title: Facile "stop codon" method reveals elevated neuronal toxicity by discrete S87p-α-synuclein oligomers.
abstract: Herein, a new method for preparing phosphorylated proteins at specific sites has been applied to α-synuclein (α-Syn). Three different α-Syn species phosphorylated at Serine 87 (S87p-α-Syn), Serine 129 (S129p-α-Syn) and Serine 87/129 (S87p,129p-α-Syn) were prepared through the 'stop codon' method and verified by LC/MS/MS and immunoblotting. Each type of phosphorylated α-Syn was tested for oligomerization trends and cellular toxicity with dopamine (DA), Cu(2+) ions and pyridoxal 5'-phosphate. Aggregation trends induced by DA or DA/Cu(2+) were similar between phosphorylated and non-phosphorylated α-Syn in SDS-PAGE. However, except for the monomer, phosphorylated oligomers showed higher toxicity than the non-phosphorylated α-Syn (Np-α-Syn) oligomers via WST-1 assays when tested on SH-SY5Y human neuroblastoma cells. In particular, S87p-α-Syn and S87p,129p-α-Syn oligomers induced by DA/Cu(2+), showed higher toxicity than did S129p-α-Syn. When α-Syn was treated with pyridoxal 5'-phosphate in the presence of DA or Cu(2+) to determine aggregation effects, high inhibition effects were shown in both non-phosphorylated and phosphorylated versions. α-Syn co-incubated with DA or DA/Cu(2+) showed less cellular toxicity upon pyridoxal 5'-phosphate treatment, especially in the case of DA-induced Np-α-syn. This study supports that phosphorylated oligomers of α-Syn at residue 87 can contribute to neuronal toxicity and the pyridoxal 5'-phosphate can be used as an inhibitor for α-Syn aggregation.
authors.affiliation: Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Yonghwang
authors.initials: Y
authors.lastname: Ha
authors.affiliation: Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Aerin
authors.initials: A
authors.lastname: Yang
authors.affiliation: Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Seyoung
authors.initials: S
authors.lastname: Lee
authors.affiliation: Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Kibong
authors.initials: K
authors.lastname: Kim
authors.affiliation: Convergent Research Consortium for Immunologic Disease I Cardiovascular Research Lab, 7105, 8102, Annex to the Seoul St. Mary's Hospital, 222 Banpo-Daero, Seocho-gu, 137-701 Seoul, Republic of Korea.
authors.forename: Hyunjeong
authors.initials: H
authors.lastname: Liew
authors.affiliation: Department of Pharmacology, College of Medicine, Seoul National University, 28 Yongon-dong, Chongno-Gu, Seoul 110-799, Republic of Korea; Korea Brain Research Institute, Daegu 700-010, Republic of Korea. Electronic address: yhsuh@snu.ac.kr.
authors.forename: Yoo-Hun
authors.initials: YH
authors.lastname: Suh
authors.affiliation: Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea. Electronic address: hspark@kaist.ac.kr.
authors.forename: Hee-Sung
authors.initials: HS
authors.lastname: Park
authors.affiliation: Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea. Electronic address: dchurchill@kaist.ac.kr.
authors.forename: David G
authors.initials: DG
authors.lastname: Churchill
mesh_terms: D002460:Cell Line; D018388:Codon, Terminator / Q000235:genetics*; D003300:Copper / Q000494:pharmacology; D004298:Dopamine / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism*; D055503:Protein Multimerization / Q000187:drug effects; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D051844:alpha-Synuclein / Q000737:chemistry* / Q000633:toxicity*
chemical_list: D018388:Codon, Terminator; D051844:alpha-Synuclein; D010768:Phosphoserine; D011732:Pyridoxal Phosphate; D003300:Copper; D004298:Dopamine
keywords: Copper; Dopamine; Oligomerization; Phosphorylation; Pyridoxal 5′-phosphate; Stop codon