scholarly journals FICD acts bi-functionally to AMPylate and de-AMPylate the endoplasmic reticulum chaperone BiP

2016 ◽  
Author(s):  
Steffen Preissler ◽  
Claudia Rato ◽  
Luke Perera ◽  
Vladimir Saudek ◽  
David Ron
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Active Site ◽  
Enzymatic Reaction ◽  
Covalent Modification ◽  
Unfolded Proteins ◽  
Unfolded Protein ◽  
Er Chaperone

Significance statementSome 25 years ago it was discovered that the activity of the ER chaperone BiP is regulated by covalent modification, the nature of which, AMPylation (not ADPribosylation, as had long been thought) and the enzyme responsible, FICD, have only recently been identified. Genetic inactivation of FICD and in vitro studies of the purified enzyme and substrate have done much to clarify the biochemical consequences of the modification and its underlying logic: As ER stress wanes, FICD uses ATP to AMPylate Thr518 of BiP locking BiP in a relatively inactive conformation. As ER stress levels re-mount the cells draw on this pool of inactive chaperone, which is de-AMPylated and restored to its fully active state.Here we report on the identity of the de-AMPylating enzyme - and with it on the surprising finding that both AMPylation and de-AMPylation of BiP are carried out by the same polypeptide (FICD) using the same active site, both in vivo and in vitro. Analysis of the reaction products reveals that de-AMPylation does not involve trivial concentration-dependent micro-reversibility of an enzymatic reaction, but rather a switch in the active site of FICD that facilitates two antagonistic thermodynamically favored reactions.Surprisingly BiP de-AMPylation (not AMPylation) is the default activity of FICD. The side-chain of a single regulatory residue, E234, toggles the enzyme between de-AMPylation and AMPylation in vitro. Our studies thereby uncover an active mechanism that must exist in the ER for coupling waning levels of unfolded protein stress to the conversion of FICD from its default de-AMPylation mode to BiP AMPylation. Whilst the details of this active switch remain to be discovered, we are able to suggest a plausible mechanism by which it may come about.Identification of the enzyme that de-modifies BiP to reactivate it will be of interest to cell biologists, whereas the novel features of FICD as a dualfunctioning enzyme with a single bi-functional active site will be of broad interest to enzymologists and molecular biologists.AbstractProtein folding homeostasis in the endoplasmic reticulum (ER) is defended by an unfolded protein response (UPR) that matches ER chaperone capacity to the burden of unfolded proteins. As levels of unfolded proteins decline, a metazoanspecific FIC-domain containing ER-localized enzyme, FICD/HYPE, rapidly inactivates the major ER chaperone BiP by AMPylating T518. Here it is shown that the single catalytic domain of FICD can also release the attached AMP, restoring functionality to BiP. Consistent with a role for endogenous FICD in de-AMPylating BiP, FICD−/− cells are hypersensitive to introduction of a constitutively AMPylating, de-AMPylation defective mutant FICD. These opposing activities hinge on a regulatory residue, E234, whose default state renders FICD a constitutive de-AMPylase in vitro. The location of E234 on a conserved regulatory helix and the mutually antagonistic activities of FICD in vivo, suggest a mechanism whereby fluctuating unfolded protein load actively switches FICD from a de-AMPylase to an AMPylase.

scholarly journals A Novel Transcription Factor, ERD15 (Early Responsive to Dehydration 15), Connects Endoplasmic Reticulum Stress with an Osmotic Stress-induced Cell Death Signal

2011 ◽  
Vol 286 (22) ◽  
pp. 20020-20030 ◽  
Author(s):  
Murilo S. Alves ◽  
Pedro A. B. Reis ◽  
Silvana P. Dadalto ◽  
Jerusa A. Q. A. Faria ◽  
Elizabeth P. B. Fontes ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Transcription Factor ◽  
Osmotic Stress ◽  
Er Stress ◽  
Unfolded Protein ◽  
Eukaryotic Organisms ◽  
Protein Response

As in all other eukaryotic organisms, endoplasmic reticulum (ER) stress triggers the evolutionarily conserved unfolded protein response in soybean, but it also communicates with other adaptive signaling responses, such as osmotic stress-induced and ER stress-induced programmed cell death. These two signaling pathways converge at the level of gene transcription to activate an integrated cascade that is mediated by N-rich proteins (NRPs). Here, we describe a novel transcription factor, GmERD15 (Glycine max Early Responsive to Dehydration 15), which is induced by ER stress and osmotic stress to activate the expression of NRP genes. GmERD15 was isolated because of its capacity to stably associate with the NRP-B promoter in yeast. It specifically binds to a 187-bp fragment of the NRP-B promoter in vitro and activates the transcription of a reporter gene in yeast. Furthermore, GmERD15 was found in both the cytoplasm and the nucleus, and a ChIP assay revealed that it binds to the NRP-B promoter in vivo. Expression of GmERD15 in soybean protoplasts activated the NRP-B promoter and induced expression of the NRP-B gene. Collectively, these results support the interpretation that GmERD15 functions as an upstream component of stress-induced NRP-B-mediated signaling to connect stress in the ER to an osmotic stress-induced cell death signal.

An oligomeric state-dependent switch in FICD regulates AMPylation and deAMPylation of the chaperone BiP

2019 ◽  
Author(s):  
Luke A. Perera ◽  
Claudia Rato ◽  
Yahui Yan ◽  
Lisa Neidhardt ◽  
Stephen H. McLaughlin ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Active Site ◽  
Covalent Modification ◽  
Energy Status ◽  
Oligomeric State ◽  
Dimer Interface ◽  
Er Chaperone ◽  
State Dependent ◽  
Repressive Effect

AbstractAMPylation is an inactivating modification that matches the activity of the major endoplasmic reticulum (ER) chaperone BiP to the burden of unfolded proteins. A single ER-localised Fic protein, FICD (HYPE), catalyses both AMPylation and deAMPylation of BiP. However, the basis for the switch in FICD’s activity is unknown. We report on the transition of FICD from a dimeric enzyme, that deAMPylates BiP, to a monomer with potent AMPylation activity. Mutations in the dimer interface or in residues tracing an inhibitory relay from the dimer interface to the enzyme’s active site favour BiP AMPylation in vitro and in cells. Mechanistically, monomerisation relieves a repressive effect allosterically-propagated from the dimer interface to the inhibitory Glu234, thereby permitting AMPylation-competent binding of MgATP. Whereas, a reciprocal signal propagated from the nucleotide binding site, provides a mechanism for coupling the oligomeric-state and enzymatic activity of FICD to the energy status of the ER.Impact StatementUnique amongst known chaperones, the endoplasmic reticulum (ER)-localized Hsp70, BiP, is subject to transient inactivation under conditions of low ER stress by reversible, covalent modification – AMPylation. The enzyme responsible for this modification, FICD, is in fact a bifunctional enzyme with a single active site capable of both AMPylation and deAMPylation. Here we elucidate, by biochemical, biophysical and structural means, the mechanism by which this enzyme is able to switch enzymatic modality: by regulation of its oligomeric state. The oligomeric state-dependent reciprocal regulation of FICD activity is, in turn, sensitive to the ATP/ADP ratio. This allosteric pathway potentially facilitates the sensing of unfolded protein load in the ER and permits the transduction of this signal into a post-translational buffering of ER chaperone activity.

scholarly journals AMPylation matches BiP activity to client protein load in the endoplasmic reticulum

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Steffen Preissler ◽  
Cláudia Rato ◽  
Ruming Chen ◽  
Robin Antrobus ◽  
Shujing Ding ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammalian Cells ◽  
Atp Hydrolysis ◽  
Covalent Modification ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Unfolded Protein ◽  
Peptide Dissociation

The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP affects protein folding homeostasis and the response to ER stress. Reversible inactivating covalent modification of BiP is believed to contribute to the balance between chaperones and unfolded ER proteins, but the nature of this modification has so far been hinted at indirectly. We report that deletion of FICD, a gene encoding an ER-localized AMPylating enzyme, abolished detectable modification of endogenous BiP enhancing ER buffering of unfolded protein stress in mammalian cells, whilst deregulated FICD activity had the opposite effect. In vitro, FICD AMPylated BiP to completion on a single residue, Thr518. AMPylation increased, in a strictly FICD-dependent manner, as the flux of proteins entering the ER was attenuated in vivo. In vitro, Thr518 AMPylation enhanced peptide dissociation from BiP 6-fold and abolished stimulation of ATP hydrolysis by J-domain cofactor. These findings expose the molecular basis for covalent inactivation of BiP.

Mechanisms of disordered neurodegenerative function: concepts and facts about the different roles of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)

2018 ◽  
Vol 29 (4) ◽  
pp. 387-415 ◽  
Author(s):  
Yasmeen M. Taalab ◽  
Nour Ibrahim ◽  
Ahmed Maher ◽  
Mubashir Hassan ◽  
Wael Mohamed ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Kinase ◽  
Neurodegenerative Diseases ◽  
Er Stress ◽  
Protein Misfolding ◽  
Disease Models ◽  
Downstream Signaling ◽  
Unfolded Protein

AbstractNeurodegenerative diseases, such as Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, prion disease, and amyotrophic lateral sclerosis, are a dissimilar group of disorders that share a hallmark feature of accumulation of abnormal intraneuronal or extraneuronal misfolded/unfolded protein and are classified as protein misfolding disorders. Cellular and endoplasmic reticulum (ER) stress activates multiple signaling cascades of the unfolded protein response (UPR). Consequently, translational and transcriptional alterations in target gene expression occur in response directed toward restoring the ER capacity of proteostasis and reestablishing the cellular homeostasis. Evidences fromin vitroandin vivodisease models indicate that disruption of ER homeostasis causes abnormal protein aggregation that leads to synaptic and neuronal dysfunction. However, the exact mechanism by which it contributes to disease progression and pathophysiological changes remains vague. Downstream signaling pathways of UPR are fully integrated, yet with diverse unexpected outcomes in different disease models. Three well-identified ER stress sensors have been implicated in UPR, namely, inositol requiring enzyme 1, protein kinase RNA-activated-like ER kinase (PERK), and activating transcription factor 6. Although it cannot be denied that each of the involved stress sensor initiates a distinct downstream signaling pathway, it becomes increasingly clear that shared pathways are crucial in determining whether or not the UPR will guide the cells toward adaptive prosurvival or proapoptotic responses. We review a body of work on the mechanism of neurodegenerative diseases based on oxidative stress and cell death pathways with emphasis on the role of PERK.

scholarly journals GnRHa protects the ovarian reserve by reducing endoplasmic reticulum stress during cyclophosphamide-based chemotherapy

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Xiaolin Li ◽  
Sixuan Liu ◽  
Xuan Chen ◽  
Run Huang ◽  
Lisi Ma ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Er Stress ◽  
Molecular Mechanisms ◽  
Ovarian Function ◽  
Mtor Signaling ◽  
Unfolded Protein ◽  
Hormone Analogs

AbstractChemotherapy-induced ovarian dysfunction is a serious adverse effect in premenopausal patients with cancer. Gonadotrophin-releasing hormone analogs (GnRHa) protect ovarian function, but its molecular mechanisms have not yet been determined. In this study, we attempted to determine the previously unknown molecular mechanism by which such protection occurs. Serum anti-Müllerian hormone (AMH) levels were tested in tumor-bearing nude mice, a series of exploratory experiments were conducted. We discovered that GnRHa protects granulosa cells from chemotherapeutic toxicity in vivo and in vitro. We also showed that CTX-induced endoplasmic reticulum stress inhibits the secretion of AMH, and treatment with GnRHa relieves ER stress and the subsequent unfolded-protein response by modulating mTOR signaling to induce autophagy. The results of mechanistic studies indicated that GnRHa-modulated mTOR signaling to induce autophagy, which alleviated CTX-induced ER stress and promoted the secretion of AMH.

scholarly journals Dehydrodiisoeugenol inhibits colorectal cancer growth by endoplasmic reticulum stress-induced autophagic pathways

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Changhong Li ◽  
Kui Zhang ◽  
Guangzhao Pan ◽  
Haoyan Ji ◽  
Chongyang Li ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Endoplasmic Reticulum ◽  
Cell Growth ◽  
Er Stress ◽  
Cancer Cells ◽  
Tumor Xenograft ◽  
Anticancer Activities ◽  
Colorectal Cancer Cells

Abstract Background Dehydrodiisoeugenol (DEH), a novel lignan component extracted from nutmeg, which is the seed of Myristica fragrans Houtt, displays noticeable anti-inflammatory and anti-allergic effects in digestive system diseases. However, the mechanism of its anticancer activity in gastrointestinal cancer remains to be investigated. Methods In this study, the anticancer effect of DEH on human colorectal cancer and its underlying mechanism were evaluated. Assays including MTT, EdU, Plate clone formation, Soft agar, Flow cytometry, Electron microscopy, Immunofluorescence and Western blotting were used in vitro. The CDX and PDX tumor xenograft models were used in vivo. Results Our findings indicated that treatment with DEH arrested the cell cycle of colorectal cancer cells at the G1/S phase, leading to significant inhibition in cell growth. Moreover, DEH induced strong cellular autophagy, which could be inhibited through autophagic inhibitors, with a rction in the DEH-induced inhibition of cell growth in colorectal cancer cells. Further analysis indicated that DEH also induced endoplasmic reticulum (ER) stress and subsequently stimulated autophagy through the activation of PERK/eIF2α and IRE1α/XBP-1 s/CHOP pathways. Knockdown of PERK or IRE1α significantly decreased DEH-induced autophagy and retrieved cell viability in cells treated with DEH. Furthermore, DEH also exhibited significant anticancer activities in the CDX- and PDX-models. Conclusions Collectively, our studies strongly suggest that DEH might be a potential anticancer agent against colorectal cancer by activating ER stress-induced inhibition of autophagy.

scholarly journals Biochanin A Alleviates Cerebral Ischemia/Reperfusion Injury by Suppressing Endoplasmic Reticulum Stress-Induced Apoptosis and p38MAPK Signaling Pathway In Vivo and In Vitro

2021 ◽  
Vol 12 ◽  
Author(s):  
Min-min Guo ◽  
Sheng-biao Qu ◽  
Hui-ling Lu ◽  
Wen-bo Wang ◽  
Mu-Liang He ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
P38 Mapk ◽  
Ischemia Reperfusion ◽  
Biochanin A ◽  
Neurological Deficits ◽  
Mapk Phosphorylation ◽  
Cerebral Ischemia Reperfusion

We have previously shown that biochanin A exhibits neuroprotective properties in the context of cerebral ischemia/reperfusion (I/R) injury. The mechanistic basis for such properties, however, remains poorly understood. This study was therefore designed to explore the manner whereby biochanin A controls endoplasmic reticulum (ER) stress, apoptosis, and inflammation within fetal rat primary cortical neurons in response to oxygen-glucose deprivation/reoxygenation (OGD/R) injury, and in a rat model of middle cerebral artery occlusion and reperfusion (MCAO/R) injury. For the OGD/R in vitro model system, cells were evaluated after a 2 h OGD following a 24 h reoxygenation period, whereas in vivo neurological deficits were evaluated following 2 h of ischemia and 24 h of reperfusion. The expression of proteins associated with apoptosis, ER stress (ERS), and p38 MAPK phosphorylation was evaluated in these samples. Rats treated with biochanin A exhibited reduced neurological deficits relative to control rats following MCAO/R injury. Additionally, GRP78 and CHOP levels rose following I/R modeling both in vitro and in vivo, whereas biochanin A treatment was associated with reductions in CHOP levels but further increases in GRP78 levels. In addition, OGD/R or MCAO/R were associated with markedly enhanced p38 MAPK phosphorylation that was alleviated by biochanin A treatment. Similarly, OGD/R or MCAO/R injury resulted in increases in caspase-3, caspase-12, and Bax levels as well as decreases in Bcl-2 levels, whereas biochanin A treatment was sufficient to reverse these phenotypes. Together, these findings thus demonstrate that biochanin A can alleviate cerebral I/R-induced damage at least in part via suppressing apoptosis, ER stress, and p38 MAPK signaling, thereby serving as a potent neuroprotective agent.

scholarly journals Activation of Mammalian Unfolded Protein Response Is Compatible with the Quality Control System Operating in the Endoplasmic Reticulum

2004 ◽  
Vol 15 (6) ◽  
pp. 2537-2548 ◽  
Author(s):  
Satomi Nadanaka ◽  
Hiderou Yoshida ◽  
Fumi Kano ◽  
Masayuki Murata ◽  
Kazutoshi Mori
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Control System ◽  
Golgi Apparatus ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Secretory Pathway ◽  
Unfolded Proteins ◽  
Unfolded Protein ◽  
Protein Response

Newly synthesized secretory and transmembrane proteins are folded and assembled in the endoplasmic reticulum (ER) where an efficient quality control system operates so that only correctly folded molecules are allowed to move along the secretory pathway. The productive folding process in the ER has been thought to be supported by the unfolded protein response (UPR), which is activated by the accumulation of unfolded proteins in the ER. However, a dilemma has emerged; activation of ATF6, a key regulator of mammalian UPR, requires intracellular transport from the ER to the Golgi apparatus. This suggests that unfolded proteins might be leaked from the ER together with ATF6 in response to ER stress, exhibiting proteotoxicity in the secretory pathway. We show here that ATF6 and correctly folded proteins are transported to the Golgi apparatus via the same route and by the same mechanism under conditions of ER stress, whereas unfolded proteins are retained in the ER. Thus, activation of the UPR is compatible with the quality control in the ER and the ER possesses a remarkable ability to select proteins to be transported in mammalian cells in marked contrast to yeast cells, which actively utilize intracellular traffic to deal with unfolded proteins accumulated in the ER.

scholarly journals Pentoxifylline Attenuates Methionine- and Choline-Deficient-Diet-Induced Steatohepatitis by Suppressing TNF-αExpression and Endoplasmic Reticulum Stress

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Min Kyung Chae ◽  
Sang Gyu Park ◽  
Sun-Ok Song ◽  
Eun Seok Kang ◽  
Bong Soo Cha ◽  
...  
Keyword(s):  
Er Stress ◽  
Deficient Diet ◽  
Group Iii ◽  
Unfolded Protein ◽  
Sd Rats ◽  
Group I ◽  
Hep3b Cells ◽  
Protein Response

Background. Pentoxifylline (PTX) anti-TNF properties are known to exert hepatoprotective effects in various liver injury models. The aim of this study was to investigate whether PTX has beneficial roles in the development of methionine- and choline-deficient-(MCD-) diet-induced NAFLD SD ratsin vivoand TNF-α-induced Hep3B cellsin vitro.Methods. SD Rats were classified according to diet (chow or MCD diet) and treatment (normal saline or PTX injection) over a period of 4 weeks: group I (chow + saline,n=4), group II (chow + PTX), group III (MCD + saline), and group IV (MCD + PTX). Hep3B cells were treated with 100 ng/ml TNF-α(24 h) in the absence or presence of PTX (1 mM).Results. PTX attenuated MCD-diet-induced serum ALT levels and hepatic steatosis. In real-time PCR and western blotting analysis, PTX decreased MCD-diet-induced TNF-alpha mRNA expression and proapoptotic unfolded protein response by ER stress (GRP78, p-eIF2, ATF4, IRE1α, CHOP, and p-JNK activation)in vivo. PTX (1 mM) reduced TNF-α-induced activation of GRP78, p-eIF2, ATF4, IRE1α, and CHOPin vitro.Conclusion. PTX has beneficial roles in the development of MCD-diet-induced steatohepatitis through partial suppression of TNF-αand ER stress.

scholarly journals Regulation of Adipsin Expression by Endoplasmic Reticulum Stress in Adipocytes

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 314
Author(s):  
Ka-Young Ryu ◽  
Eon Ju Jeon ◽  
Jaechan Leem ◽  
Jae-Hyung Park ◽  
Hochan Cho
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Peroxisome Proliferator ◽  
Adipose Tissues ◽  
Peroxisome Proliferator Activated Receptor ◽  
Er Stress Response ◽  
Stress Markers ◽  
Transcriptional Reprogramming

Adpsin is an adipokine that stimulates insulin secretion from β-cells and improves glucose tolerance. Its expression has been found to be markedly reduced in obese animals. However, it remains unclear what factors lead to downregulation of adipsin in the context of obesity. Endoplasmic reticulum (ER) stress response is activated in various tissues under obesity-related conditions and can induce transcriptional reprogramming. Therefore, we aimed to investigate the relationship between adipsin expression and ER stress in adipose tissues during obesity. We observed that obese mice exhibited decreased levels of adipsin in adipose tissues and serum and increased ER stress markers in adipose tissues compared to lean mice. We also found that ER stress suppressed adipsin expression via adipocytes-intrinsic mechanisms. Moreover, the ER stress-mediated downregulation of adipsin was at least partially attributed to decreased expression of peroxisome proliferator-activated receptor γ (PPARγ), a key transcription factor in the regulation of adipocyte function. Finally, treatment with chemical chaperones recovered the ER stress-mediated downregulation of adipsin and PPARγ in vivo and in vitro. Our findings suggest that activated ER stress in adipose tissues is an important cause of the suppression of adipsin expression in the context of obesity.

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