Endoplasmic Reticulum Chaperone BiP Research Articles

The endoplasmic reticulum protein HSPA5/BiP is essential for decidual transformation of human endometrial stromal cells

Decidualization denotes the process of inflammatory reprogramming of endometrial stromal cells (EnSC) into specialized decidual cells (DC). During this process, EnSC are subjected to endoplasmic reticulum (ER) stress as well as acute cellular senescence. Both processes contribute to the proinflammatory mid-luteal implantation window and their dysregulation has been implicated in reproductive failure. Here, we evaluated the link between ER stress, decidual differentiation and senescence. In-silico analysis identified HSPA5 gene, codifying the ER chaperone BiP, as a potentially critical regulator of cell fate divergence of decidualizing EnSC into anti-inflammatory DC and pro-inflammatory senescent decidual cells (snDC). Knockdown of HSPA5 in primary EnSC resulted both in decreased expression of DC marker genes and attenuated induction of senescence associated β-galactosidase activity, a marker of snDC. Stalling of the decidual reaction upon HSPA5 knockdown was apparent at 8 days of differentiation and was preceded by the upregulation of ER stress associated proteins IRE1α and PERK. Further, HSPA5 knockdown impaired colony-forming unit activity of primary EnSC, indicative of loss of cellular plasticity. Together, our results point to a key role for HSPA5/BiP in decidual transformation of EnSCs and highlight the importance of constraining ER stress levels during this process.

Dysregulation of FicD AMPylation causes diabetes by disrupting pancreatic endocrine homeostasis.

Bi-functional enzyme FicD regulates the endoplasmic reticulum chaperone BiP using AMPylation and deAMPylation during ER homeostasis and stress, respectively. Human FicD with an arginine-to-serine mutation disrupts FicD deAMPylation activity resulting in severe neonatal diabetes. We generated the FicD R371S mutation in mice to create a pre-clinical murine model for neonatal diabetes. We observed elevated BiP AMPylation levels across multiple tissues and signature markers for diabetes including glucose intolerance and reduced serum insulin levels. While the pancreas of FicD R371S mice appeared normal at birth, adult FicD R371S mice displayed disturbed pancreatic islet organization that progressed with age. FicD R371S mice provide a preclinical mouse model for the study of UPR associated diabetes and demonstrate the essentiality of FicD for tissue resilience.

Endoplasmic reticulum chaperone BiP regulates ER stress and cell fate of endometrial stromal cells during the decidualization program

Endoplasmic reticulum chaperone BiP regulates ER stress and cell fate of endometrial stromal cells during the decidualization program

Loss of Fic causes progressive neurodegeneration in a Drosophila model of hereditary spastic paraplegia

Hereditary Spastic Paraplegia (HSP) is a group of rare inherited disorders characterized by progressive weakness and spasticity of the legs. Recent newly discovered biallelic variants in the gene FICD were found in patients with a highly similar phenotype to early onset HSP. FICD encodes filamentation induced by cAMP domain protein. FICD is involved in the AMPylation and deAMPylation protein modifications of the endoplasmic reticulum (ER) chaperone BIP, a major constituent of the ER that regulates the unfolded protein response. Although several biochemical properties of FICD have been characterized, the neurological function of FICD and the pathological mechanism underlying HSP are unknown. We established a Drosophila model to gain mechanistic understanding of the function of FICD in HSP pathogenesis, and specifically the role of BIP in neuromuscular physiology. Our studies on Drosophila Fic null mutants uncovered that loss of Fic resulted in locomotor impairment and reduced levels of BIP in the motor neuron circuitry, as well as increased reactive oxygen species (ROS) in the ventral nerve cord of Fic null mutants. Finally, feeding Drosophila Fic null mutants with chemical chaperones PBA or TUDCA, or treatment of patient fibroblasts with PBA, reduced the ROS accumulation. The neuronal phenotypes of Fic null mutants recapitulate several clinical features of HSP patients and further reveal cellular patho-mechanisms. By modeling FICD in Drosophila, we provide potential targets for intervention for HSP, and advance fundamental biology that is important for understanding related rare and common neuromuscular diseases.

Modulating Endoplasmic Reticulum Chaperones and Mutant Protein Degradation in GABRG2(Q390X) Associated with Genetic Epilepsy with Febrile Seizures Plus and Dravet Syndrome.

A significant number of patients with genetic epilepsy do not obtain seizure freedom, despite developments in new antiseizure drugs, suggesting a need for novel therapeutic approaches. Many genetic epilepsies are associated with misfolded mutant proteins, including GABRG2(Q390X)-associated Dravet syndrome, which we have previously shown to result in intracellular accumulation of mutant GABAA receptor γ2(Q390X) subunit protein. Thus, a potentially promising therapeutic approach is modulation of proteostasis, such as increasing endoplasmic reticulum (ER)-associated degradation (ERAD). To that end, we have here identified an ERAD-associated E3 ubiquitin ligase, HRD1, among other ubiquitin ligases, as a strong modulator of wildtype and mutant γ2 subunit expression. Overexpressing HRD1 or knockdown of HRD1 dose-dependently reduced the γ2(Q390X) subunit. Additionally, we show that zonisamide (ZNS)-an antiseizure drug reported to upregulate HRD1-reduces seizures in the Gabrg2+/Q390X mouse. We propose that a possible mechanism for this effect is a partial rescue of surface trafficking of GABAA receptors, which are otherwise sequestered in the ER due to the dominant-negative effect of the γ2(Q390X) subunit. Furthermore, this partial rescue was not due to changes in ER chaperones BiP and calnexin, as total expression of these chaperones was unchanged in γ2(Q390X) models. Our results here suggest that leveraging the endogenous ERAD pathway may present a potential method to degrade neurotoxic mutant proteins like the γ2(Q390X) subunit. We also demonstrate a pharmacological means of regulating proteostasis, as ZNS alters protein trafficking, providing further support for the use of proteostasis regulators for the treatment of genetic epilepsies.

In-Depth Characterization of bZIP Genes in the Context of Endoplasmic Reticulum (ER) Stress in Brassica campestris ssp. chinensis.

Numerous studies have been conducted to investigate the genomic characterization of bZIP genes and their involvement in the cellular response to endoplasmic reticulum (ER) stress. These studies have provided valuable insights into the coordinated cellular response to ER stress, which is mediated by bZIP transcription factors (TFs). However, a comprehensive and systematic investigations regarding the role of bZIP genes and their involvement in ER stress response in pak choi is currently lacking in the existing literature. To address this knowledge gap, the current study was initiated to elucidate the genomic characteristics of bZIP genes, gain insight into their expression patterns during ER stress in pak choi, and investigate the protein-to-protein interaction of bZIP genes with the ER chaperone BiP. In total, 112 members of the BcbZIP genes were identified through a comprehensive genome-wide analysis. Based on an analysis of sequence similarity, gene structure, conserved domains, and responsive motifs, the identified BcbZIP genes were categorized into 10 distinct subfamilies through phylogenetic analysis. Chromosomal location and duplication events provided insight into their genomic context and evolutionary history. Divergence analysis estimated their evolutionary history with a predicted divergence time ranging from 0.73 to 80.71 million years ago (MYA). Promoter regions of the BcbZIP genes were discovered to exhibit a wide variety of cis-elements, including light, hormone, and stress-responsive elements. GO enrichment analysis further confirmed their roles in the ER unfolded protein response (UPR), while co-expression network analysis showed a strong relationship of BcbZIP genes with ER-stress-responsive genes. Moreover, gene expression profiles and protein-protein interaction with ER chaperone BiP further confirmed their roles and capacity to respond to ER stress in pak choi.

Atrogin-1 promotes muscle homeostasis by regulating levels of endoplasmic reticulum chaperone BiP.

Skeletal muscle wasting results from numerous pathological conditions affecting both the musculoskeletal and nervous systems. A unifying feature of these pathologies is the upregulation of members of the E3 ubiquitin ligase family, resulting in increased proteolytic degradation of target proteins. Despite the critical role of E3 ubiquitin ligases in regulating muscle mass, the specific proteins they target for degradation and the mechanisms by which they regulate skeletal muscle homeostasis remain ill-defined. Here, using zebrafish loss-of-function models combined with in vivo cell biology and proteomic approaches, we reveal a role of atrogin-1 in regulating the levels of the endoplasmic reticulum chaperone BiP. Loss of atrogin-1 resulted in an accumulation of BiP, leading to impaired mitochondrial dynamics and a subsequent loss in muscle fiber integrity. We further implicated a disruption in atrogin-1-mediated BiP regulation in the pathogenesis of Duchenne muscular dystrophy. We revealed that BiP was not only upregulated in Duchenne muscular dystrophy, but its inhibition using pharmacological strategies, or by upregulating atrogin-1, significantly ameliorated pathology in a zebrafish model of Duchenne muscular dystrophy. Collectively, our data implicate atrogin-1 and BiP in the pathogenesis of Duchenne muscular dystrophy and highlight atrogin-1's essential role in maintaining muscle homeostasis.

Oncogenic Roles of Laminin Subunit Gamma-2 in Intrahepatic Cholangiocarcinoma via Promoting EGFR Translation.

Intrahepatic cholangiocarcinoma (iCCA) is a highly lethal biliary epithelial cancer in the liver. Here, Laminin subunit gamma-2 (LAMC2) with important oncogenic roles in iCCA is discovered. In a total of 231 cholangiocarcinoma patients (82% of iCCA patients) across four independent cohorts, LAMC2 is significantly more abundant in iCCA tumor tissue compared to normal bile duct and non-tumor liver. Among 26.3% of iCCA patients, LAMC2 gene is amplified, contributing to its over-expression. Functionally, silencing LAMC2 significantly blocks tumor formation in orthotopic iCCA mouse models. Mechanistically, it promotes EGFR protein translation via interacting with nascent unglycosylated EGFR in the endoplasmic reticulum (ER), resulting in activated EGFR signaling. LAMC2-mediated EGFR translation also depends on its interaction with the ER chaperone BiP via their C-terminus. Together LAMC2 and BiP generate a binding "pocket" of nascent EGFR and facilitate EGFR translation. Consistently, LAMC2-high iCCA patients have poor prognosis in two iCCA cohorts. LAMC2-high iCCA cells are highly sensitive to EGFR tyrosine kinase inhibitors (TKIs) treatment both in vitro and in vivo. Together, these data demonstrate LAMC2 as an oncogenic player in iCCA by promoting EGFR translation and an indicator to identify iCCA patients who may benefit from available EGFR-targeted TKIs therapies.

Toll-like receptor 4 damages the intestinal epithelial cells by activating endoplasmic reticulum stress in septic rats.

The severity of acute gastrointestinal injury (AGI) is a critical determinant of survival in sepsis. However, there is no specifically interventional management for gastrointestinal dysfunction. Toll-like Receptor 4 (TLR4) is an important contributor to sepsis-induced multiple organ dysfunction syndrome. So, we investigated the effect of TLR4 on leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) + cells and goblet cells and its potential mechanism. A cecal ligation and puncture (CLP) model reflecting the development of clinical sepsis was developed. Tak-242, a TLR4 inhibitor, was administered to septic rats at a dose of 3 mg/kg via intraperitoneal injection. Immunohistochemistry was performed to detect TLR4 and Lgr5+ cells. AB-PAS staining was performed to detect goblet cells. MUC1 and MUC2 secreted by goblet cells, biomarkers of endoplasmic reticulum (ER) stress and inflammatory cytokines in the intestine were detected by western blotting and real-time PCR. We found that the upregulation of the TLR4/NF-κB signaling pathway activated intestinal inflammatory response in sepsis. Meanwhile, the structure of intestinal mucosa was destroyed, Lgr5+ cells and goblet cells count were significantly reduced, and the secretory function of goblet cells also decreased. Further studies have found that TLR4 increased the levels of activating transcription factor-6 (ATF6), XBP1, ER chaperone (Bip) and CHOP, but did not activate the protein kinase RNA (PKR)-like ER kinase (P-PERK). We concluded that the inhibition of TLR4/NF-κB signaling pathway can reduce intestinal inflammatory response, protect intestinal mucosa, protect Lgr5+ cells, goblet cells and relieve ER stress. Our findings suggest that Tak-242 protects Lgr5+ cells and goblet cells after sepsis, partly may be through the suppression of ER stress. Thus, inhibition of TLR4-mediated ER stress may be a promising therapy of septic AGI.

Understanding Heat‐Associated Adult Platichthys stellatus Mortality: Differential Transcriptome Analysis of Juvenile and Adult Starry Flounder Liver under Heat Stress

The starry flounder Platichthys stellatus, an economically important benthic fish species widely distributed throughout the North Pacific Ocean, exhibits poor heat tolerance, resulting in frequent adult mass mortality in aquaculture settings. Heat stress disrupts the energy metabolism of P. stellatus, elevating blood glucose and implying its sensitivity to rising water temperatures. In particular, the survival of adult fish in aquaculture settings during the summer season is less than that of larvae or juveniles. This study aimed to understand the heat stress‐associated survival of adults by focusing on the molecular pathways of glycolysis/gluconeogenesis using comparative transcriptome analysis. Transcriptomes were obtained by RNA sequencing of P. stellatus livers of adults exposed to 16 and 28°C, together with juveniles at the same conditions. A total of 16,121,503–18,774,222 raw paired‐end reads were obtained after trimming. The assembled unigenes (39,489 out of 127,460) were annotated in the nr, Clusters of Orthologous Groups (COG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Swiss‐Prot databases. A total of 6,858 differentially expressed genes were found in juveniles and adults at control (16°C) and high (28°C) water temperatures. Juveniles and adults had 1,856 genes downregulated and 1,548 genes upregulated at the 16°C control water temperature and 1,055 genes downregulated and 1,231 genes upregulated at 28°C, overall involving at least 47 KEGG pathways. When raising the water temperature and comparing the DEGs of juveniles and adults, respectively, we found that both groups showed similar upregulation patterns for the major heat shock protein (HSP) genes under temperature‐increasing conditions, indicating a conserved response mechanism. Additionally, both juvenile and adult fish showed similar upregulation of ER stress‐related genes, including endoplasmic reticulum chaperone BiP (BiP) and homocysteine‐responsive endoplasmic reticulum‐resident ubiquitin‐like domain member 1 (HERP), suggesting consistent transcriptional changes associated with ER stress under heat stress conditions. However, notable differences were observed in the expression of inflammation and immune response‐related genes, and juvenile P. stellatus showed upregulation of immune costimulatory protein B7‐H3 (B7H3) and immunoglobulin superfamily member 9 (IGSF9), while adults showed upregulation of high‐affinity immunoglobulin epsilon receptor subunit gamma (FCER1G), V‐type immunoglobulin domain‐containing suppressor of T‐cell activation (VSIR), and immunoglobulin superfamily DCC subclass member 3 (IGDCC3). Especially, phosphoglucomutase (PGM), glucose‐6‐phosphatase catalytic subunit (G6PC), phosphofructokinase (PFK), and bisphosphoglycerate mutase (BPGM), the enzymes involved in the glycolysis/gluconeogenesis pathway, showed significantly higher FPKM in adult P. stellatus than in juveniles. Overall, this study provides valuable insights into the transcriptional responses of P. stellatus to heat stress, which differ with age and may inform the development of strategies to improve the survival and productivity of this economically important fish species in aquaculture.

The ER chaperone, BIP protects Microglia from ER stress-mediated Apoptosis in Hyperglycemia

A major endoplasmic reticulum (ER) chaperone, binding of Immunoglobulin heavy chain protein (BIP) facilitates the assembly of newly synthesized proteins in the ER. Microglia vigorously respond to brain injuries and eliminate the damaged neuronal and apoptotic cells through phagocytosis in the central nervous system. However, the mechanism of BIP-mediated microglial function is not clear in hyperglycemia. We explored the molecular mechanism of BIP in microglial function during hyperglycemic conditions. Hyperglycemia was induced in mice by two consecutive intraperitoneal injections of streptozotocin (STZ 100/kg) and confirmed by measuring the blood glucose from day 2 to day 14. After 14 days of experimental hyperglycemia, mice were sacrificed and brains were collected for ER chaperone expression. In-vitro hyperglycemia was induced by exposing HMC3 cells to 25 mM glucose for 5 days and proteins involved in ER stress, apoptosis, and autophagy were analyzed. In hyperglycemic conditions, BIP protein expression was dramatically reduced in HMC3 cells, which led to increased apoptosis through the activation of CHOP and mitochondrial pro-apoptotic proteins (Bax, Bad, and cleaved caspase-3). The flow cytometry results indicate hyperglycemia-induced apoptosis and reactive oxygen species (ROS) production. Interestingly, the BIP inducer X restored the apoptosis in HMC3 cells by derepressing BIP expression and inhibiting ER stress. These results suggest that the ER chaperone BIP is required for the microglial function and protects from apoptosis in hyperglycemia. A better understanding of BIP's molecular mechanism and role in microglial function may contribute to developing novel therapies for microglia dysfunction-associated neurodegenerative diseases.

The synthetic oleanane triterpenoid CDDO-2P-Im binds GRP78/BiP to induce unfolded protein response-mediated apoptosis in myeloma.

Synthetic oleanane triterpenoids (SOTs) are small molecules with broad anticancer properties. A recently developed SOT, 1-[2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]-4(-pyridin-2-yl)-1H-imidazole (CDDO-2P-Im or '2P-Im'), exhibits enhanced activity and improved pharmacokinetics over CDDO-Im, a previous generation SOT. However, the mechanisms leading to these properties are not defined. Here, we show the synergy of 2P-Im and the proteasome inhibitor ixazomib in human multiple myeloma (MM) cells and 2P-Im activity in a murine model of plasmacytoma. RNA sequencing and quantitative reverse transcription PCR revealed the upregulation of the unfolded protein response (UPR) in MM cells upon 2P-lm treatment, implicating the activation of the UPR as a key step in 2P-Im-induced apoptosis. Supporting this hypothesis, the deletion of genes encoding either protein kinase R-like endoplasmic reticulum kinase (PERK) or DNA damage-inducible transcript 3 protein (DDIT3; also known as CHOP) impaired the MM response to 2P-Im, as did treatment with ISRIB, integrated stress response inhibitor, which inhibits UPR signaling downstream of PERK. Finally, both drug affinity responsive target stability and thermal shift assays demonstrated direct binding of 2P-Im to endoplasmic reticulum chaperone BiP (GRP78/BiP), a stress-inducible key signaling molecule of the UPR. These data reveal GRP78/BiP as a novel target of SOTs, and specifically of 2P-Im, and suggest the potential broader utility of this class of small molecules as modulators of the UPR.

Infancy‐onset diabetes caused by de‐regulated AMPylation of the human endoplasmic reticulum chaperone BiP

Dysfunction of the endoplasmic reticulum (ER) in insulin‐producing beta cells results in cell loss and diabetes mellitus. Here we report on five individuals from three different consanguineous families with infancy‐onset diabetes mellitus and severe neurodevelopmental delay caused by a homozygous p.(Arg371Ser) mutation in FICD. The FICD gene encodes a bifunctional Fic domain‐containing enzyme that regulates the ER Hsp70 chaperone, BiP, via catalysis of two antagonistic reactions: inhibitory AMPylation and stimulatory deAMPylation of BiP. Arg371 is a conserved residue in the Fic domain active site. The FICDR371S mutation partially compromises BiP AMPylation in vitro but eliminates all detectable deAMPylation activity. Overexpression of FICDR371S or knock‐in of the mutation at the FICD locus of stressed CHO cells results in inappropriately elevated levels of AMPylated BiP and compromised secretion. These findings, guided by human genetics, highlight the destructive consequences of de‐regulated BiP AMPylation and raise the prospect of tuning FICD's antagonistic activities towards therapeutic ends.

Full-Length Transcriptome Reconstruction Reveals the Genetic Mechanisms of Eyestalk Displacement and Its Potential Implications on the Interspecific Hybrid Crab (Scylla serrata ♀ × S. paramamosain ♂).

Simple SummaryThe eyestalk is a key organ in crustaceans that produces neurohormones and regulates a range of physiological functions. Eyestalk displacement was discovered in some first-generation (F1) offspring of the novel interspecific hybrid crab (Scylla serrata ♀ × S. paramamosain ♂). To uncover the genetic mechanism underlying eyestalk displacement and its potential implications, high-quality transcriptome was reconstructed using single-molecule real-time (SMRT) sequencing. A total of 37 significantly differential alternative splicing (DAS) events (17 up-regulated and 20 down-regulated) and 1475 significantly differential expressed transcripts (DETs) (492 up-regulated and 983 down-regulated) were detected in hybrid crabs with displaced eyestalks (DH). The most significant DAS events and DETs were annotated as being endoplasmic reticulum chaperone BiP and leucine-rich repeat protein lrrA-like isoform X2. In addition, the top ten significant gene ontology (GO) terms were related to the cuticle or chitin. Overall, this study highlights the underlying genetic mechanisms of eyestalk displacement and provide useful knowledge for mud crab (Scylla spp.) crossbreeding.The lack of high-quality juvenile crabs is the greatest impediment to the growth of the mud crab (Scylla paramamosain) industry. To obtain high-quality hybrid offspring, a novel hybrid mud crab (S. serrata ♀ × S. paramamosain ♂) was successfully produced in our previous study. Meanwhile, an interesting phenomenon was discovered, that some first-generation (F1) hybrid offspring’s eyestalks were displaced during the crablet stage I. To uncover the genetic mechanism underlying eyestalk displacement and its potential implications, both single-molecule real-time (SMRT) and Illumina RNA sequencing were implemented. Using a two-step collapsing strategy, three high-quality reconstructed transcriptomes were obtained from purebred mud crabs (S. paramamosain) with normal eyestalks (SPA), hybrid crabs with normal eyestalks (NH), and hybrid crabs with displaced eyestalks (DH). In total, 37 significantly differential alternative splicing (DAS) events (17 up-regulated and 20 down-regulated) and 1475 significantly differential expressed transcripts (DETs) (492 up-regulated and 983 down-regulated) were detected in DH. The most significant DAS events and DETs were annotated as being endoplasmic reticulum chaperone BiP and leucine-rich repeat protein lrrA-like isoform X2. In addition, the top ten significant GO terms were related to the cuticle or chitin. Overall, high-quality reconstructed transcriptomes were obtained for the novel interspecific hybrid crab and provided valuable insights into the genetic mechanisms of eyestalk displacement in mud crab (Scylla spp.) crossbreeding.

Abstract 5144: Using proteomic approach to identify potential tumor-associated antigens (TAAs) as biomarkers in Hispanic hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with high prevalence and poor prognosis. The incidence rate and mortality rate in Hispanic HCC is relative higher than other ethnic groups. Autoantibodies against tumor associated antigens (TAAs) have long been proved as useful biomarkers in cancer diagnosis. In the present study, sera from 25 Hispanic patients with HCC and 25 sera from normal individuals were used in a proteomic approach to identify HCC-related TAAs. Twenty-four immunoreactive protein spots (16 from HepG2 cells, and 8 from SUN449 cells) were excised from the two-dimensional electrophoresis (2DE) gels, digested with trypsin, and subsequently analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Of 24 immunoreactive protein spots, 21 were identified with compatible molecular weights and isoelectric points. After further analysis, a total of 9 proteins were found to be potential TAAs, including endoplasmic reticulum chaperone BiP (HSPA5), 60 kDa heat shock protein (HSP60), triosephosphate isomerase (TPI1), protein disulfide-isomerase (PDI), Actin-cytoplasmic 2 (ACTG1), calreticulin (CRP55), alpha-enolase (ENO1), heat shock 70 kDa protein (HSP70), tropomyosin alpha-3 chain (TPM3). Among these proteins, HSPA5 was corresponded to 5 protein spots on the 2DE gels, while 4 proteins (HSPA5, HSP60, TPI1 and PDI) were found in both HepG2 and SNU449 cell lines. Enrichment analysis show that these proteins are mostly responsible for protein folding in endoplasmic reticulum as well as ubiquitin ligase binding function. In the subsequent study, autoantibodies against these proteins will be detected by immunoassay, and further evaluated as biomarkers in detection of Hispanic HCC. Citation Format: Yangcheng Ma, Cuipeng Qiu, Xiaojun Zhang, Xiao Wang, Jianying Zhang. Using proteomic approach to identify potential tumor-associated antigens (TAAs) as biomarkers in Hispanic hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5144.

Cancer cells adapt FAM134B/BiP mediated ER-phagy to survive hypoxic stress

In the tumor microenvironment, cancer cells experience hypoxia resulting in the accumulation of misfolded/unfolded proteins largely in the endoplasmic reticulum (ER). Consequently, ER proteotoxicity elicits unfolded protein response (UPR) as an adaptive mechanism to resolve ER stress. In addition to canonical UPR, proteotoxicity also stimulates the selective, autophagy-dependent, removal of discrete ER domains loaded with misfolded proteins to further alleviate ER stress. These mechanisms can favor cancer cell growth, metastasis, and long-term survival. Our investigations reveal that during hypoxia-induced ER stress, the ER-phagy receptor FAM134B targets damaged portions of ER into autophagosomes to restore ER homeostasis in cancer cells. Loss of FAM134B in breast cancer cells results in increased ER stress and reduced cell proliferation. Mechanistically, upon sensing hypoxia-induced proteotoxic stress, the ER chaperone BiP forms a complex with FAM134B and promotes ER-phagy. To prove the translational implication of our mechanistic findings, we identified vitexin as a pharmacological agent that disrupts FAM134B-BiP complex, inhibits ER-phagy, and potently suppresses breast cancer progression in vivo.

Subtilase cytotoxin from Shiga-toxigenic Escherichia coli impairs the inflammasome and exacerbates enteropathogenic bacterial infection

SummarySubtilase cytotoxin (SubAB) is an AB5 toxin mainly produced by the locus of enterocyte effacement-negative Shiga-toxigenic Escherichia coli (STEC) strain such as O113:H21, yet the contribution of SubAB to STEC infectious disease is unclear. We found that SubAB reduced activation of the STEC O113:H21 infection-induced non-canonical NLRP3 inflammasome and interleukin (IL)-1β and IL-18 production in murine macrophages. Downstream of lipopolysaccharide signaling, SubAB suppressed caspase-11 expression by inhibiting interferon-β/STAT1 signaling, followed by disrupting formation of the NLRP3/caspase-1 assembly. These inhibitions were regulated by PERK/IRE1α-dependent endoplasmic reticulum (ER) stress signaling initiated by cleavage of the host ER chaperone BiP by SubAB. Our murine model of SubAB-producing Citrobacter rodentium demonstrated that SubAB promoted C. rodentium proliferation and worsened symptoms such as intestinal hyperplasia and diarrhea. These findings highlight the inhibitory effect of SubAB on the NLRP3 inflammasome via ER stress, which may be associated with STEC survival and infectious disease pathogenicity in hosts.

The endoplasmic reticulum chaperone BiP is a closure-accelerating cochaperone of Grp94

Hsp70 and Hsp90 chaperones provide protein quality control to the cytoplasm, endoplasmic reticulum (ER), and mitochondria. Hsp90 activity is often enhanced by cochaperones that drive conformational changes needed for ATP-dependent closure and capture of client proteins. Hsp90 activity is also enhanced when working with Hsp70, but, in this case, the underlying mechanistic explanation is poorly understood. Here we examine the ER-specific Hsp70/Hsp90 paralogs (BiP/Grp94) and discover that BiP itself acts as a cochaperone that accelerates Grp94 closure. The BiP nucleotide binding domain, which interacts with the Grp94 middle domain, is responsible for Grp94 closure acceleration. A client protein initiates a coordinated progression of steps for the BiP/Grp94 system, in which client binding to BiP causes a conformational change that enables BiP to bind to Grp94 and accelerate its ATP-dependent closure. Single-molecule fluorescence resonance energy transfer measurements show that BiP accelerates Grp94 closure by stabilizing a high-energy conformational intermediate that otherwise acts as an energetic barrier to closure. These findings provide an explanation for enhanced activity of BiP and Grp94 when working as a pair, and demonstrate the importance of a high-energy conformational state in controlling the timing of the Grp94 conformational cycle. Given the high conservation of the Hsp70/Hsp90 system, other Hsp70s may also serve dual roles as both chaperones and closure-accelerating cochaperones to their Hsp90 counterparts.

Influence of the Season and Region Factor on Phosphoproteome of Stallion Epididymal Sperm

Simple SummaryPhosphorylation and dephosphorylation of proteins are considered to be the most important processes in sperm maturation during the epididymal transit. We demonstrated that 27 proteins underwent phosphorylation both in and out of the breeding season. Differences in the phosphorylation status were demonstrated in the case of endoplasmic reticulum chaperone BiP, albumin, protein disulfide-isomerase A3, nesprin-1, peroxiredoxin-5, and protein bicaudal D homolog.Epididymal maturation can be defined as a scope of changes occurring during epididymal transit that prepare spermatozoa to undergo capacitation. One of the most common post-translational modifications involved in the sperm maturation process and their ability to fertilise an oocyte is the phosphorylation of sperm proteins. The aim of this study was to compare tyrosine, serine, and threonine phosphorylation patterns of sperm proteins isolated from three subsequent segments of the stallion epididymis, during and out of the breeding season. Intensities of phosphorylation signals and phosphoproteins profiles varied in consecutive regions of the epididymis. However, significant differences in the phosphorylation status were demonstrated in case of endoplasmic reticulum chaperone BiP (75 and 32 kDa), protein disulfide-isomerase A3 (50 kDa), nesprin-1 (23 kDa), peroxiredoxin-5 (17 kDa), and protein bicaudal D homolog (15 kDa) for season x type of phosphorylated residues variables. Significant differences in the phosphorylation status were also demonstrated in case of endoplasmic reticulum chaperone BiP and albumin (61 kDa), protein disulfide-isomerase A3 (50 kDa), and protein bicaudal D homolog (15 kDa) for region x type of phosphorylated residues variables.

Progressive Aggregation of 16 kDa Gamma-Zein during Seed Maturation in Transgenic Arabidopsis thaliana.

Prolamins constitute a unique class of seed storage proteins, present only in grasses. In the lumen of the endoplasmic reticulum (ER), prolamins form large, insoluble heteropolymers termed protein bodies (PB). In transgenic Arabidopsis (Arabidopsis thaliana) leaves, the major maize (Zea mays) prolamin, 27 kDa γ-zein (27γz), assembles into insoluble disulfide-linked polymers, as in maize endosperm, forming homotypic PB. The 16 kDa γ-zein (16γz), evolved from 27γz, instead forms disulfide-bonded dispersed electron-dense threads that enlarge the ER lumen without assembling into PB. We have investigated whether the peculiar features of 16γz are also maintained during transgenic seed development. We show that 16γz progressively changes its electron microscopy appearance during transgenic Arabidopsis embryo maturation, from dispersed threads to PB-like, compact structures. In mature seeds, 16γz and 27γz PBs appear very similar. However, when mature embryos are treated with a reducing agent, 27γz is fully solubilized, as expected, whereas 16γz remains largely insoluble also in reducing conditions and drives insolubilization of the ER chaperone BiP. These results indicate that 16γz expressed in the absence of the other zein partners forms aggregates in a storage tissue, strongly supporting the view that 16γz behaves as the unassembled subunit of a large heteropolymer, the PB, and could have evolved successfully only following the emergence of the much more structurally self-sufficient 27γz.