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Structural Determinants of PLD2 Inhibition by α-Synuclein
doi:10.1016/j.jmb.2004.02.014
J. Mol. Biol. (2004) 337, 1001–1009
Structural Determinants of PLD2 Inhibition by a-Synuclein Jacqueline E. Payton1,2, Richard J. Perrin2,3, Wendy S. Woods3 and Julia M. George3* 1 Department of Cell and Structural Biology, University of Illinois, Urbana, IL 61801 USA 2
Medical Scholars Program University of Illinois, Urbana IL 61801, USA 3
Department of Molecular and Integrative Physiology University of Illinois, Urbana IL 61801, USA
The presynaptic protein a-synuclein has been implicated in both neuronal plasticity and neurodegenerative disease, but its normal function remains unclear. We described the induction of an amphipathic a-helix at the N terminus (exons 2– 4) of a-synuclein upon exposure to phospholipid vesicles, and hypothesized that lipid-binding might serve as a functional switch by stabilizing a-synuclein in an active (a-helical) conformation. Others have shown that a and b-synucleins inhibit phospholipase D (PLD), an enzyme involved in lipid-mediated signaling cascades and vesicle trafficking. Here, we report that all three naturally occurring synuclein isoforms (a, b, and g-synuclein) are similarly effective inhibitors of PLD2 in vitro, as is the Parkinson’s disease-associated mutant A30P. The PD-associated mutant A53T, however, is a more potent inhibitor of PLD2 than is wild-type a-synuclein. We analyze mutations of the a-synuclein protein to identify critical determinants of human PLD2 inhibition in vitro. Deletion of residues 56 –102 (exon 4) decreases PLD2 inhibition significantly; this activity of exon 4 may require adoption of an a-helical conformation, as mutations that disrupt a-helicity also abrogate inhibition. Deletion of C-terminal residues 130– 140 (exon 6) completely abolishes inhibitory activity. In addition, PLD2 inhibition is blocked by phosphorylation at serine 129 or at tyrosine residues 125 and 136, or by mutations that mimic phosphorylation at these sites. We conclude that PLD2 inhibition by a-synuclein is mediated by a lipid-stabilized a-helical structure in exon 4 and also by residues within exon 6, and that this inhibition can be modulated by phosphorylation of specific residues in exons 5 and 6. q 2004 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: Parkinson’s disease; synaptic vesicle; a-helix; amphipathic; plasticity
Introduction The synucleins comprise a family of small, highly conserved proteins that are generally implicated in the regulation of membrane dynamics. Physiologically, a and b-synuclein are expressed in the nervous system (neocortex, hippocampus, cerebellum, striatum, and thalamus), and these Abbreviations used: PD, Parkinson’s disease; PLD, phospholipase D; PA, phosphatidic acid; PE, phosphatidylethanolamine; PI(4,5)P2, phosphoinositol 4,5-bisphosphate; PC, phosphatidylcholine; ANCOVA, analysis of covariates. E-mail address of the corresponding author: [email protected]
proteins are enriched at presynaptic terminals.1,2 In songbirds, a-synuclein is upregulated in a specific population of presynaptic terminals as they undergo learning-related synaptic rearrangement.3 a-Synuclein appears to be an activity-dependent negative regulator of dopamine neurotransmission at nigrostriatal terminals,4 and a-synuclein anti-sense or knockout leads to depletion of a reserve/distal pool of vesicles in the presynaptic terminal.5,6 g-Synuclein is expressed primarily in the peripheral nervous system and in certain tumors,7,8 and its overexpression enhances the invasive and metastatic potential of breast tumors.9 In 1998, it was discovered that point mutations in the a-synuclein protein sequence are associated
0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
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with early-onset familial Parkinson’s disease (PD),10,11 and wild-type a-synuclein was subsequently shown to accumulate within intracellular inclusions and abnormal neurites (Lewy bodies and Lewy neurites) in cases of sporadic PD.12,13 a-Synuclein aggregation has now been identified in a host of neurodegenerative diseases,14 and the term synucleinopathy has been coined to describe this broad spectrum of disorders sharing common synuclein pathology.15 Most relevant to the current study, a and b-synuclein have been identified as specific inhibitors of the phospholipase D (PLD) isoforms PLD216,17 and PLD1.17 In general, the PLD enzymes are phosphatidylcholine-specific hydrolases that produce phosphatidic acid (PA) and choline in response to various hormones, growth factors, cytokines, neurotransmitters, and bioactive lipids.18 PLD is important in the generation of choline for acetylcholine synthesis,19 and PA and its metabolites exhibit several second messenger activities, including the mediation of vesicular transport,20 mitogenesis,21 respiratory burst in neutrophils,18 receptor-mediated endocytosis,22,23 and cytoskeletal rearrangement.24 Such a wide range of activators and downstream effects suggests that PLD plays a pivotal role in signal transduction in many cell types. In the current study, we use site-directed mutagenesis to dissect the structural requirements for synuclein inhibition of PLD2.
Results hPLD2 inhibition by natural synuclein isoforms and familial PD mutants Using a baculovirus construct containing fulllength human PLD2 cDNA plus an N-terminal His6 tag,25 hPLD2 was expressed in Sf9 cells and purified as described in Materials and Methods. Staining of eluates separated by SDS-PAGE with Coomassie brilliant blue showed a 105 kDa protein in infected, but not uninfected, cells; no contaminating proteins were detected. Western blot with a PLD2-specific antibody (PLD2-internal, QCB Inc.) confirmed expression of PLD2 (data not shown). Wild-type and mutant synuclein proteins were purified as described,26 and incubated with 0.1 nM hPLD2 enzyme and 100 mM PE/PI(4,5)P2/PC (16:1.4:1 molar ratio; see Materials and Methods) vesicles. Concentration-dependence was determined by varying concentrations of synuclein proteins from 0.0001 mM to 10 mM. After incubation, lipid vesicles and proteins were precipitated and free [3H]choline was measured by scintillation counting. Increasing concentrations of wild-type hAS induced a sigmoidal decrease in hPLD2 activity (in semi-logarithmic scale, Figure 1A). The linear portion of the curve had an average slope of 2 0.111, standard error ^ 0.015. To determine the Ki of hAS, varying concen-
Synuclein Inhibition of PLD2
trations of hAS and lipid vesicles were assayed as described and the results used to generate a Lineweaver –Burke plot (Figure 1B). Intersection of the double reciprocal inhibitory curves at 1=Vmax on the Y-axis demonstrates that hAS increases the Km ; but does not affect the Vmax of hPLD2, a pattern consistent with competitive inhibition. Michaelis – Menten calculations determined the Ki to be 21 nM hAS. Human b and g-synuclein isoforms inhibited hPLD2 activity as well as hAS. Two mutations associated with early-onset, autosomal dominant Parkinson’s disease, A30P and A53T, were also tested. Interestingly, although A30P inhibition was not different from wild-type, the A53T mutant had a greater inhibitory effect (Figure 2). Mapping of the PLD2-interacting domains To identify the regions of hAS required for hPLD2 inhibition, we utilized a panel of recombinant mutant proteins that we had characterized in previous studies.26,27 We first tested a series of mutants bearing deletions of individual exons within the coding region (D2, D3, D4, D5, D6). We are now revising our exon nomenclature to reflect the genomic sequence analysis reported by Touchman et al.28 Thus, recombinant mutants D2 (residues 2– 41 deleted), D3 (residues 43 –56 deleted), D4 (residues 56 – 102 replaced by Glu), D5 (residues 103 –130 deleted), and D6 (residues 130 –140 deleted) are identical with those mutants described in our previous reports26,27 as recombinant mutants D3 – D7. Our results show that deletion of either exon 4 or exon 6 decreases inhibition significantly (Figure 2). These domains are physically separate and functionally distinct: exon 4 lies within the a-helical domain of hAS, and is sufficient to mediate lipid-binding,26 while exon 6 lies in the acidic tail, including or adjacent to candidate phosphorylation sites (S129, Y125, Y133, and Y136),29 – 33 and has no role in lipid-binding.26 Effect of a-helical secondary structure on PLD2 inhibition We examined the mechanism by which D4 causes a loss of hPLD2 inhibition. Our previous studies have shown that exons 2, 3 and 4 are each individually sufficient to mediate lipid binding, and that deletion of any of these exons causes a decrease in a-helix content, with exon 4 contributing most to both lipid-binding and helix formation (exon 4 . exon 2 . exon 3).26 In the current experiment, D4 was the only exon deletion to exhibit a significant decrement in hPLD2 inhibition, suggesting that hPLD2 inhibition could depend either upon the strength of membrane binding or upon the adoption of a particular a-helical secondary structure by synuclein. To help clarify this point, we used a pair of substitution mutants (T6K and T6E) shown to disrupt the a-helical conformation of hAS.26 T6K, in which six positively
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Synuclein Inhibition of PLD2
Figure 1. hAS is a concentration-dependent inhibitor of hPLD2. A, Varying concentrations of hAS were incubated with 0.1 nM hPLD2 and 100 mM lipid vesicles for 30 minutes at 37 8C and hPLD2 activity measured by scintillation counting of [3H]choline release. The results are plotted as a percentage of hPLD2 activity in the absence of hAS. B, Varying concentrations of lipid vesicles and of wild-type hAS were incubated with 0.1 nM hPLD2 for 30 minutes at 37 8C and [3H]choline release was measured by scintillation counting. Results were plotted for each concentration of hAS, non-linear regression was performed to determine Km and Vmax ; and a Lineweaver – Burke double-reciprocal plot was constructed from the data. Lines representing different concentrations of hAS intersect on the Y-axis at 1=Vmax :
charged lysine residues are substituted for threonine residues along the predicted hydrophobic face of the lipid-binding helix (at positions 22, 33, 44, 59, 81, and 92; see Figure 5), is inhibited in its adoption of an a-helical conformation, although it maintains the capacity to bind acidic phospholipid vesicles (probably through an electrostatic interaction).26 T6E, in which negatively charged glutamate residues are substituted at the same positions, disrupts both lipid binding and a-helix formation.26 Both T6K and T6E mutations decreased the inhibitory capacity of hAS significantly (Figure 2). These data indicate that lipid association (e.g. by T6K) is not sufficient for hPLD2 inhibition by hAS, but that a conformational transition to an a-helix is also required. However, it remains a formal possibility that one or
more of the specific threonine residues that are altered in T6E and T6K, and which lie in exon 4, may be required specifically for inhibition of PLD2 (i.e. residues T59, T81, and T92). Effect of hAS phosphorylation on hPLD2 inhibition Next, we considered the mechanism by which exon 6 contributes to PLD2 inhibition. Exon 6 lies outside of the a-helical domain of hAS, and our laboratory has shown that deletion of exon 6 has no effect on vesicle-binding.26 This evidence suggests that exon 6 participates directly in protein– protein interactions that lead to hPLD2 inhibition. We observed that several potential and
Figure 2. Inhibition of hPLD2 by synucleins. Varying concentrations of a-synuclein, b-synuclein, g-synuclein, and mutant forms of a-synuclein were assayed for hPLD2 inhibition as in Figure 1. The slope of the inhibitory curve is plotted for each protein, with decreased slope indicating increased inhibition. Means ^ SD for three or four separate experiments are shown. P values are reported relative to wild-type hAS.
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Synuclein Inhibition of PLD2
Figure 3. Phosphorylation of serine or tyrosine abolishes inhibition of hPLD2. Varying concentrations of wild-type hAS, fyn-phosphorylated hAS, a tyrosine phosphorylation null mutant (Y4F), tyrosine phosphorylation mimic mutants (DDY, DYD, DYY, and YYD; see Materials and Methods), CKII-phosphorylated hAS, a serine phosphorylation null mutant (S129A), or a serine phosphorylation mimic mutant (S129E) were assayed for hPLD2 inhibition as in Figure 2. Means ^ SD from three or four separate experiments are shown. P values are reported relative to wild-type hAS.
confirmed phosphorylation sites lie within and adjacent to exon 6,29 – 34 and sought to address the role that phosphorylation in this domain might play in regulating hPLD2 inhibition. Figure 3 demonstrates that hPLD2 inhibition is abolished completely by in vitro phosphorylation of hAS with fyn tyrosine kinase. In contrast, mutant Y4F, in which all tyrosine residues in hAS were replaced with phenylalanine (positions 39, 125, 133 and 136), did not differ from wild-type in inhibitory capacity, but lost its sensitivity to regulation by fyn. We tested mutants designed to mimic phosphorylation at specific tyrosine residues (Figure 3). Because Y125 has been reported as the major site of phosphorylation by fyn,34 we substituted aspartate at this position, either alone (DYY) or in combination with similar substitutions at Y133 (DDY) or Y136 (DYD). Of these, DYD abolished hPLD2 inhibition by hAS completely, while DDY and DYY were not significantly different from wild-type. Likewise, substitution at position 136 alone (YYD) had no significant effect on inhibition. We conclude that, for the phosphorylation mimic mutants, a double substitution at positions 125 and 136 is required. Future experiments must show whether the effects of phosphorylation require the modification of both sites. The serine residue at position 129 of hAS has been reported as a site of phosphorylation. We used casein kinase II, a serine/threonine kinase, to phosphorylate hAS in vitro at this position.29,30,33 Figure 3 shows that phosphorylation with CKII abrogates inhibition of hPLD2. We confirmed the phosphorylation site at S129 by substituting alanine at this position (S129A). This null phosphorylation mutant is not different from wild-type in its capacity to inhibit hPLD2, but it is not sensitive to CKII regulation. Again, we generated a mutant to mimic phosphorylation at position 129, by substituting glutamate for serine (S129E). As predicted, this mutant shows significant abrogation of hPLD2 inhibition (Figure 3).
To confirm that the mutants DYD and S129E were still capable of interacting with lipid vesicles and undergoing an a-helical shift, they were subjected to circular dichroism spectroscopy in the presence of phospholipid vesicles as prepared for the hPLD2 assay. Both mutants were unstructured in the absence of lipid, but assumed an a-helical conformation in the presence of vesicles, which previous studies have shown to reflect a
Figure 4. Phosphorylation mimic mutants associate with phospholipid vesicles and undergo a concomitant a-helical shift. Circular dichroism spectra (mean residue ellipticity) are shown for hAS or phosphorylation mimic mutants (0.05 mg/ml) incubated alone (fine lines) or with lipid vesicles (1 mg/ml, PE, PI(4,5)P2, PC 16:1.4:1, bold lines) in 50 mM phosphate buffer. Spectra were collected at 25 8C in a 0.1 cm path-length quartz cell. The a-helix content of mutants was estimated using the K2d algorithm:47 hAS, 77% (continuous line); S129E, 65% (dotted line); and DYD, 59% (broken line).
Synuclein Inhibition of PLD2
helix-stabilizing association with the membrane surface.35 We conclude that DYD and S129E retain the capacity for vesicle binding, although each shows a slight decrement in a-helix content as estimated from the circular dichroism spectra (Figure 4).
Discussion In this study, we find that all natural isoforms of synuclein (a, b and g) can inhibit the activity of purified recombinant hPLD2 with similar efficacy. g-Synuclein was not identified as an inhibitor of PLD2 in Jenco’s reconstitution experiments,16 possibly because the source tissue in those experiments was mouse brain, while g-synuclein is expressed primarily in the peripheral nervous system.36 The observation that PLD2 inhibition is a common property of all synuclein family members is consistent with the hypothesis that PLD inhibition is a biologically significant function of this protein family. While we did not perform a molecular dissection of the b and g-synuclein sequences, their natural variations relative to the a-synuclein sequence are informative. Figure 2 shows that exon 4 (which spans residues 56 –102) is critical for PLD2 inhibition. The corresponding region in b-synuclein is quite similar overall to the a-synuclein sequence (with 12, mostly conservative, substitutions), but notably bears a deletion corresponding to residues 73 –83 of a-synuclein.37 We conclude that the essential residues for PLD2 inhibition in exon 4 must lie between residues 56 and 70 and/or between residues 84 and 102 of a-synuclein. a-Synuclein and g-synuclein inhibit PLD2 to a similar degree, but there is considerable sequence diversity in the domains shown to be relevant for PLD2 inhibition. While differences in exon 4 consist mostly of conservative substitutions, g-synuclein has a considerably truncated C terminus, with no homology to the conserved sequences in exon 6 of a and b-synuclein, shown here to be critical for PLD2 inhibition. In particular, the residues S129 and Y125/Y136, conserved in a and b-synuclein, are absent from g-synuclein. However, the C terminus of g-synuclein maintains the general acidic motif found in a and b-synucleins, due to a preponderance of glutamate residues. We speculate that this chemical quality may contribute to PLD2 inhibition, but that g-synuclein is either not modulated by phosphorylation, or is modulated by phosphorylation at different sites. The PD-associated mutants A53T and A30P were examined in this study. A30P did not differ significantly from wild-type in its capacity to inhibit PLD2. This is not so surprising, given that deletion of exon 2, in which A30P resides, has no significant effect on PLD2 inhibition in the present experiments, indicating that the inhibitory phenomenon is not dependent upon the structure or even the
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presence of this domain. This is consistent with our previous studies demonstrating that A30P and D2 mutants are competent to bind synthetic membranes and assume a conformation that is predominantly a-helical, albeit with a lower overall helical content.26 Interestingly, however, the PD-associated mutant A53T displayed enhanced inhibition of PLD2 as compared to wild-type hAS. The significance of this difference will require further exploration, but the autosomal dominant expression of the A53T phenotype is consistent with a biochemical gainof-function, and excessive inhibition of PLD2 should be considered as a possible contributing factor in the pathology associated with this mutation. We examined possible mechanisms of PLD2 inhibition by a-synuclein. One candidate mechanism had been eliminated by Jenco et al.16 They reported that PLD2 activity is dependent upon the availability of phosphoinositol 4,5-bisphosphate (PI(4,5)P2), but that inhibition by a-synuclein could not be overcome by increasing the concentration of PI(4,5)P2 in the assay vesicles. Thus inhibition by a-synuclein is not the result of sequestration of the available PI(4,5)P2. To complement these data, we performed Lineweaver –Burke analysis (Figure 1B), varying the concentrations of a-synuclein and bulk vesicles; the result is consistent with a competitive inhibition model, wherein a-synuclein competes with the substrate for binding PLD2. However, we know that a-synuclein is itself capable of binding phospholipid membranes of similar composition27 and at comparable ionic strengths35,38 as those used in the PLD2 assay system. This leaves room for the alternative interpretation, that a-synuclein inhibits PLD2 by intercalating into the membrane and reducing the effective surface concentration of the substrate (surface dilution).39 Like competitive inhibition, surface dilution should be overcome by increasing the bulk lipid concentration, so our analysis does not distinguish these two possibilities. To discriminate between competitive inhibition and surface dilution, one should ideally use a mixed detergent/phospholipid micelle system, which allows the concentration of substrate (phosphatidylcholine) to be varied independently of the bulk lipid concentration. Unfortunately, Jenco et al. report that PLD2 does not maintain its activity in such a mixed-micelle system, somewhat complicating our experimental design. However, by analyzing the roles of different a-synuclein domains in PLD2 inhibition we can speculate about the likely mechanisms of inhibition. The sequences required for PLD2 inhibition by a-synuclein were determined by comparing the activities of a series of complementary deletion mutants covering all of the coding exons of hAS (exons 2 –6). All synuclein isoforms share a similar overall structural organization. More than 60% of the protein sequence is derived from an 11 residue
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repeating motif, which can fold as an apolipoprotein-like amphipathic a-helix.3 This helix (spanning exons 2 –4) mediates binding to membranes containing acidic phospholipid, and undergoes a conformational shift from random coil to a-helix upon membrane binding.26 This a-helical conformation is observed only when a-synuclein is associated with a surface such as a lipid vesicle or detergent micelle; the protein is natively unfolded in solution.40 The lipid-binding domain associates with the membrane as a continuous a-helix with a single break,41 and association of this helical domain with a phospholipid bilayer causes disruption of the membrane structure.38 If synuclein inhibits PLD2 primarily by surface dilution, then deletions within the surface-active amphipathic a-helical domain (D2, D3 and D4) should decrease enzyme inhibition. However, we observe significant effects only for D4, a result seemingly inconsistent with surface dilution. Likewise, the effects of the C-terminal truncation (D6) and the C-terminal phosphorylation-mimic mutants (DYD and S129E) are more consistent with competitive inhibition. We showed that exon 6 does not participate in lipid-binding, and does not undergo an a-helical shift in the presence of lipid.26 It follows that deletion of exon 6 should not affect the ability of the protein to intercalate into the membrane and dilute the surface concentration of substrate (phosphatidylcholine), yet D6 is completely devoid of inhibitory activity (Figure 2). Similarly, we show here that the mutants S129E and DYD retain their ability to interact with lipid membranes and undergo a shift to an a-helical conformation (Figure 4), yet lack the capacity to inhibit PLD2. Together with the observation by Ahn that a-synuclein and PLD2 proteins interact directly,17 we favor the conclusion that a-synuclein inhibits PLD2 primarily through a specific competitive interaction. Our conclusion that PLD2 inhibition is modulated by C-terminal phosphorylation of a-synuclein is consistent with other published reports. In transfected HEK293 cells, a-synuclein inhibits the pervanadate-induced activity of both PLD1 and PLD2, but inhibition is increased if alanine is substituted for Y125 of a-synuclein, suggesting a role for tyrosine phosphorylation in modulation of PLD inhibition.17 Phosphorylation of a-synuclein by G protein-coupled receptor kinase 5 (GRK5) has also been shown to decrease inhibition of PLD2 in vitro.29 We have extended these studies by developing site-specific phosphorylation mimic mutants that are constitutively deficient in PLD2 inhibition. Other presynaptic proteins have been found to be specific inhibitors of PLD1 and PLD2, including AP3/AP180,42 synaptojanin,43 and amphiphysin I and II.44 Localized production of phosphatidic acid by PLD promotes positive membrane curvature, which can enhance vesicle fission and endocytosis. However, PLD activity must presumably be inhibited in the newly formed vesicle to
Synuclein Inhibition of PLD2
allow for vesicle fusion with the target membrane, and the redundancy of this inhibition in the presynaptic terminal is thought to be important for rapid synaptic vesicle recycling.44 Thus, the synucleins and other synapse-specific PLD inhibitors may regulate synaptic plasticity by influencing the availability of synaptic vesicles for release. Studies in a-synuclein knockout mice support this hypothesis. Abeliovich et al. report that dopaminergic neurons in the substantia nigra of a-synuclein knockout animals display more rapid recovery from paired-pulse depression, implying more efficient replenishment of the “readilyreleasable” pool of synaptic vesicles in the absence of a-synuclein.4 In contrast, Cabin et al. report that CA1 synapses from knockout mice are rapidly depleted of a different subset of presynaptic vesicles, the “reserve” or “resting pool”, upon prolonged stimulation.6 Taken together, these observations support a model wherein a-synuclein inhibits rapid recycling of presynaptic vesicles to the “readily releasable” pool, and promotes a slower process whereby membranes are recycled to the “reserve” pool. a-Synuclein may thus serve a critical modulatory role in synaptic function by influencing synaptic strength following particular patterns of synaptic activity.
Materials and Methods Purification of hPLD2 Sf9 insect cells (Invitrogen) were maintained at 27.5 8C in Grace’s Insect Medium (Invitrogen) supplemented with 10% (v/v) insect-tested FBS (Sigma) and Pen-strepfungizone (Invitrogen). A baculovirus containing fulllength human PLD2 cDNA plus an N-terminal His6 tag was obtained from Dr Rebecca S. Arnold.25 A total of 8 £ 106 exponentially growing cells per 75 cm2 flask were infected with hPLD2 baculovirus at a multiplicity of infection of 10. Cells were incubated for 48 – 72 hours and then collected and washed in PBS (137 mM NaCl, 2.7 mM KCl, 10 nM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). Cells were solubilized in lysis buffer (50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 10 mM imidazole), centrifuged at 12,000g, and the supernatant incubated with Ni-NTA beads (Qiagen) for two hours. Beads were washed with 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 120 mM imidazole, and recombinant hPLD2 was eluted with 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 1% n-octyl b-D -glucopyranoside, 250 mM imidazole. Protein purity was analyzed by SDS-PAGE; no contaminating proteins were detectable by staining with Coomassie brilliant blue. Recombinant synuclein protein Recombinant substitution mutants engineered for this study were as follows: S129E, S129 replaced by Glu; S129A, S129 replaced by Ala; DYD, Y125 and Y136 each replaced by Asp; DDY, Y125 and Y133 each replaced by Asp; YYD, Y136 replaced by Asp. All were cloned into the vector pET28(a) (Novagen) for expression in Escherichia coli under an inducible bacteriophage T7 lac promoter, as described.26 Human a, b, and g-synuclein,26
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Synuclein Inhibition of PLD2
Figure 5. A map of hAS. The coding region of the hAS gene is represented by exons 2 – 6. Exons 2 – 4 encode seven amphipathic a-helical repeats (shaded ovals) that are required for lipid binding.26 Six threonine residues (T22, T33, T44, T59, T81, T92) lie centrally along the hydrophobic face of the modeled a-helix; substitution with charged residues at these sites (T6E and T6K) decreases lipid binding26 and abrogates hPLD2 inhibition (Figure 4). A30P and A53T describe point mutations in the protein that are associated with familial, early-onset Parkinson’s disease. Also indicated are the positions of tyrosine residues (Y39, Y125, Y133, Y136) and of S129, which are sites of phosphorylation and/or substitution in this study.
and recombinant mutants, Y4F, A30P, A53T, T6E and T6K were prepared as described.27 We have revised our nomenclature so that mutants described as D3 – D726,27 are now referred to as D2 – D6, to reflect the genomic sequence analysis reported by Touchman et al.28 Specifically, these mutants are D2 (residues 2 – 41 deleted), D3 (residues 43 – 56 deleted), D4 (residues 56 – 102 replaced by Glu), D5 (residues 103– 130 deleted), and D6 (residues 130– 140 deleted) (Figure 5). Protein purity was confirmed by staining with Coomassie brilliant blue following SDS-PAGE.
In vitro phosphorylation For serine phosphorylation, recombinant purified hAS (5 mg) was incubated with 138 milliunits of active casein kinase II (Upstate Biotechnology) in 20 mM Tris –HCl (pH 8.0), 4 mM MgCl2, 130 mM KCl, 800 mM ATP and 250 ng of poly-L -lysine (Sigma; 150– 300 kDa) at 30 8C for 60 minutes. The total reaction volume was 50 ml. For tyrosine phosphorylation, 2 mg of recombinant purified hAS was incubated with 18.75 milliunits of active fyn kinase (Upstate Biotechnology) in 50 mM Tris (pH 7.5), 10 mM EGTA, 100 mM vanadate, 75 mM MnCl2 and 300 mM ATP at 30 8C for 60 minutes. The total reaction volume was 50 ml.
hPLD2 activity and inhibition by synucleins hPLD2 activity was assayed as described.45 Briefly, 0.1 nM hPLD2 (1 ng) was added to assay buffer (50 mM Na-Hepes (pH 7.5), 3 mM EGTA, 80 mM KCl, 1 mM DTT, 3 mM MgCl2, 2 mM CaCl2) containing lipid vesicles (phosphatidylethanolamine (PE), phosphoinositol 4,5bisphosphate (PI(4,5)P2), and phosphatidylcholine (PC) (Avanti Polar Lipids) molar ratio 16:1.4:1, plus 50,000– (Amersham100,000 cpm [3H]phosphatidylcholine Pharmacia)) at a final lipid concentration of 100 mM in a total volume of 100 ml. Synucleins were added where indicated. Reactions were incubated at 37 8C for 30 minutes and then stopped with 200 ml of 10% (v/v) trichloroacetic acid and 100 ml of 10% (w/v) BSA to precipitate proteins and lipids. Free [3H]choline released by hPLD2 was measured by scintillation counting. Statistical analysis of inhibition used the General Linear Model to perform analysis of covariates (ANCOVA) to compare slopes between proteins. Statistical analysis was performed in consultation with the Illinois Statistics Office of the University of Illinois at Urbana-Champaign, using the statistical analysis package SAS.
Circular dichroism spectroscopy CD spectra were collected for hAS and the mutants DYD and S129E using a JASCO J-720 spectropolarimeter. Spectra were taken at 25 8C in a 0.1 cm path-length quartz cuvette containing the sample at 0.05 mg/ml of protein, 1 mg/ml of lipid vesicles (PE, PI(4,5)P2, and PC (Avanti Polar Lipids) molar ratio 16:1.4:1), in 20 mM sodium phosphate buffer (pH 7.4). The spectral contributions of buffer and vesicles were subtracted as appropriate. The percentage of a-helix was determined from the molar ellipticities at 222 nm,46 as well as by computer fitting to a library of CD spectra from proteins of known structure using the learning neural network program K2D, which is based on the algorithm published by Andrade et al.47
Acknowledgements This work was supported by grant R01 AG13762 from the National Institute on Aging, and by grants to J.P. from the Illinois Department of Public Health and the American Federation for Aging Research. We thank Dr Rebecca Arnold for her gift of the His-tagged hPLD2 baculovirus construct. Circular dichroism experiments were performed at the Laboratory for Fluorescence Dynamics (LFD) at the University of Illinois at UrbanaChampaign (UIUC). The LFD is supported jointly by the Division of Research Resources of the National Institutes of Health (PHS 5 P41-RR03155) and UIUC.
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during the critical period for song learning in the Zebra finch. Neuron, 15, 361– 372. Abeliovich, A., Schmitz, Y., Farinas, I., ChoiLundberg, D., Ho, W. H., Castillo, P. E. et al. (2000). Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system. Neuron, 25, 239– 252. Murphy, D. D., Rueter, S. M., Trojanowski, J. Q. & Lee, V. M. (2000). Synucleins are developmentally expressed, and alpha-synuclein regulates the size of the presynaptic vesicular pool in primary hippocampal neurons. J. Neurosci. 20, 3214– 3220. Cabin, D. E., Shimazu, K., Murphy, D., Cole, N. B., Gottschalk, W., McIlwain, K. L. et al. (2002). Synaptic vesicle depletion correlates with attenuated synaptic responses to prolonged repetitive stimulation in mice lacking alpha-synuclein. J. Neurosci. 22, 8797– 8807. Lavedan, C., Leroy, E., Dehejia, A., Buchholtz, S., Dutra, A., Nussbaum, R. L. & Polymeropoulos, M. H. (1998). Identification, localization and characterization of the human gamma-synuclein gene. Hum. Genet. 103, 106– 112. Bruening, W., Giasson, B. I., Klein-Szanto, A. J., Lee, V. M., Trojanowski, J. Q. & Godwin, A. K. (2000). Synucleins are expressed in the majority of breast and ovarian carcinomas and in preneoplastic lesions of the ovary. Cancer, 88, 2154– 2163. Jia, T., Liu, Y. E., Liu, J. & Shi, Y. E. (1999). Stimulation of breast cancer invasion and metastasis by synuclein gamma. Cancer Res. 59, 742– 747. Polymeropoulos, M. H., Lavedan, C., Leroy, E., Ide, S. E., Dehijia, A., Dutra, A. et al. (1997). Mutation in the a-synuclein gene identified in families with Parkinson’s disease. Science, 276, 2045– 2047. Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S. et al. (1998). Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson’s disease. Nature Genet. 18, 106– 108. Irizarry, M. C., Growdon, W., Gomez-Isla, T., Newell, K., George, J. M., Clayton, D. F. & Hyman, B. T. (1998). Nigral and cortical Lewy bodies and dystrophic nigral neurites in Parkinson’s disease and cortical Lewy body disease contain alphasynuclein immunoreactivity. J. Neuropathol. Expt. Neurol. 57, 334– 337. Spillantini, M. G., Crowther, R. A., Jakes, R., Hasegawa, M. & Goedert, M. (1998). Alphasynuclein in filamentous inclusions of Lewy bodies from Parkinson’s disease and dementia with Lewy bodies. Proc. Natl Acad. Sci. USA, 95, 6469– 6473. Goedert, M. (2001). Alpha-synuclein and neurodegenerative diseases. Nature Rev. Neurosci. 2, 492– 501. Galvin, J. E., Lee, V. M. & Trojanowski, J. Q. (2001). Synucleinopathies: clinical and pathological implications. Arch. Neurol. 58, 186– 190. Jenco, J. M., Rawlingson, A., Daniels, B. & Morris, A. J. (1998). Regulation of phospholipase D2: selective inhibition of mammalian phospholipase D isoenzymes by a- and b-synucleins. Biochemistry, 37, 4901– 4909. Ahn, B. H., Rhim, H., Kim, S. Y., Sung, Y. M., Lee, M. Y., Choi, J. Y. et al. (2002). alpha-Synuclein interacts with phospholipase D isozymes and inhibits pervanadate-induced phospholipase D activation in human embryonic kidney-293 cells. J. Biol. Chem. 277, 12334– 12342. Liscovitch, M., Czarny, M., Fiucci, G. & Tang, X.
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(2000). Phospholipase D: molecular and cell biology of a novel gene family. Biochem. J. 345, 401– 415. Zhao, D., Frohman, M. A. & Blusztajn, J. K. (2001). Generation of choline for acetylcholine synthesis by phospholipase D isoforms. BMC Neurosci. 2, 16. Jones, D., Morgan, C. & Cockcroft, S. (1999). Phospholipase D and membrane traffic. Potential roles in regulated exocytosis, membrane delivery and vesicle budding. Biochim. Biophys. Acta, 1439, 229–244. Venable, M. E. & Obeid, L. M. (1999). Phospholipase D in cellular senescence. Biochim. Biophys. Acta, 1439, 291– 298. Shen, Y., Xu, L. & Foster, D. A. (2001). Role for phospholipase D in receptor-mediated endocytosis. Mol. Cell. Biol. 21, 595– 602. Rizzo, M. A., Shome, K., Vasudevan, C., Stolz, D. B., Sung, T. C., Frohman, M. A. et al. (1999). Phospholipase D and its product, phosphatidic acid, mediate agonist-dependent raf-1 translocation to the plasma membrane and the activation of the mitogenactivated protein kinase pathway. J. Biol. Chem. 274, 1131–1139. Cockcroft, S. (2001). Signalling roles of mammalian phospholipase D1 and D2. Cell. Mol. Life Sci. 58, 1674– 1687. Lopez, I., Arnold, R. S. & Lambeth, J. D. (1998). Cloning and initial characterization of a human phospholipase D2 (hPLD2). ADP-ribosylation factor regulates hPLD2. J. Biol. Chem. 273, 12846– 12852. Perrin, R. J., Woods, W. S., Clayton, D. F. & George, J. M. (2000). Interaction of human alpha-synuclein and Parkinson’s disease variants with phospholipids. Structural analysis using site-directed mutagenesis. J. Biol. Chem. 275, 34393– 34398. Perrin, R. J., Woods, W. S., Clayton, D. F. & George, J. M. (2001). Exposure to long chain polyunsaturated fatty acids triggers rapid multimerization of synucleins. J. Biol. Chem. 276, 41958– 41962. Touchman, J. W., Dehejia, A., Chiba-Falek, O., Cabin, D. E., Schwartz, J. R., Orrison, B. M. et al. (2001). Human and mouse alpha-synuclein genes: comparative genomic sequence analysis and identification of a novel gene regulatory element. Genome Res. 11, 78 – 86. Pronin, A. N., Morris, A. J., Surguchov, A. & Benovic, J. L. (2000). Synucleins are a novel class of substrates for G protein-coupled receptor kinases. J. Biol. Chem. 275, 26515– 26522. Fujiwara, H., Hasegawa, M., Dohmae, N., Kawashima, A., Masliah, E., Goldberg, M. S. et al. (2002). Alpha-synuclein is phosphorylated in synucleinopathy lesions. Nature Cell. Biol. 4, 160– 164. Negro, A., Brunati, A. M., Donella-Deana, A., Massimino, M. L. & Pinna, L. A. (2002). Multiple phosphorylation of alpha-synuclein by protein tyrosine kinase Syk prevents eosin-induced aggregation. FASEB J. 16, 210– 212. Ellis, C. E., Schwartzberg, P. L., Grider, T. L., Fink, D. W. & Nussbaum, R. L. (2001). Alpha-synuclein is phosphorylated by members of the Src family of protein-tyrosine kinases. J. Biol. Chem. 276, 3879– 3884. Okochi, M., Walter, J., Koyama, A., Nakajo, S., Baba, M., Iwatsubo, T. et al. (2000). Constitutive phosphorylation of the Parkinson’s disease associated alpha-synuclein. J. Biol. Chem. 275, 390– 397. Nakamura, T., Yamashita, H., Takahashi, T. & Nakamura, S. (2001). Activated Fyn phosphorylates alpha-synuclein at tyrosine residue 125. Biochem. Biophys. Res. Commun. 280, 1085– 1092.
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35. Davidson, W. S., Jonas, A., Clayton, D. F. & George, J. M. (1998). Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes. J. Biol. Chem. 273, 9443– 9449. 36. Buchman, V. L., Hunter, H. J., Pinon, L. G., Thompson, J., Privalova, E. M., Ninkina, N. N. & Davies, A. M. (1998). Persyn, a member of the synuclein family, has a distinct pattern of expression in the developing nervous system. J. Neurosci. 18, 9335–9341. 37. Clayton, D. F. & George, J. M. (1998). The synucleins: a family of proteins involved in synaptic function, plasticity, neurodegeneration and disease. Trends Neurosci. 21, 249– 254. 38. Zhu, M., Li, J. & Fink, A. L. (2003). The association of alpha-synuclein with membranes affects bilayer structure, stability, and fibril formation. J. Biol. Chem. 278, 40186– 40197. 39. Carman, G. M., Deems, R. A. & Dennis, E. A. (1995). Lipid signaling enzymes and surface dilution kinetics. J. Biol. Chem. 270, 18711 – 18714. 40. Weinreb, P. H., Zhen, W., Poon, A. W., Conway, K. A. & Lansbury, P. T., Jr (1996). NACP, a protein implicated in Alzheimer’s disease and learning, is natively unfolded. Biochemistry, 35, 13709– 13715. 41. Bussell, R., Jr & Eliezer, D. (2003). A structural and functional role for 11-mer repeats in alpha-synuclein and other exchangeable lipid binding proteins. J. Mol. Biol. 329, 763 –778.
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42. Lee, C., Kang, H. S., Chung, J. K., Sekiya, F., Kim, J. R., Han, J. S. et al. (1997). Inhibition of phospholipase D by clathrin assembly protein 3 (AP3). J. Biol. Chem. 272, 15986– 15992. 43. Chung, J. K., Sekiya, F., Kang, H. S., Lee, C., Han, J. S., Kim, S. R. et al. (1997). Synaptojanin inhibition of phospholipase D activity by hydrolysis of phosphatidylinositol 4,5-bisphosphate. J. Biol. Chem. 272, 15980 – 15985. 44. Lee, C., Kim, S. R., Chung, J. K., Frohman, M. A., Kilimann, M. W. & Rhee, S. G. (2000). Inhibition of phospholipase D by amphiphysins. J. Biol. Chem. 275, 18751– 18758. 45. Brown, H. A., Gutowski, S., Moomaw, C. R., Slaughter, C. & Sternweis, P. C. (1993). ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity. Cell, 75, 1137– 1144. 46. Chen, Y. H., Yang, J. T. & Martinez, H. M. (1972). Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion. Biochemistry, 11, 4120– 4131. 47. Andrade, M. A., Chacon, P., Merelo, J. J. & Moran, F. (1993). Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng. 6, 383–390.
Edited by P. T. Lansbury Jr (Received 6 May 2003; received in revised form 2 December 2003; accepted 5 February 2004)
J. Mol. Biol. (2004) 337, 1001–1009
Structural Determinants of PLD2 Inhibition by a-Synuclein Jacqueline E. Payton1,2, Richard J. Perrin2,3, Wendy S. Woods3 and Julia M. George3* 1 Department of Cell and Structural Biology, University of Illinois, Urbana, IL 61801 USA 2
Medical Scholars Program University of Illinois, Urbana IL 61801, USA 3
Department of Molecular and Integrative Physiology University of Illinois, Urbana IL 61801, USA
The presynaptic protein a-synuclein has been implicated in both neuronal plasticity and neurodegenerative disease, but its normal function remains unclear. We described the induction of an amphipathic a-helix at the N terminus (exons 2– 4) of a-synuclein upon exposure to phospholipid vesicles, and hypothesized that lipid-binding might serve as a functional switch by stabilizing a-synuclein in an active (a-helical) conformation. Others have shown that a and b-synucleins inhibit phospholipase D (PLD), an enzyme involved in lipid-mediated signaling cascades and vesicle trafficking. Here, we report that all three naturally occurring synuclein isoforms (a, b, and g-synuclein) are similarly effective inhibitors of PLD2 in vitro, as is the Parkinson’s disease-associated mutant A30P. The PD-associated mutant A53T, however, is a more potent inhibitor of PLD2 than is wild-type a-synuclein. We analyze mutations of the a-synuclein protein to identify critical determinants of human PLD2 inhibition in vitro. Deletion of residues 56 –102 (exon 4) decreases PLD2 inhibition significantly; this activity of exon 4 may require adoption of an a-helical conformation, as mutations that disrupt a-helicity also abrogate inhibition. Deletion of C-terminal residues 130– 140 (exon 6) completely abolishes inhibitory activity. In addition, PLD2 inhibition is blocked by phosphorylation at serine 129 or at tyrosine residues 125 and 136, or by mutations that mimic phosphorylation at these sites. We conclude that PLD2 inhibition by a-synuclein is mediated by a lipid-stabilized a-helical structure in exon 4 and also by residues within exon 6, and that this inhibition can be modulated by phosphorylation of specific residues in exons 5 and 6. q 2004 Elsevier Ltd. All rights reserved.
*Corresponding author
Keywords: Parkinson’s disease; synaptic vesicle; a-helix; amphipathic; plasticity
Introduction The synucleins comprise a family of small, highly conserved proteins that are generally implicated in the regulation of membrane dynamics. Physiologically, a and b-synuclein are expressed in the nervous system (neocortex, hippocampus, cerebellum, striatum, and thalamus), and these Abbreviations used: PD, Parkinson’s disease; PLD, phospholipase D; PA, phosphatidic acid; PE, phosphatidylethanolamine; PI(4,5)P2, phosphoinositol 4,5-bisphosphate; PC, phosphatidylcholine; ANCOVA, analysis of covariates. E-mail address of the corresponding author: [email protected]
proteins are enriched at presynaptic terminals.1,2 In songbirds, a-synuclein is upregulated in a specific population of presynaptic terminals as they undergo learning-related synaptic rearrangement.3 a-Synuclein appears to be an activity-dependent negative regulator of dopamine neurotransmission at nigrostriatal terminals,4 and a-synuclein anti-sense or knockout leads to depletion of a reserve/distal pool of vesicles in the presynaptic terminal.5,6 g-Synuclein is expressed primarily in the peripheral nervous system and in certain tumors,7,8 and its overexpression enhances the invasive and metastatic potential of breast tumors.9 In 1998, it was discovered that point mutations in the a-synuclein protein sequence are associated
0022-2836/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
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with early-onset familial Parkinson’s disease (PD),10,11 and wild-type a-synuclein was subsequently shown to accumulate within intracellular inclusions and abnormal neurites (Lewy bodies and Lewy neurites) in cases of sporadic PD.12,13 a-Synuclein aggregation has now been identified in a host of neurodegenerative diseases,14 and the term synucleinopathy has been coined to describe this broad spectrum of disorders sharing common synuclein pathology.15 Most relevant to the current study, a and b-synuclein have been identified as specific inhibitors of the phospholipase D (PLD) isoforms PLD216,17 and PLD1.17 In general, the PLD enzymes are phosphatidylcholine-specific hydrolases that produce phosphatidic acid (PA) and choline in response to various hormones, growth factors, cytokines, neurotransmitters, and bioactive lipids.18 PLD is important in the generation of choline for acetylcholine synthesis,19 and PA and its metabolites exhibit several second messenger activities, including the mediation of vesicular transport,20 mitogenesis,21 respiratory burst in neutrophils,18 receptor-mediated endocytosis,22,23 and cytoskeletal rearrangement.24 Such a wide range of activators and downstream effects suggests that PLD plays a pivotal role in signal transduction in many cell types. In the current study, we use site-directed mutagenesis to dissect the structural requirements for synuclein inhibition of PLD2.
Results hPLD2 inhibition by natural synuclein isoforms and familial PD mutants Using a baculovirus construct containing fulllength human PLD2 cDNA plus an N-terminal His6 tag,25 hPLD2 was expressed in Sf9 cells and purified as described in Materials and Methods. Staining of eluates separated by SDS-PAGE with Coomassie brilliant blue showed a 105 kDa protein in infected, but not uninfected, cells; no contaminating proteins were detected. Western blot with a PLD2-specific antibody (PLD2-internal, QCB Inc.) confirmed expression of PLD2 (data not shown). Wild-type and mutant synuclein proteins were purified as described,26 and incubated with 0.1 nM hPLD2 enzyme and 100 mM PE/PI(4,5)P2/PC (16:1.4:1 molar ratio; see Materials and Methods) vesicles. Concentration-dependence was determined by varying concentrations of synuclein proteins from 0.0001 mM to 10 mM. After incubation, lipid vesicles and proteins were precipitated and free [3H]choline was measured by scintillation counting. Increasing concentrations of wild-type hAS induced a sigmoidal decrease in hPLD2 activity (in semi-logarithmic scale, Figure 1A). The linear portion of the curve had an average slope of 2 0.111, standard error ^ 0.015. To determine the Ki of hAS, varying concen-
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trations of hAS and lipid vesicles were assayed as described and the results used to generate a Lineweaver –Burke plot (Figure 1B). Intersection of the double reciprocal inhibitory curves at 1=Vmax on the Y-axis demonstrates that hAS increases the Km ; but does not affect the Vmax of hPLD2, a pattern consistent with competitive inhibition. Michaelis – Menten calculations determined the Ki to be 21 nM hAS. Human b and g-synuclein isoforms inhibited hPLD2 activity as well as hAS. Two mutations associated with early-onset, autosomal dominant Parkinson’s disease, A30P and A53T, were also tested. Interestingly, although A30P inhibition was not different from wild-type, the A53T mutant had a greater inhibitory effect (Figure 2). Mapping of the PLD2-interacting domains To identify the regions of hAS required for hPLD2 inhibition, we utilized a panel of recombinant mutant proteins that we had characterized in previous studies.26,27 We first tested a series of mutants bearing deletions of individual exons within the coding region (D2, D3, D4, D5, D6). We are now revising our exon nomenclature to reflect the genomic sequence analysis reported by Touchman et al.28 Thus, recombinant mutants D2 (residues 2– 41 deleted), D3 (residues 43 –56 deleted), D4 (residues 56 – 102 replaced by Glu), D5 (residues 103 –130 deleted), and D6 (residues 130 –140 deleted) are identical with those mutants described in our previous reports26,27 as recombinant mutants D3 – D7. Our results show that deletion of either exon 4 or exon 6 decreases inhibition significantly (Figure 2). These domains are physically separate and functionally distinct: exon 4 lies within the a-helical domain of hAS, and is sufficient to mediate lipid-binding,26 while exon 6 lies in the acidic tail, including or adjacent to candidate phosphorylation sites (S129, Y125, Y133, and Y136),29 – 33 and has no role in lipid-binding.26 Effect of a-helical secondary structure on PLD2 inhibition We examined the mechanism by which D4 causes a loss of hPLD2 inhibition. Our previous studies have shown that exons 2, 3 and 4 are each individually sufficient to mediate lipid binding, and that deletion of any of these exons causes a decrease in a-helix content, with exon 4 contributing most to both lipid-binding and helix formation (exon 4 . exon 2 . exon 3).26 In the current experiment, D4 was the only exon deletion to exhibit a significant decrement in hPLD2 inhibition, suggesting that hPLD2 inhibition could depend either upon the strength of membrane binding or upon the adoption of a particular a-helical secondary structure by synuclein. To help clarify this point, we used a pair of substitution mutants (T6K and T6E) shown to disrupt the a-helical conformation of hAS.26 T6K, in which six positively
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Synuclein Inhibition of PLD2
Figure 1. hAS is a concentration-dependent inhibitor of hPLD2. A, Varying concentrations of hAS were incubated with 0.1 nM hPLD2 and 100 mM lipid vesicles for 30 minutes at 37 8C and hPLD2 activity measured by scintillation counting of [3H]choline release. The results are plotted as a percentage of hPLD2 activity in the absence of hAS. B, Varying concentrations of lipid vesicles and of wild-type hAS were incubated with 0.1 nM hPLD2 for 30 minutes at 37 8C and [3H]choline release was measured by scintillation counting. Results were plotted for each concentration of hAS, non-linear regression was performed to determine Km and Vmax ; and a Lineweaver – Burke double-reciprocal plot was constructed from the data. Lines representing different concentrations of hAS intersect on the Y-axis at 1=Vmax :
charged lysine residues are substituted for threonine residues along the predicted hydrophobic face of the lipid-binding helix (at positions 22, 33, 44, 59, 81, and 92; see Figure 5), is inhibited in its adoption of an a-helical conformation, although it maintains the capacity to bind acidic phospholipid vesicles (probably through an electrostatic interaction).26 T6E, in which negatively charged glutamate residues are substituted at the same positions, disrupts both lipid binding and a-helix formation.26 Both T6K and T6E mutations decreased the inhibitory capacity of hAS significantly (Figure 2). These data indicate that lipid association (e.g. by T6K) is not sufficient for hPLD2 inhibition by hAS, but that a conformational transition to an a-helix is also required. However, it remains a formal possibility that one or
more of the specific threonine residues that are altered in T6E and T6K, and which lie in exon 4, may be required specifically for inhibition of PLD2 (i.e. residues T59, T81, and T92). Effect of hAS phosphorylation on hPLD2 inhibition Next, we considered the mechanism by which exon 6 contributes to PLD2 inhibition. Exon 6 lies outside of the a-helical domain of hAS, and our laboratory has shown that deletion of exon 6 has no effect on vesicle-binding.26 This evidence suggests that exon 6 participates directly in protein– protein interactions that lead to hPLD2 inhibition. We observed that several potential and
Figure 2. Inhibition of hPLD2 by synucleins. Varying concentrations of a-synuclein, b-synuclein, g-synuclein, and mutant forms of a-synuclein were assayed for hPLD2 inhibition as in Figure 1. The slope of the inhibitory curve is plotted for each protein, with decreased slope indicating increased inhibition. Means ^ SD for three or four separate experiments are shown. P values are reported relative to wild-type hAS.
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Synuclein Inhibition of PLD2
Figure 3. Phosphorylation of serine or tyrosine abolishes inhibition of hPLD2. Varying concentrations of wild-type hAS, fyn-phosphorylated hAS, a tyrosine phosphorylation null mutant (Y4F), tyrosine phosphorylation mimic mutants (DDY, DYD, DYY, and YYD; see Materials and Methods), CKII-phosphorylated hAS, a serine phosphorylation null mutant (S129A), or a serine phosphorylation mimic mutant (S129E) were assayed for hPLD2 inhibition as in Figure 2. Means ^ SD from three or four separate experiments are shown. P values are reported relative to wild-type hAS.
confirmed phosphorylation sites lie within and adjacent to exon 6,29 – 34 and sought to address the role that phosphorylation in this domain might play in regulating hPLD2 inhibition. Figure 3 demonstrates that hPLD2 inhibition is abolished completely by in vitro phosphorylation of hAS with fyn tyrosine kinase. In contrast, mutant Y4F, in which all tyrosine residues in hAS were replaced with phenylalanine (positions 39, 125, 133 and 136), did not differ from wild-type in inhibitory capacity, but lost its sensitivity to regulation by fyn. We tested mutants designed to mimic phosphorylation at specific tyrosine residues (Figure 3). Because Y125 has been reported as the major site of phosphorylation by fyn,34 we substituted aspartate at this position, either alone (DYY) or in combination with similar substitutions at Y133 (DDY) or Y136 (DYD). Of these, DYD abolished hPLD2 inhibition by hAS completely, while DDY and DYY were not significantly different from wild-type. Likewise, substitution at position 136 alone (YYD) had no significant effect on inhibition. We conclude that, for the phosphorylation mimic mutants, a double substitution at positions 125 and 136 is required. Future experiments must show whether the effects of phosphorylation require the modification of both sites. The serine residue at position 129 of hAS has been reported as a site of phosphorylation. We used casein kinase II, a serine/threonine kinase, to phosphorylate hAS in vitro at this position.29,30,33 Figure 3 shows that phosphorylation with CKII abrogates inhibition of hPLD2. We confirmed the phosphorylation site at S129 by substituting alanine at this position (S129A). This null phosphorylation mutant is not different from wild-type in its capacity to inhibit hPLD2, but it is not sensitive to CKII regulation. Again, we generated a mutant to mimic phosphorylation at position 129, by substituting glutamate for serine (S129E). As predicted, this mutant shows significant abrogation of hPLD2 inhibition (Figure 3).
To confirm that the mutants DYD and S129E were still capable of interacting with lipid vesicles and undergoing an a-helical shift, they were subjected to circular dichroism spectroscopy in the presence of phospholipid vesicles as prepared for the hPLD2 assay. Both mutants were unstructured in the absence of lipid, but assumed an a-helical conformation in the presence of vesicles, which previous studies have shown to reflect a
Figure 4. Phosphorylation mimic mutants associate with phospholipid vesicles and undergo a concomitant a-helical shift. Circular dichroism spectra (mean residue ellipticity) are shown for hAS or phosphorylation mimic mutants (0.05 mg/ml) incubated alone (fine lines) or with lipid vesicles (1 mg/ml, PE, PI(4,5)P2, PC 16:1.4:1, bold lines) in 50 mM phosphate buffer. Spectra were collected at 25 8C in a 0.1 cm path-length quartz cell. The a-helix content of mutants was estimated using the K2d algorithm:47 hAS, 77% (continuous line); S129E, 65% (dotted line); and DYD, 59% (broken line).
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helix-stabilizing association with the membrane surface.35 We conclude that DYD and S129E retain the capacity for vesicle binding, although each shows a slight decrement in a-helix content as estimated from the circular dichroism spectra (Figure 4).
Discussion In this study, we find that all natural isoforms of synuclein (a, b and g) can inhibit the activity of purified recombinant hPLD2 with similar efficacy. g-Synuclein was not identified as an inhibitor of PLD2 in Jenco’s reconstitution experiments,16 possibly because the source tissue in those experiments was mouse brain, while g-synuclein is expressed primarily in the peripheral nervous system.36 The observation that PLD2 inhibition is a common property of all synuclein family members is consistent with the hypothesis that PLD inhibition is a biologically significant function of this protein family. While we did not perform a molecular dissection of the b and g-synuclein sequences, their natural variations relative to the a-synuclein sequence are informative. Figure 2 shows that exon 4 (which spans residues 56 –102) is critical for PLD2 inhibition. The corresponding region in b-synuclein is quite similar overall to the a-synuclein sequence (with 12, mostly conservative, substitutions), but notably bears a deletion corresponding to residues 73 –83 of a-synuclein.37 We conclude that the essential residues for PLD2 inhibition in exon 4 must lie between residues 56 and 70 and/or between residues 84 and 102 of a-synuclein. a-Synuclein and g-synuclein inhibit PLD2 to a similar degree, but there is considerable sequence diversity in the domains shown to be relevant for PLD2 inhibition. While differences in exon 4 consist mostly of conservative substitutions, g-synuclein has a considerably truncated C terminus, with no homology to the conserved sequences in exon 6 of a and b-synuclein, shown here to be critical for PLD2 inhibition. In particular, the residues S129 and Y125/Y136, conserved in a and b-synuclein, are absent from g-synuclein. However, the C terminus of g-synuclein maintains the general acidic motif found in a and b-synucleins, due to a preponderance of glutamate residues. We speculate that this chemical quality may contribute to PLD2 inhibition, but that g-synuclein is either not modulated by phosphorylation, or is modulated by phosphorylation at different sites. The PD-associated mutants A53T and A30P were examined in this study. A30P did not differ significantly from wild-type in its capacity to inhibit PLD2. This is not so surprising, given that deletion of exon 2, in which A30P resides, has no significant effect on PLD2 inhibition in the present experiments, indicating that the inhibitory phenomenon is not dependent upon the structure or even the
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presence of this domain. This is consistent with our previous studies demonstrating that A30P and D2 mutants are competent to bind synthetic membranes and assume a conformation that is predominantly a-helical, albeit with a lower overall helical content.26 Interestingly, however, the PD-associated mutant A53T displayed enhanced inhibition of PLD2 as compared to wild-type hAS. The significance of this difference will require further exploration, but the autosomal dominant expression of the A53T phenotype is consistent with a biochemical gainof-function, and excessive inhibition of PLD2 should be considered as a possible contributing factor in the pathology associated with this mutation. We examined possible mechanisms of PLD2 inhibition by a-synuclein. One candidate mechanism had been eliminated by Jenco et al.16 They reported that PLD2 activity is dependent upon the availability of phosphoinositol 4,5-bisphosphate (PI(4,5)P2), but that inhibition by a-synuclein could not be overcome by increasing the concentration of PI(4,5)P2 in the assay vesicles. Thus inhibition by a-synuclein is not the result of sequestration of the available PI(4,5)P2. To complement these data, we performed Lineweaver –Burke analysis (Figure 1B), varying the concentrations of a-synuclein and bulk vesicles; the result is consistent with a competitive inhibition model, wherein a-synuclein competes with the substrate for binding PLD2. However, we know that a-synuclein is itself capable of binding phospholipid membranes of similar composition27 and at comparable ionic strengths35,38 as those used in the PLD2 assay system. This leaves room for the alternative interpretation, that a-synuclein inhibits PLD2 by intercalating into the membrane and reducing the effective surface concentration of the substrate (surface dilution).39 Like competitive inhibition, surface dilution should be overcome by increasing the bulk lipid concentration, so our analysis does not distinguish these two possibilities. To discriminate between competitive inhibition and surface dilution, one should ideally use a mixed detergent/phospholipid micelle system, which allows the concentration of substrate (phosphatidylcholine) to be varied independently of the bulk lipid concentration. Unfortunately, Jenco et al. report that PLD2 does not maintain its activity in such a mixed-micelle system, somewhat complicating our experimental design. However, by analyzing the roles of different a-synuclein domains in PLD2 inhibition we can speculate about the likely mechanisms of inhibition. The sequences required for PLD2 inhibition by a-synuclein were determined by comparing the activities of a series of complementary deletion mutants covering all of the coding exons of hAS (exons 2 –6). All synuclein isoforms share a similar overall structural organization. More than 60% of the protein sequence is derived from an 11 residue
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repeating motif, which can fold as an apolipoprotein-like amphipathic a-helix.3 This helix (spanning exons 2 –4) mediates binding to membranes containing acidic phospholipid, and undergoes a conformational shift from random coil to a-helix upon membrane binding.26 This a-helical conformation is observed only when a-synuclein is associated with a surface such as a lipid vesicle or detergent micelle; the protein is natively unfolded in solution.40 The lipid-binding domain associates with the membrane as a continuous a-helix with a single break,41 and association of this helical domain with a phospholipid bilayer causes disruption of the membrane structure.38 If synuclein inhibits PLD2 primarily by surface dilution, then deletions within the surface-active amphipathic a-helical domain (D2, D3 and D4) should decrease enzyme inhibition. However, we observe significant effects only for D4, a result seemingly inconsistent with surface dilution. Likewise, the effects of the C-terminal truncation (D6) and the C-terminal phosphorylation-mimic mutants (DYD and S129E) are more consistent with competitive inhibition. We showed that exon 6 does not participate in lipid-binding, and does not undergo an a-helical shift in the presence of lipid.26 It follows that deletion of exon 6 should not affect the ability of the protein to intercalate into the membrane and dilute the surface concentration of substrate (phosphatidylcholine), yet D6 is completely devoid of inhibitory activity (Figure 2). Similarly, we show here that the mutants S129E and DYD retain their ability to interact with lipid membranes and undergo a shift to an a-helical conformation (Figure 4), yet lack the capacity to inhibit PLD2. Together with the observation by Ahn that a-synuclein and PLD2 proteins interact directly,17 we favor the conclusion that a-synuclein inhibits PLD2 primarily through a specific competitive interaction. Our conclusion that PLD2 inhibition is modulated by C-terminal phosphorylation of a-synuclein is consistent with other published reports. In transfected HEK293 cells, a-synuclein inhibits the pervanadate-induced activity of both PLD1 and PLD2, but inhibition is increased if alanine is substituted for Y125 of a-synuclein, suggesting a role for tyrosine phosphorylation in modulation of PLD inhibition.17 Phosphorylation of a-synuclein by G protein-coupled receptor kinase 5 (GRK5) has also been shown to decrease inhibition of PLD2 in vitro.29 We have extended these studies by developing site-specific phosphorylation mimic mutants that are constitutively deficient in PLD2 inhibition. Other presynaptic proteins have been found to be specific inhibitors of PLD1 and PLD2, including AP3/AP180,42 synaptojanin,43 and amphiphysin I and II.44 Localized production of phosphatidic acid by PLD promotes positive membrane curvature, which can enhance vesicle fission and endocytosis. However, PLD activity must presumably be inhibited in the newly formed vesicle to
Synuclein Inhibition of PLD2
allow for vesicle fusion with the target membrane, and the redundancy of this inhibition in the presynaptic terminal is thought to be important for rapid synaptic vesicle recycling.44 Thus, the synucleins and other synapse-specific PLD inhibitors may regulate synaptic plasticity by influencing the availability of synaptic vesicles for release. Studies in a-synuclein knockout mice support this hypothesis. Abeliovich et al. report that dopaminergic neurons in the substantia nigra of a-synuclein knockout animals display more rapid recovery from paired-pulse depression, implying more efficient replenishment of the “readilyreleasable” pool of synaptic vesicles in the absence of a-synuclein.4 In contrast, Cabin et al. report that CA1 synapses from knockout mice are rapidly depleted of a different subset of presynaptic vesicles, the “reserve” or “resting pool”, upon prolonged stimulation.6 Taken together, these observations support a model wherein a-synuclein inhibits rapid recycling of presynaptic vesicles to the “readily releasable” pool, and promotes a slower process whereby membranes are recycled to the “reserve” pool. a-Synuclein may thus serve a critical modulatory role in synaptic function by influencing synaptic strength following particular patterns of synaptic activity.
Materials and Methods Purification of hPLD2 Sf9 insect cells (Invitrogen) were maintained at 27.5 8C in Grace’s Insect Medium (Invitrogen) supplemented with 10% (v/v) insect-tested FBS (Sigma) and Pen-strepfungizone (Invitrogen). A baculovirus containing fulllength human PLD2 cDNA plus an N-terminal His6 tag was obtained from Dr Rebecca S. Arnold.25 A total of 8 £ 106 exponentially growing cells per 75 cm2 flask were infected with hPLD2 baculovirus at a multiplicity of infection of 10. Cells were incubated for 48 – 72 hours and then collected and washed in PBS (137 mM NaCl, 2.7 mM KCl, 10 nM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). Cells were solubilized in lysis buffer (50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 10 mM imidazole), centrifuged at 12,000g, and the supernatant incubated with Ni-NTA beads (Qiagen) for two hours. Beads were washed with 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 120 mM imidazole, and recombinant hPLD2 was eluted with 50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, 1% n-octyl b-D -glucopyranoside, 250 mM imidazole. Protein purity was analyzed by SDS-PAGE; no contaminating proteins were detectable by staining with Coomassie brilliant blue. Recombinant synuclein protein Recombinant substitution mutants engineered for this study were as follows: S129E, S129 replaced by Glu; S129A, S129 replaced by Ala; DYD, Y125 and Y136 each replaced by Asp; DDY, Y125 and Y133 each replaced by Asp; YYD, Y136 replaced by Asp. All were cloned into the vector pET28(a) (Novagen) for expression in Escherichia coli under an inducible bacteriophage T7 lac promoter, as described.26 Human a, b, and g-synuclein,26
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Synuclein Inhibition of PLD2
Figure 5. A map of hAS. The coding region of the hAS gene is represented by exons 2 – 6. Exons 2 – 4 encode seven amphipathic a-helical repeats (shaded ovals) that are required for lipid binding.26 Six threonine residues (T22, T33, T44, T59, T81, T92) lie centrally along the hydrophobic face of the modeled a-helix; substitution with charged residues at these sites (T6E and T6K) decreases lipid binding26 and abrogates hPLD2 inhibition (Figure 4). A30P and A53T describe point mutations in the protein that are associated with familial, early-onset Parkinson’s disease. Also indicated are the positions of tyrosine residues (Y39, Y125, Y133, Y136) and of S129, which are sites of phosphorylation and/or substitution in this study.
and recombinant mutants, Y4F, A30P, A53T, T6E and T6K were prepared as described.27 We have revised our nomenclature so that mutants described as D3 – D726,27 are now referred to as D2 – D6, to reflect the genomic sequence analysis reported by Touchman et al.28 Specifically, these mutants are D2 (residues 2 – 41 deleted), D3 (residues 43 – 56 deleted), D4 (residues 56 – 102 replaced by Glu), D5 (residues 103– 130 deleted), and D6 (residues 130– 140 deleted) (Figure 5). Protein purity was confirmed by staining with Coomassie brilliant blue following SDS-PAGE.
In vitro phosphorylation For serine phosphorylation, recombinant purified hAS (5 mg) was incubated with 138 milliunits of active casein kinase II (Upstate Biotechnology) in 20 mM Tris –HCl (pH 8.0), 4 mM MgCl2, 130 mM KCl, 800 mM ATP and 250 ng of poly-L -lysine (Sigma; 150– 300 kDa) at 30 8C for 60 minutes. The total reaction volume was 50 ml. For tyrosine phosphorylation, 2 mg of recombinant purified hAS was incubated with 18.75 milliunits of active fyn kinase (Upstate Biotechnology) in 50 mM Tris (pH 7.5), 10 mM EGTA, 100 mM vanadate, 75 mM MnCl2 and 300 mM ATP at 30 8C for 60 minutes. The total reaction volume was 50 ml.
hPLD2 activity and inhibition by synucleins hPLD2 activity was assayed as described.45 Briefly, 0.1 nM hPLD2 (1 ng) was added to assay buffer (50 mM Na-Hepes (pH 7.5), 3 mM EGTA, 80 mM KCl, 1 mM DTT, 3 mM MgCl2, 2 mM CaCl2) containing lipid vesicles (phosphatidylethanolamine (PE), phosphoinositol 4,5bisphosphate (PI(4,5)P2), and phosphatidylcholine (PC) (Avanti Polar Lipids) molar ratio 16:1.4:1, plus 50,000– (Amersham100,000 cpm [3H]phosphatidylcholine Pharmacia)) at a final lipid concentration of 100 mM in a total volume of 100 ml. Synucleins were added where indicated. Reactions were incubated at 37 8C for 30 minutes and then stopped with 200 ml of 10% (v/v) trichloroacetic acid and 100 ml of 10% (w/v) BSA to precipitate proteins and lipids. Free [3H]choline released by hPLD2 was measured by scintillation counting. Statistical analysis of inhibition used the General Linear Model to perform analysis of covariates (ANCOVA) to compare slopes between proteins. Statistical analysis was performed in consultation with the Illinois Statistics Office of the University of Illinois at Urbana-Champaign, using the statistical analysis package SAS.
Circular dichroism spectroscopy CD spectra were collected for hAS and the mutants DYD and S129E using a JASCO J-720 spectropolarimeter. Spectra were taken at 25 8C in a 0.1 cm path-length quartz cuvette containing the sample at 0.05 mg/ml of protein, 1 mg/ml of lipid vesicles (PE, PI(4,5)P2, and PC (Avanti Polar Lipids) molar ratio 16:1.4:1), in 20 mM sodium phosphate buffer (pH 7.4). The spectral contributions of buffer and vesicles were subtracted as appropriate. The percentage of a-helix was determined from the molar ellipticities at 222 nm,46 as well as by computer fitting to a library of CD spectra from proteins of known structure using the learning neural network program K2D, which is based on the algorithm published by Andrade et al.47
Acknowledgements This work was supported by grant R01 AG13762 from the National Institute on Aging, and by grants to J.P. from the Illinois Department of Public Health and the American Federation for Aging Research. We thank Dr Rebecca Arnold for her gift of the His-tagged hPLD2 baculovirus construct. Circular dichroism experiments were performed at the Laboratory for Fluorescence Dynamics (LFD) at the University of Illinois at UrbanaChampaign (UIUC). The LFD is supported jointly by the Division of Research Resources of the National Institutes of Health (PHS 5 P41-RR03155) and UIUC.
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Edited by P. T. Lansbury Jr (Received 6 May 2003; received in revised form 2 December 2003; accepted 5 February 2004)