Endoplasmic Reticulum Chaperone Research Articles

Targeted Inhibition of P4HB Promotes Cell Sensitivity to Gemcitabine in Urothelial Carcinoma of the Bladder.

BackgroundBladder cancer (BC) is a common malignancy worldwide that accounts for 3% of global cancer diagnoses. Chemotherapy resistance limits the therapeutic effect of chemotherapeutic agents in patients with BC. Prolyl 4-hydroxylase, beta polypeptide (P4HB) is an endoplasmic reticulum (ER) chaperone that is upregulated in bladder cancer tissues (The Cancer Genome Atlas, TCGA datasets). Knockdown or suppression of P4HB exerts anticancer activity and sensitizes cells to chemotherapy in various types of cancer.PurposeWe aimed to investigate whether the inhibition of P4HB enhances the anticancer efficacy of gemcitabine (GEM) in BC cells and to study the underlying molecular mechanisms.Patients and MethodsThe P4HB mRNA expression levels of 411 BC patients from the TCGA database and P4HB expression level of eighty BC paraffin-embedded samples detected by immunohistochemistry (IHC) staining were used for clinical feature and prognostic analyses. Bioinformatics analysis was utilized for the mechanistic investigation. Highly P4HB-expressed BC cell lines (T24 and 5637) treated with P4HB inhibitor (Bacitracin, BAC) were used to study the effects of BAC on the sensitivity of BC cells to GEM and the potential mechanism. P4HB inhibition experiments were performed in highly P4HB-expressed BC cells, and cell viability, colony formation, cell cycle, reactive oxygen species (ROS), apoptosis and pathway proteins were assessed in T24 and 5637 cells.ResultsWestern blot analysis showed that P4HB expression was significantly higher in BC tissues than in paired normal tissues. IHC showed that patients with high P4HB expression had a poorer overall survival (OS) rate than those with low P4HB expression. Furthermore, increased P4HB expression was demonstrated to be an independent prognostic marker for BC. Functionally, P4HB inhibition by BAC decreased the cell proliferation ability in vitro. Moreover, BAC treatment sensitized BC cells to GEM. Molecular mechanism analysis indicated that inhibition of P4HB by BAC treatment enhanced the anticancer effects of GEM through increasing cellular ROS content and promoting cell apoptosis and PERK/eIF2α/ATF4/CHOP signaling.ConclusionHigh P4HB expression was significantly correlated with poor prognosis in BC patients. Inhibition of P4HB by BAC decreased the cell proliferation ability and sensitized BC cells to GEM by activating apoptosis and the PERK/eIF2α/ATF4/CHOP pathways.

Abstract MP22: GRP78 Protects Against Angiotensin II-induced Protein Aggregation, Profibrotic VSMC Phenotype, And Vascular Remodeling

Arterial stiffening is a critical risk factor contributing to the development of cardiovascular disease and end organ damage. Vascular smooth muscle cell (VSMC) hypercontractility, vessel fibrosis, and proinflammatory responses are involved. The renin angiotensin system plays critical roles in the development of VSMC stiffness phenotype, as well as CVD pathophysiology. Angiotensin II (Ang II) stimulation induces these stiffness-promoting mechanisms, however, the involvement of dysfunctional proteostasis governing Ang II contribution to vascular stiffness remains underdefined. While endoplasmic reticulum (ER) stress has been implicated in the development of arterial stiffness and hypertension, it is unknown whether the protein aggregation potentially due to maladapted unfolded protein response (UPR) in VSMCs is involved. We hypothesize AngII induced ER stress initiates protein misfolding and effects VSMC stiffness phenotype. We identify a significant increase in protein aggregate size (P<0.001: from 1,000 to 4,500 pixel/aggregate) and number per cell (P<0.01: from 20 to 100 aggregate #/cell) in rat VSMCs stimulated with AngII, which is attenuated with adenoviral overexpression of resident ER chaperone, GRP78 (P<0.01: 2000 pixels/aggregate and P<0.05: 25 aggregate #/cell). Furthermore, UPRosome associated IRE1α phosphorylation induced at 3 and 6 h following AngII stimulation is reduced in GRP78 overexpressing VSMCs (P<0.05), which may contribute to reduced monocyte adhesion (P<0.001), assessed utilizing THP-1 cells. In mice, pharmacological chaperone 3-HNA reduced AngII induced vascular hypertrophy (P=0.056), cardiac perivascular fibrosis (P=0.065), and pulse propagation velocity from 0.35 m/s to 0.23 m/s (P<0.05). A trend in pre-amyloid oligomer accumulation was seen in the medial layer of thoracic aortas following 2 weeks of AngII infusion, which was reduced with 3-HNA treatment. Similar findings were investigated in a SM22α cre-GRP78 mouse model, where a trend in reduction of these parameters are also seen. Overall, GRP78 regulation over VSMC proteostasis influences vascular stiffness phenotype and therefore, these findings may elude new pharmacological intervention for arterial stiffness and aging associated CVD.

Cell surface GRP78 signaling: An emerging role as a transcriptional modulator in cancer.

Cancer cells acquire dysregulated gene expression to establish specific transcriptional dependencies and their underlying mechanisms that are ultimately responsible for this addictions have not been fully elucidated. Glucose-regulated protein 78 (GRP78) is a stress-inducible, multifunctional, prosurvival, endoplasmic reticulum chaperone in the heat shock protein 70 family. Expression of cell surface GRP78 (CS-GRP78) is associated with increased malignant behavior and resistance to chemotherapy and radiotherapy by endowing various cancer cells with increased proliferative ability, altered metabolism, improved survival, and augmented invasive and metastatic potential. Emerging evidence has highlighted an unusual role of CS-GRP78 in regulating transcription factors (TFs) by mediating various signaling pathways involved in malignant transformation, metabolic reprogramming, and tumor progression. During the last decade, we targeted CS-GRP78 with C38 monoclonal antibody (C38 Mab) in numerous studies, which have highlighted the epigenetic interplay between CS-GRP78 and various TFs including c-MYC, Yes-associated protein/transcriptional coactivator with PDZ-binding motif, c-Fos, and histone acetylation to potentiate subsequent modulation of tumorigenesis, invasion, and metastasis. Here, we summarize the current state of knowledge about the role of CS-GRP78 in cancer development and progression, including epigenetic regulation and sheds light on CS-GRP78 as vulnerable target for cancer therapy. Overall, this review focuses on the mechanisms of TFs that are behind the transcriptional dysregulation in cancer and lays the groundwork for rational therapeutic use of C38 Mab based on CS-GRP78 biology.

An ALS-linked mutation in TDP-43 disrupts normal protein interactions in the motor neuron response to oxidative stress

TDP-43 pathology is a key feature of amyotrophic lateral sclerosis (ALS), but the mechanisms linking TDP-43 to altered cellular function and neurodegeneration remain unclear. We have recently described a mouse model in which human wild-type or mutant TDP-43 are expressed at low levels and where altered stress granule formation is a robust phenotype of TDP-43M337V/− expressing cells. In the present study we use this model to investigate the functional connectivity of human TDP-43 in primary motor neurons under resting conditions and in response to oxidative stress. The interactome of human TDP-43WT or TDP-43M337V was compared by mass spectrometry, and gene ontology enrichment analysis identified pathways dysregulated by the M337V mutation. We found that under normal conditions the interactome of human TDP-43WT was enriched for proteins involved in transcription, translation and poly(A)-RNA binding. In response to oxidative stress, TDP-43WT recruits proteins of the endoplasmic reticulum and endosomal-extracellular transport pathways, interactions which are reduced in the presence of the M337V mutation. Specifically, TDP-43M337V impaired protein-protein interactions involved in stress granule formation including reduced binding to the translation initiation factors Poly(A)-binding protein and Eif4a1 and the endoplasmic reticulum chaperone Grp78. The M337V mutation also affected interactions involved in endosomal-extracellular transport and this this was associated with reduced extracellular vesicle secretion in primary motor neurons from TDP-43M337V/− mice and in human iPSCs-derived motor neurons. Taken together, our analysis highlights a TDP-43 interaction network in motor neurons and demonstrates that an ALS associated mutation may alter the interactome to drive aberrant pathways involved in the pathogenesis of ALS.

Protective Effects of Glucose-Related Protein 78 and 94 on Cisplatin-Mediated Ototoxicity.

Cisplatin is a widely used chemotherapeutic drug for treating various solid tumors. Ototoxicity is a major dose-limiting side effect of cisplatin, which causes progressive and irreversible sensorineural hearing loss. Here, we examined the protective effects of glucose-related protein (GRP) 78 and 94, also identified as endoplasmic reticulum (ER) chaperone proteins, on cisplatin-induced ototoxicity. Treating murine auditory cells (HEI-OC1) with 25 μM cisplatin for 24 h increased cell death resulting from excessive intracellular reactive oxygen species (ROS) accumulation and caspase-involved apoptotic signaling pathway activation with subsequent DNA fragmentation. GRP78 and GRP94 expression was increased in cells treated with 3 nM thapsigargin or 0.1 μg/mL tunicamycin for 24 h, referred to as mild ER stress condition. This condition, prior to cisplatin exposure, attenuated cisplatin-induced ototoxicity. The involvement of GRP78 and GRP94 induction was demonstrated by the knockdown of GRP78 or GRP94 expression using small interfering RNAs, which abolished the protective effect of mild ER stress condition on cisplatin-induced cytotoxicity. These results indicated that GRP78 and GRP94 induction plays a protective role in remediating cisplatin-ototoxicity.

1021 – BIOLOGY AND THERAPEUTIC TARGETING OF MUTANT CALRETICULIN INDUCED MPN

Recurrent mutations in calreticulin (CALR) are present in approximately 20-25% of patients with myeloproliferative neoplasms (MPN). Since its discovery in 2013, we now have a more precise understanding of how mutant CALR, an endoplasmic reticulum chaperone protein, activates the JAK/STAT signaling pathway via a pathogenic binding interaction with the thrombopoietin receptor, MPL, to induce MPN. A series of investigations from several groups, including ours, have since established the biologic requirements for mutant CALR-induced MPN which include: (i) expression of MPL and its N-glycosylation sites (ii) the mutant-specific C-terminus of mutant CALR and in particular its positive electrostatic charge (iii) a physical interaction between mutant CALR and MPL and (iv) the lectin-dependent function of mutant CALR. Building on this work, our group is focused on exploiting the mechanism understanding of mutant CALR-driven MPN for therapeutic gain with a particular focus on identifying and targeting unique molecular dependencies and on immunological approaches. Recurrent mutations in calreticulin (CALR) are present in approximately 20-25% of patients with myeloproliferative neoplasms (MPN). Since its discovery in 2013, we now have a more precise understanding of how mutant CALR, an endoplasmic reticulum chaperone protein, activates the JAK/STAT signaling pathway via a pathogenic binding interaction with the thrombopoietin receptor, MPL, to induce MPN. A series of investigations from several groups, including ours, have since established the biologic requirements for mutant CALR-induced MPN which include: (i) expression of MPL and its N-glycosylation sites (ii) the mutant-specific C-terminus of mutant CALR and in particular its positive electrostatic charge (iii) a physical interaction between mutant CALR and MPL and (iv) the lectin-dependent function of mutant CALR. Building on this work, our group is focused on exploiting the mechanism understanding of mutant CALR-driven MPN for therapeutic gain with a particular focus on identifying and targeting unique molecular dependencies and on immunological approaches.

HLA-B27 Subtypes Predisposing to Ankylosing Spondylitis Accumulate in an Endoplasmic Reticulum-Derived Compartment Apart From the Peptide-Loading Complex.

It was previously shown that HLA-B27 subtypes predisposing to spondyloarthritis (SpA), i.e., B*27:02, B*27:05, and B*27:07, displayed an increased propensity to form intracellular oligomers and to accumulate at a high density in cytoplasmic vesicles, as compared to the non-SpA-associated HLA-B*07:02 and HLA-B*27:06. This study was undertaken to characterize the nature and content of HLA-B-containing vesicles and to further examine their relevance to SpA predisposition. Vesicles containing HLA-B proteins were detected in transfected HeLa cells and in cells from SpA patients or HLA-B27/human β2 -microglobulin (hβ2 m)-transgenic rats, by microscopy. The nature and content of HLA-B-containing vesicles were characterized in colocalization experiments with appropriate markers. The SpA-associated HLA-B*27:04 subtype accumulated at higher levels (P < 10-5 ) in cytoplasmic vesicles compared to HLA-B*27:06, from which it differs only by 2 substitutions, reinforcing the correlation between vesicle formation and SpA predisposition. Colocalization studies showed that those vesicles contained misfolded HLA-B heavy chain along with β2 m and endoplasmic reticulum (ER) chaperones (calnexin, calreticulin, BiP, glucose-regulated protein 94-kd) and belonged to the ER but were distinct from the peptide-loading complex (PLC). Similar vesicles were observed in immune cells from HLA-B27+ SpA patients, in greater abundance than in healthy controls (P < 0.01), and in dendritic cells from HLA-B27/hβ2 m transgenic rats, correlating with SpA susceptibility. Accumulation of misfolded HLA-B heavy chain along with β2 m and ER chaperones into ER-derived vesicles distinct from the PLC is a characteristic feature of HLA-B27 subtypes predisposing to SpA. This phenomenon could contribute to HLA-B27 pathogenicity, via a noncanonical mechanism.

Urinary ERdj3 and mesencephalic astrocyte-derived neutrophic factor identify endoplasmic reticulum stress in glomerular disease

Podocyte injury and endoplasmic reticulum (ER) stress have been implicated in the pathogenesis of various glomerular diseases. ERdj3 (DNAJB11) and mesencephalic astrocyte-derived neurotrophic factor (MANF) are ER chaperones lacking the KDEL motif, and may be secreted extracellularly. Since podocytes reside in the urinary space, we examined if podocyte injury is associated with secretion of KDEL-free ER chaperones from these cells into the urine, and if chaperones in the urine reflect ER stress in glomerulonephritis. In cultured podocytes, ER stress increased ERdj3 and MANF intracellularly and in culture medium, whereas GRP94 (KDEL chaperone) increased only intracellularly. ERdj3 and MANF secretion was blocked by the secretory trafficking inhibitor, brefeldin A. Urinary ERdj3 and MANF increased in rats injected with tunicamycin (in the absence of proteinuria). After induction of passive Heymann nephritis (PHN) and puromycin aminonucleoside nephrosis (PAN), there was an increase in glomerular ER stress, and appearance of ERdj3 and MANF in the urine, coinciding with the onset of proteinuria. Rats with PHN were treated with the chemical chaperone, 4-phenyl butyrate (PBA), starting at the time of disease induction, or after disease was established. In both protocols, 4-PBA reduced proteinuria and urinary ER chaperone secretion, compared with PHN rats treated with saline (control). In conclusion, urinary ERdj3 and MANF reflect glomerular ER stress. 4-PBA protected against complement-mediated podocyte injury and the therapeutic response could be monitored by urinary ERdj3 and MANF.

N-glycan in the scavenger receptor cysteine-rich domain of hepsin promotes intracellular trafficking and cell surface expression

The group A scavenger receptor cysteine-rich (SRCR) domain is a conserved module present in numerous proteins involved in diverse biological processes. Hepsin, a hepatic protease implicated in many cancers, consists of a cytoplasmic tail, a transmembrane domain and an extracellular regions with a group A SRCR domain and a serine protease domain. Like in many SRCR-containing proteins, the SRCR domain in hepsin has an N-glycosylation site, but its functional significance is unknown. In this study, we confirmed N-glycosylation at Asn112 in hepsin by glycosidase digestion and site-directed mutagenesis in human hepatoma cells. In Western blotting, fluorogenic substrate assay, flow cytometry, and protein-chase experiments, we found that Asn112 to Gln (N112Q) mutation inhibited hepsin intracellular trafficking, cell surface expression, and zymogen activation. By immunofluorescent staining, we found that the N112Q mutant was more abundant than wild-type hepsin in the endoplasmic reticulum (ER). Further co-immunoprecipitation studies indicated increased binding of the N112Q mutant to calnexin and binding-immunoglobulin protein (BiP), two ER chaperones. Our results indicate that the N-glycan in the SRCR domain of hepsin promotes intracellular trafficking and cell surface expression, possibly by a calnexin-dependent mechanism in facilitating ER exiting.

Ultrastructural variation and key ER chaperones response induced by heat stress in intestinal cells of sea cucumber Apostichopus japonicus

The unfolded protein response (UPR) is an important protective and compensatory strategy used during endoplasmic reticulum stress caused by factors including glucose starvation, low pH, or heat shock. However, there is very little information on the possible role(s) of the UPR under adverse conditions experienced by marine invertebrates. We observed that rough endoplasmic reticulum (ER) was dramatically expanded and numerous autophagosomes were accumulated in the intestinal cells of sea cucumbers, Apostichopus japonicus, under heat stress (4 h at 25°C compared with 15°C controls). Moreover, heat stress led to sharp increases in the relative transcript and protein expression levels of two primary ER chaperones: the endoplasmic reticulum resident protein 29-like (ERP29) and protein disulfide-isomerase A6-like (PDIA6). These results suggest a potential adaptive mechanism to deal with heat-induced stress in sea cucumber intestine.

51-OR: Loss of Critical ER Chaperone GRP78 Leads to Beta-Cell Death by a JNK Dependent Mechanism

Endoplasmic reticulum (ER) health is an important determinant of pancreatic beta cell function or failure. Adaptive ER stress leads to expansion of beta cell number through proliferation, but decompensated ER stress results in beta cell loss and contributes to both T1D and T2D. Therefore, understanding the mechanisms of ER-stress-induced beta cell death could help identify novel strategies for the prevention or treatment of diabetes. Glucose responsive protein GRP78/HSPA5 is a key ER chaperone. Loss of GRP78 causes cell death in many cell types; including, in our own prior work, the beta cell. To study the mechanism by which GRP78 loss leads to beta cell death we devised an ex vivo model of GRP78 knockdown by transducing Grp78-flox mouse islets with adenovirus expressing Cre recombinase. To study human islets, we used adenovirus expressing shRNA against GRP78. Using these tools, we successfully reduced GRP78 RNA and protein in primary mouse and human islet cells. Loss of GRP78 strongly activated the ATF6, IRE1 and PERK pathways of the unfolded protein response (UPR). GRP78 knockdown decreased beta cell number due to increased cell death as measured by Annexin V and TUNEL staining. Probing the mechanisms of cell loss, we found that loss of GRP78 activated multiple death-related pathways, notably autophagy and apoptosis; cell death effectors Chop, caspase 9 and cleaved caspase 3 were increased. Additionally, GRP78 deletion increased stress kinase JNK (c-Jun-N-terminal kinase) which has been implicated in cellular proliferation as well as cell death. Inhibition of JNK kinase using JNK-IN-8 inhibitor during GRP78 loss conditions decreased phosphorylation of JNK and rescued beta cells from cell death as measured with Annexin V and TUNEL. Therefore, we conclude that ER stress related cell death induced by loss of GRP78 (ER stress) involves JNK activation, and that modulation of JNK under certain circumstances may help maintain beta cell mass to protect against diabetes. Disclosure R.B. Sharma: None. C.O. Darko: None. B. Gablaski: None. A.S. Lee: None. L.C. Alonso: Consultant; Self; Fairbanks Pharmaceuticals Inc. Research Support; Self; American Diabetes Association. Funding National Institute of Diabetes and Digestive and Kidney Diseases (5R01DK113300-02)

2136-PUB: GLP-1 Agonist Exendin-4 Protects Endothelial Cells against Hyperhomocysteinemia by Alleviating ER Stress and Enhancing Protein Folding Machinery

Hyperhomocysteinemia (HHcy) is a risk factor for higher mortality in type 2 diabetic patients. HHcy induces endothelial dysfunction, which precedes overt hyperglycemia in type 2 diabetic patients. In addition, endothelial dysfunction predisposes diabetic patients to cardiovascular disorders, such as atherosclerotic cardiovascular diseases and retinopathy. HHcy induces oxidative stress and endoplasmic reticulum (ER) stress to impair endothelial cells. GLP-1 agonist exendin-4 was shown to attenuate ER stress in endothelial cells although detailed mechanism remains elusive. The current study intends to reveal the beneficial effects of exendin-4 on HHcy-impaired endothelial cells. HHcy was induced by feeding C57BL/6 mice a high methionine low folate diet for 12 weeks. To assess endothelial function, mouse aortae and mesenteric arteries were mounted on wire myograph to determine acetylcholine-induced endothelium-dependent relaxations (EDRs). OGTT and ITT were conducted to study in vivo glucose metabolism. Dihydroethidium staining and lucigenin-enhanced chemiluminescence were used to measure ROS production. The ER stress markers expression was measured by biochemical assays. The role of ER chaperone was studied by adenoviral overexpression and siRNA-mediated knockdown. A 4-week subcutaneous injection of exendin-4 significantly improved glucose tolerance, insulin sensitivity and EDRs in arteries of diet-induced HHcy mice. Exendin-4 inhibited ROS production and ER stress markers expression in mouse aortae and human endothelial cells. Such inhibitions were ablated by compound C, an AMPK inhibitor. By AMPK activation, exendin-4 up-regulated endothelial expression of ER oxidoreductase (ERO1α), an ER chaperone. In short, through AMPK-dependent upregulation of ERO1α in endothelial cells, GLP-1 agonist exendin-4 lowers HHcy-induced ER stress and oxidative stress to restore endothelial function. Disclosure C. Cheng: None. J. Luo: None. C. Lau: None. X. Tian: None. Y. Huang: None. Funding Health and Medical Research Fund (05162906); Early Career Scheme (24122318); Hong Kong Research Grants Council (14112919); Collaborative Research Fund (C4024-16W)

Dkk3/REIC, an N-glycosylated Protein, Is a Physiological Endoplasmic Reticulum Stress Inducer in the Mouse Adrenal Gland

Dickkopf 3 (Dkk3) is a secreted protein belonging to the Dkk family and encoded by the orthologous gene of REIC. Dkk3/REIC is expressed by mouse and human adrenal glands, but the understanding of its roles in this organ is still limited. To determine the functions of Dkk3 in the mouse adrenal gland, we first identified that the mouse Dkk3 protein is N-glycosylated in the adrenal gland as well as in the brain. We performed proteome analysis on adrenal glands from Dkk3-null mice, in which exons 5 and 6 of the Dkk3 gene are deleted. Twodimensional polyacrylamide gel electrophoresis of adrenal proteins from wild-type and Dkk3-null mice revealed 5 protein spots whose intensities were altered between the 2 genotypes. Mass spectrometry analysis of these spots identified binding immunoglobulin protein (BiP), an endoplasmic reticulum (ER) chaperone. To determine whether mouse Dkk3 is involved in the unfolded protein response (UPR), we carried out a reporter assay using ER-stress responsive elements. Forced expression of Dkk3 resulted in the induction of distinct levels of reporter expression, showing the UPR initiated by the ER membrane proteins of activating transcription factor 6 (ATF6) and inositol-requring enzyme 1 (IRE1). Thus, it is possible that Dkk3 is a physiological ER stressor in the mouse adrenal gland.

Heat Shock Protein 47 Maintains Cancer Cell Growth by Inhibiting the Unfolded Protein Response Transducer IRE1α.

HSP47 is a collagen-specific protein chaperone expressed in fibroblasts, myofibroblasts, and stromal cells. HSP47 is also expressed in and involved in growth of cancer cells in which collagen levels are extremely low. However, its role in cancer remains largely unclear. Here, we showed that HSP47 maintains cancer cell growth via the unfolded protein response (UPR), the activation of which is well known to be induced by endoplasmic reticulum (ER) stress. We observed that HSP47 forms a complex with both the UPR transducer inositol-requiring enzyme 1α (IRE1α) and ER chaperone BiP in cancer cells. Moreover, HSP47 silencing triggered dissociation of BiP from IRE1α and IRE1α activation, followed by an increase in the intracellular level of reactive oxygen species (ROS). Increase in ROS induced accumulation of 4-hydroxy-2-nonenal-protein adducts and activated two UPR transducers, PKR-like ER kinase (PERK) and activating transcription factor 6α (ATF6α), resulting in impaired cancer cell growth. Our work indicates that HSP47 expressed in cancer cells relieves the ER stress arising from protein synthesis overload within these cells and tumor environments, such as stress induced by hypoxia, low glucose, and pH. We also propose that HSP47 has a biological role that is distinct from its normal function as a collagen-specific chaperone. IMPLICATIONS: HSP47 maintains cancer cell growth by inhibiting IRE1α.

SHQ1 is an ER stress response gene that facilitates chemotherapeutics-induced apoptosis via sensitizing ER-stress response

SHQ1 was reported to control the biogenesis and assembly of H/ACA ribonucleoprotein particles (RNPs). It was independently isolated as a growth suppressor, GRIM1, in a genetic screen. Recent studies have indicated that SHQ1 inhibits prostate cancer growth and metastasis. SHQ1 facilitates MYC RNA splicing to promote T-acute lymphoblastic leukemia (T-ALL) development. Thus, the mechanisms of SHQ1 in cancers remain largely unknown. We report here that SHQ1 promotes tumor apoptosis and chemo-sensitivity in hepatocellular carcinoma (HCC) cells. In HCC tissues from patients, expression of SHQ1 was significantly decreased in the tumor compared to adjacent tissues. Experiments with HCC xenograft models revealed that restoring SHQ1 levels enhanced the anti-tumor activity of the endoplasmic reticulum (ER) stress inducer tunicamycin (TM) and common chemotherapy drug paclitaxel (PTX). Mechanistically, SHQ1 is an ER-stress response gene which is regulated by p50ATF6 and XBP1s through an ER stress response like element located on the SHQ1 promoter. SHQ1 interacts with the ER chaperone GRP78 to release ER sensors PERK/IRE1α/ATF6 from GRP78/ER-sensor complexes, leading to hyper-activation of unfolded protein response (UPR). In the persistent ER stress conditions of a HepG2 xenograft tumor model, SHQ1-mediated hyper-activation of ER-sensor signaling induces apoptosis. Our study thus demonstrates a SHQ1-mediated ER-stress response feedback loop that promotes tumor sensitivity to chemotherapeutics.

Factor VIII exhibits chaperone-dependent and glucose-regulated reversible amyloid formation in the endoplasmic reticulum

AbstractHemophilia A, an X-linked bleeding disorder caused by deficiency of factor VIII (FVIII), is treated by protein replacement. Unfortunately, this regimen is costly due to the expense of producing recombinant FVIII as a consequence of its low-level secretion from mammalian host cells. FVIII expression activates the endoplasmic reticulum (ER) stress response, causes oxidative stress, and induces apoptosis. Importantly, little is known about the factors that cause protein misfolding and aggregation in metazoans. Here, we identified intrinsic and extrinsic factors that cause FVIII to form aggregates. We show that FVIII forms amyloid-like fibrils within the ER lumen upon increased FVIII synthesis or inhibition of glucose metabolism. Significantly, FVIII amyloids can be dissolved upon restoration of glucose metabolism to produce functional secreted FVIII. Two ER chaperone families and their cochaperones, immunoglobulin binding protein (BiP) and calnexin/calreticulin, promote FVIII solubility in the ER, where the former is also required for disaggregation. A short aggregation motif in the FVIII A1 domain (termed Aggron) is necessary and sufficient to seed β-sheet polymerization, and BiP binding to this Aggron prevents amyloidogenesis. Our findings provide novel insight into mechanisms that limit FVIII secretion and ER protein aggregation in general and have implication for ongoing hemophilia A gene-therapy clinical trials.

GRP78 expression and clinical outcomes in pancreatic cancer: A single institution review.

e16767 Background: Pancreatic Ductal Adenocarcinoma (PDAC) has one of the lowest 5-year survival rates of all cancers. Poor prognosis can be attributed in part to late detection and chemoresistance. Perioperative chemotherapy with or without radiation is the standard treatment for resectable PDAC, however recurrence rates remain high. GRP78 (HSPA5/BiP) is an endoplasmic reticulum (ER) chaperone protein involved in regulation of the ER stress response that leads to pro-survival pathway activation and chemoresistance in tumor cells. Prior studies of suppression of GRP78 expression in PDAC cell lines led to increased pancreatic cell apoptosis in response to gemcitabine. In vivo studies of human PDAC xenografts showed that tumors with higher GRP78 expression were associated with increased chemoresistance. Methods: We conducted a single institution retrospective review of 53 patients who underwent resection of PDAC from 1/1/2009-7/1/2016. Archived tumor tissue expression of GRP78 was assessed by immunohistochemistry (IHC) and scored as 0-3+. GRP78 IHC scores of 0-1+ were categorized as low and scores of 2-3+ as high. Corresponding clinical data were collected. Adjusted effects of GRP78 expression on the hazard of treatment failure or death were estimated using Cox proportional hazards models. Type III Wald Chi-square p-values are reported for the overall effect in each model. Results: GRP78 expression level demonstrated a significant effect on disease free survival (DFS) in unadjusted analyses (HR 2.00, p = 0.05), as well as after adjusting for performance status (p = 0.03), initial overall stage (p = 0.05), pathological tumor stage (p = 0.02), pathological node stage (p &lt; 0.01), resection margins (p = 0.05), and neoadjuvant therapy (p = 0.05) in separate models. In unadjusted analysis, high GRP78 expression showed a trend towards worsened overall survival (OS), but did not reach statistical significance (HR 1.8, p = 0.11). The effect on OS was significant when adjusting for pathological tumor stage (p = 0.04) and pathological node stage (p = 0.01). (575). Conclusions: Consistent with prior animal studies, our retrospective review revealed that high GRP78 expression was associated with worsened DFS in patients with resectable PDAC. When adjusting for pathological staging, high GRP78 expression had a similar impact on OS. These findings warrant further evaluation of the role of GRP78 in prognosis, response to therapy and as a potential treatment target.

ERp29 as a regulator of Insulin biosynthesis.

The environment within the Endoplasmic Reticulum (ER) influences Insulin biogenesis. In particular, ER stress may contribute to the development of Type 2 Diabetes (T2D) and Cystic Fibrosis Related Diabetes (CFRD), where evidence of impaired Insulin processing, including elevated secreted Proinsulin/Insulin ratios, are observed. Our group has established the role of a novel ER chaperone ERp29 (ER protein of 29 kDa) in the biogenesis of the Epithelial Sodium Channel, ENaC. The biogenesis of Insulin and ENaC share may key features, including their potential association with COP II machinery, their cleavage into a more active form in the Golgi or later compartments, and their ability to bypass such cleavage and remain in a less active form. Given these similarities we hypothesized that ERp29 is a critical factor in promoting the efficient conversion of Proinsulin to Insulin. Here, we confirmed that Proinsulin associates with the COP II vesicle cargo recognition component, Sec24D. When Sec24D expression was decreased, we observed a corresponding decrease in whole cell Proinsulin levels. In addition, we found that Sec24D associates with ERp29 in co-precipitation experiments and that ERp29 associates with Proinsulin in co-precipitation experiments. When ERp29 was overexpressed, a corresponding increase in whole cell Proinsulin levels was observed, while depletion of ERp29 decreased whole cell Proinsulin levels. Together, these data suggest a potential role for ERp29 in regulating Insulin biosynthesis, perhaps in promoting the exit of Proinsulin from the ER via Sec24D/COPII vesicles.

Isolation and characterization of a novel nanobody for detection of GRP78 expressing cancer cells.

Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER) chaperone that has been shown that is overexpressed in cancer cells. Overexpression of GRP78 on cancer cells makes this molecule a suitable candidate for cancer detection and targeted therapy. VHH is the binding fragment of camelid heavy-chain antibodies also known as "nanobody." The aim of this study is to isolate and produce a new recombinant nanobody using phage display technique to detect cancer cells. Using the c-terminal domain of GRP78 (CGRP) as an antigen, four rounds of biopanning were performed, and high-affinity binders were selected by ELISA. Their affinity and functionality were characterized by surface plasmon resonance (SPR) cell ELISA and immunocytochemistry. A unique nanobody named V80 was purified. ELISA and SPR showed that this antibody had high specificity and affinity to the GRP78. Immunofluorescence analysis showed that V80 could specifically bind to the HepG2 and A549 cancer cell lines. This novel recombinant nanobody could bind to the cell surface of different cancer cells. After further evaluation, this nanobody can be used as a new tool for cancer detection and tumor therapy.

Review: Protein misfolding diseases - the rare case of Marinesco-Sjögren syndrome.

Secretory and cell membrane proteins are synthesized in the endoplasmic reticulum (ER), where a network of molecular chaperones and folding factors ensure correct protein folding and export to post-ER compartments. Failure of this process leads to accumulation of unfolded/misfolded proteins, ER stress, and activation of the unfolded protein response (UPR), a complex signalling pathway aimed at restoring ER homeostasis, whose failure eventually leads to cell death. Suppressor of Ire1/Lhs1 double mutant (SIL1) is a nucleotide exchange factor for immunoglobulin binding protein, the main ER chaperone and primary sensor of ER stress. Loss of SIL1 function causes Marinesco-Sjögren syndrome (MSS), a rare multisystem disease of early infancy for which there is no cure. This review, examines the current understanding of SIL1 activities in the ER, and reviews experimental data describing the consequences of SIL1 deficiency in cell and animal models. We discuss the evidence supporting a role of the UPR - particularly the protein kinase RNA-like endoplasmic reticulum kinase branch - in the pathogenesis of MSS, and how this may be pharmacologically manipulated for treatment.