HBO1 catalyzes lysine lactylation and mediates histone H3K9la to regulate gene transcription.

Lysine lactylation (Kla) links metabolism and gene regulation and plays a key role in multiple biological processes. However, the regulatory mechanism and functional consequence of Kla remain to be explored. Here, we report that HBO1 functions as a lysine lactyltransferase to regulate transcription. We show that HBO1 catalyzes the addition of Kla in vitro and intracellularly, and E508 is a key site for the lactyltransferase activity of HBO1. Quantitative proteomic analysis further reveals 95 endogenous Kla sites targeted by HBO1, with the majority located on histones. Using site-specific antibodies, we find that HBO1 may preferentially catalyze histone H3K9la and scaffold proteins including JADE1 and BRPF2 can promote the enzymatic activity for histone Kla. Notably, CUT&Tag assays demonstrate that HBO1 is required for histone H3K9la on transcription start sites (TSSs). Besides, the regulated Kla can promote key signaling pathways and tumorigenesis, which is further supported by evaluating the malignant behaviors of HBO1- knockout (KO) tumor cells, as well as the level of histone H3K9la in clinical tissues. Our study reveals HBO1 serves as a lactyltransferase to mediate a histone Kla-dependent gene transcription.

Maleic Anhydride Labeling-Based Approach for Quantitative Proteomics and Successive Derivatization of Peptides.

Chemical derivatization is a simple approach for stable-isotope covalent labeling of proteins in quantitative proteomics. Herein we describe the development of a novel maleyl-labeling-based approach for protein quantification. Under optimized conditions, maleic anhydride can serve as a highly efficient reagent to label the amino groups of tryptic peptides. Furthermore, "click chemistry" was successfully applied to obtain the second modification of maleylated peptides via thiol-Michael addition reaction. Accurate quantification was further achieved via the first or/and second step stable-isotope labeling in this study. Our data thus demonstrate that the maleyl-labeling-based method is simple, accurate, and reliable for quantitative proteomics. The developed method not only enables an enhanced sequence coverage of proteins by improving the identification of small and hydrophilic peptides, but also enables a controllable, successive, second derivatization of labeled peptides or proteins, and therefore holds a very promising potential for in-depth analysis of protein structures and dynamics.

Analytical strategies used to identify the readers of histone modifications: A review.

The so-called "readers" of histone post-translational modifications (HPTMs) refer to proteins or complexes that are recruited to HPTMs thus eventually regulate gene transcription. To identify these "readers", mass spectrometry plays an essential role following various enriching strategies. These enriching methods include the use of modified histone peptides/proteins or chemically synthesized histones/nucleosomes containing desired HPTMs to enrich the readers of HPTMs. Despite the peptide- or protein-based assay is straightforward and easy to perform for most labs, this strategy has limited applications for those weak or combinational interactions among various HPTMs and false-positive results are a potential big problem. While the results derived from synthesized histone proteins/nucleosomes is more reliable as it mimics the real chromatic conditions thus is able to analyze the binders of those cross-talked HPTMs, usually the synthesis is so difficult that their applications are impeded for high throughput analysis. In this review, an overview of these analytical techniques is provided and their advantages and disadvantages are discussed.