Biology subjects

Shimogawa, M.

Publications and source records attributed to Shimogawa, M..

4 recordsLinked to original sources

bioRxiv2026

Genetic Code Expansion, Enzymatic Modification, and C-Terminal Labeling Enable Facile Production of Highly Modified α-Synuclein

-Synuclein (S), a protein that plays a central role in Parkinsons disease and related synucleinopathies, is an intrinsically disordered protein (IDP) whose functional interactions and aggregation behavior can be strongly influenced by post-translational modifications (PTMs). Phosphorylation, acetylation, and other PTMs regulate Ss interactions with lipid membranes and binding partners, whereas their dysregulation is associated with aggregation and neuronal toxicity. Despite significant progress through chemical and semi-synthetic approaches, investigating the combinatorial effects of PTMs has remained challenging due to the lack of accessible, site-specific methods. Here, we present an integrated strategy combining genetic code expansion, enzymatic modification, and intein-mediated click chemistry to generate S variants bearing multiple defined PTMs and a C-terminal fluorescent label. The resulting constructs enable direct evaluation of how individual and combined PTMs influence S structure, lipid binding, and cellular internalization. Our approach expands the molecular toolkit for dissecting PTM crosstalk in S and other aggregation-prone IDPs, advancing mechanistic understanding and supporting the development of therapeutic strategies for neurodegenerative disease.

biochemistry↗

bioRxiv2025

Genetic Code Expansion and Enzymatic Modifications as Accessible Methods for Studying Site-Specific Post-Translational Modifications of Alpha-Synuclein and Tau

Alpha-synuclein (S) and tau play important roles in the pathology of Parkinsons Disease and Alzheimers Disease, respectively, as well as numerous other neurodegenerative diseases. Both proteins are classified as intrinsically disordered proteins (IDPs), as they have no stable structure that underlies their function in healthy tissue, and both proteins are prone to aggregation in disease states. There is substantial interest in understanding the roles that post-translational modifications (PTMs) play in regulating the structural dynamics and function of S and tau monomers, as well as their propensity to aggregate. While there have been many valuable insights into site-specific effects of PTMs garnered through chemical synthesis and semi-synthesis, these techniques are often outside of the expertise of biochemistry and biophysics laboratories wishing to study S and tau. Therefore, we have assembled a primer on genetic code expansion and enzymatic modification approaches to installing PTMs into S and tau site-specifically, including isotopic labeling for NMR and fluorescent labeling for biophysics and microscopy experiments. These methods should be enabling for those wishing to study authentic PTMs in S or tau as well as the broader field of IDPs and aggregating proteins.

biochemistry↗

bioRxiv2025

Investigation of All Disease-Relevant Lysine Acetylation Sites in α-Synuclein Enabled by Non-canonical Amino Acid Mutagenesis

Aggregates of -synuclein (S) are hallmarks of synucleinopathies, including Parkinsons Disease (PD) and Multiple System Atrophy (MSA). We have recently shown that S lysine acetylation in the soluble monomer pool varies between healthy controls, PD, and MSA patients. We used non-canonical amino acid (ncAA) mutagenesis to express and purify all 12 currently known disease-relevant variants of S and studied their binding to membranes as well as their aggregation propensities. We found that acetylation of lysine 12, 43, and 80 had particularly strong effects. To understand the implications for acetylation of monomeric S found in healthy cells, we performed NMR experiments to study protein conformation and fluorescence correlation spectroscopy experiments to quantify lipid binding. We also investigated the effects of acetylation at lysine 12, 43, and 80 on fibril seeding in neurons. Collectively, our biochemical and cell biological investigations indicated that acetylation of K80 could inhibit aggregation without conferring negative effects on monomer function in healthy cells. Therefore, we studied the structures of fibrils with K80 acetylation through cryo-electron microscopy to uncover the structural basis for these effects. Finally, we identified inhibition of HDAC8 as a way of potentially increasing acetylation at K80 and other inhibitory sites for therapeutic benefit.

biochemistry↗

bioRxiv2023

Semi-synthetic CoA-alpha-Synuclein Constructs Trap N-terminal Acetyltransferase NatB for Binding Mechanism Studies

N-terminal acetylation is a chemical modification carried out by N-terminal acetyltransferases (NATs). A major member of this enzyme family, NatB, acts on much of the human proteome, including -synuclein (S), a synaptic protein that mediates vesicle trafficking. NatB acetylation of S modulates its lipid vesicle binding properties and amyloid fibril formation, which underlies its role in the pathogenesis of Parkinsons disease. Although the molecular details of the interaction between human NatB (hNatB) and the N-terminus of S have been resolved, whether the remainder of the protein plays a role in interacting with the enzyme is unknown. Here we execute the first synthesis, by native chemical ligation, of a bisubstrate inhibitor of NatB consisting of coenzyme A and full-length human S, additionally incorporating two fluorescent probes for studies of conformational dynamics. We use cryo-electron microscopy (cryo-EM) to characterize the structural features of the hNatB/inhibitor complex and show that, beyond the first few residues, S remains disordered when in complex with hNatB. We further probe changes in the S conformation by single molecule Forster resonance energy transfer (smFRET) to reveal that the C-terminus expands when bound to hNatB. Computational models based on the cryo-EM and smFRET data help to explain the conformational changes and their implications for hNatB substrate recognition and specific inhibition of the interaction with S. Beyond the study of S and NatB, these experiments illustrate valuable strategies for the study of challenging structural biology targets through a combination of protein semi-synthesis, cryo-EM, smFRET, and computational modeling.

biochemistry↗