ABSTRACT
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. Herein we describe the existence of Lys and Arg modifications on histones by a 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 marked disruption of H2B acetylation and ubiquitylation without affecting H2A, H3, and H4 modifications. Using RNA sequencing, we show that MGO is capable of altering gene transcription, most notably in cells lacking GLO1. Finally, we show that the deglycase DJ-1 protects histones from adduction by MGO. Collectively, our findings demonstrate the existence of a previously undetected histone modification derived from glycolysis, which may have far-reaching implications for the control of gene expression and protein transcription linked to metabolism.
Subject(s)
Arginine/metabolism , Histones/metabolism , Lactoylglutathione Lyase/metabolism , Protein Processing, Post-Translational/drug effects , Pyruvaldehyde , Transcription, Genetic/drug effects , HEK293 Cells , Humans , Pyruvaldehyde/metabolism , Pyruvaldehyde/pharmacologyABSTRACT
Lysophospholipids (LysoPLs) are bioactive lipid species involved in cellular signaling processes and the regulation of cell membrane structure. LysoPLs are metabolized through the action of lysophospholipases, including lysophospholipase A1 (LYPLA1) and lysophospholipase A2 (LYPLA2). A new X-ray crystal structure of LYPLA2 compared with a previously published structure of LYPLA1 demonstrated near-identical folding of the two enzymes; however, LYPLA1 and LYPLA2 have displayed distinct substrate specificities in recombinant enzyme assays. To determine how these in vitro substrate preferences translate into a relevant cellular setting and better understand the enzymes' role in LysoPL metabolism, CRISPR-Cas9 technology was utilized to generate stable KOs of Lypla1 and/or Lypla2 in Neuro2a cells. Using these cellular models in combination with a targeted lipidomics approach, LysoPL levels were quantified and compared between cell lines to determine the effect of losing lysophospholipase activity on lipid metabolism. This work suggests that LYPLA1 and LYPLA2 are each able to account for the loss of the other to maintain lipid homeostasis in cells; however, when both are deleted, LysoPL levels are dramatically increased, causing phenotypic and morphological changes to the cells.
Subject(s)
Homeostasis , Lysophospholipids/metabolism , Signal Transduction , Thiolester Hydrolases/metabolism , Amino Acid Sequence , Cell Differentiation , Cell Line , Gene Knockout Techniques , Humans , Hydrolysis , Models, Molecular , Neurons/cytology , Protein Conformation , Thiolester Hydrolases/chemistry , Thiolester Hydrolases/deficiency , Thiolester Hydrolases/geneticsABSTRACT
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.
Subject(s)
Arginine/analysis , Chromatography, High Pressure Liquid/methods , Histones/chemistry , Lysine/analysis , Peptides/chemistry , Protein Processing, Post-Translational , Tandem Mass Spectrometry/methods , HEK293 Cells , Humans , Hydrolysis , Jumonji Domain-Containing Histone Demethylases/chemistry , Recombinant Proteins/chemistryABSTRACT
Prostaglandin glycerol esters (PG-Gs) are produced as a result of the oxygenation of the endocannabinoid, 2-arachidonoylglycerol, by cyclooxygenase 2. Understanding the role that PG-Gs play in a biological setting has been difficult because of their sensitivity to enzymatic hydrolysis. By comparing PG-G hydrolysis across human cancer cell lines to serine hydrolase activities determined by activity-based protein profiling, we identified lysophospholipase A2 (LYPLA2) as a major enzyme responsible for PG-G hydrolysis. The principal role played by LYPLA2 in PGE2-G hydrolysis was confirmed by siRNA knockdown. Purified recombinant LYPLA2 hydrolyzed PG-Gs in the following order of activity: PGE2-G > PGF2α-G > PGD2-G; LYPLA2 hydrolyzed 1- but not 2-arachidonoylglycerol or arachidonoylethanolamide. Chemical inhibition of LYPLA2 in the mouse macrophage-like cell line, RAW264.7, elicited an increase in PG-G production. Our data indicate that LYPLA2 serves as a major PG-G hydrolase in human cells. Perturbation of this enzyme should enable selective modulation of PG-Gs without alterations in endocannabinoids, thereby providing a means to decipher the unique functions of PG-Gs in biology and disease.
Subject(s)
Gene Expression Regulation, Neoplastic , Glycerol/metabolism , Macrophages/enzymology , Prostaglandins/metabolism , Thiolester Hydrolases/metabolism , Animals , Arachidonic Acids/metabolism , Cell Line, Tumor , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Endocannabinoids/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Esters , Glycerides/metabolism , Humans , Hydrolysis , Kinetics , Macrophages/cytology , Mice , Polyunsaturated Alkamides/metabolism , Prostaglandins/chemistry , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction , Substrate Specificity , Thiolester Hydrolases/antagonists & inhibitors , Thiolester Hydrolases/geneticsABSTRACT
Herein, we report the structure-activity relationship of a chiral morpholine-based scaffold, which led to the identification of a potent and selective dopamine 4 (D4) receptor antagonist. The 4-chlorobenzyl moiety was identified, and the compound was designated an MLPCN probe molecule, ML398. ML398 is potent against the D4 receptor with IC50 = 130 nM and K i = 36 nM and shows no activity against the other dopamine receptors tested (>20 µM against D1, D2S, D2L, D3, and D5). Further in vivo studies showed that ML398 reversed cocaine-induced hyperlocomotion at 10 mg/kg.