[Heparan sulphates, amyloidosis and neurodegeneration]. / Heparan sulfatos, amiloidosis y neurodegeneracion.

INTRODUCTION: A number of neurodegenerative disorders have been linked directly to the accumulation of amyloid fibres. These fibres are made up of proteins or peptides with altered structures and which join together in vivo in association with heparan sulphate-type polysaccharides. AIMS: To examine the most recent concepts in the biology of heparan sulphates and their role in the aggregation of the peptide Abeta, of tau protein, of alpha-synuclein and of prions. The study also seeks to analyse their implications in neurodegenerative disorders such as Alzheimer's and Parkinson's disease and prion diseases. DEVELOPMENT: In vitro, heparan sulphates have played an important role in the process of oligomerisation and fibrillation of amyloidogenic proteins or peptides, in the stabilisation of these bodies and their resistance to proteolysis, thereby participating in the formation of a wide range of amyloid fibres. Heparan sulphates have also been related to the internalisation of pro-amyloid fibres during the process of intercellular propagation (spreading), which is considered to be crucial in the development of proteinopathies, the best example of which is Alzheimer's disease. CONCLUSION: This study suggests that the fine structures of heparan sulphates, their localisation in cells and tissues, together with their local concentration, may regulate the amyloidosis processes. The advances made in the understanding of this area of glyconeurobiology will make it possible to improve the understanding of the cell and molecular mechanisms underlying the neurodegenerative process.

TITLE: Heparan sulfatos, amiloidosis y neurodegeneracion.Introduccion. Numerosos trastornos neurodegenerativos se han asociado directamente a la acumulacion de fibras amiloides. Estas fibras estan formadas por proteinas o peptidos con conformaciones alteradas y que se agregan in vivo en asociacion con polisacaridos de tipo heparan sulfatos. Objetivos. Examinar los conceptos mas recientes sobre la biologia de los heparan sulfatos y su papel en la agregacion del peptido Abeta, de la proteina tau, de la alfa-sinucleina y de los priones, y analizar sus implicaciones en trastornos neurodegenerativos como las enfermedades de Alzheimer y de Parkinson y las enfermedades prionicas. Desarrollo. In vitro, los heparan sulfatos han desempeñado un papel importante en el proceso de oligomerizacion y fibrilacion de proteinas o peptidos amiloidogenos, en la estabilizacion de estos cuerpos y su resistencia a la proteolisis, participando asi en la formacion de una gran variedad de fibras amiloides. Los heparan sulfatos se han relacionado tambien con el proceso de internalizacion de fibras proamiloides durante el proceso de propagacion intercelular (spreading) considerado como central en la evolucion de las proteinopatias, cuyo mejor ejemplo es la enfermedad de Alzheimer. Conclusion. Este trabajo sugiere que las estructuras finas de los heparan sulfatos, sus localizaciones celulares y tisulares, asi como sus concentraciones locales, pueden regular los procesos de amiloidosis. Avances en la comprension de esta area de la gliconeurobiologia permitiran mejorar la comprension de los mecanismos celulares y moleculares del proceso neurodegenerativo.

Early stages of LDL oxidation: apolipoprotein B structural changes monitored by infrared spectroscopy.

Changes in the conformation of apoliprotein B-100 in the early stages of copper-mediated low density lipoprotein oxidation have been monitored by infrared spectroscopy. During the lag phase no variation in structure is observed, indicating that copper binding to the protein does not significantly affect its structure. In the propagation phase, while hydroperoxides are formed but the protein is not modified, no changes in secondary structure are observed, but the thermal profile of the band corresponding to alpha-helix is displaced in frequency, indicating changes in tertiary structure associated with this conformation but not with beta-sheet components. When aldehyde formation starts, a decrease of approximately 3% in the area of bands corresponding to alpha-helix and beta-sheet is produced, concomitantly with an increase in beta-turns and unordered structure. The two bands corresponding to beta-turns vary as well under these conditions, indicating changes in these structures. Also at this stage the thermal profile shows variations in frequency for the bands corresponding to both alpha-helix and beta-sheet. The results are consistent with the hypothesis that as soon as the polyunsaturated fatty acids from the particle core are modified, this change is reflected at the surface, in the alpha-helical components contacting the monolayer.

Protein-induced fusion of phospholipid vesicles of heterogeneous sizes.

We have investigated the fusion of phospholipid vesicles induced by lysozyme and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Vesicles were composed of dimyristoylphosphatidylcholine/dioleoylphosphatidylethanolamine/ cholesterol (DMPC:DOPE:Chol, 2:1:1). Small unilamellar vesicles (SUV, diameter ca. 30 nm) obtained by extensive sonication or large unilamellar vesicles (LUV, diameters ranged from 100 to 400 nm) obtained by extrusion methods were used. Fusion of LUV induced by lysozyme and GAPDH was drastically decreased when the diameter of the vesicles increased over a value of 100 nm. Lysozyme effect was stopped at the aggregation step while GAPDH effect was stopped at the fusion (lipid mixing) step. Fusion of heterogeneous vesicle populations (SUV with LUV) was observed only with GAPDH and this happened only when the lipids were in the liquid-crystalline state.

Tyrosine hydroxylase binds tetrahydrobiopterin cofactor with negative cooperativity, as shown by kinetic analyses and surface plasmon resonance detection.

Kinetic studies of tetrameric recombinant human tyrosine hydroxylase isoform 1 (hTH1) have revealed properties so far not reported for this enzyme. Firstly, with the natural cofactor (6R)-Lerythro-5,6,7, 8-tetrahydrobiopterin (H4biopterin) a time-dependent change (burst) in enzyme activity was observed, with a half-time of about 20 s for the kinetic transient. Secondly, nonhyperbolic saturation behaviour was found for H4biopterin with a pronounced negative cooperativity (0.39 < h < 0.58; [S]0.5 = 24 +/- 4 microM). On phosphorylation of Ser40 by protein kinase A, the affinity for H4biopterin increased ([S]0.5 = 11 +/- 2 microM) and the negative cooperativity was amplified (h = 0.27 +/- 0.03). The dimeric C-terminal deletion mutant (Delta473-528) of hTH1 also showed negative cooperativity of H4biopterin binding (h = 0.4). Cooperativity was not observed with the cofactor analogues 6-methyl-5,6,7,8-tetrahydropterin (h = 0.9 +/- 0.1; Km = 62.7 +/- 5.7 microM) and 3-methyl-5,6,7, 8-tetrahydropterin (H43-methyl-pterin)(h = 1.0 +/- 0.1; Km = 687 +/- 50 microM). In the presence of 1 mM H43-methyl-pterin, used as a competitive cofactor analogue to BH4, hyperbolic saturation curves were also found for H4biopterin (h = 1.0), thus confirming the genuine nature of the kinetic negative cooperativity. This cooperativity was confirmed by real-time biospecific interaction analysis by surface plasmon resonance detection. The equilibrium binding of H4biopterin to the immobilized iron-free apoenzyme results in a saturable positive resonance unit (DeltaRU) response with negative cooperativity (h = 0.52-0.56). Infrared spectroscopic studies revealed a reduced thermal stability both of the apo-and the holo-hTH1 on binding of H4biopterin and Lerythro-dihydrobiopterin (H2biopterin). Moreover, the ligand-bound forms of the enzyme also showed a decreased resistance to limited tryptic proteolysis. These findings indicate that the binding of H4biopterin at the active site induces a destabilizing conformational change in the enzyme which could be related to the observed negative cooperativity. Thus, our studies provide new insight into the regulation of TH by the concentration of H4biopterin which may have significant implications for the physiological regulation of catecholamine biosynthesis in neuroendocrine cells.

pH-dependent thermal transitions of lentil lectin.

The thermal stability of lentil lectin in the 5.0-10.0 pH range was studied by high-sensitivity differential scanning calorimetry and infrared spectroscopy. The thermally induced transitions for protein were irreversible and strongly dependent upon the scan rate at all pH values, suggesting that the denaturation is under kinetic control. It is shown that process of lentil lectin denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme, N-->D, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation, N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.

Differential transmembrane diffusion of triiodothyronine and thyroxine in liposomes: regulation by lipid composition.

How thyroid hormones move across biological or model membranes is a subject of controversy. The passage of the 3,5,3'triiodo l-thyronine and 3,5,3',5' tetraiodo l-thyronine across model membranes was evaluated by the addition of the hormones to liposomes containing 2, 4,6-trinitrobenzene sulfonic acid. Results indicate that hormones can react with an amino-reactive compound pre-encapsulated into phosphatidylcholine liposomes. The transversal motions of thyroid hormones were characterized by using physiological concentration levels of (125I) 3,5,3'triiodo l-thyronine and (125I) 3,5,3',5' tetraiodo l-thyronine. The hormone distribution between the two monolayers was time-dependent and kinetic data were fitted to a single exponential. Results obtained show that 3,5,3' triiodo l-thyronine can permeate phospholipid membranes and the diffusion time increases in the gel and liquid-ordered phase. On the contrary, 3,5,3', 5' tetraiodo l-thyronine could not diffuse the liposomal membrane from dimyristoyl and dipalmitoyl phosphatidylcholine in gel phase and egg yolk phosphatidylcholine:cholesterol in the liquid-ordered phase. Our results in the liquid-ordered phase suggest that diffusion movement of thyroid hormones across cell membranes depends on the amount of cholesterol in the bilayer.

Thermal and pH-induced conformational changes of a beta-sheet protein monitored by infrared spectroscopy.

The stability of a lentil lectin, an all-beta protein, has been perturbed by changes in pH and temperature. In the pH interval 5.0 --> 10.0, the overall secondary structure does not undergo significant changes. However, if the individual components of the infrared amide I band are considered, changes in band components attributed to variations in beta-sheet and beta-turns cross-interactions are detected. The combined effects of pH and temperature reveal that the protein is more compact at pH 7.5 with lower denaturation temperatures at pH 5.0 or 10.0, indicating a less stable protein under those conditions. According to our results, the structural stability of the beta-sheet would depend not only on the intermolecular interactions among the strands but also on the conformation of the segments connecting these strands. The protein infrared band assignment has also been examined since the three-dimensional structure of the lentil lectin protein is known from X-ray diffraction studies. Two of the bands observed are attributed to beta-sheet. The one at 1620 cm-1, not affected if the medium is deuterated, is assigned to hairpins composed by two strands connected by a rigid turn whereas that located at 1633 cm-1 corresponds to strands associated by more flexible segments. The band appearing at 1645 cm-1 in H2O corresponds to the open, flexible loops that are connecting the beta-strands. The simplest assumption of the various secondary structure components having identical IR extinction coefficients is enough to provide IR-derived data that are in good agreement with the structure solved by X-ray diffraction.

Domain structure and stability of human phenylalanine hydroxylase inferred from infrared spectroscopy.

We have studied the conformation and thermal stability of recombinant human phenylalanine hydroxylase (hPAH) and selected truncated forms, corresponding to distinct functional domains, by infrared spectroscopy. The secondary structure of wild-type hPAH was estimated to be 48% alpha-helix, 28% extended structures, 12% beta-turns and 12% non-structured conformations. The catalytic C-terminal domain (residues 112-452) holds most of the regular secondary structure elements, whereas the regulatory N-terminal domain (residues 2-110) adopts mainly an extended and disordered, flexible conformation. Thermal stability studies of the enzyme forms indicate the existence of interactions between the two domains. Our results also demonstrate that the conformational events involved in the activation of hPAH by its substrate (L-Phe) are mainly related to changes in the tertiary/quaternary structure. The activating effect of phosphorylation, however, affects the secondary structure of the N-terminal domain of the protein.

Differential effect of triiodothyronine and thyroxine on liposomes containing cholesterol: physiological speculations.

The effect of thyroid hormones on the steady-state fluorescence polarization and on the release of the liposomal content was analyzed in liposomes composed of egg phosphatidylcholine and egg phosphatidyl choline:cholesterol in different molar ratios. Depending on liposome cholesterol composition, a dual effect of triiodothyronine was found. The fluorescence polarization of 1,6 diphenyl 1,3,5 hexatriene or 1-(4-trimethylaminophenyl) 6 phenyl-1, 3, 5 hexatriene decreased by the addition of the hormone when cholesterol content was in the range from 0 to 30 moles %, while it increased with cholesterol from 30 to 50 moles %. In the release experiments, the effect of triiodothyronine was also biphasic; the leakage was the highest at 0% and 50% and the lowest at 30 moles % of cholesterol. On the contrary, thyroxine was without effect on liposomes containing cholesterol from 30 to 50 mol %. This fact correlated with a lower incorporation of thyroxine, compared with that of triiodothyronine in liposomes containing up to 30 moles % of cholesterol. The fact that the above differential incorporation of thyroid hormones was also observed at physiological concentration and that most of the mammalian membrane cells have more than 25 moles % of cholesterol have for physiological implications to the observations reported here.

Differential effect of triiodothyronine and thyroxine on the liposomal membrane in liquid-crystalline and gel state.

The effect of thyroid hormones on the degree of order or fluidity of dimyristoyl, dipalmitoyl or egg yolk phosphatidyl choline liposomes was evaluated by fluorescence spectroscopy methods. The freedom of molecular motion above the phase transition temperature was decreased, while below the transition, the mobility was actually increased by the incorporation of triiodothyronine to liposomes. While thyroxine decreases the fluidity in the liquid crystalline state, it cannot increase the fluidity in the gel state. A differential effect of triiodothyronine and thyroxine on the release of the liposomal content was found, depending on the liquid crystalline or gel state of the liposomes. These facts were correlated with the differential incorporation of the hormones to liposomes above and below the phase transition temperature of dimyristoyl and dipalmitoyl phospholipid choline. In gel state, a low incorporation of thyroxine compared with triiodothyronine was found.