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Practice using J. Am. Chem. Soc. 2018, 140, 4757-4760\cite{Bos_2018}

A Chemical Probe for Protein Crotonylation

Abstract: Protein lysine crotonylation has emerged as an important post-translational modification (PTM) in the regulation of gene transcription through epigenetic mechanism. Here, we introduce a chemical probe, based on a water-soluble phosphine warhead, which reacts with the crotonyl modification. We show that this reagent is complementary to antibody-based tools allowing detection of endogenous cellular proteins such as histones carrying the crotonylation PTM. The tool is also used to show the histone acylation activity of the transcriptional coactivator, p300, can be activated by pre-existing lysine crotonylation through a positive feedback mechanism. This reagent provides a versatile and sensitive probe for the analysis of this PTM.
Eukaryotic chromosomes are decorated with a diverse set of histone PTMs that function to regulate access to genomic DNA and that, as a consequence, are critical for the establishment and maintenance of cellular identity. An abundant PTM found on histones involves acetylation of e-amino group of lysine side-chains (Kac). This archetypal "epigenetic mark" acts a positive regulator of gene transcription and functions either by directly altering chromatin structure, or through the recruitment of a myriad of trans-acting nuclear factors. Recent advances in mass spectrometry have revealed several other nonacetyl histone acetylation, classified as sort chain Lys acylations. Thiese include propionylation butyrylation, b-hydroxybutyrylation and crotonylation. Among this group, crotonylation has received the most attention since its initial discovery; it is found on all histones (H2A, H2B, H3, H4 and H1)and functions as a strong transcriptional activator. Intriguingly, the level of histone crotonylation appear to mediate a crosstalk between the metabolic status of the cell, i.e. crotonyl-CoA levels, and gene expression. Moreover, recent proteomics studies indicate that treatment of cells with sodium crotonate, which elevates crotonyl-CoA levels, leads to the crotonylation of numerous nonhistone nuclear proteins. Many questions remain regarding the functional implications of these protein crotonylation events. 
The crotonyl group is unique among known short-chain lysine acylations in that it contains an \(\alpha,\beta\)-unsaturated functionality. In the context of histones, this feature has been shown to mediate the interaction with protein "reader" domains through either a \(\pi-\pi-\pi\) stacking mode, or a distinct hydrophobic encapsulation and hydrogen bonding mechanism. The unique chemical structure of the Kcr mark, in principle, endows it with the ability to undergo conjugate addition reactions with suitable nucleophiles. However, the very fact that thte PTM exists argues that is largely unreactive to the common nucleophiles found in the cell, for example abundant thiol-containing modelcules such as glutathione, Here we show that the crotonyl mark is highly reactive toward phosphine nucleophiles containing a pendent carboxylic acid group. Based on this observation, we go on to develop a covalent chemoproteomic probe for the detection and functional analysis of protein crotonylation. This simple reagent provides a verstaile and cost-effective complement to antibody-based tools for the analysis of this PTM. 
In the course of our investigations into the chemical synrthesis of crotonylated proteins, we observed a major side reaction between crotonylated lysine residues and tris(2-carboxyethyl)phosphine (TCEP, 1a), a commonly used water-soluble reducing agent. An adduct of ~251 Da was observed that was attributed to the covalent addition of TCEP to crotonylated lysine residues. We note that a similar addition has been observed between TCEP and alkyne and acrylamide containing biomolecules, with the latter developed into a bioconjugation reaction. To further investigate the ability of TCEP to covalently add to crotonylated lysine residues in aqueous solution, a series of model compounds, 2a-d, were prepared and used in a set exploratory reactions. Treatment of 2a with TCEP at pH 8 led to clean conversion into a single product (3a) whoes mass was consistent with conjugate addition of TCEP to the crotonyl group. This assignment was confirmed by a series NMR studies using model compound 2b, which indicated the generation of an apparent \(\beta\)-phosphnium species (3b) during the course of the reaction with concomitant loss of the olefin signals from the crotonyl group. Other studies using model peptide 2c revealed that the reaction proceeded smoothly over the pH range 6-10 and was tolerant to a range of aditives commonly used in protein biochemistry, for example chaotropes and detergents. Kinetic studies indicated that the reaction at pH 8 proceeded with an associated second order rate constant of 6 x 10-4 M-1 s-1. In keeping with a conjugate addition type process, we observed no reaction between TCEP and control compound 2d, containing the 3-butenoyl acyl group. Moreover, the oxidized version of TCEP, 1b, was unreactive toward the crotonyl group. 
We were curious as to the driving force for this phosphine addition reaction. Phosphines are commonly used in organic chemistry as catalysts in conjugate addition reactions where they generate a \(\beta\)-phosphonium enolate species that acts as a strong base to deprotonate pronucleophiles. Indeed trimethylphosphine has been shown to catalyze the reaction of water with methyl vinyl ketones. It is thus somewhat surprising that \(\beta\)-phosphonium cation would be generated as the final product in the aqueous reaction between TCEP and the acyl crotonyl moiety. Consequenctly, we wondered whether there was something special about the TCEP structure that might account for this. With this in mind, we treated model compound 2a with several other water-soluble phosphines. In no case did we observe any reaction. Note that prolonged exposure of 2a to high concentration of reduced glutathione did lead to trace amount of an adduct. A unique feature of TCEP compared to the other unreactive phosphines tested is the presence of the three pendent carboxylate groups: these render the molecular water-soluble, but also influence the pKa of the phosphorus. In a key experiment,. we found that the previously described tris-methyl ester analog of TCEP, 1x, was completely unreactive with compound 2a. This was unexpected since, 1c, acts a nucleophilic reducing agent over an extended pH range, and is in fact more reactive than TCEP itself at acidic pH. Notably, during the course of the incubation at pH 8 we observed slow hydrolysis of 1c to give the mono- and bis-methyl ester species. Unlike the parent triester compound, these hydrolysis product reacted with 2a to give the corresponding phosphnium addducts. Collectively, these observations indicate that the presence of a free carboxylate group within the nucleophilic phosphine reagent facilitates the addition reaction. Concievably, the negatively charged barboxylated group might sabilize the developing phosphonium cation through purely electrostatic means. However, other explanations are certainly possible, or example H-bonding interactions or even the involvement of a transient cyclic phosphorane species.
The observation that mono- and bis-ester derivatives of TCEP react with the crotonyl group suggested a strategy or the development of a chemical pro be for detecting protein crotonylation. Specifically, we introduced a biotin moiety onto one of the arms of TCEP to give probe compound 4. Modelstudies indicated that this modification did not significantly impact reactivity with the crotonyl group. We imaginged that this molecule would allow for both the enrichment and detection of crotonylated proteins using standard avidin/streptavidin based affinity reagents. As an initial test of this idea, we generated semisynthetic mono nucleosomes containing either a single Kcr mark at position 18 on histone H3 or several Kcr marks on the tail of H4. Treatmnet o these mononucleosomes with probe 4 led to robust incorporation of biotin into the crotonylated histone, as read out by streptavidin blotting. Detection of the biotin signal was observed after as little as 1 h reaction at 37'c and was dependent upon the presence o he reactive phosphine warhead. Importnatly, no biotin was incorporated into unm odified nucleosomes, or nucleosomes with preinstalled Kac marks, even after overnight incubation. To furtyher validate the chemical probe indeed reacts with the crotonylated lysine residue , H3K18Cr mononucleosome were treated with the probe and subjected to ArgC digestion followed by LC-MS/MS analysis. This confirmed that the probe reacted with the preinstalled crotonyl unit within the H3K18Cr protein. Thus, probe 4 is able to detect crotonylation on purified histones. 
Next, we  asked whether probe 4 could be used to analyze crotonylated proteins within a complex proteome., As an initial step, we showed that our probe could detect semisynthetic H3K18Cr spiked into an Escherichia coli lysates at nanomolar concentrations. Moreover, we were able to affinity capture the crotonylated protein, but not an unmodified control, from this lysate using streptavidin beads. Encouraged by these results, we moved on to the detection of crotonylated proteins in mammalian cells. Indeed, we were able to detect endogenous crotonylated histone protein in HEK293 cells using our probe. Morever, treatment of these cells with sodium corotonate, which as noted earlier up-regulateds crotonyl-CoA levels, led to a substantial increase in the number of proteins reactive toward our probe. 
We were also keen to explore the utility of our probe for studying the biochemistry surrounding histone crotonylation. Specifically, we asked whether the histone acyltransferase activity of the transcriptional coactivator, p300, which is known to crotonylate histone, is sensitive to the presence of pre-existing crotonylation marks on nucleosome. This was motivated by previous tudies that revealed a positive feedback mechanism for histone acetylation by p300. Accordingly, we generated 12-mer nucleosome arrays containing either unmodified histones, histones crotonylated on H3, or histones acetylated on H4. These chromatin substrates were then treated with recombiant full-length p300 supplemented with crotonyl-CoA. Analysis of the reaction mixtures using our probe revealed that the crotonlyated and acetylated arrays were superior substrates compared to unmodified chromatin, as indicated by the level of de novo crotonylation of all histones. Analoguous experiments were performed using acetyl-CoA as the cofactor, revealing that p300-mediated acetylation of the histone is stimulated by the presence of both Kcr and Kac marks on H4. Thus, there appears to be a cross-stimulation of p300 acetyltransfeerase activity by pre-existing Kcr or Kac marks on the chromatin substrate. Additional studies will be needed to acertain whether this stimulation operates through a simple enzyme recruitment mechanims, or involves a more complex allosteric mode of activation.
Finally, in the course of our investigations we noticed that the pan anti-Kcr antibody cross-reacted with acetylated nucleosomes. In principle, this cross-reactivity could complicate biochemical or proteomics studies relying on this reagent. We reasoned that treatment of crotonylated histones, but not their acetylated counterparts, with TCEP should selectively mask the "mark" from antibody detection, due to the drastic changes in steric bulk and charge that accoumpanies the reaction. Indeed, treatment of crotonylated nucleosomes with TCEP blocked the recognition of the crotonyl mark by the anti-Kcr antibody, wherease the ability of the pan anti-Kac antibody to recognize acetylated nucleosomes was unaffected by TCEP treatment. Thus TCEP can be used in simple control reaction to verify that signals obtained from immunoblotting originated from the crotonylation mark. Based on this idea, it should be possible to selectively elute crotonylated proteins following immunoprecipitation of cellular extract using anti-Kcr antiboies. Thus, we imaging that the TCEP-crotonyl reaction will prove useful in the context of existing antibody-based proteomics workflows. 
The crotonyl group bears a unique structure among known protein PTMs in that it contains an \(\alpha,\beta\)-unsaturated functionality in conjugation with a carbonyl group. Here we exploit the serendipitous discovery that this PTM undergoes a conjugate addition reaction with the reducing agent TCEP to give a stable adduct. This unexpected reactivity of TCEP was traced to the presence of its pendent carboxylate groups, which we speculate stabilize the phosphonium species in the product. On the basis of this insight, we developed a sensitive and selective chemical probe of protein crotonylation and desmonstrate its utility in the context of protemics efforts and mechanismtic biochemical studies. The new probe complenents existing antibody-based tools for studying crotonylation. For example, the ability to covalently attach a biotin moiety to the modified protein would allow for the adoption of regourous enrichement protocols within proteomics workflows employing readily available streptavidin reagents. Moreover, the chemical probe is unlikely to be sensitive to the local sequence or TPM environment around the crotonyl mark, and is thus expected to be less susceptible to epitope occlusion effect compared to antibodies. Thus, we postulate that our chemical probe will find utility in the study of this important PTM.