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Methylglyoxal - A Glycolytic Byproduct that Adducts Histones


By: Carol A. Rouzer, VICB Communications
Published:  August 30, 2018


Adduction of histone lysine and arginine residues by methylglyoxal occurs in vivo and may lead to chromatin dysfunction.


Histones are proteins that play a major role in DNA structure and dynamics. They package DNA into nucleosomes, the core particles of chromatin (Figure 1), and are intimately involved in the modulation of gene transcription. Much of the regulatory function of histones is based upon the presence of numerous post-translational modifications (PTMs) on lysine and arginine residues, frequently located in the protein's N-terminal tail. Most of these PTMs are added and removed enzymatically by writer and eraser proteins, respectively, and they serve as binding sites for reader proteins that facilitate the interaction of effector molecules involved in DNA processing (Figure 2). Increasing evidence indicates that the lysine and arginine residues of histones may also be modified nonenzymatically through reaction with a variety of electrophilic molecules. These nonenzymatic PTMs may block the site of enzymatic PTM addition, lead to inappropriate reader function, and evade removal by erasers with potentially toxic consequences. This led Vanderbilt Institute of Chemical Biology member Larry Marnett, Research Assistant Professor Jim Galligan (now at the University of Arizona), and the Marnett lab to investigate the potential of methylglyoxal (MGO), a ubiquitous electrophilic byproduct of glycolysis, to form nonenzymatic PTMs in histones [J. Galligan, et al., Proc. Natl. Acad. Sci. U.S.A., (2018) published online August 27, DOI: 10.1073/pnas.1802901115].



FIGURE 1. Two views of the nucleosome core particle, crystal structure (PDB ID: 1EQZ) obtained from Wikimedia Commons. Histones H2A, H2B, H3, and H4 as well as DNA are colored as indicated. Image taken from the Protein Data Bank (ID 1EQZ) [Harp, J.M., Hanson, B.L., Timm, D.E., Bunick, G.J. (2000) Acta Crystallogr., Sect.D, 56, 1513] created with Jmol (an open-source Java viewer for chemical structures in 3D.




FIGURE 2. (A) Interactions between writers, readers, and erasers. Once a writer has placed one or more PTMs on histones within a nucleosome, reader proteins can then bind. In some cases, readers provide a binding site for additional writers or erasers, enabling them to add more or remove PTMs. (B) Example of how a histone PTM can modulate chromatin function. The PTM is a methyl group on lysine-9 of H3 (H3K9me). The reader in this case is heterochromatin protein 1 (HP1), a dimer that binds to H3K9me groups on two adjacent nucleosomes. The HP1 dimer then provides a binding site for various effector proteins. Figure reproduced by permission from Macmillan Publishers, Ltd./Springer Nature from R. C. Allshire, & H.D. Madhani (2018) Nat. Rev. Mol. Cell Biol., 19, 229. Copyright 2018, Springer Nature.



The researchers' hypothesis that MGO could form PTMs in histones was based on prior reports of its ability to react with lysine, yielding Nε-(carboxyethyl)lysine (CEL), and arginine to form three hydroimidazolones that have been designated MG-H1, MG-H2, and MG-H3 (Figure 3). MG-H3 is subject to hydrolysis to form carboxyethylarginine (CEA), which is the adduct routinely identified in biological samples. To test their hypothesis, the researchers developed a mass spectrometry-based assay to detect all potential MGO-protein adducts with high sensitivity and specificity. They used this assay to search for the adducts in chromatin isolated from seven different cell lines that had been maintained in medium containing low concentrations (5 mM) of glucose. MG-H1 and CEA were present in the samples from all of the cell lines in quantities similar to those of some enzymatically formed PTMs that are known to modulate chromatin function. They also detected CEL in their samples, but it was present at very low concentrations. Of particular interest were the results of subsequent studies that demonstrated increased levels of CEA in cells grown in the presence of medium containing high concentrations (25 mM) of glucose.



FIGURE 3. (A) Reaction of MGO with lysine to form CEL [-(carboxyethyl)lysine]. (B) Reaction of MGO with arginine to form the hydroimidazolone adducts MG-H1, MG-H2, and MG-H3. Hydrolysis of MG-H3 produces CEA (carboxyelthylarginine). Figure reproduced with permission from J. Galligan, et al., Proc. Natl. Acad. Sci. U.S.A., (2018) published online August 27, DOI: 10.1073/pnas.1802901115. Copyright 2018, J. Galligan, et al.


Due to MGO's potentially toxic nature, cells have evolved a detoxification pathway (Figure 4). MGO reacts nonenzymatically and reversibly with the sulfhydryl of glutathione to form a hemithioacetal. Subsequent isomerization of this intermediate catalyzed by the enzyme glyoxylase 1 (GLO1) yields lactoyl-glutathione, which is then hydrolyzed by glyoxylase 2 (GLO2) to lactate and glutathione. The investigators hypothesized that inactivation of this pathway would impact levels of MGO-mediated histone PTMs. To test this hypothesis, they used CRISPR/Cas technology to create an HEK293 cell line lacking the gene for GLO1 (GLO1-/-). They were surprised to find that loss of GLO1 had no effect on baseline levels of MGO in cells grown under control culture conditions; however it did lead to increased susceptibility to MGO-mediated cytotoxicity. This was consistent with the finding that GLO1-/- cells exposed to exogenous MGO exhibited a much higher peak intracellular MGO concentration than wild-type cells, although levels eventually returned to baseline after about 12 h. GLO1-/- cells also exhibited higher levels of MGO adducts in their chromatin than wild-type cells, but only if they had been exposed to exogenous MGO.



FIGURE 4. The glyoxylase cycle. MGO (red) reacts with glutathione to form a hemithioacetal, which is then isomerized to lactoyl-glutathione by GLO1. GLO2 subsequently hydrolyzes lactoyl-glutathione to glutathione and lactate. Thus, the overall effect is to convert MGO to lactate.


The researchers used chromatin from GLO1-/- cells that had been treated with MGO to isolate and characterize specific adducts within histones. They identified 17 MG-H, 6 CEA, and 5 CEL adducts. Notably, these were not restricted to the N-terminal tail of the histones where most enzymatic PTMs are found. Of particular interest was the finding that four of the adducts, located on Arg-23 of H4, Arg-55 of H4, Arg-92 of H2B, and Arg-72 of H3, were positioned near DNA (Figure 5). In addition, adducts at Arg-53 of H3 had the potential to disrupt nucleosomal stability, based on the effects of enzymatic acetylation at that position.


FIGURE 5. (Left) Identified sites of modification are mapped (in red) onto the crystal structure of the nucleosome core particle. (Right) Boxes show magnified views of H4R23, H4R55, and H3R72, all of which lie within close proximity to DNA. Figure reproduced with permission from J. Galligan, et al., Proc. Natl. Acad. Sci. U.S.A., (2018) published online August 27, DOI: 10.1073/pnas.1802901115. Copyright 2018, J. Galligan, et al.



Having confirmed the presence of MGO adducts in histones, the researchers next hypothesized that these PTMs might interfere with the normal function of writer, eraser, and/or reader proteins. In support of this hypothesis, they found that GLO1-/- cells exposed to exogenous MGO exhibited reduced levels of histone acetylation and ubiquitination (common writer-generated PTMs), particularly on H2B. This finding suggested the possibility that MGO adducts could lead to aberrant histone-mediated transcription regulation. Consistently, RNA-Seq analysis revealed that, compared to wild-type cells, GLO1-/- cells incubated in the absence of exogenous MGO exhibited altered gene transcription, characterized by 88 down-regulated and 59 up-regulated genes. Upon exposure to 50 μM MGO, the number of down- and up-regulated genes in GLO1-/- cells increased to 164 and 140, respectively, using wild-type control cells for comparison. Exposure of GLO1-/- cells to 500 μM MGO led to changes in >1000 genes, but the researchers concluded that this was likely due to generalized toxicity. Gene ontology analysis of transcription changes in GLO1-/- cells cultured in the presence of 50 μM MGO revealed that the observed modifications did not appear to be enriched in any particular metabolic pathways. In particular, there was no evident stimulation or inhibition of transcription in the antioxidant, endoplasmic reticulum stress, or heat shock pathways that cells routinely use to deal with toxic exposures. These findings suggested that the effects of MGO adduction on transcription were randomly distributed as a result of an unregulated chemical reaction.


The deglycase DJ-1 repairs MGO-derived modifications to guanine in DNA and prevents MGO-mediated modifications of lysine and arginine in proteins (Figure 6). To see if DJ-1 plays a role in protecting histones from MGO adduction, the researchers used CRISPR/Cas to create wild-type and GLO1-/- cells lacking the gene for DJ-1. They discovered that knockout of DJ-1 only had little effect on MGO-mediated PTMs in histones, even in cells exposed to exogenous MGO. However, cells lacking both GLO1 and DJ-1 exhibited higher levels of histone MGO adducts on arginine following MGO exposure than cells lacking only GLO1. These findings suggest that DJ-1 plays a role in regulating levels of MGO-arginine adducts in histones.




FIGURE 6. DJ-1 protects cells from MGO-mediated adduction by hydrolyzing the aminocarbinol intermediate formed during MG-H formation. Figure reproduced with permission from J. Galligan, et al., Proc. Natl. Acad. Sci. U.S.A., (2018) published online August 27, DOI: 10.1073/pnas.1802901115. Copyright 2018, J. Galligan, et al.



As MGO is a byproduct of glucose metabolism, its levels may be elevated in diabetes. Indeed, data from human patients suggest a correlation between elevated MGO levels and diabetic nephropathy, one of the major complications of diabetes that can lead to serious morbidity or death. A link between MGO and diabetic nephropathy is further supported by the finding that the severity of the disease increases as levels of GLO1 decrease. MGO concentrations are also elevated in other diseases, including cancer, cardiovascular disease, and renal failure. Clearly, the ability of MGO to form adducts in histones, potentially altering chromatin function, may be an important mechanism by which this ubiquitous electrophile exerts toxic effects. Future research will address this important question.



View PNAS article: Methylglyoxal-derived posttranslational arginine modifications are abundant histone marks









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