Glutathionylation involves reversible protein cysteine modification that regulates the function of numerous proteins in response to redox stimuli, thereby altering cellular processes. Herein we developed a selective and versatile approach to identifying glutathionylation by using a mutant of glutathione synthetase (GS). GS wild-type catalyzes coupling of γGlu-Cys to Gly to form glutathione. We generated a GS mutant that catalyzes azido-Ala in place of Gly with high catalytic efficiency and selectivity. Transfection of this GS mutant (F152A/S151G) and incubation of azido-Ala in cells efficiently afford the azide-containing glutathione derivative, γGlu-Cys-azido-Ala. Upon H2O2 treatment, clickable glutathione allowed for selective and sensitive detection of glutathionylated proteins by Western blotting or fluorescence after click reaction with biotin-alkyne or rhodamine-alkyne. This approach affords the efficient metabolic tagging of intracellular glutathione with small clickable functionality, providing a versatile handle for characterizing glutathionylation.

Background: Lipolysis contributes to adipose inflammation. Results: Lipolysis up-regulates sphingosine kinase 1 (SphK1) in adipocytes. Modulation of SphK1 regulates adipose lipolysisstimulated interleukin 6 production. Conclusion: SphK1 plays a pivotal role in adipose inflammation. Significance: Identification of a role for SphK1 in lipolysis-triggered adipose inflammation. Targeting SphK1 provides a novel intervention for adipose inflammation and associated metabolic syndromes.

Reactive oxygen species (ROS) are important signaling molecules, but their overproduction is associated with many cardiovascular diseases, including cardiomyopathy. ROS induce various oxidative modifications, among which glutathionylation is one of the significant protein oxidations that occur under oxidative stress. Despite previous efforts, direct and site-specific identification of glutathionylated proteins in cardiomyocytes has been limited. In this report, we used a clickable glutathione approach in a HL-1 mouse cardiomyocyte cell line under exposure to hydrogen peroxide, finding 1,763 glutathionylated peptides with specific Cys modification sites, which include many muscle-specific proteins. Bioinformatic and cluster analyses found 125 glutathionylated proteins, whose mutations or dysfunctions are associated with cardiomyopathy, many of which include sarcomeric structural and contractile proteins, chaperone, and other signaling or regulatory proteins. We further provide functional implication of glutathionylation for several identified proteins, including CSRP3/MLP and complex I, II, and III, by analyzing glutathionylated sites in their structures. Our report establishes a chemoselective method for direct identification of glutathionylated proteins and provides potential target proteins whose glutathionylation may contribute to muscle diseases.

Protein S‐glutathionylation is one of the important cysteine oxidation events that regulate various redox‐mediated biological processes. Despite several existing methods, there are few proteomic approaches to identify and quantify specific cysteine residues susceptible to S‐glutathionylation. We previously developed a clickable glutathione approach that labels intracellular glutathione with azido‐Ala by using a mutant form of glutathione synthetase. In this study, we developed a quantification strategy with clickable glutathione by using isotopically labeled heavy and light derivatives of azido‐Ala, which provides the relative quantification of glutathionylated peptides in mass spectrometry‐based proteomic analysis. We applied isotopically labeled clickable glutathione to HL‐1 cardiomyocytes, quantifying relative levels of 1398 glutathionylated peptides upon addition of hydrogen peroxide. Importantly, we highlight elevated levels of glutathionylation on sarcomere‐associated muscle proteins while validating glutathionylation of two structural proteins, α‐actinin and desmin. Our report provides a chemical proteomic strategy to quantify specific glutathionylated cysteines.

Excessive adipocyte lipolysis generates lipid mediators and triggers inflammation in adipose tissue. However, the specific roles of lipolysis-generated mediators in adipose inflammation remain to be elucidated. In the present study, cultured 3T3-L1 adipocytes were treated with isoproterenol to activate lipolysis and the fatty acyl lipidome of released lipids was determined by using LC-MS/MS. We observed that ␤-adrenergic activation elevated levels of approximately fifty lipid species, including metabolites of cyclooxygenases, lipoxygenases, epoxygenases, and other sources. Moreover, we found that ␤-adrenergic activation induced cyclooxygenase 2 (COX-2), not COX-1, expression in a manner that depended on activation of hormone-sensitive lipase (HSL) in cultured adipocytes and in the epididymal white adipose tissue (EWAT) of C57BL/6 mice. We found that lipolysis activates the JNK/NFB signaling pathway and inhibition of the JNK/NFB axis abrogated the lipolysis-stimulated COX-2 expression. In addition, pharmacological inhibition of COX-2 activity diminished levels of COX-2 metabolites during lipolytic activation. Inhibition of COX-2 abrogated the induction of CCL2/MCP-1 expression by ␤-adrenergic activation and prevented recruitment of macrophage/monocyte to adipose tissue. Collectively, our data indicate that excessive adipocyte lipolysis activates the JNK/NFB pathway leading to the up-regulation of COX-2 expression and recruitment of inflammatory macrophages.