# Shiga Toxin Therapeutics: Beyond Neutralization
## 8 The Ribotoxic Response  
In addition to the UPR, damage to domains V or VI of the ribosomal 28S rRNA has been found to initiate a stress response signaling cascade termed the ribonucleoxic stress response (RSR) [107]. The activity of ribotoxins such as ricin, sarcin, anisomycin, and deoxynivalenol (DON) leads to the activation of double-stranded-RNA-activated kinase R (PKR), likely through the homodimerization and transphosphorylation of ribosome-associated PKR [108]. PKR phosphorylates the translation factor eIF2a to inactivate it and initiate a signaling cascade leading to variable activation of classical p38 MAP kinases, ERK, and JNK depending on the cell type. It should be noted that both the UPR and the ribotoxic response involve the phosphorylation of eIF2a, but in cell culture experiments the ribotoxic stress response could be elicited at low levels of translational inhibition by ribotoxins. Furthermore, ribotoxins, such as emetine and pactamycin, that act on ribosomal regions other than domain V or VI fail to elicit the activation of PKR with downstream kinase activation despite their ability to inhibit protein synthesis [107]. These findings suggest that the RSR is an independent stress pathway that is distinct from the UPR and is not reliant on translational inhibition. Hematopoietic cell kinase (Hck) was found to associate with PKR and the 40S ribosome in human monocytic U937 cells, and the pharmacological inhibition of Hck activity prevented p38 MAPK phosphorylation and IL-8 secretion in response to DON, suggesting that Hck is also a key sensor of ribosomal damage necessary for downstream effector kinase  
Figure 2: **Therapeutic targeting of the unfolded protein response (UPR) during Shiga toxicity. ** Following ribosomal inhibition by Shiga toxin A subunits, an accumulation of unfolded and misfolded proteins is detected by sensor proteins to initiate the UPR. Therapeutics enhancing the early UPR seek to increase cellular capacity to resolve endoplasmic reticulum stress via the restoration of protein homeostasis. A chronic activation of UPR results in apoptosis or cellular dysfunction via the activity of CHOP-ATF4 heterodimers. Therapies targeting the late UPR seek to inhibit the activity or formation of CHOP-ATF4 heterodimers or inhibit initiators of apoptosis to preserve cellular function. signaling [109]. While the details of the signaling pathway between PKR and downstream effector kinase activity remain unclear, the zipper sterile alpha motif kinase (ZAK) has been identified as a critical component to signaling via pharmacological and siRNA knockdown studies in vitro. An abrogation of ZAK activity prevents the activation of p38 MAPK, JNK, and ERK in Hct-8 and Vero cells incubated with STX1 [81]. The ribotoxic stress response may also lead to cell death if chronic stimulation occurs, with NLRP3 inflammasome activation and pyrotposis via caspase 1 activation documented in THP-1 cells incubated with active STX1 in a concentration-dependent manner [41]. The in vitro impact of the RSR varies depending on the cell type, but is generally characterized by an upregulation of inflammatory cytokine and chemokine transcription and translation. Differentiated macrophage-like THP-1 cells secrete TNF\(\alpha\), IL-1\(\beta\), IL-8, and MIP-1\(\alpha\) in response to stimulation with STXs, and the response could be blunted through an inhibition of JNK, p38 MAPK, and ERK with variance in effect based on the kinase inhibited [43; 110; 111; 112]. Human intestinal epithelial Hct-8 cells secreted the chemokine IL-8 when incubated with STX1 or the ribotoxic antibiotic anisomycin, and concurrent incubation with _E. coli_ flagella and STX2 led to a superinduction of IL-8 secretion compared to flagella alone [113; 114]. Interleukin 8 secretion was reduced through the inhibition of ERK, p38 MAPK, or JNK signaling or through use of a ZAK inhibitor.