Hudmon, A. | Search bioRxiv

bioRxiv2022

Substrate selectivity of the PRDM9 lysine methyltransferase domain.

Lysine methylation is a dynamic, post-translational mark that regulates the function of histone and non-histone proteins. Many of the enzymes that mediate lysine methylation, known as lysine methyltransferases (KMTs), were originally identified to modify histone proteins but have also been discovered to methylate non-histone proteins. In this work, we investigate the substrate selectivity of the lysine methyltransferase PRDM9 to identify both potential histone and non-histone substrates. Though normally expressed in germ cells, PRDM9 is significantly upregulated across many cancer types. The methyltransferase activity of PRDM9 is essential for double-strand break formation during meiotic recombination. PRDM9 has been reported to methylate histone H3 at lysine residues 4 and 36; however, PRDM9 KMT activity had not previously been evaluated on non-histone proteins. Using lysine-oriented peptide (K-OPL) libraries to screen potential substrates of PRDM9, we determined that PRDM9 preferentially methylates peptide sequences not found in any histone protein. We confirmed PRDM9 selectivity through in vitro KMT reactions using peptides with substitutions at critical positions. A multisite {lambda}-dynamics computational analysis provided a structural rationale for the observed PRDM9 selectivity. The substrate selectivity profile was then used to identify putative non-histone substrates, which were tested by peptide spot array. Finally, PRDM9 methylation non-histone substrates were validated at the protein level by in vitro KMT assays on recombinant proteins. The selectivity profile of PRDM9 will be useful in identifying putative PRDM9 substrates in different cellular contexts, and future studies are required to determine whether PRDM9 methylates non-histone proteins in the context of meiotic recombination or cancer.

biochemistry↗

bioRxiv2020

Calmodulin binds to the N-terminal domain of the cardiac sodium channel Nav1.5

The cardiac voltage-gated sodium channel Nav1.5 conducts the rapid inward sodium current crucial for cardiomyocyte excitability. Loss-of-function mutations in its gene SCN5A are linked to cardiac arrhythmias such as Brugada Syndrome (BrS). Several BrS-associated mutations in the Nav1.5 N-terminal domain exert a dominant-negative effect (DNE) on wild-type channel function, for which mechanisms remain poorly understood. We aim to contribute to the understanding of BrS pathophysiology by characterizing three mutations in the Nav1.5 N-terminal domain (NTD): Y87C-here newly identified-, R104W and R121W. In addition, we hypothesize that the calcium sensor protein calmodulin is a new NTD binding partner. Recordings of whole-cell sodium currents in TsA-201 cells expressing WT and variant Nav1.5 showed that Y87C and R104W but not R121W exert a DNE on WT channels. Biotinylation assays revealed reduction in fully glycosylated Nav1.5 at the cell surface and in whole-cell lysates. Localization of Nav1.5 WT channel with the ER however did not change in the presence of variants, shown by transfected and stained rat neonatal cardiomyocytes. We next demonstrated that calmodulin binds Nav1.5 N-terminus using in silico modeling, SPOTS, pull-down and proximity ligation assays. This binding is impaired in the R121W variant and in a Nav1.5 construct missing residues 80-105, a predicted calmodulin binding site. In conclusion, we present the first evidence that calmodulin binds to the Nav1.5 NTD, which seems to be a determinant for the DNE.

physiology↗