Non-enzymatic Covalent Modifications Research Articles

Covalent adduct formation of histone with organophosphorus pesticides in vitro

It is established that organophosphorus pesticide (OPP) toxicity results from modification of amino acids in active sites of target proteins. OPPs can also modify unrelated target proteins such as histones and such covalent histone modifications can alter DNA-binding properties and lead to aberrant gene expression. In the present study, we report on non-enzymatic covalent modifications of calf thymus histones adducted to selected OPPs and organophosphate flame retardants (OPFRs) in vitro using a bottom-up proteomics method approach. Histones were not found to form detectable adducts with the two tested OPFRs but were avidly modified by a few of the seven OPPs that were tested in vitro. Dimethyl phosphate (or diethyl phosphate) adducts were identified on Tyr, Lys and Ser residues. Most of the dialkyl phosphate adducts were identified on Tyr residues. Methyl and ethyl modified histones were also detected. Eleven amino residues in histones showed non-enzymatic covalent methylation by exposure of dichlorvos and malathion. Our bottom-up proteomics approach showing histone-OPP adduct formation warrants future studies on the underlying mechanism of chronic illness from exposure to OPPs.

Histone Deacetylase 1 Inhibition by Peptides Containing a DNA Damage-Induced, Nonenzymatic, Histone Covalent Modification.

Treatment of HeLa cells with the DNA damaging agent, bleomycin (BLM), results in the formation of a nonenzymatic 5-methylene-2-pyrrolone histone covalent modification on lysine residues (KMP). KMP is much more electrophilic than other N-acyllysine covalent modifications and post-translational modifications, including N-acetyllysine (KAc). Using histone peptides containing KMP, we show that this modification inhibits the class I histone deacetylase, HDAC1, by reacting with a conserved cysteine (C261) located near the active site. HDAC1 is inhibited by histone peptides whose corresponding N-acetylated sequences are known deacetylation substrates, but not one containing a scrambled sequence. The HDAC1 inhibitor, trichostatin A, competes with covalent modification by the KMP-containing peptides. HDAC1 is also covalently modified by a KMP-containing peptide in a complex milieu. These data indicate that peptides containing KMP are recognized by HDAC1 and are bound in the active site. The effects on HDAC1 indicate that KMP formation in cells may contribute to the biological effects of DNA damaging agents, such as BLM, that form this nonenzymatic covalent modification.

Bottom-up proteomics analysis for adduction of the broad-spectrum herbicide atrazine to histone

Histones are the major proteinaceous components of chromatin in eukaryotic cells and an important part of the epigenome. The broad-spectrum herbicide atrazine (2-chloro-4-[ethylamino]-6-[isopropylamino]-1, 3, 5-triazine) and its metabolites are known to form protein adducts, but the formation of atrazine–histone adducts has not been studied. In this study, a bottom-up proteomics analysis method was optimized and applied to identify histone adduction by atrazine in vitro. Whole histones of calf thymus or human histone H3.3 were incubated with atrazine. After solvent-based protein precipitation, the protein was digested by trypsin/Glu-C and the resulting peptides were analyzed by high-resolution mass spectrometry using an ultra-high-performance liquid chromatograph interfaced with a quadrupole Exactive-Orbitrap mass spectrometer. The resulting tryptic/Glu-C peptide of DTNLCAIHAK from calf thymus histone H3.1 or human histone H3.3 was identified with an accurate mass shift of +179.117 Da in atrazine incubated samples. It is deduced that a chemical group with an elemental composition of C8H13N5 (179.1171 Da) from atrazine adducted with calf thymus histone H3.1 or human histone H3.3. It was confirmed by MS/MS analysis that the adduction position was at its cysteine 110 residue. Time- and concentration-dependent assays also confirmed the non-enzymatic covalent modification of histone H3.3 by atrazine in vitro. Thus, the potential exists that atrazine adduction may lead to the alteration of histones that subsequently disturbs their normal function.Graphical abstract

Chemical Labeling and Enrichment of Histone Glyoxal Adducts.

Because of their long half-lives and highly nucleophilic tails, histones are particularly susceptible to accumulating nonenzymatic covalent modifications, such as glycation. The resulting modifications can have profound effects on cellular physiology due to the regulatory role histones play in all DNA-templated processes; however, the complexity of Maillard chemistry on proteins makes tracking and enriching for glycated proteins a challenging task. Here, we characterize glyoxal (GO) modifications on histones using quantitative proteomics and an aniline-derived GO-reactive probe. In addition, we leverage this chemistry to demonstrate that the glycation regulatory proteins DJ-1 and GLO1 reduce levels of histone GO adducts. Finally, we employ a two-round pull-down method to enrich histone H3 GO glycation and map these adducts to specific chromatin regions.

An Azidoribose Probe to Track Ketoamine Adducts in Histone Ribose Glycation.

Reactive cellular metabolites can modify macromolecules and form adducts known as nonenzymatic covalent modifications (NECMs). The dissection of the mechanisms, regulation, and consequences of NECMs, such as glycation, has been challenging due to the complex and often ambiguous nature of the adducts formed. Specific chemical tools are required to directly track the formation of these modifications on key targets in order to uncover their underlying physiological importance. Here, we present the novel chemoenzymatic synthesis of an active azido-modified ribose analog, 5-azidoribose (5-AR), as well as the synthesis of an inactive control derivative, 1-azidoribose (1-AR), and their application toward understanding protein ribose-glycation in vitro and in cellulo. With these new probes we found that, similar to methylglyoxal (MGO) glycation, ribose glycation specifically accumulates on histones. In addition to fluorescent labeling, we demonstrate the utility of the probe in enriching modified targets, which were identified by label-free quantitative proteomics and high-resolution MS/MS workflows. Finally, we establish that the known oncoprotein and hexose deglycase, fructosamine 3-kinase (FN3K), recognizes and facilitates the removal of 5-AR glycation adducts in live cells, supporting the dynamic regulation of ribose glycation as well as validating the probe as a new platform to monitor FN3K activity. Altogether, we demonstrate this probe's utilities to uncover ribose-glycation and deglycation events as well as track FN3K activity toward establishing its potential as a new cancer vulnerability.

Reversible histone glycation is associated with disease-related changes in chromatin architecture

Cellular proteins continuously undergo non-enzymatic covalent modifications (NECMs) that accumulate under normal physiological conditions and are stimulated by changes in the cellular microenvironment. Glycation, the hallmark of diabetes, is a prevalent NECM associated with an array of pathologies. Histone proteins are particularly susceptible to NECMs due to their long half-lives and nucleophilic disordered tails that undergo extensive regulatory modifications; however, histone NECMs remain poorly understood. Here we perform a detailed analysis of histone glycation in vitro and in vivo and find it has global ramifications on histone enzymatic PTMs, the assembly and stability of nucleosomes, and chromatin architecture. Importantly, we identify a physiologic regulation mechanism, the enzyme DJ-1, which functions as a potent histone deglycase. Finally, we detect intense histone glycation and DJ-1 overexpression in breast cancer tumors. Collectively, our results suggest an additional mechanism for cellular metabolic damage through epigenetic perturbation, with implications in pathogenesis.

(De)Toxifying the Epigenetic Code.

Cells are continuously subjected to an array of reactive/toxic chemical species which are produced both endogenously through metabolic pathways and taken up exogenously by diet and exposure to drugs or toxins. As a result, proteins often undergo non-enzymatic covalent modifications (NECMs) by these species, which can alter protein structure, function, stability, and binding partner affinity. NECMs accumulate over time and are linked to various diseases such as Alzheimer's disease, cancer, and diabetes. In the cellular proteome, histones have some of the longest half-lives, making them prime targets for NECMs. In addition, histones have emerged as key regulators of transcription, a function that is primarily controlled by modificationoftheir tails. These modifications are usually installed or removed enzymatically, but recent evidence suggests that some may also occur non-enzymatically. Despite the vast knowledge detailing the relationship between histone modifications and gene regulation, NECMs on histones remain poorly explored. A major reason for this difference stems from the fact that, unlike their enzymatically installed counterparts, NECMs are difficult to both control and test in vivo. Here, we review advances in our understanding of the effect non-enzymatic covalent modifications (NECMs) have on the epigenetic landscape, cellular fate, and their implications in disease. Cumulatively, this illustrates how the epigenetic code is directly toxified by chemicals and detoxified by corresponding eraser enzymes.

Mass spectrometry profiling of non-enzymatic deamidation of articular cartilage components suggests slower protein turnover in deep regions and in hips compared with knees

Mass spectrometry profiling of non-enzymatic deamidation of articular cartilage components suggests slower protein turnover in deep regions and in hips compared with knees

Identification and relative quantification of specific glycation sites in human serum albumin

Glycation (or non-enzymatic glycosylation) is a common non-enzymatic covalent modification of human proteins. Glucose, the highest concentrated monosaccharide in blood, can reversibly react with amino groups of proteins to form Schiff bases that can rearrange to form relatively stable Amadori products. These can be further oxidized to advanced glycation end products (AGEs). Here, we analyzed the glycation patterns of human serum albumin (HSA) in plasma samples obtained from five patients with type 2 diabetes mellitus. Therefore, glycated peptides from a tryptic digest of plasma were enriched with m-aminophenylboronic acid (mAPBA) affinity chromatography. The glycated peptides were then further separated in the second dimension by RP-HPLC coupled on-line to an electrospray ionization (ESI) tandem mass spectrometer (MS/MS). Altogether, 18 Amadori peptides, encompassing 40% of the HSA sequence, were identified. The majority of the peptides were detected and relatively quantified in all five samples with a high reproducibility among the replicas. Eleven Lys-residues were glycated at similar quantities in all samples, with glycation site Lys(549) (K(Am(Glc))QTALVELVK) being the most abundant. In conclusion, the established mAPBA/nanoRP-HPLC-ESI-MS/MS approach could reproducibly identify and quantify glycation sites in plasma samples, potentially useful in diagnosis and therapeutic control.

Non-enzymatic covalent modifications of proteins: mechanisms, physiological consequences and clinical applications.

Given the complexity of the biosynthetic machinery and the delicate chemical composition of proteins, it is remarkable that cells manage to produce and maintain normally functioning proteins under most conditions. However, it is now well known that proteins are susceptible to various non-enzymatic covalent modifications (NECM) under physiological conditions. Such modifications can be of no or little importance to the protein or they can be absolutely detrimental. Often NECM are difficult to study due to the complex and technically demanding methods required to identify many of these modifications. Thus, the role of NECM has not yet been adequately resolved but recent research has allowed a better understanding of such modifications. The present review outlines the various forms of NECM that involve covalent modifications of proteins, and discusses their relevance, biological impact and potential applications in the study of protein turnover and diagnosis of disease.

Diabetes, Oxidative Stress, and Platelet Activation

Diabetes mellitus is a major source of morbidity in developed countries and, among its comorbid conditions, atherosclerosis is perhaps the most important. Since the availability of insulin, up to three fourths of all deaths among diabetics can be directly attributed to coronary artery disease (CAD).1 In adult patients with diabetes, the risk of CAD is 3- to 5-fold greater in than nondiabetics despite controlling for other known CAD risk factors.1 In patients with IDDM, up to one third will die of CAD by the age of 50 years.2 A number of known risk factors for CAD, such as hypertension, central obesity, and dyslipidemia, are more common in diabetics than in the general population.1 Despite this prevalence of risk factors, no more than 25% of the excess CAD risk in diabetes can be accounted for by known risk factors.2 Thus diabetes represents a major contributing factor to the CAD burden in the developed world, and most of the excess attributed risk of CAD in diabetics cannot be readily quantified with the use of traditional risk factor analysis. Diabetes is associated with a variety of metabolic abnormalities, principle among which is hyperglycemia. The relation between hyperglycemia and CAD is the subject of considerable debate because serum glucose does not consistently predict the existence of CAD.2 Presumably, this confusion stems from the reliance on a single blood glucose measurement, as recent prospective data have clearly established a link between a marker for chronic average glucose levels (HbA1C) and cardiovascular morbidity and mortality.3 There is considerable controversy with respect to the precise mechanism by which hyperglycemia may contribute to the development of CAD in diabetes. Established sequelae of hyperglycemia, such as cytotoxicity, increased extracellular matrix production, and vascular dysfunction, have all been implicated in the pathogenesis …