GlutathioneS-Transferase P-Mediated Protein S-Glutathionylation of Resident Endoplasmic Reticulum Proteins Influences Sensitivity to Drug-Induced Unfolded Protein Response

Abstract

Aims: S-glutathionylation of cysteine residues, catalyzed by glutathione S-transferase Pi (GSTP), alters structure/function characteristics of certain targeted proteins. Our goal is to characterize how S-glutathionylation of proteins within the endoplasmic reticulum (ER) impact cell sensitivity to ER-stress inducing drugs. Results: We identify GSTP to be an ER-resident protein where it demonstrates both chaperone and catalytic functions. Redox based proteomic analyses identified a cluster of proteins cooperatively involved in the regulation of ER stress (immunoglobulin heavy chain-binding protein [BiP], protein disulfide isomerase [PDI], calnexin, calreticulin, endoplasmin, sarco/endoplasmic reticulum Ca 2+ -ATPase [SERCA]) that individually coimmunoprecipitated with GSTP (implying protein complex formation) and were subject to reactive oxygen species (ROS) induced S-glutathionylation. S-glutathionylation of each of these six proteins was attenuated in cells (liver, embryo fibroblasts or bone marrow dendritic) from mice lacking GSTP (Gstp1/p2 -/-) compared to wild type (Gstp1/p2 +/+ ). Moreover, Gstp1/p2 -/-cells were significantly more sensitive to the cytotoxic effects of the ER-stress inducing drugs, thapsigargin (7-fold) and tunicamycin (2-fold). Innovation: Within the family of GST isozymes, GSTP has been ascribed the broadest range of catalytic and chaperone functions. Now, for the first time, we identify it as an ER resident protein that catalyzes S-glutathionylation of critical ER proteins within this organelle. Of note, this can provide a nexus for linkage of redox based signaling and pathways that regulate the unfolded protein response (UPR). This has novel importance in determining how some drugs kill cancer cells. Conclusions: Contextually, these results provide mechanistic evidence that GSTP can exert redox regulation in the oxidative ER environment and indicate that, within the ER, GSTP influences the cellular consequences of the UPR through S-glutathionylation of a series of key interrelated proteins. Antioxid. Redox Signal. 26, 247-261.

S-Glutathionylation of estrogen receptor α affects dendritic cell function

“…This is exemplified by the IRE1/XBP1 pathway response, where Gstp1/p2 -/-BMDDC exhibited a more marked activation compared to WT ( Figure 4E). These results were similar to our earlier studies (24), where both thapsigargin and tunicamycin caused a more pronounced UPR response in GSTp1/p2 -/-BMDDC compared to wild type. IRE1/XBP1 activation has been shown to be essential for the development of DC and regulate innate immune responses in macrophages (Iwakoshi et al, 2007;Martinon et al, 2010).…”

“…There is evidence that BMDDC functions may also be influenced by endoplasmic reticulum induced stress, particularly as they relate as precursors of the unfolded protein response (39)(40)(41)(42). We previously showed that markers for UPR, including IRE1 and ATF6 are constitutively higher in cells from Gstp1/p2 -/-mice and that these animals have a narrower threshold of tolerance to oxidative stress compared to wild type (24). In this regard, such data indicate that Gstp1/p2 -/-mice are under higher levels of constitutive oxidative stress.…”

“…This catalysis is facilitated by GSTP forming a temporal, reversible association with ERα allowing for the transfer of the -SG moiety from the enzyme's "G" site (30). Whether GSTP and ERα form a direct or indirect (part of a larger protein cluster) complex (24,31), it is apparent that the latter is a substrate for S-glutathionylation. Since S-glutathionylation has a profound impact on the structure, function and sub-cellular distribution of most proteins (13,32), it seems reasonable to suggest that ERα will be similarly impacted.…”

Deletion of the Glutaredoxin-2 Gene Protects Mice from Diet-Induced Weight Gain, Which Correlates with Increased Mitochondrial Respiration and Proton Leaks in Skeletal Muscle

“…In addition, complex I has multiple S-glutathionylation sites, which have been postulated to fine tune its activity and rate of reactive oxygen species (ROS) release in response to different physiological cues and changes in mitochondrial redox state (27). It is important to acknowledge that glutathione S-transferases (GST) have also been shown to catalyze S-glutathionylation reactions in the cytoplasm and endoplasmic reticulum (31,48). In addition, it is now apparent that GST plays a significant role in mediating these reactions in mammalian cells.…”

Crosstalk between calcium and reactive oxygen species signaling in cancer

“…This highlights that the GSH/GSSH pool within cells is an important determinant for protein redox modification. S-glutahtionlylation of SERCA was also recently shown to occur during the unfolded protein response, with loss of the ER glutathione S -transferase Pi expression resulting in cells being more sensitive to ER-stress causing agents [344]. SERCA Tyrosine nitration was also demonstrated in models of cardiovascular disease [345, 346].…”