An Integrated Analytical Approach for Screening Functional Post-Translational Modification Sites in Metabolic Enzymes

Post-translational modifications (PTMs) are pivotal in the orchestration of diverse physiological and pathological processes. Despite this, the identification of functional PTM sites within the vast amount of data remains challenging. Conventionally, those PTM sites are discerned through labor-intensive and time-consuming experiments. Here, we developed an integrated analytical approach for the identification of functional PTM sites on metabolic enzymes via a screening process. Through gene ontology (GO) analysis, we identified 269 enzymes with lysine 2-hydroxyisobutyrylation (Khib) from our proteomics data set of Escherichia coli. The first round of screening was performed based on the enzyme structures/predicted structures using the TM-score engineer, a tool designed to evaluate the impact of PTM on the protein structure. Subsequently, we examined the influence of Khib on the enzyme–substrate interactions through both static and dynamic analyses, molecular docking, and molecular dynamics simulation. Ultimately, we identified NfsB K181hib and ThiF K83hib as potential functional sites. This work has established a novel analytical approach for the identification of functional protein PTM sites, thereby contributing to the understanding of Khib functions

An Integrated Analytical Approach for Screening Functional Post-Translational Modification Sites in Metabolic Enzymes

Post-translational modifications (PTMs) are pivotal in the orchestration of diverse physiological and pathological processes. Despite this, the identification of functional PTM sites within the vast amount of data remains challenging. Conventionally, those PTM sites are discerned through labor-intensive and time-consuming experiments. Here, we developed an integrated analytical approach for the identification of functional PTM sites on metabolic enzymes via a screening process. Through gene ontology (GO) analysis, we identified 269 enzymes with lysine 2-hydroxyisobutyrylation (Khib) from our proteomics data set of Escherichia coli. The first round of screening was performed based on the enzyme structures/predicted structures using the TM-score engineer, a tool designed to evaluate the impact of PTM on the protein structure. Subsequently, we examined the influence of Khib on the enzyme–substrate interactions through both static and dynamic analyses, molecular docking, and molecular dynamics simulation. Ultimately, we identified NfsB K181hib and ThiF K83hib as potential functional sites. This work has established a novel analytical approach for the identification of functional protein PTM sites, thereby contributing to the understanding of Khib functions

An Integrated Approach Based on a DNA Self-Assembly Technique for Characterization of Crosstalk among Combinatorial Histone Modifications

Combinatorial histone post-translational modifications (HPTMs) form a complex epigenetic code that can be decoded by specific binding proteins, termed as readers. Their specific interplays have been thought to determine gene expression and downstream biological functions. However, it is still a big challenge to analyze such interactions due to various limitations including rather weak, transient, and complicated interactions between HPTMs and readers, the high dynamic property of HPTMs, and the low abundance of reader proteins. Here we sought to take advantage of DNA-templated and photo-cross-linking techniques to design a group of combinatorial histone PTM peptide probes for the identification of multivalent interactions among histone PTMs and readers. By use of trimethylation on histone H3K4 (H3K4me3) and phosphorylation on H3T3, we demonstrated that this approach can be successfully utilized for identification of the PTM crosstalk on the same histone. By use of H3K4me3 and acetylation on H4K16, we showed the potential application of the probe in the multivalent interactions among PTMs on different histones. Thus, this new chemical proteomics tool combined with mass spectrometry holds a promising potential in profiling of the readers of combinatorial HPTMs and characterization of crosstalk among multiple PTMs on histones and can be adapted for broad biomedical applications

An Integrated Approach Based on a DNA Self-Assembly Technique for Characterization of Crosstalk among Combinatorial Histone Modifications

Combinatorial histone post-translational modifications (HPTMs) form a complex epigenetic code that can be decoded by specific binding proteins, termed as readers. Their specific interplays have been thought to determine gene expression and downstream biological functions. However, it is still a big challenge to analyze such interactions due to various limitations including rather weak, transient, and complicated interactions between HPTMs and readers, the high dynamic property of HPTMs, and the low abundance of reader proteins. Here we sought to take advantage of DNA-templated and photo-cross-linking techniques to design a group of combinatorial histone PTM peptide probes for the identification of multivalent interactions among histone PTMs and readers. By use of trimethylation on histone H3K4 (H3K4me3) and phosphorylation on H3T3, we demonstrated that this approach can be successfully utilized for identification of the PTM crosstalk on the same histone. By use of H3K4me3 and acetylation on H4K16, we showed the potential application of the probe in the multivalent interactions among PTMs on different histones. Thus, this new chemical proteomics tool combined with mass spectrometry holds a promising potential in profiling of the readers of combinatorial HPTMs and characterization of crosstalk among multiple PTMs on histones and can be adapted for broad biomedical applications