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Interactions of Synphilin-1 with phospholipids and lipid membranes
FEBS Letters 580 (2006) 4479–4484
Interactions of Synphilin-1 with phospholipids and lipid membranes Tetsuya Takahashi, Hiroshi Yamashita*, Yoshito Nagano, Takeshi Nakamura, Tatsuo Kohriyama, Masayasu Matsumoto Department of Clinical Neuroscience and Therapeutics, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Hiroshima 734-8551, Japan Received 9 June 2006; revised 5 July 2006; accepted 5 July 2006 Available online 17 July 2006 Edited by Judit Ova´di
Abstract Synphilin-1 is an a-synuclein binding protein that is involved in the pathogenesis of Parkinson’s disease. The present study investigated the phospholipid-binding capacity of Synphilin-1. The C-terminus of Synphilin-1 was found to selectively bind to acidic phospholipids, including phosphatidic acid, phosphatidylserine, and phosphatidylglycerol, but not to naturally charged phospholipids. Synphilin-1 was targeted to cytoplasmic lipid droplets in mammalian cells. The amino acid sequence 610–640 was found to represent the primary determinant site for phospholipid binding. Moreover, the R621C mutation identified in Parkinson’s disease abolished Synphilin-1 association with lipid droplets. The lipophilicity of Synphilin-1 might prove relevant to its physiologic function. 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Synphilin-1; a-Synuclein; Parkinson’s disease; Phospholipid; Lipid droplet
1. Introduction Parkinson’s disease represents the second most common neurodegenerative disorder, pathologically characterized by loss of dopaminergic neurons in the substantia nigra pars compacta and appearance of Lewy bodies in remaining neurons [1]. Autosomal dominant Parkinson’s disease, i.e. PARK1 and PARK4, is caused by missense mutations and triplication of the gene for a-synuclein, one of the main components of Lewy bodies [2]. Several studies suggest association of a-synuclein with phospholipids and lipid membranes [3]. a-Synuclein is involved in vesicle trafficking and lipid droplet accumulation [4]. Synphilin-1, an a-synuclein-interacting protein, is a component of the core region of Lewy bodies. The mutation in the Synphilin-1 gene leading to a substitution from arginine (R) to cysteine (C) at position 621 (R621C) has been identified in Parkinson’s disease [5]. Synphilin-1 contains six ankyrin repeats, a coiled-coil domain, and a well conserved region * Corresponding author. Fax: +81 82 505 0490. E-mail address: [email protected] (H. Yamashita).
Abbreviations: CL, cardiolipin; GST, glutathione S-transferase; LC, lysophosphatidylcholine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phsophatidylinositol; PS, phosphatidylserine; SM, sphingomyeline; MLVs, multilamellar vesicles
(amino acids (aa) 612–689) of unknown function [5]. Lipid fractions of brain extracts contain Synphilin-1 [6–8]. Synphilin-1 is predominantly expressed in neurons, where it localizes to the cytoplasm as well as presynaptic nerve terminals, and associates with synaptic vesicles. Accordingly, intracellular localization of Synphilin-1 is similar to that of a-synuclein. The present study investigated the binding capacity of Synphilin-1 with phospholipids and lipid membranes.
2. Materials and methods 2.1. Phospholipids and reagents Lysophosphatidylcholine (LC), sphingomyeline (SM), phosphatidylcholine (PC), phosphatidic acid (PA), phsophatidylinositol (PI), phosphatidylserine (PS), cardiolipin (CL), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and dioleoylphosphocholine were obtained from Sigma. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphate, 1-palmitoyl-2-oleoyl-snglycero-3-[phospho-L -serine] were purchased from Avanti Polar Lipid. Oleic acid, fatty acid-free BSA, and oil red O were purchased from Sigma. 2.2. Vectors and preparation of GST-fusion proteins The coding region of human Synphilin-1 cDNA was subcloned into a pcDNA3 vector (BD Biosciences) in frame with the HA tag sequence (pcDNA3-HA-Synphilin-1), as previously described [9]. The R621C point mutation of Synphilin-1 was generated using a transformer sitedirected mutagenesis kit (BD Biosciences) with the following synthetic oligonucleotide: 5 0 -CTG ATA AAA TCT TAT GCC AGT TAT TGG G-3 0 . The mutation was confirmed by DNA sequencing. DNA fragments obtained from PCR were subcloned into a pcDNA3 vector in frame with the HA tag sequence (pcDNA3-HA-Synphilin-1 R621C). To generate Synphilin-1 proteins fused with glutathione S-transferase (GST) at the N-terminus, corresponding cDNA fragments for various truncations of Synphilin-1, including the N-terminus (aa 1–202, GST-Syp-N1), the middle N-terminus plus the ankyrin repeats from 1–3 (aa 87–458, GST-Syp-N2), the ankyrin repeats 1–4 plus the coiled-coil domain (aa 349–617, GST-Syp-ANK), the ankyrin repeats 5–6 plus the C-terminus (aa 611–919, GST-Syp-C), and serial C-terminal truncations (aa 610–675, GST-Syp-C1; aa 641–730, GST-Syp-C2; aa 706–806, GST-Syp-C3; aa 771–880, GST-Syp-C4; and aa 851– 919, GST-Syp-C5) were subcloned into a pGEX6P-1 vector (Amersham), as previously described [9]. The sequences of all constructs were confirmed by DNA sequencing. GST-fusion proteins were produced in Escherichia coli BL21 via isopropyl-b-D -thiogalactopyranoside (Sigma) induction and purified using glutathione–sepharose 4B (Amersham), as previously described [9–14]. 2.3. Cell culture and immunofluorescent staining COS7 cells were obtained from the American Type Culture Collection and grown in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum. Cells seeded and grown on glass coverslips in the medium were transfected with pcDNA3-HA-Synphilin-1 or pcDNA3-HA-Synphilin-1 R621C using FuGENE 6 (Roche), as previously described
0014-5793/$32.00 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.07.019
4480 [11,12]. At a time of 24 h post-transfection, cells were grown in the presence of 600 lM oleic acid and 100 lM fatty acid-free BSA in the medium for 14 h to induce cytoplasmic lipid droplets. Adherent cells on the glass coverslips were fixed, permeabilized, and immunostained using an anti-HA antibody (Sigma), followed by the Alexa Fluor 488 anti-rabbit IgG antibody (Invitrogen), as previously described [12]. Cells on the same glass coverslips were incubated with the oil red O solution (3 mg/ml in 36% triethylphosphate) for 0.5 h to visualize cytoplasmic lipid droplets. Preparations were examined with a Zeiss LSM510 confocal microscope. 2.4. Protein–lipid overlay assay A protein–lipid overlay assay was performed using the GST-fusion proteins, as previously described [15]. To make a membrane pre-spotted with a concentration gradient of phospholipids (the membrane phospholipid array), the phospholipid solutions, each at 12.5 lg/ll (1·), were serially diluted from 2-fold (2·) up to 32-fold (32·) with chloroform/methanol/distilled water (1:2:0.8). Each 1 ll of serial dilutions of phospholipids was spotted onto a nitrocellulose membrane (Schleicher and Schuell) and dried to allow each spot to contain the following dose of phospholipid: 12.5 lg/1· spot, 6.25 lg/2· spot, 3.13 lg/4· spot, 1.56 lg/8· spot, 0.78 lg/16· spot, and 0.39 lg/32· spot. For a protein–lipid overlay assay, the membrane phospholipid array was blocked with TBS-T (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20) plus 3% (w/v) fatty acid-free BSA for 1 h at room
T. Takahashi et al. / FEBS Letters 580 (2006) 4479–4484 temperature, followed by incubation with GST-fusion proteins (2 lg/ ml) or proteins in TBS-T plus 3% (w/v) fatty acid-free BSA at 4 C overnight. After the membrane was washed 6 times with TBS-T, at 5 min per washing, GST-fusion proteins bound to phospholipids spotted on the membrane were analyzed with immunoblotting with an antiGST monoclonal antibody (1:1000). The blots were developed using enhanced chemiluminescence reagents (Amersham). Alternatively, COS7 cells seeded on a 6 cm-dish (1.5 · 106 cells per dish) were transfected with empty pcDNA3-HA vectors, pcDNA3HA-Synphilin-1 or pcDNA3-HA-Synphilin-1 R621 C. After 24 h post transfection, cells were washed with ice-cold PBS and lysed in ice-cold lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, and protease inhibitors) and centrifuged. After the lysate protein contents were measured using a protein assay kit (Bio-Rad, Hercules, CA, USA), the lysate proteins were diluted at a concentration of 2 lg/ml in TBS-T plus 3% (w/v) fatty acid-free BSA, and subjected to a protein– lipid overlay assay using the membrane phospholipid array in the same manner. 2.5. Binding of Synphilin-1 to synthetic phospholipid vesicles 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate, or 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L -serine] was each mixed with dioleoylphosphocholine (molar ratio 1:1) in chloroform. The lipid was deposited as a film on the wall of a glass test tube by solvent evaporation with nitrogen stream, followed by pumping for 2 h under vacuum. The resulting
Fig. 1. Association of Synphilin-1 protein with phospholipids. (A) Schematic representation of the primary structures of glutathione S-transferase (GST)-fusion proteins utilized for the assay. A series of truncations in the conserved region of Synphilin-1 (Syp), including six ankyrin repeats (ANK1-6) or the coiled-coil (CC) domain, was fused with GST at the N-terminus. (right) GST-fusion protein used for the assay was detected by Coomassie staining. Molecular mass markers are indicated on the left. (B) Protein–lipid overlay assay. To generate the membrane phospholipid array, serial dilutions of phospholipids, including lysophosphatidylcholine (LC), sphingomyeline (SM), phosphatidylcholine (PC), phosphatidic acid (PA), phsophatidylinositol (PI), phosphatidylserine (PS), cardiolipin (CL), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG), were spotted onto a nitrocellulose membrane to allow each spot to contain the following doses: 12.5 lg/1· spot, 6.25 lg/2· spot, 3.13 lg/4· spot, 1.56 lg/ 8· spot, 0.78 lg/16· spot, and 0.39 lg/32· spot. The membrane phospholipid arrays were incubated with 2 lg/ml of GST-fusion proteins (input) or vehicle (none) and bound proteins were detected by immunoblotting with an anti-GST antibody.
T. Takahashi et al. / FEBS Letters 580 (2006) 4479–4484
4481 (mass ratio 1:20) in TE (10 mM Tris–HCl (pH 8.0) and 1 mM EDTA) and incubated for 2 h, followed by ultracentrifugation at 164 000 · g for 0.5 h. The pellet of MLVs was washed five times with TE and dissolved in sample buffer. GST-fusion proteins bound to MLVs were analyzed by immunoblotting with an anti-GST antibody (Sigma).
3. Results and discussion
Fig. 2. Binding of Synphilin-1 with phospholipid-containing vesicles. Multilamellar vesicles (MLVs) containing phosphatidic acid (PA), phosphatidylserine (PS), and phosphatidylethanolamine (PE) were prepared. Incubations with various GST-fusion Synphilin-1 proteins or GST alone (input) were performed with MLVs or vehicle (N), followed by ultracentrifugation. Proteins bound to MLVs in the pellet, as well as input proteins, were analyzed by immunoblotting with an anti-GST antibody.
lipid film was re-hydrated in HBS (10 mM HEPES–NaOH (pH 7.4) and 150 mM NaCl) followed by vortexing for 20 min to form multilamellar vesicles (MLVs). MLVs were mixed with GST-fusion proteins
3.1. The C-terminus of Synphilin-1 has a specific capacity to bind to acidic phospholipids To investigate whether or not Synphilin-1 interacts with phospholipids in cells, nine different biologically abundant phospholipids found in intracellular membranes, that is LC, SM, PC, PA, PI, PS, CL, PE, and PG were chosen for the assay. A protein–lipid overlay assay was performed using the membrane phospholipid array with recombinant GST-fusion proteins containing various truncations of Synphilin-1 (Fig. 1A). The binding affinity of proteins for phospholipids was also evaluated at six dilution levels from 1· to 32·. The PA-binding affinity of GST alone, as well as GST-Syp-N1, -N2, or -ANK was only detected at the 1· to 2· dilution level (Fig. 1), suggesting that such binding is non-specific. In contrast, the binding of GST-Syp-C to PA was detected in a
Fig. 3. The C-terminus of Synphilin-1 associates with phospholipids. (A) Schematic representation of the constructs of GST-fusion Synphilin-1 (Syp) proteins utilized for the assay. The C-terminus of Syp (GST-Syp-C in Fig. 1A) was divided into five parts and a series of truncations was fused with GST. (right) GST-fusion protein used for the assay was detected by Coomassie staining. Molecular mass markers are indicated on the left. (B) Protein–lipid overlay assay. The membrane phospholipid arrays were incubated with GST-fusion proteins (2 lg/ml, input) in the same manner as in Fig. 1. GST-fusion proteins bound to phospholipids spotted on the membrane were analyzed by immunoblotting with an anti-GST antibody.
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Fig. 4. Phospholipid-binding property of Synphilin-1. (A) A protein–lipid overlay assay using COS7 cells transfected with empty pcDNA3-HA vectors (Cont), pcDNA3-HA-Synphilin-1 wild type (WT), or pcDNA3-HA-Synphilin-1 R621C (R621C) was performed. (left upper) Wild type and mutant HA-tagged Synphilin-1 (HA-Synphilin-1) proteins in lysates proteins from transfected cells were analyzed by immunoblotting with an antiHA antibody. 0.2 lg of cell lysate protein was loaded per each lane. (left lower and right) A protein–lipid overlay assay. The cell lysate proteins were diluted at a concentration of 2 lg/ml in TBS-T plus 3% (w/v) fatty acid-free BSA, and subjected to a protein–lipid overlay assay using the membrane phospholipid array. HA-Synphilin-1 proteins bound to phospholipids were analyzed by immunoblotting with an anti-HA antibody. (B) Wild type Synphilin-1 localization around lipid droplets in mammalian cells. COS7 cells transfected with expression vectors for wild type (WT) or R621C mutant HA-Synphilin-1 were incubated with oleic acid (600 lM) in medium for 14 h. The cells were processed for double fluorescence confocal microscopy for HA-Synphilin-1 and lipid droplets using an anti-HA antibody and oil red O, respectively. Arrows represent co-localization of HASynphilin-1 and lipid droplets. Bars, 10 lm.
dose-dependent manner (Fig. 1B), with binding affinity detected at the 32· dilution level, suggesting that the Synphilin1 C-terminus possesses capacity to bind to PA. In addition, no binding between phospholipids, except PA and all GST-fusion proteins other than GST-Syp-C, was detected (Fig. 1B). GST-Syp-C also showed a high affinity for PS at the 16· dilution level, as well as a low affinity for CL and PG at the 2· dilution level (Fig. 1B). GST-Syp-C did not interact with PC, SM, PC, PI, or PE. All assays were replicated three times with the same results. Accordingly, the C-terminus (aa 610–919) confers phospholipid-binding capacity to Synphilin-1. The theoretical isoelectric point (pI) for other regions of Synphilin-1 is lower than 7.4 (4.5 for Syp-N1, 4.9 for Syp-N2, and 5.3 for Syp-ANK) and is thus unlikely to interact with acidic phospholipids. On the other hand, the pI for Syp-C was 9.5, consis-
tent with an affinity to acidic phospholipids. The C-terminus might represent a primary determinant of phospholipid-binding capacity.
3.2. The C-terminus of Synphilin-1 interacts with multilamellar vesicles containing PA and PS Since the C-terminus of Synphilin-1 showed affinity for PA and PS, a pull-down assay was performed using MLVs containing such phospholipids to confirm the interaction. As a control, MLVs containing PE were also subjected to the assay. MLVs containing PA, PS, and PE were prepared using their palmitoylated derivatives, i.e. 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-snglycero-3-phosphate, and 1-palmitoyl-2-oleoyl-sn-glycero-3-
T. Takahashi et al. / FEBS Letters 580 (2006) 4479–4484
[phospho-L -serine]. MLVs were first incubated with GST-fusion Synphilin-1 proteins, then bound proteins were analyzed by immunoblotting with anti-GST antibodies. MLVs containing PA and PS, but not PE, precipitated GST-Syp-C (Fig. 2). On the other hand, no MLVs precipitated GST alone or GST-Syp-N1, -N2, or -ANK. No GST-fusion protein was precipitated in the absence of MLVs (Fig. 2). Such selective interaction agreed with the results of the protein–lipid overlay assays. 3.3. The primary determinant site for phospholipid binding To identify the region responsible for phospholipid binding within the C-terminus, GST-fusion proteins containing serial C-terminal truncations, i.e. GST-Syp-C1, -C2, -C3, -C4, and -C5, were generated (Fig. 3A). GST-Syp-C (Fig. 1B) and GST-Syp-C1 (aa 610–675) showed similar binding to phospholipids, including PA, PS, CL, and PG, while GST-Syp-C2 (aa 641–730) had a low binding affinity to only PS. Such results suggest that 30 amino acids (aa 610–640), which includes the ankyrin repeat 5, represent the primary determinant site for phospholipid binding. Interestingly, the R621C mutation found in Parkinson’s disease also resides within this sequence. It should also be noted that GST-Syp-C4 and -C5 also exhibited binding affinity to PS, CL, and PG (Fig. 3B). All assays were replicated three times with the same results. Since the calculated pI of aa 675–730 is 5.1, it is likely that the acidic nature of this region inhibits phospholipid binding. However, such phenomenon was not observed. Therefore, simple chargebased interactions may not be the sole influence on in vitro binding. 3.4. Synphilin-1 was localized around lipid droplets The R621C mutation in the Synphilin-1 protein has been reported in Parkinson’s disease [5]. Moreover, amino acid residue 621 localizes to the middle of the ankyrin repeat 5. To investigate whether or not the R621C mutation in Synphilin1 affects the phospholipid-binding specificity, a protein–lipid overlay assay was performed using lysate proteins from COS7 cells transfected with HA-tagged expression vectors for wild type or R621C mutant Synphilin-1, as well as empty expression vectors. At a time of 24 h post-transfection, proteins in cell lysates (Fig. 4A) were incubated with the membrane phospholipid array and bound proteins were detected with immunoblotting with an anti-HA antibody. The phospholipid-binding specificity of wild type Synphilin-1 was similar to that of GST-Syp-C or GST-Syp-C1 (Fig. 4A). In contrast, the R621C mutation inhibited Synphilin-1 binding to PS (Fig. 4A), suggesting that a single amino acid substitution in the primary determinant site could change the phospholipid-binding specificity. Repeated immunofluorescent staining revealed that wild type Synphilin-1 preferentially localized around lipid droplets (Fig. 4B). The appearance of Synphilin-1 localized to the vicinity of lipid droplets was similar to previous descriptions, namely a ring-like configuration [9]. In contrast, R621C mutant Synphilin-1 did not localize to the vicinity of lipid droplets and both entities were independently distributed in the cytoplasm (Fig. 4B). It is noteworthy that a single substitution in the Syp-C1 region abolished the ability of Synphlilin-1 to interact with lipid droplets.
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3.5. Concluding remarks A number of proteins with affinity to phospholipids, such as adipose differentiation-related protein perilipin or a-synuclein, are present on the surface of lipid droplets that are coated by a phospholipid monolayer originating from endoplasmic reticulum [4,16]. In a previous study, we found that Synphilin-1 exhibits a ring-like configuration in the cytoplasm [9]. Overexpression of Synphilin-1 leads to formation of membranebound inclusions that contain multi-layered or lamellar-like phospholipid accumulations [17]. The core region of Lewy bodies is immunolabeled intensely with anti-Synphilin-1 antibody [18] and enriched with lipids [19]. In addition, a-synuclein immunoreactivity concentrates around lipid droplets, which are a component of neuromelanin in the substantia nigra [20]. Such findings indicate that the lipophilic properties of both Synphilin-1 and a-synuclein may be relevant to not only the pathogenesis of Parkinson’s disease, but also physiologic function. Acknowledgements: This work was supported by grants-in-aid for the Encouragement of Young Scientists (2005, 2006) and for Scientific Research (2005, 2006) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology. We thank Mrs. Y. Furuno and M. Sasanishi for technical assistance and Dr. C.J. Hurt for manuscript corrections.
References [1] Forno, L.S. (1996) Neuropathology of Parkinson’s disease. J. Neuropathol. Exp. Neurol. 55, 259–272. [2] Spillantini, M.G., Schmidt, M.L., Lee, V.M., Trojanowski, J.Q., Jakes, R. and Goedert, M. (1997) Alpha-synuclein in Lewy bodies. Nature 388, 839–840. [3] Kubo, S., Nemani, V.M., Chalkley, R.J., Anthony, M.D., Hattori, N., Mizuno, Y., Edwards, R.H. and Fortin, D.L. (2005) A combinatorial code for the interaction of alphasynuclein with membranes. J. Biol. Chem. 280, 31664–31672. [4] Cole, N.B., Murphy, D.D., Grider, T., Rueter, S., Brasaemle, D. and Nussbaum, R.L. (2002) Lipid droplet binding and oligomerization properties of the Parkinson’s disease protein alphasynuclein. J. Biol. Chem. 277, 6344–6352. [5] Marx, F.P., Holzmann, C., Strauss, K.M., Li, L., Eberhardt, O., Gerhardt, E., Cookson, M.R., Hernandez, D., Farrer, M.J., Kachergus, J., Engelender, S., Ross, C.A., Berger, K., Schols, L., Schulz, J.B., Riess, O. and Kruger, R. (2003) Identification and functional characterization of a novel R621C mutation in the synphilin-1 gene in Parkinson’s disease. Hum. Mol. Genet. 12, 1223–1231. [6] Kruger, R. (2004) The role of synphilin-1 in synaptic function and protein degradation. Cell Tissue Res. 318, 195–199. [7] Murray, I.J., Medford, M.A., Guan, H.P., Rueter, S.M., Trojanowski, J.Q. and Lee, V.M. (2003) Synphilin in normal human brains and in synucleinopathies: studies with new antibodies. Acta Neuropathol. (Berl.) 105, 177–184. [8] Ribeiro, C.S., Carneiro, K., Ross, C.A., Menezes, J.R. and Engelender, S. (2002) Synphilin-1 is developmentally localized to synaptic terminals, and its association with synaptic vesicles is modulated by alpha-synuclein. J. Biol. Chem. 277, 23927–23933. [9] Nagano, Y., Yamashita, H., Takahashi, T., Kishida, S., Nakamura, T., Iseki, E., Hattori, N., Mizuno, Y., Kikuchi, A. and Matsumoto, M. (2003) Siah-1 facilitates ubiquitination and degradation of synphilin-1. J. Biol. Chem. 278, 51504–51514. [10] Nagano, Y., Yamashita, H., Nakamura, T., Takahashi, T., Kondo, E. and Nakamura, S. (2001) Lack of binding observed between human alpha-synuclein and Bcl-2 protein family. Neurosci. Lett. 316, 103–107. [11] Nakamura, T., Yamashita, H., Nagano, Y., Takahashi, T., Avraham, S., Avraham, H., Matsumoto, M. and Nakamura, S. (2002) Activation of Pyk2/RAFTK induces tyrosine phosphorylation
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[12] [13]
[14]
[15] [16]
T. Takahashi et al. / FEBS Letters 580 (2006) 4479–4484 of alpha-synuclein via Src-family kinases. FEBS Lett. 521, 190–194. Nakamura, T., Yamashita, H., Takahashi, T. and Nakamura, S. (2001) Activated Fyn phosphorylates alpha-synuclein at tyrosine residue 125. Biochem. Biophys. Res. Commun. 280, 1085–1092. Takahashi, T., Yamashita, H., Nagano, Y., Nakamura, T., Ohmori, H., Avraham, H., Avraham, S., Yasuda, M. and Matsumoto, M. (2003) Identification and characterization of a novel Pyk2/related adhesion focal tyrosine kinase-associated protein that inhibits alpha-synuclein phosphorylation. J. Biol. Chem. 278, 42225–42233. Takahashi, T., Yamashita, H., Nakamura, T., Nagano, Y. and Nakamura, S. (2002) Tyrosine 125 of alpha-synuclein plays a critical role for dimerization following nitrative stress. Brain Res. 938, 73–80. Dowler, S., Kular, G. and Alessi, D.R. (2002) Protein lipid overlay assay. Sci. STKE 2002, PL6. Tauchi-Sato, K., Ozeki, S., Houjou, T., Taguchi, R. and Fujimoto, T. (2002) The surface of lipid droplets is a phospholipid monolayer with a unique fatty acid composition. J. Biol. Chem. 277, 44507–44512.
[17] O’Farrell, C., Murphy, D.D., Petrucelli, L., Singleton, A.B., Hussey, J., Farrer, M., Hardy, J., Dickson, D.W. and Cookson, M.R. (2001) Transfected synphilin-1 forms cytoplasmic inclusions in HEK293 cells. Brain Res. Mol. Brain Res. 97, 94–102. [18] Iseki, E., Takayama, N., Furukawa, Y., Marui, W., Nakai, T., Miura, S., Ueda, K. and Kosaka, K. (2002) Immunohistochemical study of synphilin-1 in brains of patients with dementia with Lewy bodies – synphilin-1 is non-specifically implicated in the formation of different neuronal cytoskeletal inclusions. Neurosci. Lett. 326, 211–215. [19] Gai, W.P., Yuan, H.X., Li, X.Q., Power, J.T., Blumbergs, P.C. and Jensen, P.H. (2000) In situ and in vitro study of colocalization and segregation of alpha-synuclein, ubiquitin, and lipids in Lewy bodies. Exp. Neurol. 166, 324– 333. [20] Halliday, G.M., Ophof, A., Broe, M., Jensen, P.H., Kettle, E., Fedorow, H., Cartwright, M.I., Griffiths, F.M., Shepherd, C.E. and Double, K.L. (2005) Alpha-synuclein redistributes to neuromelanin lipid in the substantia nigra early in Parkinson’s disease. Brain 128, 2654–2664.
Interactions of Synphilin-1 with phospholipids and lipid membranes Tetsuya Takahashi, Hiroshi Yamashita*, Yoshito Nagano, Takeshi Nakamura, Tatsuo Kohriyama, Masayasu Matsumoto Department of Clinical Neuroscience and Therapeutics, Hiroshima University Graduate School of Biomedical Sciences, 1-2-3 Kasumi, Hiroshima 734-8551, Japan Received 9 June 2006; revised 5 July 2006; accepted 5 July 2006 Available online 17 July 2006 Edited by Judit Ova´di
Abstract Synphilin-1 is an a-synuclein binding protein that is involved in the pathogenesis of Parkinson’s disease. The present study investigated the phospholipid-binding capacity of Synphilin-1. The C-terminus of Synphilin-1 was found to selectively bind to acidic phospholipids, including phosphatidic acid, phosphatidylserine, and phosphatidylglycerol, but not to naturally charged phospholipids. Synphilin-1 was targeted to cytoplasmic lipid droplets in mammalian cells. The amino acid sequence 610–640 was found to represent the primary determinant site for phospholipid binding. Moreover, the R621C mutation identified in Parkinson’s disease abolished Synphilin-1 association with lipid droplets. The lipophilicity of Synphilin-1 might prove relevant to its physiologic function. 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Synphilin-1; a-Synuclein; Parkinson’s disease; Phospholipid; Lipid droplet
1. Introduction Parkinson’s disease represents the second most common neurodegenerative disorder, pathologically characterized by loss of dopaminergic neurons in the substantia nigra pars compacta and appearance of Lewy bodies in remaining neurons [1]. Autosomal dominant Parkinson’s disease, i.e. PARK1 and PARK4, is caused by missense mutations and triplication of the gene for a-synuclein, one of the main components of Lewy bodies [2]. Several studies suggest association of a-synuclein with phospholipids and lipid membranes [3]. a-Synuclein is involved in vesicle trafficking and lipid droplet accumulation [4]. Synphilin-1, an a-synuclein-interacting protein, is a component of the core region of Lewy bodies. The mutation in the Synphilin-1 gene leading to a substitution from arginine (R) to cysteine (C) at position 621 (R621C) has been identified in Parkinson’s disease [5]. Synphilin-1 contains six ankyrin repeats, a coiled-coil domain, and a well conserved region * Corresponding author. Fax: +81 82 505 0490. E-mail address: [email protected] (H. Yamashita).
Abbreviations: CL, cardiolipin; GST, glutathione S-transferase; LC, lysophosphatidylcholine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phsophatidylinositol; PS, phosphatidylserine; SM, sphingomyeline; MLVs, multilamellar vesicles
(amino acids (aa) 612–689) of unknown function [5]. Lipid fractions of brain extracts contain Synphilin-1 [6–8]. Synphilin-1 is predominantly expressed in neurons, where it localizes to the cytoplasm as well as presynaptic nerve terminals, and associates with synaptic vesicles. Accordingly, intracellular localization of Synphilin-1 is similar to that of a-synuclein. The present study investigated the binding capacity of Synphilin-1 with phospholipids and lipid membranes.
2. Materials and methods 2.1. Phospholipids and reagents Lysophosphatidylcholine (LC), sphingomyeline (SM), phosphatidylcholine (PC), phosphatidic acid (PA), phsophatidylinositol (PI), phosphatidylserine (PS), cardiolipin (CL), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and dioleoylphosphocholine were obtained from Sigma. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphate, 1-palmitoyl-2-oleoyl-snglycero-3-[phospho-L -serine] were purchased from Avanti Polar Lipid. Oleic acid, fatty acid-free BSA, and oil red O were purchased from Sigma. 2.2. Vectors and preparation of GST-fusion proteins The coding region of human Synphilin-1 cDNA was subcloned into a pcDNA3 vector (BD Biosciences) in frame with the HA tag sequence (pcDNA3-HA-Synphilin-1), as previously described [9]. The R621C point mutation of Synphilin-1 was generated using a transformer sitedirected mutagenesis kit (BD Biosciences) with the following synthetic oligonucleotide: 5 0 -CTG ATA AAA TCT TAT GCC AGT TAT TGG G-3 0 . The mutation was confirmed by DNA sequencing. DNA fragments obtained from PCR were subcloned into a pcDNA3 vector in frame with the HA tag sequence (pcDNA3-HA-Synphilin-1 R621C). To generate Synphilin-1 proteins fused with glutathione S-transferase (GST) at the N-terminus, corresponding cDNA fragments for various truncations of Synphilin-1, including the N-terminus (aa 1–202, GST-Syp-N1), the middle N-terminus plus the ankyrin repeats from 1–3 (aa 87–458, GST-Syp-N2), the ankyrin repeats 1–4 plus the coiled-coil domain (aa 349–617, GST-Syp-ANK), the ankyrin repeats 5–6 plus the C-terminus (aa 611–919, GST-Syp-C), and serial C-terminal truncations (aa 610–675, GST-Syp-C1; aa 641–730, GST-Syp-C2; aa 706–806, GST-Syp-C3; aa 771–880, GST-Syp-C4; and aa 851– 919, GST-Syp-C5) were subcloned into a pGEX6P-1 vector (Amersham), as previously described [9]. The sequences of all constructs were confirmed by DNA sequencing. GST-fusion proteins were produced in Escherichia coli BL21 via isopropyl-b-D -thiogalactopyranoside (Sigma) induction and purified using glutathione–sepharose 4B (Amersham), as previously described [9–14]. 2.3. Cell culture and immunofluorescent staining COS7 cells were obtained from the American Type Culture Collection and grown in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum. Cells seeded and grown on glass coverslips in the medium were transfected with pcDNA3-HA-Synphilin-1 or pcDNA3-HA-Synphilin-1 R621C using FuGENE 6 (Roche), as previously described
0014-5793/$32.00 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.07.019
4480 [11,12]. At a time of 24 h post-transfection, cells were grown in the presence of 600 lM oleic acid and 100 lM fatty acid-free BSA in the medium for 14 h to induce cytoplasmic lipid droplets. Adherent cells on the glass coverslips were fixed, permeabilized, and immunostained using an anti-HA antibody (Sigma), followed by the Alexa Fluor 488 anti-rabbit IgG antibody (Invitrogen), as previously described [12]. Cells on the same glass coverslips were incubated with the oil red O solution (3 mg/ml in 36% triethylphosphate) for 0.5 h to visualize cytoplasmic lipid droplets. Preparations were examined with a Zeiss LSM510 confocal microscope. 2.4. Protein–lipid overlay assay A protein–lipid overlay assay was performed using the GST-fusion proteins, as previously described [15]. To make a membrane pre-spotted with a concentration gradient of phospholipids (the membrane phospholipid array), the phospholipid solutions, each at 12.5 lg/ll (1·), were serially diluted from 2-fold (2·) up to 32-fold (32·) with chloroform/methanol/distilled water (1:2:0.8). Each 1 ll of serial dilutions of phospholipids was spotted onto a nitrocellulose membrane (Schleicher and Schuell) and dried to allow each spot to contain the following dose of phospholipid: 12.5 lg/1· spot, 6.25 lg/2· spot, 3.13 lg/4· spot, 1.56 lg/8· spot, 0.78 lg/16· spot, and 0.39 lg/32· spot. For a protein–lipid overlay assay, the membrane phospholipid array was blocked with TBS-T (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20) plus 3% (w/v) fatty acid-free BSA for 1 h at room
T. Takahashi et al. / FEBS Letters 580 (2006) 4479–4484 temperature, followed by incubation with GST-fusion proteins (2 lg/ ml) or proteins in TBS-T plus 3% (w/v) fatty acid-free BSA at 4 C overnight. After the membrane was washed 6 times with TBS-T, at 5 min per washing, GST-fusion proteins bound to phospholipids spotted on the membrane were analyzed with immunoblotting with an antiGST monoclonal antibody (1:1000). The blots were developed using enhanced chemiluminescence reagents (Amersham). Alternatively, COS7 cells seeded on a 6 cm-dish (1.5 · 106 cells per dish) were transfected with empty pcDNA3-HA vectors, pcDNA3HA-Synphilin-1 or pcDNA3-HA-Synphilin-1 R621 C. After 24 h post transfection, cells were washed with ice-cold PBS and lysed in ice-cold lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, and protease inhibitors) and centrifuged. After the lysate protein contents were measured using a protein assay kit (Bio-Rad, Hercules, CA, USA), the lysate proteins were diluted at a concentration of 2 lg/ml in TBS-T plus 3% (w/v) fatty acid-free BSA, and subjected to a protein– lipid overlay assay using the membrane phospholipid array in the same manner. 2.5. Binding of Synphilin-1 to synthetic phospholipid vesicles 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate, or 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L -serine] was each mixed with dioleoylphosphocholine (molar ratio 1:1) in chloroform. The lipid was deposited as a film on the wall of a glass test tube by solvent evaporation with nitrogen stream, followed by pumping for 2 h under vacuum. The resulting
Fig. 1. Association of Synphilin-1 protein with phospholipids. (A) Schematic representation of the primary structures of glutathione S-transferase (GST)-fusion proteins utilized for the assay. A series of truncations in the conserved region of Synphilin-1 (Syp), including six ankyrin repeats (ANK1-6) or the coiled-coil (CC) domain, was fused with GST at the N-terminus. (right) GST-fusion protein used for the assay was detected by Coomassie staining. Molecular mass markers are indicated on the left. (B) Protein–lipid overlay assay. To generate the membrane phospholipid array, serial dilutions of phospholipids, including lysophosphatidylcholine (LC), sphingomyeline (SM), phosphatidylcholine (PC), phosphatidic acid (PA), phsophatidylinositol (PI), phosphatidylserine (PS), cardiolipin (CL), phosphatidylethanolamine (PE), and phosphatidylglycerol (PG), were spotted onto a nitrocellulose membrane to allow each spot to contain the following doses: 12.5 lg/1· spot, 6.25 lg/2· spot, 3.13 lg/4· spot, 1.56 lg/ 8· spot, 0.78 lg/16· spot, and 0.39 lg/32· spot. The membrane phospholipid arrays were incubated with 2 lg/ml of GST-fusion proteins (input) or vehicle (none) and bound proteins were detected by immunoblotting with an anti-GST antibody.
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4481 (mass ratio 1:20) in TE (10 mM Tris–HCl (pH 8.0) and 1 mM EDTA) and incubated for 2 h, followed by ultracentrifugation at 164 000 · g for 0.5 h. The pellet of MLVs was washed five times with TE and dissolved in sample buffer. GST-fusion proteins bound to MLVs were analyzed by immunoblotting with an anti-GST antibody (Sigma).
3. Results and discussion
Fig. 2. Binding of Synphilin-1 with phospholipid-containing vesicles. Multilamellar vesicles (MLVs) containing phosphatidic acid (PA), phosphatidylserine (PS), and phosphatidylethanolamine (PE) were prepared. Incubations with various GST-fusion Synphilin-1 proteins or GST alone (input) were performed with MLVs or vehicle (N), followed by ultracentrifugation. Proteins bound to MLVs in the pellet, as well as input proteins, were analyzed by immunoblotting with an anti-GST antibody.
lipid film was re-hydrated in HBS (10 mM HEPES–NaOH (pH 7.4) and 150 mM NaCl) followed by vortexing for 20 min to form multilamellar vesicles (MLVs). MLVs were mixed with GST-fusion proteins
3.1. The C-terminus of Synphilin-1 has a specific capacity to bind to acidic phospholipids To investigate whether or not Synphilin-1 interacts with phospholipids in cells, nine different biologically abundant phospholipids found in intracellular membranes, that is LC, SM, PC, PA, PI, PS, CL, PE, and PG were chosen for the assay. A protein–lipid overlay assay was performed using the membrane phospholipid array with recombinant GST-fusion proteins containing various truncations of Synphilin-1 (Fig. 1A). The binding affinity of proteins for phospholipids was also evaluated at six dilution levels from 1· to 32·. The PA-binding affinity of GST alone, as well as GST-Syp-N1, -N2, or -ANK was only detected at the 1· to 2· dilution level (Fig. 1), suggesting that such binding is non-specific. In contrast, the binding of GST-Syp-C to PA was detected in a
Fig. 3. The C-terminus of Synphilin-1 associates with phospholipids. (A) Schematic representation of the constructs of GST-fusion Synphilin-1 (Syp) proteins utilized for the assay. The C-terminus of Syp (GST-Syp-C in Fig. 1A) was divided into five parts and a series of truncations was fused with GST. (right) GST-fusion protein used for the assay was detected by Coomassie staining. Molecular mass markers are indicated on the left. (B) Protein–lipid overlay assay. The membrane phospholipid arrays were incubated with GST-fusion proteins (2 lg/ml, input) in the same manner as in Fig. 1. GST-fusion proteins bound to phospholipids spotted on the membrane were analyzed by immunoblotting with an anti-GST antibody.
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Fig. 4. Phospholipid-binding property of Synphilin-1. (A) A protein–lipid overlay assay using COS7 cells transfected with empty pcDNA3-HA vectors (Cont), pcDNA3-HA-Synphilin-1 wild type (WT), or pcDNA3-HA-Synphilin-1 R621C (R621C) was performed. (left upper) Wild type and mutant HA-tagged Synphilin-1 (HA-Synphilin-1) proteins in lysates proteins from transfected cells were analyzed by immunoblotting with an antiHA antibody. 0.2 lg of cell lysate protein was loaded per each lane. (left lower and right) A protein–lipid overlay assay. The cell lysate proteins were diluted at a concentration of 2 lg/ml in TBS-T plus 3% (w/v) fatty acid-free BSA, and subjected to a protein–lipid overlay assay using the membrane phospholipid array. HA-Synphilin-1 proteins bound to phospholipids were analyzed by immunoblotting with an anti-HA antibody. (B) Wild type Synphilin-1 localization around lipid droplets in mammalian cells. COS7 cells transfected with expression vectors for wild type (WT) or R621C mutant HA-Synphilin-1 were incubated with oleic acid (600 lM) in medium for 14 h. The cells were processed for double fluorescence confocal microscopy for HA-Synphilin-1 and lipid droplets using an anti-HA antibody and oil red O, respectively. Arrows represent co-localization of HASynphilin-1 and lipid droplets. Bars, 10 lm.
dose-dependent manner (Fig. 1B), with binding affinity detected at the 32· dilution level, suggesting that the Synphilin1 C-terminus possesses capacity to bind to PA. In addition, no binding between phospholipids, except PA and all GST-fusion proteins other than GST-Syp-C, was detected (Fig. 1B). GST-Syp-C also showed a high affinity for PS at the 16· dilution level, as well as a low affinity for CL and PG at the 2· dilution level (Fig. 1B). GST-Syp-C did not interact with PC, SM, PC, PI, or PE. All assays were replicated three times with the same results. Accordingly, the C-terminus (aa 610–919) confers phospholipid-binding capacity to Synphilin-1. The theoretical isoelectric point (pI) for other regions of Synphilin-1 is lower than 7.4 (4.5 for Syp-N1, 4.9 for Syp-N2, and 5.3 for Syp-ANK) and is thus unlikely to interact with acidic phospholipids. On the other hand, the pI for Syp-C was 9.5, consis-
tent with an affinity to acidic phospholipids. The C-terminus might represent a primary determinant of phospholipid-binding capacity.
3.2. The C-terminus of Synphilin-1 interacts with multilamellar vesicles containing PA and PS Since the C-terminus of Synphilin-1 showed affinity for PA and PS, a pull-down assay was performed using MLVs containing such phospholipids to confirm the interaction. As a control, MLVs containing PE were also subjected to the assay. MLVs containing PA, PS, and PE were prepared using their palmitoylated derivatives, i.e. 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-snglycero-3-phosphate, and 1-palmitoyl-2-oleoyl-sn-glycero-3-
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[phospho-L -serine]. MLVs were first incubated with GST-fusion Synphilin-1 proteins, then bound proteins were analyzed by immunoblotting with anti-GST antibodies. MLVs containing PA and PS, but not PE, precipitated GST-Syp-C (Fig. 2). On the other hand, no MLVs precipitated GST alone or GST-Syp-N1, -N2, or -ANK. No GST-fusion protein was precipitated in the absence of MLVs (Fig. 2). Such selective interaction agreed with the results of the protein–lipid overlay assays. 3.3. The primary determinant site for phospholipid binding To identify the region responsible for phospholipid binding within the C-terminus, GST-fusion proteins containing serial C-terminal truncations, i.e. GST-Syp-C1, -C2, -C3, -C4, and -C5, were generated (Fig. 3A). GST-Syp-C (Fig. 1B) and GST-Syp-C1 (aa 610–675) showed similar binding to phospholipids, including PA, PS, CL, and PG, while GST-Syp-C2 (aa 641–730) had a low binding affinity to only PS. Such results suggest that 30 amino acids (aa 610–640), which includes the ankyrin repeat 5, represent the primary determinant site for phospholipid binding. Interestingly, the R621C mutation found in Parkinson’s disease also resides within this sequence. It should also be noted that GST-Syp-C4 and -C5 also exhibited binding affinity to PS, CL, and PG (Fig. 3B). All assays were replicated three times with the same results. Since the calculated pI of aa 675–730 is 5.1, it is likely that the acidic nature of this region inhibits phospholipid binding. However, such phenomenon was not observed. Therefore, simple chargebased interactions may not be the sole influence on in vitro binding. 3.4. Synphilin-1 was localized around lipid droplets The R621C mutation in the Synphilin-1 protein has been reported in Parkinson’s disease [5]. Moreover, amino acid residue 621 localizes to the middle of the ankyrin repeat 5. To investigate whether or not the R621C mutation in Synphilin1 affects the phospholipid-binding specificity, a protein–lipid overlay assay was performed using lysate proteins from COS7 cells transfected with HA-tagged expression vectors for wild type or R621C mutant Synphilin-1, as well as empty expression vectors. At a time of 24 h post-transfection, proteins in cell lysates (Fig. 4A) were incubated with the membrane phospholipid array and bound proteins were detected with immunoblotting with an anti-HA antibody. The phospholipid-binding specificity of wild type Synphilin-1 was similar to that of GST-Syp-C or GST-Syp-C1 (Fig. 4A). In contrast, the R621C mutation inhibited Synphilin-1 binding to PS (Fig. 4A), suggesting that a single amino acid substitution in the primary determinant site could change the phospholipid-binding specificity. Repeated immunofluorescent staining revealed that wild type Synphilin-1 preferentially localized around lipid droplets (Fig. 4B). The appearance of Synphilin-1 localized to the vicinity of lipid droplets was similar to previous descriptions, namely a ring-like configuration [9]. In contrast, R621C mutant Synphilin-1 did not localize to the vicinity of lipid droplets and both entities were independently distributed in the cytoplasm (Fig. 4B). It is noteworthy that a single substitution in the Syp-C1 region abolished the ability of Synphlilin-1 to interact with lipid droplets.
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3.5. Concluding remarks A number of proteins with affinity to phospholipids, such as adipose differentiation-related protein perilipin or a-synuclein, are present on the surface of lipid droplets that are coated by a phospholipid monolayer originating from endoplasmic reticulum [4,16]. In a previous study, we found that Synphilin-1 exhibits a ring-like configuration in the cytoplasm [9]. Overexpression of Synphilin-1 leads to formation of membranebound inclusions that contain multi-layered or lamellar-like phospholipid accumulations [17]. The core region of Lewy bodies is immunolabeled intensely with anti-Synphilin-1 antibody [18] and enriched with lipids [19]. In addition, a-synuclein immunoreactivity concentrates around lipid droplets, which are a component of neuromelanin in the substantia nigra [20]. Such findings indicate that the lipophilic properties of both Synphilin-1 and a-synuclein may be relevant to not only the pathogenesis of Parkinson’s disease, but also physiologic function. Acknowledgements: This work was supported by grants-in-aid for the Encouragement of Young Scientists (2005, 2006) and for Scientific Research (2005, 2006) from the Japanese Ministry of Education, Culture, Sports, Science, and Technology. We thank Mrs. Y. Furuno and M. Sasanishi for technical assistance and Dr. C.J. Hurt for manuscript corrections.
References [1] Forno, L.S. (1996) Neuropathology of Parkinson’s disease. J. Neuropathol. Exp. Neurol. 55, 259–272. [2] Spillantini, M.G., Schmidt, M.L., Lee, V.M., Trojanowski, J.Q., Jakes, R. and Goedert, M. (1997) Alpha-synuclein in Lewy bodies. Nature 388, 839–840. [3] Kubo, S., Nemani, V.M., Chalkley, R.J., Anthony, M.D., Hattori, N., Mizuno, Y., Edwards, R.H. and Fortin, D.L. (2005) A combinatorial code for the interaction of alphasynuclein with membranes. J. Biol. Chem. 280, 31664–31672. [4] Cole, N.B., Murphy, D.D., Grider, T., Rueter, S., Brasaemle, D. and Nussbaum, R.L. (2002) Lipid droplet binding and oligomerization properties of the Parkinson’s disease protein alphasynuclein. J. Biol. Chem. 277, 6344–6352. [5] Marx, F.P., Holzmann, C., Strauss, K.M., Li, L., Eberhardt, O., Gerhardt, E., Cookson, M.R., Hernandez, D., Farrer, M.J., Kachergus, J., Engelender, S., Ross, C.A., Berger, K., Schols, L., Schulz, J.B., Riess, O. and Kruger, R. (2003) Identification and functional characterization of a novel R621C mutation in the synphilin-1 gene in Parkinson’s disease. Hum. Mol. Genet. 12, 1223–1231. [6] Kruger, R. (2004) The role of synphilin-1 in synaptic function and protein degradation. Cell Tissue Res. 318, 195–199. [7] Murray, I.J., Medford, M.A., Guan, H.P., Rueter, S.M., Trojanowski, J.Q. and Lee, V.M. (2003) Synphilin in normal human brains and in synucleinopathies: studies with new antibodies. Acta Neuropathol. (Berl.) 105, 177–184. [8] Ribeiro, C.S., Carneiro, K., Ross, C.A., Menezes, J.R. and Engelender, S. (2002) Synphilin-1 is developmentally localized to synaptic terminals, and its association with synaptic vesicles is modulated by alpha-synuclein. J. Biol. Chem. 277, 23927–23933. [9] Nagano, Y., Yamashita, H., Takahashi, T., Kishida, S., Nakamura, T., Iseki, E., Hattori, N., Mizuno, Y., Kikuchi, A. and Matsumoto, M. (2003) Siah-1 facilitates ubiquitination and degradation of synphilin-1. J. Biol. Chem. 278, 51504–51514. [10] Nagano, Y., Yamashita, H., Nakamura, T., Takahashi, T., Kondo, E. and Nakamura, S. (2001) Lack of binding observed between human alpha-synuclein and Bcl-2 protein family. Neurosci. Lett. 316, 103–107. [11] Nakamura, T., Yamashita, H., Nagano, Y., Takahashi, T., Avraham, S., Avraham, H., Matsumoto, M. and Nakamura, S. (2002) Activation of Pyk2/RAFTK induces tyrosine phosphorylation
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[12] [13]
[14]
[15] [16]
T. Takahashi et al. / FEBS Letters 580 (2006) 4479–4484 of alpha-synuclein via Src-family kinases. FEBS Lett. 521, 190–194. Nakamura, T., Yamashita, H., Takahashi, T. and Nakamura, S. (2001) Activated Fyn phosphorylates alpha-synuclein at tyrosine residue 125. Biochem. Biophys. Res. Commun. 280, 1085–1092. Takahashi, T., Yamashita, H., Nagano, Y., Nakamura, T., Ohmori, H., Avraham, H., Avraham, S., Yasuda, M. and Matsumoto, M. (2003) Identification and characterization of a novel Pyk2/related adhesion focal tyrosine kinase-associated protein that inhibits alpha-synuclein phosphorylation. J. Biol. Chem. 278, 42225–42233. Takahashi, T., Yamashita, H., Nakamura, T., Nagano, Y. and Nakamura, S. (2002) Tyrosine 125 of alpha-synuclein plays a critical role for dimerization following nitrative stress. Brain Res. 938, 73–80. Dowler, S., Kular, G. and Alessi, D.R. (2002) Protein lipid overlay assay. Sci. STKE 2002, PL6. Tauchi-Sato, K., Ozeki, S., Houjou, T., Taguchi, R. and Fujimoto, T. (2002) The surface of lipid droplets is a phospholipid monolayer with a unique fatty acid composition. J. Biol. Chem. 277, 44507–44512.
[17] O’Farrell, C., Murphy, D.D., Petrucelli, L., Singleton, A.B., Hussey, J., Farrer, M., Hardy, J., Dickson, D.W. and Cookson, M.R. (2001) Transfected synphilin-1 forms cytoplasmic inclusions in HEK293 cells. Brain Res. Mol. Brain Res. 97, 94–102. [18] Iseki, E., Takayama, N., Furukawa, Y., Marui, W., Nakai, T., Miura, S., Ueda, K. and Kosaka, K. (2002) Immunohistochemical study of synphilin-1 in brains of patients with dementia with Lewy bodies – synphilin-1 is non-specifically implicated in the formation of different neuronal cytoskeletal inclusions. Neurosci. Lett. 326, 211–215. [19] Gai, W.P., Yuan, H.X., Li, X.Q., Power, J.T., Blumbergs, P.C. and Jensen, P.H. (2000) In situ and in vitro study of colocalization and segregation of alpha-synuclein, ubiquitin, and lipids in Lewy bodies. Exp. Neurol. 166, 324– 333. [20] Halliday, G.M., Ophof, A., Broe, M., Jensen, P.H., Kettle, E., Fedorow, H., Cartwright, M.I., Griffiths, F.M., Shepherd, C.E. and Double, K.L. (2005) Alpha-synuclein redistributes to neuromelanin lipid in the substantia nigra early in Parkinson’s disease. Brain 128, 2654–2664.