Cellular responses to endoplasmic reticulum stress and apoptosis

The endoplasmic reticulum (ER) is the cell organelle where secretory and membrane proteins are synthesized and folded. Correctly folded proteins exit the ER and are transported to the Golgi and other destinations within the cell, but proteins that fail to fold properly—misfolded proteins—are retained in the ER and their accumulation may constitute a form of stress to the cell—ER stress. Several signaling pathways, collectively known as unfolded protein response (UPR), have evolved to detect the accumulation of misfolded proteins in the ER and activate a cellular response that attempts to maintain homeostasis and a normal flux of proteins in the ER. In certain severe situations of ER stress, however, the protective mechanisms activated by the UPR are not sufficient to restore normal ER function and cells die by apoptosis. Most research on the UPR used yeast or mammalian model systems and only recently Drosophila has emerged as a system to study the molecular and cellular mechanisms of the UPR. Here, we review recent advances in Drosophila UPR research, in the broad context of mammalian and yeast literature.     

BiP Availability Distinguishes States of Homeostasis and Stress in the Endoplasmic Reticulum of Living Cells

Accumulation of misfolded secretory proteins causes cellular stress and induces the endoplasmic reticulum (ER) stress pathway, the unfolded protein response (UPR). Although the UPR has been extensively studied, little is known about the molecular changes that distinguish the homeostatic and stressed ER. The increase in levels of misfolded proteins and formation of complexes with chaperones during ER stress are predicted to further crowd the already crowded ER lumen. Surprisingly, using live cell fluorescence microscopy and an inert ER reporter, we find the crowdedness of stressed ER, treated acutely with tunicamycin or DTT, either is comparable to homeostasis or significantly decreases in multiple cell types. In contrast, photobleaching experiments revealed a GFP-tagged variant of the ER chaperone BiP rapidly undergoes a reversible quantitative decrease in diffusion as misfolded proteins accumulate. BiP mobility is sensitive to exceptionally low levels of misfolded protein stressors and can detect intermediate states of BiP availability. Decreased BiP availability temporally correlates with UPR markers, but restoration of BiP availability correlates less well. Thus, BiP availability represents a novel and powerful tool for reporting global secretory protein misfolding levels and investigating the molecular events of ER stress in single cells, independent of traditional UPR markers.     

RESETing ER proteostasis: selective stress pathway hidden in the secretory route

The efficient folding of membrane and secreted proteins relies on the unfolded protein response (UPR) to buffer fluctuations in the load of misfolded proteins. Although the UPR is thought to operate on a generic manner to maintain ER proteostasis, a recent study revealed the existence of a novel mechanism to eliminate misfolded GPI‐anchored proteins via the secretory pathway, termed ‘rapid ER stress‐induced export’ (RESET) (Satpute‐Krishnan et al, 2014 ). RESET involves the export of misfolded GPI proteins to the plasma membrane for subsequent degradation by the lysosome.     

The impact of the unfolded protein response on human disease

A central function of the endoplasmic reticulum (ER) is to coordinate protein biosynthetic and secretory activities in the cell. Alterations in ER homeostasis cause accumulation of misfolded/unfolded proteins in the ER. To maintain ER homeostasis, eukaryotic cells have evolved the unfolded protein response (UPR), an essential adaptive intracellular signaling pathway that responds to metabolic, oxidative stress, and inflammatory response pathways. The UPR has been implicated in a variety of diseases including metabolic disease, neurodegenerative disease, inflammatory disease, and cancer. Signaling components of the UPR are emerging as potential targets for intervention and treatment of human disease.     

Kar2p availability defines distinct forms of endoplasmic reticulum stress in living cells

Accumulation of misfolded secretory proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) stress pathway. To enhance secretory protein folding and promote adaptation to stress, the UPR upregulates ER chaperone levels, including BiP. Here we describe chromosomal tagging of KAR2, the yeast homologue of BiP, with superfolder green fluorescent protein (sfGFP) to create a multifunctional endogenous reporter of the ER folding environment. Changes in Kar2p-sfGFP fluorescence levels directly correlate with UPR activity and represent a robust reporter for high-throughput analysis. A novel second feature of this reporter is that photobleaching microscopy (fluorescence recovery after photobleaching) of Kar2p-sfGFP mobility reports on the levels of unfolded secretory proteins in individual cells, independent of UPR status. Kar2p-sfGFP mobility decreases upon treatment with tunicamycin or dithiothreitol, consistent with increased levels of unfolded proteins and the incorporation of Kar2p-sfGFP into slower-diffusing complexes. During adaptation, we observe a significant lag between down-regulation of the UPR and resolution of the unfolded protein burden. Finally, we find that Kar2p-sfGFP mobility significantly increases upon inositol withdrawal, which also activates the UPR, apparently independent of unfolded protein levels. Thus Kar2p mobility represents a powerful new tool capable of distinguishing between the different mechanisms leading to UPR activation in living cells.     

Endoplasmic reticulum stress and fungal pathogenesis

The gateway to the secretory pathway is the endoplasmic reticulum (ER), an organelle that is responsible for the accurate folding, post-translational modification and final assembly of up to a third of the cellular proteome. When secretion levels are high, errors in protein biogenesis can lead to the accumulation of abnormally folded proteins, which threaten ER homeostasis. The unfolded protein response (UPR) is an adaptive signaling pathway that counters a buildup in misfolded and unfolded proteins by increasing the expression of genes that support ER protein folding capacity. Fungi, like other eukaryotic cells that are specialized for secretion, rely upon the UPR to buffer ER stress caused by fluctuations in secretory demand. However, emerging evidence is also implicating the UPR as a central regulator of fungal pathogenesis. In this review, we discuss how diverse fungal pathogens have adapted ER stress response pathways to support the expression of virulence-related traits that are necessary in the host environment.     

The therapeutic effects of 4-phenylbutyric acid in maintaining proteostasis

Recently, there has been an increasing amount of literature published on the effects of 4-phenylbutyric acid (4-PBA) in various biological systems. 4-PBA is currently used clinically to treat urea cycle disorders under the trade name Buphenyl. Recent studies however have explored 4-PBA in the context of a low weight molecular weight chemical chaperone. Its properties as a chemical chaperone prevent misfolded protein aggregation and alleviate endoplasmic reticulum (ER) stress. As the ER is responsible for folding proteins targeted for use in membranes or secreted out of the cell, failure of maintaining adequate ER homeostasis may lead to protein misfolding and subsequent cell and organ pathology. Accumulation of misfolded proteins within the ER activates the unfolded protein response (UPR), a molecular repair response. The activation of the UPR aims to restore ER and cellular proteostasis by regulating the rate of synthesis of newly formed proteins as well as initiating molecular programs aimed to help fold or degrade misfolded proteins. If proteostasis is not restored, the UPR may initiate pro-apoptotic pathways. It is suggested that 4-PBA may help fold proteins in the ER, attenuating the activation of the UPR, and thus potentially alleviating various pathologies. This review discusses the biomedical research exploring the potential therapeutic effects of 4-PBA in various in vitro and in vivo model systems and clinical trials, while also commenting on the possible mechanisms of action.     

Aerobic exercise training rescues cardiac protein quality control and blunts endoplasmic reticulum stress in heart failure rats

Cardiac endoplasmic reticulum (ER) stress through accumulation of misfolded proteins plays a pivotal role in cardiovascular diseases. In an attempt to reestablish ER homoeostasis, the unfolded protein response (UPR) is activated. However, if ER stress persists, sustained UPR activation leads to apoptosis. There is no available therapy for ER stress relief. Considering that aerobic exercise training (AET) attenuates oxidative stress, mitochondrial dysfunction and calcium imbalance, it may be a potential strategy to reestablish cardiac ER homoeostasis. We test the hypothesis that AET would attenuate impaired cardiac ER stress after myocardial infarction (MI). Wistar rats underwent to either MI or sham surgeries. Four weeks later, rats underwent to 8 weeks of moderate‐intensity AET. Myocardial infarction rats displayed cardiac dysfunction and lung oedema, suggesting heart failure. Cardiac dysfunction in MI rats was paralleled by increased protein levels of UPR markers (GRP78, DERLIN‐1 and CHOP), accumulation of misfolded and polyubiquitinated proteins, and reduced chymotrypsin‐like proteasome activity. These results suggest an impaired cardiac protein quality control. Aerobic exercise training improved exercise capacity and cardiac function of MI animals. Interestingly, AET blunted MI‐induced ER stress by reducing protein levels of UPR markers, and accumulation of both misfolded and polyubiquinated proteins, which was associated with restored proteasome activity. Taken together, our study provide evidence for AET attenuation of ER stress through the reestablishment of cardiac protein quality control, which contributes to better cardiac function in post‐MI heart failure rats. These results reinforce the importance of AET as primary non‐pharmacological therapy to cardiovascular disease.     

The unfolded protein response and its role in intestinal homeostasis and inflammation

The unfolded protein response (UPR) is a signaling pathway from the endoplasmic reticulum (ER) to the nucleus that protects cells from the stress caused by misfolded or unfolded proteins [1, 2]. As such, ER stress is an ongoing challenge for all cells given the central biologic importance of secretion as part of normal physiologic functions. This is especially the case for cells that are highly dependent upon secretory function as part of their major duties. Within mucosal tissues, the intestinal epithelium is especially dependent upon an intact UPR for its normal activities [3]. This review will discuss the UPR and the special role that it provides in the functioning of the intestinal epithelium and, when dysfunctional, its implications for understanding mucosal homeostasis and intestinal inflammation, as occurs in inflammatory bowel disease (IBD).     

Cotranslocational degradation protects the stressed endoplasmic reticulum from protein overload

The ER's capacity to process proteins is limited, and stress caused by accumulation of unfolded and misfolded proteins (ER stress) contributes to human disease. ER stress elicits the unfolded protein response (UPR), whose components attenuate protein synthesis, increase folding capacity, and enhance misfolded protein degradation. Here, we report that P58(IPK)/DNAJC3, a UPR-responsive gene previously implicated in translational control, encodes a cytosolic cochaperone that associates with the ER protein translocation channel Sec61. P58(IPK) recruits HSP70 chaperones to the cytosolic face of Sec61 and can be crosslinked to proteins entering the ER that are delayed at the translocon. Proteasome-mediated cytosolic degradation of translocating proteins delayed at Sec61 is cochaperone dependent. In P58(IPK-/-) mice, cells with a high secretory burden are markedly compromised in their ability to cope with ER stress. Thus, P58(IPK) is a key mediator of cotranslocational ER protein degradation, and this process likely contributes to ER homeostasis in stressed cells.     

JAMP optimizes ERAD to protect cells from unfolded proteins

Clearance of misfolded proteins from the ER is central for maintenance of cellular homeostasis. This process requires coordinated recognition, ER-cytosol translocation, and finally ubiquitination-dependent proteasomal degradation. Here, we identify an ER resident seven-transmembrane protein (JAMP) that links ER chaperones, channel proteins, ubiquitin ligases, and 26S proteasome subunits, thereby optimizing degradation of misfolded proteins. Elevated JAMP expression promotes localization of proteasomes at the ER, with a concomitant effect on degradation of specific ER-resident misfolded proteins, whereas inhibiting JAMP promotes the opposite response. Correspondingly, a jamp-1 deleted Caenorhabditis elegans strain exhibits hypersensitivity to ER stress and increased UPR. Using biochemical and genetic approaches, we identify JAMP as important component for coordinated clearance of misfolded proteins from the ER.     

High ER stress in beta-cells stimulates intracellular degradation of misfolded insulin

Endoplasmic reticulum (ER) stress, which is caused by the accumulation of misfolded proteins in the ER, elicits an adaptive response, the unfolded protein response (UPR). One component of the UPR, the endoplasmic reticulum-associated protein degradation (ERAD) system, has an important function in the survival of ER stressed cells. Here, we show that HRD1, a component of the ERAD system, is upregulated in pancreatic islets of the Akita diabetes mouse model and enhances intracellular degradation of misfolded insulin. High ER stress in beta-cells stimulated mutant insulin degradation through HRD1 to protect beta-cells from ER stress and ensuing death. If HRD1 serves the same function in humans, it may serve as a target for therapeutic intervention in diabetes.     

疱疹病毒与内质网应激

内质网是分泌型蛋白和膜蛋白折叠及翻译后修饰的主要场所.病毒感染所引起的宿主细胞内环境的改变可使细胞或病毒的未折叠和/或错误折叠蛋白在内质网中大量聚集,使内质网处于生理功能紊乱的应激状态.为了缓解这种应激压力,细胞会启动未折叠蛋白反应(UPR),并通过一系列分子的信号转导维持内质网稳态;同时病毒也会通过对UPR的精密调控...