Cell into which protein, which can serve as polymerization nucleus of protein polymer, or polymer thereof is introduced, and method for production of the cell
The present invention has the object of providing a cell into which a protein, which can serve as a polymerization nucleus of a protein polymer, or polymer thereof is introduced, and a method for producing the cell. The invention relates to a cell into which a protein, which can serve as a polymerization nucleus of a protein polymer, or a polymer thereof is introduced, a method for producing the cell, and a method of screening for a compound inhibiting an intracellular accumulation of a protein containing fibril structures, wherein the method comprises bringing a candidate substance into contact with the cell.
1. A method for producing a cellular model of α-synuclein inclusion formation by polymerization, wherein the method comprises:
introducing into a neuron or a glial cell, by use of a lipid transport reagent, an α-synuclein fibril formed from α-synuclein of SEQ ID NO: 2 or a mutant thereof at A30P, A53T, or E46K or a variant thereof with portion(s) deleted in any or all of the amino acid residues at positions 10 to 15, 21 to 26, 32 to 37, 43 to 48, and 58 to 63, said α-synuclein fibril being a suitable polymerization nucleus of filamentous α-synuclein inclusions;
introducing into a neuron or a glial cell an expression vector encoding a recombinant α-synuclein of SEQ ID NO: 2 or a mutant thereof at A30P, A53T, or E46K or a variant thereof with portion(s) deleted in any or all of the amino acid residues at positions 10 to 15, 21 to 26, 32 to 37, 43 to 48, and 58 to 63; and
thereby causing interaction between the α-synuclein fibril and the recombinant α-synuclein generated by expression of the vector forming filamentous α-synuclein inclusions in the cell by polymerization,
wherein the α-synuclein fibril which can serve as a polymerization nucleus and the recombinant α-synuclein generated in the cell are phosphorylated in the cell.
2. The method according to claim 1 , wherein the expression plasmid comprises the polynucleotide having the sequence set forth in SEQ ID NO: 1, which encodes the α-synuclein having the polypeptide sequence set forth in SEQ ID NO: 2.
3. The method according to claim 1 , wherein the α-synuclein fibril or the recombinant α-synuclein comprises a variant of α-synuclein having amino acid residue 30 of SEQ ID NO: 2 substituted with proline; having amino acid residue 46 of SEQ ID NO: 2 substituted with lysine; or having amino acid residue 53 of SEQ ID NO: 2 substituted with threonine.
4. The method according to claim 1 , wherein the lipid transport regent is a 3:1 (w/w) formulation of the polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and the neutral lipid dioleoyl phosphatidylethanolamine (DOPE).