RESUMO
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 >5500 cysteine sites across ~3000 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.
Assuntos
Proteômica , RNA , Humanos , RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sítios de Ligação , Peptídeos/metabolismoRESUMO
The mechanisms of general anesthetics have been debated in the literature for many years and continue to be of great interest. As anesthetic molecules are notoriously difficult to study due to their low binding affinities and multitude of binding partners, it is advantageous to have additional tools to study these interactions. Fropofol is a hydroxyl to fluorine-substituted propofol analogue that is able to antagonize the actions of propofol. Understanding fropofol's ability to antagonize propofol would facilitate further characterization of the binding interactions of propofol that may contribute to its anesthetic actions. However, the study of fropofol's molecular interactions has many of the same difficulties as its parent compound. Here, we present the synthesis and characterization of ortho-azi-fropofol (AziFo) as a suitable photoaffinity label (PAL) of fropofol that can be used to covalently label proteins of interest to characterize fropofol's binding interactions and their contribution to general anesthetic antagonism.
Assuntos
Anestésicos Gerais , Propofol , Diazometano , Propofol/farmacologiaRESUMO
Several additional examples of cascade cyclizations of α,ß-unsaturated thioesters proceeding are reported, which proceed via two distinct mechanistic pathways: enantioselective acyl transfer promoted by amidine-based catalysts (ABCs) and a racemic chain mechanism mediated by a thiolate nucleophile.
RESUMO
The highly Lewis basic amidine-based catalyst DHIP promotes the rearrangement of S-phenacyl thiocinnamate and related thioesters into dihydrothiophene derivatives. In contrast to previously explored rearrangements of thioesters, the reaction proceeds via a novel Dieckmann-like cyclization pathway. An alternative two-component synthesis of the same products has also been developed.