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-synuclein
* Department of Biology and
Department of Chemical Sciences, University of Padova, Padova, Italy
1Correspondence: Department of Biology, University of Padova, Via U. Bassi 58B, 35121, Padova, Italy. E-mail: luigi.bubacco{at}unipd.it
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ABSTRACT |
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-Synuclein is an intrinsically unfolded protein that can adopt a partially helical structure when it interacts with different lipid membranes. Its pathological relevance is linked to its involvement in several neurodegenerative disorders including Parkinson’s disease, Alzheimer’s disease, and dementia with Lewy bodies. Typical of such ailments is the presence of -synuclein aggregates in a β-structure that can be soluble or precipitate. This review focuses on the structural knowledge acquired in recent years on the various conformations accessible to -synuclein and to its pathologically relevant mutants. Furthermore, the role of the different variables of the chemical environments that govern the equilibria among the accessible conformations is also reviewed. The hypotheses that rationalize the relevance of the individual structural features and conformations for the physiological function of the protein or for its purported pathological role are described and compared.—Bisaglia, M., Mammi, S., Bubacco, L. Structural insights on physiological functions and pathological effects of -synuclein.
Key Words: Parkinson’s disease • structural analysis • membranes • fibrils • oligomers
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INTRODUCTION |
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-synuclein (syn) started with a novel protein family, relevant to several neural processes, that emerged between the end of the 1980s and the early 1990s (1)
. The almost concomitant and independent discoveries of the proteins, which were later recognized to belong to this family, explain the collection of different names that appeared in the literature.
The three genes for the brain synucleins were discovered independently by four different laboratories before researchers had a chance to compare their findings (2
3
4
5
6)
. The earliest report in which the information was united is a paper by Jakes et al. (7)
, which led to a clear classification: -, β-, and 
-synuclein (1)
. The relevance of the name originated from a supposed nuclear localization. It serendipitously resulted to be very pertinent when evidence showed that synucleins seem to nucleate the insoluble deposits typical of Alzheimer’s disease and Parkinson’s disease (PD). By now, a plethora of experimental evidence supports a role of syn in the pathology of PD, the second most common neurodegenerative disorder, which affects 
1–2% of the population above age 65 and 4–5% above age 85. The protein has also been shown to play a major role in the pathology of dementia with Lewy bodies and multiple-system atrophy, prompting the use of the term "synucleinopathies," introduced to indicate a major class of human neurodegenerative disorders (8)
. The peptide corresponding to the sequence 61–95, originally termed NAC (non-amyloid-β component), was observed in amyloid plaques associated to Alzheimer’s disease (5)
.
This review examines the structural studies recently published on syn and the proposed implications for both its physiological function and its potential role in the etiology of PD. The reaction pathways and the structural equilibria accessible to syn that will be described in the following paragraphs are shown in Fig. 1
.
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PRIMARY STRUCTURE OF SYN
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Syn is a small (14.5 kDa, 140 amino acids), highly acidic, natively unfolded protein. As depicted in Fig. 2
, the amino-terminal sequence of syn is almost entirely composed of variants of an imperfect 11 amino acid repeat (XKTKEGVXXXX, where bold characters refer to the consensus motif), which strongly resembles that found in the amphipathic helices of the exchangeable apolipoproteins (9
, 10)
. The sequence 61–95 is the most hydrophobic portion of the protein, and several studies have defined this region as responsible for syn aggregation and β-sheet formation (11
, 12)
. The carboxy terminus of syn, which is rich in acidic residues, has been shown to regulate fibril formation (13)
. Seeding experiments, under conditions where wild-type (wt) syn alone did not readily aggregate, revealed that carboxy-truncated syn fragments 1–102 and 1–110, but not 1–120, were efficient in promoting wt syn aggregation. The negatively charged residues 104, 105, and 114, 115 in the carboxy terminus were suggested to be responsible for the reduced aggregation and the lack of seeding of wt syn (13)
.
|
Three syn missense mutations, linked to autosomal dominant early-onset PD, have been reported in the literature. The first to be discovered is a A53T point substitution found in one Italian and in one Greek family (14)
. The second point mutation, A30P, was detected in a German family (15)
. More recently, a third E46K variant has been described in a Spanish family (16)
. In concordance with the suggestion that high expression levels of wt synuclein are associated with disease, familiar forms of PD have been found with triplication (17)
and duplications (18)
of the entire synuclein locus.
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STRUCTURAL ANALYSIS OF THE MONOMERIC -SYN
|
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syn has been defined by Weinreb et al. (19)
, requires the characterization of an ensemble of structures rather than the determination of a unique conformation. While natively unfolded proteins may lack static conformers containing extended regions of -helix and β-sheet or β-strands, it is possible that they adopt an ensemble of dynamically interchanging regular secondary structures.
Most of the biophysical methods currently available have been applied to the issue of defining the solution structure of monomeric syn, which makes this protein a paradigm in the field. In this context, NMR methods emerged as the most effective tools to approach the structural investigation of monomeric syn. The intrinsic heterogeneity of disordered proteins prevents the use of conventional nuclear Overhauser effect (NOE) if the protein contacts that generate these signals are rare in the conformer population. These problems can be circumvented by the paramagnetic relaxation enhancement (PRE) method that has been fully exploited to study the solution structure of syn. This technique employs a nitroxide spin-label attached to a chosen position in the syn sequence by the introduction of a Cys residue. The acquired paramagnetic center enhances the relaxation of the NMR signals of nearby nuclei, which causes signal broadening in a 1H-15N HSQC spectrum. Since this effect extends as far as 20 Å from the unpaired electron, it is possible to detect interactions that are much shorter-lived than those detectable by NOEs. The known r–6 relation between the distance of the observed nuclei from the paramagnetic probe and the peak intensity allows the transient short interactions to manifest sizeable effects.
Using site-directed spin labeling and PRE methodology, three different studies demonstrated the presence of long-range interactions. In the first one, syn mutants containing a cysteine residue at positions Q24, S42, Q62, S87, and N103 were used. In addition to local effects of the spin label, long-range contacts between the C-terminal tail and the central region of the protein were observed. Specifically, contacts between residues 120–140 and residues 30–100 were detected (20)
. Shortly after the study by Dedmon et al. (20)
, a paper by Bertoncini et al. (21)
explored the effect of both temperature and polyamine binding on these long-range interactions, using the PRE approach. While the paramagnetic effects at 15 and 37°C were very similar and confirmed the earlier conclusions, the C terminus of syn was found to adopt an almost fully extended conformation at 47°C that was also induced by polyamine binding (21)
. This observation led the authors to suggest that a release of long–range interactions in the syn monomer to a fully unfolded state renders the hydrophobic patches of the NAC region accessible, triggering oligomerization and aggregation. Analogous conclusions were reached more recently by a third group by comparing the PRE effects obtained at supercooled temperature (–10°C) on human and mouse syn (22)
. Based on C and Cβ secondary shifts, the authors also showed a mild propensity toward the β conformation for residues 30 to 140 of human syn (22)
. In agreement with this conclusion, Kim et al. (23)
demonstrated that, on the one hand, at –15°C, the N-terminal region (residues 1 to 38) did not show any propensity for either -helix or β-sheet, inferring a substantially unstructured N terminus. On the other hand, the region between residues 39 and 98, the key player in fibril formation, showed both secondary shifts and 3J(HN,H) coupling values typical of β conformations, which the authors took as evidence that "the central domain of syn transiently populates the β region in the Ramachandran plot." In contrast to the behavior observed at supercooled temperatures, in the first NMR work published on syn, the N-terminal region up to residue 100 was shown to have a tendency toward -helical 
and 
torsion angles, as indicated by a preponderance of positive C secondary shifts in this region. A continuous stretch of positive C shifts was observed between residues 6 and 37, an indication of a nascent or transiently populated helix (24)
. Later, a quantitative analysis of the C secondary chemical shifts in this region indicated that, on average, helical conformations are populated 10% of the time (25)
.
Most structural studies on syn described here were performed in vitro in dilute solutions. This finding raises the issue of the physiological relevance of the conformation determined in such an environment. By acquiring NMR spectra directly in Escherichia coli without any isolation step, two groups demonstrated that the protein retains the unfolded structure also in a crowded environment (26
, 27)
. Nevertheless, at present it is not known whether any genuinely unfolded syn exists when it is expressed in mammalian cells.
The NMR structural information discussed above provides a framework to rationalize the data that emerged from the application of other biophysical techniques, such as time-resolved fluorescence energy-transfer, Raman, and FTIR. Together, these techniques suggested a highly dynamic distribution of secondary structures for syn, interchanging on the microsecond time scale (28
29
30)
. Very recently, atomic force microscopy-based single-molecule mechanical unfolding methodology has been applied to characterize the conformational heterogeneity of monomeric syn (31)
. This methodology can explore the full conformational space of a protein at a single molecule level, detecting even poorly populated conformers and measuring their distribution in a variety of biologically important conditions. Three distinct classes of structures in equilibrium were identified: random coil, β-like structures, and conformations stabilized by short- and long-distance mechanically weak interactions. Their native-like populations were found to be 38.2, 7.3, and 54.5%, respectively (31)
. These mechanically weak interactions were tentatively attributed to intermolecular contacts between -helical syn and surrounding titin modules. If confirmed, these observations would be the only indication of an ordered secondary structure adopted by syn interacting with a different protein.
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FUNCTIONAL IMPLICATIONS OF THE MONOMERIC SYN CONFORMATION
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syn has been proposed to belong to the intrinsically unstructured protein (IUP) family (32
, 33)
. The lack of a well-defined secondary or tertiary structure in IUPs seems to play a key role in the molecular recognition of their partners. The major advantages of structural disorder are the potential binding strength with low specificity, increased speed of interaction, effective regulation by degradation, and ability to bind distinct partners. The functional importance of structural disorder is related to signal transduction, cell-cycle regulation, gene expression, and chaperone activity (33)
. In agreement with such properties, syn has been described to specifically interact with numerous proteins involved in signal transduction, the ubiquitin-proteasome system, vesicular recycling, synaptic functionality, regulation of oxidative stress, and mitochondrial function (34
, 35)
. Considering the ability of syn to adopt an ordered conformation on binding to membranes or within fibrils, it is conceivable that protein folding may also occur on binding with its partners. To date, however, no modification of the conformation of syn induced by interactions with other proteins has been described.
The sequence homology of syn with the phospho-dependent signaling chaperone proteins 14-3-3, in addition to the discovery that syn can interact with these proteins (36)
, have fueled a chaperone-hypothesis for syn function (36
37
38
39)
. Like 14-3-3 proteins, syn can interact with and modulate the activity of tyrosine hydroxylase, the enzyme involved in the rate-limiting step of dopamine synthesis (40)
. This observation suggests a role for the protein in the metabolism of dopamine. In agreement with this functional hypothesis are the ability of syn to inhibit aromatic amino acid decarboxylase activity in dopaminergic cells (41)
and further lines of evidence to be described.
While the physiological role of syn in its monomeric solution state is not entirely clear, more convincing is its role in the complex network of equilibria that, when altered, may lead to pathology. The monomeric form of syn can take part in a complex series of chemical modifications spanning from a site-specific phosphorylation mediated by cytoplasmatic enzymes to a redox chemistry mediated by dopamine-derived quinones, hydrogen peroxide, nitric oxide, or metals. These possibilities have been reviewed recently (42
, 43)
and will not be discussed further here.
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SYN OLIGOMERIZATION AND FIBRIL FORMATION
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syn in the etiology of PD. An early indication was the description of one of the pathological hallmarks of PD, i.e., the presence of intraneuronal proteinaceous cytoplasmic inclusions, called Lewy bodies (LBs). LBs also occur in a number of other neurological disorders including dementia with LBs (44)
, Alzheimer’s disease (45)
and Down syndrome (46)
. LBs are spherical eosinophilic cytoplasmic protein aggregates that contain ubiquitin and fibrils of syn (47)
. LBs are >15 µm in diameter and have an organized structure containing a dense hyaline core surrounded by a clear halo. EM analyses reveal a dense granulo-vesicular core surrounded by a ring of radiating 8–10 nm fibrils (48)
. In vitro studies on recombinant syn demonstrated that the purified protein aggregates into fibrils resembling those found in LBs (49)
. Nevertheless, it has been shown that the morphology of syn aggregates is highly sensitive to the solution conditions under which they are formed, implying that the fibrillar state does not necessarily represent the predominant or most significant aggregated state in vivo (50)
.
Since then, important efforts have been made to understand the structural properties of these fibrils and the nature of the fibrillation process. The first direct indication of a transition for syn to a cross-β conformation was obtained by X-ray diffraction. Syn fibers gave a cross-β pattern consisting of a 0.47 nm spacing between main chain -strands and a 1.0 to 1.1 nm spacing between the β-sheets making up the polymer (49)
. An EPR analysis on fibrils grown from a vast collection of site-directed spin-labeled syns suggested the presence of a well-ordered central region comprising 70 residues, with parallel, inregister β-strands (51)
. Organized, distinct domains were identified within syn fibrils, as well as strong parallelism in the core region. These were the first elements toward understanding the mechanism of fibrillogenesis and led the authors to propose a structural scheme in which the parallel polypeptide core region is characterized by several turn-and-bend regions with the N- and C-terminal ends completely unstructured. A second site-directed spin-labeled EPR study by the same group focused on syn115, a deletion mutant that has a faster rate of fibrillation (52)
. A more resolved structural model of fibrils was presented (Fig. 3A
), which was based on oxygen accessibility measurements. Most of the core region of syn fibrils, from residue 36 to 98, is characterized by stacking interactions and strong immobilization. However, some regions within the core, such as those around residues 62–67, show significant mobility. The authors suggest a less tightly packed structure for this stretch, which coincides with the beginning of the NAC region, probably with a loop or turn conformation. As observed for the wt protein, an increased mobility and almost no stacking interactions were also detected for residues before 35 and after 99, beyond the N- and C-terminal boundaries of the core region. The increased sensitivity of the accessibility measurements allowed the identification of several regions with a 2-fold periodicity characteristic of β-structures (regions 35–40, 51–54, 69–87, and 95–98) (52)
.
|
A more detailed structural model of the individual syn molecule within the fibrils emerged from two NMR studies. In the first one, fibrils obtained from syn were studied by solid-state, magic-angle spinning NMR spectroscopy, leading to the characterization of distinct regions in the fibrillized protein, as shown in Fig. 3B
(53)
. The β-sheet-rich core region was confirmed to comprise residues 38–95 and to contain turns or loops along with extended portions of β-strands. The N-terminal backbone is rigid from at least residue 22 onward with some degree of static disorder. The C terminus is unstructured and mobile, starting from at least residue 107. Two different classes of fibers were observed, indicating that at least two distinct fibril nucleation mechanisms exist for syn. In agreement with these results, in the second work, the presence of two distinct structural entities was demonstrated by hydrogen/deuterium exchange measured by solution-state NMR on syn fibrils and by high-resolution cryo-electron microscopy (54)
. Specifically, the investigators pointed out the presence of twisted and straight fibrils. By means of solid-state NMR, they also indicated a β-strand secondary structure for residues 37–43, 53–59, 62–66, 68–77, and 90–95 in the fibril core of syn (Fig. 3C
). In Fig. 4
, a proposed fold of syn fibrils is shown. The picture that emerges is still far from atomic resolution, but it provides some clues to analyze the multitude of experimental data on the fibrillation process, leading to the definition of a plausible molecular mechanism for fibrillation in the near future.
|
In addition to the tools developed to study the morphology of fibrils, considerable effort has been put into the development of effective methods to evaluate the kinetic parameters of fibrillation. Among these are thioflavin T assays that show sigmoidal line shapes for fibril formation, suggesting a nucleation-dependent lag phase in the aggregation mechanism (55
, 56)
. Although it is now clear that transient, soluble oligomeric species are present during the lag phase (Fig. 4
), the early steps of the aggregation process of syn remain poorly characterized. The kinetic behavior of the early aggregates has been studied mainly by fluorescence, which appears to be the technique of choice in this context (57
, 58)
. The growth of syn oligomers starts soon after the beginning of incubation. They typically reach a maximum concentration of 15% of the total protein toward the end of the lag period. Subsequently, they decline in concentration as the speed of fibril growth increases.
Three discrete nonfibrillar syn oligomers have been observed by AFM: "spheres" of several heights, chains that appear made of linearly associated 4–5 nm spheres, and rings, apparently comprising annular chains (56)
. The first-formed oligomeric species seem to be predominantly spherical with heights varying between 2.5–4.2 nm. Prolonged incubation of spheres results in the appearance of a more compact population. Further incubation of the latter species finally produces annular structures (59)
. It is still unclear whether the transient oligomers are on the direct pathway to fibrils or are off-pathway dead ends but in equilibrium with the monomer that can add directly to growing fibrils. It has been proposed that annular oligomers themselves are not on the direct monomer-fibril pathway but are supposed to "reopen" to become part of the growing fibrils (59
, 60)
. Based on experimental results, it has been suggested that prefibrillar oligomers, rather than fibrils themselves, may be the pathogenic species (61)
. In agreement with this hypothesis is the fact that dopaminergic neurons that contain LBs appear to be healthier than neighboring neurons (62
, 63)
. Moreover, some studies indicate that syn annular oligomers can modify the permeability of vesicles by formation of pores similar to those generated by pore-forming bacterial toxins (64
, 65)
.
Considering the link between syn aggregates and the onset and progression of PD, it is not surprising that many efforts are currently directed toward finding molecules that inhibit syn oligomer and fibril formation and in developing methods for rapid screening of potential inhibitors (66
67
68
69
70)
.
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SYN INTERACTION WITH MEMBRANES
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Syn has been reported to bind to acidic synthetic membranes such as PC/PS, PC/PA, PC/PG, PE/PS, PE/PA, PE/PG, and PE/PI vesicles and to undergo a conformational transition to a helical state (10
, 71
, 72)
. Several factors seem to play a central role in modulating the binding equilibrium of syn to membranes. First, the protein-lipid interaction has been demonstrated to be very sensitive to the chemical properties of the membrane (10
, 71)
. Second, the interaction with lipids has also been shown to be strongly dependent on the ionic strength of the solution with a decrease in the amount of helix with increasing salt concentration, although the binding is not completely absent even at 1.5 M NaCl. Thus, electrostatic forces play a key role in the binding process, but additional forces, presumably hydrophobic in nature, contribute to stabilize the protein-lipid interaction (10
, 73)
.
The vesicle size, or more precisely the curvature of the phospholipid surface, has been proposed to be another important determinant of the syn-lipid interaction. Incubation experiments were performed using comparable amounts of protein and vesicles composed of PC/PA phospholipids. Less syn was found to bind to large unilamellar vesicles (125 nm diameter) with respect to small unilamellar vesicles (25 nm diameter), indicating a higher affinity of the protein for vesicles with a higher surface curvature (10)
. However, fluorescence correlation spectroscopy failed to reproduce the substantial preference of syn for the more highly curved surface of the smaller vesicles (74)
. These results showed that the fractions of syn bound as a function of total accessible lipid for 100% PS vesicles with a measured diameter of either 60 or 120 nm were very similar. Furthermore, strong binding of syn to large unilamellar vesicles or to giant unilamellar vesicles (diameter up to 50 µm) has also been reported (75
, 76)
.
Finally, significantly different effects were observed as a function of the mass ratio of syn to lipid (72)
. At relatively high protein/lipid weight ratios (5:1), only a slightly folded conformation is detected and increased fibril formation is observed, in comparison to the lipid-free solution. In contrast, as the protein to lipid mass ratio is lowered to 1:1, the amount of helical protein increases to 30% and fibril formation is markedly slowed down. At a 1:5 weight ratio, the helical fraction becomes 70–80%, and fibril formation is completely inhibited (72)
.
The syn-lipid interaction has been thoroughly studied in vitro. On the contrary, the interaction with membranes in cells is less known. Using a binding assay to characterize the association of syn with cell membranes, it was found that syn colocalizes with protein and lipid components that are suggested to be part of detergent-resistant membrane microdomains, known as lipid rafts. This association to rafts seems to be required for the localization on the nerve terminal (77)
. The same investigators demonstrated that syn binds with high affinity to detergent-resistant membranes isolated from HeLa cells or synaptic vesicles. The association seems to be mediated by lipid rather than protein interactions and, unlike the binding to artificial membranes, it is completely independent of ionic interactions (78
, 79)
.
|
MEMBRANE-BOUND STRUCTURE OF SYN
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syn. In the first NMR study published, it was clearly demonstrated that, while the first 100 residues of the N-terminal region of syn are involved in the interaction with SDS micelles or PA/PC vesicles, the 40 C-terminal residues remain substantially unstructured (24)
. However, based on fluorescence and limited proteolysis experiments, two parallel studies have proposed that, in the presence of calcium and SDS micelles, the C-terminal tail is likely to interact with the lipid surface, implying a certain degree of rigidity and structure (80
, 81)
.
The ability of different syn fragments to bind to vesicles of different phospholipid composition has been studied by CD spectroscopy, and a cooperative action of the repeats in selecting the lipid composition of vesicles with which syn can interact has been suggested (82)
. Unfortunately, the NMR structure of syn in the presence of vesicles cannot be determined because the syn residues associated with the vesicle tumble at the same slow rate, leading to undetectably broad NMR lines. As a consequence, the three-dimensional structure was determined in the presence of SDS micelles.
The complete analysis of short-range NOEs, diagnostic for different types of secondary structure, was made difficult by strong resonance overlap; the structure was calculated mostly relying on RDCs measured in negatively charged stretched polyacrylamide gels (83)
. The structure of micelle-bound syn consists of two curved -helices (Fig. 5
), between residues Val3-Val37 and Lys45-Thr92, connected by a short linker in an antiparallel arrangement, followed by a short extended region (Gly93-Lys97) and a predominantly unstructured mobile tail (Asp98–Ala140). The helix-helix connector forms an extended conformation with a turn propensity near its midpoint.
|
The analysis of the backbone dynamic processes on a fast timescale (picoseconds to nanoseconds) revealed the presence of three distinct helical regions, Ala30-Val37, Asn65-Val70, and Glu83-Ala89, with greater mobility with respect to the other helical fragments. These regions also showed the highest solvent exchange rate, indicating a less stable helical structure (83)
. Similar results were described in two other studies. In the first one, on the basis of H and C chemical shift deviations, helix fraying was suggested in the sequences spanning residues 30–42, 60–65, and 82–100 (84)
. In the second one, a helical break was proposed in the region 36–45 and around residues 66 and 86 on the basis of dynamic analyses and H chemical shift deviations (85)
. The NMR structure of the NAC fragment alone in the presence of SDS micelles, determined on the basis of NOE measurements, contains three distinct helices separated by two flexible stretches around residues 66 and 85 (86)
. Despite only one full three-dimensional characterization of SDS-bound syn, several topological models of the interaction between syn and lipids have been proposed. The NAC region of the protein was suggested to be partially inserted into the membrane (85
, 87)
, although no evidence has been found for a transmembrane arrangement (86
, 88
, 89)
. In another model, the N-terminal region has been described as a noncanonical conformation, the 11/3 helix, to allow a better arrangement of the basic and acidic residues to favor membrane binding (87
, 90)
. Specifically, these authors found that micelle-resident paramagnetic spin labels induced a periodic pattern of broadening in the lipid-binding region of syn that correlates with a helical model possessing 11/3 periodicity but not with a canonical -helical model with 18/5 periodicity. The 11/3 periodicity was also evident, in the absence of any spin label, in amide proton chemical shift deviations, which reflect variations in hydrogen bond lengths caused by helix curvature, an intrinsic structural feature of micelle-bound proteins (87)
. This periodicity was also indicated in a recent study based on molecular dynamics simulations (91)
and in a work based on EPR measurements, obtained using 47 different singly spin-labeled syn mutants bound to small unilamellar vesicles (89)
. In the latter work, the authors emphasized the absence, under their experimental conditions, of the previously described helical break around residues 42–43, proposing that it might be induced by the high curvature strain of SDS micelles. Hence, the presence or absence of the helical break appears to be the more controversial structural feature of syn when bound to lipids. Very recently, three independent works appeared, based on pulsed dipolar EPR spectroscopy, with contradictory results (92
93
94)
. The strategic placement of the spin labels on different points of the two helical stretches allowed the generation of a matrix of distances used as restraints. In the first study, the distance distribution obtained was compatible with the presence of conformational disorder in the region 35–43 of the protein (92)
. Consistently, the second study showed that syn adopts a two-helix, antiparallel arrangement on vesicles large enough to accommodate an extended helix, suggesting that the bent structure is the preferred conformation of syn also on larger vesicles (93)
. On the contrary, the data presented by the third study is consistent with an extended helical conformation (94)
. The investigators also suggested the idea that the protein can interconvert between the broken and extended helical forms and this new picture could account for the experimental discrepancies.
|
PHYSIOLOGICAL FUNCTION OF MEMBRANE-BOUND SYN
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syn (95)
and its ability to interact with membranes seem to be the key factors to understand its elusive physiological functions. Numerous results obtained mainly on syn knockout mice suggest that, although not essential for synapse formation or cell survival, syn plays an important role in presynaptic dopamine recruitment and synaptic transmission (96
97
98
99
100)
. Although the precise mechanism of how syn functions in synaptic transmission is not clear, accumulating evidence suggests that syn may have a role in brain lipid metabolism and vesicle trafficking. A genome-wide screening in yeast showed that nearly one-third of the genes that enhance the toxicity of syn are functionally related to lipid metabolism and vesicle trafficking (101)
.
In a different model organism, transgenic Drosophila melanogaster, the expression of lipid and membrane transport genes was shown to be associated with that of syn (102)
. Syn has also been suggested to function as a fatty acid binding protein (FABP) in the central nervous system. Syn resembles FABPs in size, but more importantly, short amino acid stretches in its N and C termini share 55% and 67% homology, respectively, with a fatty acid-binding motif found in FABPs (103)
. Although the direct binding of fatty acids remains controversial (103
, 104)
, lack of syn in the brain depresses palmitic, arachidonic, and docosahexaenoic acid uptake and alters their metabolism and turnover in brain phospholipid pools (104
105
106)
. Consistent with the observation that syn may limit choline glycerophospholipid fatty acids turnover in vivo is the ability of the protein to interact and inhibit the action of phospholipase D2 (107
, 108)
. Phospholipase D2, which is selective for choline glycerophospholipids, is a membrane-bound enzyme with a presumed role in vesicle trafficking and formation (109)
. Like synapsin 1, a synaptic vesicle-associated phosphoprotein that has been implicated in synaptic vesicle recycling (110)
, syn might be an important presynaptic regulator of the vesicle cycle. In fact, immunogold EM measurements have shown considerably less synapsin 1 or syn associated with synaptic vesicles proximal to the presynaptic membrane compared with distal ones. Furthermore, after high K+ stimulation, both proteins redistributed in a similar way from the distal part of the terminal into the active zone along with the synaptic vesicles (111)
. A direct interaction between synapsin 1a and syn in the helical conformation has also been described (112)
. However, syn was not present in all terminals, indicating that it may not be involved in essential functions at all synapses but rather play a more subtle role than synapsin 1 in modifying synaptic responses (111)
. The data reported above may be rationalized by the hypothesis that syn regulates vesicle trafficking and release by altering lipid membrane stability, intracellular lipid uptake, and metabolism. Finally, in addition to the mechanisms previously emphasized, syn also appears to be involved in the regulation of cytoplasmic dopamine concentration. This can be achieved by an action of syn either on the anabolic pathway that leads to the synthesis of dopamine or on the permeability of the plasma membrane to dopamine by modulation of the dopamine transporter (113
114
115)
. It is worth mentioning that most of the studies examined the effects of syn on dopamine probably because PD is characterized by the specific loss of dopaminergic neurons. Nevertheless, it has been shown that syn can directly regulate the activity of serotonine and norepinephrine transporters. The ability of syn to modulate three different monoamine transporters, thereby regulating synaptic neurotransmission of the corresponding monoamines, highlights a previously unappreciated homeostatic role for the protein, implicating its probable participation in numerous mental conditions linked to these neurotransmitters (116)
.
|
STRUCTURAL ASPECTS OF THE PATHOGENIC FORMS OF SYN
|
|---|
syn (14
15
16)
. These discoveries were crucial in shifting the etiological concepts of the disease, moving from a nearly exclusively environmentally mediated disease toward a complex disorder with important genetic contributions. It also increased scientific interest in syn. Nevertheless, the molecular mechanisms by which these mutations give rise to PD have not yet been fully elucidated. In vitro studies have indicated that all of the mutations alter the kinetics of syn fibrillation: the rate is increased for the A53T and the E46K substitutions (117
, 118)
, while it is decreased in the case of A30P (56)
. Moreover, in comparison with the wt protein, both the A53T and the A30P mutations promote accumulation of prefibrillar oligomeric species (56)
, while the E46K protein reduces the formation of such aggregates (119)
. As described previously, based on the results obtained with the A53T and A30P mutants, a model of cytotoxicity has been proposed in which the toxic species were represented by prefibrillar intermediates with pore-like activity (61
, 64)
. The data obtained with the E46K variant cause some doubt about this model and demonstrate that the mechanism of neurotoxicity should not be oversimplified. Nevertheless, consistent with the proposed model, it was observed that E46K syn generates pore-like soluble aggregates similar in size and morphology to annular species isolated for wt, A53T, and A30P syn and displays permeabilizing activity when in the oligomeric form (119)
.
The structural studies on the point-mutated syn variants were carried out mainly on A53T and A30P proteins, the first mutations to be discovered. NMR analyses indicated that A30P and A53T mutations have no global effects on the structural properties of the protein or on the dynamic behavior of the syn backbone (25)
. Nevertheless, the secondary structure propensity for the free disordered state of the wt protein was altered by the two PD-linked variants. Specifically, the analysis of the C secondary shifts revealed that the A30P mutation attenuates the helical propensity found in the N-terminal region of wt syn, whereas the A53T mutation leaves this region unperturbed, inducing a more subtle and local preference for extended β-sheet-like conformations around the site of mutation (25)
. More recently, an additional NMR study indicated that the E46K mutation results in only very minor conformational changes in the free state of syn (119)
, but, unfortunately, a C secondary shift analysis was missing. As in the case of wt syn, more detailed conformational information came from NMR residual dipolar coupling and PRE measurements. Bertoncini et al. (120)
demonstrated that the two familial PD-associated mutants A30P and A53T perturb the ensemble of syn conformers. The two variants show increased backbone flexibility and the absence of the long-range interactions that were previously observed in the wt protein. Their results point toward a reduced shielding of the hydrophobic NAC region in A30P and A53T mutants and an increased range of conformations available. The possibility to overcome the energetic barrier for self-association more easily has been suggested by the authors as the reason for the increased tendency of these proteins to aggregate. A modified distribution of conformers, compared to the wt protein, was also observed for A30P in a single-molecule AFM study (31)
.
The effect of the PD-linked mutations on the interactions of syn with lipids has been investigated by a number of groups. While the A53T mutation seems to have little effect on membrane binding (121
, 122)
, several reports indicate that the A30P mutation decreases the extent of lipid interactions in vitro (121
, 123)
and in vivo (124)
. On the contrary, the third point mutation, E46K, increases the ability of the protein to bind to negatively charged liposomes (118)
. Structural analyses on syn mutants were performed only in the presence of SDS micelles. In the first study, the C chemical shift deviations of A30P and A53T syn variants were compared with those found for wt syn (122)
. The A53T data are nearly identical to those for the wt protein, indicating that this mutation has no detectable effect on the structure of micelle-bound syn. On the contrary, the A30P mutation appears to destabilize the helical structure of the protein around the site of the mutation. A similar NMR analysis of the E46K variant in its helical state indicated structural modifications that propagate well beyond the site of the mutation. According to the authors, the alterations observed are not large enough to suggest a disruption of the secondary structure of the protein. Rather, they seem to suggest some rearrangement of the helical structure with respect to its surrounding environment (119)
.
The description of the three-dimensional conformations of the A53T and A30P mutants did not change the picture that emerged from the above discussion. The structure and dynamics of the A53T variant were found to be indistinguishable from those of wt syn. In the case of the A30P mutation, the introduction of a Pro residue causes the N-terminal helix to terminate at A27 instead of A37, but the two helices of A30P syn are again found in an antiparallel orientation (125)
. Structural studies on familial PD-linked mutations in a more physiologically relevant membrane-mimetic system are still lacking, and future progress in this field is anticipated.
In a recent study, wt synuclein and E46K, A53T mutants were found competent to form helix-based ion channels with well-defined conductance states in membranes on application of a trans-negative potential (126)
. The basic character of KXKE repeats are proposed to allow voltage sensing, serving as energy-transducer elements to drive the helices into a trans-membrane orientation. The observation that the A30P mutant did not form ion channels was rationalized by assuming that the helix break due to the mutation close to the apex of the helix hairpin would increase the energy cost of this hairpin movement across the hydrophobic core of the membrane. This study, which may require further independent validation from other research groups, opens a new avenue of investigation.
|
ACKNOWLEDGMENTS |
|---|
syn oligomers and fibrils. This work was supported by grants from the Italian Ministry of Education, University and Research (PRIN and FIRB). Received for publication August 13, 2008. Accepted for publication September 25, 2008.
|
REFERENCES |
|---|
-synuclein. FEBS Lett. 576,363-368[CrossRef][Medline]
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INTRODUCTION
