Getting the Right Grip? How Understanding Electrophile Selectivity Profiles Could Illuminate Our Understanding of Redox Signaling

Abstract

Significance: Electrophile signaling is coming into focus as a bona fide cell signaling mechanism. The electrophilic regulation occurs typically through a sensing event (i.e., labeling of a protein) and a signaling event (the labeling event having an effect of the proteins activity, association, etc.). Recent Advances: Herein, we focus on the first step of this process, electrophile sensing. Electrophile sensing is typically a deceptively simple reaction between the thiol of a protein cysteine, of which there are around 200,000 in the human proteome, and a Michael acceptor, of which there are numerous flavors, including enals and enones. Recent data overall paint a picture that despite being a simple chemical reaction, electrophile sensing is a discerning process, showing labeling preferences that are often not in line with reactivity of the electrophile. Critical Issues: With a view to trying to decide what brings about highly electrophile-reactive protein cysteines, and how reactive these sensors may be, we discuss aspects of the thermodynamics and kinetics of covalent/noncovalent binding. Data made available by several laboratories indicate that it is likely that specific proteins exhibit highly stereo- and chemoselective electrophile sensing, which we take as good evidence for recognition between the electrophile and the protein before forming a covalent bond. Future Directions: We propose experiments that could help us gain a better and more quantitative understanding of the mechanisms through which sensing comes about. We further extoll the importance of performing more detailed experiments on labeling and trying to standardize the way we assess protein-specific electrophile sensing.

Keywords: affinity; covalent labeling; electrophile signaling; kinetic control; mechanism; stereoselectivity.

FIG. 1.

Comparison of covalent bond formation and noncovalent binding in terms of approximate free energy (ΔG) or enthalpy (ΔH) of binding at ambient temperature (RT = 8.314 × 298). For nitromethane coupling with cinnamaldehyde, an approximate average of all four calculated ΔG values in the cited literature (52) was presented; and for amine conjugate addition to parthenolides, the value shown is an approximate mean of 3 different six-membered-cyclic amines (62).

FIG. 2.

Thermodynamics and kinetics of ligand–target interactions. (A) Change in ΔG as a function of K_d; color-coded from dark to light gray corresponding to: metal-enzyme interactions, covalent binding, high-affinity binders such as biotin, highest-affinity inhibitors such as Immucillin H, and finally down to lower-affinity binders. (B) Half-life of bimolecular reactions (e.g., covalent bond formation) versus rate constant for different concentrations of reagents, assuming equal concentrations between the two reacting species. Five concentrations are chosen arbitrarily to represent high (20 μM) and low (0.05 μM) concentrations of reagent and protein (40). The arrow beside the y-axis denotes an estimate of the lifetime of HNE in cells. The arrow below the x-axis refers to the lower limit threshold of second-order rate constants considered to lead to biologically relevant enzyme-catalyzed reactions. Fully dark shapes indicate a likely biologically relevant signaling process (half-life <10 min); half-dark, half-gray shapes indicate a possibly biologically relevant signaling process (half-life between 10 and 80 min); and gray shapes indicate a likely nonbiologically relevant signaling process (half-life >80 min). HNE, 4-hydroxynonenal.

FIG. 3.

Conformational flexibilities of select LDEs and divergent targets reported for HNE and 15d-PG J2. (A) Chemical structures of HNE, ONE, 15d-PG J2, and acrolein. Relevant s-trans or s-cis conformations are highlighted in red and purple, respectively. Shaded regions highlight unique structural features of these molecules discussed in the review. (B) Data available in the literature (56) are plotted using GraphPad Prism8. Human MDA-MB-231 proteome was treated with 100 μM HNE or 15d-PG J2. Reactivity of cysteines in proteome was studied with isoTOP-ABPP platform. Cysteines with R values (isoTOP-ABPP ratios) greater than 5 were considered to be highly sensitive to compound added. In both graphs, the resulting hits from experiments using HNE versus 15d-PG J2 are labeled, respectively, in blue and red. 15d-PG J2, 15-deoxy-delta(12,14)-prostaglandin J2; isoTOP-ABPP, isotopic tandem orthogonal proteolysis–activity-based protein profiling; LDE, lipid-derived electrophiles; ONE, 4-oxononenal. Color images are available online.

FIG. 4.

Representative indirect and direct profiling of LDE-modified proteins. (A) Competitive isoTOP-ABPP platform. The proteomes are treated with either an LDE or DMSO and then treated with IA-alkyne, which serves as a proxy electrophilic probe (56). The cysteines reactive to LDE are blocked from IA-alkyne labeling (figure drawn assuming that IA-alkyne also reacts faithfully with all LDE-reactive cysteines—see: DMSO-treated samples), so they are “lost” in the LDE-treated proteome compared with DMSO-treated proteome. (Note: figure further reflects the fact that not all remaining cysteines that have not been modified by LDE react with IA-alkyne—see: LDE-treated samples.) The IA-alkyne labeled proteins are conjugated to isotopically labeled TEV-cleavable biotin tag, and proteomes labeled with light and heavy isotopes are mixed in a 1:1 ratio. After enrichment with streptavidin beads and on-bead trypsin digestion, the probe-labeled peptides are quantified by LC-MS/MS analysis. (B) A quantitative protein carbonylation profiling method using aniline-derived probe m-APA (10). Proteome of interest is treated with m-APA. LDE-modified proteins (where carbonyl groups are still retained) can be labeled with m-APA and conjugated to acid-cleavable biotin tags. The labeled proteins are enriched with streptavidin pulldown, followed by trypsin digestion and acid cleavage. Target profiling is performed using LC-MS/MS analysis. DMSO, dimethyl sulfoxide; IA-alkyne, iodoacetamide-alkyne; LC-MS/MS, liquid chromatography–tandem mass spectrometry; LDE, lipid-derived electrophile; m-APA, m-aminophenylacetylene; TEV, tobacco etch virus protease.

FIG. 5.

T-REX method interrogates individual protein- and LDE-chemotype-specific cellular response. (A) T-REX. Cells expressing HaloTagged-POI are treated with a photocaged precursor of LDE of interest, housing a HaloTag-targeting motif (gray triangle) that stoichiometrically and irreversibly binds to HaloTag, and unbound precursors are washed out. Light exposure releases LDE within the proximity of POI. If POI is a privileged sensor of the LDE, the POI will be labeled specifically. Downstream signaling events triggered by labeling of POI by the LDE, as well as the identity of modified residue(s) and LDE-ligand occupancy can be analyzed (39, 42, 54). (B) Chemical structures of T-REX photocaged precursor of LDEs, and two representative LDEs (HNE and CHE) (27). CHE, 6-(prop-2-yn-1-yl)cyclohex-2-en-1-one; POI, protein of interest; T-REX, targetable reactive electrophiles and oxidants.

FIG. 6.

Sensing sites on Keap1 vary under different experimental conditions, although T-REX faithfully labels specific sites regardless of N- or C-terminus Halo fusion and shows chemotype-specific changes of sensing site within Keap1; Akt-isoform specific sensing arises from a single cysteine residue within Akt3 linker region. (A) Domain structure of human Keap1 protein. Cysteines detected to be modified by HNE or CHE under indicated conditions are illustrated (27, 38). (B) Domain structure of human Akt1, Akt2, and Akt3. The amino acid sequences of the linker region are shown. Akt2(C124) (bolded and underlined) is a peroxide-sensing residue (57). Akt3(C119) (bolded and underlined) is an HNE-sensing residue (31, 32). Domain structures were generated with software DOG 2.0 (45).

FIG. 7.

Comparison between external administration of reactive molecules and in situ release in G-REX method (61).