Geniposide Increases Unfolded Protein Response-Mediating HRD1 Expression to Accelerate APP Degradation in Primary Cortical Neurons

Altered proteostasis induced by amyloid peptide aggregation and hyperphosphorylation of tau protein, is a prominent feature of Alzheimer’s disease, which highlights the occurrence of endoplasmic reticulum stress and triggers the activation of the unfolded protein response (UPR), a signaling pathway that enforces adaptive programs to sustain proteostasis. In this study, we investigated the role of geniposide in the activation of UPR induced by high glucose in primary cortical neurons. We found that high glucose induced a significant activation of UPR, and geniposide enhanced the effect of high glucose on the phosphorylation of IRE1α, the most conserved UPR signaling branch. We observed that geniposide induced the expression of HRD1, an ubiquitin-ligase E3 in a time dependent manner, and amplified the expression of HRD1 induced by high glucose in primary cortical neurons. Suppression of IRE1α activity with STF-083010, an inhibitor of IRE1 phosphorylation, prevented the roles of geniposide on the expression of HRD1 and APP degradation in high glucose-treated cortical neurons. In addition, the results from RNA interfere on HRD1 revealed that HRD1 was involved in geniposide regulating APP degradation in cortical neurons. These data suggest that geniposide might be benefit to re-establish proteostasis by enhancing the UPR to decrease the load of APP in neurons challenged by high glucose.     

Armet, a UPR-upregulated protein, inhibits cell proliferation and ER stress-induced cell death

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress that initiates the unfolded protein response (UPR). UPR activates both adaptive and apoptotic pathways, which contribute differently to disease pathogenesis. To further understand the functional mechanisms of UPR, we identified 12 commonly UPR-upregulated genes by expression microarray analysis. Here, we describe characterization of Armet/MANF, one of the 12 genes whose function was not clear. We demonstrated that the Armet/MANF protein was upregulated by various forms of ER stress in several cell lines as well as by cerebral ischemia of rat. Armet/MANF was localized in the ER and Golgi and was also a secreted protein. Silencing Armet/MANF by siRNA oligos in HeLa cells rendered cells more susceptible to ER stress-induced death, but surprisingly increased cell proliferation and reduced cell size. Overexpression of Armet/MANF inhibited cell proliferation and improved cell viability under glucose-free conditions and tunicamycin treatment. Based on its inhibitory properties for both proliferation and cell death we have demonstrated, Armet is, thus, a novel secreted mediator of the adaptive pathway of UPR.     

SEL1L Regulates Adhesion,Proliferation and Secretion of Insulin by Affecting Integrin Signaling

SEL1L, a component of the endoplasmic reticulum associated degradation (ERAD) pathway, has been reported to regulate the (i) differentiation of the pancreatic endocrine and exocrine tissue during the second transition of mouse embryonic development, (ii) neural stem cell self-renewal and lineage commitment and (iii) cell cycle progression through regulation of genes related to cell-matrix interaction. Here we show that in the pancreas the expression of SEL1L is developmentally regulated, such that it is readily detected in developing islet cells and in nascent acinar clusters adjacent to basement membranes, and becomes progressively restricted to the islets of Langherans in post-natal life. This peculiar expression pattern and the presence of two inverse RGD motifs in the fibronectin type II domain of SEL1L protein indicate a possible interaction with cell adhesion molecules to regulate islets architecture. Co-immunoprecipitation studies revealed SEL1L and ß1-integrin interaction and, down-modulation of SEL1L in pancreatic ß-cells, negatively influences both cell adhesion on selected matrix components and cell proliferation likely due to altered ERK signaling. Furthermore, the absence of SEL1L protein strongly inhibits glucose-stimulated insulin secretion in isolated mouse pancreatic islets unveiling an important role of SEL1L in insulin trafficking. This phenotype can be rescued by the ectopic expression of the ß1-integrin subunit confirming the close interaction of these two proteins in regulating the cross-talk between extracellular matrix and insulin signalling to create a favourable micro-environment for ß-cell development and function.     

ER signaling in unfolded protein response

Abnormally folded proteins are susceptible to aggregation and accumulation in cells, ultimately leading to cell death. To protect cells against such dangers, expression of various genes including molecular chaperones can be induced and ER-associated protein degradation (ERAD) activated in response to the accumulation of unfolded protein in the endoplasmic reticulum (ER). This is known as the unfolded protein response (UPR). ERAD requires retrograde transport of unfolded proteins from the ER back to the cytosol via the translocon for degradation by the ubiquitin-proteasome system. Hrd1p is a UPR-induced ER membrane protein that acts as a ubiquitin ligase (E3) in the ERAD system. Hrd3p interacts with and stabilizes Hrd1p. We have isolated and identified human homologs (HRD1 and SEL1/HRD3) of Saccharomyces cerevisiae Hrd1p and Hrd3p. Human HRD1 and SEL1 were up-regulated in response to ER stress and overexpression of human IRE1 and ATF6, which are ER stress-sensor molecules in the ER. HEK293T cells overexpressing HRD1 showed resistance to ER stress-induced cell death. These results suggest that HRD1 and SEL1 are up-regulated by the UPR and contribute to protection against the ER stress-induced cell death by degrading unfolded proteins accumulated in the ER.     

Palmitate and thapsigargin have contrasting effects on ER membrane lipid composition and ER proteostasis in neuronal cells

The endoplasmic reticulum (ER) is an organelle that performs several key functions such as protein synthesis and folding, lipid metabolism and calcium homeostasis. When these functions are disrupted, such as upon protein misfolding, ER stress occurs. ER stress can trigger adaptive responses to restore proper functioning such as activation of the unfolded protein response (UPR). In certain cells, the free fatty acid palmitate has been shown to induce the UPR. Here, we examined the effects of palmitate on UPR gene expression in a human neuronal cell line and compared it with thapsigargin, a known depletor of ER calcium and trigger of the UPR. We used a Gaussia luciferase-based reporter to assess how palmitate treatment affects ER proteostasis and calcium homeostasis in the cells. We also investigated how ER calcium depletion by thapsigargin affects lipid membrane composition by performing mass spectrometry on subcellular fractions and compared this to palmitate. Surprisingly, palmitate treatment did not activate UPR despite prominent changes to membrane phospholipids. Conversely, thapsigargin induced a strong UPR, but did not significantly change the membrane lipid composition in subcellular fractions. In summary, our data demonstrate that changes in membrane lipid composition and disturbances in ER calcium homeostasis have a minimal influence on each other in neuronal cells. These data provide new insight into the adaptive interplay of lipid homeostasis and proteostasis in the cell.     

过量表达内质网小分子热激蛋白增强番茄的衣霉素抗性

真核细胞内质网腔内未折叠蛋白的过度积累会引起内质网胁迫（ER胁迫）,继而激活未折叠蛋白应答（UPR）信号途径,诱导内质网定位的分子伴侣的大量表达（如BiP和calnexin等）。本工作将CaMV35S启动子驱动的内质网小分子热激蛋白基因（ER-sHSP）导入番茄,发现ER-sHSP的过量表达提高了转基因番茄整株对衣霉素的抗性。衣霉素处理使未转基因番茄中BiP和calnexin基因的表达迅速升高,转基因番茄中这两个基因的表达也有增加,但表达强度明显低于未转基因番茄。说明ER-sHSP能够减轻ER胁迫,并可能参与UPR信号转导途径。     

The unfolded protein response regulates an angiogenic response by the kidney epithelium during ischemic stress

Ischemic injuries permanently affect kidney tissue and challenge cell viability, promoting inflammation and fibrogenesis. Ischemia results in nutrient deprivation, which triggers endoplasmic reticulum stress, ultimately resulting in the unfolded protein response (UPR). The aim of this study was to test whether the UPR could promote an angiogenic response independently of the HIF-1α pathway during ischemic stress in the human kidney epithelium. Glucose deprivation induced the secretion of vascular endothelial growth factor A (VEGFA), basic fibroblast growth factor (bFGF) and angiogenin (ANG) in human kidney epithelial cells independently of HIF-1α. Glucose deprivation, but not hypoxia, triggered endoplasmic reticulum stress and activated the UPR. RNA interference-mediated inhibition of the gene encoding the kinase PERK decreased VEGFA and bFGF expression, but neither gene was affected by the inhibition of IRE1α or ATF6. Furthermore, we show that the expression of angiogenin, which inhibits protein synthesis, is regulated by both IRE1α and PERK, which could constitute a complementary function of the UPR in the repression of translation. In a rat model of acute ischemic stress, we show that the UPR is activated in parallel with VEGFA, bFGF, and ANG expression and independently of HIF-1α.     

Endoplasmic reticulum proteostasis impairment in aging

Perturbed neuronal proteostasis is a salient feature shared by both aging and protein misfolding disorders. The proteostasis network controls the health of the proteome by integrating pathways involved in protein synthesis, folding, trafficking, secretion, and their degradation. A reduction in the buffering capacity of the proteostasis network during aging may increase the risk to undergo neurodegeneration by enhancing the accumulation of misfolded proteins. As almost one‐third of the proteome is synthetized at the endoplasmic reticulum (ER), maintenance of its proper function is fundamental to sustain neuronal function. In fact, ER stress is a common feature of most neurodegenerative diseases. The unfolded protein response (UPR) operates as central player to maintain ER homeostasis or the induction of cell death of chronically damaged cells. Here, we discuss recent evidence placing ER stress as a driver of brain aging, and the emerging impact of neuronal UPR in controlling global proteostasis at the whole organismal level. Finally, we discuss possible therapeutic interventions to improve proteostasis and prevent pathological brain aging.     

The endoplasmic reticulum proteostasis network profoundly shapes the protein sequence space accessible to HIV envelope

The sequence space accessible to evolving proteins can be enhanced by cellular chaperones that assist biophysically defective clients in navigating complex folding landscapes. It is also possible, at least in theory, for proteostasis mechanisms that promote strict quality control to greatly constrain accessible protein sequence space. Unfortunately, most efforts to understand how proteostasis mechanisms influence evolution rely on artificial inhibition or genetic knockdown of specific chaperones. The few experiments that perturb quality control pathways also generally modulate the levels of only individual quality control factors. Here, we use chemical genetic strategies to tune proteostasis networks via natural stress response pathways that regulate the levels of entire suites of chaperones and quality control mechanisms. Specifically, we upregulate the unfolded protein response (UPR) to test the hypothesis that the host endoplasmic reticulum (ER) proteostasis network shapes the sequence space accessible to human immunodeficiency virus-1 (HIV-1) envelope (Env) protein. Elucidating factors that enhance or constrain Env sequence space is critical because Env evolves extremely rapidly, yielding HIV strains with antibody- and drug-escape mutations. We find that UPR-mediated upregulation of ER proteostasis factors, particularly those controlled by the IRE1-XBP1s UPR arm, globally reduces Env mutational tolerance. Conserved, functionally important Env regions exhibit the largest decreases in mutational tolerance upon XBP1s induction. Our data indicate that this phenomenon likely reflects strict quality control endowed by XBP1s-mediated remodeling of the ER proteostasis environment. Intriguingly, and in contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display enhanced mutational tolerance when XBP1s is induced, hinting at a role for host proteostasis network hijacking in potentiating antibody escape. These observations reveal a key function for proteostasis networks in decreasing instead of expanding the sequence space accessible to client proteins, while also demonstrating that the host ER proteostasis network profoundly shapes the mutational tolerance of Env in ways that could have important consequences for HIV adaptation.

The host cell’s endoplasmic reticulum proteostasis network has a profound, constraining impact on the protein sequence space accessible to HIV’s envelope protein, which is a major target of the host’s adaptive immune system; in particular, upregulation of stringent quality control pathways appears to restrict the viability of destabilizing envelope variants.     

Divergent Effects of PERK and IRE1 Signaling on Cell Viability

Protein misfolding in the endoplasmic reticulum (ER) activates a set of intracellular signaling pathways, collectively termed the Unfolded Protein Response (UPR). UPR signaling promotes cell survival by reducing misfolded protein levels. If homeostasis cannot be restored, UPR signaling promotes cell death. The molecular basis for the switch between prosurvival and proapoptotic UPR function is poorly understood. The ER-resident proteins, PERK and IRE1, control two key UPR signaling pathways. Protein misfolding concomitantly activates PERK and IRE1 and has clouded insight into their contributions toward life or death cell fates. Here, we employed chemical-genetic strategies to activate individually PERK or IRE1 uncoupled from protein misfolding. We found that sustained PERK signaling impaired cell proliferation and promoted apoptosis. By contrast, equivalent durations of IRE1 signaling enhanced cell proliferation without promoting cell death. These results demonstrate that extended PERK and IRE1 signaling have opposite effects on cell viability. Differential activation of PERK and IRE1 may determine life or death decisions after ER protein misfolding.     

Inhibition of endoplasmic reticulum associated degradation reduces endoplasmic reticulum stress and alters lysosomal morphology and distribution

Disturbances in proteostasis are observed in many neurodegenerative diseases. This leads to activation of protein quality control to restore proteostasis, with a key role for the removal of aberrant proteins by proteolysis. The unfolded protein response (UPR) is a protein quality control mechanism of the endoplasmic reticulum (ER) that is activated in several neurodegenerative diseases. Recently we showed that the major proteolytic pathway during UPR activation is via the autophagy/lysosomal system. Here we investigate UPR induction if the other major proteolytic pathway of the ER -ER associated degradation (ERAD)-is inhibited. Surprisingly, impairment of ERAD results in decreased UPR activation and protects against ER stress toxicity. Autophagy induction is not affected under these conditions, however, a striking relocalization of the lysosomes is observed. Our data suggest that a protective UPR-modulating mechanism is activated if ERAD is inhibited, which involves lysosomes. Our data provide insight in the cross-talk between proteolytic pathways involved in ER proteostasis. This has implications for neurodegenerative diseases like Alzheimer’s disease where disturbed ER proteostasis and proteolytic impairment are early phenomena in the pathology.     

RNA metabolism: putting the brake on the UPR

The unfolded protein response (UPR) is a major signaling cascade that determines cell fate under conditions of endoplasmic reticulum (ER) stress. The kinetics and amplitude of UPR responses are tightly controlled by several feedback loops and the expression of positive and negative regulators. In this issue of EMBO Reports, the Wilkinson lab uncovers a novel function of nonsense‐mediated RNA decay (NMD) in fine‐tuning the UPR 1 . NMD is an mRNA quality control mechanism known to destabilize aberrant mRNAs that contain premature termination codons. In this work, NMD was shown to determine the threshold of stress necessary to activate the UPR, in addition to adjusting the amplitude of downstream responses and the termination phase. These effects were mapped to the control of the mRNA stability of IRE1, a major ER stress transducer. This study highlights the dynamic crosstalk between mRNA metabolism and the proteostasis network demonstrating the physiological relevance of normal mRNA regulation by the NMD pathway.     

RNA-mediated interference indicates that SEL1L plays a role in pancreatic beta-cell growth

The specificity of SEL1L expression and promoter activity for the pancreatic cell population, its chromosomal location, as well as its similarities to the yeast Hrd3p protein, a component of HRD complex which is responsible for endoplasmic reticulum (ER)-associated degradation of numerous ER-resident proteins, prompted us to study its effects on beta cell function. In this study we show that lowering SEL1L expression, by using the short interfering RNAs technology as well as antisense transfection, resulted in severe perturbation of betaTC-3 growth and metabolic activity. We hypothesize that SEL1L may exert its function by protecting the cells from ER stress and could counteract immune responses.     

A Radial Glia Gene Marker,Fatty Acid Binding Protein 7 (FABP7), Is Involved in Proliferation and Invasion of Glioblastoma Cells

Glioblastoma multiforme (GBM) is among the most deadly cancers. A number of studies suggest that a fraction of tumor cells with stem cell features (Glioma Stem-like Cells, GSC) might be responsible for GBM recurrence and aggressiveness. GSC similarly to normal neural stem cells, can form neurospheres (NS) in vitro, and seem to mirror the genetic features of the original tumor better than glioma cells growing adherently in the presence of serum. Using cDNA microarray analysis we identified a number of relevant genes for glioma biology that are differentially expressed in adherent cells and neurospheres derived from the same tumor. Fatty acid-binding protein 7 (FABP7) was identified as one of the most highly expressed genes in NS compared to their adherent counterpart. We found that down-regulation of FABP7 expression in NS by small interfering RNAs significantly reduced cell proliferation and migration. We also evaluated the potential involvement of FABP7 in response to radiotherapy, as this treatment may cause increased tumor infiltration. Migration of irradiated NS was associated to increased expression of FABP7. In agreement with this, in vivo reduced tumorigenicity of GBM cells with down-regulated expression of FABP7 was associated to decreased expression of the migration marker doublecortin. Notably, we observed that PPAR antagonists affect FABP7 expression and decrease the migration capability of NS after irradiation. As a whole, the data emphasize the role of FABP7 expression in GBM migration and provide translational hints on the timing of treatment with anti-FABP7 agents like PPAR antagonists during GBM evolution.