A close connection between the PERK and IRE arms of the UPR and the transcriptional regulation of autophagy.

Endoplasmic reticulum (ER) stress is known to lead to activation of both the unfolded protein response (UPR) and autophagy. Although regulatory connections have been identified between the UPR and autophagy, it is still unclear to what extent the UPR regulates the genes involved at the different stages of the autophagy pathway. Here, we carried out a microarray analysis of HCT116 cells subjected to ER stress and observed the transcriptional upregulation of a large cohort of autophagy-related genes. Of particular interest, we identified the transcriptional upregulation of the autophagy receptor genes SQSTM1/p62, NBR1 and BNIP3L/NIX in response to ER stress and show that the inhibition of the UPR transmembrane receptors, PERK and IRE1, abrogates this upregulation.

ER stress responses in the absence of apoptosome: a comparative study in CASP9 proficient vs deficient mouse embryonic fibroblasts.

Cells respond to endoplasmic reticulum (ER) stress through the unfolded protein response (UPR), autophagy and cell death. In this study we utilized casp9(+/+) and casp9(-/-) MEFs to determine the effect of inhibition of mitochondrial apoptosis pathway on ER stress-induced-cell death, UPR and autophagy. We observed prolonged activation of UPR and autophagy in casp9(-/-) cells as compared with casp9(+/+) MEFs, which displayed transient activation of both pathways. Furthermore we showed that while casp9(-/-) MEFs were resistant to ER stress, prolonged exposure led to the activation of a non-canonical, caspase-mediated mode of cell death.

Stress-induced self-cannibalism: on the regulation of autophagy by endoplasmic reticulum stress.

Macroautophagy (autophagy) is a cellular catabolic process which can be described as a self-cannibalism. It serves as an essential protective response during conditions of endoplasmic reticulum (ER) stress through the bulk removal and degradation of unfolded proteins and damaged organelles; in particular, mitochondria (mitophagy) and ER (reticulophagy). Autophagy is genetically regulated and the autophagic machinery facilitates removal of damaged cell components and proteins; however, if the cell stress is acute or irreversible, cell death ensues. Despite these advances in the field, very little is known about how autophagy is initiated and how the autophagy machinery is transcriptionally regulated in response to ER stress. Some three dozen autophagy genes have been shown to be required for the correct assembly and function of the autophagic machinery; however; very little is known about how these genes are regulated by cellular stress. Here, we will review current knowledge regarding how ER stress and the unfolded protein response (UPR) induce autophagy, including description of the different autophagy-related genes which are regulated by the UPR.

HSP72 protects cells from ER stress-induced apoptosis via enhancement of IRE1alpha-XBP1 signaling through a physical interaction.

Endoplasmic reticulum (ER) stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR). Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1alpha, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1alpha or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1alpha. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1alpha in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1alpha enhances IRE1alpha/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response.

Correction: Inhibition of IRE1α RNase activity reduces NLRP3 inflammasome assembly and processing of pro-IL1ß.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Inhibition of IRE1α RNase activity reduces NLRP3 inflammasome assembly and processing of pro-IL1ß.

The inflammasome is a multiprotein complex assembled in response to Pathogen Associated Molecular Patterns (PAMPs) and Danger Associated Molecular Patterns (DAMPs). Inflammasome activation occurs through a two-step mechanism, with the first signal facilitating priming of inflammasome components while the second signal triggers complex assembly. Once assembled, the inflammasome recruits and activates pro-caspase-1, which in turn processes pro-interleukin (IL)-18 and pro-IL-1ß into their bio-active forms. Owing to its key role in the regulation of innate immune responses, the inflammasome has emerged as a therapeutic target for the treatment of inflammatory conditions. In this study we demonstrate that IRE1α, a key component of the Unfolded Protein Response, contributes to assembly of the NLRP3 inflammasome. Blockade of IRE1α RNase signaling lowered NLRP3 inflammasome assembly, caspase-1 activation and pro-IL-1ß processing. These results underscore both the importance and potential therapeutic relevance of targeting IRE1α signaling in conditions of excessive inflammasome formation.

The Unfolded Protein Response in Breast Cancer.

In 2018, in the US alone, it is estimated that 268,670 people will be diagnosed with breast cancer, and that 41,400 will die from it. Since breast cancers often become resistant to therapies, and certain breast cancers lack therapeutic targets, new approaches are urgently required. A cell-stress response pathway, the unfolded protein response (UPR), has emerged as a promising target for the development of novel breast cancer treatments. This pathway is activated in response to a disturbance in endoplasmic reticulum (ER) homeostasis but has diverse physiological and disease-specific functions. In breast cancer, UPR signalling promotes a malignant phenotype and can confer tumours with resistance to widely used therapies. Here, we review several roles for UPR signalling in breast cancer, highlighting UPR-mediated therapy resistance and the potential for targeting the UPR alone or in combination with existing therapies.

A microfluidic fluidized bed to capture, amplify and detect bacteria from raw samples.

Bacterial contamination and subsequent infections are a major threat to human health. An early detection in the food chain, clinics or the environment, is key to limit this threat. We present a new concept to develop low-cost hand-held devices for the ultra-sensitive and specific detection of bacteria in a one-step process of 2-8h, directly from complex raw samples. This approach is based on a novel microfluidic magnetic fluidized bed. It reaches a 4CFU (colony forming unit) sensitivity with high quantification accuracy in a large dynamic range of 100-107CFU/mL. The versatility of the approach was demonstrated with the detection of different bacteria strains, among which Salmonella Typhimurium and E. coli O157:H15. Additionally, the method is sensitive to infectious bacteria only, a criterion requested by main applications and currently requiring additional culture steps of one to several days.

A new microfluidic approach for the one-step capture, amplification and label-free quantification of bacteria from raw samples.

A microfluidic method to specifically capture and detect infectious bacteria based on immunorecognition and proliferative power is presented. It involves a microscale fluidized bed in which magnetic and drag forces are balanced to retain antibody-functionalized superparamagnetic beads in a chamber during sample perfusion. Captured cells are then cultivated in situ by infusing nutritionally-rich medium. The system was validated by the direct one-step detection of Salmonella Typhimurium in undiluted unskimmed milk, without pre-treatment. The growth of bacteria induces an expansion of the fluidized bed, mainly due to the volume occupied by the newly formed bacteria. This expansion can be observed with the naked eye, providing simple low-cost detection of only a few bacteria and in a few hours. The time to expansion can also be measured with a low-cost camera, allowing quantitative detection down to 4 cfu (colony forming unit), with a dynamic range of 100 to 107 cfu ml-1 in 2 to 8 hours, depending on the initial concentration. This mode of operation is an equivalent of quantitative PCR, with which it shares a high dynamic range and outstanding sensitivity and specificity, operating at the live cell rather than DNA level. Specificity was demonstrated by controls performed in the presence of a 500× excess of non-pathogenic Lactococcus lactis. The system's versatility was demonstrated by its successful application to the detection and quantitation of Escherichia coli O157:H15 and Enterobacter cloacae. This new technology allows fast, low-cost, portable and automated bacteria detection for various applications in food, environment, security and clinics.

Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions.

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.

NOXA contributes to the sensitivity of PERK-deficient cells to ER stress.

PKR-like ER kinase (PERK) deficient mouse embryonic fibroblasts (MEFs) are hypersensitive to ER stress-induced apoptosis. However, the molecular determinants of increased sensitivity of PERK(-/-) MEFs are not clearly understood. Here we show that induction of several Unfolded Protein Response (UPR) target genes is attenuated in PERK(-/-) MEFs. We also report elevated expression of the BH3-only protein, NOXA in PERK(-/-) MEFs. Further, shRNA-mediated knockdown of NOXA rescued the hypersensitivity of PERK(-/-) MEFs to ER stress-induced apoptosis. Taken together our results suggest that compromised induction of UPR and increased NOXA expression contributes to hypersensitivity of PERK(-/-) MEFs to ER stress-induced apoptosis.

Assays for detecting the unfolded protein response.

The endoplasmic reticulum (ER) is the site for folding of membrane and secreted proteins in the cell. Physiological or pathological processes that disturb protein folding in the ER cause ER stress and activate a set of signaling pathways termed the unfolded protein response (UPR). The UPR leads to transcriptional activation of genes encoding ER-resident chaperones, oxidoreductases, and ER-associated degradation (ERAD) components. Thus, UPR promotes cellular repair and adaptation by enhancing protein-folding capacity, reducing the secretory protein load, and promoting degradation of misfolded proteins. In mammalian cells, the UPR also triggers apoptosis, perhaps when adaptive responses fail. Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field. Here, we describe the experimental methods that we have used to study UPR in tissue culture cells. These methods can be used by researchers to plan and interpret experiments aimed at evaluating whether the UPR and related processes are activated or not. It is important to note that these are general guidelines for monitoring the UPR and not all assays will be appropriate for every model system.

Methods for monitoring endoplasmic reticulum stress and the unfolded protein response.

The endoplasmic reticulum (ER) is the site of folding of membrane and secreted proteins in the cell. Physiological or pathological processes that disturb protein folding in the endoplasmic reticulum cause ER stress and activate a set of signaling pathways termed the Unfolded Protein Response (UPR). The UPR can promote cellular repair and sustained survival by reducing the load of unfolded proteins through upregulation of chaperones and global attenuation of protein synthesis. Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field. There are also many researchers not working directly on ER stress, but who wish to determine whether this response is activated in the system they are studying: thus, it is important to list a standard set of criteria for monitoring UPR in different model systems. Here, we discuss approaches that can be used by researchers to plan and interpret experiments aimed at evaluating whether the UPR and related processes are activated. We would like to emphasize that no individual assay is guaranteed to be the most appropriate one in every situation and strongly recommend the use of multiple assays to verify UPR activation.