Biology subjects

Miranda-Castrodad, P.

Publications and source records attributed to Miranda-Castrodad, P..

2 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

Scouring the human Hsp70 network uncovers diverse chaperone safeguards buffering TDP-43 toxicity

Cytoplasmic aggregation and concomitant dysfunction of the prion-like, RNA-binding protein TDP-43 underpin several fatal neurodegenerative diseases, including amyotrophic lateral sclerosis. To elucidate endogenous defenses, we systematically scoured the entire human Hsp70 network for buffers of TDP-43 toxicity. We identify 30 J-domain proteins (2 DNAJAs, 10 DNAJBs, 18 DNAJCs), 6 Hsp70s, and 5 nucleotide-exchange factors that mitigate TDP-43 toxicity. Specific chaperones reduce TDP-43 aggregate burden and detoxify diverse synthetic or disease-linked TDP-43 variants. Sequence-activity mapping unveiled unexpected, modular mechanisms of chaperone-mediated protection. Typically, DNAJBs collaborate with Hsp70 to suppress TDP-43 toxicity, whereas DNAJCs act independently. In human cells, specific chaperones increase TDP-43 solubility and enhance viability under proteotoxic stress. Strikingly, spliceosome-associated DNAJC8 and DNAJC17 retain TDP-43 in the nucleus and promote liquid-phase behavior. Thus, we disambiguate a diverse chaperone arsenal embedded in the human proteostasis network that counters TDP-43 toxicity and illuminate mechanistic gateways for therapeutic intervention in TDP-43 proteinopathies.

biochemistry↗