Posttranslational modifications (PTMs) play a crucial role in a wide range of biological processes. Lysine crotonylation (Kcr) is a newly discovered histone PTM that is enriched at active gene promoters and potential enhancers in mammalian cell genomes. However, the cellular enzymes that regulate the addition and removal of Kcr are unknown, which has hindered further investigation of its cellular functions. Here we used a chemical proteomics approach to comprehensively profile 'eraser' enzymes that recognize a lysine-4 crotonylated histone H3 (H3K4Cr) mark. We found that Sirt1, Sirt2, and Sirt3 can catalyze the hydrolysis of lysine crotonylated histone peptides and proteins. More importantly, Sirt3 functions as a decrotonylase to regulate histone Kcr dynamics and gene transcription in living cells. This discovery not only opens opportunities for examining the physiological significance of histone Kcr, but also helps to unravel the unknown cellular mechanisms controlled by Sirt3, that have previously been considered solely as a deacetylase.

Post-translational modifications (PTMs) have key roles in regulating protein-protein interactions in living cells. However, it remains a challenge to identify these PTM-mediated interactions. Here we develop a new lysine-based photo-reactive amino acid, termed photo-lysine. We demonstrate that photo-lysine, which is readily incorporated into proteins by native mammalian translation machinery, can be used to capture and identify proteins that recognize lysine PTMs, including 'readers' and 'erasers' of histone modifications.

Tag! You′re it! MalAM‐yne is a chemical reporter for malonylation of lysines within proteins (see scheme), a newly identified posttranslational modification. MalAM‐yne is cell‐permeable and metabolically incorporated into proteins in living cells. Subsequent bioorthogonal tag conjugation allows the fluorescent visualization of cellular malonylation and profiling of malonylated proteins.

No abstract

Eukaryotic translation initiation factor 4E (eIF4E) binds the m7GTP cap structure at the 5′-end of mRNAs, stimulating the translation of proteins implicated in cancer cell growth and metastasis. eIF4E is a notoriously challenging target, and most of the reported inhibitors are negatively charged guanine analogues with negligible cell permeability. To overcome these challenges, we envisioned a covalent targeting strategy. As there are no cysteines near the eIF4E cap binding site, we developed a covalent docking approach focused on lysine. Taking advantage of a "make-on-demand" virtual library, we used covalent docking to identify arylsulfonyl fluorides that target a noncatalytic lysine (Lys162) in eIF4E. Guided by cocrystal structures, we elaborated arylsulfonyl fluoride 2 to 12, which to our knowledge is the first covalent eIF4E inhibitor with cellular activity. In addition to providing a new tool for acutely inactivating eIF4E in cells, our computational approach may offer a general strategy for developing selective lysine-targeted covalent ligands.

New chemical tools to ‘trap’ post translational modification (PTM)-mediated protein–protein interactions.

Protein fatty-acylation is important for the regulation of membrane association, trafficking, subcellular localization and activity of many cellular proteins. While significant progress has been made on our understanding of the two major forms of protein fatty-acylation, N-myristoylation and S-palmitoylation, studies of fatty-acylation of protein lysine residues have lagged behind. This is in large part due to a lack of knowledge about the enzymes that regulate the addition and removal of lysine fatty-acylation. To fill this knowledge gap, here we demonstrate the use of integrative chemical biology approaches to examine human sirtuins as a de-fatty-acylase in vitro and in cells. We used a photo-cross-linking-based chemical approach to investigate enzymes that recognize lysine fatty-acylation. This approach enables the identification of human Sirt2 as a robust lysine de-fatty-acylase in vitro. Using a bioorthogonal chemical reporter for protein fatty-acylation, we show that Sirt2 can regulate protein lysine fatty-acylation in cells. This finding not only opens a new avenue to investigate the biological significances of protein lysine fatty-acylation, but also may help to unravel unrecognized cellular functions of Sirt2 that has been considered solely as a deacetylase until now. Furthermore, the photo-cross-linking-based chemical approach used in this study should also be useful to examine the interactions between other protein posttranslational modifications and their ‘erasers’.

No abstract