Maintain Endoplasmic Reticulum Homeostasis Research Articles

The role of sphingolipids in endoplasmic reticulum stress.

The endoplasmic reticulum (ER) is an important intracellular compartment in eukaryotic cells and has diverse functions, including protein synthesis, protein folding, lipid metabolism and calcium homeostasis. ER functions are disrupted by various intracellular and extracellular stimuli that cause ER stress, including the inhibition of glycosylation, disulphide bond reduction, ER calcium store depletion, impaired protein transport to the Golgi, excessive ER protein synthesis, impairment of ER-associated protein degradation and mutated ER protein expression. Distinct ER stress signalling pathways, which are known as the unfolded protein response, are deployed to maintain ER homeostasis, and a failure to reverse ER stress triggers cell death. Sphingolipids are lipids that are structurally characterized by long-chain bases, including sphingosine or dihydrosphingosine (also known as sphinganine). Sphingolipids are bioactive molecules long known to regulate various cellular processes, including cell proliferation, migration, apoptosis and cell-cell interaction. Recent studies have uncovered that specific sphingolipids are involved in ER stress. This review summarizes the roles of sphingolipids in ER stress and human diseases in the context of pathogenic events.

EDEM1 Drives Misfolded Protein Degradation via ERAD and Exploits ER-Phagy as Back-Up Mechanism When ERAD Is Impaired

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the main mechanism of targeting ER proteins for degradation to maintain homeostasis, and perturbations of ERAD lead to pathological conditions. ER-degradation enhancing α-mannosidase-like (EDEM1) was proposed to extract terminally misfolded proteins from the calnexin folding cycle and target them for degradation by ERAD. Here, using mass-spectrometry and biochemical methods, we show that EDEM1 is found in auto-regulatory complexes with ERAD components. Moreover, the N-terminal disordered region of EDEM1 mediates protein–protein interaction with misfolded proteins, whilst the absence of this domain significantly impairs their degradation. We also determined that overexpression of EDEM1 can induce degradation, even when proteasomal activity is severely impaired, by promoting the formation of aggregates, which can be further degraded by autophagy. Therefore, we propose that EDEM1 maintains ER homeostasis and mediates ERAD client degradation via autophagy when either dislocation or proteasomal degradation are impaired.

Vacuole Membrane Protein 1 Deficiency Promotes the Development of Pancreatitis Through Autophagy Impairment and Endoplasmic Reticulum Stress

Pancreatitis is a potentially fatal inflammatory disease with significant morbidity and mortality but the exact molecular mechanisms remain largely obscure. We investigated the role of vacuole membrane protein 1 (VMP1), an endoplasmic reticulum (ER) resident protein, in the pathogenesis of pancreatitis. We examined VMP1 expression and ER stress in human pancreatitis samples. We also determined VMP1 level and ER stress in experimental pancreatitis induced by cerulein as well as in pancreatic acinar cell‐specific VMP1 knockout (VMP1Δacinar) mice. We found that VMP1 expression was significantly lower in human pancreatitis samples associated with extensive ER stress. In a mouse experimental pancreatitis model, treatment with ceruleint markedly decreased VMP1 expression but increased levels of several ER stress markers such as p‐elf2α, XBP1s and CHOP. Western blot and immune histochemical analysis of human and experimental mouse pancreatitis samples showed impaired autophagy with accumulation of p62, an autophagy substrate protein that generally is degraded by autophagy. Intriguingly, VMP1Δacinar mice developed spontaneous pancreatitis with pathological features of chronic pancreatitis. In addition, VMP1Δacinar caused accumulation of p62 as well as selective ER‐phagy receptor FAM134B. Moreover, VMP1Δacinar mice had increased ER tress accompanied with massive cell death, suggesting VMP1 may serve as a key mediator of pancreatic ER homeostasis. Collectively, these data indicate a critical role of VMP1 in maintaining ER homeostasis in acinar cells and decreased VMP1 expression may contribute to the development of pancreatitis.Support or Funding InformationR01 AA020518, R01 DK102142, U01 AA024733 and P30GM118247 (W.X.D).

SWI/SNF chromatin remodelling complex contributes to clearance of cytoplasmic protein aggregates and regulates unfolded protein response in Saccharomycescerevisiae.

Chromatin remodelling complexes are multi-subunit assemblies, each containing a catalytic ATPase and translocase that is capable of mobilizing nucleosomes to alter the chromatin structure. SWI/SNF remodelling complexes with higher DNA translocation efficiency evict histones or slide the nucleosomes away from each other making DNA accessible for transcription and repair machinery. Chromatin remodelling at the promoter of stress-responsive genes by SWI/SNF becomes necessary during the heat and proteotoxic stress. While the involvement of SWI/SNF in transcription of stress-responsive genes has been studied extensively, the regulation of proteostasis by SWI/SNF is not well understood. This study demonstrates critical functions of SWI/SNF in response to cadmium-induced proteotoxic stress. Deletion of either ATPase-translocase subunit of SWI/SNF complex (Swi2/Snf2) or a regulatory subunit Swi3 abrogates the clearance of cadmium-induced protein aggregates. Our results suggest that Snf2 and Swi3 regulate the protein folding in endoplasmic reticulum (ER) that reduces the chances of forming unfolded protein aggregates under the proteotoxic stress of cadmium. The Ire1-mediated unfolded protein response (UPR) maintains ER homeostasis by upregulating the expression of chaperones and ER-associated degradation (ERAD) components. We found that Snf2 maintains normal oxidative environment essential for Ire1 activity. Deletion of SNF2 reduced the Ire1 activity and UPR, indicating involvement of Snf2 in Ire1-mediated ER proteostasis. Together, these findings suggest that SWI/SNF complex regulates ER homeostasis and protein folding crucial for tolerating proteotoxic stress.

ERO1α Knockdown Attenuates Palmitic Acid-Inhibited Testosterone Secretion by Endoplasmic Reticulum Stress in Testicular Leydig Cells

Background and objectivePalmitic acid (PA), the most common saturated free fatty acid (FFA) in food, is related to obesity-related male infertility. The possible mechanism is PA-mediated inhibition of testosterone secretion. Endoplasmic reticulum (ER) oxidoreductin-1alpha (ERO1α), an oxidase that is localized in the ER, plays an essential role in maintaining ER homeostasis and is related to hormone secretion. However, the role and underlying mechanisms of ERO1α in PA-mediated inhibition of testosterone secretion have not been reported. Material and methodsMurine Leydig tumor cell line 1 (MLTC-1) cells were treated with different doses of PA. Cell viability, testosterone secretion, and ERO1α expression were measured by the Cell Counting Kit 8 (CCK-8) assay, enzyme-linked immunesorbent assay (ELISA), and Western blotting, respectively. Moreover, the expres-sion of ER stress marker proteins (glucose-regulated protein 78 [GRP78] and CCAAT/enhancer-binding protein homologous protein [CHOP]) was also measured after treatment. Subsequently, the expression ofERO1α was knocked down, and cell viability, testosterone secretion, and ER stress were measured after treatment with the PA or the ER stress agonist thapsigargin (TG, an ER stress inducer). Also, testosterone secretion was measured by ELISA when ER stress was inhibited by 4-phenylbutyric acid (4-PBA, an ER stress inhibitor). ResultsPA treatment reduced cell viability, induced ERO1α expression, and enhanced the expression of the ER stress marker GRP78 and CHOP, while ERO1α knockdown inhibits ER stress marker expression, promotes testosterone secretion, and enhances cell viability in PA-treated MLTC-1 cells. In addition, ERO1α knock-down rescued the TG-induced the decrease in testosterone secretion and cell viability. ConclusionsThese findings suggest that PA inhibits testosterone secretion via ER stress and that ERO1α knockdown ameliorates PA-induced decreases in testosterone via ER stress in testicular Leydig cells. Our results indi-cate the necessity of exploring the potential applications of ERO1α as a target gene for restoring fertility in obese men.

The Impact of the ER Unfolded Protein Response on Cancer Initiation and Progression: Therapeutic Implications.

Cellular stress induced by the accumulation of misfolded proteins in the endoplasmic reticulum (ER) activates an elaborate signalling network termed the unfolded protein response (UPR). This adaptive response is mediated by the transmembrane signal transducers IRE1, PERK, and ATF6 to decide cell fate of recovery or death. In malignant cells, UPR signalling may be required to maintain ER homeostasis and survival in the tumor microenvironment characterized by oxidative stress, hypoxia, lactic acidosis and compromised protein folding. Here we provide an overview of the ER response to cellular stress and how the sustained activation of this network enables malignant cells to develop tumorigenic, metastatic and drug-resistant capacities to thrive under adverse conditions. Understanding the complexity of ER stress responses and how to target the UPR in disease will have significant potential for novel future therapeutics.

Intracellular XBP1-IL-24 axis dismantles cytotoxic unfolded protein response in the liver

Endoplasmic reticulum (ER) stress-associated cell death is prevalent in various liver diseases. However, the determinant mechanism how hepatocytes survive unresolved stress was still unclear. Interleukin-24 (IL-24) was previously found to promote ER stress-mediated cell death, and yet its expression and function in the liver remained elusive. Here we identified an antiapoptotic role of IL-24, which transiently accumulated within ER-stressed hepatocytes in a X-box binding protein 1 (XBP1)-dependent manner. Disruption of IL-24 increased cell death in the CCL4- or APAP-challenged mouse liver or Tm-treated hepatocytes. In contrast, pharmaceutical blockade of eukaryotic initiation factor 2α (eIF2α) or genetical ablation of C/EBP homologous protein (CHOP) restored hepatocyte function in the absence of IL-24. In a clinical setting, patients with acute liver failure manifested a profound decrease of hepatic IL-24 expression, which was associated with disease progression. In conclusion, intrinsic hepatocyte IL-24 maintains ER homeostasis by restricting the eIF2α-CHOP pathway-mediated stress signal, which might be exploited as a bio-index for prognosis or therapeutic intervention in patients with liver injury.

Disposing of misfolded ER proteins: A troubled substrate's way out of the ER

Secreted, plasma membrane, and resident proteins of the secretory pathway are synthesized in the endoplasmic reticulum (ER) where they undergo post-translational modifications, oxidative folding, and subunit assembly in tightly monitored processes. An ER quality control (ERQC) system oversees protein maturation and ensures that only those reaching their native state will continue trafficking into the secretory pathway to reach their final destinations. Those that fail must be recognized and eliminated to maintain ER homeostasis. Two cellular mechanisms have been identified to rid the ER of terminally unfolded, misfolded, and aggregated proteins. ER-associated degradation (ERAD) was discovered nearly 30 years ago and entails the identification of improperly matured secretory pathway proteins and their retrotranslocation to the cytosol for degradation by the ubiquitin-proteasome system. ER-phagy has been more recently described and caters to larger, more complex proteins and protein aggregates that are not readily handled by ERAD. This pathway has unique upstream components and relies on the same downstream effectors of autophagy used in other cellular processes to deliver clients to lysosomes for degradation. In this review, we describe the main elements of ERQC, ERAD, and ER-phagy and focus on recent advances in these fields.

The various shades of ER-phagy.

Endoplasmic reticulum (ER) is a large and dynamic cellular organelle. ER morphology consists of sheets, tubules, matrixes, and contact sites shared with other membranous organelles. The capacity of the ER to fulfill its numerous biological functions depends on its continuous remodeling and the quality control of its proteome. Selective turnover of the ER by autophagy, termed ER‐phagy, plays an important role in maintaining ER homeostasis. ER network integrity and turnover rely on specific ER‐phagy receptors, which influence and coordinate alterations in ER morphology and the degradation of ER contents and membranes via the lysosome, by interacting with the LC3/GABARAP family. In this commentary, we discuss general principles and identify the major players in this recently characterized form of selective autophagy, while simultaneously highlighting open questions in the field.

A Tense Situation: Maintaining ER Homeostasis during Lipid Droplet Budding

In this issue of Developmental Cell, Chorlay etal. (2019) provide evidence that asymmetric membrane surface tension determines the directionality of lipid droplet (LD) emergence. Furthermore, phospholipid synthesis "refills" the outer leaflet of the endoplasmic reticulum (ER) membrane to maintain cytosolic LD emergence and prevent disruptions to ER homeostasis.

Translation Control of HAC1 by Regulation of Splicing in Saccharomyces cerevisiae.

Hac1p is a key transcription factor regulating the unfolded protein response (UPR) induced by abnormal accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) in Saccharomyces cerevisiae. The accumulation of unfolded/misfolded proteins is sensed by protein Ire1p, which then undergoes trans-autophosphorylation and oligomerization into discrete foci on the ER membrane. HAC1 pre-mRNA, which is exported to the cytoplasm but is blocked from translation by its intron sequence looping back to its 5’UTR to form base-pair interaction, is transported to the Ire1p foci to be spliced, guided by a cis-acting bipartite element at its 3’UTR (3’BE). Spliced HAC1 mRNA can be efficiently translated. The resulting Hac1p enters the nucleus and activates, together with coactivators, a large number of genes encoding proteins such as protein chaperones to restore and maintain ER homeostasis and secretary protein quality control. This review details the translation regulation of Hac1p production, mediated by the nonconventional splicing, in the broad context of translation control and summarizes the evolution and diversification of the UPR signaling pathway among fungal, metazoan and plant lineages.

Is selenoprotein K required for Borrelia burgdorferi infection within the tick vector Ixodes scapularis?

BackgroundTick selenoproteins are involved in regulating oxidative and endoplasmic reticulum stress during prolonged tick feeding on mammalian hosts. How selenoproteins are activated upon tick-borne pathogen infection is yet to be defined.MethodsTo examine the functional role of selenoprotein K in Borrelia burgdorferi infection within the tick host Ixodes scapularis, RNA interference (RNAi)-based gene silencing was performed.ResultsSelenoprotein K is an endoplasmic reticulum (ER)-resident protein and a component of the ERAD complex involved in ER homeostasis. A qRT-PCR assay revealed the significant upregulation of selenogene K (selenoK) expression in B. burgdorferi-infected tick tissues. Silencing of the selenoK transcript significantly depleted B. burgdorferi copies within the infected tick tissues. Upon selenoK knockdown, another component of the ERAD complex, selenoprotein S (selenoS), was significantly upregulated, suggesting a compensatory mechanism to maintain ER homeostasis within the tick tissues. Knockdown of selenoK also upregulated ER stress-related unfolded protein response (UPR) pathway components, ATF6 and EIF2.ConclusionsThe exact mechanisms that contribute to depletion of B. burgdorferi upon selenoK knockdown is yet to be determined, but this study suggests that selenoK may play a vital role in the survival of B. burgdorferi within the tick host.

An N‐terminal truncated isoform of the ER‐phagy receptor FAM134B (FAM134B‐2) regulates starvation‐induced ER‐to‐lysosome protein degradation

Autophagy is a conserved system that adapts to nutrient starvation, after which proteins and organelles are degraded to recycle amino acids in response to starvation. Recently, the endoplasmic reticulum (ER) was added to the list of targets of autophagic degradation. Autophagic degradation of the ER (ER‐phagy) is considered a key mechanism in maintaining ER homeostasis. Four ER‐resident proteins (FAM134B, CCPG1, SEC62, and RTN3) have been identified as ER‐phagy cargo receptors, which contain LC3‐interacting regions. In this study we identified an N‐terminal truncated isoform of FAM134B (FAM134B‐2) that contributes to starvation‐induced ER‐phagy. FAM134B‐2 but not full length FAM134B (FAM134B‐1) is expressed in a fed state. Starvation drastically induces FAM134B‐2 but no other ER‐phagy cargo receptors through transcriptional activation by C/EBPβ. C/EBPβ overexpression increases FAM134B‐2 recruitment into autophagosomes and lysosomal degradation. In addition, FAM134B‐2 is preferentially localized in the smooth ER. This study demonstrates that the C/EBPβ‐FAM134B‐2 axis regulates starvation‐induced smooth ER‐phagy.Support or Funding InformationR01DK096030, R01HL117062, R01HL133545 and R01 HL132318.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Targeting the Zinc Transporter ZIP7 in the Treatment of Insulin Resistance and Type 2 Diabetes.

Type 2 diabetes mellitus (T2DM) is a disease associated with dysfunctional metabolic processes that lead to abnormally high levels of blood glucose. Preceding the development of T2DM is insulin resistance (IR), a disorder associated with suppressed or delayed responses to insulin. The effects of this response are predominately mediated through aberrant cell signalling processes and compromised glucose uptake into peripheral tissue including adipose, liver and skeletal muscle. Moreover, a major factor considered to be the cause of IR is endoplasmic reticulum (ER) stress. This subcellular organelle plays a pivotal role in protein folding and processes that increase ER stress, leads to maladaptive responses that result in cell death. Recently, zinc and the proteins that transport this metal ion have been implicated in the ER stress response. Specifically, the ER-specific zinc transporter ZIP7, coined the “gate-keeper” of zinc release from the ER into the cytosol, was shown to be essential for maintaining ER homeostasis in intestinal epithelium and myeloid leukaemia cells. Moreover, ZIP7 controls essential cell signalling pathways similar to insulin and activates glucose uptake in skeletal muscle. Accordingly, ZIP7 may be essential for the control of ER localized zinc and mechanisms that disrupt this process may lead to ER-stress and contribute to IR. Accordingly, understanding the mechanisms of ZIP7 action in the context of IR may provide opportunities to develop novel therapeutic options to target this transporter in the treatment of IR and subsequent T2DM.

The intramembrane protease SPP impacts morphology of the endoplasmic reticulum by triggering degradation of morphogenic proteins

The endoplasmic reticulum (ER), as a multifunctional organelle, plays crucial roles in lipid biosynthesis and calcium homeostasis as well as the synthesis and folding of secretory and membrane proteins. Therefore, it is of high importance to maintain ER homeostasis and to adapt ER function and morphology to cellular needs. Here, we show that signal peptide peptidase (SPP) modulates the ER shape through degradation of morphogenic proteins. Elevating SPP activity induces rapid rearrangement of the ER and formation of dynamic ER clusters. Inhibition of SPP activity rescues the phenotype without the need for new protein synthesis, and this rescue depends on a pre-existing pool of proteins in the Golgi. With the help of organelle proteomics, we identified certain membrane proteins to be diminished upon SPP expression and further show that the observed morphology changes depend on SPP-mediated cleavage of ER morphogenic proteins, including the SNARE protein syntaxin-18. Thus, we suggest that SPP-mediated protein abundance control by a regulatory branch of ER-associated degradation (ERAD-R) has a role in shaping the early secretory pathway.

The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1.

The protein folding capacity of the endoplasmic reticulum (ER) is tightly regulated by a network of signaling pathways, known as the unfolded protein response (UPR). UPR sensors monitor the ER folding status to adjust ER folding capacity according to need. To understand how the UPR sensor IRE1 maintains ER homeostasis, we identified zero-length crosslinks of RNA to IRE1 with single nucleotide precision in vivo. We found that IRE1 specifically crosslinks to a subset of ER-targeted mRNAs, SRP RNA, ribosomal and transfer RNAs. Crosslink sites cluster in a discrete region of the ribosome surface spanning from the A-site to the polypeptide exit tunnel. Moreover, IRE1 binds to purified 80S ribosomes with high affinity, indicating association with ER-bound ribosomes. Our results suggest that the ER protein translocation and targeting machineries work together with the UPR to tune the ER's protein folding load.

Redox-Mediated Regulatory Mechanisms of Endoplasmic Reticulum Homeostasis.

The endoplasmic reticulum (ER) is a dynamic organelle responsible for many cellular functions in eukaryotic cells. Proper redox conditions in the ER are necessary for the functions of many luminal pathways and the maintenance of homeostasis. The redox environment in the ER is oxidative compared with that of the cytosol, and a network of oxidoreductases centering on the protein disulfide isomerase (PDI)-Ero1α hub complex is constructed for efficient electron transfer. Although these oxidizing environments are advantageous for oxidative folding for protein maturation, electron transfer is strictly controlled by Ero1α structurally and spatially. The ER redox environment shifts to a reductive environment under certain stress conditions. In this review, we focus on the reducing reactions that maintain ER homeostasis and introduce their significance in an oxidative ER environment.

Two novel EIF2S3 mutations associated with syndromic intellectual disability with severe microcephaly, growth retardation, and epilepsy

The vast majority of proteins that a cell secretes or displays on its surface first enter the endoplasmic reticulum (ER), where they fold and assemble. Only properly assembled proteins advance from the ER to the cell surface. The ER coordinates protein biosynthetic and secretory activities in the cell. Alterations in ER homeostasis cause accumulation of misfolded/unfolded proteins in the ER. To maintain ER homeostasis, eukaryotic cells have evolved the unfolded protein response (UPR), an essential adaptive intracellular signaling pathway that responds to metabolic, oxidative stress, and inflammatory response pathways. The UPR has been implicated in a variety of diseases including metabolic disease, neurodegenerative disease, inflammatory disease, and cancer. Signaling components of the UPR are emerging as potential targets for intervention and treatment of human disease. MEHMO syndrome (OMIM# 300148) is a recently described disorder characterized by X-linked intellectual disability, epileptic seizures, hypogonadism, hypogenitalism, microcephaly, and obesity (2). It is caused my mutations in the EIF2S3. The EIF2S3 gene encodes the alpha subunit of eukaryotic translation initiation factor 2 (eIF2), crucial for initiation of protein synthesis and regulation of the ER stress response. MEMHO syndrome patients may have multiple endocrine manifestations including short stature, hypogonadism and obesity. In addition the manuscript by Stanik J et al report neonatal hypoglycaemia, hypopituitarism and neonatal diabetes mellitus. Thus MEMHO syndrome comes in the category of defects in the ER stress response and includes disorders such as Walcott-Rallison syndrome, Wolfram syndrome, Microcephaly, Epilepsy, and Diabetes Syndrome (MEDS) and the syndromic form of intellectual disability and diabetes caused by mutations in the PPP1R15B (3-6).

Endoplasmic reticulum stress signalling - from basic mechanisms to clinical applications.

The endoplasmic reticulum (ER) is a membranous intracellular organelle and the first compartment of the secretory pathway. As such, the ER contributes to the production and folding of approximately one‐third of cellular proteins, and is thus inextricably linked to the maintenance of cellular homeostasis and the fine balance between health and disease. Specific ER stress signalling pathways, collectively known as the unfolded protein response (UPR), are required for maintaining ER homeostasis. The UPR is triggered when ER protein folding capacity is overwhelmed by cellular demand and the UPR initially aims to restore ER homeostasis and normal cellular functions. However, if this fails, then the UPR triggers cell death. In this review, we provide a UPR signalling‐centric view of ER functions, from the ER's discovery to the latest advancements in the understanding of ER and UPR biology. Our review provides a synthesis of intracellular ER signalling revolving around proteostasis and the UPR, its impact on other organelles and cellular behaviour, its multifaceted and dynamic response to stress and its role in physiology, before finally exploring the potential exploitation of this knowledge to tackle unresolved biological questions and address unmet biomedical needs. Thus, we provide an integrated and global view of existing literature on ER signalling pathways and their use for therapeutic purposes.

Abstract 448: The novel role of ecdysoneless (ECD) in maintain ER Ca2+ homeostasis

Abstract Background and Significance: Calcium (Ca2+) is an essential signal transduction element involved in the regulation of several cellular activities such as gene transcription, proliferation and apoptosis. Endoplasmic Reticulum (ER) is the Ca2+ reservoir inside the cell that strictly orchestrate Ca2+ gradient across the Plasma Membrane(PM). Importantly, intracellular Ca2+ flux, is a key to orderly regulate cell proliferation, if altered can promote uncontrolled proliferation leading to cancer. Ca2+ entry through the [Ca2+]PM channels during [Ca2+]ER depletion is called the Store Operated Ca2+ Entry (SOCE). Impairment of endogenous SOCE leads to sustained [Ca2+]ER depletion and results in accumulation of unfolded proteins in the ER (known as ER stress). Understanding the basic molecular mechanism regulating PM to ER Ca2+ flow and developing novel strategies to target ER Ca2+ signaling could be beneficial from cancer therapeutic perspective. We have identified Ecdysoneless (ECD), an evolutionary conserved novel cell cycle regulator protein. ECD is overexpressed in several cancers, including breast and pancreatic cancers and its overexpression in breast cancer correlates with poor prognosis and short survival of patients. Recently our lab demonstrates that ECD is a negative regulator of ER stress; however, there is no mechanistic insights of how ECD senses and acts to maintain ER homeostasis. Experimental Design: We have utilized mouse embryonic fibroblasts (MEFs) from Tet(O)-FhECD-IRES-eGFP-rtTA and Ecdflox/flox mice transgenic mice to regulate ECD levels in cells to assess the role of ECD in Ca2+ signaling in a highly controlled system. Structured illumination microscopy(SIM) and biochemical fractionation techniques were performed to identify localization of ECD. Live cell imaging of cells stained with Ca2+ binding dyes like Fluo4-AM and Fluo5N-AM were used to access [Ca2+]Cyto and [Ca2+]ER levels respectively. Fluorescence resonance energy transfer (FRET) and immunoprecipitation methods were conducted to analyze SOCE pathway. Western blotting was used to assess the levels of various proteins involved in Ca2+ signaling. Results: We demonstrate several novel findings: (i) ECD is a smooth ER associated protein; (ii) ECD regulates assembly of ER-PM protein complex to drive Ca2+ entry through SOCE into the ER, maintaining [Ca2+]ER homeostasis; iii) ECD is overexpressed upon Ca2+ depletion and provides survival benefit to cells; v) ECD regulates Ca2+ mediated cell proliferation via CaMKII phosphorylation. Conclusion: Our results demonstrate ECD is a sensor of ER stress, induced by Ca2+deprivation, and overexpressed ECD promotes cell proliferation and survival under ER stress. These findings suggest ECD could be a potential target of Cancer therapy. Citation Format: Aniruddha Sarkar, Irfana Saleem, Sameer Mirza, Keshore Bidasee, Rajini Rao, Hamid Band, Vimla Band. The novel role of ecdysoneless (ECD) in maintain ER Ca2+ homeostasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 448.