Chemical tool to synthesize and investigate glutathionylated proteins

Daniel Oppong

doi:10.17918/00010907

Cellular biological processes are regulated by reactive oxygen species (ROS). On the other hand, oxidative damage to biomolecules caused by elevated ROS levels has been linked to multiple illnesses, including cancer, metabolic abnormalities, and inflammation. When ROS are generated in cells, protein cysteine is highly vulnerable to oxidations, particularly S-glutathionylation (SSG). Protein S-glutathionylation is a reversible post-translational modification that involves disulfide bond formation between intracellular glutathione and protein cysteine residues. Although methods for detecting protein glutathionylation have been established, it remains challenging to do a biological functional analysis of glutathionylated proteins after their identifications. Here, we develop a chemical biology approach to study the biological functional effects of glutathionylation of proteins of interest (POI). The first part of this approach introduces glutathionylation modification on a POI in vitro. The second is to deliver the glutathionylated POI to cells for downstream functional analysis. Dehydroglutathione (dhG), a derivative of glutathione (GSH), was envisioned to form a stable (thioether) mimic of glutathionylation on POI. We anticipated that the POI modified by dhG would maintain the glutathionylation modification in the reducing environment of cells. To illustrate our approach, we chose fatty acid-binding protein 5 (FABP5), a key molecular target in cancer progression. We demonstrated that FABP5 is glutathionylated at position C127 by dhG and that this modification is stable both in vitro and in cells. In contrast to non-glutathionylated FABP5 (KD = 1-5 [mu]M), we have shown that FABP5 glutathionylation by dhG enhances its binding to linoleic acid (KD = 0.6-0.8 [mu]M). Also, after treatment with linoleic acid, dhG-modified FABP5 was translocated to the nucleus to activate PPAR[beta]/[delta] and cause migration of MCF7 cancer cells. We also extended our dhG approach to study the effect of glutathionylation on SET and MYND domain-containing protein 2 (SMYD2). We showed that dhG modifies SMYD2 at the C13 residue, resembling S-glutathionylation. In vitro, functional analysis of SMYD2 showed that dhG-mediated glutathionylation does not reduce SMYD2 methyltransferase activity. Our research develops a glutathione-based new tool and demonstrates that the dhG modification mimics protein S-glutathionylation, which can help study the effect of protein glutathionylation.

Chemical tool to synthesize and investigate glutathionylated proteins

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Chemical tool to synthesize and investigate glutathionylated proteins

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