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Perturbed secretion of insulin and other pancreatic islet hormones is the main cause of type 2 diabetes (T2D). The islets harbor five cell types that are potentially altered differently by T2D. Whole-islet transcriptomics and single-cell RNA-sequencing (scRNAseq) studies have revealed differentially expressed genes without reaching consensus. Here, we demonstrate that further insights into T2D disease mechanisms can be obtained by network-based analysis of scRNAseq data from individual cell types. We developed differential gene coordination network analysis (dGCNA) and analyzed islet SmartSeq2 scRNAseq data from 16 T2D and 16 non-T2D individuals. dGCNA reveals T2D-induced cell type-specific networks of dysregulated genes with remarkable ontological specificity, thus allowing for a comprehensive and unbiased functional classification of genes involved in T2D. In beta cells eleven networks of genes are detected, revealing that mitochondrial electron transport chain, glycolysis, cytoskeleton organization, cell proliferation, unfolded protein response and three networks of beta cell transcription factors are perturbed, whereas exocytosis, lysosomal regulation and insulin translation programs are instead enhanced in T2D. Furthermore, we validated the ability of dGCNA to reveal disease mechanisms and predict the functional context of genes by showing that TMEM176A/B regulates beta cell microfilament organization and that CEPBG is an important regulator of the unfolded protein response. In addition, when comparing beta- and alpha cells, we found substantial differences, reproduced across independent datasets, confirming cell type-specific alterations in T2D. We conclude that analysis of networks of differentially coordinated genes provides detailed insight into cell type-specific gene function and T2D pathophysiology. Single cell transcriptomics can be used to identify genes associated with type 2 diabetes (T2D) and computational models can be used to identify gene networks. Here the authors identify networks of dysregulated genes in T2D and the biological processes involved, further demonstrating the functional context of the identified genes.

Cellular stress induced by the abnormal accumulation of unfolded or misfolded proteins at the endoplasmic reticulum (ER) is emerging as a possible driver of human diseases, including cancer, diabetes, obesity and neurodegeneration. ER proteostasis surveillance is mediated by the unfolded protein response (UPR), a signal transduction pathway that senses the fidelity of protein folding in the ER lumen. The UPR transmits information about protein folding status to the nucleus and cytosol to adjust the protein folding capacity of the cell or, in the event of chronic damage, induce apoptotic cell death. Recent advances in the understanding of the regulation of UPR signalling and its implications in the pathophysiology of disease might open new therapeutic avenues.The unfolded protein response (UPR) comprises a network of signalling pathways that reprogramme transcription, translation and protein modifications to relieve the load of unfolded or misfolded proteins in the endoplasmic reticulum lumen and restore proteostasis. Understanding the regulation of the UPR and the role it has in the pathophysiology of various cell types and organs might open new therapeutic avenues.

The unfolded protein response (UPR) is a cellular homeostatic mechanism that is activated in many human cancers and plays pivotal roles in tumor progression and therapy resistance. However, the molecular mechanisms for UPR activation and regulation in cancer cells remain elusive. Here, we show that oncogenic MYC regulates the inositol-requiring enzyme 1 (IRE1)/X-box binding protein 1 (XBP1) branch of the UPR in breast cancer via multiple mechanisms. We found that MYC directly controls IRE1 transcription by binding to its promoter and enhancer. Furthermore, MYC forms a transcriptional complex with XBP1, a target of IRE1, and enhances its transcriptional activity. Importantly, we demonstrate that XBP1 is a synthetic lethal partner of MYC. Silencing of XBP1 selectively blocked the growth of MYC-hyperactivated cells. Pharmacological inhibition of IRE1 RNase activity with small molecule inhibitor 8866 selectively restrained the MYC-overexpressing tumor growth in vivo in a cohort of preclinical patient-derived xenograft models and genetically engineered mouse models. Strikingly, 8866 substantially enhanced the efficacy of docetaxel chemotherapy, resulting in rapid regression of MYC-overexpressing tumors. Collectively, these data establish the synthetic lethal interaction of the IRE1/XBP1 pathway with MYC hyperactivation and provide a potential therapy for MYC-driven human breast cancers.

Longevity is regulated by a network of closely linked metabolic systems. We used a combination of mouse population genetics and RNA interference in Caenorhabditis elegans to identify mitochondrial ribosomal protein S5 ( Mrps5 ) and other mitochondrial ribosomal proteins as metabolic and longevity regulators. MRP knockdown triggers mitonuclear protein imbalance, reducing mitochondrial respiration and activating the mitochondrial unfolded protein response. Specific antibiotics targeting mitochondrial translation and ethidium bromide (which impairs mitochondrial DNA transcription) pharmacologically mimic mrp knockdown and extend worm lifespan by inducing mitonuclear protein imbalance, a stoichiometric imbalance between nuclear and mitochondrially encoded proteins. This mechanism was also conserved in mammalian cells. In addition, resveratrol and rapamycin, longevity compounds acting on different molecular targets, similarly induced mitonuclear protein imbalance, the mitochondrial unfolded protein response and lifespan extension in C. elegans . Collectively these data demonstrate that MRPs represent an evolutionarily conserved protein family that ties the mitochondrial ribosome and mitonuclear protein imbalance to the mitochondrial unfolded protein response, an overarching longevity pathway across many species. Mitochondrial ribosomal proteins have been identified as longevity regulators in C. elegans and mammalian systems, their role in longevity is linked to mitonuclear protein imbalance and the mitochondrial unfolded protein response. MRP gene family linked to longevity Animals vary dramatically in lifespan, but why is not clear. Here Johan Auwerx and colleagues report how natural variation in mitochondrial ribosome protein expression translates to lifespan extension in mice and worms, and suggest a unified mechanism behind the effects of metabolic perturbations on longevity. They looked for genetic variation linked to longevity in the BXD genetic reference population of inbred mouse strains. Longevity mapped to mitochondrial ribosomal proteins. Using mouse population genetics and RNA interference experiments in Caenorhabditis elegans , mitochondrial ribosomal protein S5 (Mrps5) and other mitochondrial ribosomal proteins were identified as metabolic and longevity regulators.

Systemic manifestation of preeclampsia (PE) is associated with circulating factors, including inflammatory cytokines and damage-associated molecular patterns (DAMPs), or alarmins. However, it is unclear whether the placenta directly contributes to the increased levels of these inflammatory triggers. Here, we demonstrate that pyroptosis, a unique inflammatory cell death pathway, occurs in the placenta predominantly from early onset PE, as evidenced by elevated levels of active caspase-1 and its substrate or cleaved products, gasdermin D (GSDMD), IL-1β, and IL-18. Using cellular models mimicking pathophysiological conditions (e.g., autophagy deficiency, hypoxia, and endoplasmic reticulum (ER) stress), we observed that pyroptosis could be induced in autophagy-deficient human trophoblasts treated with sera from PE patients as well as in primary human trophoblasts exposed to hypoxia. Exposure to hypoxia elicits excessive unfolded protein response (UPR) and ER stress and activation of the NOD-like receptor pyrin-containing 3 (NLRP3) inflammasome in primary human trophoblasts. Thioredoxin-interacting protein (TXNIP), a marker for hyperactivated UPR and a crucial signaling molecule linked to NLRP3 inflammasome activation, is significantly increased in hypoxia-treated trophoblasts. No evidence was observed for necroptosis-associated events. Importantly, these molecular events in hypoxia-treated human trophoblasts are significantly observed in placental tissue from women with early onset PE. Taken together, we propose that placental pyroptosis is a key event that induces the release of factors into maternal circulation that possibly contribute to severe sterile inflammation and early onset PE pathology.

Cancer progression is characterized by rapidly proliferating cancer cells that are in need of increased protein synthesis. Therefore, enhanced endoplasmic reticulum (ER) activity is required to facilitate the folding, assembly and transportation of membrane and secretory proteins. These functions are carried out by ER chaperones. It is now becoming clear that the ER chaperones have critical functions outside of simply facilitating protein folding. For example, cancer progression requires glucose regulated protein (GRP) 78 for cancer cell survival and proliferation, as well as angiogenesis in the microenvironment. GRP78 can translocate to the cell surface acting as a receptor regulating oncogenic signaling and cell viability. Calreticulin, another ER chaperone, can translocate to the cell surface of apoptotic cancer cells and induce immunogenic cancer cell death and antitumor responses in vivo . Tumor-secreted GRP94 has been shown to elicit antitumor immune responses when used as antitumor vaccines. Protein disulfide isomerase is another ER chaperone that demonstrates pro-oncogenic and pro-survival functions. Because of intrinsic alterations of cellular metabolism and extrinsic factors in the tumor microenvironment, cancer cells are under ER stress, and they respond to this stress by activating the unfolded protein response (UPR). Depending on the severity and duration of ER stress, the signaling branches of the UPR can activate adaptive and pro-survival signals, or induce apoptotic cell death. The protein kinase RNA-like ER kinase signaling branch of the UPR has a dual role in cancer proliferation and survival, and is also required for ER stress-induced autophagy. The activation of the inositol-requiring kinase 1α branch promotes tumorigenesis, cancer cell survival and regulates tumor invasion. In summary, perturbance of ER homeostasis has critical roles in tumorigenesis, and therapeutic modulation of ER chaperones and/or UPR components presents potential antitumor treatments.

Acute protein folding stress in the mitochondrial matrix activates both increased chaperone availability within the matrix and reduced matrix-localized protein synthesis through translational inhibition. Mammalian mitochondrial stress responses The mitochondrial unfolded protein response (UPR mt ) pathway has been studied in detail in the Caenorhabditis elegans roundworm, where it has been shown to sense protein misfolding within the mitochondrial matrix and to induce a program of nuclear gene expression to counteract this stress. How mammalian cells respond to unfolded matrix proteins has remained much less clear. Christian Münch and Wade Harper used pharmacological inhibitors to induce acute protein folding stress in the mitochondrial matrix, and performed transcriptional and quantitative proteomic analysis to examine the response of mammalian cells. They observed widespread induction of nuclear genes, including matrix-localized proteins involved in folding, pre-RNA processing and translation. This was accompanied by a rapid reduction in the matrix-localized protein synthesis through translational inhibition. The work could spur further investigation of the mammalian UPR mt . The mitochondrial matrix is unique in that it must integrate the folding and assembly of proteins derived from the nuclear and mitochondrial genomes. In Caenorhabditis elegans , the mitochondrial unfolded protein response (UPR mt ) senses matrix protein misfolding and induces a program of nuclear gene expression, including mitochondrial chaperonins, to promote mitochondrial proteostasis 1 , 2 , 3 . While misfolded mitochondrial-matrix-localized ornithine transcarbamylase induces chaperonin expression 4 , 5 , 6 , our understanding of mammalian UPR mt is rudimentary 7 , reflecting a lack of acute triggers for UPR mt activation. This limitation has prevented analysis of the cellular responses to matrix protein misfolding and the effects of UPR mt on mitochondrial translation to control protein folding loads. Here we combine pharmacological inhibitors of matrix-localized HSP90/TRAP1 (ref. 8 ) or LON protease 9 , which promote chaperonin expression, with global transcriptional and proteomic analysis to reveal an extensive and acute response of human cells to UPR mt . This response encompasses widespread induction of nuclear genes, including matrix-localized proteins involved in folding, pre-RNA processing and translation. Functional studies revealed rapid but reversible translation inhibition in mitochondria occurring concurrently with defects in pre-RNA processing caused by transcriptional repression and LON-dependent turnover of the mitochondrial pre-RNA processing nuclease MRPP3 (ref. 10 ). This study reveals that acute mitochondrial protein folding stress activates both increased chaperone availability within the matrix and reduced matrix-localized protein synthesis through translational inhibition, and provides a framework for further dissection of mammalian UPR mt .

Idiopathic pulmonary fibrosis (IPF), the most common form of idiopathic interstitial pneumonia, is a progressive, irreversible, and typically lethal disease characterized by an abnormal fibrotic response involving vast areas of the lungs. Given the poor knowledge of the mechanisms underpinning IPF onset and progression, a better understanding of the cellular processes and molecular pathways involved is essential for the development of effective therapies, currently lacking. Besides a number of established IPF-associated risk factors, such as cigarette smoking, environmental factors, comorbidities, and viral infections, several other processes have been linked with this devastating disease. Apoptosis, senescence, epithelial-mesenchymal transition, endothelial-mesenchymal transition, and epithelial cell migration have been shown to play a key role in IPF-associated tissue remodeling. Moreover, molecules, such as chemokines, cytokines, growth factors, adenosine, glycosaminoglycans, non-coding RNAs, and cellular processes including oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, hypoxia, and alternative polyadenylation have been linked with IPF development. Importantly, strategies targeting these processes have been investigated to modulate abnormal cellular phenotypes and maintain tissue homeostasis in the lung. This review provides an update regarding the emerging cellular and molecular mechanisms involved in the onset and progression of IPF.

Key Points The unfolded protein response (UPR) has an important role in the differentiation and maturation of various immune cells and is crucial for immune cell function, such as cytokine production by macrophages and cross-presentation by dendritic cells, for example. Innate immune signalling differentially affects the three arms of the UPR to optimize inflammatory responses, while simultaneously inhibiting the activation of the terminal UPR, which is associated with cell death. This allows the cell to survive and manage temporary increases in protein production during immune responses to pathogens. In complex autoimmune diseases, chronic activation of the UPR can function as the nidus for the development of inflammation. UPR activation triggers inflammatory responses mainly through nuclear factor-κB (NF-κB) activation, phosphorylation of JUN N-terminal kinase (JNK), activation of the inflammasome and direct interaction of downstream UPR targets with the promoters of inflammatory cytokine genes. UPR activation in cancer cells may interfere with antitumour immunity, which indicates that manipulating UPR signalling could boost antitumour immune responses. The UPR is amenable to therapeutic manipulation to either promote its beneficial homeostasis-inducing properties and/or inhibit its inflammation-inducing activities in the setting of unresolved endoplasmic reticulum (ER) stress. The unfolded protein response pathway that is induced by endoplasmic reticulum (ER) stress has important roles in immune cell development and function, which have led to new insights into the pathogenesis of inflammatory diseases. The unfolded protein response (UPR) is a highly conserved pathway that allows the cell to manage endoplasmic reticulum (ER) stress that is imposed by the secretory demands associated with environmental forces. In this role, the UPR has increasingly been shown to have crucial functions in immunity and inflammation. In this Review, we discuss the importance of the UPR in the development, differentiation, function and survival of immune cells in meeting the needs of an immune response. In addition, we review current insights into how the UPR is involved in complex chronic inflammatory diseases and, through its role in immune regulation, antitumour responses.

Key Points Alterations in the function of the endoplasmic reticulum (ER) can result in the accumulation of unfolded or misfolded proteins, a cellular condition referred to as ER stress. ER stress engages the unfolded protein response (UPR), an adaptive reaction that reduces unfolded protein load to maintain cell viability and function. Conditions of chronic or irreversible ER stress trigger cell death by apoptosis, a process that involves the activation of the canonical mitochondrial pathway, which is controlled by the B cell lymphoma 2 (BCL-2) protein family. Chronic ER stress has been linked to the occurrence of many diseases, including cancer, neurodegeneration, ischaemia and diabetes. The UPR is a complex signal transduction pathway that is initiated by the activation of at least three UPR stress sensors: inositol-requiring protein 1 (IRE1), protein kinase RNA-like ER kinase (PERK) and activating transcription factor 6 (ATF6). These sensors controls adaptive process through both transcriptional and non-transcriptional responses, affecting almost every aspect of the secretory pathway, including protein folding, ER biogenesis, ER-associated degradation (ERAD), protein entry to the ER, autophagy and secretion, among others. UPR signalling has distinct kinetics, intensities and downstream consequences depending on the nature and intensity of the stimuli and the cell type involved. These effects may be explained by the modulation of UPR stress sensor activity through interactions with several modulators and adaptor proteins, which possibly results in the assembly of dynamic signalling platforms that have been referred to as 'UPRosomes'. Recent advances in the UPR field indicate that this pathway has important functions in many physiological processes that are not directly related to protein folding. For example, UPR signalling modules crosstalk with signalling pathways that are key in the control of lipid and energy metabolism, innate immunity and cell differentiation programmes. The unfolded protein response (UPR) buffers protein-folding stress at the endoplasmic reticulum (ER). Research has provided insight into the regulatory mechanisms of UPR branches initiated by the stress sensors protein kinase RNA-like ER kinase (PERK), inositol-requiring protein 1α (IRE1α) and activating transcription factor 6 (ATF6), and into outcomes of the UPR that are not directly related to protein misfolding. Protein-folding stress at the endoplasmic reticulum (ER) is a salient feature of specialized secretory cells and is also involved in the pathogenesis of many human diseases. ER stress is buffered by the activation of the unfolded protein response (UPR), a homeostatic signalling network that orchestrates the recovery of ER function, and failure to adapt to ER stress results in apoptosis. Progress in the field has provided insight into the regulatory mechanisms and signalling crosstalk of the three branches of the UPR, which are initiated by the stress sensors protein kinase RNA-like ER kinase (PERK), inositol-requiring protein 1α (IRE1α) and activating transcription factor 6 (ATF6). In addition, novel physiological outcomes of the UPR that are not directly related to protein-folding stress, such as innate immunity, metabolism and cell differentiation, have been revealed.