**Alpha-Synuclein Interactions with Membranes**

Katja Pirc1 and Nataša Poklar Ulrih1,2

*1University of Ljubljana, Biotechnical faculty 2Centre of excellence CIPKeBiP Slovenia* 

#### **1. Introduction**

86 Etiology and Pathophysiology of Parkinson's Disease

Wolozin, B., et al., 2008. Investigating convergent actions of genes linked to familial

Wszolek, ZK et al., 2004. Autosomal dominant parkinsonism associated with variable

Yao, C., et al., 2010. LRRK2-mediated neurodegeneration and dysfunction of dopaminergic

Zimprich, A., et al., 2004. Mutations in LRRK2 cause autosomal-dominant parkinsonism

neurons in a Caenorhabditis elegans model of Parkinson's disease. Neurobiol Dis.

Parkinson's disease. Neurodegener Dis. 5, 182-5.

with pleomorphic pathology. Neuron. 44, 601-7.

40, 73-81.

synuclein and tau pathology. Neurology. 62(9):1619-22.

Synucleinopathies are a group of neurodegenerative disorders that share common pathological intracellular deposits that contain aggregates of the protein α-synuclein. Substantial evidence suggests that fibril formation by α-synuclein is a critical step in the development of Parkinson's disease (PD). Indeed, *in vitro*, α-synuclein forms fibrils with morphologies and staining characteristics similar to those extracted from disease-affected brains. Also, three single-point mutations and duplication or triplication of the α-synuclein locus correlate with early onset of PD.

However, the function of α-synuclein remains unknown. A significant portion of αsynuclein is localized within membrane fractions, and especially synaptic vesicles associated with vesicular transport processes. These observations suggest that α-synuclein has a role in vesicular trafficking. Although α-synuclein belongs to a group of natively unfolded proteins, there is strong evidence that the membrane affinity of the protein induces an αhelical conformation. A large number of studies have investigated α-synuclein–lipid interactions in the search for a physiological function, as well as to understand this potential membrane involvement in the pathogenesis of α-synuclein. In this review, we will predominantly focus on current opinion about the native wild-type α-synuclein–lipid interactions and the structure of α-synuclein that is established at the membrane surface. However, it should be noted that membranes have been reported to both accelerate and inhibit the fibril formation of α-synuclein, although this will not be the focus of the present review.

#### **2. Intrinsically disordered proteins**

A significant number of proteins involved in protein deposition diseases have been seen to be intrinsically disordered proteins. Well-known examples include amyloid β-protein and tau protein in Alzheimer's disease, prion protein (PrP) in prion diseases, exon 1 region of huntingtin in Huntington's disease, and α-synuclein in PD (Fink, 2005).

It has been estimated that more than 30% of eukaryotic proteins have disordered regions that are greater than 50 consecutive residues (Dunker et al., 2001). This term "disordered protein" refers to proteins that in their purified state at neutral pH, have been either shown experimentally or predicted to lack an ordered structure; such proteins are also known as natively unfolded, or intrinsically unstructured. Disordered proteins, or disordered regions

Alpha-Synuclein Interactions with Membranes 89

In brain homogenates, α-synuclein represents 0.5% to 1% of the total protein (Iwai et al., 1995). Northern blotting and *in-situ* hybridization in human and mice have show relatively high expression of α-synuclein in a restricted number of brain regions, one of which is the *substantia nigra* (Abeliovich et al., 2000; Lavedan, 1998). Here, α-synuclein is localized in the presynaptic terminals (George et al., 1995; Iwai et al., 1995), with about 15% found in the membrane fraction (Lee et al., 2002); after synaptosomal lysis, α-synuclein is in the soluble

Although the normal physiological function of α-synuclein remains unknown, it appears to be involved in maintenance of the synaptic vesicle reserve pool of the brain (Davidson et al., 1998; Fortin et al., 2004; Iwai et al., 1995; Nuscher et al., 2004). However, other roles for αsynuclein have been considered: roles in lipid metabolism and transport (Scherzer et al., 2003; Sharon et al., 2001; Willingham et al., 2003), vesicle docking at the membrane (Larsen et al., 2006), exocytosis (Srivastava et al., 2007), lipid organisation (Madine et al., 2006) and prevention of oxidation of unsaturated lipids (Zhu et al., 2006). To date, no conclusive evidence showing the precise role of α-synuclein in cell physiology has been provided.

Alpha-synuclein is a small (140 amino acid; 14 kDa) highly acidic protein (Figure 1), and it is intrinsically disordered under physiological conditions *in vitro* (Bisaglia et al., 2009; Fink, 2006). The first 89 residues are composed almost entirely of seven 11-amino-acid imperfect repeats, with a consensus sequence of KTKEGV (George et al., 1995). This strongly resembles sequence motifs found in exchangeable apolipoproteins, which are believed to constitute amphipathic helical lipid-binding domains (Segrest et al., 1992). This 11-residue periodicity is broken in one point by the insertion of four uncharged amino acids, separating units 4 and 5.

The structure of α-synuclein can be divided into three regions (Figure 1). The N-terminal domain (residues 1-60) is positively charged and contains five of the imperfect repeats (Fink, 2006; George et al., 1995). The sequence 61-95 is the most hydrophobic portion of the protein, and this was originally described as the "non-amyloid-beta component" (NAC) of Alzheimer´s disease plaques (Takeda et al., 1998). Several studies have defined this region as responsible for α-synuclein aggregation and β-sheet formation (Bodles et al., 2001; Giasson et al., 2001). The homologous β-synuclein, which is distinct from α-synuclein by the absence of the central hydrophobic sequence, is much less prone to self-aggregation. The interaction between β-synuclein and α-synuclein has been argued to inhibit aggregation (Park & Lansbury, 2003). The highly acidic C-terminal domain of α-synuclein is rich in Pro and acidic residues, with a predominance of Glu residues over Asp (George et al., 1995). This domain contains three of the four Tyr residues, at positions 125, 133 and 136; the fourth Tyr residue is at position 39. It has been shown that monomeric α-synuclein has a more compact structure than expected for a completely unfolded polypeptide, and this compactness has been linked to its inhibition of fibril formation due to burial of the hydrophobic NAC domain (Bertoncini et al., 2005; Dedmon et al., 2005). In addition, it has been shown that the 1–102 and 1–110 C-terminal-truncated α-synuclein fragments, but not that of 1–120, are efficient in the promotion of α-synuclein aggregation. The negatively charged 104, 105, 114 and 115 residues in the C-terminus have been suggested to be responsible for reduced αsynuclein aggregation and a lack of seeding of wild-type α-synuclein (Murray et al., 2003).

There are no Cys or Trp residues in the α-synuclein sequence (George et al., 1995).

**4. Alpha-synuclein** 

fraction (Iwai et al., 1995).

**4.1 Primary sequence** 

of a protein, lack specific tertiary structure and can be composed of an ensemble of conformations (Fink, 2005). Intrinsically unstructured proteins are frequently involved in important regulatory functions in the cell, and the lack of intrinsic structure is in many cases removed when the protein binds to its target molecule. Some functional advantages of these proteins might be an ability to bind to several different targets, the precise control of their binding thermodynamics, and their involvement in cell-cycle control and both transcriptional and translational regulation (Wright & Dyson, 1999). Gunasekaran et al. proposed that disordered proteins provide a simple solution to the need for large intermolecular interfaces while maintaining smaller proteins, and hence a smaller genome and a smaller cell size. For monomeric proteins with extensive intermolecular interfaces, such proteins would need to be 2-3-fold larger, and this would either increase intracellular crowding or enlarge the size of the cell by some 15% to 30%, owing to the increase in the sequence length (Gunasekaran et al., 2003).
