Methodological approach to investigate peroxiredoxin 1 deacetylation by histone deacetylase 6.

Post-translational acetylation of cytoplasmic proteins has emerged as a critical regulatory mechanism in neurological disease. Peroxiredoxin 1 (Prdx1), a key antioxidant enzyme, undergoes reversible lysine acetylation that modulates its enzymatic activity. Histone Deacetylase 6 (HDAC6), a predominantly cytoplasmic deacetylase, has been identified as a regulator of Prdx1 acetylation, linking deacetylase activity to redox homeostasis. Accurate detection of Prdx1 deacetylation requires methodological strategies capable of preserving endogenous acetylation states and selectively enriching acetylated protein fractions. This chapter describes a reproducible workflow for assessing HDAC6-mediated Prdx1 deacetylation in cortical tissue which integrates optimized protein extraction under acetylation-preserving conditions, acetyl-lysine-based co-immunoprecipitation, and immunoblot detection of Prdx1. Parallel assessment of total Prdx1 and established HDAC6 substrates enables normalization and validation of deacetylase activity. Quantitative densitometric analysis provides comparative evaluation of acetylation levels across experimental conditions. This methodology offers a practical and translationally applicable approach for investigating non-histone deacetylation mechanisms and can be adapted to other HDAC6-regulated substrates in neurodegenerative research.