Molecular interaction of α-synuclein with tubulin influences on the polymerization of microtubule in vitro and structure of microtubule in cells
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- Zhou, R.M., Huang, Y.X., Li, X.L. et al. Mol Biol Rep (2010) 37: 3183. doi:10.1007/s11033-009-9899-2
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Microtubule dynamics is essential for many vital cellular processes such as in intracellular transport, metabolism, and cell division. Evidences demonstrate that α-synuclein may associate with microtubular cytoskeleton and its major component, tubulin. In the present study, the molecular interaction between α-synuclein and tubulin was confirmed by GST pull-down assay and co-immunoprecipitation. The interacting regions within α-synuclein with tubulin were mapped at the residues 60–100 of α-synuclein that is critical for the binding activity with tubulin. Microtubule assembly assays and sedimentation tests demonstrated that α-synuclein influenced the polymerization of tubulin in vitro, revealing an interacting region-dependent feature. Confocal microscopy detected that exposures of α-synuclein proteins inhibited microtubule formation in the cultured cells, with a length-dependent phenomenon. Our data highlight a potential role of α-synuclein in regulating the microtubule dynamics in neurons. The association of α-synuclein with tubulin may further provide insight into the biological and pathophysiological function of synuclein.
Keywordsα-SynucleinTubulinMolecular interactionMicrotubule dynamicsSynucleinopathies
α-Synuclein (SNCP) is a kind of presynaptic proteins, distributing widely in brain tissues . Increasing evidences suggest that α-synuclein is a common pathogenic molecule in several neurodegenerative disorders, in which deposition of α-synuclein as fibrillary aggregates in neurons or glial cells is a hallmark lesion . Those disorders include Parkinson’s disease , dementia with Lewy bodies [4, 5], Lewy body variant of Alzheimer’s disease  and multiple system atrophy , collectively referred to as synucleinopathies . However, the physiological function of α-synuclein remains to be largely unclear. It was reported that α-synuclein is functional microtubule-associated protein (MAP), and that tubulin co-localizes with α-synuclein in Lewy bodies and influences the formation of α-synuclein aggregation [9, 10]. Thus, the association of α-synuclein with microtubular network may be related to physiological or pathologic activities of α-synuclein in the neurons.
Microtubules are cellular structures that play a central role in intracellular transport, metabolism, and cell division. A wide range of chemicals or proteins are able to interrupt microtubule function, which can be categorized as microtubule stabilizers and microtubule depolymerizers [11, 12]. Our previous studies have demonstrated that PrP, a cellular protein related with transmissible spongiform encephalopathy or prion disease, affects the formation of microtubule through interacting with tubulin [13, 14]. Interference with microtubule dynamics leads to mitotic arrest and initiation of apoptosis. Previous studies reported that tubulin plays a protagonist role in α-synuclein oligomer and fibril formation , and that α-synuclein can facilitate tubulin polymerization . However, there was a different report that α-synuclein inhibits tubulin polymerization . The discrepancy of the effectiveness of α-synuclein on microtubule indicates a need of more experimental data.
In this study, we propose the molecular evidence of recombinant human α-synuclein interacting with native tubulin. The residues 60–100 within α-synuclein are responsible for recognizing tubulin, except for the C-terminus of α-synuclein (residues 96–102 and 131–140) reported previously . Transiently expressed α-synuclein co-locates well with the endogenous microtubules in HeLa cells. We also confirm that the full-length α-synuclein, as well as the segments for interacting with tubulin, have marked influence on the polymerization of tubulin in vitro and the structure of microtubule in cultured cells.
Materials and methods
Plasmid construction and protein purification
Total RNA of a human neuroblastoma cell line SH-SY5Y was extracted and the single-stranded cDNA was synthesized. With a PCR technique, the cDNA sequence encoding human α-synuclein (SNCP) was amplified with the primers Hu-SNCA-F (5′-GTCGACATGGATGTATTCATGAAAGG-3′, with a SalI site underlined) and Hu-SNCA-B (5′-GGATCCTTAGGCTTCAGGTTCGTAGTA-3′, with a BamHI site underlined). After verified by DNA sequencing, the full-length human SNCP cDNA was cloned into T-vector (pT-SNCP1-140) and subcloned into a GST-fusion protein expression vector pQE-30-GST, designated pGST-SNCP1-140. To construct the mammalian expressing plasmid encoding the full-length SNCP, SNCP sequence was released from pT-SNCP1-140 and subcloned into vector pcDNA3.1, generating recombinant plasmid pcDNA-SNCP1-140. Various lengths of sequences encoding truncated mutants of SNCP, including SNCP1-60, SNCP61-100, SNCP31-100 and SNCP1-100, were generated by PCR using the plasmid pT-SNCP1-140 as the templates. The PCR product was subsequently inserted into a prokaryotic expressing vector pGEX-2T, generating recombinant plasmids pGST-SNCP1-60, pGST-SNCP61-100, pGST-SNCP31-100 and pGST-SNCP1-100.
The recombinant prokaryotic proteins tagged with GST were bacterially expressed in E. coli M15 and purified with Glutathione Sepharose 4B Agarose (Amersham Pharmacia) according to the protocol described in our previous study . Each purity of the protein was verified by SDS-PAGE and immunoblotting, showing to be >95%.
The commercial rabbit tubulin protein was purchased from Sigma. Native hamsters’ tubulin was purified from fresh prepared hamster brains according to the procedure of microtubules preparation kit (Sigma). Before experiments tubulin preparations were thawed from −80°C and centrifuged at 20,000×g at 4°C for 30 min. The obtained supernatants were used in subsequent tests.
Anti-α-tubulin monoclonal antibody (mAb) and anti-SNCP polyclonal antibody (pAb) were purchased from Santa Cruz Biotechnology. Anti-His, anti-GST mAb and anti-α-tubulin pAb were purchased from Tiangen Biotechnology, China.
Preparation of brain tissue homogenates
Normal hamsters’ brain tissues were homogenized in nine volumes (10% w/v) of lysis buffer (100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, 0.5% sodium deoxycholate in 10 mM Tris-Cl, pH 7.4). The brain homogenates were centrifuged at 20,000×g for 30 min, and the supernatants were collected for subsequent experiments.
To identify the potential interaction between SNCP and tubulin, 5 μM various SNCP proteins was incubated with 5 μM tubulin or 0.1 ml of the supernatant of normal hamster brain homogenates in 500 μl binding solution containing 20 mM Tris-Cl, 200 mM NaCl, 10 mM aprotinin, pH 8.0, at 4°C for 4 h, while equal amount of GST protein was used as control. Ten microliter of Glutathione Sepharose 4B beads were added to the reaction solution and incubated at 37°C for 30 min with end-over-end mixing. After centrifugation at 2,000 rpm for 2 min, the supernatants were discarded and beads were washed with 500 μl washing buffer (50 mM Tris-Cl, 300 mM NaCl, pH 8.0) for three times. The complexes were separated on 15% SDS-PAGE and transferred to nitrocellulose filter blocked with 5% nonfat milk in PBST (135 mM NaCl, 1.3 mM KCl, 3.2 mM Na2HPO4, 0.5 mM KH2PO4, 0.05% Tween 20, pH 7.4). Tubulin reactive band was detected with 1:4000 anti-α-tubulin mAb as the primary antibody and 1:4000 HRP-conjugated anti-mouse IgG (Santa Cruz Biotechnology) as the second one, using ECL method (Perkin–Elmer).
Five micromolar of various SNCP protein were separately incubated with 5 μM tubulin or 0.1 ml of the supernatant of normal hamster brain homogenates in 500 μl binding solution containing 20 mM Tris-Cl, 200 mM NaCl, 10 mM aprotinin, pH 8.0, at 4°C for 4 h, while equal amount of GST protein was employed as control in parallel. After incubated with anti-GST mAb (Tiangen Biotechnology) for 2 h, 10 μl Protein G Sepharose (Roche) was applied into the reactions and incubated for further 2 h. The Sepharose beads were precipitated at 2,000 rpm for 5 min and washed with 500 μl washing buffer (50 mM Tris-Cl, 300 mM NaCl, pH 8.0) for three times. The bound complexes were separated by 15% SDS-PAGE and transferred to nitrocellulose membranes. The bound tubulin was detected by anti-α-tubulin pAb in Western blot.
Microtubule assembly assay
Microtubule was assembled in vitro from the purified tubulin at 3 mg/ml in the polymerization buffer contained 80 mM PIPES (pH 6.9), 0.5 mM MgCl2, 1 mM GTP, 5% Glycerol, and the turbidity of the solution was monitored continuously after mixture based on the protocol described previously . The assay ran for 30 min while measuring the turbidity every 5 min at 340 nm in a spectrophotometer.
Samples containing 3 mg/ml tubulin were incubated for 30 min and subsequently centrifuged for 10 min at 14,000×g. The pellets were resuspended in a volume of deionized water equal to the volume of the supernatants and analyzed by 15% SDS-PAGE.
Cell culture, transfection and immunofluorescence microscopy
HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum. Cells were transiently transfected with the pcDNA-SNCP1-140 vector using Lipofectamin 2000 Reagent (Qiagen) according to the manufacture’s protocol. One day after transfection, cells were fixed with 4% paraformaldehyde and 10% methanol in PBS for 30 min at room temperature. After blocking with 5% fetal bovine serum, 0.1% Triton X-100 in PBS for 30 min, cells were incubated with anti-α-synuclein (1:400), anti-α-tubulin (1:500) antibodies overnight at 4°C. After washing with PBS, cells were stained with Alexa Fluor 546 conjugated anti-rabbit IgG (1:500, Invitrogen) for α-synuclein or Alexa Fluor 488 conjugated anti-mouse IgG (1:500, Invitrogen) for α-tubulin for 1 h at room temperature. Fluorescently stained cells were analyzed using a confocal laser scanning microscope (Bio-Rad).
Formation of microtubule in the cultured cells
To see the influence on the formation of microtubule in the cultured cells, 10 μM of each SNCP was added to the cells for 24 h. In parallel, 10 μM GST protein and 10 μM colchicines were employed as control. After incubation, cells were extensively washed and fixed in 4% paraformaldehyde. In order to allow antibodies to penetrate the cell membrane, monoclonal tubulin antibodies and Alexa488 (green) Fluor-conjugated anti-rabbit IgG was added with 0.1% Triton X-100. Fluorescently stained cells were analyzed using a confocal laser scanning microscope (Bio-Rad).
Cell viability determination
HeLa cells were plated as normal in 96-well trays and maintained in DMSM containing 10% BSA. Various materials were applied directly to the wells according to different experiments. Viability was assessed by the conversion of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma, USA) to a formazan product. Absorbance was measured at 492 nm in a spectrophotometer.
Recombinant full-length α-synuclein interacts with tubulin in vitro
α-Synuclein co-locates with microtubule in transiently transfected HeLa cells
The interacting domains of α-synuclein with tubulin are located at SNCP spanning residues 60–100
α-Synuclein increases turbidity of tubulin solution and induces sedimentation of tubulin
To see the influence of different SNCP truncated peptides on the changes of turbidities of tubulin solution, various recombinant SNCP proteins were subjected into the solutions (molar ratio of tubulin to various SNCP was 8:1), while the preparations with GST were used as control. GST-SNCP1-140, GST-SNCP61-100, GST-SNCP31-100 and GST-SNCP1-100 showed significant influence on the solution turbidities compared with the control, while the solution turbidity of GST-SNCP1-60 have no significant change (Fig. 5b, c). It suggests that the region influencing the turbidity of tubulin solution is likely located at SNCP spanning residues 60–100.
α-Synuclein affects the formation of microtubules in the culture cells
α-Synuclein affects the cell viability
In this study co-immunoprecipitation and pull-down assay provide reliable evidence again that α-synuclein interacts with both the native tubulin presenting in 10% hamsters’ brains homogenate and the purified tubulin from rabbit brains in vitro. α-synuclein is the major constituent of pathological intracellular inclusion bodies, which is a common feature of several neurodegenerative diseases. In Lewy body, aggregation and deposits of α-synuclein usually co-localize with tubulin [9, 16, 17]. The interaction data of α-synuclein and tubulin supply credible molecular basis for co-deposit of these two proteins in the pathological intracellular inclusion bodies. In addition, well co-localization of endogenous microtubule and transiently expressed α-synuclein in cells also implies that accumulation of α-synuclein in cytoplasm may easily bind to cellular microtubule.
The residues 61–95 of α-synuclein, also known as non-Aβ component region, is believed to by itself highly amyloidogenic, and residues 31–109 of α-synuclein may represent the core unit of α-synuclein filaments, which contributes to the structural stability of these filaments [18, 19]. Previous study has proposed that the potential binding sites of α-synuclein to tubulin locate at its C-terminal region, between residues 96 and 102, as well as 131 and 140 . Our study confirms that the segment of α-synuclein spanning residues 60–100 also has remarkable binding capacity with native tubulin. Unfortunately, the expression of segment of SNCP101-140 is not successful, which prohibits us to carry out further assay. Existences of several binding sites for tubulin within α-synuclein may somehow reflect a relative strong interference between two proteins.
Although α-synuclein segments of residues 61–100 and 31–100 show comparable binding activities with tubulin and similar influences on the tubulin polymerization in vitro as the full-length protein, their capacities in destroying the structure of microtubule in the cultured cells are almost undetectable. However, the segment of residues 1–100 reveals clear disrupting activity on the structure of microtubule in cells, although its disrupting activity seems to be lower than the full-length α-synuclein. These results highlight a length-dependent manner for influence of structure of cellular microtubule induced by α-synuclein, which imply that besides the interacting sites other potential domains or structures in the context of whole α-synuclein protein contribute to its microtubule disruption. Certainly, the experimental odds can not be excluded, e.g. the different proteolysis pathways or speeds in cells. Possibly, the shorter peptides are easier to be degraded than the larger one.
Tubulin forms the monomeric subunit of microtubules that are responsible for eukaryotic cell structure in the formation of the cytoskeleton . Microtubules can be stabilized by structural microtubule-associated proteins (MAPs), such as tau that stimulates microtubule assembly [11, 21]. Microtubule networks are critical elements in a variety of fundamental functions, including intracellular scaffolding, cell division, secretory processes, regulatory of cellular motility and transport. Typically, dimers of α- and β-tubulin continually undergo polymerization and de-polymerization by the addition and dissociation of tubulin subunits, thereby affording the cell’s ability to move or change functions [22–24]. In neurodegenerative diseases, one of the common characteristics is deposit of abnormal pathologic proteins in brains, which may form plaques. Interestingly, many pathologic proteins and their precursors show the abilities to form protein complexes with tubulin and affect the structures of microtubule in cells, e.g. prion protein [13, 14, 25], amyloid precursor protein , tau [27, 28] and α-synuclein. Moreover, disfunction of microtubule has been repeatedly described in neurodegenerative diseases [29, 30]. Therefore, interfering with the normal function of tubulin and normal structure of microtubule of neurons due to deposits of pathologic proteins may be the essential hallmark in the pathogenesis of neurodegenerative diseases.
This work was supported by Chinese National Natural Science Foundation Grants 30771914 and 30800975, National Science and Technology Task Force Project (2006BAD06A13-2), Institution Technique R&D Grant (2008EG150300), National Basic Research Program of China (973 Program) (2007CB310505), China Mega-Project for Infectious Disease (2009ZX10004-101) and the SKLID development Grant (2008SKLID102 and 2008SKLID202).