Study on the cellular regulation and function of lysine malonylation, glutarylation and crotonylation

Abstract

Protein posttranslational modifications are covalent chemical modifications occurring on proteins after their biosynthesis. Accumulating evidence has linked protein PTMs to normal cell physiology and the abnormalities in the regulation of PTMs can often lead to human diseases such as cancer and developmental disorder. To date, hundreds of protein PTMs have been identified, however, only a handful of them have been extensively studied. The cellular regulatory mechanisms of many PTMs, particularly newly identified ones, remain poorly understood. It is therefore necessary to characterize these PTMs. During the last four years, I focused on the study of three newly identified PTMs, lysine malonylation, glutarylation and crotonylation, with respect to profiling modified proteins, unveiling the biological functions and identifying regulating enzymes.

In Chapter 2, we developed a chemical reporter (MalAM-yne) for the detection and identification of malonylated proteins. We demonstrated that this alkyne-functionalized chemical reporter could be metabolically incorporated into cellular proteins. And the subsequent bioorthogonal conjugation with an azide-fluorescent dye or an affinity purification tag enables the visualization and profiling of malonylated proteins, respectively. Using MalAM-yne, we identified 361 more candidates of malonylated proteins in addition to 14 out of 17 known malonylated proteins. We subsequently confirmed the modification of MalAM-yne at lysine residues of both known and newly identified malonylated proteins.

In Chapter 3, we identified and validated lysine glutarylation as a new type of histones modifications. There were 27 glutarylated lysine sites identified on human histones. To unveil its biological significance, in Chapter 4, we focused on histone H4 lysine 91 site (H4K91), which lies at the interface between histone H2A/H2B dimer and H3/H4 tetramer and forms a salt bridge with histone H2B to stabilize nucleosome structure. We found that the replacement of lysine with glutamic acid, mimicking lysine glutarylation, resulted in significant delay of DNA replication and hypersensitivity to DNA damage reagents. We further demonstrated that the substitution enhanced the sensitivity of chromatin to micrococcal nuclease digestion, indicating that the incorporation of glutaryl group at H4 Lys 91 could lead to a more relaxed chromatin state.

In Chapter 5, we used a chemical proteomics approach to comprehensively profile ‘eraser’ enzymes for lysine crotonylation, a newly discovered histone posttranslational modification enriched at active gene promoters and potential enhancers in mammalian cell genomes. We focused on the crotonyl mark at histone H3 lysine (H3K4Cr) and found that Sirt1, Sirt2 and Sirt3 exhibit hydrolysis activity towards both lysine crotonylated histone peptides and proteins in vitro in a nicotinamide adenine dinucleotide (NAD)-dependent manner. More importantly, we identified Sirt3 as an endogenous decrotonylation enzyme to regulate the dynamics of histone lysine crotonylation.