A Novel Method for Creating a Synthetic L-DOPA Proteome and In Vitro Evidence of Incorporation.

Joel Ricky Steele,Matthew P Padula,Natalie Strange,Kenneth J Rodgers

doi:10.3390/proteomes9020024

Abstract

Proteinopathies are protein misfolding diseases that have an underlying factor that affects the conformation of proteoforms. A factor hypothesised to play a role in these diseases is the incorporation of non-protein amino acids into proteins, with a key example being the therapeutic drug levodopa. The presence of levodopa as a protein constituent has been explored in several studies, but it has not been examined in a global proteomic manner. This paper provides a proof-of-concept method for enzymatically creating levodopa-containing proteins using the enzyme tyrosinase and provides spectral evidence of in vitro incorporation in addition to the induction of the unfolded protein response due to levodopa.

Highlights

It is known that free L-DOPA can induce oxidative stress, resulting in reactive oxygen species (ROS) generation via interaction with iron and copper to produce Fenton reaction products [27], which leads to further hydroxylation of free or protein incorporated tyrosine, thereby producing free and Protein-bound DOPA (PB-DOPA) [5,22]
The following reagents were sourced from Sigma-Aldrich (Saint Louis, MI, USA): L-DOPA (Cat #D9628); urea (Cat #U5378); thiourea (Cat #T7875); C7BzO (Cat #C0856); Dulbecco’s Modified Eagle Medium (DMEM; Cat#D6429) (New York, NY, USA); cOmpleteTM
We demonstrate the first use of the tyrosinase reaction to convert wholesample peptide for subsequent proteomic analysis, providing spectral evidence

Summary

Introduction

The mechanisms of L-DOPA-induced neurodegeneration include induction of oxidative stress and the misincorporation of L-DOPA into proteins in place of L-tyrosine, resulting in protein misfolding and the formation of proteolysis-resistant aggregates [3,5,7,17,18,19]. Protein-bound DOPA (PB-DOPA) can form via several mechanisms, including direct incorporation of free L-DOPA in place of tyrosine into a growing polypeptide chain [21]. It is known that free L-DOPA can induce oxidative stress, resulting in ROS generation via interaction with iron and copper to produce Fenton reaction products [27], which leads to further hydroxylation of free or protein incorporated tyrosine, thereby producing free and PB-DOPA [5,22]

Methods

Results

Discussion

Conclusion