Cysteine-reactive ligand discovery in proteomes

Abstract

Cells produce electrophilic products with the potential to modify and affect the function of proteins. Chemoproteomic methods have provided a means to qualitatively inventory proteins targeted by endogenous electrophiles; however, ascertaining the potency and specificity of these reactions to identify the most sensitive sites in the proteome to electrophilic modification requires more quantitative methods. Here, we describe a competitive activity-based profiling method for quantifying the reactivity of electrophilic compounds against 1000+ cysteines in parallel in the human proteome. Using this approach, we identify a select set of proteins that constitute “hot spots” for modification by various lipid-derived electrophiles, including the oxidative stress product 4-hydroxynonenal (HNE). We show that one of these proteins, ZAK kinase, is labeled by HNE on a conserved, active site-proximal cysteine, resulting in enzyme inhibition to create a negative feedback mechanism that can suppress the activation of JNK pathways by oxidative stress.

1 - 15 . (canceled) 
     
     
         16 . A protein identified as a target of a lipid-derived electrophile by the method comprising:
 (a) contacting a first set of proteins of a mammalian cell with the lipid-derived electrophile, wherein the lipid-derived electrophile is a cysteine-reactive lipid-derived electrophile to generate an alkylated set of proteins;   (b) contacting the alkylated set of proteins with an alkynylated iodoacetamide probe, followed by reaction with an azido compound comprising a first isotopic marker, to provide an isotopically-marked alkylated set of proteins, wherein the reaction with the azido compound is carried out using a copper-catalyzed azide-alkyne cycloaddition reaction;   (c) contacting the first set of proteins of a mammalian cell of step (a), not exposed to the lipid-derived electrophile, with an alkynylated iodoacetamide probe, followed by reaction with an azido compound comprising a second isotopic marker, to provide an isotopically-marked control set of proteins, wherein the reaction with the azido compound is carried out using a copper-catalyzed azide-alkyne cycloaddition reaction;   (d) combining the isotopically-marked alkylated set of proteins and the isotopically-marked control set of proteins to provide a combined sample;   (e) quantifying reactivities of the isotopically-marked alkylated set of proteins and the isotopically-marked control set of proteins to identify a protein target by comparing the abundance of the first isotopic marker and the abundance of the second isotopic marker for each protein of the combined sample; and   (g) identifying the protein target and sites of modification of the electrophile.   
     
     
         17 . The protein of  claim 16 , wherein the first set of proteins includes two or more proteins. 
     
     
         18 . The protein of  claim 16 , wherein the lipid-derived electrophile is a stress-induced electrophile. 
     
     
         19 . The protein of  claim 16 , wherein lipid-derived electrophile comprises a Michael acceptor having an α,β-unsaturated carbonyl group. 
     
     
         20 . The protein of  claim 16 , wherein the lipid-derived electrophile undergoes a Michael conjugate addition reaction with a cysteine residue of one or more proteins of the first set of proteins. 
     
     
         21 . The protein of  claim 16 , wherein the lipid-derived electrophile is 4-hydroxynonenal (HNE) or 15-deoxy-Al 2,14-prostaglandin J2 (15d-PGJ2). 
     
     
         22 . The protein of  claim 16 , wherein the first isotopic marker and the second isotopic marker are isotopically-differentiated azide-biotin tags. 
     
     
         23 . The protein of  claim 16  wherein the protein target possess at least 5-fold or higher ratio of the second isotopic marker to the first isotopic marker among the proteins of the combined sample. 
     
     
         24 . The protein of  claim 16  wherein the site of cysteine modification by a lipid-derived electrophile is at a non-active site. 
     
     
         25 . The protein of  claim 16  wherein the protein is a kinase. 
     
     
         26 . The protein of  claim 25 , wherein the kinase is ZAK kinase.