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Phosphorylation of the neuronal Tau protein is implicated in both the regulation of its physiological function of microtubule stabilization and its pathological propensity to aggregate into the fibers that characterize Alzheimer's diseased neurons. However, how specific phosphorylation events influence both aspects of Tau biology remains largely unknown. In this study, we address the structural impact of phosphorylation of the Tau protein by Nuclear Magnetic Resonance (NMR) spectroscopy on a functional fragment of Tau (Tau[Ser208-Ser324] = TauF4). TauF4 was phosphorylated by the proline-directed CDK2/CycA3 kinase on Thr231 (generating the AT180 epitope), Ser235, and equally on Thr212 and Thr217 in the Proline-rich region (Tau[Ser208-Gln244] or PRR). These modifications strongly decrease the capacity of TauF4 to polymerize tubulin into microtubules. While all the NMR parameters are consistent with a globally disordered Tau protein fragment, local clusters of structuration can be defined. The most salient result of our NMR analysis is that phosphorylation in the PRR stabilizes a short α-helix that runs from pSer235 till the very beginning of the microtubule-binding region (Tau[Thr245-Ser324] or MTBR of TauF4). Phosphorylation of Thr231/Ser235 creates a N-cap with helix stabilizing role while phosphorylation of Thr212/Thr217 does not induce modification of the local transient secondary structure, showing that the stabilizing effect is sequence specific. Using paramagnetic relaxation experiments, we additionally show a transient interaction between the PRR and the MTBR, observed in both TauF4 and phospho-TauF4. Proteins 2011;. © 2011 Wiley Periodicals, Inc.

Nucleolin is a major nucleolar protein involved in fundamental processes of ribosome biogenesis, regulation of cell proliferation and growth. Nucleolin is known to shuttle between nucleus, cytoplasm and cell surface. We have previously found that nucleolin undergoes complex N- and O-glycosylations in extra-nuclear isoforms. We found that surface nucleolin is exclusively glycosylated and that N-glycosylation is required for its expression on the cells. Interestingly, the two N-glycans are located in the RNA-binding domains (RBDs) which participate in the self-association properties of nucleolin. We hypothesized that the occupancy of RBDs by N-glycans plays a role in these self-association properties. Here, owing to the inability to quantitatively produce full-size nucleolin, we expressed four N-glycosylation nucleolin variants lacking the N-terminal acidic domain in a baculovirus/insect cell system. As assessed by heptafluorobutyrate derivatization and mass spectrometry, this strategy allowed the production of proteins bearing or not paucimannosidic-type glycans on either one or two of the potential N-glycosylation sites. Their structure was investigated by circular dichroism and fluorimetry, and their ability to self-interact was analyzed by electrophoresis and surface plasmon resonance. Our results demonstrate that all nucleolin-derived variants are able to self-interact and that N-glycosylation on both RBD1 and RBD3, or RBD3 alone, but not RBD1 alone, modifies the structure of the N-terminally truncated nucleolin and enhances its self-association properties. In contrast, N-glycosylation does not modify interaction with lactoferrin, a ligand of cell surface nucleolin. Our results suggest that the occupancy of the N-glycosylation sites may contribute to expression and functions of surface nucleolin. Structured digital abstract • NCT binds to NCT by surface plasmon resonance (View interaction) • R3CT binds to R3CT by surface plasmon resonance (View interaction) • RCT binds to RCT by surface plasmon resonance (View interaction) • RCT binds to RCT by blue native page (View interaction) • NCT binds to lactoferrin by surface plasmon resonance (View interaction) • R3CT binds to R3CT by blue native page (View interaction)

Thymine-DNA glycosylases (TDGs) initiate base excision repair by debasification of the erroneous thymine or uracil nucleotide in G.T and G.U mispairs which arise at high frequency through spontaneous or enzymatic deamination of methylcytosine and cytosine, respectively. Human TDG has furthermore been shown to have a functional role in transcription and epigenetic regulation through the interaction with transcription factors from the nuclear receptor superfamily, transcriptional coregulators, and a DNA methyltransferase. The TDG N-terminus encodes regulatory functions, as it assures both G.T versus G.U specificity and contains the sites for interaction and posttranslational modification by transcription-related activities. While the molecular function of the evolutionarily conserved central catalytic domain of TDG in base excision repair has been elucidated by determination of its three-dimensional structure, the mechanisms by which the N-terminus exerts its regulatory roles, as well as the function of TDG in transcription regulation, remain to be understood. We describe here the residual structure of the TDG N-terminus in both contexts of the isolated domain and the entire protein. These studies lead to the characterization of a small structural domain in the TDG N-terminal region preceding the catalytic core and coinciding with the region of functional regulation of TDG's activities. This regulatory domain exhibits a small degree of organization and is implicated in dynamic molecular interactions with the catalytic domain and nonselective interactions with double-stranded DNA, providing a molecular explanation for the evolutionarily acquired G.T mismatch processing activity of TDG.

Whereas the interaction between Tau and the microtubules has been studied in great detail both by macroscopic techniques (cosedimentation, cryo-electron microscopy, and fluorescence spectroscopy) using the full-length protein or by peptide mapping assays, no detailed view at the level of individual amino acids has been presented when using the full-length protein. Here, we present a nuclear magnetic resonance (NMR) study of the interaction between the full-length neuronal protein Tau and paclitaxel-stabilized microtubules (MTs). As signal disappearance in the heteronuclear 1H-15N correlation spectra of isotope-labeled Tau in complex with MTs is due to direct association of the corresponding residue with the solid-like MT wall, we can map directly the fragment in interaction with the MT surface, and obtain a molecular picture of the precise interaction zones. The N-terminal region projects from the microtubule surface, and the lack of chemical shift variations when compared with free Tau proves that this region can regulate microtubular separation without adopting a stable conformation. Amino acids in the four microtubule binding repeats (MTBRs) lose all of their intensity, underscoring their immobilization upon binding to the MTs. The same loss of NMR intensity was observed for the proline-rich region starting at Ser214, underscoring its importance in the Tau:MT interaction. Fluorescence resonance energy transfer (FRET) experiments were used to obtain thermodynamic binding parameters, and led to the conclusion that the NMR defined fragment indeed is the major player in the interaction. When the same Ser214 is phosphorylated by the PKA kinase, the Tau:MT interaction strength decreases by 2 orders of magnitude, but the proline-rich region including the phospho-Ser214 does not gain sufficient mobility in the complex to make it observable by NMR spectroscopy. The presence of an intramolecular disulfide bridge, on the contrary, does lead to a partial detachment of the C-terminus of Tau, and decreases significantly the overloading of Tau on the MT surface.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and its interaction with the human chaperone cyclophilin A are both targets for highly potent and promising antiviral drugs that are in the late stages of clinical development. Despite its high interest in regards to the development of drugs to counteract the worldwide HCV burden, NS5A is still an enigmatic multifunctional protein poorly characterized at the molecular level. NS5A is required for HCV RNA replication and is involved in viral particle formation and regulation of host pathways. Thus far, no enzymatic activity or precise molecular function has been ascribed to NS5A that is composed of a highly structured domain 1 (D1), as well as two intrinsically disordered domains 2 (D2) and 3 (D3), representing half of the protein. Here, we identify a short structural motif in the disordered NS5A-D2 and report its NMR structure. We show that this structural motif, a minimal Pro314–Trp316 turn, is essential for HCV RNA replication, and its disruption alters the subcellular distribution of NS5A. We demonstrate that this Pro-Trp turn is required for proper interaction with the host cyclophilin A and influences its peptidyl-prolyl cis/trans isomerase activity on residue Pro314 of NS5A-D2. This work provides a molecular basis for further understanding of the function of the intrinsically disordered domain 2 of HCV NS5A protein. In addition, our work highlights how very small structural motifs present in intrinsically disordered proteins can exert a specific function. 290;31

The neuronal Tau protein is involved in stabilizing microtubules but is also the major component of the paired helical filaments (PHFs), the intracellular aggregates that characterize Alzheimer's disease (AD) in neurons. In vitro, Tau can be induced to form AD-like aggregates by adding polyanions such as heparin. While previous studies have identified the microtubule binding repeats (MTBRs) as the major player in Tau aggregation, the fact that the full-length protein does not aggregate by itself indicates the presence of inhibitory factors. Charge and conformational changes are of uttermost importance near the second (R2) and third (R3) MTBR that are thought to be involved directly in the nucleation of the aggregation. Recently, the positively charged regions flanking the MTBR were proposed to inhibit PHF assembly, where hyperphosphorylation neutralizes these basic inhibitory domains, enabling Tau-Tau interactions. Here we present results of an NMR study on the interaction between intact full-length Tau and small heparin fragments of well-defined size, under conditions where no aggregation occurs. Our findings reveal (i) micromolar affinity of heparin to residues in R2 and R3, (ii) two zones of strong interaction within the positively charged inhibitory regions flanking the MTBR, and (iii) another interaction site upstream of the two inserts encoded by exons 2 and 3. Three-dimensional heteronuclear NMR experiments demonstrate that the interaction with heparin induces beta-strand structure in several regions of Tau that might act as nucleation sites for its aggregation but indicate as well alpha-helical structure in regions outside the core of PHF. In the PHF, the residues outside of the core maintain sufficient mobility for NMR detection and recover their unbound chemical shift values after an overnight incubation at 37 degrees C with heparin. Heparin thus becomes integrated into the rigid core region of the PHF, probably providing the charge compensation for the lysine-rich stretches that form upon the in-register, parallel stacking of the repeat regions. The neuronal Tau protein is involved in stabilizing microtubules but is also the major component of the paired helical filaments (PHFs), the intracellular aggregates that characterize Alzheimer's disease (AD) in neurons. In vitro, Tau can be induced to form AD-like aggregates by adding polyanions such as heparin. While previous studies have identified the microtubule binding repeats (MTBRs) as the major player in Tau aggregation, the fact that the full-length protein does not aggregate by itself indicates the presence of inhibitory factors. Charge and conformational changes are of uttermost importance near the second (R2) and third (R3) MTBR that are thought to be involved directly in the nucleation of the aggregation. Recently, the positively charged regions flanking the MTBR were proposed to inhibit PHF assembly, where hyperphosphorylation neutralizes these basic inhibitory domains, enabling Tau-Tau interactions. Here we present results of an NMR study on the interaction between intact full-length Tau and small heparin fragments of well-defined size, under conditions where no aggregation occurs. Our findings reveal (i) micromolar affinity of heparin to residues in R2 and R3, (ii) two zones of strong interaction within the positively charged inhibitory regions flanking the MTBR, and (iii) another interaction site upstream of the two inserts encoded by exons 2 and 3. Three-dimensional heteronuclear NMR experiments demonstrate that the interaction with heparin induces beta-strand structure in several regions of Tau that might act as nucleation sites for its aggregation but indicate as well alpha-helical structure in regions outside the core of PHF. In the PHF, the residues outside of the core maintain sufficient mobility for NMR detection and recover their unbound chemical shift values after an overnight incubation at 37 degrees C with heparin. Heparin thus becomes integrated into the rigid core region of the PHF, probably providing the charge compensation for the lysine-rich stretches that form upon the in-register, parallel stacking of the repeat regions. The neuronal Tau protein is involved in stabilizing microtubules but is also the major component of the paired helical filaments (PHFs), the intracellular aggregates that characterize Alzheimer's disease (AD) in neurons. In vitro, Tau can be induced to form AD-like aggregates by adding polyanions such as heparin. While previous studies have identified the microtubule binding repeats (MTBRs) as the major player in Tau aggregation, the fact that the full-length protein does not aggregate by itself indicates the presence of inhibitory factors. Charge and conformational changes are of uttermost importance near the second (R2) and third (R3) MTBR that are thought to be involved directly in the nucleation of the aggregation. Recently, the positively charged regions flanking the MTBR were proposed to inhibit PHF assembly, where hyperphosphorylation neutralizes these basic inhibitory domains, enabling Tau-Tau interactions. Here we present results of an NMR study on the interaction between intact full-length Tau and small heparin fragments of well-defined size, under conditions where no aggregation occurs. Our findings reveal (i) micromolar affinity of heparin to residues in R2 and R3, (ii) two zones of strong interaction within the positively charged inhibitory regions flanking the MTBR, and (iii) another interaction site upstream of the two inserts encoded by exons 2 and 3. Three-dimensional heteronuclear NMR experiments demonstrate that the interaction with heparin induces beta-strand structure in several regions of Tau that might act as nucleation sites for its aggregation but indicate as well alpha-helical structure in regions outside the core of PHF. In the PHF, the residues outside of the core maintain sufficient mobility for NMR detection and recover their unbound chemical shift values after an overnight incubation at 37 degrees C with heparin. Heparin thus becomes integrated into the rigid core region of the PHF, probably providing the charge compensation for the lysine-rich stretches that form upon the in-register, parallel stacking of the repeat regions.

The mechanisms involved in the virulence of Yersinia pestis , the plague pathogen, are not fully understood. In previous research, we found that a Y. pestis mutant lacking the HicB3 (YPO3369) putative orphan antitoxin was attenuated for virulence in a murine model of bubonic plague. Toxin-antitoxin systems (TASs) are widespread in prokaryotes. Most bacterial species possess many TASs of several types. In type II TASs, the toxin protein is bound and neutralized by its cognate antitoxin protein in the cytoplasm. Here we identify the hicA3 gene encoding the toxin neutralized by HicB3 and show that HicA3-HicB3 constitutes a new functional type II TAS in Y. pestis . Using biochemical and mutagenesis-based approaches, we demonstrate that the HicA3 toxin is an RNase with a catalytic histidine residue. HicB3 has two functions: it sequesters and neutralizes HicA3 by blocking its active site, and it represses transcription of the hicA3B3 operon. Gel shift assays and reporter fusion experiments indicate that the HicB3 antitoxin binds to two operators in the hicA3B3 promoter region. We solved the X-ray structures of HicB3 and the HicA3-HicB3 complex; thus, we present the first crystal structure of a TA complex from the HicAB family. HicB3 forms a tetramer that can bind two HicA3 toxin molecules. HicA3 is monomeric and folds as a double-stranded-RNA-binding domain. The HicB3 N-terminal domain occludes the HicA3 active site, whereas its C-terminal domain folds as a ribbon-helix-helix DNA-binding motif.

Maritime pine (Pinus pinaster)-seed oil contains two Δ5 unsaturated polymethylene interrupted fatty acids (allcis-5,9,12–18:3 and allcis-5,11,14–20:3 acids) one of which resembles eicosapentaenoic acid. The goal of the present study was to test whether maritime pine-seed oil consumption affects HDL and apolipoprotein (Apo) A-I levels as well as the ability of serum to promote efflux of cholesterol from cultured cells. To this end, wild type (WT) non-transgenic mice and transgenic mice expressing human ApoA-I (HuA-ITg) were fed on isoenergetic diet containing either 200 g maritime pine-seed oil/kg or 200 g lard/kg for 2 weeks. WT and HuA-ITg mice fed maritime pine-seed oil had lower cholesterol, HDL-cholesterol, LDL-cholesterol and HuA-ITg mice had lower human ApoA-I than those fed lard. The differences in cholesterol (P< 0·0001) and HDL-cholesterol (P< 0·003) levels between mice fed on the two diets were more pronounced in the HuA-ITg than in the WT mice. The ability of HuA-ITg serum to promote cholesterol efflux in cultured cells was greater (P< 0·008) than that of WT animals. However, the maritime pine-seed oil diet was associated with lower (P< 0·005)in vitrocholesterol efflux ability than the lard diet in both mice genotypes. This suggests a negative effect of the maritime pine-seed oil on reverse cholesterol transport. Cholesterol efflux was correlated with serum free or esterified cholesterol and phospholipid levels. The slope of the regression line was smaller in the HuA-ITg than in the WT mice indicating that overexpression of human ApoA-I reduces the negative impact of maritime pine-seed oil on cholesterol efflux. In conclusion, maritime pine-seed oil diet lowers HDL-cholesterol and diminishesin vitrocholesterol efflux. This potentially detrimental effect is attenuated by overexpression of human ApoA-I in mice.

To gain insight into the nature of the lecithin-cholesterol acyltransferase inhibitory factor(s), we separated and collected the oxidation products from oxidized lipoproteins after lipoxygenase treatment. Isolated fractions identified by chemiluminescence, as hydroperoxides of phosphatidylcholine, were found to produce a significant reduction of lecithin-cholesterol acyltransferase activity. The reaction kinetics of lecithin-cholesterol acyltransferase with reconstitued high density lipoproteins were studied in the presence of 0.6 and 1.2 μM hydroperoxides of phosphatidylcholine. No significant changes in the apparent Vmax were observed but a concentration-dependent increase in slope of the reciprocal plots and in the apparent Km values was observed with increasing hydroperoxide concentrations. These results show that the active site of lecithin-cholesterol acyltransferase is not affected by the presence of phosphatidylcholine hydroperoxides. Nevertheless, hydroperoxides of phosphatidylcholine altered the reactivity of lecithin-cholesterol acyltransferase for reconstitued high density lipoproteins suggesting either an alteration of the binding of lecithin-cholesterol acyltransferase to the reconstitued high density lipoproteins or a competitive inhibition mechanism.

Fibrates are widely used as lipid lowering drugs acting as peroxisome proliferator-activated receptors α (PPARα) agonists and modulating the expression of several genes involved in lipid and lipoprotein metabolism. Much less is known on the effect of fibrates in HDL structure and composition. Therefore, we examined whether fenofibrate induces quantitative and/or qualitative modifications in HDL metabolism in the rabbit, an animal that, contrary to rodents and similar to humans, is less sensitive to peroxisome proliferators. We first demonstrated that 3-week treatment with fenofibrate (250 mg/kg/day) induced an important increase in serum apolipoprotein A-I, HDL-cholesterol and HDL-phospholipids concentrations and a relative enrichment in HDL cholesteryl ester content. Moreover, the fatty acid profiles from fenofibrate-treated rabbits displayed a dramatic increase in the serum or HDL C18:3 ω6 to C18:2 ω6 ratio suggesting higher Δ6 desaturase activity. In addition, HDL from fenofibrate-treated animals exhibited higher relative proportions of sphingomyelin, phosphatidylinositol and phosphatidylethanolamine. We then reported that fenofibrate induced major changes in the physical characteristics of HDL, mainly a higher size and a faster mobility on agarose gel electrophoresis. Finally, serum or HDL from treated rabbits exhibited higher capacity to promote cholesterol efflux from Scavenger receptor class B type I (SR-BI)-rich Fu5AH cells compared to controls. Our findings demonstrate that fenofibrate has beneficial effects in rabbits by increasing the mass of the circulating HDL pool and by modifying their composition transforming them as better acceptors of cellular cholesterol through SR-BI pathway. These effects of fenofibrate might contribute to its benefits on the prevention and treatment of atherosclerosis.

International audience; We describe our efforts to combine in vitro enzymatic reactions with recombinant kinases to phosphorylate the neuronal tau protein, and NMR spectroscopy to unravel the resulting phosphorylation pattern in both qualitative and quantitative manners. This approach, followed by functional assays with the same samples, gives access to the complex phosphorylation code of tau. As a result, we propose a novel hypothesis for the link between tau (hyper)phosphorylation and aggregation.

The effect of n-propanol on the secondary and tertiary structure of human apolipoprotein A-I (apoA-I), an interfacial protein, was investigated using near and far ultraviolet (UV)-circular dichroism (CD) and fluorescence spectroscopy, as well as limited proteolytic digestion with trypsin, and cross-linking with bis(sulfosuccinimidyl) suberate. The structure of apoA-I in n-propanol (30%, v/v) was compared with that in Tris buffer and in reconstituted, spherical or discoidal, high density lipoproteins (rHDL). Addition of n-propanol to apoA-I in Tris buffer induces major changes in its near and far CD spectra: α-helical content increases by 27% and the near UV-CD spectrum becomes very similar to that of apoA-I in rHDL particles. Fluorescence spectral, lifetime, and polarization results, and quenching by KI confirm that major structural changes occur in the N-terminal half of apoA-I as n-propanol is added: the Trp residues become more exposed to solvent than in buffer alone or in rHDL. Higher concentrations of guanidine hydrochloride or urea are required to denature apoA-I in n-propanol than in buffer alone, but a similar free energy of unfolding is observed. The N-terminus of apoA-I is relatively resistant to trypsin digestion and the C-terminus has equivalent digestion sites for apoA-I in the three states, but the kinetics of digestion are much slower in n-propanol and in rHDL compared to apoA-I in Tris buffer. Cross-linking experiments reveal that dimers of apoA-I exist in n-propanol, in contrast to dimers plus multimeric aggregates in Tris buffer. From these results we conclude that in 30% n-propanol the structure of apoA-I approaches that of ‘native’ lipid-bound apoA-I, in contrast to its structure in the aqueous Tris buffer.

International audience; Nonstructural protein 5A (NS5A) is essential for Hepatitis C Virus (HCV) replication and constitutes an attractive target for antiviral drug development. While structural data for its in-plane membrane anchor and domain D1 are available, the structure of domains 2 (D2) and 3 (D3) remain poorly defined. We report here a comparative molecular characterization of the NS5A-D3 domains of the HCV JFH-1 (genotype 2a) and Con1 (genotype 1b) strains. Combining gel filtration, circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy analyses, we show that NS5A-D3 is natively unfolded. However, NS5A-D3 domains from both JFH-1 and Con1 strains exhibit propensity to partially fold into an alpha-helix. NMR analysis identifies two putative alpha-helices, for which a molecular model could be obtained. The amphipathic nature of the first helix and its conservation in all genotypes suggest that it might correspond to a molecular recognition element (MoRE), and as such promote the interaction with relevant biological partner(s). As mutations conferring resistance to cyclophilin inhibitors have been mapped into NS5A-D3, we also investigated the functional interaction between NS5A-D3 and Cyclophilin A (CypA). CypA indeed interacts with NS5A-D3 and this interaction is completely abolished by Cyclosporin A (CsA). NMR heteronuclear exchange experiments demonstrate that CypA has in vitro PPIase activity toward some, but not all, of the peptidyl-prolyl bonds in NS5A-D3. These studies lead to novel insights into the structural features of NS5A-D3 and its relationships with CypA.

International audience; Phosphorylation of the microtubule-associated Tau protein plays a major role in the regulation of its activity of tubulin polymerization and/or stabilization of microtubule assembly. A dysregulation of the phosphorylation/dephosphorylation balance leading to the hyperphosphorylation of Tau proteins in neurons is thought to favor their aggregation into insoluble filaments. This in turn might underlie neuronal death as encountered in many neurodegenerative disorders, including Alzheimer's disease. Another post-translational modification, the O-linked β-N-acetylglucosaminylation (O-GlcNAcylation), controls the phosphorylation state of Tau, although the precise mechanism is not known. Moreover, analytical difficulties have hampered the precise localization of the O-GlcNAc sites on Tau, except for the S400 site that was very recently identified on the basis of ETD-FT-MS. Here, we identify three O-GlcNAc sites by screening a library of small peptides sampling the proline-rich, the microtubule-associated repeats and the carboxy-terminal domains of Tau as potential substrates for the O-β-N-acetylglucosaminyltransferase (OGT). The in vitro activity of the nucleocytoplasmic OGT was assessed by tandem mass spectrometry and NMR spectroscopy. Using phosphorylated peptides, we establish the relationship between phosphate and O-GlcNAc incorporation at these sites. Phosphorylation of neighboring residues S396 and S404 was found to decrease significantly S400 O-GlcNAcylation. Reciprocally, S400 O-GlcNAcylation reduces S404 phosphorylation by the CDK2/cyclinA3 kinase and interrupts the GSK3β-mediated sequential phosphorylation process.

International audience; Alzheimer disease neurons are characterized by extraneuronal plaques formed by aggregated amyloid-β peptide and by intraneuronal tangles composed of fibrillar aggregates of the microtubule-associated Tau protein. Tau is mostly found in a hyperphosphorylated form in these tangles. Glycogen synthase kinase 3β (GSK3β) is a proline-directed kinase generally considered as one of the major players that (hyper)phosphorylates Tau. The kinase phosphorylates mainly (Ser/Thr)-Pro motifs and is believed to require a priming activity by another kinase. Here, we use an in vitro phosphorylation assay and NMR spectroscopy to characterize in a qualitative and quantitative manner the phosphorylation of Tau by GSK3β. We find that three residues can be phosphorylated (Ser-396, Ser-400, and Ser-404) by GSK3β alone, without priming. Ser-404 is essential in this process, as its mutation to Ala prevents all activity of GSK3β. However, priming enhances the catalytic efficacy of the kinase, as initial phosphorylation of Ser-214 by the cAMP-dependent protein kinase (PKA) leads to the rapid modification by GSK3β of four regularly spaced additional sites. Because the regular incorporation of negative charges by GSK3β leads to a potential parallel between phospho-Tau and heparin, we investigated its interaction with the heparin/low density lipoprotein receptor binding domain of human apolipoprotein E. We indeed observed an interaction between the GSK3β-promoted regular phospho-pattern on Tau and the apolipoprotein E fragment but none in the absence of phosphorylation or the presence of an irregular phosphorylation pattern by the prolonged activity of PKA. Apolipoprotein E is therefore able to discriminate and interact with specific phosphorylation patterns of Tau.

International audience; NMR spectroscopy was used to explore the different aspects of the normal and pathological functions of tau, but proved challenging because the protein contains 441 amino acids and has poor signal dispersion. We have set out to dissect the phosphorylation patterns of tau in order to understand better its role in the aggregation process and microtubule-binding regulation. Our current knowledge on the functional consequences of specific phosphorylations is still limited, mainly because producing and assessing quantitatively phosphorylated tau samples is far from straightforward, even in vitro. We use NMR spectroscopy as a proteomics tool to characterize the phosphorylation patterns of tau, after in vitro phosphorylation by recombinant kinases. The phosphorylated tau can next be use for functional assays or interaction assays with phospho-dependent protein partners, such as the prolyl cis-trans isomerase Pin1.

Rabbit plasma low density lipoprotein (LDL) contains one major apolipoprotein of apparent molecular weight of 320 kDa, designated apolipoprotein (apo) Bh, while another component termed apoB1 of apparent molecular weight of 220 kDa is found in chylomicrons. The fragments generated by thrombin digestion of the protein moieties of rabbit and human LDL were separated by polyacrylamide gradient gel electrophoresis and compared. As in the human species, the enzyme produced limited cleavage patterns of rabbit LDL apoB. Within the first 2 h, two fragments (Tr1 and Tr2, with apparent molecular weights 280,000 and 44,000, respectively) appeared. Longer incubations led to the production of two additional peptides, Tr3 and Tr4 (apparent molecular weights 180,000 and 96,000, respectively). Ten monoclonal antibodies, developed against rabbit LDL and designated P01 to P10, were found to react with rabbit apoB. Some also cross-reacted with human apoB. Epitope mapping, performed with these antibodies, showed that Tr3 and Tr4 were derived from the further degradation of Tr1. The rabbit is one of the most frequently used animals in atherosclerosis research. Its LDL receptor has been characterized and there exists a strain of homozygous LDL receptor-deficient rabbits referred to as WHHL rabbits. Despite this, little has been done to characterize the structure of rabbit apoB; only a short region has been sequenced and shown to be the carboxyl-terminal region, the rabbit apoB1. The molecular weight of human apoB (550,000) is much larger than rabbit apoBh. In both species, a primary and secondary thrombin cleavage occur, but the size of the fragments produced is very different between the two species. Identification of the thrombolytic fragments of the rabbit apoB have afforded the opportunity to compare the structures of both apoB species.

The observation by NMR spectroscopy of microinjected (15)N-labelled proteins into Xenopus laevis oocytes might open the way to link structural and cellular biology. We show here that embedding the oocytes into a 20% Ficoll solution maintains their structural integrity over extended periods of time, allowing for the detection of nearly physiological protein concentrations. We use these novel conditions to study the neuronal Tau protein inside the oocytes. Spectral reproducibility and careful comparison of the spectra of Tau before and after cell homogenization is presented. When injecting Tau protein into immature oocytes, we show that both its microtubule association and different phosphorylation events can be detected.

The crucial role of the neuronal Tau protein in microtubule stabilization and axonal transport suggests that too little or too much Tau might lead to neuronal dysfunction. The presence of a hyper phosphorylated but non aggregated molecule as a toxic species that might sequester normal Tau is discussed. We present recent in vitro results that might allow us to dissect the role of individual phosphorylation sites on its structure and function. We also discuss in this review the role of phosphorylation for the aggregation of the neuronal Tau protein, and compare it to the aggregation induced by external poly anions.

The neuronal Tau protein, whose physiological role is to stabilize the microtubules, is found under the form of aggregated filaments and tangles in Alzheimer's diseased neurons. Until recently detailed structural analysis of the natively unfolded Tau protein has been hindered due to its shear size and unfavourable amino acid composition. We review here the recent progress in the assignments of the full-length polypeptide using novel methods of product planes and peptide NMR mapping, and indicate the structural insights that can be obtained from this assignment. Preliminary NMR data on the fibers show that the assignment enables a precise mapping of the rigid core. Future NMR experiments should allow one to gain more insight into the conformational aspects of this intriguing protein.

The phosphorylation of the neuronal Tau protein modulates both its physiological role of microtubule binding and its aggregation into paired helical fragments observed in Alzheimer's diseased neurons. However, detailed knowledge of the role of phosphorylation at specific sites has been hampered by the analytical difficulties to evaluate the level of site-specific phosphate incorporation. Even with recombinant kinases, mass spectrometry and immunodetection are not evident for determining the full phosphorylation pattern in a qualitative and quantitative manner. We show here that heteronuclear NMR spectroscopy on a 15N labeled Tau sample modified by the cAMP dependent kinase allows identification of all phosphorylation sites, measures their level of phosphate integration, and yields kinetic data for the enzymatic modification of the individual sites. Filtering through the 15N label discards the necessity of any further sample purification and allows the in situ monitoring of kinase activity at selected sites. We finally demonstrate that the NMR approach can equally be used to evaluate potential kinase inhibitors in a straightforward manner.

We have studied the mature Alzheimer-like fibers of tau by fluorescence and NMR spectroscopy. Assembly of the protein into paired helical filaments after incubation with heparin at 37 degrees C was verified by electron microscopy and size-exclusion chromatography. NMR spectroscopy on these mature fibers revealed different regions of residual mobility for tau: the N-terminal domain was found to maintain solution-like dynamics and was followed by a large domain of decreasing mobility; finally the core region was distinguished by a solid-like character. Heteronuclear-NOE data indicate that the decreasing mobility is due to both a slowing down of the rapid nanosecond movements and the introduction of slower movements that lead to exchange broadening. Fluorescence spectroscopy confirmed the presence of this rigid core, and some degree of protection from hydrogen exchange for those residues was observed. Hence, our data give a more precise picture of the dynamics of tau when it is integrated into mature filaments and should provide further understanding of the molecular processes that govern aggregation.

A combined strategy to obtain a partial NMR assignment of the neuronal Tau protein is presented. Confronted with the extreme spectral degeneracy that the spectrum of this 441 amino acid long unstructured protein presents, we have introduced a graphical procedure based on residue type-specific product planes. Combining this strategy with the search for pairwise motifs, and combining the spectra of different Tau isoforms and even of peptides derived from the native sequence, we arrive at a partial assignment that is sufficient to map the interactions of Tau with its molecular partners. The obtained assignments equally confirm the absence of regular secondary structure in the isolated protein.

The interaction between the neuronal Tau protein and the Pin1 prolyl cis/trans-isomerase is dependent on the phosphorylation state of the former. The interaction site was mapped to the unique phospho-Thr231-Pro232 motif, despite the presence of many other Thr/Ser-Pro phosphorylation sites in Tau and structural evidence that the interaction site does not significantly extend beyond those very two residues. We demonstrate here by NMR and fluorescence mapping that the Alzheimer's disease specific epitope centered around the phospho-Thr212-Pro213 motif is also an interaction site, and that the sole phospho-Thr-Pro motif is already sufficient for interaction. Because a detectable fraction of the Pro213 amide bond in the peptide centered around the phospho-Thr212-Pro213 motif is in the cis conformation, catalysis of the isomerization by the catalytic domain of Pin1 could be investigated via NMR spectroscopy.

We studied the effects of n-propanol and pH on the structure of the apolipoprotein E3 N-terminal receptor binding domain, apo E3(1-191), to determine whether conditions similar to those occurring near lipid surfaces (decreased dielectric constant and pH) can mimic lipid-induced conformational changes in apo E3. The addition of 30% n-propanol, at pH 7, induces a conformational change in apo E3(1-191) as shown by changes in the intrinsic tryptophan fluorescence and by an increase in the Stokes radius of the majority of the protein from 3.0 to 4.1 nm, although the protein remains monomeric as shown by chemical cross-linking. These changes are accompanied by increased resistance to limited proteolysis with trypsin, chymotrypsin, subtilisin and endoproteinase glu-C, as is the case for apo E3(1-191) reconstituted into phospholipid/cholesterol lipid bicelles. Far and near UV circular dichroism showed that n-propanol increases the amount of calculated alpha-helical structure (42-65%) and alters the tertiary structure of the protein although not as much as when apo E3(1-191) is incorporated into lipid bicelles. In the absence of n-propanol, lowering the pH to 4.5 decreases the Stokes radius of the majority of the protein somewhat, with little effect upon the secondary and the tertiary structures. The addition of 30% n-propanol at pH 4.5 increases the Stokes radius of apo E3(1-191) from 2.2 to 5.0 nm, even more than at pH 7 (3.0-4.1 nm) although the protein still remains predominantly monomeric. There is increased resistance to limited proteolysis with endoproteinase glu-C. As assessed by far and near UV circular dichroism, the addition of 30% n-propanol at pH 4.5, in contrast to pH 7, markedly increases the alpha-helical structure and changes the tertiary structure of the protein similarly to that resulting from the incorporation of apo E3(1-191) into lipid bicelles. The results suggest that a combination of n-propanol and low pH in aqueous solutions may be useful as a simple model system for studying conformational changes in apo E3 similar to those, which occur upon interaction of the protein with lipids.

HRMAS NMR of tau paired helical fragments assembled with heparin show an intensity decrease for those amino acids that are incorporated into the rigid core region, whereas the N-terminal amino acids maintain their full mobility.

Apolipoprotein A-I-containing lipoproteins represent a heterogeneous group of lipoparticles. Recent studies suggest that a specific subpopulation within the lipoprotein (AI) subclass may be more effective than other lipoproteins in promoting cholesterol efflux from cells and in the different steps of reverse cholesterol transport. This review describes the different apolipoprotein A-I-containing HDL subfractions, their role in reverse cholesterol transport, and their metabolic interconversions and relationship with lipoprotein-modifying enzymes.

To elucidate the molecular details of the conformation of apolipoprotein AI (apo AI), we have developed an approach related to the solubilization of this protein in 30% n-propanol. We have previously reported the promotion of a native-like structure for apo AI solubilized in n-propanol, as depicted by circular dichroism, fluorescence, and limited proteolytic digestion as compared to the lipid associated form of apo AI. In the present study, we labeled the Lys residues of apo AI with 13C by reductive methylation and used 13C NMR to confirm the formation of a native-like structure of apo AI in this environment. Furthermore, by the above criteria (circular dichroism and 13C NMR) and by using urea and temperature as denaturing agents, we show that the denaturation of the native-like structure of apo AI in n-propanol is a biphasic process. These studies show that in 30% n-propanol, apo AI contains two independently folded structural domains, of markedly different stabilities that might correspond to the amino-terminal and the carboxy-terminal halves of the molecule.

NMR spectroscopy of the full-length neuronal Tau protein has proved to be difficult due to the length of the protein and the unfavorable amino acid composition. We show that the random-coil chemical shift values and their dependence on the presence of a proline residue in the (i+1) position can successfully be exploited to assign all proline-directed phosphorylation sites. This is a first step toward the study of the phosphorylation of Tau by NMR spectroscopy.

This study describes the development of an enzyme-linked immunosorbent assay for human apolipoprotein E (apo E). A mouse monoclonal IgG1 antibody named E01 against apolipoprotein E was selected from five antibodies secreted by hybridomas. This antibody had a high affinity for apo E ((K a 1.2 × 107 L.M−1 for purified apo E and K = 1.05 × 107 L.M−1 for native apo E in very low density lipoproteins) in liquid phase and recognized every isoform of apo E but not other proteins in VLDL. Competition experiments with 125I apo E showed that its binding affinity for the E in every density class (VLDL, HDL, LDL) and in serum was the same. This antibody was used for the quantification of human apo E in serum by enzyme linked immunoassay. E01 was coated on microtiter plates and a polyclonal peroxidase-conjugate was used as second antibody. A good correlation was observed between the values obtained for apo E using both monoclonal and polyclonal antibodies.

Two monoclonal antibodies against human apolipoprotein A-I were characterized to recognize rabbit apo A-I. By immunoblotting we determined that while neither antibody reacted with the other rabbit apolipoproteins they both recognized all rabbit apo A-I isoforms. Cotitration revealed that each antibody bound to different apo A-I epitopes. Then we developed a noncompetitive enzyme-linked immunosorbent assay (ELISA) to measure the concentration of total apolipoprotein A-I in rabbit serum. The ELISA curves of the different lipoprotein class and serum showed the similarity of the expression of the apo A-I epitopes recognized in each of them and in serum. This assay, as opposed to other techniques, offers several advantages such as sensitivity, specificity, and simplicity and avoids the use of radioisotope.

Rabbit apolipoprotein A-I (apo A-I) of molecular weight 27,612 contained 241 amino acids. In contrast to its human counterpart which has 3 methionine residues, the rabbit protein possesses only one and therefore produces 2 fragments after CNBr cleavage (CNBr I and II, NH2- and COOH-terminal, respectively). From a series of monoclonal antibodies raised against human apo A-I, 2 (A05 and A16) cross-reacted with rabbit apo A-I. In the present study, we show that A05 recognizes the rabbit CNBr I fragment while the integrity of the intermediate region between the 2 CNBr fragments (including the methionine residue) is required for the expression of the A16 antigenic determinant. Competition experiments were performed between human 125I-labelled high density lipoprotein (HDL) and a variety of preparations of human and rabbit apo A-I (including the purified and delipidated protein, complexes of dimyristoylphosphatidylcholine (DMPC) containing apo A-I, HDL subfractions and whole serum). The A05 antigenic determinant was expressed identically in all these fractions of both species. In contrast the A16 showed poor reactivity with delipidated apo A-I, the apparent affinity constant being about 100 times less than for HDL. These data suggest that phospholipids improve the recognition of apo A-I by the A16 antibody. The similar immunoreactivity of the human and rabbit proteins in the present study is consistent with the view that the NH2-terminal region contains the major portion activating lecithin:cholesterol acyltransferase.