Editor | Xi Signal transduction in cells depends largely on the post-translational modification status of protein amino acid side chains [1]. When post-translational modifications occur at different sites, occupy different proportions, and produce diverse modification combination

Editor | 西

Signal transduction in cells depends to a large extent on the post-translational modification state of protein amino acid side chains【1】. When post-translational modifications occur at different sites, occupy different proportions, and produce diverse modification combinations, the same substrate protein will be "dressed" into protein variants with huge differences in conformation, function, binding partners, and positioning. This has inspired researchers to study protein post-translational modifications. In recent years, people have gained an in-depth understanding of classic post-translational modifications such as phosphorylated , glycosylated , acetylated , ubiquitinated , methylated , etc. However, what is interesting is that there are still new acylation modifications on the lysine residue, such as crotonylation [2] , butyrylation [3] , malonylation [4] , succinylated [5] was discovered. Also on lysine residues, in 2019 University of Chicago Professor Zhao Yingming’s research group first reported the discovery of lactylated on histones (see BioArt report for details: Nature Highlights | Zhao Yingming’s group discovered lactylation of histones New modification) [6] . This study demonstrates that histone lactylation modification is derived from lactic acid, and that this modification has transcription and regulatory functions that do not overlap with histone acetylation in different biological scenarios. This is undoubtedly an important discovery that explains how cells sense metabolic changes and initiate transcriptional regulation mechanisms.

But interesting questions remain to be answered: Is lactylation a post-translational modification that is widespread in human cells and tissues? Can lactylation occur at lysine residues in human nonhistone proteins? What is the level of lactylation modification of non-histone proteins, and does it have a biological regulatory effect?

In order to answer these questions, on June 27, 2022, China Pharmaceutical University Hao Haiping / Ye Hui team and Nanjing University of Chinese Medicine Professor Wang Nanxi published an article on Nature Methods Cyclic immonium ion of lactyllysine reveals widespread lactylation in the human proteome. This work first identified and confirmed the characteristic cyclic imine ion produced by the polypeptide carrying lactylated lysine, and applied this ion from the existing non-enriched, large-scale human proteome data. Information on the new lactylation-modified substrate protein and site was unearthed from the resources, and by introducing lactylation modification to metabolic enzymes at specific sites, it was initially confirmed that lactylation occurs on human non-histone substrates as well. It has important regulatory functions.

This study was inspired by a summary of the rules of proteome post-translational modification research: phosphorylation, acetylation and other post-translational modifications can produce characteristic ions with diagnostic significance. Does lactylation modification also produce diagnostic ions?

In order to verify this conjecture, the team proposed to explore whether there are new substrates for lactylation modification in the massive shared human proteome database. However, the false positive rate of and for retrieving modification sites from non-enriched proteome data is extremely high. If modification-specific characteristic ions can be found, spectral screening can significantly reduce the risk of modified lysine sites. The false positive rate reveals the true modification target and guides subsequent biological function exploration. Based on this need, the team synthesized and studied the spectra of model lactylated peptides. discovered for the first time that polypeptides carrying lactylated lysine will form chain imine ions after secondary fragmentation in the mass spectrometry collision chamber. , the ion undergoes deamination cyclization to form secondary fragments—cyclic imine ions. The team used to analyze chemical modifications and positive lactylated peptides enriched in biological samples, and then used nearly 100,000 unmodified synthetic peptide spectra of the human proteome as negative controls to confirm the identification of cyclic imines. The sensitivity and specificity of ion index lactylation modification can be used as the gold standard for identifying new lactylation modification sites obtained through database searches.

Based on this diagnostic ion strategy, researchers have mined a large amount of new lactylation modification substrate protein and site information from existing non-enriched, large-scale human proteome data resources, especially from the 2020 Nature Methods [7] published the proteome thermostability Meltome Atlas data resource of various human cell lines and found that lactylation modification is highly enriched in metabolic enzymes of the glycolysis pathway. Among them, the lactylation-modified metabolic enzyme ALDOA is conserved in various human tumor cell lines and has a high modification occupancy ratio, leading to the conjecture that lactylation modification can regulate metabolic enzyme activities and other functions, thereby regulating the glycolysis pathway. .

Hao Haiping and Ye Hui’s team further collaborated with Wang Nanxi’s research group and used the advanced chemical biology technology - gene codon expansion technology to introduce lactylation modification to the target protein ALDOA at a specific site for the first time. It was found that the enzyme activity was significantly reduced after the modification. , revealed the feedback regulation mechanism of inhibiting glycolytic activity by covalently modifying upstream metabolic enzymes in the glycolytic pathway after lactate accumulation, supplementing the existing "end product inhibition" regulatory model in the field of biochemistry.

In summary, This study shows that lactylation is a non-histone-specific post-translational modification that is widely present in human tissues and cells, and it also has regulatory functions on non-histone substrate proteins. This analysis strategy can reveal more covalent modification targets of lactic acid, elucidate the causal relationship between the dynamic changes of lactylation modification and the important role of lactic acid disorders in the occurrence and development of major chronic diseases such as inflammation and tumors, and then discover new ones. Targets for disease treatment provide clues.

Wanning, a 2019 doctoral student, and Wang Nian, a 2018 master's student, are the co-first authors of this paper. Researcher Ye Hui, Professor Hao Haiping, and Professor Wang Nanxi are the co-corresponding authors of this article.

The Hao Haiping/Ye Hui team has long-term recruitment of doctoral/master students with backgrounds in bioinformatics, , metabolic regulation, target discovery, etc. Resumes can be sent to the following email addresses: [email protected] and [email protected]; Students who are interested in pharmacy, microbiology and biochemical pharmacy are welcome to apply for doctoral/master's degree under Professor Wang Nanxi. Please send your resume to [email protected].

Original link:

https://www.nature.com/articles/s41592-022-01523-1

References

[1] Knorre DG, Kudryashova NV, Godovikova TS. Chemical and functional aspects of posttranslational modification of proteins. Acta Naturae . 2009, 1(3):29-51

[2] Tan M, Luo H, Lee S, et al. Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification. Cell. 2011, 146(6 ):1016-28.

[3] Chen Y, Sprung R, Tang Y, et al. Lysine propionylation and butyrylation are novel post-translational modifications in histones. Mol Cell Proteomics. 2007, 6(5):812-9.

[4] Peng C, Lu Z, Xie Z, et al. The first identification of lysine malonylation substrates and its regulatory enzyme. Mol Cell Proteomics. 2011, 10(12):M111. 012658.

[5] Hirschey MD, Zhao Y. Metabolic regulation by lysine malonylation, succinylation, and glutarylation. Mol Cell Proteomics. 2015, 14(9):2308-15.

[6] Zhang D, Tang Z, Huang H, et al. Metabolic regulation of gene expression by histone lactylation. Nature. 2019, 574(7779):575-580.

[7] Jarzab A, Kurzawa N, Hopf T, et al. Meltome atlas-thermal proteome stability across the tree of life. Nat Methods. 2020, 17(5):495-503.

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