Calcium Sensors ALG-2 and Peflin Bind ER Exit Sites in Alternate States to Modulate Secretion in Response to Calcium Signaling

2020 ◽  
Author(s):  
John Sargeant ◽  
Tucker Costain ◽  
Corina Madreiter-Sokolowski ◽  
David E. Gordon ◽  
Andrew A. Peden ◽  
...  
Keyword(s):  
Expression Ratio ◽  
Linked Gene ◽  
Unfolded Protein ◽  
Calcium Sensors ◽  
Ef Hand ◽  
Er Exit Sites ◽  
Alternate States ◽  
Stressed Cells ◽  
Er Export ◽  
Protein Response

ABSTRACTPenta EF-hand (PEF) proteins apoptosis-linked gene 2 (ALG-2) and peflin are cytoplasmic Ca2+ sensors with emerging functions in secretion. Here we demonstrate that adjustment of the ALG-2:peflin expression ratio can modulate ER export rates up or down by 107% of the basal rate. Through their ALG-2 subunit, ALG-2-peflin hetero-oligomers are shown to bind ERES to inhibit ER export of multiple cargo types, including collagen I. Conversely, without peflin, ALG-2 binds ERES to stimulate ER export. In a novel physiological response to sustained, agonist-driven ER Ca2+ signaling, PEF protein rearrangements at ERES alter the COPII outer coat to sharply decrease ER export rates. Though it is assumed that this response is pro-survival in the short term, in highly Ca2+-stressed cells, peflin’s suppressive role promotes pro-apoptotic unfolded protein response (UPR) signaling. Regulation of secretion by PEF protein subcomplexes in response to Ca2+ signals thus impacts cellular decisions relevant to many diseases.

Alterations in an IRE1-RNA complex in the mammalian unfolded protein response

2001 ◽  
Vol 114 (17) ◽  
pp. 3207-3212
Author(s):  
Anne Bertolotti ◽  
David Ron
Keyword(s):  
Unfolded Protein Response ◽  
Mammalian Cells ◽  
Dynamic Change ◽  
Rna Molecules ◽  
Unfolded Protein ◽  
Rna Fragments ◽  
Stressed Cells ◽  
Rna Complex ◽  
Protein Response

IRE1 proteins mediate cellular responses to accumulation of malfolded proteins in the endoplasmic reticulum in the yeast and mammalian unfolded protein responses. A sensitive in vivo u.v. crosslinking assay showed that IRE1 proteins are intimately associated with RNA in mammalian cells. The IRE1-associated RNA fragments recovered by this assay were different in stressed and unstressed cells. The amount of RNA associated with IRE1 that could be revealed by end-labeling with T4 kinase was greater in IRE1-containing complexes isolated from stressed cells. Furthermore, the RNA fragments recovered from complexes found in stressed cells were shorter than those from unstressed cells, revealing a dynamic change in the IRE1-RNA complex during the UPR. Formation of the complex between IRE1 and RNA was dependent on both the kinase and endonuclease domains of IRE1, and involved pre-existing RNA species. When viewed in the context of the known importance of Ire1p-HAC1 mRNA interactions to the yeast unfolded protein response, these findings suggest that full-length mammalian IRE1s also engage RNA molecules as downstream effectors.

scholarly journals Regulated Ire1-dependent mRNA decay requires no-go mRNA degradation to maintain endoplasmic reticulum homeostasis in S. pombe

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Nicholas R Guydosh ◽  
Philipp Kimmig ◽  
Peter Walter ◽  
Rachel Green
Keyword(s):  
Endoplasmic Reticulum ◽  
Critical Role ◽  
Mrna Degradation ◽  
Ribosome Profiling ◽  
Unfolded Protein ◽  
Selective Degradation ◽  
Stressed Cells ◽  
Novel Target ◽  
The Unfolded Protein Response ◽  
Protein Response

The unfolded protein response (UPR) monitors and adjusts the protein folding capacity of the endoplasmic reticulum (ER). In S. pombe, the ER membrane-resident kinase/endoribonuclease Ire1 utilizes a mechanism of selective degradation of ER-bound mRNAs (RIDD) to maintain homeostasis. We used a genetic screen to identify factors critical to the Ire1-mediated UPR and found several proteins, Dom34, Hbs1 and Ski complex subunits, previously implicated in ribosome rescue and mRNA no-go-decay (NGD). Ribosome profiling in ER-stressed cells lacking these factors revealed that Ire1-mediated cleavage of ER-associated mRNAs results in ribosome stalling and mRNA degradation. Stalled ribosomes iteratively served as a ruler to template precise, regularly spaced upstream mRNA cleavage events. This clear signature uncovered hundreds of novel target mRNAs. Our results reveal that the UPR in S. pombe executes RIDD in an intricate interplay between Ire1, translation, and the NGD pathway, and establish a critical role for NGD in maintaining ER homeostasis.

ER stress and the unfolded protein response in intestinal inflammation

2010 ◽  
Vol 298 (6) ◽  
pp. G820-G832 ◽  
Author(s):  
Michael A. McGuckin ◽  
Rajaraman D. Eri ◽  
Indrajit Das ◽  
Rohan Lourie ◽  
Timothy H. Florin
Keyword(s):  
Er Stress ◽  
Unfolded Protein Response ◽  
Protein Misfolding ◽  
Intestinal Inflammation ◽  
Secretory Cells ◽  
Unfolded Protein ◽  
Bowel Diseases ◽  
Stressed Cells ◽  
The Unfolded Protein Response ◽  
Protein Response

Endoplasmic reticulum (ER) stress is a phenomenon that occurs when excessive protein misfolding occurs during biosynthesis. ER stress triggers a series of signaling and transcriptional events known as the unfolded protein response (UPR). The UPR attempts to restore homeostasis in the ER but if unsuccessful can trigger apoptosis in the stressed cells and local inflammation. Intestinal secretory cells are susceptible to ER stress because they produce large amounts of complex proteins for secretion, most of which are involved in mucosal defense. This review focuses on ER stress in intestinal secretory cells and describes how increased protein misfolding could occur in these cells, the process of degradation of misfolded proteins, the major molecular elements of the UPR pathway, and links between the UPR and inflammation. Evidence is reviewed from mouse models and human inflammatory bowel diseases that ties ER stress and activation of the UPR with intestinal inflammation, and possible therapeutic approaches to ameliorate ER stress are discussed.

scholarly journals Steady-state Regulation of Secretory Cargo Export by Inositol Trisphosphate Receptors and Penta EF Hand Proteins

2020 ◽  
Author(s):  
Aaron Held ◽  
Jennet Hojanazarova ◽  
John Sargeant ◽  
Corina Madreiter-Sokolowski ◽  
Roland Mali ◽  
...  
Keyword(s):  
Steady State ◽  
Inositol Trisphosphate ◽  
State Regulation ◽  
Outer Coat ◽  
Inositol Trisphosphate Receptors ◽  
Golgi Transport ◽  
Ef Hand ◽  
Er Exit Sites ◽  
Er Export ◽  
Partial Depletion

ABSTRACTER Ca2+ regulates ER-to-Golgi transport machinery. Sustained Ca2+ signaling by inositol trisphosphate receptors (IP3Rs) leads to depression of cargo export through activation of penta EF hand protein (PEF) ALG-2 which reduces outer COPII coat at ER exit sites (ERES). However, we do not know whether tonic Ca2+ signals during steady-state conditions affect ER export rates and if so by what mechanisms. Here we report that partial depletion of IP3 receptors from NRK epithelial cells causes a marked increase of basal ER export of the transmembrane glycoprotein cargo VSV-G. The increased ER-to-Golgi transport required ALG-2 and was actuated by decreased peflin and increased ALG-2 at ER exit sites (ERES) – a condition previously demonstrated to stimulate COPII-dependent ER export. Upon IP3R depletion the amount of outer coat at ERES increased, the opposite to what occurs during ALG-2-dependent inhibition of secretion during agonist-driven Ca2+ signaling. The increased ER export correlated with reduced spontaneous cytosolic Ca2+ oscillations caused by the reduced number of Ca2+ release channels. IP3R depletion also unexpectedly resulted in partial depletion of ER luminal Ca2+ stores. The low Ca2+ conditions appeared to decrease both ALG-2 and peflin expression levels somewhat, but these were the only detectable expression changes in COPII trafficking machinery and the Ca2+ decrease had no detectable impact on ER stress. We conclude that at steady state, IP3Rs produce tonic Ca2+ signals that suppress the basal rate of ER export by maintaining lower outer coat targeting to ERES.

scholarly journals Localization of GRP78 to mitochondria under the unfolded protein response

2006 ◽  
Vol 396 (1) ◽  
pp. 31-39 ◽  
Author(s):  
Fang-Chun Sun ◽  
Shou Wei ◽  
Chia-Wei Li ◽  
Yuo-Sheng Chang ◽  
Chih-Chung Chao ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Unfolded Protein Response ◽  
Calcium Ionophore ◽  
Ionophore A23187 ◽  
Intermembrane Space ◽  
Atpase Inhibitor ◽  
Unfolded Protein ◽  
Stressed Cells ◽  
Protein Response ◽  
In Cells

The ubiquitously expressed molecular chaperone GRP78 (78 kDa glucose-regulated protein) generally localizes to the ER (endoplasmic reticulum). GRP78 is specifically induced in cells under the UPR (unfolded protein response), which can be elicited by treatments with calcium ionophore A23187 and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor TG (thapsigargin). By using confocal microscopy, we have demonstrated that GRP78 was concentrated in the perinuclear region and co-localized with the ER marker proteins, calnexin and PDI (protein disulphide-isomerase), in cells under normal growth conditions. However, treatments with A23187 and TG led to diminish its ER targeting, resulting in redirection into a cytoplasmic vesicular pattern, and overlapping with the mitochondrial marker MitoTracker. Cellular fractionation and protease digestion of isolated mitochondria from ER-stressed cells suggested that a significant portion of GRP78 is localized to the mitochondria and is protease-resistant. Localizations of GRP78 in ER and mitochondria were confirmed by using immunoelectron microscopy. In ER-stressed cells, GRP78 mainly localized within the mitochondria and decorated the mitochondrial membrane compartment. Submitochondrial fractionation studies indicated further that the mitochondria-resided GRP78 is mainly located in the intermembrane space, inner membrane and matrix, but is not associated with the outer membrane. Furthermore, radioactive labelling followed by subcellular fractionation showed that a significant portion of the newly synthesized GRP78 is localized to the mitochondria in cells under UPR. Taken together, our results indicate that, at least under certain circumstances, the ER-resided chaperone GRP78 can be retargeted to mitochondria and thereby may be involved in correlating UPR signalling between these two organelles.

scholarly journals The Unfolded Protein Response: An Overview

Biology ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 384
Author(s):  
Adam Read ◽  
Martin Schröder
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Unfolded Protein Response ◽  
Protein Homeostasis ◽  
Unfolded Proteins ◽  
Unfolded Protein ◽  
Altered Glycosylation ◽  
Stressed Cells ◽  
The Unfolded Protein Response ◽  
Protein Response

The unfolded protein response is the mechanism by which cells control endoplasmic reticulum (ER) protein homeostasis. Under normal conditions, the UPR is not activated; however, under certain stresses, such as hypoxia or altered glycosylation, the UPR can be activated due to an accumulation of unfolded proteins. The activation of the UPR involves three signaling pathways, IRE1, PERK and ATF6, which all play vital roles in returning protein homeostasis to levels seen in non-stressed cells. IRE1 is the best studied of the three pathways, as it is the only pathway present in Saccharomyces cerevisiae. This pathway involves spliceosome independent splicing of HAC1 or XBP1 in yeast and mammalians cells, respectively. PERK limits protein synthesis, therefore reducing the number of new proteins requiring folding. ATF6 is translocated and proteolytically cleaved, releasing a NH2 domain fragment which is transported to the nucleus and which affects gene expression. If the UPR is unsuccessful at reducing the load of unfolded proteins in the ER and the UPR signals remain activated, this can lead to programmed cell death.

scholarly journals Dynamic changes in complexes of IRE1α, PERK, and ATF6α during endoplasmic reticulum stress

2018 ◽  
Vol 29 (11) ◽  
pp. 1376-1388 ◽  
Author(s):  
Arunkumar Sundaram ◽  
Suhila Appathurai ◽  
Rachel Plumb ◽  
Malaiyalam Mariappan
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Negative Regulator ◽  
Secretory Proteins ◽  
Unfolded Proteins ◽  
Native Page ◽  
Unfolded Protein ◽  
Blue Native Page ◽  
Stressed Cells ◽  
Protein Response

The endoplasmic reticulum (ER) localized unfolded protein response (UPR) sensors, IRE1α, PERK, and ATF6α, are activated by the accumulation of misfolded proteins in the ER. It is unclear how the endogenous UPR sensors are regulated by both ER stress and the ER luminal chaperone BiP, which is a negative regulator of UPR sensors. Here we simultaneously examined the changes in the endogenous complexes of UPR sensors by blue native PAGE immunoblotting in unstressed and stressed cells. We found that all three UPR sensors exist as preformed complexes even in unstressed cells. While PERK complexes shift to large complexes, ATF6α complexes are reduced to smaller complexes on ER stress. In contrast, IRE1α complexes were not significantly increased in size on ER stress, unless IRE1α is overexpressed. Surprisingly, depletion of BiP had little impact on the endogenous complexes of UPR sensors. In addition, overexpression of BiP did not significantly affect UPR complexes, but suppressed ER stress mediated activation of IRE1α, ATF6α and, to a lesser extent, PERK. Furthermore, we captured the interaction between IRE1α and misfolded secretory proteins in cells, which suggests that the binding of unfolded proteins to preformed complexes of UPR sensors may be crucial for activation.

scholarly journals Understanding Thyroid Cell Stress

2019 ◽  
Vol 105 (3) ◽  
pp. e66-e69
Author(s):  
Syed A Morshed ◽  
Terry F Davies
Keyword(s):  
Stress Responses ◽  
Thyroid Cell ◽  
Thyroid Cells ◽  
Unfolded Protein ◽  
Intracellular Signals ◽  
Damage Response ◽  
Strong Inflammatory Response ◽  
Stressed Cells ◽  
The Unfolded Protein Response ◽  
Protein Response

Abstract Understanding the regulatory mechanisms that control intracellular stress has fundamental importance since its failure results in cell death. Evidence has emerged indicating that the intracellular signals that are induced in response to diverse stresses include the deoxyribonucleic acid damage response, the unfolded protein response, the mitochondrial and/or endoplasmic reticulum stress responses, and the autophagy signals to degrade dangerous protein aggregates. These signals bring changes to the stressed cells that may support systemic homeostasis or contribute to disease pathology. In normal thyroid cells, both reactive oxygen species (ROS) and antioxidant (AOD) activity is low. An increase in ROS balanced by AOD leads only to mild inflammation, but unopposed increases in ROS lead to a strong inflammatory response and may result in apoptosis. A balance between ROS and AOD is, therefore, needed to maintain thyrocyte homeostasis. This perspective describes how thyroid cells are subjected to multiple insults and how they try to protect themselves using these different cellular responses.

scholarly journals Dynamic changes in oligomeric complexes of UPR sensors induced by misfolded proteins in the ER

2017 ◽  
Author(s):  
Arunkumar Sundaram ◽  
Suhila Appathurai ◽  
Malaiyalam Mariappan
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Mammalian Cells ◽  
Negative Regulator ◽  
Misfolded Proteins ◽  
Dynamic Changes ◽  
Unfolded Protein ◽  
Stressed Cells ◽  
Protein Response

AbstractThe endoplasmic reticulum (ER) localized unfolded protein response (UPR) sensors, IRE1α, PERK, and ATF6α, are activated upon accumulation of misfolded proteins caused by ER stress. It is debated whether these UPR sensors are activated either by the release of their negative regulator BiP chaperone or directly binding to misfolded proteins during ER stress. Here we simultaneously examined oligomerization and activation of all three endogenous UPR sensors. We found that UPR sensors existed as preformed oligomers even in unstressed cells, which shifted to large oligomers for PERK and small oligomers for ATF6α, but little changed for IRE1α upon ER stress. Neither depletion nor overexpression of BiP had significant effects on oligomeric complexes of UPR sensors both in unstressed and stressed cells. Thus, our results find less evidence for the BiP-mediated activation of UPR sensors in mammalian cells and support that misfolded proteins bind and activate the preformed oligomers of UPR sensors.

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