The endoplasmic reticulum (ER) is a membranous intracellular organelle and the first compartment of the secretory pathway. As such, the ER contributes to the production and folding of approximately one-third of cellular proteins, and is thus inextricably linked to the maintenance of cellular homeostasis and the fine balance between health and disease. Specific ER stress signalling pathways, collectively known as the unfolded protein response (UPR), are required for maintaining ER homeostasis. The UPR is triggered when ER protein folding capacity is overwhelmed by cellular demand and the UPR initially aims to restore ER homeostasis and normal cellular functions. However, if this fails, then the UPR triggers cell death. In this review, we provide a UPR signalling-centric view of ER functions, from the ER's discovery to the latest advancements in the understanding of ER and UPR biology. Our review provides a synthesis of intracellular ER signalling revolving around proteostasis and the UPR, its impact on other organelles and cellular behaviour, its multifaceted and dynamic response to stress and its role in physiology, before finally exploring the potential exploitation of this knowledge to tackle unresolved biological questions and address unmet biomedical needs. Thus, we provide an integrated and global view of existing literature on ER signalling pathways and their use for therapeutic purposes.

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The communication between the cysteine, Cys439, at the substrate site and the tyrosyl radical, Tyr122, in ribonucleotide reductase is studied by quantum chemical models at the DFT-B3LYP level. Recent theoretical and experimental studies have indicated that an electron transfer between these sites is highly unlikely. Instead, a model based on the hydrogen atom transfer (HAT) mechanism is investigated. In this mechanism both the proton and electron are moved in each step to avoid a costly charge separation. It is found that the hydrogen atom transfer steps required for communication between Cys439 in R1 and Trp48 in the region of the iron dimer in R2 all have quite low energy barriers. The radical transfer between Tyr731 and Tyr730 has a barrier of 4.9 kcal/mol, while the one between Tyr730 to Cys439 has a barrier of 8.1 kcal/mol. An interesting aspect of these transfers is that the dielectric contribution from the protein is very small, indicating very small charge separations. The radical transfer from Tyr122 to Trp48 over the iron dimer is considerably more complicated. A model is suggested where this transfer occurs in essentially one step by a hydrogen atom transfer from a water ligand of the iron dimer to Tyr122. In this process an electron is transferred between Trp48 to the hydroxyl ligand of iron over the Trp48-Asp237-His241 chain leading to a cationic tryptophan radical.

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Density functional methods, alone and together with molecular mechanics, are used to study the catalytic mechanism of galactose oxidase. This enzyme catalyzes the conversion of primary alcohols to the corresponding aldehydes, coupled with reduction of dioxygen to hydrogen peroxide. It is shown that the proposed mechanism for this enzyme is energetically feasible. In particular the barrier for the postulated rate-limiting hydrogen atom transfer between the substrate and the tyrosyl radical, located at equatorial Tyr272, is very plausible. We propose that the radical site, prior to the initial proton transfer step, is located at the axial tyrosine (Tyr495). The radical is transferred to the equatorial tyrosine (Tyr272) simultaneously with the proton transfer. It is, furthermore, argued that the electron transfer from the ketyl radical intermediate to Cu(II) cannot be very exothermic, because this would render the oxygen reduction steps rate-limiting. Finally, the cysteine cross-link on the active site tyrosine is shown to have very minor effects on the energetics of the reaction.

The extracellular matrix (ECM) plays an instrumental role in determining the spatial orientation of epithelial polarity and the formation of lumens in glandular tissues during morphogenesis. Here, we show that the Endoplasmic Reticulum (ER)-resident protein anterior gradient-2 (AGR2), a soluble protein-disulfide isomerase involved in ER protein folding and quality control, is secreted and interacts with the ECM. Extracellular AGR2 (eAGR2) is a microenvironmental regulator of epithelial tissue architecture, which plays a role in the preneoplastic phenotype and contributes to epithelial tumorigenicity. Indeed, eAGR2, is secreted as a functionally active protein independently of its thioredoxin-like domain (CXXS) and of its ER-retention domain (KTEL), and is sufficient, by itself, to promote the acquisition of invasive and metastatic features. Therefore, we conclude that eAGR2 plays an extracellular role independent of its ER function and we elucidate this gain-of-function as a novel and unexpected critical ECM microenvironmental pro-oncogenic regulator of epithelial morphogenesis and tumorigenesis.DOI: http://dx.doi.org/10.7554/eLife.13887.001

Pyruvate formate-lyase (PFL) is a glycyl radical containing enzyme that catalyzes the reversible CoA-dependent conversion of pyruvate into acetyl-CoA and formate. We have studied the catalytic mechanism of this enzyme by means of accurate quantum chemical methods. It is shown that an overall homolytic radical mechanism is very feasible. In particular, the formation of a tetrahedral radical intermediate, by addition of thiyl radical to pyruvate, is supported by the calculated reaction energies and barriers. Furthermore, we propose that the thioester exchange between active site cysteine and CoA proceeds via a radical mechanism. This is made possible by the quenching of the formate radical by Cys418, and not Gly734, as previously proposed.