Project description:Lipoic acid is an essential enzyme cofactor in central metabolic pathways. Due to its claimed antioxidant properties, racemic (R/S)-lipoic acid is used as a food supplement but is also investigated as a pharmaceutical in over 180 clinical trials covering a broad range of diseases. Moreover, (R/S)-lipoic acid is an approved drug for the treatment of diabetic neuropathy. However, its mechanism of action remains elusive. Here, we performed chemoproteomics-aided target deconvolution of lipoic acid and its active close analog lipoamide. We find that histone deacetylases HDAC1, HDAC2, HDAC3, HDAC6, HDAC8, and HDAC10 are molecular targets of the reduced form of lipoic acid and lipoamide. Importantly, only the naturally occurring (R)-enantiomer inhibits HDACs at physiologically relevant concentrations and leads to hyperacetylation of HDAC substrates. The inhibition of HDACs by (R)-lipoic acid and lipoamide explain why both compounds prevent stress granule formation in cells and may also provide a molecular rationale for many other phenotypic effects elicited by lipoic acid.
Project description:Proteomic methods for RNA interactome capture (RIC) rely principally on crosslinking native or labeled cellular RNA to enrich and investigate RNA-binding protein (RBP) composition and function in cells. The ability to measure RBP activity at individual binding sites by RIC, however, has been more challenging due to the heterogenous nature of peptide adducts derived from the RNA-protein crosslinked site. Here, we present an orthogonal strategy that utilizes clickable electrophilic purines to directly quantify protein-RNA interactions on proteins through photoaffinity competition with 4-thiouridine (4SU)-labeled RNA in cells. Our photo-activatable-competition and chemoproteomic enrichment (PACCE) method facilitated detection of >5,500 cysteine sites across ~3,000 proteins displaying RNA-sensitive alterations in probe binding. Importantly, PACCE enabled functional profiling of canonical RNA-binding domains as well as discovery of moonlighting RNA binding activity in the human proteome. Collectively, we present a chemoproteomic platform for global quantification of protein-RNA binding activity in living cells.
Project description:Quantitative LC MS/MS data from chemoproteomic pulldown experiments using biotinylated macrocycles to enrich the HTT HAP40 complex and associated proteins from HEK293T cells and human neural progenitor cells with varying HTT CAG repeat lengths.
Project description:Here we show that biotin-labelled miR-34a can be loaded to AGO2, and AGO2 immunoprecipitation can pulldown biotinylated miR-34a (Bio-miR pulldown). RNA-sequencing (RNA-seq) of the Bio-miR pulldown RNAs efficiently identified miR-34a mRNA targets, which could be verified with luciferase assays. In contrast to the approach of Bio-miR pulldown, RNA-seq of miR-34a overexpression samples had limited value in identifying direct targets of miR-34a. It seems that pulldown of 30 -Biotin-tagged miRNA can identify bona fide microRNA targets at least for miR34a.
Project description:We used dose-dependent chemoproteomic competition assays to profile the proteome-wide target binding and selectivity of the kinase inhibitor TAK285.