Dann, G. P. | Search bioRxiv

bioRxiv2024

An Unbiased Proteomic Platform for Activity-based Arginylation Profiling

Protein arginylation is an essential posttranslational modification (PTM) catalyzed by arginyl-tRNA-protein transferase 1 (ATE1) in mammalian systems. Arginylation features a post-translational conjugation of an arginyl to a protein, making it extremely challenging to differentiate from translational arginine residues with the same mass in a protein sequence. Here we present a general ATE1-based arginylation profiling platform for the unbiased discovery of arginylation substrates and their precise modification sites. This method integrates isotopic arginine labeling into an ATE1 assay utilizing biological lysates (ex vivo) rather than live cells, thus eliminating translational bias derived from the ribosomal activity and enabling bona fide arginylation identification using isotopic features. The method has been successfully applied to an array of peptide, protein, cell, patient, and animal tissue samples using 20 {micro}g sample input, with 235 unique arginylation sites revealed from human proteomes. Representative sites were validated and followed up for their biological functions. The developed platform is globally applicable to the aforementioned sample types and therefore paves the way for functional studies of this difficult-to-characterize protein modification.

biochemistry↗

bioRxiv2019

JASPer controls interphase histone H3S10 phosphorylation by chromosomal kinase JIL-1 in Drosophila

In flies, the chromosomal kinase JIL-1 is responsible for most interphase histone H3S10 phosphorylation and has been proposed to protect active chromatin from acquiring heterochromatic marks, like dimethylated histone H3K9 (H3K9me2) and HP1. Here, we show that JIL-1s targeting to chromatin depends on a new PWWP domain-containing protein JASPer (JIL-1 Anchoring and Stabilizing Protein). The JASPer-JIL-1 (JJ)-complex is the major form of the kinase in vivo and is targeted to active genes and telomeric transposons via binding of the PWWP domain of JASPer to H3K36me3 nucleosomes. Put in place, the complex modulates the transcriptional output. JIL-1 and JJ-complex depletion in cycling cells lead to small changes in H3K9me2 distribution at active genes and telomeric transposons. Finally, we identified many new interactors of the endogenous JJ-complex and propose that JIL-1 not only prevents heterochromatin formation, but also coordinates chromatin-based regulation in the transcribed part of the genome.

molecular biology↗