# Shiga Toxin Therapeutics: Beyond Neutralization
## 5 The Unfolded Protein Response (UPR)  
The UPR is a eukaryotic cellular stress response triggered by the accumulation of unfolded or improperly folded peptides within the lumen of ER, also known as ER stress. The UPR is initiated by several proteins located within the ER lumen that sense unfolded proteins and are then activated to initiate signaling cascades that attempt to restore protein homeostasis. The best characterized sensors of ER stress are protein kinase R-like endoplasmic reticulum kinase (PERK), inositol-requiring protein 1\(\alpha\) (IRE1\(\alpha\)), and activating transcription factor 6\(\alpha\) (ATF6\(\alpha\)) [73]. IRE1\(\alpha\) is an ER membrane-bound protein that activates its endoribonuclease activity via the dissociation of the inhibitory chaperone protein GRP78 during ER stress. IRE1\(\alpha\) cleaves a 26 nucleotide segment from xbox binding protein 1 (XBP1) mRNA. The frameshift created by this cleavage leads to the translation of the active form of XBP1, which acts as a transcription factor for protein folding, protein transport, protein degradation, and mRNA degradation-associated genes. Concurrent with IRE1\(\alpha\) activation, the ER stress sensor protein ATF6\(\alpha\) moves from the ER to the Golgi for processing into its active form. Active ATF6\(\alpha\) travels to the nucleus to upregulate the transcription of chaperone proteins. The combination of ATF6\(\alpha\) and IRE1\(\alpha\) activation leads to an upregulation of protein-folding and degradation machinery to restore protein homeostasis [73; 74]. The ER membrane-bound kinase PERK is activated to initiate a separate signaling pathway during ER stress via phosphorylation and the inactivation of eukaryotic translation initiation factor 2a (eIF2a). The inactivation of eIF2a inhibits translation to allow cellular machinery time to properly fold or degrade misfolded proteins. Phosphorylated eIF2a simultaneously upregulates several transcripts coding for proteins involved in protein degradation, mRNA degradation, chaperone proteins to aid in peptide folding, and protein transporters. A key known transcriptional target of phosphorylated eIF2a is activating transcription factor 4 (ATF4). ATF4 is a transcription factor for antioxidant and amino acid biosynthesis genes as well as CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP). CHOP forms heterodimers with ATF4 to upregulate the transcription of additional UPR targets, including growth arrest and DNA-damage-inducible protein 34 (GADD34). GADD34 is aphosphatase that dephosphorylor plates eIF2a to reinitiate ribosomal translation [73; 74; 75]. The activation of ATF6\(\alpha\) and IRE1\(\alpha\) occurs transiently during ER stress, but the activation of PERK persists until ER stress is resolved [76]. While a short term activation of the UPR promotes survival of the cell, chronic UPR activation eventually leads to cellular apoptosis. The transcription factor CHOP concurrently suppresses anti-apoptotic Bcl-2 expression and enhances pro-apoptotic factor expression if chronically present in the cell. CHOP activates ER oxidase 1\(\alpha\) (ERO1\(\alpha\)), a protein that facilitates disulfide bond formation in the ER [77]. In the context of unresolved ER stress, the reactive oxygen species generated by disulfide bond formation lead to increased Ca\({}^{+2}\) efflux from the ER, which in turn causes mitochondrial stress. Resuming ribosomal translation via GADD34 phosphatase activity on eIF2a in the context of unresolved ER stress leads to further generation of reactive oxygen species and ATP depletion [75]. CHOP appears to be the key factor in the progression of the UPR to apoptosis, as the knockout of CHOP in cultured cells and mice protects from apoptosis in various models of chronic UPR [77; 78]. A chronic upregulation of CHOP leads to an upregulation of Bcl-2 interacting mediator of cell death (BIM) protein expression with a concurrent downregulation of the anti-apoptotic protein Bcl-2.