Alessandra Gliozzi

Archaeal lipids and their biotechnological applications

The lipids of Archaea, based on glycerol isopranoid ethers, can be used taxonomically to distinguish between phenotypic subgroups of the domain to delineate them clearly from all other organisms. This review is a general survey of the structural features of archaeal lipids and how they relate to survival in the harsh environments in which the Archaea live. The molecular organization of archaeal lipids in monolayers, artificial black membranes and vesicles and the unique properties and possible biotechnological applications of liposomes of the lipids are presented. The results with these liposomes are compared with similar data obtained with synthetic compounds which mimic the structure of archaeal lipids. Studies on computer simulation are also reported.

Collagen Plays an Active Role in the Aggregation of β2-Microglobulin under Physiopathological Conditions of Dialysis-related Amyloidosis

Dialysis-related amyloidosis is characterized by the deposition of insoluble fibrils ofβ2-microglobulin (β2-m) in the musculoskeletal system. Atomic force microscopy inspection of ex vivo amyloid material reveals the presence of bundles of fibrils often associated to collagen fibrils. Aggregation experiments were undertaken in vitro with the aim of reproducing the physiopathological fibrillation process. To this purpose, atomic force microscopy, fluorescence techniques, and NMR were employed. We found that in temperature and pH conditions similar to those occurring in periarticular tissues in the presence of flogistic processes, β2-m fibrillogenesis takes place in the presence of fibrillar collagen, whereas no fibrils are obtained without collagen. Moreover, the morphology ofβ2-m fibrils obtained in vitro in the presence of collagen is extremely similar to that observed in the ex vivo sample. This result indicates that collagen plays a crucial role in β2-m amyloid deposition under physiopathological conditions and suggests an explanation for the strict specificity of dialysis-related amyloidosis for the tissues of the skeletal system. We hypothesize that positively charged regions along the collagen fiber could play a direct role inβ2-m fibrillogenesis. This hypothesis is sustained by aggregation experiments performed by replacing collagen with a poly-l-lysine-coated mica surface. As shown by NMR measurements, no similar process occurs when poly-l-lysine is dissolved in solution with β2-m. Overall, the findings are consistent with the estimates resulting from a simplified collagen model whereby electrostatic effects can lead to high local concentrations of oppositely charged species, such as β2-m, that decay on moving away from the fiber surface.

Heparin Strongly Enhances the Formation of β2-Microglobulin Amyloid Fibrils in the Presence of Type I Collagen

The tissue specificity of fibrillar deposition in dialysis-related amyloidosis is most likely associated with the peculiar interaction of β2-microglobulin (β2-m) with collagen fibers. However, other co-factors such as glycosaminoglycans might facilitate amyloid formation. In this study we have investigated the role of heparin in the process of collagen-driven amyloidogenesis. In fact, heparin is a well known positive effector of fibrillogenesis, and the elucidation of its potential effect in this type of amyloidosis is particularly relevant because heparin is regularly given to patients subject to hemodialysis to prevent blood clotting. We have monitored by atomic force microscopy the formation of β2-m amyloid fibrils in the presence of collagen fibers, and we have discovered that heparin strongly accelerates amyloid deposition. The mechanism of this effect is still largely unexplained. Using dynamic light scattering, we have found that heparin promotes β2-m aggregation in solution at pH 6.4. Morphology and structure of fibrils obtained in the presence of collagen and heparin are highly similar to those of natural fibrils. The fibril surface topology, investigated by limited proteolysis, suggests that the general assembly of amyloid fibrils grown under these conditions and in vitro at low pH is similar. The exposure of these fibrils to trypsin generates a cleavage at the C-terminal of lysine 6 and creates the 7–99 truncated form of β2-m (ΔN6β2-m) that is a ubiquitous constituent of the natural β2-m fibrils. The formation of this β2-m species, which has a strong propensity to aggregate, might play an important role in the acceleration of local amyloid deposition.

Monolayer black membranes from bipolar lipids of archaebacteria and their temperature-induced structural changes.

SummaryThe membrane ofCaldariella acidophila, an extreme thermophilic archaebacterium, is characterized by unusual bipolar complex lipids. They consist of two nonequivalent polar heads, linked by a C40 alkylic component. The molecular organization of these lipids in the plasma membrane is still a matter of study. In this paper, we present current-voltage measurements on artificial bipolar lipid membranes, indicating that molecules are indeed organized as a covalently bound bilayer, in which each molecule is completely stretched and spans its entire thickness. Furthermore, conformational transitions of these artificial membranes (which could be formed only above 70°C from a lipid/squalene dispersion) are analyzed in the 80 to 15°C temperature range. Abrupt variations in capacitance and valinomycin-induced conductance seem to indicate the occurrence of at least two structural changes. Measurements are also extended to different solvent systems. Results are consistent with the picture of a monolayer bipolar lipid membrane in which few solvent molecules align themselves parallel to the lipophilic chains. The amount of solvent as well as the temperature at which conformational transitions occur, depend on the solvent system in which the lipid is dispersed.

Electroporation in symmetric and asymmetric membranes

We present results of electrical measurements performed both on symmetric and asymmetric membranes in current-clamp conditions. The current-voltage characteristic curve of the membranes shows a reversible conductance transition to a higher level above a critical potential Vc. The experimental results are interpreted in the light of the electroporation theory, which allows estimates of the line tension to be made. These estimates are compared to previous experimental findings or theoretical calculations. The behaviour of symmetric membranes of different chain lengths or consisting of mixtures of short and long chains indicates a strong dependence of Vc on the chain composition and on the presence of charges on the polar head. The electroporation process is also analyzed in asymmetric bilayers consisting of a charged and an uncharged monolayer, a condition which mimics that of natural membranes. Therefore it is possible to analyze the electrical forces acting on the uncharged monolayer due to the presence of charges on the other one, under several ionic-strength conditions. It is shown that the instability arises in the uncharged monolayer, while the coupling between the two monolayers triggers the electroporation process.

Structure and permeability properties of biomimetic membranes of bolaform archaeal tetraether lipids

This review is a general survey of the properties of membranes formed by tetraether lipids extracted from microorganisms living in extreme conditions, named Archaea. After describing the unusual structure and physico-chemical properties of the membrane-spanning lipids, which allow Archaea to maintain membrane integrity in harsh environments, we consider their molecular organization in model systems such as monolayers, artificial black membranes and liposomes. The latter, due to their remarkable thermal stability, can lead to attractive biotechnological applications. Membrane permeability, local membrane fluidity and packing characteristics are reviewed with the aim of correlating structural features to permeability properties. In particular, studies on the very low passive proton permeation and leakage of entrapped molecules are reported and discussed in terms of membrane structure. Work on synthetic tetraether compounds is also reported.

The two-fold aspect of the interplay of amyloidogenic proteins with lipid membranes

Investigating the pathways leading to the formation of amyloid protein aggregates and the mechanism of their cytotoxicity is fundamental for a deeper understanding of a broad range of human diseases. Increasing evidence indicates that early aggregates are responsible for the cytotoxic effects. This paper addresses the catalytic role of lipid surfaces in promoting aggregation of amyloid proteins and the permeability changes that these aggregates induce on lipid membranes. Effects of amyloid aggregates on model systems such as monolayers, vesicles, liposomes and supported lipid bilayers are reviewed. In particular, the relevance of atomic force microscopy in detecting both kinetics of amyloid formation and amyloid-membrane interactions is emphasized.

Effect of Tetracyclines on the Dynamics of Formation and Destructuration of β2-Microglobulin Amyloid Fibrils

The discovery of methods suitable for the conversion in vitro of native proteins into amyloid fibrils has shed light on the molecular basis of amyloidosis and has provided fundamental tools for drug discovery. We have studied the capacity of a small library of tetracycline analogues to modulate the formation or destructuration of β2-microglobulin fibrils. The inhibition of fibrillogenesis of the wild type protein was first established in the presence of 20% trifluoroethanol and confirmed under a more physiologic environment including heparin and collagen. The latter conditions were also used to study the highly amyloidogenic variant, P32G. The NMR analysis showed that doxycycline inhibits β2-microglobulin self-association and stabilizes the native-like species through fast exchange interactions involving specific regions of the protein. Cell viability assays demonstrated that the drug abolishes the natural cytotoxic activity of soluble β2-microglobulin, further strengthening a possible in vivo therapeutic exploitation of this drug. Doxycycline can disassemble preformed fibrils, but the IC50 is 5-fold higher than that necessary for the inhibition of fibrillogenesis. Fibril destructuration is a dynamic and time-dependent process characterized by the early formation of cytotoxic protein aggregates that, in a few hours, convert into non-toxic insoluble material. The efficacy of doxycycline as a drug against dialysis-related amyloidosis would benefit from the ability of the drug to accumulate just in the skeletal system where amyloid is formed. In these tissues, the doxycycline concentration reaches values several folds higher than those resulting in inhibition of amyloidogenesis and amyloid destructuration in vitro.

A Major Role for Side-Chain Polyglutamine Hydrogen Bonding in Irreversible Ataxin-3 Aggregation

The protein ataxin-3 consists of an N-terminal globular Josephin domain (JD) and an unstructured C-terminal region containing a stretch of consecutive glutamines that triggers the neurodegenerative disorder spinocerebellar ataxia type 3, when it is expanded beyond a critical threshold. The disease results from misfolding and aggregation, although the pathway and structure of the aggregation intermediates are not fully understood. In order to provide insight into the mechanism of the process, we monitored the aggregation of a normal (AT3Q24) ataxin-3, an expanded (AT3Q55) ataxin-3, and the JD in isolation. We observed that all of them aggregated, although the latter did so at a much slower rate. Furthermore, the expanded AT3Q55 displayed a substantially different behavior with respect to the two other variants in that at the latest stages of the process it was the only one that did the following: i) lost its reactivity towards an anti-oligomer antibody, ii) generated SDS-insoluble aggregates, iii) gave rise to bundles of elongated fibrils, and iv) displayed two additional bands at 1604 and 1656 cm−1 in FTIR spectroscopy. Although these were previously observed in other aggregated polyglutamine proteins, no one has assigned them unambiguously, yet. By H/D exchange experiments we show for the first time that they can be ascribed to glutamine side-chain hydrogen bonding, which is therefore the hallmark of irreversibly SDS-insoluble aggregated protein. FTIR spectra also showed that main-chain intermolecular hydrogen bonding preceded that of glutamine side-chains, which suggests that the former favors the latter by reorganizing backbone geometry.

Conductance transition induced by an electric field in lipid bilayers

A cooperative phenomenon showing a structural change in the organization of bilayer lipid membranes at a critical value of the applied electric field is presented. The transition is characterized by a sharp increase in conductance. The phenomenon can be observed under current-clamp conditions (rather than the usual voltage-clamp conditions) to avoid rupturing the membrane. At a critical potential value the conductance increases and therefore the potential decreases to keep the current constant. Results refer to membranes made of egg phosphatidylcholine (PC), diphytanoylphosphatidylcholine and cholesterol/egg PC. It is found that the critical potential at which the transition occurs depends dramatically on pH and ionic concentration, indicating that the electrical properties of the external surface determine the major characteristics of such a transition.