PUBLICATIONS
Methylglyoxal-Derived Posttranslational Arginine Modifications are Abundant Histone Marks
Galligan et al., Proceedings of the National Academy of Sciences, 2018
Histone posttranslational modifications (PTMs) regulate chromatin dynamics, DNA accessibility, and transcription to expand the genetic code. Many of these PTMs are produced through cellular metabolism to offer both feedback and feedforward regulation. Here, we describe the existence of Lys and Arg modifications on histones by the glycolytic by-product, methylglyoxal (MGO). Our data demonstrate that adduction of histones by MGO is an abundant modification, present at the same order of magnitude as Arg methylation. These modifications were detected on all four core histones at critical residues involved in both nucleosome stability and reader domain binding. In addition, MGO treatment of cells lacking the major detoxifying enzyme, glyoxalase 1, results in a marked disruption in H2B acetylation and ubiquitylation, without affecting H2A, H3, and H4 modifications. Using RNA-Seq, we show that MGO is capable of altering gene transcription, most notably in cells lacking GLO1. Lastly, we show that the deglycase, DJ-1, protects histones from adduction by MGO. Collectively, we demonstrate the existence of a previously undetected histone modification derived from glycolysis, which may have far reaching implications on the control of gene expression and protein transcription linked to metabolism.
(A) Methylglyoxal generates stable Arg protein adducts. Three isoforms of MG-H are possible with MG-H3 undergoing hydrolysis to generate CEA. Only MG-H1 and CEA are observed in physiological settings. Cells were cultured for 24 h in low-glucose medium (5 mM) and (B) cellular MGO was quantified. Chromatin was extracted from the indicated cells and subjected to QuARKMod, demonstrating basal levels of ADMA (C), MG-H1 (D), and CEA (E). Basal levels of MGO (F), chromatin ADMA (G), MG-H1 (H), and CEA (I) were also evaluated in tissues isolated from mice. Data are presented as the mean ± S.D. of three measurements.
Identification of 28 site-specific MGO modifications on histones. Proteomic interrogation of site-specific MGO PTMs reveals the N-terminus of H3 and the globular domain (bold) of H2B to be heavily susceptible to modification by MGO.
DJ-1 protects chromatin from modification by MGO. (A) DJ-1 hydrolyzes the intermediate aminocarbinol product of MGO and Arg. (B) Western blotting demonstrates complete knockout of DJ-1 in both WT and GLO1-/- cell lines (DKO). (C-E) QuARKMod was performed on chromatin fractions isolated from each cohort, demonstrating significant increases in MG-H1 and CEA, and not CEL, in DKO cells compared to GLO1-/- alone. Treatments were for 6 h. Data are presented as the mean ± S.D., N = 3, and statistical significance was determined via two-way ANOVA (*** p < 0.001).
Quantitative Analysis and Discovery of Lysine and Arginine Modifications
Galligan et al., Analytical Chemistry, 2017
Post-translational modifications (PTMs) affect protein function, localization, and stability, yet very little is known about the ratios of these modifications. Here, we describe a novel method to quantitate and assess the relative stoichiometry of Lys and Arg modifications (QuARKMod) in complex biological settings. We demonstrate the versatility of this platform in monitoring recombinant protein modification of peptide substrates, PTMs of individual histones, and the relative abundance of these PTMs as a function of subcellular location. Lastly, we describe a product ion scanning technique that offers the potential to discover unexpected and possibly novel Lys and Arg modifications. In summary, this approach yields accurate quantitation and discovery of protein PTMs in complex biological systems without the requirement of high mass accuracy instrumentation.
A generalized scheme of the QuARKMod platform. QuARKMod has been validated for use with a broad range of cell and tissue samples. Following protein digestion, the absolute concentrations of PTMs can be determined in a given sample. In addition, using data-dependent scanning, we outline a method for the discovery of novel or low-abundance Lys and Arg PTMs in the same biological samples.
Application of QuARKMod for the study of in vitro enzyme activity. QuARKMod was used to investigate the overall methylation status of a synthetic peptide (2 nmol) containing a single me3Lys following incubation with a recombinant Lys demethylase, JMJD2A. A quantitative yield was observed in total methylated Lys following incubation with JMJD2A with me2Lys being the predominant PTM. The peptide sequence was as follows: ARTKQTARKme3STGGKA.
Quantitation of Lys and Arg PTMs on isolated histones. (a) To measure histone-specific changes, histones were purified using an offline HPLC method, resulting in significant separation of each core histone. (b) Purified histones collected from HEK293 cells demonstrate varying degrees of Lys and Arg PTMs.
Stable Histone Adduction by 4-oxo-2-nonenal: A Potential Link Between Oxidative Stress and Epigenetics
Galligan et al., Journal of the American Chemical Society, 2014
Lipid electrophiles modify cellular targets altering their function. Here, we described histones as major targets for modification by 4-oxo-2-nonenal, resulting in a stable Lys modification structurally analogous to other histone Lys acylations. Seven adducts were identified in chromatin isolated from intact cells - four 4-ketoamides to Lys and 3 Michael adducts to His; modification of histones H3 and H4 were found to prevent nucleosome assembly.
Scheme 1. 4-hydroxy-2-nonenal (4-HNE) and 4-oxo-2-nonenal (4-ONE) are electrophilic molecules that adduct both DNA and protein, resulting in altered structure and function. Although similar in structure, 4-HNE and 4-ONE differ in reactivity in that 4-ONE can form stable ketoamide adducts via 1,2 addition to Lys
Figure 1. (A) Alkynyl-4-ONE preferentially modifies histone proteins in chromatin isolated from RKO cells. (B) Time-course of histone adduction with a4-ONE.
Figure 2. 4-ONE adducts are located on surface-exposed amino acids. (A) Crystal structure of the nucleosome. Adducts are located at (B) H4K79, (C) H2BH82, H109 and K116, (D) H3K23 and K27 and (E) H2AH123. Structure obtained from PDB:1AOI.