Annalisa Relini

A causative link between the structure of aberrant protein oligomers and their toxicity

The aberrant assembly of peptides and proteins into fibrillar aggregates proceeds through oligomeric intermediates that are thought to be the primary pathogenic species in many protein deposition diseases. We describe two types of oligomers formed by the HypF-N protein that are morphologically and tinctorially similar, as detected with atomic force microscopy and thioflavin T assays, though one is benign when added to cell cultures whereas the other is toxic. Structural investigation at a residue-specific level using site-directed labeling with pyrene indicated differences in the packing of the hydrophobic interactions between adjacent protein molecules in the oligomers. The lower degree of hydrophobic packing was found to correlate with a higher ability to penetrate the cell membrane and cause an influx of Ca(2+) ions. Our findings suggest that structural flexibility and hydrophobic exposure are primary determinants of the ability of oligomeric assemblies to cause cellular dysfunction and its consequences, such as neurodegeneration.

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.

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.

Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein oligomers

Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded oligomers preformed from three different proteins added extracellularly to cultured cells. All the chaperones were found to decrease oligomer toxicity significantly, even at very low chaperone/protein molar ratios, provided that they were added extracellularly rather than being overexpressed in the cytosol. Infrared spectroscopy and site-directed labeling experiments using pyrene ruled out structural reorganizations within the discrete oligomers. Rather, confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and chaperones. Moreover, atomic force microscopy imaging indicated that larger assemblies of oligomers are formed in the presence of the chaperones. This suggests that the chaperones bind to the oligomers and promote their assembly into larger species, with consequent shielding of the reactive surfaces and a decrease in their diffusional mobility. Overall, the data indicate a generic ability of chaperones to neutralize extracellular misfolded oligomers efficiently and reveal that further assembly of protein oligomers into larger species can be an effective strategy to neutralize such extracellular species.

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.

Aβ(1-42) Aggregates into Non-Toxic Amyloid Assemblies in the Presence of the Natural Polyphenol Oleuropein Aglycon

Amyloid aggregation starts with the initial misfolding of peptide/protein precursors, with subsequent structural rearrangement into oligomers and protofibrils; the latter eventually organize into fibrils with shared basic structural features, found deposited in amyloid diseases. Mounting evidence indicates early oligomers as the most toxic amyloid species; accordingly, the search of inhibitors of their growth is considered a promising target to prevent amyloid toxicity. We recently showed that oleuropein aglycon, a polyphenol abundant in the extra virgin olive oil, interferes with the aggregation of amylin (involved in type-2 diabetes), eliminating its cytotoxicity. Here we report that oleuropein aglycon also hinders amyloid aggregation of Aβ(1-42) and its cytotoxicity, suggesting a general effect of such polyphenol. In particular, by using a wide panel of different spectroscopic, immunologic, cell viability and imaging techniques we provide a more detailed description of Aβ(1-42) structural modifications arising in the presence of the inhibitor and the resulting cytotoxicity. We here report that the polyphenol eliminates the appearance of early toxic oligomers favouring the formation of stable harmless protofibrils, structurally different from the typical Aβ(1-42) fibrils. We also show that oleuropein aglycon is maximally effective when is present at the beginning of the aggregation process; furthermore, when added to preformed fibrils, it does not induce the release of toxic oligomers but, rather, neutralizes any residual toxicity possibly arising from the residual presence of traces of soluble oligomers and other toxic aggregates. The possible use of this polyphenol as anti-aggregation molecule is discussed in the light of these data.

Kinetic analysis of amyloid formation in the presence of heparan sulfate: faster unfolding and change of pathway.

A number of human diseases are associated with the conversion of proteins from their native state into well defined fibrillar aggregates, depositing in the extracellular space and generally termed amyloid fibrils. Heparan sulfate (HS), a glycosaminoglycan normally present in the extracellular matrix, has been found to be universally associated with amyloid deposits and to promote amyloid fibril formation by all studied protein systems. We have studied the impact of HS on the amyloidogenesis of human muscle acylphosphatase, monitoring the process with an array of techniques, such as normal and stopped-flow far-UV circular dichroism, thioflavin T fluorescence, static and dynamic light scattering, and atomic force microscopy. The results show that HS accelerates the conversion of the studied protein from the native state into the amyloidogenic, yet monomeric, partially folded state. They also indicate that HS does not simply accelerate the conversion of the resulting partially folded state into amyloid species but splits the process into two distinct pathways occurring in parallel: a very fast phase in which HS interacts with a fraction of protein molecules, causing their rapid aggregation into ThT-positive and β-sheet containing oligomers, and a slow phase resulting from the normal aggregation of partially folded molecules that cannot interact with HS. The HS-mediated aggregation pathway is severalfold faster than that observed in the absence of HS. Two aggregation phases are generally observed when proteins aggregate in the presence of HS, underlying the importance of a detailed kinetic analysis to fully understand the effect of this glycosaminoglycan on amyloidogenesis.