Attenuation of yeast UPR is essential for survival and is mediated by IRE1 kinase

The unfolded protein response (UPR) activates Ire1, an endoplasmic reticulum (ER) resident transmembrane kinase and ribonuclease (RNase), in response to ER stress. We used an in vivo assay, in which disappearance of the UPR-induced spliced HAC1 messenger ribonucleic acid (mRNA) correlates with the recovery of the ER protein-folding capacity, to investigate the attenuation of the UPR in yeast. We find that, once activated, spliced HAC1 mRNA is sustained in cells expressing Ire1 carrying phosphomimetic mutations within the kinase activation loop, suggesting that dephosphorylation of Ire1 is an important step in RNase deactivation. Additionally, spliced HAC1 mRNA is also sustained after UPR induction in cells expressing Ire1 with mutations in the conserved DFG kinase motif (D828A) or a conserved residue (F842) within the activation loop. The importance of proper Ire1 RNase attenuation is demonstrated by the inability of cells expressing Ire1-D828A to grow under ER stress. We propose that the activity of the Ire1 kinase domain plays a role in attenuating its RNase activity when ER function is recovered.

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Adaptation to Endoplasmic Reticulum Stress Requires Transphosphorylation within the Activation Loop of Protein Kinases Kin1 and Kin2, Orthologs of Human Microtubule Affinity-Regulating Kinase

Molecular and Cellular Biology ◽

10.1128/mcb.00266-18 ◽

2018 ◽

Vol 38 (23) ◽

Author(s):

Chandrima Ghosh ◽

Leena Sathe ◽

Joel David Paprocki ◽

Valerica Raicu ◽

Madhusudan Dey

Keyword(s):

Endoplasmic Reticulum ◽

Er Stress ◽

Cellular Membrane ◽

Kinase Domain ◽

Activation Loop ◽

Content Type ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response ◽

Er Homeostasis

ABSTRACT Perturbations in endoplasmic reticulum (ER) homeostasis, a condition termed ER stress, activate the unfolded protein response (UPR), an intracellular network of signaling pathways. Recently, we have shown that protein kinase Kin1 and its paralog, Kin2, in the budding yeast Saccharomyces cerevisiae (orthologs of microtubule affinity-regulating kinase in humans) contribute to the UPR function. These Kin kinases contain a conserved kinase domain and an autoinhibitory kinase-associated 1 (KA1) domain separated by a long undefined domain. Here, we show that Kin1 or Kin2 protein requires minimally a kinase domain and an adjacent kinase extension region (KER) for UPR function. We also show that the functional mini-Kin2 protein is predominantly visualized inside the cells and precipitated with the cellular membrane fraction, suggesting its association with the cellular endomembrane system. Furthermore, we show that transphosphorylation of the Kin1 residue T302 and the analogous Kin2 residue T281 within the activation loop are important for full kinase activity. Collectively, our data suggest that, during ER stress, the Kin kinase domain is released from its autoinhibitory KA1 domain and is activated by transphosphorylation.

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Phosphoregulation of Ire1 RNase splicing activity

Nature Communications ◽

10.1038/ncomms4554 ◽

2014 ◽

Vol 5 (1) ◽

Cited By ~ 61

Author(s):

Filippo Prischi ◽

Piotr R. Nowak ◽

Marta Carrara ◽

Maruf M. U. Ali

Keyword(s):

Activation Loop ◽

Kinase Activation ◽

Unfolded Protein ◽

Xbp1 Mrna ◽

Phosphorylation Reaction ◽

The Unfolded Protein Response ◽

Protein Response ◽

Kinase Phosphorylation

Abstract Ire1 is activated in response to accumulation of misfolded proteins within the endoplasmic reticulum as part of the unfolded protein response (UPR). It is a unique enzyme, possessing both kinase and RNase activity that is required for specific splicing of Xbp1 mRNA leading to UPR activation. How phosphorylation impacts on the Ire1 splicing activity is unclear. In this study, we isolate distinct phosphorylated species of Ire1 and assess their effects on RNase splicing both in vitro and in vivo. We find that phosphorylation within the kinase activation loop significantly increases RNase splicing in vitro. Correspondingly, mutants of Ire1 that cannot be phosphorylated on the activation loop show decreased specific Xbp1 and promiscuous RNase splicing activity relative to wild-type Ire1 in cells. These data couple the kinase phosphorylation reaction to the activation state of the RNase, suggesting that phosphorylation of the activation loop is an important step in Ire1-mediated UPR activation.

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IRE1β negatively regulates IRE1α signaling in response to endoplasmic reticulum stress

The Journal of Cell Biology ◽

10.1083/jcb.201904048 ◽

2020 ◽

Vol 219 (2) ◽

Cited By ~ 4

Author(s):

Michael J. Grey ◽

Eva Cloots ◽

Mariska S. Simpson ◽

Nicole LeDuc ◽

Yevgeniy V. Serebrenik ◽

...

Keyword(s):

Epithelial Cells ◽

Er Stress ◽

Unfolded Protein Response ◽

Kinase Domain ◽

Dominant Negative ◽

Hek293 Cells ◽

Stress Sensor ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

IRE1β is an ER stress sensor uniquely expressed in epithelial cells lining mucosal surfaces. Here, we show that intestinal epithelial cells expressing IRE1β have an attenuated unfolded protein response to ER stress. When modeled in HEK293 cells and with purified protein, IRE1β diminishes expression and inhibits signaling by the closely related stress sensor IRE1α. IRE1β can assemble with and inhibit IRE1α to suppress stress-induced XBP1 splicing, a key mediator of the unfolded protein response. In comparison to IRE1α, IRE1β has relatively weak XBP1 splicing activity, largely explained by a nonconserved amino acid in the kinase domain active site that impairs its phosphorylation and restricts oligomerization. This enables IRE1β to act as a dominant-negative suppressor of IRE1α and affect how barrier epithelial cells manage the response to stress at the host–environment interface.

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Unfolded Protein Response and Crohn’s Diseases: A Molecular Mechanism of Wound Healing in the Gut

10.20944/preprints202012.0578.v2 ◽

2021 ◽

Author(s):

Chao Li

Keyword(s):

Er Stress ◽

Unfolded Protein Response ◽

Immune Cell ◽

Macrophage Polarization ◽

Intestinal Epithelial ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

Endoplasmic reticulum (ER) stress triggers a series of signaling and transcriptional events termed the unfolded protein response (UPR). Severe ER stress is associated with the development of fibrosis in different organs including lung, liver, kidney, heart, and intestine. ER stress is an essential response of epithelial and immune cells in the pathogenesis of inflammatory bowel disease (IBD) including Crohn’s disease. Intestinal epithelial cells are susceptible to ER stress-mediated damage due to secretion of a large amount of proteins that are involved in mucosal defense. In other cells, ER stress is linked to myofibroblast activation, extracellular matrix production, macrophage polarization, and immune cell differentiation. This review focuses on the role of UPR in the pathogenesis in IBD from an immunologic perspective. The roles of macrophage and mesenchymal cells in the UPR from in vitro and in vivo animal models are discussed. The links between ER stress and other signaling pathways such as senescence and autophagy are introduced. Recent advances in the understanding of the epigenetic regulation of UPR signaling are also updated here. The future directions of development of the UPR research and therapeutic strategies to manipulate ER stress levels are also reviewed.

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Activation of the IRE1α Arm, but not the PERK Arm, of the Unfolded Protein Response Contributes to Fumonisin B1-Induced Hepatotoxicity

Toxins ◽

10.3390/toxins12010055 ◽

2020 ◽

Vol 12 (1) ◽

pp. 55 ◽

Cited By ~ 1

Author(s):

Xiaoyi Liu ◽

Enxiang Zhang ◽

Shutao Yin ◽

Chong Zhao ◽

Lihong Fan ◽

...

Keyword(s):

Oxidative Stress ◽

Er Stress ◽

Unfolded Protein Response ◽

Liver Toxicity ◽

Fumonisin B1 ◽

Critical Event ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

Previous studies by us or others have shown that endoplasmic reticulum (ER) stress was activated by fumonisin 1 (FB1) exposure, which is considered to be a critical event in the FB1-induced toxic effect. However, the detailed mechanisms underlying FB1-induced ER stress-mediated liver toxicity remain elusive. The objectives of the present study were designed to address the following issues: (1) the contribution of each arm of the unfolded protein response (UPR); (2) the downstream targets of ER stress that mediated FB1-induced liver toxicity; and (3) the relationship between ER stress and oxidative stress triggered by FB1. We also investigated whether the inhibition of ER stress by its inhibitor could offer protection against FB1-induced hepatotoxicity in vivo, which has not been critically addressed previously. The results showed that the activation of the IRE1α axis, but not of the PERK axis, of UPR contributed to FB1-induced ER stress-mediated hepatocyte toxicity; the activation of the Bax/Bak-mediated mitochondrial pathway lay downstream of IRE1α to trigger mitochondrial-dependent apoptosis in response to FB1; FB1-induced oxidative stress and ER stress augmented each other through a positive feedback mechanism; tauroursodeoxycholic acid (TUDCA)-mediated ER stress inactivation is an effective approach to counteract FB1-induced hepatotoxicity in vivo. The data of the present study allow us to better understand the mechanisms of FB1-induced hepatotoxicity.

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AMP-Activated Protein Kinase Activation during Cardioplegia-Induced Hypoxia/Reoxygenation Injury Attenuates Cardiomyocytic Apoptosis via Reduction of Endoplasmic Reticulum Stress

Mediators of Inflammation ◽

10.1155/2010/130636 ◽

2010 ◽

Vol 2010 ◽

pp. 1-9 ◽

Cited By ~ 26

Author(s):

Chi-Hsiao Yeh ◽

Tzu-Ping Chen ◽

Yao-Chang Wang ◽

Yu-Min Lin ◽

Shu-Wen Fang

Keyword(s):

Er Stress ◽

Tunel Staining ◽

Ampk Activation ◽

Kinase Activation ◽

Antiapoptotic Proteins ◽

Unfolded Protein ◽

Protein Kinase Activation ◽

The Unfolded Protein Response ◽

Protein Response ◽

Amp Activated Protein Kinase

Cardioplegic-induced H/R injury results in cardiomyocytic apoptosis. AMPK has been shown to reduce ER stress and the unfolded protein response (UPR). Whether AMPK activation can attenuate cardiomyocytic apoptosis after cardioplegia-induced H/R injury is unknown. Cardiomyocytes were exposed to simulated ischemia by incubation in a hypoxic chamber with intermittent cold cardioplegia solution infusion at 20-minute intervals and subsequently reoxygenated in a normoxic environment. Various doses of AMPK activators (AICAR or metformin) were given 2 days before H/R injury. The cardiomyocytes were harvested after reoxygenation for subsequent examination. With both AMPK activators, the antiapoptotic genes of ER stress and UPR, the subsequent production of proapoptotic proteins was attenuated, and the antiapoptotic proteins were elevated. The activity of the apoptotic effectors of ER stress was also reduced with AMPK activation. Moreover, TUNEL staining showed that AMPK activation significantly reduced the percentage of apoptotic cardiomyocytes after cardioplegia-induced H/R injury. Our results revealed that AMPK activation during cardioplegia-induced H/R injury attenuates cardiomyocytic apoptosis, via enhancement of antiapoptotic and reduction of proapoptotic responses, resulting from lessening ER stress and the UPR. AMPK activation may serve as a future pharmacological target to reduce H/R injury in the clinical setting.

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CDNF rescues motor neurons in three animal models of ALS by targeting ER stress

10.1101/2020.05.05.078618 ◽

2020 ◽

Author(s):

Francesca De Lorenzo ◽

Patrick Lüningschrör ◽

Jinhan Nam ◽

Federica Pilotto ◽

Emilia Galli ◽

...

Keyword(s):

Er Stress ◽

Motor Neurons ◽

Motor Behavior ◽

Great Promise ◽

Disease Etiology ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

AbstractThe role of chronic endoplasmic reticulum (ER) stress in the pathophysiology of Amyotrophic lateral sclerosis (ALS), as well as a potential drug target, has received increasing attention. Here, we investigated the mode of action and therapeutic effect of the ER resident protein cerebral dopamine neurotrophic factor (CDNF) in preclinical models of ALS harboring different genetic mutations. We identify that intracerebroventricular (i.c.v.) administration of CDNF significantly halts the progression of the disease and improves motor behavior in TDP43-M337V and SOD1-G93A rodent models of ALS. CDNF rescues motor neurons (MNs) in vitro and in vivo from ER stress associated cell death and its beneficial effect is independent of genetic disease etiology. Notably, CDNF regulates the unfolded protein response (UPR) initiated by transducers IRE1α, PERK, and ATF6, thereby enhancing MN survival. Thus, CDNF holds great promise for the design of new rational treatments for ALS.

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Targeting IRE1 with small molecules counteracts progression of atherosclerosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1621188114 ◽

2017 ◽

Vol 114 (8) ◽

pp. E1395-E1404 ◽

Cited By ~ 84

Author(s):

Ozlem Tufanli ◽

Pelin Telkoparan Akillilar ◽

Diego Acosta-Alvear ◽

Begum Kocaturk ◽

Umut Inci Onat ◽

...

Keyword(s):

Er Stress ◽

Atherosclerotic Plaque ◽

Plasma Lipid ◽

Inflammasome Activation ◽

Low Grade ◽

Unfolded Protein ◽

Cytokines And Chemokines ◽

The Unfolded Protein Response ◽

Protein Response

Metaflammation, an atypical, metabolically induced, chronic low-grade inflammation, plays an important role in the development of obesity, diabetes, and atherosclerosis. An important primer for metaflammation is the persistent metabolic overloading of the endoplasmic reticulum (ER), leading to its functional impairment. Activation of the unfolded protein response (UPR), a homeostatic regulatory network that responds to ER stress, is a hallmark of all stages of atherosclerotic plaque formation. The most conserved ER-resident UPR regulator, the kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1), is activated in lipid-laden macrophages that infiltrate the atherosclerotic lesions. Using RNA sequencing in macrophages, we discovered that IRE1 regulates the expression of many proatherogenic genes, including several important cytokines and chemokines. We show that IRE1 inhibitors uncouple lipid-induced ER stress from inflammasome activation in both mouse and human macrophages. In vivo, these IRE1 inhibitors led to a significant decrease in hyperlipidemia-induced IL-1β and IL-18 production, lowered T-helper type-1 immune responses, and reduced atherosclerotic plaque size without altering the plasma lipid profiles in apolipoprotein E-deficient mice. These results show that pharmacologic modulation of IRE1 counteracts metaflammation and alleviates atherosclerosis.

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Unfolded Protein Response and Crohn’s Diseases: A Molecular Mechanism of Wound Healing in the Gut

Gastrointestinal Disorders ◽

10.3390/gidisord3010004 ◽

2021 ◽

Vol 3 (1) ◽

pp. 31-43

Author(s):

Chao Li

Keyword(s):

Er Stress ◽

Unfolded Protein Response ◽

Immune Cell ◽

Macrophage Polarization ◽

Intestinal Epithelial ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

Endoplasmic reticulum (ER) stress triggers a series of signaling and transcriptional events termed the unfolded protein response (UPR). Severe ER stress is associated with the development of fibrosis in different organs, including lung, liver, kidney, heart, and intestine. ER stress is an essential response of epithelial and immune cells in the pathogenesis of Inflammatory Bowel Disease (IBD), including Crohn’s disease (CD). Intestinal epithelial cells are susceptible to ER stress-mediated damage due to secretion of a large amount of proteins that are involved in mucosal defense. In other cells, ER stress is linked to myofibroblast activation, extracellular matrix production, macrophage polarization, and immune cell differentiation. This review focuses on the role of the UPR in the pathogenesis in IBD from an immunologic perspective. The roles of macrophage and mesenchymal cells in the UPR from in vitro and in vivo animal models are discussed. The links between ER stress and other signaling pathways, such as senescence and autophagy, are introduced. Recent advances in the understanding of the epigenetic regulation of the UPR signaling are also updated here. The future directions of development of the UPR research and therapeutic strategies to manipulate ER stress levels are also reviewed.

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The Role of Mitochondrial Dysfunction and ER Stress in TDP-43 and C9ORF72 ALS

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2021.653688 ◽

2021 ◽

Vol 15 ◽

Author(s):

Ruxandra Dafinca ◽

Paola Barbagallo ◽

Kevin Talbot

Keyword(s):

Er Stress ◽

Mitochondrial Dysfunction ◽

Unfolded Protein Response ◽

Nucleocytoplasmic Transport ◽

Cellular Processes ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the motor system with complex determinants, including genetic and non-genetic factors. Despite this heterogeneity, a key pathological signature is the mislocalization and aggregation of specific proteins in the cytoplasm, suggesting that convergent pathogenic mechanisms focusing on disturbances in proteostasis are important in ALS. In addition, many cellular processes have been identified as potentially contributing to disease initiation and progression, such as defects in axonal transport, autophagy, nucleocytoplasmic transport, ER stress, calcium metabolism, the unfolded protein response and mitochondrial function. Here we review the evidence from in vitro and in vivo models of C9ORF72 and TDP-43-related ALS supporting a central role in pathogenesis for endoplasmic reticulum stress, which activates an unfolded protein response (UPR), and mitochondrial dysfunction. Disruption in the finely tuned signaling between the ER and mitochondria through calcium ions may be a crucial trigger of mitochondrial deficits and initiate an apoptotic signaling cascade, thus acting as a point of convergence for multiple upstream disturbances of cellular homeostasis and constituting a potentially important therapeutic target.

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