117 / 2023-10-10 00:18:18

Semisynthesis of Phosphorylated α-Synuclein at Different Sites

protein semisynthesis,protein phosphorylation

Phosphorus Chemistry and Life & Health science > 1) Phosphorus chemistry in chemical biology

α-synuclein (α-syn) plays critical roles in multiple neurodegenerative disease, including Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). The aggregates of α-synuclein are regarded as an important hallmark in the process of these diseases. As one of the most common posttranslational modifications, phosphorylation exists in patient samples and is proposed to regulate some properties of α-synuclein, including aggregation, membrane binding, and cytotoxicity. However, it remains unknown how phosphorylation at specific sites regulates these processes.

Previous studies have utilized some negatively charged residues as phosphorylation mimicks, like aspartic acid or glutamic acid. These mimicks gave some clue about effects of phosphorylation, but have been proved to possess distinct properties compared with phosphorylated protein in vitro. Another general enzymatic method using kinase is lack of selectivity. There are usually many possible sites for kinase, thus it is not suitable for single-site studies. Considering the above, we chose to synthesize homogenous α-synuclein with phosphorylation by protein semisynthesis.

We synthesized three different α-synuclein protein with phosphorylation at Tyr39, Ser87, and Ser129 respectively. Phosphorylation at these sites was reported to appear and might play some roles in disease process, and they are located in three different regions of α-synuclein, N-terminus, midst, and C-terminus.

The phosphorylation was introduced to peptides through solid-phase peptide synthesis. The long segments were recombinantly expressed. Then we performed N-to-C sequential native chemical ligation on all the segments. After radical-catalyzed desulfurization, the Cys residues on ligation sites were transformed into Ala to get final phosphorylated product.

Overall, we proposed a semisynthetic strategy to obtain homogenous α-synuclein with single-site phosphorylation. The phosphorylated proteins have been applied in related works.