Work

Alpha-centractin is a novel substrate of SETD3 methyltransferase in vitro

Year: 2025

Type: article

Abstract: Background: The SETD3 enzyme, a protein histidine methyltransferase, catalyzes the Nτ-methylation of the histidine 73 residue in β-actin. This post-translational modification is important for maintaining cytoskeleton integrity, and actin remains the only known substrate of this methyltransferase to date. However, SETD3 was also postulated to play a role in the regulation of processes that are not directly related to actin homeostasis, such as cell cycle control and response to hypoxic conditions. These findings suggest that actin may not be the sole substrate of SETD3 methyltransferase. Here, we demonstrate that SETD3 methylates additional proteins in human cells, and α-centractin (ACTR1A) may be one of them. Methods: Three different human SETD3 knockout cell lines (HAP1, HeLa, HEK293T) were generated with the CRISPR/Cas9 method and used as a source of SETD3 substrates. Fluorography was used to detect the SETD3-dependent methylation of proteins present in cell lysates, while the TurboID biotin ligase proximity labeling technique was used to isolate proteins that interact with SETD3. The molecular identity of the proteins was determined by mass spectrometry and the activity of recombinant SETD3 towards potential substrates was tested using a radiochemical assay. Results: methylation target, suggesting that SETD3 potentially regulates not only actin cytoskeleton dynamics but also dynein-mediated intracellular transport.

Source: PeerJ

DOI: 10.7717/peerj.20195