Brucella Induces an Unfolded Protein Response via TcpB That Supports Intracellular Replication in Macrophages

Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other intracellular bacteria that rely on host physiologic stress responses for replication.     

The putative Arabidopsis zinc transporter ZTP29 is involved in the response to salt stress

Salt stress leads to a stress response, called the unfolded protein response (UPR), in the endoplasmic reticulum (ER). UPR is also induced in a wide range of organisms by zinc deficiency. However, it is not clear whether regulation of zinc levels is involved in the initiation of the UPR in plant response to salt stress. In this study, a putative zinc transporter, ZTP29, was identified in Arabidopsis thaliana. ZTP29 localizes to the ER membrane and is expressed primarily in hypocotyl and cotyledon tissues, but its expression can be induced in root tissue by salt stress. T-DNA insertion into the ZTP29 gene led to NaCl hypersensitivity in seed germination and seedling growth, leaf etiolation, and widening of cells in the root elongation zone. In addition, in ztp29 mutant plants, salt stress-induced upregulation of the UPR pathway genes BiP2 and bZIP60 was inhibited. Furthermore, under conditions of salt stress, upregulation of BiP2 and bZIP60 was inhibited by treatment with high concentrations of zinc in both control and ztp29 plants. However, zinc chelation restored salt stress-induced BiP2 and bZIP60 upregulation in ztp29 mutant plants. These experimental results suggest that ZTP29 is involved in the response to salt stress, perhaps through regulation of zinc levels required to induce the UPR pathway.     

The Effects of Melatonin on Transcriptional Profile of Unfolded Protein Response Genes Under Endoplasmic Reticulum Stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

When the load of secretory pathway is increased or folding capacity in the endoplasmic reticulum (ER) is insufficient, unfolded proteins might accumulate in ER lumen causing a phenomenon called ER stress. During ER stress, normal cell functions are suppressed and unfolded protein response (UPR) is induced. Studies in animal systems suggest that melatonin alleviates the detrimental effects of ER stress; however, there is no study in plants in this respect. Hence, in this study, we investigated the possible role of melatonin on alleviation of ER stress in model plant Arabidopsis thaliana. Tunicamycin (Tm) was used to specifically induce ER stress. Melatonin treatment (10 and 25 μM but not 1 μM) increased root growth under Tm treatment, but it did not reach control levels. ER stress induced the expressions of ER stress sensor/transducer genes, ER chaperones and folding helper genes, ER-associated degradation (ERAD) genes, and ER stress-associated apoptosis genes in roots and shoots (a total of 16 genes). Among them, the expressions of ER stress sensor/transducer bZIP17, bZIP28, IRE1A, IRE1B, ERAD-related SEL1, and apoptosis genes AGB1 were decreased back to control levels with 25 μM melatonin under ER stress in roots. Moreover, Tm?+?melatonin treatments decreased the expressions of these genes when compared to only Tm-treated plants. Downregulation of UPR components with increased concentrations of melatonin under Tm treatment demonstrated that melatonin alleviated the detrimental effects of ER stress.     

Localization and function in endoplasmic reticulum stress tolerance of ERdj3, a new member of Hsp40 family protein

Heat shock protein 40 (Hsp40) family proteins are known to bind to Hsp70 through their J-domain and regulate the function of Hsp70 by stimulating its adenosine triphosphatase activity. In the endoplasmic reticulum (ER), there are 5 Hsp40 family proteins known so far, 3 of which were recently identified. In this report, one of the novel Hsp40 cochaperones, ERdj3, was characterized in terms of its subcellular localization, stress response, and stress tolerance of cells. By using ERdj3-specific polyclonal antibody, endogenous ERdj3 protein was shown to reside in the ER as gene transfer–mediated exogenous ERdj3. Analysis of the expression level of endogenous ERdj3 protein revealed its moderate induction in response to various ER stressors, indicating its possible action as a stress protein in the ER. Subsequently, we analyzed whether this molecule was involved in ER stress tolerance of cells, as was the case with the ER-resident Hsp70 family protein BiP. Although overexpression of ERdj3 by gene transfection could not strengthen ER stress tolerance of neuroblastoma cells, reduction of ERdj3 expression by small interfering ribonucleic acid decreased the tolerance of cells, indicating that ERdj3 might have just a marginal role in the ER stress resistance of neuroblastoma cells. In contrast, overexpression of ERdj3 notably suppressed vero toxin–induced cell death. These data suggest that ERdj3 might have diverse roles in the ER, including that of the molecular cochaperone of BiP and an as yet unknown protective action against vero toxin.     

Cell cycle arrest mediated by WEE1 is involved in the unfolded protein response in plants

Activation of the unfolded protein response (UPR) in mammalian cells leads to cell cycle arrest at the G1 phase (Thomas et al., J Biol Chem 288:7606–7617, 2013). However, how UPR signaling affects cell cycle arrest remains largely unknown in plants. Here, we examined UPR and endoreduplication in Col-0, wee1, and ER stress sensing-deficient ire1a&b plants during DNA replication and ER stress conditions. We found that WEE1, an essential negative regulator of the cell cycle, is involved in the maintenance of ER homeostasis during genotoxic stress and the ER stress hypersensitivity of ire1a&b is alleviated by loss-of-function mutation in WEE1. WEE1-mediated cell cycle arrest was required for IRE1–bZIP60 pathway activation during ER stress. In contrast, loss-of-function mutation in WEE1 caused increased expression of UPR-related genes during DNA replication stress. WEE1 and IRE1 were required for endoreduplication during DNA replication stress and ER stress, respectively. Taken together, these findings suggest that cell cycle regulation is associated with UPR activation in different manners during ER stress and DNA replication stress in Arabidopsis.     

Endogenous BiP reporter system for simultaneous identification of ER stress and antibody production in Chinese hamster ovary cells

As the biopharmaceutical industry expands, improving the production of therapeutic proteins using Chinese hamster ovary (CHO) cells is important. However, excessive and complicated protein production causes protein misfolding and triggers endoplasmic reticulum (ER) stress. When ER stress occurs, cells mediate the unfolded protein response (UPR) pathway to restore protein homeostasis and folding capacity of the ER. However, when the cells fail to control prolonged ER stress, UPR induces apoptosis. Therefore, monitoring the degree of UPR is required to achieve high productivity and the desired quality. In this study, we developed a fluorescence-based UPR monitoring system for CHO cells. We integrated mGFP into endogenous HSPA5 encoding BiP, a major ER chaperone and the primary ER stress activation sensor, using CRISPR/Cas9-mediated targeted integration. The mGFP expression level changed according to the ER stress induced by chemical treatment and batch culture in the engineered cell line. Using this monitoring system, we demonstrated that host cells and recombinant CHO cell lines with different mean fluorescence intensities (MFI; basal expression levels of BiP) possess a distinct capacity for stress culture conditions induced by recombinant protein production. Antibody-producing recombinant CHO cell lines were generated using site-specific integration based on host cells equipped with the BiP reporter system. Targeted integrants showed a strong correlation between productivity and MFI, reflecting the potential of this monitoring system as a screening readout for high producers. Taken together, these data demonstrate the utility of the endogenous BiP reporter system for the detection of real-time dynamic changes in endogenous UPR and its potential for applications in recombinant protein production during CHO cell line development.     

HCV causes chronic endoplasmic reticulum stress leading to adaptation and interference with the unfolded protein response

Background

The endoplasmic reticulum (ER) is the cellular site for protein folding. ER stress occurs when protein folding capacity is exceeded. This stress induces a cyto-protective signaling cascades termed the unfolded protein response (UPR) aimed at restoring homeostasis. While acute ER stress is lethal, chronic sub-lethal ER stress causes cells to adapt by attenuation of UPR activation. Hepatitis C virus (HCV), a major human pathogen, was shown to cause ER stress, however it is unclear whether HCV induces chronic ER stress, and if so whether adaptation mechanisms are initiated. We wanted to characterize the kinetics of HCV-induced ER stress during infection and assess adaptation mechanisms and their significance.

Methods and Findings

The HuH7.5.1 cellular system and HCV-transgenic (HCV-Tg) mice were used to characterize HCV-induced ER stress/UPR pathway activation and adaptation. HCV induced a wave of acute ER stress peaking 2–5 days post-infection, which rapidly subsided thereafter. UPR pathways were activated including IRE1 and EIF2α phosphorylation, ATF6 cleavage and XBP-1 splicing. Downstream target genes including GADD34, ERdj4, p58ipk, ATF3 and ATF4 were upregulated. CHOP, a UPR regulated protein was activated and translocated to the nucleus. Remarkably, UPR activity did not return to baseline but remained elevated for up to 14 days post infection suggesting that chronic ER stress is induced. At this time, cells adapted to ER stress and were less responsive to further drug-induced ER stress. Similar results were obtained in HCV-Tg mice. Suppression of HCV by Interferon-α 2a treatment, restored UPR responsiveness to ER stress tolerant cells.

Conclusions

Our study shows, for the first time, that HCV induces adaptation to chronic ER stress which was reversed upon viral suppression. These finding represent a novel viral mechanism to manipulate cellular response pathways.     

High levels of the molecular chaperone Mdg1/ERdj4 reflect the activation state of endothelial cells

Mdg1/ERdj4, a mammalian chaperone that belongs to the HSP40 protein family, has been reported to be located in the endoplasmic reticulum (ER), is induced by ER stress, and protects ER stressed cells from apoptosis. Here we show that under normal physiological conditions, Mdg1/ERdj4 is expressed at various levels in the vasculature due to different activation states of the endothelium. To elucidate the stimuli that induce ER stress and thus upregulate Mdg1/ERdj4, we investigated the effect of several endothelium specific stressors on its expression. Mdg1/ERdj4 mRNA is induced by activated macrophages, by nitric oxide (NO) and heat shock, and during terminal cell differentiation, whereas shear stress does not affect Mdg1/ERdj4 expression levels. While the mRNA stability of BiP/GRP78 is unaffected in ER stressed cells, the stability of Mdg1/ERdj4 mRNA is prolonged during ER stress resulting in rapid increases and high levels of Mdg1/ERdj4 mRNA. Mdg1/ERdj4 protein is localized in the ER under control conditions. While heat shock induces a rapid translocation of Mdg1/ERdj4 to the nucleoli, no translocation could be observed during ER stress. This indicates that Mdg1/ERdj4 protein has diverse mechanisms to protect stressed cells from apoptosis.     

Medicago falcata MfSTMIR,an E3 ligase of endoplasmic reticulum‐associated degradation,is involved in salt stress response

Recent studies on E3 of endoplasmic reticulum (ER)‐associated degradation (ERAD) in plants have revealed homologs in yeast and animals. However, it remains unknown whether the plant ERAD system contains a plant‐specific E3 ligase. Here, we report that MfSTMIR, which encodes an ER‐membrane‐localized RING E3 ligase that is highly conserved in leguminous plants, plays essential roles in the response of ER and salt stress in Medicago. MfSTMIR expression was induced by salt and tunicamycin (Tm). mtstmir loss‐of‐function mutants displayed impaired induction of the ER stress‐responsive genes BiP1/2 and BiP3 under Tm treatment and sensitivity to salt stress. MfSTMIR promoted the degradation of a known ERAD substrate, CPY*. MfSTMIR interacted with the ERAD‐associated ubiquitin‐conjugating enzyme MtUBC32 and Sec61‐translocon subunit MtSec61γ. MfSTMIR did not affect MtSec61γ protein stability. Our results suggest that the plant‐specific E3 ligase MfSTMIR participates in the ERAD pathway by interacting with MtUBC32 and MtSec61γ to relieve ER stress during salt stress.     

AtIRE1A/AtIRE1B and AGB1 independently control two essential unfolded protein response pathways in Arabidopsis

The endoplasmic reticulum (ER) has the ability to maintain the balance between demand for and synthesis of secretory proteins. To ensure protein‐folding homeostasis in the ER, cells invoke signaling pathways known as the unfolded protein response (UPR). To initiate UPR, yeasts largely rely on a conserved sensor, IRE1. In metazoans, there are at least three independent UPR signalling pathways. Some UPR transducers have been identified in plants, but no genetic interaction among them has yet been examined. The Arabidopsis genome encodes two IRE1 sequence homologs, AtIRE1A and AtIRE1B. Here we provide evidence that AtIRE1A and AtIRE1B have overlapping functions that are essential for the plant UPR. A double mutant of AtIRE1A and AtIRE1B, atire1a atire1b, showed reduced ER stress tolerance and a compromised UPR activation phenotype. We have also established that Arabidopsis AGB1, a subunit of the ubiquitous heterotrimeric GTP‐binding protein family, and AtIRE1A/AtIRE1B independently control two essential plant UPR pathways. By demonstrating that atire1a atire1b has a short root phenotype that is enhanced by an agb1 loss‐of‐function mutation, we have identified a role for UPR transducers in organ growth regulation.     

Deficiency of the BiP cochaperone ERdj4 causes constitutive endoplasmic reticulum stress and metabolic defects

Endoplasmic reticulum–localized DnaJ 4 (ERdj4) is an immunoglobulin-binding protein (BiP) cochaperone and component of the endoplasmic reticulum–associated degradation (ERAD) pathway that functions to remove unfolded/misfolded substrates from the ER lumen under conditions of ER stress. To elucidate the function of ERdj4 in vivo, we disrupted the ERdj4 locus using gene trap (GT) mutagenesis, leading to hypomorphic expression of ERdj4 in mice homozygous for the trapped allele (ERdj4^GT/GT). Approximately half of ERdj4^GT/GT mice died perinatally associated with fetal growth restriction, reduced hepatic glycogen stores, and hypoglycemia. Surviving adult mice exhibited evidence of constitutive ER stress in multiple cells/tissues, including fibroblasts, lung, kidney, salivary gland, and pancreas. Elevated ER stress in pancreatic β cells of ERdj4^GT/GT mice was associated with β cell loss, hypoinsulinemia, and glucose intolerance. Collectively these results suggest an important role for ERdj4 in maintaining ER homeostasis during normal fetal growth and postnatal adaptation to metabolic stress.