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Many lines of evidence suggest that α-synuclein can be secreted from cells and can penetrate into them, although the detailed mechanism is not known. In this study, we investigated the amino acid sequence motifs required for the membrane translocation of α-synuclein, and the mechanistic features of the phenomenon. We first showed that not only α-synuclein but also β- and γ-synucleins penetrated into live cells, indicating that the conserved N-terminal region might be responsible for the membrane translocation. Using a series of deletion mutants, we demonstrated that the 11-amino acid imperfect repeats found in synuclein family members play a critical role in the membrane translocation of these proteins. We further demonstrated that fusion peptides containing the 11-amino acid imperfect repeats of α-synuclein can transverse the plasma membrane, and that the membrane translocation efficiency is optimal when the peptide contains two repeat motifs. α-Synuclein appeared to be imported rapidly and efficiently into cells, with detectable protein in the cytoplasm within 5 min after exogenous treatment. Interestingly, the import of α-synuclein at 4°C was comparable with the import observed at 37°C. Furthermore, membrane translocation of α-synuclein was not significantly affected by treatment with inhibitors of endocytosis. These results suggest that the internalization of α-synuclein is temperature-insensitive and occurs very rapidly via a mechanism distinct from normal endocytosis.
α-Synuclein is an acidic neuronal protein which is highly expressed in brain tissues and is primarily localized at the presynaptic terminals of neurons (Ueda et al. 1993; Jakes et al. 1994; Lavedan 1998). It is also expressed in hematopoietic cells (Hashimoto et al. 1997; Shin et al. 2000) and in other tissues, such as the heart, skeletal muscle, pancreas, and placenta, but it is less abundant than in the brain (Ueda et al. 1993; Jakes et al. 1994). In addition to α-synuclein, β- and γ-synucleins constitute the synuclein family in humans (Jakes et al. 1994; Maroteaux et al. 1988; Ji et al. 1997). α-Synuclein has been identified as a major component of intracellular fibrillar protein deposits (Lewy bodies) in several neurodegenerative diseases, including Parkinson's disease (PD), diffuse Lewy body disease and multiple systemic atrophy (Spillantini et al. 1997, 1998; Takeda et al. 1998; Wakabayashi et al. 1998). Particularly, accumulating evidence suggests that aggregation of α-synuclein may contribute to disease pathogenesis (reviewed in Lücking and Brice 2000; Rajagopalan and Andersen 2001; Ericksen et al. 2003). Although significant progress has been made in understanding the pathological role of α-synuclein in neurodegenerative diseases (El-Agnaf et al. 1998; da Costa et al. 2000; Hsu et al. 2000; Saha et al. 2000), the biological function of α-synuclein remains to be clarified. Recent studies suggest that α-synuclein may function in the regulation of synaptic plasticity, neural differentiation, and the regulation of dopamine synthesis (George et al. 1995; Kholodilov et al. 1999; Abeliovich et al. 2000; van der Putten et al. 2000; Stefanis et al. 2001).
Many studies have shown that α-synuclein can be secreted from cells, although the protein has no conventional signal sequence for secretion. For example, Borghi et al. (2000) showed that full-length α-synuclein might be released from neurons into the extracellular space as part of its normal cellular processing. Furthermore, α-synuclein can be detected in serum from both normal and PD subjects (El-Aganaf et al. 2003; Miller et al. 2004). Recently, transfection studies also demonstrated that a portion of α-synuclein can be constitutively secreted from cells through an unconventional exocytic pathway (Lee et al. 2005; Sung et al. 2005). α-Synuclein is also known to penetrate into cells by an unknown mechanism. In previous work, we demonstrated that α-synuclein could penetrate inside neuronal cells by Rab5A-dependent endocytosis and induce cell death (Sung et al. 2001), and that α-synuclein could penetrate into platelets and subsequently inhibit α-granule release upon stimulation (Park et al. 2002). In addition, Forloni et al. (2004) showed that the non-Aβ component of Alzheimer's disease amyloid (NAC) peptide derived from α-synuclein can penetrate inside cells and accumulate in the perinulcear region.
As described above, many lines of evidence suggest that α-synuclein can be secreted from cells and can penetrate into them, although details of the mechanism are not known. In this study, we investigated the amino acid sequence motifs required for the membrane translocation of α-synuclein using a series of deletion mutants and recombinant peptides. We also addressed the mechanistic features of the cellular import of α-synuclein.
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- Materials and methods
Many proteins with no signal sequence can be secreted through an unconventional exocytosis pathway independent of the ER/Golgi pathway (reviewed in Kuchler 1993; Nickel 2003). α-Synuclein is also known to be secreted into CSF and plasma (Borghi et al. 2000; El-Aganaf et al. 2003; Miller et al. 2004). Furthermore, exogenous α-synuclein can be imported into cells (Sung et al. 2001; Park et al. 2002). In this study, we demonstrated that the N-terminal amphipathic (amino acid residues 1–60) and the NAC peptide (amino acid residues 61–95) regions are responsible for the membrane translocation of α-synuclein, although the NAC is less effective than the N-terminal region. Particularly, the 11-amino acid imperfect repeat sequences in these regions appear to mediate the import of α-synuclein into cells. These sequence motifs are distinct from those of other protein transduction domain (PTD) containing proteins, including Tat and VP22 (discussed below in detail). However, mechanistic features of the membrane translocation of α-synuclein appear to be very similar to other PTD-containing proteins. These results extend our understanding of the secretary proteins lacking signal sequences, particularly of the PTD-containing protein family.
Although α-synuclein does not possess a hydrophobic N-terminal signal sequence for secretion, earlier studies demonstrated that α-synuclein is secreted in both PD patients and in normal subjects (Borghi et al. 2000; El-Aganaf et al. 2003; Miller et al. 2004). Secreted α-synuclein can be detected at nanomolar concentrations in the CSF and blood. Interestingly, the blood levels of α-synuclein have been shown to be increased in familial PD patients with an α-synuclein gene triplication (Miller et al. 2004). α-Synuclein secretion has also been demonstrated in vitro by transfection studies (Lee et al. 2005; Sung et al. 2005). Particularly, Lee et al. (2005) demonstrated that a portion of cellular α-synuclein is present in vesicles and is secreted from cells through an unconventional exocytic pathway in a constitutive manner. Secretion of α-synuclein was temperature sensitive, but was not affected by Brefeldin A treatment, suggesting that an unconventional exocytosis mechanism might be involved.
In this study, we showed that α-synuclein can be translocated into HeLa cells, neuronal cells (SH-SY5Y), hematopoietic cells, and Chinese hamster ovary cells (CHO-K1). Previous studies also showed that α-synuclein can penetrate into undifferentiated neuronal cells and platelets (Sung et al. 2001; Park et al. 2002). These results suggest that the membrane translocation of α-synuclein is not specific to certain cell types. If α-synuclein uses a specific receptor for its import into cells, penetration of α-synuclein should be limited to cell types expressing the receptor(s). Therefore, it seems highly likely that α-synuclein may bind to common molecules on the cell surface. As the N-terminal region of α-synuclein is known to interact with lipid layers in vitro as well as in vivo (Perrin et al. 2000; Bussel and Eliezer 2003; Jao et al. 2004), we propose that the interaction between α-synuclein and the plasma membrane is an essential step for the membrane translocation of α-synuclein. Transfection studies demonstrated that the secretion of α-synuclein is also not specific to certain cell types (Lee et al. 2005; Sung et al. 2005). Over-expressed α-synuclein can even be secreted from yeast (Dixon et al. 2005) and from E. coli. (authors' unpublished results). Interestingly, Lee and colleagues reported that a portion of α-synuclein is stored in the lumen of vesicles in the cytoplasm, and that the α-synuclein in vesicles might be secreted through an unconventional exocytosis pathway. These results suggest that the interaction between α-synuclein and vesicle membranes is critical for the translocation of α-synuclein into vesicles, and presumably for the subsequent secretion process. Thus, our data clearly indicate that the 11-amino acid imperfect repeat motifs are responsible for the membrane translocation of α-synuclein, i.e. both secretion and penetration.
The 11-amino acid repeat motifs contain a well-conserved core sequence of KTKEGV, and these repeats are also present in the N-terminal region of β- and γ-synuclein. The 11-mer repeats of α-synuclein are supposed to form amphiphatic α-helices when the protein is bound to lipid molecules (Bussel and Eliezer 2003). Although no significant sequence homology is found, the repeat region is structurally homologous to the lipid binding domain of exchangeable apolipoproteins, in which the repeat sequence motifs also form amphipathic α-helices (Segrest et al. 1992). In this study, all the α-synuclein deletion mutants and recombinant peptides that contained one or more of the repeat sequence motif(s) appeared to translocate the cell membrane (Figs 2, 4 and 5). However, Syn96–140 and control proteins, which have no such motif, did not permeate into cells (Fig. 2). Taken together, the data suggest that the repeat sequence motifs bind to the lipid bilayer, and the binding interaction might be critical for the membrane translocation of synuclein proteins.
We demonstrated that the cellular uptake of α-synuclein could be detected within 5 min, and that this uptake was not inhibited when cells were incubated at 4°C. It is well established that receptor-mediated endocytosis is blocked by incubation at 4°C (Pastan and Willingham 1981). The cellular uptake of α-synuclein also appeared to be insensitive to treatment with the general endocytosis inhibitors, Brefeldin A and Cytochalasin D. Brefeldin A is known to disrupt the Golgi apparatus and inhibit transport through the Golgi (Lippincott Schwartz et al. 1990), whereas Cytochalasin D is a microfilament-disrupting drug (Elliott and O'Hare 1997). Therefore, these results suggest that internalization of α-synuclein is temperature insensitive and occurs via a route distinct from normal endocytosis, as is the case for other PTDs.
Basic peptides derived from translocatory proteins, such as the Tat protein, the Antennapedia protein and VP22, and even many arginine-rich peptides, have been reported to have a membrane permeability and a carrier function for intracellular cargo delivery (reviewed in Futaki 2002; Leifert and Whitton 2003; Zhao and Weissleder 2004). These peptides are called protein transduction domains (PTDs). Like other translocatory proteins and PTDs derived from them, α-synuclein appears to pass through the cell membrane in an energy-independent, non-endocytic manner, at temperatures as low as 4°C. Unlike other translocatory proteins, however, α-synuclein does not appear to penetrate into the nucleus. Translocated α-synuclein is localized primarily in the cytoplasm, although it is not clear whether the internalized protein is localized in vesicles or in particular organelles at this stage. Furthermore, the amino acid sequence of the α-synuclein's PTD (STD) is distinct from those of other PTDs. No significant amino acid sequence homology exists between STD and other PTDs, but a common feature is that they are all basic peptides. STD is composed of 11-mer repeats that contain no arginine residues. Instead, each 11-mer repeat includes one or two lysine residues. Rather, the 11-mer repeats are structurally homologous to those found in apolipoproteins, but it is not known whether the apolipoproteins are actually able to tanslocate across cell membranes.
In summary, not only α-synuclein, but also β- and γ-synucleins can penetrate into live cells, and the 11-amino acid imperfect repeats of synuclein family members appear to play a critical role in the membrane translocation of these proteins. Fusion peptides containing the 11-amino acid imperfect repeats of α-synuclein (STD) can transverse the plasma membrane, and the membrane translocation efficiency is optimal when the peptide contains two repeat motifs. Internalization of α-synuclein is temperature insensitive and occurs very rapidly via a route distinct from normal endocytosis. These features suggest that the synuclein proteins will create a useful model for analysing unconventional import and export pathways in mammalian cells. Furthermore, STD could be a potential carrier for the efficient delivery of peptides that do not permeate living cells.