Polycation-induced oligomerization and accelerated fibrillation of human alpha-synuclein in vitro

The aggregation and fibrillation of alpha-synuclein has been implicated as a causative factor in Parkinson's disease and several other neurodegenerative disorders known as synucleinopathies. The effect of different factors on the process of fibril formation has been intensively studied in vitro. We show here that alpha-synuclein interacts with different unstructured polycations (spermine, polylysine, polyarginine, and polyethyleneimine) to form specific complexes. In addition, the polycations catalyze alpha-synuclein oligomerization. The formation of alpha-synuclein-polycation complexes was not accompanied by significant structural changes in alpha-synuclein. However, alpha-synuclein fibrillation was dramatically accelerated in the presence of polycations. The magnitude of the accelerating effect depended on the nature of the polymer, its length, and concentration. The results illustrate the potential critical role of electrostatic interactions in protein aggregation, and the potential role of naturally occurring polycations in modulating alpha-synuclein aggregation.     

Peroxidative aggregation of alpha-synuclein requires tyrosines

Alpha-synuclein is the main component of the intracellular protein aggregates in neurons of patients with Parkinson's disease. The occurrence of the disease is associated with oxidative damage. Although it is known that peroxidative chemistry leads to the aggregation of alpha-synuclein in vitro, the specific amino acid types of alpha-synuclein involved in this type of aggregation have not been identified. We show, using human cytochrome c plus H(2)O(2) as the source oxidative stress, that the tyrosines of alpha-synuclein are required for aggregation. The studies reveal the chemical basis for a crucial step in the aggregation process.     

Calcium(II) selectively induces alpha-synuclein annular oligomers via interaction with the C-terminal domain

Alpha-synuclein filaments are the major component of intracytoplasmic inclusion bodies characteristic of Parkinson's disease and related disorders. The process of alpha-synuclein filament formation proceeds via intermediate or protofibrillar species, each of which may be cytotoxic. Because high levels of calcium(II) and other metal ions may play a role in disease pathogenesis, we investigated the influence of calcium and other metals on alpha-synuclein speciation. Here we report that calcium(II) and cobalt(II) selectively induce the rapid formation of discrete annular alpha-synuclein oligomeric species. We used atomic force microscopy to monitor the aggregation state of alpha-synuclein after 1 d at 4 degrees C in the presence of a range of metal ions compared with the filament formation pathway in the absence of metal ions. Three classes of effect were observed with different groups of metal ions: (1) Copper(II), iron(III), and nickel(II) yielded 0.8-4 nm spherical particles, similar to alpha-synuclein incubated without metal ions; (2) magnesium(II), cadmium(II), and zinc(II) gave larger, 5-8 nm spherical oligomers; and, (3) cobalt(II) and calcium(II) gave frequent annular oligomers, 70-90 nm in diameter with calcium(II) and 22-30 nm in diameter with cobalt(II). In the absence of metal ions, annular oligomers ranging 45-90 nm in diameter were observed after 10 d incubation, short branched structures appeared after a further 3 wk and extended filaments after 2-3 mo. Previous studies have shown that alpha-synuclein calcium binding is mediated by the acidic C terminus. We found that truncated alpha-synuclein (1-125), lacking the C-terminal 15 amino acids, did not form annular oligomers upon calcium addition, indicating the involvement of the calcium-binding domain.     

Characterization of conformational and dynamic properties of natively unfolded human and mouse alpha-synuclein ensembles by NMR: implication for aggregation

Conversion of human α-synuclein (aS) from the free soluble state to the insoluble fibrillar state has been implicated in the etiology of Parkinson's disease. Human aS is highly homologous in amino acid sequence to mouse aS, which contains seven substitutions including the A53T that has been linked to familial Parkinson's disease, and including five substitutions in the C-terminal region. It has been shown that the rate of fibrillation is highly dependent on the exact sequence of the protein, and mouse aS is reported to aggregate more rapidly than human aS in vitro. Nuclear magnetic resonance experiments of mouse and human aS at supercooled temperatures (263 K) are used to understand the effect of sequence on conformational fluctuations in the disordered ensembles and to relate these to differences in propensities to aggregate. We show that both aS are natively unfolded at low temperature with different propensities to secondary structure, backbone dynamics and long-range contacts across the protein. Mouse aS exhibits a higher propensity to helical conformation around the C-terminal substitutions as well as the loss of transient long-range contacts from the C- to the N-terminal end and hydrophobic central regions of the protein relative to human aS. Lack of back-folding from the C-terminal end of mouse aS exposes the N-terminal region, which is shown, by ¹⁵N relaxation experiments, to be very restricted in mobility relative to human aS. We propose that the restricted mobility in the N-terminal region may arise from transient interchain interactions, suggesting that the N-terminal KTK(E/Q)GV repeats may serve as initiation sites for aggregation in mouse aS. These transient interchain interactions coupled with a non-Aβ amyloid component (NAC) region that is both more exposed and has a higher propensity to β structure may accelerate the rate of fibril formation of aS.     

Assignment of cytosine N3 resonances in nucleic acids via intrabase three-bond coupling to amino protons

Coherences were observed between ¹⁵N3 of cytosine and its trans amino proton (H42) using a modified gradient-based heteronuclear single quantum coherence (HSQC) pulse sequence optimized for three-bond proton-nitrogen couplings. The method is demonstrated with a 22-nucleotide RNA fragment of the P5abc region of a group I intron uniformly labeled with ¹⁵N. Use of intraresidue ¹⁵ N3-amino proton couplings to assign cytosine ¹⁵ N3 signals complements the recently proposed J_NN HNN COSY [Dingley, A.J. and Grzesiek, S. (1998) J. Am. Chem. Soc., 120, 8293–8297] method of identifying hydrogen-bonded base pairs in RNA.     

Probing the local conformational change of alpha1-antitrypsin

The native form of serpins (serine protease inhibitors) is a metastable conformation, which converts into a more stable form upon complex formation with a target protease. It has been suggested that movement of helix-F (hF) and the following loop connecting to strand 3 of beta-sheet A (thFs3A) is critical for such conformational change. Despite many speculations inferred from analysis of the serpin structure itself, direct experimental evidence for the mobilization of hF/thFs3A during the inhibition process is lacking. To probe the mechanistic role of hF and thFs3A during protease inhibition, a disulfide bond was engineered in alpha(1)-antitrypsin, which would lock the displacement of thFs3A from beta-sheet A. We measured the inhibitory activity of each disulfide-locked mutant and its heat stability against loop-sheet polymerization. Presence of a disulfide between thFs3A and s5A but not between thFs3A and s3A caused loss of the inhibitory activity, suggesting that displacement of hF/thFs3A from strand 5A but not from strand 3A is required during the inhibition process. While showing little influence on the inhibitory activity, the disulfide between thFs3A and s3A retarded loop-sheet polymerization significantly. This successful protein engineering of alpha(1)-antitrypsin is expected to be of value in clinical applications. Based on our current studies, we propose that the reactive-site loop of a serpin glides through between s5A and thFs3A for the full insertion into beta-sheet A while a substantial portion of the interactions between hF and s3A is kept intact.     

Catalysis by Glomerella cingulata cutinase requires conformational cycling between the active and inactive states of its catalytic triad

Cutinase belongs to a group of enzymes that catalyze the hydrolysis of esters and triglycerides. Structural studies on the enzyme from Fusarium solani have revealed the presence of a classic catalytic triad that has been implicated in the enzyme's mechanism. We have solved the crystal structure of Glomerella cingulata cutinase in the absence and in the presence of the inhibitors E600 (diethyl p-nitrophenyl phosphate) and PETFP (3-phenethylthio-1,1,1-trifluoropropan-2-one) to resolutions between 2.6 and 1.9 Å. Analysis of these structures reveals that the catalytic triad (Ser136, Asp191, and His204) adopts an unusual configuration with the putative essential histidine His204 swung out of the active site into a position where it is unable to participate in catalysis, with the imidazole ring 11 Å away from its expected position. Solution-state NMR experiments are consistent with the disrupted configuration of the triad observed crystallographically. H204N, a site-directed mutant, was shown to be catalytically inactive, confirming the importance of this residue in the enzyme mechanism. These findings suggest that, during its catalytic cycle, cutinase undergoes a significant conformational rearrangement converting the loop bearing the histidine from an inactive conformation, in which the histidine of the triad is solvent exposed, to an active conformation, in which the triad assumes a classic configuration.     

Specific structure appears at the N terminus in the sub-millisecond folding intermediate of the alpha subunit of tryptophan synthase, a TIM barrel protein

Competing views of the products of sub-millisecond folding reactions observed in many globular proteins have been ascribed either to the formation of discrete, partially folded states or to the random collapse of the unfolded chain under native-favoring conditions. To test the validity of these alternative interpretations for the stopped-flow burst-phase reaction in the (betaalpha)8, TIM barrel motif, a series of alanine replacements were made at five different leucine or isoleucine residues in the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli. This protein has been proposed to fold, in the sub-millisecond time range, to an off-pathway intermediate with significant stability and approximately 50% of the far-UV circular dichroism (CD) signal of the native conformation. Individual alanine replacements at any of three isoleucine or leucine residues in either alpha1, beta2 or beta3 completely eliminate the off-pathway species. These variants, within 5 ms, access an intermediate whose properties closely resemble those of an on-pathway equilibrium intermediate that is highly populated at moderate urea concentrations in wild-type alphaTS. By contrast, alanine replacements for leucine residues in either beta4 or beta6 destabilize but preserve the off-pathway, burst-phase species. When considered with complementary thermodynamic and kinetic data, this mutational analysis demonstrates that the sub-millisecond appearance of CD signal for alphaTS reflects the acquisition of secondary structure in a distinct thermodynamic state, not the random collapse of an unfolded chain. The contrasting results for replacements in the contiguous alpha1/beta2/beta3 domain and the C-terminal beta4 and beta6 strands imply a heterogeneous structure for the burst-phase species. The alpha1/beta2/beta3 domain appears to be tightly packed, and the C terminus appears to behave as a molten-globule-like structure whose folding is tightly coupled to that of the alpha1/beta2/beta3 domain.     

Role of alpha-synuclein in autophagy modulation of primary human T lymphocytes

It has been demonstrated that α-synuclein can aggregate and contribute to the pathogenesis of some neurodegenerative diseases and it is capable of hindering autophagy in neuronal cells. Here, we investigated the implication of α-synuclein in the autophagy process in primary human T lymphocytes. We provide evidence that: (i) knocking down of the α-synuclein gene resulted in increased autophagy, (ii) autophagy induction by energy deprivation was associated with a significant decrease of α-synuclein levels, (iii) autophagy inhibition by 3-methyladenine or by ATG5 knocking down led to a significant increase of α-synuclein levels, and (iv) autophagy impairment, constitutive in T lymphocytes from patients with systemic lupus erythematosus, was associated with abnormal accumulation of α-synuclein aggregates. These results suggest that α-synuclein could be considered as an autophagy-related marker of peripheral blood lymphocytes, potentially suitable for use in the clinical practice.     

Structure and topology of the non-amyloid-beta component fragment of human alpha-synuclein bound to micelles: implications for the aggregation process

Human alpha-synuclein is a small soluble protein abundantly expressed in neurons. It represents the principal constituent of Lewy bodies, the main neuropathological characteristic of Parkinson's disease. The fragment corresponding to the region 61-95 of the protein, originally termed NAC (non-amyloid-beta component), has been found in amyloid plaques associated with Alzheimer's disease, and several reports suggest that this region represents the critical determinant of the fibrillation process of alpha-synuclein. To better understand the aggregation process of alpha-synuclein and the role exerted by the biological membranes, we studied the structure and the topology of the NAC region in the presence of SDS micelles, as membrane-mimetic environment. To overcome the low solubility of this fragment, we analyzed a recombinant polypeptide corresponding to the sequence 57-102 of alpha-synuclein, which includes some charged amino acids flanking the NAC region. Three distinct helices are present, separated by two flexible stretches. The first two helices are located closer to the micelle surface, whereas the last one seems to penetrate more deeply into the micelle. On the basis of the structural and topological results presented, a possible pathway for the aggregation process is suggested. The structural information described in this work may help to identify the appropriate target to reduce the formation of pathological alpha-synuclein aggregation.     

Structure and serological analysis of the Hafnia alvei 481-L O-specific polysaccharide containing phosphate in the backbone chain

The lipopolysaccharide was extracted from cells of Hafnia alvei 481-L bacterial strain and, after mild acid hydrolysis, the O-specific polysaccharide was isolated and characterised. On the basis of chemical analyses and NMR spectroscopic studies of the polysaccharide and oligosaccharides obtained after Smith degradation, or hydrogen fluoride treatment, it was found that the repeating unit of the O-specific polysaccharide is a phosphorylated hexasaccharide: [see text]. The biological repeating unit of the H. alvei 481-L O-antigen has galactose phosphate at the nonreducing terminus. Serological tests indicate that this strain represents an individual serotype in the H. alvei genus.     

Single molecule analyses of the conformational substates of calmodulin bound to the phosphorylase kinase complex

The four integral delta subunits of the phosphorylase kinase (PhK) complex are identical to calmodulin (CaM) and confer Ca(2+) sensitivity to the enzyme, but bind independently of Ca(2+). In addition to binding Ca(2+), an obligatory activator of PhK's phosphoryltransferase activity, the delta subunits transmit allosteric signals to PhK's remaining alpha, beta, and gamma subunits in activating the enzyme. Under mild conditions about 10% of the delta subunits can be exchanged for exogenous CaM. In this study, a CaM double-mutant derivatized with a fluorescent donor-acceptor pair (CaM-DA) was exchanged for delta to assess the conformational substates of PhKdelta by single molecule fluorescence resonance energy transfer (FRET) +/-Ca(2+). The exchanged subunits were determined to occupy distinct conformations, depending on the absence or presence of Ca(2+), as observed by alterations of the compact, mid-length, and extended populations of their FRET distance distributions. Specifically, the combined predominant mid-length and less common compact conformations of PhKdelta became less abundant in the presence of Ca(2+), with the delta subunits assuming more extended conformations. This behavior is in contrast to the compact forms commonly observed for many of CaM's Ca(2+)-dependent interactions with other proteins. In addition, the conformational distributions of the exchanged PhKdelta subunits were distinct from those of CaM-DA free in solution, +/-Ca(2+), as well as from exogenous CaM bound to the PhK complex as delta'. The distinction between delta and delta' is that the latter binds only in the presence of Ca(2+), but stoichiometrically and at a different location in the complex than delta.     

Increase in the conformational flexibility of beta 2-microglobulin upon copper binding: a possible role for copper in dialysis-related amyloidosis

A key pathological event in dialysis-related amyloidosis is the fibril formation of beta(2)-microglobulin (beta 2-m). Because beta 2-m does not form fibrils in vitro, except under acidic conditions, predisposing factors that may drive fibril formation at physiological pH have been the focus of much attention. One factor that may be implicated is Cu(2+) binding, which destabilizes the native state of beta 2-m and thus stabilizes the amyloid precursor. To address the Cu(2+)-induced destabilization of beta 2-m at the atomic level, we studied changes in the conformational dynamics of beta 2-m upon Cu(2+) binding. Titration of beta 2-m with Cu(2+) monitored by heteronuclear NMR showed that three out of four histidines (His13, His31, and His51) are involved in the binding at pH 7.0. (1)H-(15)N heteronuclear NOE suggested increased backbone dynamics for the residues Val49 to Ser55, implying that the Cu(2+) binding at His51 increased the local dynamics of beta-strand D. Hydrogen/deuterium exchange of amide protons showed increased flexibility of the core residues upon Cu(2+) binding. Taken together, it is likely that Cu(2+) binding increases the pico- to nanosecond fluctuation of the beta-strand D on which His51 exists, which is propagated to the core of the molecule, thus promoting the global and slow fluctuations. This may contribute to the overall destabilization of the molecule, increasing the equilibrium population of the amyloidogenic intermediate.     

Solution structure of the hypothetical protein Mth677 from Methanobacterium thermoautotrophicum: a novel alpha+beta fold

The structure of Mth677, a hypothetical protein from Methanobacterium thermoautotrophicum (Mth), has been determined by using heteronuclear nuclear magnetic resonance (NMR) methods on a double-labeled (15)N-(13)C sample. Mth677 adopts a novel alpha+beta fold, consisting of two alpha-helices (one N terminal and one C terminal) packed on the same side of a central beta-hairpin. This structure is likely shared by its three orthologs, detected in three other Archaebacteria. There are no clear features in the sequences of these proteins or in the genome organization of Mth to make a reliable functional assignment to this protein. However, the structural similarity to Escherichia coli MinE, the protein which controls that division occurs at the midcell site, lends support to the proposal that Mth677 might be, in Mth, the counterpart of the topological specificity domain of MinE in E. coli.     

A simple algorithm locates beta-strands in the amyloid fibril core of alpha-synuclein, Abeta, and tau using the amino acid sequence alone

Fibrillar inclusions are a characteristic feature of the neuropathology found in the alpha-synucleinopathies such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy. Familial forms of alpha-synucleinopathies have also been linked with missense mutations or gene multiplications that result in higher protein expression levels. In order to form these fibrils, the protein, alpha-synuclein (alpha-syn), must undergo a process of self-assembly in which its native state is converted from a disordered conformer into a beta-sheet-dominated form. Here, we have developed a novel polypeptide property calculator to locate and quantify relative propensities for beta-strand structure in the sequence of alpha-syn. The output of the algorithm, in the form of a simple x-y plot, was found to correlate very well with the location of the beta-sheet core in alpha-syn fibrils. In particular, the plot features three peaks, the largest of which is completely absent for the nonfibrillogenic protein, beta-syn. We also report similar significant correlations for the Alzheimer's disease-related proteins, Abeta and tau. A substantial region of alpha-syn is capable [corrected] of converting from its disordered conformation into a long [corrected] alpha-helical protein. We have developed the aforementioned algorithm to locate and quantify the alpha-helical hydrophobic moment in the amino acid sequence of alpha-syn. As before, the output of the algorithm, in the form of a simple x-y plot, was found to correlate very well with the location of alpha-helical structure in membrane bilayer-associated alpha-syn.     

Elimination of a cis-Proline-Containing Loop and Turn Optimization Stabilizes a Protein and Accelerates Its Folding

In the N2 domain of the gene-3-protein of phage fd, two consecutive β-strands are connected by a mobile loop of seven residues (157-163). The stability of this loop is low, and the Asp160-Pro161 bond at its tip shows conformational heterogeneity with 90% being in the cis and 10% in the trans form. The refolding kinetics of N2 are complex because the molecules with cis or trans isomers at Pro161 both fold to native-like conformations, albeit with different rates. We employed consensus design to shorten the seven-residue irregular loop around Pro161 to a four-residue type I′ turn without a proline. This increased the conformational stability of N2 by almost 10 kJ mol^− 1 and abolished the complexity of the folding kinetics. Turn sequences obtained from in vitro selections for increased stability strongly resembled those derived from the consensus design. Two other type I′ turns of N2 could also be stabilized by consensus design. For all three turns, the gain in stability originates from an increase in the rate of refolding. The turns form native-like structures early during refolding and thus stabilize the folding transition state. The crystal structure of the variant with all three stabilized turns confirms that the 157-163 loop was in fact shortened to a type I′ turn and that the other turns maintained their type I′ conformation after sequence optimization.     

Synthesis and NMR characterization of the methyl esters of eicosapentaenoic acid monoepoxides

The activities of cytochrome P450-derived epoxide metabolites of omega-6 polyunsaturated fatty acids (PUFAs) in cellular homeostasis have generated considerable topical interest, but there is less information on the effects of omega-3 PUFA epoxides. Mass spectroscopic data on the epoxides of the omega-3 PUFA eicosapentaenoic acid (EPA) have been reported but the absence of corresponding NMR data currently hinders their biological assessment. In the present study five monoepoxy derivatives of EPA methyl ester were synthesized by treating EPA methyl ester with m-chloroperbenzoic acid. The individual regioisomers were purified by normal-phase chromatography and characterized by LC-MS/MS and a combination of NMR approaches including ¹H-, ¹³C-, ¹H-¹H-COSY, ¹H-¹³C-HSQC, and ¹H-¹³C-HMBC. The chromatographic properties for these monoepoxides were studied in normal-phase and reversephase-HPLC systems and the MS/MS fragmentation patterns using electrospray ionization were established. This paper also focuses on the NMR characterization of epoxide, olefinic and methylenic moieties and the complete assignments of the isomers.     

Ligand-induced changes in dynamics in the RT loop of the C-terminal SH3 domain of Sem-5 indicate cooperative conformational coupling

We report the effects of peptide binding on the (15)N relaxation rates and chemical shifts of the C-SH3 of Sem-5. (15)N spin-lattice relaxation time (T(1)), spin-spin relaxation time (T(2)), and ((1)H)-(15)N NOE were obtained from heteronuclear 2D NMR experiments. These parameters were then analyzed using the Lipari-Szabo model free formalism to obtain parameters that describe the internal motions of the protein. High-order parameters (S(2) > 0.8) are found in elements of regular secondary structure, whereas some residues in the loop regions show relatively low-order parameters, notably the RT loop. Peptide binding is characterized by a significant decrease in the (15)N relaxation in the RT loop. Concomitant with the change in dynamics is a cooperative change in chemical shifts. The agreement between the binding constants calculated from chemical shift differences and that obtained from ITC indicates that the binding of Sem-5 C-SH3 to its putative peptide ligand is coupled to a cooperative conformational change in which a portion of the binding site undergoes a significant reduction in conformational heterogeneity.     

Dynamic conformational equilibria in the physiological function of the Bombyx mori pheromone-binding protein

The Bombyx mori pheromone-binding protein (BmorPBP) undergoes a pH-dependent conformational transition from a form at basic pH, which contains an open cavity suitable for ligand binding (BmorPBP^B), to a form at pH 4.5, where this cavity is occupied by an additional helix (BmorPBP^A). This helix α7 is formed by the C-terminal dodecapeptide 131-142, which is flexibly disordered on the protein surface in BmorPBP^B and in its complex with the pheromone bombykol. Previous work showed that the ligand-binding cavity cannot accommodate both bombykol and helix α7. Here we further investigated mechanistic aspects of the physiologically crucial ejection of the ligand at lower pH values by solution NMR studies of the variant protein BmorPBP(1-128), where the C-terminal helix-forming tetradecapeptide is removed. The NMR structure of the truncated protein at pH 6.5 corresponds closely to BmorPBP^B. At pH 4.5, BmorPBP(1-128) maintains a B-type structure that is in a slow equilibrium, on the NMR chemical shift timescale, with a low-pH conformation for which a discrete set of ¹⁵N-¹H correlation peaks is NMR unobservable. The full NMR spectrum was recovered upon readjusting the pH of the protein solution to 6.5. These data reveal dual roles for the C-terminal tetradecapeptide of BmorPBP in the mechanism of reversible pheromone binding and transport, where it governs dynamic equilibria between two locally different protein conformations at acidic pH and competes with the ligand for binding to the interior cavity.