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The phosphorylation of eIF2α is essential for the endoplasmic reticulum (ER) stress response, the formation of stress granules, as well as macroautophagy. Several successful anticancer chemotherapeutics have the property to induce immunogenic cell death (ICD), thereby causing anticancer immune responses. ICD is accompanied by the translocation of calreticulin (CALR) from the ER lumen to the plasma membrane, which facilitates the transfer of tumor-associated antigens to dendritic cells. Here we systematically investigated the capacity of anticancer chemotherapeutics to induce signs of ER stress. ICD inducers including anthracyclines and agents that provoke tetraploidization were highly efficient in enhancing the phosphorylation of eIF2α, yet failed to stimulate other signs of ER stress including the transcriptional activation of activating transcription factor 4 (ATF4), the alternative splicing of X-box binding protein 1 (XBP1s) mRNA and the proteolytic cleavage of activating transcription factor 6 (ATF6) both in vitro and in cancers established in mice. Systematic analyses of clinically used anticancer chemotherapeutics revealed that only eIF2α phosphorylation, but none of the other signs of ER stress, correlated with CALR exposure. eIF2α phosphorylation induced by mitoxantrone, a prototype ICD-inducing anthracyline, was mediated by eIF2α kinase-3 (EIF2AK3). Machine-learning approaches were used to determine the physicochemical properties of drugs that induce ICD, revealing that the sole ER stress response relevant to the algorithm is eIF2α phosphorylation with its downstream consequences CALR exposure, stress granule formation and autophagy induction. Importantly, this approach could reduce the complexity of compound libraries to identify ICD inducers based on their physicochemical and structural characteristics. In summary, it appears that eIF2α phosphorylation constitutes a pathognomonic characteristic of ICD.

The transcription factor Blimp-1 is required for the differentiation of activated B cells into plasmablasts. Nutt and colleagues show that plasma cells need Blimp-1 to maintain antibody production by regulating the kinase mTOR and unfolded-protein-response pathways. Plasma cell differentiation requires silencing of B cell transcription, while it establishes antibody-secretory function and long-term survival. The transcription factors Blimp-1 and IRF4 are essential for the generation of plasma cells; however, their function in mature plasma cells has remained elusive. We found that while IRF4 was essential for the survival of plasma cells, Blimp-1 was dispensable for this. Blimp-1-deficient plasma cells retained their transcriptional identity but lost the ability to secrete antibody. Blimp-1 regulated many components of the unfolded protein response (UPR), including XBP-1 and ATF6. The overlap in the functions of Blimp-1 and XBP-1 was restricted to that response, with Blimp-1 uniquely regulating activity of the kinase mTOR and the size of plasma cells. Thus, Blimp-1 was required for the unique physiological ability of plasma cells that enables the secretion of protective antibody.

Mitofusin 2 (Mfn2) is a key protein in mitochondrial fusion and it participates in the bridging of mitochondria to the endoplasmic reticulum (ER). Recent data indicate that Mfn2 ablation leads to ER stress. Here we report on the mechanisms by which Mfn2 modulates cellular responses to ER stress. Induction of ER stress in Mfn2‐deficient cells caused massive ER expansion and excessive activation of all three Unfolded Protein Response (UPR) branches (PERK, XBP‐1, and ATF6). In spite of an enhanced UPR, these cells showed reduced activation of apoptosis and autophagy during ER stress. Silencing of PERK increased the apoptosis of Mfn2‐ablated cells in response to ER stress. XBP‐1 loss‐of‐function ameliorated autophagic activity of these cells upon ER stress. Mfn2 physically interacts with PERK, and Mfn2‐ablated cells showed sustained activation of this protein kinase under basal conditions. Unexpectedly, PERK silencing in these cells reduced ROS production, normalized mitochondrial calcium, and improved mitochondrial morphology. In summary, our data indicate that Mfn2 is an upstream modulator of PERK. Furthermore, Mfn2 loss‐of‐function reveals that PERK is a key regulator of mitochondrial morphology and function. Mitochondrial–ER bridging protein mitofusin 2 (Mfn2) orchestrates mitochondrial metabolism and the UPR by suppressing PERK.

Although stem cell populations mediate regeneration of rapid turnover tissues, such as skin, blood, and gut, a stem cell reservoir has not been identified for some slower turnover tissues, such as the pancreatic islet. Despite lacking identifiable stem cells, murine pancreatic β cell number expands in response to an increase in insulin demand. Lineage tracing shows that new β cells are generated from proliferation of mature, differentiated β cells; however, the mechanism by which these mature cells sense systemic insulin demand and initiate a proliferative response remains unknown. Here, we identified the β cell unfolded protein response (UPR), which senses insulin production, as a regulator of β cell proliferation. Using genetic and physiologic models, we determined that among the population of β cells, those with an active UPR are more likely to proliferate. Moreover, subthreshold endoplasmic reticulum stress (ER stress) drove insulin demand-induced β cell proliferation, through activation of ATF6. We also confirmed that the UPR regulates proliferation of human β cells, suggesting that therapeutic UPR modulation has potential to expand β cell mass in people at risk for diabetes. Together, this work defines a stem cell-independent model of tissue homeostasis, in which differentiated secretory cells use the UPR sensor to adapt organ size to meet demand.

The adhesion G-protein-coupled receptor (GPCR) latrophilin 3 (ADGRL3) has been associated with increased risk of attention deficit hyperactivity disorder (ADHD) and substance use in human genetic studies. Knockdown in multiple species leads to hyperlocomotion and altered dopamine signaling. Thus, ADGRL3 is a potential target for treatment of neuropsychiatric disorders that involve dopamine dysfunction, but its basic signaling properties are poorly understood. Identification of adhesion GPCR signaling partners has been limited by a lack of tools to acutely activate these receptors in living cells. Here, we design a novel acute activation strategy to characterize ADGRL3 signaling by engineering a receptor construct in which we could trigger acute activation enzymatically. Using this assay, we found that ADGRL3 signals through G12/G13 and Gq, with G12/13 the most robustly activated. Gα 12/13 is a new player in ADGRL3 biology, opening up unexplored roles for ADGRL3 in the brain. Our methodological advancements should be broadly useful in adhesion GPCR research. Among the adhesion receptor class of GPCRs, which are understudied, the adhesion receptor ADGRL3 can be activated by its own tethered agonist and couples to G protein G12/13 and somewhat to Gq.

Background Activating transcription factor 6 (ATF6) is an endoplasmic reticulum (ER)-localised protein and member of the leucine zipper family of transcription factors. Best known for its role in transducing signals linked to stress to the endoplasmic reticulum, the 50 kDa activated form of ATF6 is now emerging as a major regulator of organogenesis and tissue homeostasis. Responsible for the correct folding, secretion and membrane insertion of a third of the proteome in eukaryotic cells, the ER encompasses a dynamic, labyrinthine network of regulators, chaperones, foldases and cofactors. Such structures are crucial to the extensive protein synthesis required to undergo normal development and maintenance of tissue homeostasis. When an additional protein synthesis burden is placed on the ER, ATF6, in tandem with ER stress transducers inositol requiring enzyme 1 (IRE1) and PKR-like endoplasmic reticulum kinase (PERK), slows the pace of protein translation and induces the production of stress-reducing chaperones and foldases. Main Text In the context of development and tissue homeostasis, however, distinct cellular impacts have been attributed to ATF6. Drawing on data published from human, rodent, fish, goat and bovine research, this review first focuses on ATF6-mediated regulation of osteo- and chondrogenesis, ocular development as well as neuro- and myelinogenesis. The purported role of ATF6 in development of the muscular and reproductive systems as well as adipo- and lipogenesis is then described. With relevance to cardiac disease, cancer and brain disorders, the importance of ATF6 in maintaining tissue homeostasis is the subject of the final section. Conclusion In conclusion, the review encourages further elucidation of ATF6 regulatory operations during organogenesis and tissue homeostasis, to spawn the development of ATF6-targeted therapeutic strategies.

Endoplasmic reticulum (ER) stress is associated with diabetic nephropathy (DN), but its pathophysiological relevance and the mechanisms that compromise adaptive ER signalling in podocytes remain unknown. Here we show that nuclear translocation of the transcription factor spliced X-box binding protein-1 (sXBP1) is selectively impaired in DN, inducing activating transcription factor-6 (ATF6) and C/EBP homology protein (CHOP). Podocyte-specific genetic ablation of XBP1 or inducible expression of ATF6 in mice aggravates DN. sXBP1 lies downstream of insulin signalling and attenuating podocyte insulin signalling by genetic ablation of the insulin receptor or the regulatory subunits phosphatidylinositol 3-kinase (PI3K) p85α or p85β impairs sXBP1 nuclear translocation and exacerbates DN. Corroborating our findings from murine DN, the interaction of sXBP1 with p85α and p85β is markedly impaired in the glomerular compartment of human DN. Thus, signalling via the insulin receptor, p85, and XBP1 maintains podocyte homeostasis, while disruption of this pathway impairs podocyte function in DN. Diabetic kidney disease is associated with ER stress in podocytes. Here the authors use various genetically modified mouse models to study ER-stress-related signalling pathways and propose a mechanistic framework that links insulin signalling with ER stress in podocytes of diabetic mice.

Endoplasmic reticulum (ER) stress and Unfolded Protein Response (UPR) signaling promote the pathology of many human diseases. Loss-of-function variants of the UPR regulator Activating Transcription Factor 6 (ATF6) cause severe congenital vision loss diseases such as achromatopsia by unclear pathomechanisms. To investigate this, we generated retinal organoids from achromatopsia patient induced pluripotent stem cells carrying ATF6 disease variants and from gene-edited ATF6 null hESCs. We found that achromatopsia patient and ATF6 null retinal organoids failed to form cone structures concomitant with loss of cone phototransduction gene expression, while rod photoreceptors developed normally. Adaptive optics retinal imaging of achromatopsia patients carrying ATF6 variants also showed absence of cone inner/outer segment structures but preserved rod structures, mirroring the defect in cone formation observed in our retinal organoids. These results establish that ATF6 is essential for human cone development. Interestingly, we find that a selective small molecule ATF6 signaling agonist restores the transcriptional activity of some ATF6 disease-causing variants and stimulates cone growth and gene expression in patient retinal organoids carrying these variants. These findings support that pharmacologic targeting of the ATF6 pathway can promote human cone development and should be further explored for blinding retinal diseases.

The molecular pathomechanisms underlying idiopathic pulmonary fibrosis (IPF) are elusive, but chronic epithelial injury has recently been suggested as key event. We investigated the possible implication of endoplasmic reticulum (ER) stress-mediated apoptosis in sporadic IPF. We analyzed peripheral explanted lung tissues from patients with sporadic IPF (n = 24), chronic obstructive pulmonary disease (COPD) (n = 9), and organ donors (n = 12) for expression of major ER stress mediators and apoptosis markers by means of immunoblotting, semiquantitative reverse transcription-polymerase chain reaction, immunohistochemistry, and the TUNEL method. Compared with COPD and donor lungs, protein levels of ER stress mediators, such as processed p50 activating transcription factor (ATF)-6 and ATF-4 and the apoptosis-inductor CHOP (C/EBP-homologous protein), as well as transcript levels of spliced X-box binding protein (XBP)-1, were significantly elevated in lung homogenates and type II alveolar epithelial cells (AECIIs) of IPF lungs. Proapoptotic, oligomeric forms of Bax, which play a key role in ER stress-mediated apoptosis downstream of CHOP induction, as well as caspase-3 cleavage, could be detected in IPF lungs. By means of immunohistochemistry, exclusive induction of active ATF-6, ATF-4, and CHOP in AECIIs was encountered in IPF but not in COPD or donor lungs. Immunoreactivity was most prominent in the epithelium near dense zones of fibrosis and fibroblast foci, where these ER stress markers colocalized with markers of apoptosis (TUNEL, cleaved caspase-3). Severe ER stress response in the AECIIs of patients with sporadic IPF may underlie the apoptosis of this cell type and development of fibrosis in this disease.

Properly balancing microbial responses by the innate immune system through pattern recognition receptors (PRRs) is critical for intestinal immune homeostasis. Ring finger protein 186 (RNF186) genetic variants are associated with inflammatory bowel disease (IBD). However, functions for the E3 ubiquitin ligase RNF186 are incompletely defined. We found that upon stimulation of the PRR nucleotide-binding oligomerization domain containing 2 (NOD2) in human macrophages, RNF186 localized to the ER, formed a complex with ER stress sensors, ubiquitinated the ER stress sensor activating transcription factor 6 (ATF6), and promoted the unfolded protein response (UPR). These events, in turn, led to downstream signaling, cytokine secretion, and antimicrobial pathway induction. Importantly, RNF186-mediated ubiquitination of K152 on ATF6 was required for these outcomes, highlighting a key role for ATF6 ubiquitination in PRR-initiated functions. Human macrophages transfected with the rare RNF186-A64T IBD risk variant and macrophages from common rs6426833 RNF186 IBD risk carriers demonstrated reduced NOD2-induced outcomes, which were restored by rescuing UPR signaling. Mice deficient in RNF186 or ATF6 demonstrated a reduced UPR in colonic tissues, increased weight loss, and less effective clearance of bacteria with dextran sodium sulfate-induced injury and upon oral challenge with Salmonella Typhimurium. Therefore, we identified that RNF186 was required for PRR-induced, UPR-associated signaling leading to key macrophage functions; defined that RNF186-mediated ubiquitination of ATF6 was essential for these functions; and elucidated how RNF186 IBD risk variants modulated these outcomes.