Inhibition of the mitochondrial unfolded protein response by acetylcholine alleviated hypoxia/reoxygenation-induced apoptosis of endothelial cells

The mitochondrial unfolded protein response (UPR^mt) is involved in numerous diseases that have the common feature of mitochondrial dysfunction. However, its pathophysiological relevance in the context of hypoxia/reoxygenation (H/R) in endothelial cells remains elusive. Previous studies have demonstrated that acetylcholine (ACh) protects against cardiomyocyte injury by suppressing generation of mitochondrial reactive oxygen species (mtROS). This study aimed to explore the role of UPR^mt in endothelial cells during H/R and to clarify the beneficial effects of ACh. Our results demonstrated that H/R triggered UPR^mt in endothelial cells, as evidenced by the elevation of heat shock protein 60 and LON protease 1 protein levels, and resulted in release of mitochondrial pro-apoptotic proteins, including cytochrome C, Omi/high temperature requirement protein A 2 and second mitochondrial activator of caspases/direct inhibitor of apoptosis-binding protein with low PI, from the mitochondria to cytosol. ACh administration markedly decreased UPR^mt by inhibiting mtROS and alleviating the mitonuclear protein imbalance. Consequently, ACh alleviated the release of pro-apoptotic proteins and restored mitochondrial ultrastructure and function, thereby reducing the number of terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL)-positive cells. Intriguingly, 4-diphenylacetoxy-N-methylpiperidine methiodide, a type-3 muscarinic ACh receptor (M3AChR) inhibitor, abolished the ACh-elicited attenuation of UPR^mt and TUNEL positive cells, indicating that the salutary effects of ACh were likely mediated by M3AChR in endothelial cells. In conclusion, our studies demonstrated that UPR^mt might be essential for triggering the mitochondrion-associated apoptotic pathway during H/R. ACh markedly suppressed UPR^mt by inhibiting mtROS and alleviating the mitonuclear protein imbalance, presumably through M3AChR.     

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.     

Inhibition of protein translocation at the endoplasmic reticulum promotes activation of the unfolded protein response

Selective small-molecule inhibitors represent powerful tools for the dissection of complex biological processes. ES(I) (eeyarestatin I) is a novel modulator of ER (endoplasmic reticulum) function. In the present study, we show that in addition to acutely inhibiting ERAD (ER-associated degradation), ES(I) causes production of mislocalized polypeptides that are ubiquitinated and degraded. Unexpectedly, our results suggest that these non-translocated polypeptides promote activation of the UPR (unfolded protein response), and indeed we can recapitulate UPR activation with an alternative and quite distinct inhibitor of ER translocation. These results suggest that the accumulation of non-translocated proteins in the cytosol may represent a novel mechanism that contributes to UPR activation.     

The unfolded protein response is shaped by the NMD pathway

Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), an essential adaptive intracellular pathway that relieves the stress. Although the UPR is an evolutionarily conserved and beneficial pathway, its chronic activation contributes to the pathogenesis of a wide variety of human disorders. The fidelity of UPR activation must thus be tightly regulated to prevent inappropriate signaling. The nonsense-mediated RNA decay (NMD) pathway has long been known to function in RNA quality control, rapidly degrading aberrant mRNAs, and has been suggested to regulate subsets of normal mRNAs. Here, we report that the NMD pathway regulates the UPR. NMD increases the threshold for triggering the UPR in vitro and in vivo, thereby preventing UPR activation in response to normally innocuous levels of ER stress. NMD also promotes the timely termination of the UPR. We demonstrate that NMD directly targets the mRNAs encoding several UPR components, including the highly conserved UPR sensor, IRE1α, whose NMD-dependent degradation partly underpins this process. Our work not only sheds light on UPR regulation, but demonstrates the physiological relevance of NMD''s ability to regulate normal mRNAs.     

Endoplasmic reticulum: the unfolded protein response is tangled in neurodegeneration

The endoplasmic reticulum (ER) is involved in the folding and maturation of membrane-bound and secreted proteins. Disturbed homeostasis in the ER can lead to accumulation of misfolded proteins, which trigger a stress response called the unfolded protein response (UPR). In neurodegenerative diseases that are classified as tauopathies, activation of the UPR coincides with the pathogenic accumulation of the microtubule associated protein tau. Several lines of evidence indicate that UPR activation contributes to increased levels of phosphorylated tau, a prerequisite for the formation of tau aggregates. Increased understanding of the crosstalk between signaling pathways involved in protein quality control in the ER and tau phosphorylation will support the development of new therapeutic targets that promote neuronal survival.     

Hepatitis C virus-induced autophagy is independent of the unfolded protein response

Hepatitis C virus (HCV) has been shown to induce autophagy and the unfolded protein response (UPR), but the mechanistic link between the induction of these two cellular processes remains unclear. We demonstrate here that HCV infection induces autophagy, as judged by accumulation of lipidated LC3-II, and that this induction occurs rapidly after infection, preceding the stimulation of the UPR, which occurs only at later stages, after the viral envelope glycoproteins have been expressed to high levels. Furthermore, both genotype 1b and 2a subgenomic replicons expressing nonstructural (NS3-5B) proteins and JFH-1 virus lacking the envelope glycoproteins potently induced autophagy in the absence of detectable UPR. This ability was also shared by a subgenomic replicon derived from the related GB virus B (GBV-B). We also show that small interfering RNA (siRNA)-mediated silencing of the key UPR inducer, Ire1, has no effect on HCV genome replication or the induction of autophagy, further demonstrating that the UPR is not required for these processes. Lastly, we demonstrate that the HCV replicase does not colocalize with autophagosomes, suggesting that the induction of autophagy is not required to generate the membrane platform for HCV RNA replication.     

Zinc homeostasis is involved in unfolded protein response under salt stress

Accumulation of unfolded protein or misfolded protein causes endoplasmic reticulum (ER) stress. Increased salt concentration activates a stress response pathway in the ER in Arabidopsis thaliana to induce the expression of several salt stress response genes, leading to a more optimal protein folding environment in the ER. In addition, some salt stress-regulated proteins require zinc for their activity, including some zinc-dependent DNA binding proteins and zinc-finger proteins. In a recent study, we reported that ZTP29, a putative zinc transporter at the ER membrane, is involved in the response to salt stress through regulation of zinc level in the ER to induce the UPR pathway. In this addendum, we propose a testable hypothesis for the role of ZTP29 in the response to salt stress via the regulation of zinc levels in the ER.Key words: zinc, ER stress, unfolded protein response, salt stress, arabidopsisHigh salinity is a common abiotic stress that adversely affects plant growth and crop production.¹ Plants must sense the stress and transduce stress signals to activate response pathways leading to adaptation to, or tolerance of, the abiotic stress in salt environment.² Salt stress activates a stress response pathway in the endoplasmic reticulum (ER) in Arabidopsis thaliana, indicating that the adaptation of plants to salt stress involves ER stress signal regulation.^3,4 There is limited understanding of molecular mechanisms on ER stress in plants, as compared to yeast and mammalian cells. bZIP60, bZIP28, bZIP17 are three membrane-associated basic domain/leucine zipper (bZIP) factors, which have been reported as candidates for ER-folding proteins in plants.^5–7 BiP acts as a general chaperone in the ER lumen, due to its ability to discriminate between properly folded and unfolded protein structures.⁸ Unfolded or misfolded proteins are retained in the ER and form stable complexes with BiP and other ER resident chaperones.⁹ Zinc deficiency induces unfolded protein response (UPR) in most eukaryotes.¹⁰ Zinc is an important trace element, which participates in physiological and biochemical process in vivo. The requirement of zinc for proper ER function is evolutionarily conserved.     

Sphingolipids and the unfolded protein response

The unfolded protein response (UPR) is a response by the endoplasmic reticulum to stress, classically caused by any disruption to cell homeostasis that results in an accumulation in unfolded proteins. However, there is an increasing body of research demonstrating that the UPR can also be activated by changes in lipid homeostasis, including changes in sphingolipid metabolism. Sphingolipids are a family of bioactive lipids with important roles in both the formation and integrity of cellular membranes, and regulation of key cellular processes, including cell proliferation and apoptosis. Bi-directional interactions between sphingolipids and the UPR have now been observed in a range of diseases, including cancer, diabetes and liver disease. Determining how these two key cellular components influence each other could play an important role in deciphering the causes of these diseases and potentially reveal new therapeutic approaches.     

Involvement of the Aspergillus nidulans protein kinase C with farnesol tolerance is related to the unfolded protein response

Previously, we demonstrated that the Aspergillus nidulans calC2 mutation in protein kinase C pkcA was able to confer tolerance to farnesol (FOH), an isoprenoid that has been shown to inhibit proliferation and induce apoptosis. Here, we investigate in more detail the role played by A. nidulans pkcA in FOH tolerance. We demonstrate that pkcA overexpression during FOH exposure causes increased cell death. FOH is also able to activate several markers of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Our results suggest an intense cross-talk between PkcA and the UPR during FOH-induced cell death. Furthermore, the overexpression of pkcA increases both mRNA accumulation and metacaspases activity, and there is a genetic interaction between PkcA and the caspase-like protein CasA. Mutant analyses imply that MAP kinases are involved in the signal transduction in response to the effects caused by FOH.     

The isolated major homology region of the HIV capsid protein is mainly unfolded in solution and binds to the intact protein

Assembly of the mature human immunodeficiency virus type 1 (HIV-1) capsid involves the oligomerization of the capsid protein, CA. During retroviral maturation, the CA protein undergoes structural changes and forms exclusive intermolecular interfaces in the mature capsid shell, different from those in the immature precursor. The most conserved region of CA, the major homology region (MHR), is located in the C-terminal domain of CA (CTD). The MHR is involved in both immature and mature virus assembly; however, its exact function during both assembly stages is unknown. To test its conformational preferences and to provide clues on its role during CA assembly, we have used a minimalist approach by designing a peptide comprising the whole MHR (MHRpep, residues Asp152 to Ala174). Isolated MHRpep is mainly unfolded in aqueous solution, with residual structure at its C terminus. MHRpep binds to monomeric CTD with an affinity of ~30μM (as shown by fluorescence and ITC); the CTD binding region comprises residues belonging to α-helices 10 and 11. In the immature virus capsid, the MHR and α-helix 11 regions of two CTD dimers also interact [Briggs JAG, Riches JD, Glass B, Baratonova V, Zanetti G and Kr?usslich H-G (2009) Proc. Natl. Acad. Sci. USA 106, 11090-11095]. These results can be considered a proof-of-concept that the conformational preferences and binding features of isolated peptides derived from virus proteins could be used to mimic early stages of virus assembly.     

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.     

Inhibition of protein synthesis sensitizes thermotolerant cells to heat shock induced apoptosis

Hyperthermia is a potent inducer of apoptosis in many cell lines. A brief exposure to mildly elevated temperatures elicits a transient state of augmented resistance to subsequent thermal stress. Here we show that a hyperthermic treatment of 43°C for 1 h is sufficient to induce apoptosis in the cell line HL-60. This observation is based on morphologic evaluation and on comet assay results (an extremely sensitive method of detecting and quantifying apoptotic DNA fragmentation in individual cells). The thermotolerance phenomenon was also verified in the same manner by giving the cells a brief 30 min sub-lethal heat conditioning treatment at 43°C followed by a 6 h incubation time prior to the administration of a lethal heat load (43°C for 1 h). We observed a dramatic decrease in resultant apoptoses in the thermotolerized cells in comparison to unconditioned cells. We assessed the necessity of de novo protein synthesis in the protective phenomenon. When the conditioned cells were given a cycloheximide treatment prior to heat conditioning we saw a sensitization of the conditioned cells to secondary thermal injury. This revised version was published online in June 2006 with corrections to the Cover Date.