Chemoproteomic profiling of targets of lipid-derived electrophiles by bioorthogonal aminooxy probe

Abstract

Redox imbalance in cells induces lipid peroxidation and generates a class of highly reactive metabolites known as lipid-derived electrophiles (LDEs) that can modify proteins and affects their functions. Identifying targets of LDEs is critical to understand how such modifications are functionally implicated in oxidative-stress associated diseases. Here we report a quantitative chemoproteomic method to globally profile protein targets and sites modified by LDEs. In this strategy, we designed and synthesized an alkyne-functionalized aminooxy probe to react with LDE-modified proteins for imaging and proteomic profiling. Using this probe, we successfully quantified >4000 proteins modified by 4-hydroxy-2-nonenal (HNE) of high confidence in mammalian cell lysate and combined with a tandem-orthogonal proteolysis activity-based protein profiling (TOP-ABPP) strategy, we identified ~400 residue sites targeted by HNE including reactive cysteines in peroxiredoxins, an important family of enzymes with anti-oxidant roles. Our method expands the toolbox to quantitatively profile protein targets of endogenous electrophiles and the enlarged inventory of LDE-modified proteins and sites will contribute to functional elucidation of cellular pathways affected by oxidative stress.

Getting the Message? Native Reactive Electrophiles Pass Two Out of Three Thresholds to be Bona Fide Signaling Mediators

“…Recently, a variation on competitive ABPP has emerged that does not rely on a reporter electrophile. This method uses an aminooxy probe to label RES‐modified proteins through the carbonyl moiety remaining post‐RES adduction . One attractive feature of this approach is its expanded scope of electrophile adduct detection: in contrast to “classic” ABPP where reactivity of the reporter electrophile limits detection of hits to cysteine adducts, this method can also detect lysine and histidine adducts.…”

Electrophile Signaling and Emerging Immuno- and Neuro-modulatory Electrophilic Pharmaceuticals

“…Setup in lysates lacks biological context, and analyses are inherently biased toward the detection of high-occupancy LDE modifications. Recent advances in proteomic profiling techniques have attempted to obviate some of the limitations of traditional ABPP through, for instance, elimination of the need for proxy probe treatment (Chen et al, 2017(Chen et al, , 2018.…”

Gsta4 controls apoptosis of differentiating adult oligodendrocytes during homeostasis and remyelination via the mitochondria-associated Fas/Casp8/Bid-axis

“…Staining for 4-HNE in CC revealed the DA Gsta4 to have a reduced load of 4-HNE compared to DA Wt (Fig.3a, Fig.S2a). To identify transcripts potentially being directly affected by Gsta4/4-HNE we analyzed the RNA-seq data of CC derived O4 + OLs and used the following criteria to identify targets: significant differential expression (P=<0.001) between DA Wt and DA Gsta4 (Supplementary Table 2) and, the potential of the corresponding peptide to be modified by 4-8 HNE, proven by high performance liquid chromatography screening 59,60 (Supplementary Table 3). There were a 10-fold increase in transcripts being upregulated in DA Gsta4 OLs compared to DA Wt in naïve state ( Fig.3b left), however there was no difference in the proportion of transcripts of potentially 4-HNE modified peptides (Fig.3b green).…”