Eleonora Nicolai

Low density lipoprotein misfolding and amyloidogenesis

In early atherogenesis, subendothelial retention of lipidic droplets is associated with an inflammatory response‐to‐injury, culminating in the formation of foam cells and plaque. Low density lipoprotein (LDL) is the main constituent of subendothelial lipidic droplets. The process is believed to occur following LDL modification. Searching for a modified LDL in plasma, electronegative LDL [LDL(—)] was identified and found to be associated with major risk biomarkers. The apoprotein in LDL(—) is misfolded, and we show here that this modification primes the aggregation of native LDL, conforming to the typical pattern of protein amyloidogenesis. After a lag phase, whose length depends on LDL(—) concentration, light scattering and atomic force microscopy reveal early exponential growth of intermediate globules, which evolve into fibrils. These globules are remarkably similar to subendothelial droplets in atheromatous lesions and different from those produced by oxidation or biochemical manipulation. During aggregation, ellipticity and tryptophan fluorescence measurements reveal a domino‐style spread of apoprotein misfolding from LDL(—) to all of the LDL. Computational analysis of the apoprotein primary sequence predicts an unstable, aggregation‐prone domain in the regulatory α2 region. Apoprotein misfolding well represents an LDL modification able to transform this cholesterol carrier into a trigger for a response‐to‐injury in the artery wall.—Parasassi, T., De Spirito, M., Mei, G., Brunelli, R., Greco, G., Lenzi, L., Maulucci, G., Nicolai, E., Papi, M., Arcovito, G., Tosatto, S. C. E., Ursini, F. Low density lipoprotein misfolding and amyloidogenesis. FASEB J. 22, 2350–2356 (2008)

Structural Properties of Plant and Mammalian Lipoxygenases. Temperature-Dependent Conformational Alterations and Membrane Binding Ability †

Lipoxygenases form a heterogeneous family of lipid peroxidizing enzymes, which have been implicated in the synthesis of inflammatory mediators, in cell development and in the pathogenesis of various diseases with major health and political relevance (atherosclerosis, osteoporosis). The crystal structures of various lipoxygenase-isoforms have been reported, and X-ray coordinates for enzyme-ligand complexes are also available. Although the 3D-structures of plant and animal lipoxygenase-isoforms are very similar, recent small-angle X-ray scattering data suggested a higher degree of motional flexibility of mammalian isozymes in aqueous solutions. To explore the molecular basis for these differences we performed dynamic fluorescence measurements that allowed us to study temperature-induced conformational changes arising from three-dimensional fluctuations of the protein matrix. For this purpose, we first investigated the impact of elevated temperature on activity, secondary structure, tertiary structure dynamics and conformational alterations. Applying fluorescence resonance energy transfer we also tested the membrane binding properties of the two lipoxygenase-isoforms, and compared their binding parameters. Taken together, our results indicate that the rabbit 12/15-lipoxygenase is more susceptible to temperature-induced structural alterations than the soybean enzyme. Moreover, the rabbit enzyme exhibits a higher degree of conformational flexibility of the entire protein molecule (global flexibility) and offers the possibility of augmented substrate movement at the catalytic center (local flexibility).

Dynamics and Flexibility of Human Aromatase Probed by FTIR and Time Resolved Fluorescence Spectroscopy

Human aromatase (CYP19A1) is a steroidogenic cytochrome P450 converting androgens into estrogens. No ligand-free crystal structure of the enzyme is available to date. The crystal structure in complex with the substrate androstenedione and the steroidal inhibitor exemestane shows a very compact conformation of the enzyme, leaving unanswered questions on the conformational changes that must occur to allow access of the ligand to the active site. As H/D exchange kinetics followed by FTIR spectroscopy can provide information on the conformational changes in proteins where solvent accessibility is affected, here the amide I region was used to measure the exchange rates of the different elements of the secondary structure for aromatase in the ligand-free form and in the presence of the substrate androstenedione and the inhibitor anastrozole. Biphasic exponential functions were found to fit the H/D exchange data collected as a function of time. Two exchange rates were assigned to two populations of protons present in different flexible regions of the protein. The addition of the substrate androstenedione and the inhibitor anastrozole lowers the H/D exchange rates of the α-helices of the enzyme when compared to the ligand-free form. Furthermore, the presence of the inhibitor anastrozole lowers exchange rate constant (k1) for β-sheets from 0.22±0.06 min−1 for the inhibitor-bound enzyme to 0.12±0.02 min−1 for the free protein. Dynamics effects localised in helix F were studied by time resolved fluorescence. The data demonstrate that the fluorescence lifetime component associated to Trp224 emission undergoes a shift toward longer lifetimes (from ≈5.0 to ≈5.5 ns) when the substrate or the inhibitor are present, suggesting slower dynamics in the presence of ligands. Together the results are consistent with different degrees of flexibility of the access channel and therefore different conformations adopted by the enzyme in the free, substrate- and inhibitor-bound forms.

Tight association of N-terminal and catalytic subunits of rabbit 12/15-lipoxygenase is important for protein stability and catalytic activity.

12/15-Lipoxygenases (12/15-LOXs) have been implicated in inflammatory and hyperproliferative diseases but the structural biology of these enzymes is not well developed. Most LOXs constitute single polypeptide chain proteins that fold into a two-domain structure. In the crystal structure the two domains are tightly associated, but small angle X-ray scattering data and dynamic fluorescence studies suggested a high degree of structural flexibility involving movement of the N-terminal domain relative to catalytic subunit. When we inspected the interdomain interface we have found a limited number of side-chain contacts which are involved in interactions of these two structural subunits. One of such contact points involves tyrosine 98 of N-terminal domain. This aromatic amino acid is invariant in vertebrate LOXs regardless of overall sequence identity. To explore in more detail the role of aromatic interactions in interdomain association we have mutated Y98 to various residues and quantified the structural and functional consequences of these alterations. We have found that loss of an aromatic moiety at position 98 impaired the catalytic activity and membrane binding capacity of the mutant enzymes. Although CD and fluorescence emission spectra of wild-type and mutant enzyme species were indistinguishable, the mutation led to enlargement of the molecular shape of the enzyme as detected by analytic gel filtration and this structural alteration was shown to be associated with a loss of protein thermal stability. The possible role of tight interdomain association for the enzymes structural performance is discussed.

Estradiol Binding Prevents ApoB-100 Misfolding in Electronegative LDL(−)

Seeking for a modified lipoprotein present in plasma that could account for the atherogenic effect of high cholesterol, several years ago electronegative LDL(-) was identified. The peculiar feature of LDL(-) is an apoprotein misfolding that triggers the formation of aggregates, perfectly fitting in size the subendothelial droplets observed in early phases of atherogenesis. Apoprotein misfolding was therefore proposed as a possible atherogenic modification. LDL(-) can be spontaneously produced in vitro by plasma incubation through phospholipid hydrolysis catalyzed by the activity of endogenous phospholipases. As a consequence, apoprotein is misfolded. 17beta-Estradiol (E2), a specific ligand to apoB-100, was used to unravel the relationship between negative charge of the lipoprotein and apoprotein structural/conformational shift. Although E2 addition to plasma does not prevent LDL(-) generation nor phospholipase activity, it deeply stabilizes apoB-100 structure, thus preventing its structural and conformational shift. Apoprotein stabilization extends to lipids. Indeed, while a loosening of lipid packing is observed together with apoprotein misfolding, conversely, when E2 stabilizes apoprotein, lipid structure is preserved. Finally, even in the presence of LDL(-), the E2-stabilized LDL is resistant to aggregation, unambiguously demonstrating that misfolding, but not negative charge, primes aggregation. In conclusion, electronegative charge and misfolding are independent and distinct features of LDL(-), and apoprotein misfolding rather than the increase in the negative charge emerges both as a valid biomarker and as an appealing pharmacological and nutritional target.

Closing the Gate to the Active Site EFFECT OF THE INHIBITOR METHOXYARACHIDONYL FLUOROPHOSPHONATE ON THE CONFORMATION AND MEMBRANE BINDING OF FATTY ACID AMIDE HYDROLASE

Fatty acid amide hydrolase (FAAH) is a dimeric, membranebound enzyme that degrades neuromodulatory fatty acid amides and esters and is expressed in mammalian brain and peripheral tissues. The cleavage of ≈30 amino acids from each subunit creates an FAAH variant that is soluble and homogeneous in detergent-containing buffers, opening the avenue to the in vitro mechanistic and structural studies. Here we have studied the stability of FAAH as a function of guanidinium hydrochloride concentration and of hydrostatic pressure. The unfolding transition was observed to be complex and required a fitting procedure based on a three-state process with a monomeric intermediate. The first transition was characterized by dimer dissociation, with a free energy change of ≈11 kcal/mol that accounted for ≈80% of the total stabilization energy. This process was also paralleled by a large change in the solvent-accessible surface area, because of the hydration occurring both at the dimeric interface and within the monomers. As a consequence, the isolated subunits were found to be much less stable (ΔG ≈3 kcal/mol). The addition of methoxyarachidonyl fluorophosphonate, an irreversible inhibitor of FAAH activity, enhanced the stability of the dimer by ≈2 kcal/mol, toward denaturant- and pressure-induced unfolding. FAAH inhibition by methoxyarachidonyl fluorophosphonate also reduced the ability of the protein to bind to the membranes. These findings suggest that local conformational changes at the level of the active site might induce a tighter interaction between the subunits of FAAH, affecting the enzymatic activity and the interaction with membranes.

The fine-tuning of TRAF2-GSTP1-1 interaction: effect of ligand binding and in situ detection of the complex.

We provide the first biochemical evidence of a direct interaction between the glutathione transferase P1-1 (GSTP1-1) and the TRAF domain of TNF receptor-associated factor 2 (TRAF2), and describe how ligand binding modulates such an equilibrium. The dissociation constant of the heterocomplex is Kd=0.3 μM; however the binding affinity strongly decreases when the active site of GSTP1-1 is occupied by the substrate GSH (Kd≥2.6 μM) or is inactivated by oxidation (Kd=1.7 μM). This indicates that GSTP1-1’s TRAF2-binding region involves the GSH-binding site. The GSTP1-1 inhibitor NBDHEX further decreases the complex’s binding affinity, as compared with when GSH is the only ligand; this suggests that the hydrophobic portion of the GSTP1-1 active site also contributes to the interaction. We therefore hypothesize that TRAF2 binding inactivates GSTP1-1; however, analysis of the data, using a model taking into account the dimeric nature of GSTP1-1, suggests that GSTP1-1 engages only one subunit in the complex, whereas the second subunit maintains the catalytic activity or binds to other proteins. We also analyzed GSTP1-1’s association with TRAF2 at the cellular level. The TRAF2–GSTP1-1 complex was constitutively present in U-2OS cells, but strongly decreased in S, G2 and M phases. Thus the interaction appears regulated in a cell cycle-dependent manner. The variations in the levels of individual proteins seem too limited to explain the complex’s drastic decline observed in cells progressing from the G0/G1 to the S–G2–M phases. Moreover, GSH’s intracellular content was so high that it always saturated GSTP1-1. Interestingly, the addition of NBDHEX maintains the TRAF2–GSTP1-1 complex at low levels, thus causing a prolonged cell cycle arrest in the G2/M phase. Overall, these findings suggest that a reversible sequestration of TRAF2 into the complex may be crucial for cell cycle progression and that multiple factors are involved in the fine-tuning of this interaction.

Characterization of monomeric substates of ascorbate oxidase.

Ascorbate oxidase (AAO) is a large, multidomain, dimeric protein whose folding/unfolding pathway is characterized by a complex, multistep process. Here we used fluorescence correlation spectroscopy to demonstrate the formation of partially folded monomers by pH‐induced full dissociation into subunits. Hence, the structural features of monomeric AAO could be studied by fluorescence and CD spectroscopy. We found that the monomers keep their secondary structure, whereas subtle conformational changes in the tertiary structure become apparent. AAO dissociation has also been studied when unfolding the protein by high hydrostatic pressure at different pH values. A strong protein concentration dependence was observed at pH 8, whereas the enzyme was either monomeric or dimeric at pH 10 and 6, respectively. The calculated volume change associated with the unfolding of monomeric AAO, ΔV ∼ −55 mL·mol−1, is in the range observed for most proteins of the same size. These findings demonstrate that partially folded monomeric species might populate the energy landscape of AAO and that the overall AAO stability is crucially controlled by a few quaternary interactions at the subunits’ interface.

Physico-chemical properties of molten dimer ascorbate oxidase

The possible presence of dimeric unfolding intermediates might offer a clue to understanding the relationship between tertiary and quaternary structure formation in dimers. Ascorbate oxidase is a large dimeric enzyme that displays such an intermediate along its unfolding pathway. In this study the combined effect of high pressure and denaturing agents gave new insight on this intermediate and on the mechanism of its formation. The transition from native dimer to the dimeric intermediate is characterized by the release of copper ions forming the tri‐nuclear copper center located at the interface between domain 2 and 3 of each subunit. This transition, which is pH‐dependent, is accompanied by a decrease in volume, probably associated to electrostriction due to the loosening of intra‐subunit electrostatic interactions. The dimeric species is present even at 3 × 108 Pa, providing evidence that mechanically or chemically induced unfolding lead to a similar intermediate state. Instead, dissociation occurs with an extremely large and negative volume change (ΔV ≈ −200 mL·mol−1) by pressurization in the presence of moderate amounts of denaturant. This volume change can be ascribed to the elimination of voids at the subunit interface. Furthermore, the combination of guanidine and high pressure uncovers the presence of a marginally stable (ΔG ≈ 2 kcal·mol−1) monomeric species (which was not observed in previous equilibrium unfolding measurements) that might be populated in the early folding steps of ascorbate oxidase. These findings provide new aspects of the protein folding pathway, further supporting the important role of quaternary interactions in the folding strategy of large dimeric enzymes.

Role of dietary antioxidant (-)-epicatechin in the development of β-lactoglobulin fibrils.

Under specific physico-chemical conditions β-lactoglobulin is seen to form fibrils structurally highly similar to those that are formed by the amyloid-like proteins associated with neurodegenerative disorders, such as Alzheimer and Parkinson diseases. In the present study we provide insights on the possible role that the dietary flavonoid (-)-epicatechin plays on β-lactoglobulin fibril formation. Fibril formation is induced by keeping β-lactoglobulin solutions at pH2.0 and at a temperature of 80°C for 24h. Atomic Force Microscopy measurements suggest that, by adding (-)-epicatechin in the solution, fibrils density is visibly lowered. This last observation is confirmed by Fluorescence Correlation Spectroscopy experiments. With the use of Fourier Transform IR spectroscopy we monitored the relative abundances of the secondary structures components during the heating process. We observed that in the presence of (-)-epicatechin the spectral-weight exchange between different secondary structures is partially inhibited. Molecular Dynamics simulations have been able to provide an atomistic explanation of this experimental observation, showing that (-)-epicatechin interacts with β-lactoglobulin mainly via the residues that, normally in the absence of (-)-epicatechin, are involved in β-sheet formation. Unveiling this molecular mechanism is an important step in the process of identifying suitable molecules apt at finely tuning fibril formation like it is desirable to do in food industry applications.