Endoplasmic Reticulum Chaperone Research Articles

Kuanoniamine C stimulates bortezomib-induced cell death via suppression of glucose-regulated protein 78 in osteosarcoma

Osteosarcoma is the most frequent and intractable malignancy of the bone in children and young adults. Surgical operation requires extensive excision of the cancer tissue and neighboring normal tissues. In addition, anticancer drugs and radiation therapy are thought to be almost ineffective. Glucose-regulated protein 78 (GRP78), a cell-protective endoplasmic reticulum (ER) chaperone protein, is one of the most promising anticancer targets for osteosarcoma. Here, by analyzing the molecular mechanisms of kuanoniamine C, we report that kuanoniamine C suppresses GRP78 expression via GRP78 mRNA degradation in an ER stress response-independent manner. Interestingly, kuanoniamine C-induced cell death and downregulation of GRP78 expression was regulated by p53 signaling. Moreover, co-treatment with bortezomib, which is a newly identified anticancer drug for osteosarcoma, and kuanoniamine C suppressed GRP78 protein expression, which is essential for the stimulation of bortezomib-induced cell death. These results suggest that co-treatment with bortezomib and kuanoniamine C is a novel therapeutic strategy for the treatment of osteosarcoma that enhances bortezomib-dependent cell death by the downregulation of GRP78, and this combination selectively targets the major cell population of osteosarcoma, which expresses wild-type p53.

Sigma-1 Receptor Positron Emission Tomography: A New Molecular Imaging Approach Using (S)-(-)-[18F]Fluspidine in Glioblastoma.

Glioblastoma multiforme (GBM) is the most devastating primary brain tumour characterised by infiltrative growth and resistance to therapies. According to recent research, the sigma-1 receptor (sig1R), an endoplasmic reticulum chaperone protein, is involved in signaling pathways assumed to control the proliferation of cancer cells and thus could serve as candidate for molecular characterisation of GBM. To test this hypothesis, we used the clinically applied sig1R-ligand (S)-(−)-[18F]fluspidine in imaging studies in an orthotopic mouse model of GBM (U87-MG) as well as in human GBM tissue. A tumour-specific overexpression of sig1R in the U87-MG model was revealed in vitro by autoradiography. The binding parameters demonstrated target-selective binding according to identical KD values in the tumour area and the contralateral side, but a higher density of sig1R in the tumour. Different kinetic profiles were observed in both areas, with a slower washout in the tumour tissue compared to the contralateral side. The translational relevance of sig1R imaging in oncology is reflected by the autoradiographic detection of tumour-specific expression of sig1R in samples obtained from patients with glioblastoma. Thus, the herein presented data support further research on sig1R in neuro-oncology.

Aggregates of RNA Binding Proteins and ER Chaperones Linked to Exosomes in Granulovacuolar Degeneration of the Alzheimer’s Disease Brain

Granulovacuolar degeneration (GVD) occurs in Alzheimer's disease (AD) brain due to compromised autophagy. Endoplasmic reticulum (ER) function and RNA binding protein (RBP) homeostasis regulate autophagy. We observed that the ER chaperones Glucose - regulated protein, 78 KDa (GRP78/BiP), Sigma receptor 1 (SigR1), and Vesicle-associated membrane protein associated protein B (VAPB) were elevated in many AD patients' subicular neurons. However, those neurons which were affected by GVD showed lower chaperone levels, and there was only minor co-localization of chaperones with GVD bodies (GVBs), suggesting that neurons lacking sufficient chaperone-mediated proteostasis enter the GVD pathway. Consistent with this notion, granular, incipient pTau aggregates in human AD and pR5 tau transgenic mouse neurons were regularly co-localized with increased chaperone immunoreactivity, whereas neurons with mature neurofibrillary tangles lacked both the chaperone buildup and significant GVD. On the other hand, APP/PS1 (APPswe/PSEN1dE9) transgenic mouse hippocampal neurons that are devoid of pTau accumulation displayed only few GVBs-like vesicles, which were still accompanied by prominent chaperone buildup. Identifying a potential trigger for GVD, we found cytoplasmic accumulations of RBPs including Matrin 3 and FUS as well as stress granules in GVBs of AD patient and pR5 mouse neurons. Interestingly, we observed that GVBs containing aggregated pTau and pTDP-43 were consistently co-localized with the exosomal marker Flotillin 1 in both AD and pR5 mice. In contrast, intraneuronal 82E1-immunoreactive amyloid-β in human AD and APP/PS1 mice only rarely co-localized with Flotillin 1-positive exosomal vesicles. We conclude that altered chaperone-mediated ER protein homeostasis and impaired autophagy manifesting in GVD are linked to both pTau and RBP accumulation and that some GVBs might be targeted to exocytosis.

Mesencephalic astrocyte–derived neurotrophic factor is an ER-resident chaperone that protects against reductive stress in the heart

We have previously demonstrated that ischemia/reperfusion (I/R) impairs endoplasmic reticulum (ER)-based protein folding in the heart and thereby activates an unfolded protein response sensor and effector, activated transcription factor 6α (ATF6). ATF6 then induces mesencephalic astrocyte-derived neurotrophic factor (MANF), an ER-resident protein with no known structural homologs and unclear ER function. To determine MANF's function in the heart in vivo, here we developed a cardiomyocyte-specific MANF-knockdown mouse model. MANF knockdown increased cardiac damage after I/R, which was reversed by AAV9-mediated ectopic MANF expression. Mechanistically, MANF knockdown in cultured neonatal rat ventricular myocytes (NRVMs) impaired protein folding in the ER and cardiomyocyte viability during simulated I/R. However, this was not due to MANF-mediated protection from reactive oxygen species generated during reperfusion. Because I/R impairs oxygen-dependent ER protein disulfide formation and such impairment can be caused by reductive stress in the ER, we examined the effects of the reductive ER stressor DTT. MANF knockdown in NRVMs increased cell death from DTT-mediated reductive ER stress, but not from nonreductive ER stresses caused by thapsigargin-mediated ER Ca2+ depletion or tunicamycin-mediated inhibition of ER protein glycosylation. In vitro, recombinant MANF exhibited chaperone activity that depended on its conserved cysteine residues. Moreover, in cells, MANF bound to a model ER protein exhibiting improper disulfide bond formation during reductive ER stress but did not bind to this protein during nonreductive ER stress. We conclude that MANF is an ER chaperone that enhances protein folding and myocyte viability during reductive ER stress.

Heat shock protein gp96 controls regulatory T cell lineage stability via IL-2/pSTAT5 signaling

Abstract Gp96 is an essential endoplasmic reticulum chaperone for various proteins including GARP, TLRs, integrins, Wnt co-receptor LRP6 and the platelet glycoprotein Ib/IX/V complex. Our laboratory has previously shown that loss of gp96 in murine Foxp3+ regulatory T cells (Tregs) results in their instability as determined by FOXP3 downregulation, impaired immunosuppressive function and production of effector cytokines (Zhang, et al., J Clin Invest., 2015, 125: 859–869). However, the mechanism underlying Treg dysfunction upon gp96 deletion remains unknown. Using tamoxifen-inducible Treg-specific gp96 gene (encoded by Hsp90b1) knockout (KO) mice (Foxp3-ERT2creHsp90b1f/f), we first phenotypically characterized gp96-deleted Tregs in mouse spleen and peripheral lymph nodes (pLNs). Gp96 deficient Tregs showed decreased frequency of TCF1−CD62L− effector population, but were enriched with short-lived KLRG1+ and Helios+ subsets, in both spleen and pLNs. We also confirmed that the expression of Foxp3 was decreased in splenic gp96-deleted Tregs, which correlated with a reduction of cell surface CD25. Intriguingly, both Foxp3 and surface CD25 levels stayed high in gp96-null Tregs in pLNs. In addition, gp96-null Tregs in mouse spleen, but not in pLNs, demonstrated compromised suppressive function. We further found that IL-2/CD25 signaling, as assessed by STAT5 phosphorylation, was more activated in gp96-null Tregs in pLNs, compared to that in spleen. In vitro IL-2 treatment was able to rescue the attenuated Foxp3 expression in gp96-null Tregs. Our work indicates that IL-2/pSTAT-5 signaling may be a key factor in maintaining Treg Foxp3 expression and its lineage stability upon gp96 deletion.

Thyrocyte cell survival and adaptation to chronic endoplasmic reticulum stress due to misfolded thyroglobulin

The large secretory glycoprotein thyroglobulin is the primary translation product of thyroid follicular cells. This difficult-to-fold protein is susceptible to structural alterations that disable export of the misfolded thyroglobulin from the endoplasmic reticulum (ER), which is a known cause of congenital hypothyroidism characterized by severe chronic thyrocyte ER stress. Nevertheless, individuals with this disease commonly grow a goiter, indicating thyroid cell survival and adaptation. To model these processes, here we continuously exposed rat PCCL3 thyrocytes to tunicamycin, which causes a significant degree of ER stress that is specifically attributable to thyroglobulin misfolding. We found that, in response, PCCL3 cells down-regulate expression of the "tunicamycin transporter" (major facilitator superfamily domain containing-2A, Mfsd2a). Following CRISPR/Cas9-mediated Mfsd2a deletion, PCCL3 cells could no longer escape the chronic effects of high-dose tunicamycin, as demonstrated by persistent accumulation of unglycosylated thyroglobulin; nevertheless, these thyrocytes survived and grew. A proteomic analysis of these cells adapted to chronic ER protein misfolding revealed many hundreds of up-regulated proteins, indicating stimulation of ER chaperones, oxidoreductases, stress responses, and lipid biosynthesis pathways. Further, we noted increased phospho-AMP-kinase, suggesting up-regulated AMP-kinase activity, and decreased phospho-S6-kinase and protein translation, suggesting decreased mTOR activity. These changes are consistent with conserved cell survival/adaptation pathways. We also observed a less-differentiated thyrocyte phenotype with decreased PAX8, FOXE1, and TPO protein levels, along with decreased thyroglobulin mRNA levels. In summary, we have developed a model of thyrocyte survival and growth during chronic continuous ER stress that recapitulates features of congenital hypothyroid goiter caused by mutant thyroglobulin.

Calnexin cycle - structural features of the ER chaperone system.

The endoplasmic reticulum (ER) is the major folding compartment for secreted and membrane proteins and is the site of a specific chaperone system, the calnexin cycle, for folding N‐glycosylated proteins. Recent structures of components of the calnexin cycle have deepened our understanding of quality control mechanisms and protein folding pathways in the ER. In the calnexin cycle, proteins carrying monoglucosylated glycans bind to the lectin chaperones calnexin and calreticulin, which recruit a variety of function‐specific chaperones to mediate protein disulfide formation, proline isomerization, and general protein folding. Upon trimming by glucosidase II, the glycan without an inner glucose residue is no longer able to bind to the lectin chaperones. For proteins that have not yet folded properly, the enzyme UDP‐glucose:glycoprotein glucosyltransferase (UGGT) acts as a checkpoint by adding a glucose back to the N‐glycan. This allows the misfolded proteins to re‐associate with calnexin and calreticulin for additional rounds of chaperone‐mediated refolding and prevents them from exiting the ERs. Here, we review progress in structural studies of the calnexin cycle, which reveal common features of how lectin chaperones recruit function‐specific chaperones and how UGGT recognizes misfolded proteins.

Characterization of Endoplasmic Reticulum (ER) in Human Pluripotent Stem Cells Revealed Increased Susceptibility to Cell Death upon ER Stress.

Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), have a well-orchestrated program for differentiation and self-renewal. However, the structural features of unique proteostatic-maintaining mechanisms in hPSCs and their features, distinct from those of differentiated cells, in response to cellular stress remain unclear. We evaluated and compared the morphological features and stress response of hPSCs and fibroblasts. Compared to fibroblasts, electron microscopy showed simpler/fewer structures with fewer networks in the endoplasmic reticulum (ER) of hPSCs, as well as lower expression of ER-related genes according to meta-analysis. As hPSCs contain low levels of binding immunoglobulin protein (BiP), an ER chaperone, thapsigargin treatment sharply increased the gene expression of the unfolded protein response. Thus, hPSCs with decreased chaperone function reacted sensitively to ER stress and entered apoptosis faster than fibroblasts. Such ER stress-induced apoptotic processes were abolished by tauroursodeoxycholic acid, an ER-stress reliever. Hence, our results revealed that as PSCs have an underdeveloped structure and express fewer BiP chaperone proteins than somatic cells, they are more susceptible to ER stress-induced apoptosis in response to stress.

Sequential Interferon β-Cisplatin Treatment Enhances the Surface Exposure of Calreticulin in Cancer Cells via an Interferon Regulatory Factor 1-Dependent Manner.

Immunogenic cell death (ICD) refers to a unique form of cell death that activates an adaptive immune response against dead-cell-associated antigens. Accumulating evidence indicates that the efficacy of conventional anticancer agents relies on not only their direct cytostatic/cytotoxic effects but also the activation of antitumor ICD. Common anticancer ICD inducers include certain chemotherapeutic agents (such as anthracyclines, oxaliplatin, and bortezomib), radiotherapy, photodynamic therapy (PDT), and oncolytic virotherapies. However, most chemotherapeutic reagents are inefficient or fail to trigger ICD. Therefore, better understanding on the molecular determinants of chemotherapy-induced ICD will help in the development of more efficient combinational anticancer strategies through converting non- or relatively weak ICD inducers into bona fide ICD inducers. In this study, we found that sequential, but not concurrent, treatment of cancer cells with interferon β (IFNβ), a type I IFN, and cisplatin (an inefficient ICD inducer) can enhance the expression of ICD biomarkers in cancer cells, including surface translocation of an endoplasmic reticulum (ER) chaperone, calreticulin (CRT), and phosphorylation of the eukaryotic translation initiation factor alpha (eIF2α). These results suggest that exogenous IFNβ may activate molecular determinants that convert cisplatin into an ICD inducer. Further bioinformatics and in vitro experimental analyses found that interferon regulatory factor 1 (IRF1) acted as an essential mediator of surface CRT exposure by sequential IFNβ-cisplatin combination. Our findings not only help to design more effective combinational anticancer therapy using IFNβ and cisplatin, but also provide a novel insight into the role of IRF1 in connecting the type I IFN responses and ICD.

Congenital Tufting Enteropathy-Associated Mutant of Epithelial Cell Adhesion Molecule Activates the Unfolded Protein Response in a Murine Model of the Disease.

Congenital tufting enteropathy (CTE) is a rare chronic diarrheal disease of infancy caused by mutations in epithelial cell adhesion molecule (EpCAM). Previously, a murine CTE model showed mis-localization of EpCAM away from the basolateral cell surface in the intestine. Here we demonstrate that mutant EpCAM accumulated in the endoplasmic reticulum (ER) where it co-localized with ER chaperone, GRP78/BiP, revealing potential involvement of ER stress-induced unfolded protein response (UPR) pathway in CTE. To investigate the significance of ER-localized mutant EpCAM in CTE, activation of the three UPR signaling branches initiated by the ER transmembrane protein components IRE1, PERK, and ATF6 was tested. A significant reduction in BLOS1 and SCARA3 mRNA levels in EpCAM mutant intestinal cells demonstrated that regulated IRE1-dependent decay (RIDD) was activated. However, IRE1 dependent XBP1 mRNA splicing was not induced. Furthermore, an increase in nuclear-localized ATF6 in mutant intestinal tissues revealed activation of the ATF6-signaling arm. Finally, an increase in both the phosphorylated form of the translation initiation factor, eIF2α, and ATF4 expression in the mutant intestine provided support for activation of the PERK-mediated pathway. Our results are consistent with a significant role for UPR in gastrointestinal homeostasis and provide a working model for CTE pathophysiology.

CXL146, a Novel 4H-Chromene Derivative, Targets GRP78 to Selectively Eliminate Multidrug-Resistant Cancer Cells.

The 78-kDa glucose-regulated protein (GRP78), an endoplasmic reticulum (ER) chaperone, is a master regulator of the ER stress. A number of studies revealed that high levels of GRP78 protein in cancer cells confer multidrug resistance (MDR) to therapeutic treatment. Therefore, drug candidate that reduces GRP78 may represent a novel approach to eliminate MDR cancer cells. Our earlier studies showed that a set of 4H-chromene derivatives induced selective cytotoxicity in MDR cancer cells. In the present study, we elucidated its selective mechanism in four MDR cancer cell lines with one lead candidate (CXL146). Cytotoxicity results confirmed the selective cytotoxicity of CXL146 toward the MDR cancer cell lines. We noted significant overexpression of GRP78 in all four MDR cell lines compared with the parental cell lines. Unexpectedly, CXL146 treatment rapidly and dose-dependently reduced GRP78 protein in MDR cancer cell lines. Using human leukemia (HL) 60/mitoxantrone (MX) 2 cell line as the model, we demonstrated that CXL146 treatment activated the unfolded protein response (UPR); as evidenced by the activation of inositol-requiring enzyme 1α, protein kinase R-like ER kinase, and activating transcription factor 6. CXL146-induced UPR activation led to a series of downstream events, including extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase activation, which contributed to CXL146-induced apoptosis. Targeted reduction in GRP78 resulted in reduced sensitivity of HL60/MX2 toward CXL146. Long-term sublethal CXL146 exposure also led to reduction in GRP78 in HL60/MX2. These data collectively support GRP78 as the target of CXL146 in MDR treatment. Interestingly, HL60/MX2 upon long-term sublethal CXL146 exposure regained sensitivity to mitoxantrone treatment. Therefore, further exploration of CXL146 as a novel therapy in treating MDR cancer cells is warranted. SIGNIFICANCE STATEMENT: Multidrug resistance is one major challenge to cancer treatment. This study provides evidence that cancer cells overexpress 78-kDa glucose-regulated protein (GRP78) as a mechanism to acquire resistance to standard cancer therapies. A chromene-based small molecule, CXL146, selectively eliminates cancer cells with GRP78 overexpression via activating unfolded protein response-mediated apoptosis. Further characterization indicates that CXL146 and standard therapies complementarily target different populations of cancer cells, supporting the potential of CXL146 to overcome multidrug resistance in cancer treatment.

Endoplasmic reticulum stress in forebrain subfornical organ excitatory neurons during diet‐induced obesity

Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) in the brain are emerging as key mechanisms in obesity‐related cardiovascular and metabolic diseases. For example, we recently demonstrated that inhibition of brain ER stress rescues hypertension and hepatic steatosis during high fat diet (HFD) feeding. Additional findings pointed to robust HFD‐induced ER stress in the subfornical organ (SFO), a circumventricular nucleus located outside of the blood‐brain‐barrier that is crucial for cardiometabolic regulation. The neuron population and anatomical location within the SFO that experiences ER stress during obesity remains unknown. Based on this, we comprehensively investigated UPR activation in the SFO during diet‐induced obesity. Six wk old male C57Bl/6J mice were placed on a HFD (n=4) or remained on normal chow (n=5) for 11 wks. Using serial cryosections, immunohistochemistry revealed marked upregulation of the ER chaperone glucose‐regulated peptide 78 kDa (GRP78) in the SFO of HFD fed mice, relative to normal chow controls (positive cells/section: 102±8 vs 177±9, normal chow vs HFD, p<0.05). This was evident throughout the rostral to caudal extent of the SFO (fold normal chow: rostral, 1.4±0.2; medial, 1.8±0.2; caudal, 1.8±0.1; all p<0.05). Similar findings were obtained when examining the ER chaperone protein disulfide isomerase (e.g. positive cells/section: 75±8 vs 107±4, normal chow vs HFD, p<0.05). Accumulating evidence indicates involvement of the PERK‐eIF2α pathway (protein kinase R like endoplasmic reticulum kinase‐eukaryotic initiation factor 2α) in cardiometabolic diseases and in line with this, the number of PERK expressing cells (cells/section: 32±10 vs 89±5, normal chow vs HFD, p<0.05) and downstream phosphorylated eIF2α positive cells (cells/section: 51±12 vs 125±24, normal chow vs HFD, p<0.05) was markedly elevated in the SFO following HFD feeding. The SFO can be anatomically subdivided into “core” and “shell” zones, which have distinct efferent projections to downstream nuclei involved in fluid, cardiovascular, and energy homeostasis; UPR activation was evident in both the core and shell zones. Given that a significant portion of SFO neurons that project to cardiometabolic nuclei are glutamatergic, we next investigated HFD‐induced ER stress in excitatory SFO neurons using double immunohistochemistry for UPR markers and the neuronal indicator calcium/calmodulin‐dependent kinase II (CAMKII; n=7/group). While the number of CAMKII positive neurons in the SFO was similar between HFD and normal chow groups, UPR activation was clearly evident in excitatory neurons during obesity as indicated by increased co‐localization of CAMKII with GRP78 (46+2 vs 59+3%, normal chow vs HFD, p<0.05), PDI (30+1 vs 49+3%, normal chow vs HFD, p<0.05), and phosphorylated eIF2α (40+2 vs 55+3%, normal chow vs HFD, p<0.05). Collectively, these findings indicate that obesity is associated with widespread anatomical ER stress throughout the SFO, including within excitatory neurons – suggesting that SFO ER stress during obesity has the potential to influence diverse downstream nuclei and thus physiological processes.Support or Funding Information1R01DK117007, 1R01HL141393

The sigma‐1 agonist PRE‐084 protects against energy depletion induced barrier disruption and also enhances glycolysis in HUVECs

The sigma‐1 receptor (σ1) is a well‐known endoplasmic reticulum (ER) chaperone that resides at the mitochondria‐associated ER membrane, having an important role in calcium signaling between ER and mitochondria. Expression of σ1 is present in all human tissues with specific predominance in arteries, liver, fibroblasts and brain. Our laboratory has shown that σ1 activation in lymphatic vessels elicits nitric oxide‐dependent lymphatic relaxation, a finding that suggested the potential important role of σ1 in endothelium. Other recent work has highlighted that σ1 may contribute to healthy endothelial barrier function, although little is known about the mechanisms involved. We hypothesized that σ1 helps protect the endothelial barrier disruption by stabilizing cellular bioenergetics. Cultured human umbilical vein endothelial cell (HUVEC) monolayers were used to model the endothelial barrier. Electric cell‐substrate impedance sensing (ECIS) was used to study the contribution of σ1 to endothelial barrier function. Western blotting was used to quantify σ1 protein expression. Endothelial bioenergetics were determined with extracellular flux analysis using glycolytic rate and ATP rate assays. The endothelial‐specific contribution of σ1 was assessed using with the σ1‐selective agonist PRE‐084 and by specific siRNA knockdown of σ1. The mitochondrial un‐coupler Carbonyl cyanide m‐chlorophenyl hydrazine (CCCP) was used to model mitochondrial dysfunction in HUVEC. Our results show that activation of σ1 with PRE‐084 50 μM, 100 μM, 150 μM, 200 μM significantly enhances barrier function of HUVEC monolayers in a concentration‐dependent manner. Knockdown of σ1 with siRNA reduces baseline barrier function and diminishes the ability of PRE‐084 to enhance TER. The decrease in TER elicited by CCCP (10 μM) is significantly amplified in cells with siRNA‐mediated knockdown of σ1 expression, and is reversed by PRE‐084 (100 μM) in cells with normal σ1 expression. HUVEC treated with PRE‐084 display higher levels of glycolysis and glycolytic ATP production than control cells. This response to PRE‐084 was not evident in the presence of σ1 knockdown. Collectively, our results suggest that σ1 plays an important role in maintenance of baseline endothelial barrier function, and that activation of σ1 can enhance barrier function. Moreover, σ1 activation also can rescue endothelial barrier dysfunction caused by disruption of mitochondrial ATP production, likely due to its ability enhance glycolysis. Thus, σ1 may represent a future therapeutic target to rescue endothelial barrier dysfunction.Support or Funding InformationThis work was supported by NIH Grant R01GM120774.

Preparation of Biodegradable PLGA-Nanoparticles Used for pH-Sensitive Intracellular Delivery of an Anti-inflammatory Bacterial Toxin to Macrophages.

Poly(D,L-lactide-co-glycolic) acid (PLGA) is a synthetic copolymer that has been used to design micro/nanoparticles as a carrier for macromolecules, such as protein and nucleic acids, that can be internalized by the endocytosis pathway. However, it is difficult to control the intracellular delivery to target organelles. Here we report an intracellular delivery system of nanoparticles modified with bacterial cytotoxins to the endoplasmic reticulum (ER) and anti-inflammatory activity of the nanoparticles. Subtilase cytotoxin (SubAB) is a bacterial toxin in certain enterohemorrhagic Escherichia coli (EHEC) strains that cleaves the host ER chaperone BiP and suppresses nuclear factor-kappaB (NF-κB) activation and nitric oxide (NO) generation in macrophages at sub-lethal concentration. PLGA-nanoparticles were modified with oligo histidine-tagged (6 × His-tagged) recombinant SubAB (SubAB-PLGA) through a pH-sensitive linkage, and their translocation to the ER in macrophage cell line J774.1 cells, effects on inducible NO synthase (iNOS), and levels of tumor necrosis factor (TNF)-α cytokine induced by lipopolysaccharide (LPS) were examined. Compared with free SubAB, SubAB-PLGA was significantly effective in BiP cleavage and the induction of the ER stress marker C/EBP homologous protein (CHOP) in J774.1 cells. Furthermore, SubAB-PLGA attenuated LPS-stimulated induction of iNOS and TNF-α. Our findings provide useful information for protein delivery to macrophages and may encourage therapeutic applications of nanoparticles to the treatment of inflammatory diseases.

GRP94 regulates M1 macrophage polarization and insulin resistance.

Macrophage polarization contributes to obesity-induced insulin resistance. Glucose-regulated protein 94 (GRP94) is an endoplasmic reticulum (ER) chaperone specialized for folding and quality control of secreted and membrane proteins. To determine the role of GRP94 in macrophage polarization and insulin resistance, macrophage-specific GRP94 conditional knockout (KO) mice were challenged with a high-fat diet (HFD). Glucose tolerance, insulin sensitivity, and macrophage composition were compared with control mice. KO mice showed better glucose tolerance and increased insulin sensitivity. Adipose tissues from HFD-KO mice contained lower numbers of M1 macrophages, with lower expression of M1 macrophage markers, than wild-type (WT) mice. In vitro, WT adipocytes cocultured with KO macrophages retained insulin sensitivity, whereas those cultured with WT macrophages did not. In addition, compared with WT bone marrow-derived macrophages (BMDMs), BMDMs from GRP94 KO mice exhibited lower expression of M1 macrophage marker genes following stimulation with LPS or IFN-γ, and exhibited partially increased expression of M2 macrophage marker genes following stimulation with interleukin-4. These findings identify GRP94 as a novel regulator of M1 macrophage polarization and insulin resistance and inflammation.

Perturbation of the Relative Contribution of Molecular Chaperones in the Endoplasmic Reticulum.

We demonstrate the preferential orders of molecular chaperones glucose-regulated protein 94 (GRP94), binding immunoglobulin protein (BiP), and calreticulin (CRT) in an endoplasmic reticulum (ER) fraction from rat liver using columns conjugated with denatured myoglobin, RNase A, or β-lactoglobulin as client proteins in the presence or absence of ATP. The results showed that BiP, CRT, and GRP94 preferentially contributed myoglobin, RNase A, and β-lactoglobulin, respectively, in the presence of ATP. In the absence of ATP, GRP94 and CRT preferentially recognized misfolded myoglobin (α-helix-rich protein), whereas BiP preferentially recognized misfolded RNase A (α-helix/β-sheet mixed protein) and β-lactoglobulin (β-sheet-rich protein). The preferential order of ER chaperones may be dynamically regulated by ER conditions and the higher-order structure of client proteins.

Clinical spectrum, prognosis and estimated prevalence of DNAJB11-kidney disease

Monoallelic mutations of DNAJB11 were recently described in seven pedigrees with atypical clinical presentations of autosomal dominant polycystic kidney disease. DNAJB11 encodes one of the main cofactors of the endoplasmic reticulum chaperon BiP, a heat-shock protein required for efficient protein folding and trafficking. Here we conducted an international collaborative study to better characterize the DNAJB11-associated phenotype. Thirteen different loss-of-function variants were identified in 20 new pedigrees (54 affected individuals) by targeted next-generation sequencing, whole-exome sequencing or whole-genome sequencing. Amongst the 77 patients (27 pedigrees) now in total reported, 32 reached end stage kidney disease (range, 55-89 years, median age 75); without a significant difference between males and females. While a majority of patients presented with non-enlarged polycystic kidneys, renal cysts were inconsistently identified in patients under age 45. Vascular phenotypes, including intracranial aneurysms, dilatation of the thoracic aorta and dissection of a carotid artery were present in four pedigrees. We accessed Genomics England 100,000 genomes project data, and identified pathogenic variants of DNAJB11 in nine of 3934 probands with various kidney and urinary tract disorders. The clinical diagnosis was cystic kidney disease for eight probands and nephrocalcinosis for one proband. No additional pathogenic variants likely explaining the kidney disease were identified. Using the publicly available GnomAD database, DNAJB11 genetic prevalence was calculated at 0.85/10.000 individuals. Thus, establishing a precise diagnosis in atypical cystic or interstitial kidney disease is crucial, with important implications in terms of follow-up, genetic counseling, prognostic evaluation, therapeutic management, and for selection of living kidney donors.

Endothelial calreticulin deletion impairs endothelial function in aged mice.

Discrete calcium signals within the vascular endothelium decrease with age and contribute to impaired endothelial-dependent vasodilation. Calreticulin (Calr), a multifunctional calcium binding protein and endoplasmic reticulum (ER) chaperone, can mediate calcium signals and vascular function within the endothelial cells (ECs) of small resistance arteries. We found Calr protein expression significantly decreases with age in mesenteric arteries and examined the functional role of EC Calr in vasodilation and calcium mobilization in the context of aging. Third-order mesenteric arteries from mice with or without EC Calr knockdown were examined for calcium signals and constriction to phenylephrine (PE) or vasodilation to carbachol (CCh) after 75 wk of age. PE constriction in aged mice with or without EC Calr was unchanged. However, calcium signals and vasodilation to endothelial-dependent agonist carbachol were significantly impaired in aged EC Calr knockdown mice. Ex vivo incubation of arteries with the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) significantly improved vasodilation in mice lacking EC Calr. Our data suggests diminished vascular Calr expression with age can contribute to the detrimental effects of aging on endothelial calcium regulation and vasodilation.NEW & NOTEWORTHY Calreticulin (Calr) is responsible for key physiological processes in endoplasmic reticulum, especially in aging tissue. In particular, endothelial Calr is crucial to vascular function. In this study, we deleted Calr from the endothelium and aged the mice up to 75 wk to examine changes in vascular function. We found two key differences: 1) calcium events in endothelium were severely diminished after muscarinic stimulation, which 2) corresponded with a dramatic decrease in muscarinic vasodilation. Remarkably, we were able to rescue the effect of Calr deletion on endothelial-dependent vasodilatory function using tauroursodeoxycholic acid (TUDCA), an inhibitor of endoplasmic reticulum stress that is currently in clinical trials.

Sulforaphane attenuates apoptosis of hippocampal neurons induced by high glucose via regulating endoplasmic reticulum

Diabetic encephalopathy (DE) has been defined as one of the major complications of diabetes, characterized by neurochemical and neurodegenerative changes. However, the molecular mechanism of DE are not fully elucidated at present. Here, the primary hippocampal neurons were cultured in vitro with high glucose (HG) to induce diabetes-like effects, and mice were given streptozotocin (STZ) to induce a model of type 1 diabetes mellitus (T1D) mice. The administration of sulforaphane (SF) were used to observe the protective effects on the hippocampal neurons. We found that the expression of glucose-regulated protein 78 (GRP78), a typical endoplasmic reticulum chaperone, showed a trend of increasing in the early phase but decreasing in the late phase of both HG-induced primary hippocampal neurons and T1D mice. However, SF suppressed the apoptosis induced by HG in vitro and in vivo through TUNEL assay and caspase-3 immunohistochemistry staining. Meanwhile, the administration of SF suppressed the upregulation of CHOP, Bax and p-JNK protein and the downregulation of Bcl-2 protein induced by HG in hippocampal neurons in vitro and in vivo. The caspase-12 gene was upregulated only at 4 weeks in T1D mice compared with control mice, and the upregulation was suppressed by SF. In addition, the combined administration of SF and PX12, which is an inhibitor of thioredoxin (Trx), eliminated the protective effects of SF. We conclude that HG induced the development of endoplasmic reticulum stress (ERS) in hippocampal neurons, eventually leading to the apoptosis of neurons. SF prevented the ERS and attenuates the hippocampal neuron apoptosis induced by HG both in vitro and in vivo. The underlying mechanism may be involved in the suppression of the CHOP-Bax/Bcl-2, JNK and caspase-12 signaling pathways by SF through the Trx-1 target protein.

Helicobacter pylori infection impairs chaperone-assisted maturation of Na-K-ATPase in gastric epithelium.

Helicobacter pylori infection always induces gastritis, which may progress to ulcer disease or cancer. The mechanisms underlying mucosal injury by the bacteria are incompletely understood. Here, we identify a novel pathway for H. pylori-induced gastric injury, the impairment of maturation of the essential transport enzyme and cell adhesion molecule, Na-K-ATPase. Na-K-ATPase comprises α- and β-subunits that assemble in the endoplasmic reticulum (ER) before trafficking to the plasma membrane. Attachment of H. pylori to gastric epithelial cells increased Na-K-ATPase ubiquitylation, decreased its surface and total levels, and impaired ion balance. H. pylori did not alter degradation of plasmalemma-resident Na-K-ATPase subunits or their mRNA levels. Infection decreased association of α- and β-subunits with ER chaperone BiP and impaired assembly of α/β-heterodimers, as was revealed by quantitative mass spectrometry and immunoblotting of immunoprecipitated complexes. The total level of BiP was not altered, and the decrease in interaction with BiP was not observed for other BiP client proteins. The H. pylori-induced decrease in Na-K-ATPase was prevented by BiP overexpression, stopping protein synthesis, or inhibiting proteasomal, but not lysosomal, protein degradation. The results indicate that H. pylori impairs chaperone-assisted maturation of newly made Na-K-ATPase subunits in the ER independently of a generalized ER stress and induces their ubiquitylation and proteasomal degradation. The decrease in Na-K-ATPase levels is also seen in vivo in the stomachs of gerbils and chronically infected children. Further understanding of H. pylori-induced Na-K-ATPase degradation will provide insights for protection against advanced disease.NEW & NOTEWORTHY This work provides evidence that Helicobacter pylori decreases levels of Na-K-ATPase, a vital transport enzyme, in gastric epithelia, both in acutely infected cultured cells and in chronically infected patients and animals. The bacteria interfere with BiP-assisted folding of newly-made Na-K-ATPase subunits in the endoplasmic reticulum, accelerating their ubiquitylation and proteasomal degradation and decreasing efficiency of the assembly of native enzyme. Decreased Na-K-ATPase expression contributes to H. pylori-induced gastric injury.