateral pressure). With the aim to understand details of the interactions of c-Fos at the membrane level, we studied the surface packing, dipole potential, compressibility and topography of mixed films of the protein with PIP2. We show that c-Fos changes the packing of liquid-expanded PIP2 monolayers, in a different manner with respect to its effect on the similarly liquid-expanded dilauroylphosphatidylcholine monolayers. The changes at the local molecular level are transduced to long-range inhomogeneities of the surface, detected by Brewster angle (BAM) and epifluorescence microscopy (EFM). Our results highlight the capacity of c-Fos to alter the packing and dipole potential of the lipid-protein interface. This involves variations of the surface in-plane elasticity and lateral segregation of phase domains. These dynamic, reversible alterations of surface organization provide a basis by which c-Fos may transduce molecular information at the membrane level.

Membranes, Artificial↗

PubMed2005

Many length scales surface fractality in monomolecular films of whole myelin lipids and proteins.

Monomolecular films prepared with all the lipid and protein components of myelin were spread at the air/aqueous buffer interface from isolated bovine spinal cord myelin fully dissolved in chloroform:methanol (2:1) or by surface free energy shock of myelin membrane microvesicles. These monolayers show indistinguishable surface behavior, with similar compositional phase coexistence through all the compression isotherm on several subphase conditions. The domains were observed through epifluorescence and Brewster angle microscopy on the air/water interface and on Langmuir-Blodgett films. Their thickness was measured ellipsometrically. Under molecular packing conditions resembling those found in the natural membrane, the morphology and size of the domains are highly self-similar, displaying no characteristic length scale. These properties are the hallmark of fractal objects. The fractality extends at least three orders of magnitudes, from the micrometer to the millimeter range, the fractal dimension being about 1.7. A possible implication of fractality in membrane structure and/or function is demonstrated through the high fluctuation of the propagation of signals through constrained diffusion in corrals formed by domains in the plane of the monolayer, which restricts the diffusion of a fluorescent probe over many length scale domains.

Animals↗

PubMed2004

Shape transitions and lattice structuring of ceramide-enriched domains generated by sphingomyelinase in lipid monolayers.

Sphingomyelinases (SMases) hydrolyze the membrane constituent sphingomyelin (SM) to phosphocholine and ceramide (Cer). Growing evidence supports that SMase-induced SM-->Cer conversion leads to the formation of lateral Cer-enriched domains which drive structural reorganization in lipid membranes. We previously provided visual evidence in real-time for the formation of Cer-enriched domains in SM monolayers through the action of the neutral Bacillus cereus SMase. In this work, we disclose a succession of discrete morphologic transitions and lateral organization of Cer-enriched domains that underlay the SMase-generated surface topography. We further reveal how these structural parameters couple to the generation of two-dimensional electrostatic fields, based upon the specific orientation of the lipid dipole moments in the Cer-enriched domains. Advanced image processing routines in combination with time-resolved epifluorescence microscopy on Langmuir monolayers revealed: 1), spontaneous nucleation and circular growth of Cer-enriched domains after injection of SMase into the subphase of the SM monolayer; 2), domain-intrinsic discrete transitions from circular to periodically undulating shapes followed by a second transition toward increasingly branched morphologies; 3), lateral superstructure organization into predominantly hexagonal domain lattices; 4), formation of super-superstructures by the hexagonal lattices; and 5), rotationally and laterally coupled domain movement before domain border contact. All patterns proved to be specific for the SMase-driven system since they could not be observed with Cer-enriched domains generated by defined mixtures of SM/Cer in enzyme-free monolayers at the same surface pressure (pi = 10 mN/m). Following the theories of lateral shape transitions, dipolar electrostatic interactions of lipid domains, and direct determinations of the monolayer dipole potential, our data show that SMase induces a domain-specific packing and orientation of the molecular dipole moments perpendicular to the air/water interface. In consequence, protein-driven generation of specific out-of-equilibrium states, an accepted concept for maintenance of transmembrane lipid asymmetry, must also be considered on the lateral level. Lateral enzyme-specific out-of-equilibrium organization of lipid domains represents a new level of signal transduction from local (nm) to long-range (microm) scales. The cross-talk between lateral domain structures and dipolar electrostatic fields adds new perspectives to the mechanisms of SMase-mediated signal transduction in biological membranes.

Animals↗

PubMed2004

Lipid composition-dependent incorporation of multiple membrane proteins into liposomes.

Membrane proteins from bacteria Pasteurella multocida were used as a model for studying its incorporation into liposomes. An important step to achieve efficient high yield protein incorporation in proteoliposomes is the study of the more suitable lipid composition. To this end, we compared the amount of total protein, reconstituted by co-solubilization methods, into liposomes of phospholipids with different polar head groups and acyl chain lengths. The liposomes and proteoliposomes were characterised by isopycnic centrifugation in sucrose gradient and by dynamic light scattering. Experimental and theoretical results were compared considering the effects exerted through the hydrocarbon chain length, volume, and optimal cross-sectional area of the phospholipid (combined in the geometrical critical packing parameter, lipid-protein matching), critical spontaneous radius of curvature of the bilayer vesicle, phase transition temperature of the lipid and ratio of lipid-protein molecules present in the vesicles. The highest incorporation of multiple proteins was found with dipalmitoylphosphatidylcholine (DPPC), reaching a yield of 93% compared to the lower relative amounts incorporated in proteoliposomes of the other lipids. The incorporation of multiple proteins induces a proportional enhancement of vesicular dimension, since DPPC-proteoliposomes have an average diameter of 1850A, compared to the 1430A for pure DPPC vesicles.

Blotting, Western↗

PubMed2004

Phospholipase activity is modulated by c-Fos through substrate expansion and hyperpolarization.

c-Fos, a component of AP-1 transcription factors, has been shown to have marked amphitropic properties and to regulate phospholipase activity against lipid monolayers. In agreement with its high surface activity, it has also been found to associate to membranes of the endoplasmic reticulum and to activate phospholipid metabolism in vivo. All these findings point to an involvement of this oncoprotein within a membrane environment. We have previously shown that c-Fos modulates in different manners the activity of phospholipase A2 and phospholipase C against monolayers of dilauroylphosphatidylcholine (PC). In this work, we have studied the possible molecular mechanism underlying the phosphohydrolytic modulation. Our results show that c-Fos expands and hyperpolarizes PC, indicating that its effects on these enzymatic activities are due to the changes it induces on the interfacial organization of the substrate.

Animals↗

PubMed2004

Surface activity and interaction of StarD7 with phospholipid monolayers.

StarD7 protein forms stable Gibbs and Langmuir monolayers at the air-buffer interface showing marked surface activity. The latter is enhanced by penetration into phospholipid films at an initial surface pressure above the protein's own equilibrium adsorption surface pressure to a lipid-free interface. The protein-phospholipid stabilizing interactions at the interface depend on the lipid, with preference for phosphatidylserine, cholesterol, and phosphatidylglycerol, and the increases of lateral surface pressure generated are comparable to those of other membrane-active proteins. The surface activity of StarD7 is strong enough to thermodynamically drive and retain StarD7 at the lipid membrane interface where it may undergo lipid-dependent reorganization as indicated by changes of surface pressure and electrostatics.

Carrier Proteins↗

PubMed2004

Favorable and unfavorable lateral interactions of ceramide, neutral glycosphingolipids and gangliosides in mixed monolayers.

Interactions among four natural neutral sphingolipids (ceramide, glucosyl-ceramide, lactosyl-ceramide and asialo-GM1) and six gangliosides (GM3, GM2, GM1, GD3, GD1a and GT1b) were studied in binary Langmuir monolayers at the air-buffer interface in terms of their molecular packing, compressibility, dipole potential and mixing behavior. The changes of surface organization can be grouped into three sets: (a) binary films of neutral GSLs, and of the latter with ceramide, exhibit thermodynamically unfavorable mixing with mean molecular area expansions and dipole moment hyperpolarization; (b) mixed monolayers of ceramide, or of GlcCer, and gangliosides occur with thermodynamically favorable interactions leading to mean molecular area condensation and depolarisation; (c) binary mixtures of LacCer or Gg4Cer with gangliosides, and all ganglioside species among them, revealed molecular immiscibility characterized by additive mean molecular area and dipole potential, with composition-independent constant collapse pressure. These results disclose basic tendencies of GSLs to molecularly mix or demix, leading to their surface segregation, which may underlay vectorial separation of their specific biosynthetic pathways.

Animals↗

PubMed2003

Interactions of chicken liver basic fatty acid-binding protein with lipid membranes.

The interactions of chicken liver basic fatty acid-binding protein (Lb-FABP) with large unilamellar vesicles (LUVs) of palmitoyloleoyl phosphatidylcholine (POPC) and palmitoyloleoyl phosphatidylglycerol (POPG) were studied by binding assays, Fourier transform infrared (FT-IR) spectroscopy, monolayers at air-water interface, and low-angle X-ray diffraction. Lb-FABP binds to POPG LUVs at low ionic strength but not at 0.1 M NaCl. The infrared (IR) spectra of the POPG membrane-bound protein showed a decrease of the band corresponding to beta-structures as compared to the protein in solution. In addition, a cooperative decrease of the beta-edge band above 70 degrees C in solution was also evident, while the transition was less cooperative and took place at lower temperature for the POPG membrane-bound protein. Low- and wide-angle X-ray diffraction experiments with lipid multilayers indicate that binding of the protein produces a rearrangement of the membrane structure, increasing the interlamellar spacing and decreasing the compactness of the lipids.

Animals↗

PubMed2003

Ceramide modulates the lipid membrane organization at molecular and supramolecular levels.

The role of lipids in membranes has changed rapidly from static to dynamic and emphasized their involvement in information transduction, linking temporal and topological structuring through compositionally driven effects. Ceramide has been described as an important modulator of different membrane functions. In mixtures with ganglioside GM1, the condensation induced by ceramide increases intermolecular interactions, leading to an increase of the phase transition temperature and size of the self-assembled structure. In mixtures with phosphatidylcholines, ceramide segregates laterally in the gel state, forming domains whose thickness depend on global concentration and chain asymmetry of the sphingolipid.

Ceramides↗

PubMed2003

Surface behavior, microheterogeneity and adsorption equilibrium of myelin at the air-water interface.

Interfacial films of whole myelin membrane adsorb at the air-water interface from myelin vesicles. The films show a liquid state and their equilibrium spreading pressure is equal to the collapse pressure (about 47 mN/m). The films appear microheterogeneous as seen by epifluorescence microscopy, consisting in two liquid phases over all the adsorption isotherm, starting with rounded liquid expanded domains (low surface pressure) immersed in a cholesterol enriched phase and reaching a fractal pattern at high surface pressure similar to those previously observed by compressing the film. Vesicles adsorb to the interfacial film mainly at the lateral interfaces. The high surface pressure at equilibrium (almost equal to the collapse pressure) indicates the formation of surface multilayers, also shown by fluorescence microscopy.

Adsorption↗