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.     

Calcium trafficking integrates endoplasmic reticulum function with mitochondrial bioenergetics

Calcium homeostasis is central to all cellular functions and has been studied for decades. Calcium acts as a critical second messenger for both extracellular and intracellular signaling and is fundamental in cell life and death decisions (Berridge et al., 2000) [1]. The calcium gradient in the cell is coupled with an inherent ability of the divalent cation to reversibly bind multiple target biological molecules to generate an extremely versatile signaling system [2]. Calcium signals are used by the cell to control diverse processes such as development, neurotransmitter release, muscle contraction, metabolism, autophagy and cell death. “Cellular calcium overload” is detrimental to cellular health, resulting in massive activation of proteases and phospholipases leading to cell death (Pinton et al., 2008) [3]. Historically, cell death associated with calcium ion perturbations has been primarily recognized as necrosis. Recent evidence clearly associates changes in calcium ion concentrations with more sophisticated forms of cellular demise, including apoptosis (Kruman et al., 1998; Tombal et al., 1999; Lynch et al., 2000; Orrenius et al., 2003) , ,  and . Although the endoplasmic reticulum (ER) serves as the primary calcium store in the metazoan cell, dynamic calcium release to the cytosol, mitochondria, nuclei and other organelles orchestrate diverse coordinated responses. Most evidence supports that calcium transport from the ER to mitochondria plays a significant role in regulating cellular bioenergetics, production of reactive oxygen species, induction of autophagy and apoptosis. Recently, molecular identities that mediate calcium traffic between the ER and mitochondria have been discovered (Mallilankaraman et al., 2012a; Mallilankaraman et al., 2012b; Sancak et al., 2013)[8–10]. The next questions are how they are regulated for exquisite tight control of ER–mitochondrial calcium dynamics. This review attempts to summarize recent advances in the role of calcium in regulation of ER and mitochondrial function. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.     

Proteostasis regulation at the endoplasmic reticulum: a new perturbation site for targeted cancer therapy

To deal with the constant challenge of protein misfolding in the endoplasmic reticulum (ER), eukaryotic cells have evolved an ER protein quality control (ERQC) mechanism that is integrated with an adaptive stress response. The ERQC pathway is comprised of factors residing in the ER lumen that function in the identification and retention of aberrantly folded proteins, factors in the ER membrane for retrotranslocation of misfolded polypeptides, and enzymes in the cytosol that degrade retrotranslocated proteins. The integrated stress response (termed ER stress or unfolded protein response, UPR) contains several signaling branches elicited from the ER membrane, which fine-tune the rate of protein synthesis and entry into the ER to match the ER folding capacity. The fitness of the cell, particularly those bearing a high secretory burden, is critically dependent on functional integrity of the ER, which in turn relies on these stress-attenuating mechanisms to maintain protein homeostasis, or proteostasis. Aberrant proteostasis can trigger cellular apoptosis, making these adaptive stress response systems attractive targets for perturbation in treatment of cell malignancies. Here, we review our current understanding of how the cell preserves ER proteostasis and discuss how we may harness the mechanistic information on this process to develop new cancer therapeutics.     

Unfolded Protein Response Signaling and Metabolic Diseases

The endoplasmic reticulum (ER) is a central organelle for protein biosynthesis, folding, and traffic. Perturbations in ER homeostasis create a condition termed ER stress and lead to activation of the complex signaling cascade called the unfolded protein response (UPR). Recent studies have documented that the UPR coordinates multiple signaling pathways and controls various physiologies in cells and the whole organism. Furthermore, unresolved ER stress has been implicated in a variety of metabolic disorders, such as obesity and type 2 diabetes. Therefore, intervening in ER stress and modulating signaling components of the UPR would provide promising therapeutics for the treatment of human metabolic diseases.     

Corrupted ER‐mitochondrial calcium homeostasis promotes the collapse of proteostasis

Aging and age‐related diseases are associated with a decline of protein homeostasis (proteostasis), but the mechanisms underlying this decline are not clear. In particular, decreased proteostasis is a widespread molecular feature of neurodegenerative diseases, such as Alzheimer's disease (AD). Familial AD is largely caused by mutations in the presenilin encoding genes; however, their role in AD is not understood. In this study, we investigate the role of presenilins in proteostasis using the model system Caenorhabditis elegans. Previously, we found that mutations in C. elegans presenilin cause elevated ER to mitochondria calcium signaling, which leads to an increase in mitochondrial generated oxidative stress. This, in turn, promotes neurodegeneration. To understand the cellular mechanisms driving neurodegeneration, using several molecular readouts of protein stability in C. elegans, we find that presenilin mutants have widespread defects in proteostasis. Markedly, we demonstrate that these defects are independent of the protease activity of presenilin and that reduction in ER to mitochondrial calcium signaling can significantly prevent the proteostasis defects observed in presenilin mutants. Furthermore, we show that supplementing presenilin mutants with antioxidants suppresses the proteostasis defects. Our findings indicate that defective ER to mitochondria calcium signaling promotes proteostatic collapse in presenilin mutants by increasing oxidative stress.     

ER stress and its functional link to mitochondria: role in cell survival and death

The endoplasmic reticulum (ER) is the primary site for synthesis and folding of secreted and membrane-bound proteins. Proteins are translocated into ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to assist in proper folding. Properly folded proteins traffic from the ER to the Golgi apparatus; misfolded proteins are targeted to degradation. Unfolded protein response (UPR) is a highly regulated intracellular signaling pathway that prevents accumulation of misfolded proteins in the ER lumen. UPR provides an adaptive mechanism by which cells can augment protein folding and processing capacities of the ER. If protein misfolding is not resolved, the UPR triggers apoptotic cascades. Although the molecular mechanisms underlying ER stress-induced apoptosis are not completely understood, increasing evidence suggests that ER and mitochondria cooperate to signal cell death. Mitochondria and ER form structural and functional networks (mitochondria-associated ER membranes [MAMs]) essential to maintain cellular homeostasis and determine cell fate under various pathophysiological conditions. Regulated Ca(2+) transfer from the ER to the mitochondria is important in maintaining control of prosurvival/prodeath pathways. We discuss the signaling/communication between the ER and mitochondria and focus on the role of the mitochondrial permeability transition pore in these complex processes.     

Integrating stress signals at the endoplasmic reticulum: The BCL-2 protein family rheostat

The assembling of distinct signaling protein complexes at the endoplasmic reticulum (ER) membrane controls several stress responses related to calcium homeostasis, autophagy, ER morphogenesis and protein folding. Diverse pathological conditions interfere with the function of the ER altering protein folding, a condition known as “ER stress”. Adaptation to ER stress depends on the activation of the unfolded protein response (UPR) and protein degradation pathways such as autophagy. Under chronic or irreversible ER stress, cells undergo apoptosis, where the BCL-2 protein family plays a crucial role at the mitochondria to trigger cytochrome c release and apoptosome assembly. Several BCL2 family members also regulate physiological processes at the ER through dynamic interactomes. Here we provide a comprehensive view of the roles of the BCL-2 family of proteins in mediating the molecular crosstalk between the ER and mitochondria to initiate apoptosis, in addition to their emerging functions in adaptation to stress, including autophagy, UPR, calcium homeostasis and organelle morphogenesis. We envision a model where BCL-2-containing complexes may operate as stress rheostats that, beyond their known apoptosis functions at the mitochondria, determine the amplitude and kinetics of adaptive responses against ER-related injuries. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

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.     

Role of the unfolded protein response in determining the fate of tumor cells and the promise of multi-targeted therapies

Although there have been advances in our understanding of carcinogenesis and development of new treatments, cancer remains a common cause of death. Many regulatory pathways are incompletely understood in cancer development and progression, with a prime example being those related to the endoplasmic reticulum (ER). The pathological sequelae that arise from disruption of ER homeostasis are not well defined. The ER is an organelle that is responsible for secretory protein biosynthesis and the quality control of protein folding. The ER triggers an unfolded protein response (UPR) when misfolded proteins accumulate, and while the UPR acts to restore protein folding and ER homeostasis, this response can work as a switch to determine the death or survival of cells. The treatment of cancer with agents that target the UPR has shown promising outcomes. The UPR has wide crosstalk with other signaling pathways. Multi-targeted cancer therapies which target the intersections within signaling networks have shown synergistic tumoricidal effects. In the present review, the basic cellular and signaling pathways of the ER and UPR are introduced; then the crosstalk between the ER and other signaling pathways is summarized; and ultimately, the evidence that the UPR is a potential target for cancer therapy is discussed. Regulation of the UPR downstream signaling is a common therapeutic target for different tumor types. Tumoricidal effects achieved from modulating the UPR downstream signaling could be enhanced by phosphodiesterase 5 (PDE5) inhibitors. Largely untapped by Western medicine for cancer therapies are Chinese herbal medicines. This review explores and discusses the value of some Chinese herbal extracts as PDE5 inhibitors.     

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.     

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.     

酿酒酵母细胞中内质网应激与未折叠蛋白反应的研究进展

内质网应激激活的未折叠蛋白反应(Unfolded protein response,UPR)途径在酿酒酵母和哺乳动物细胞中是非常保守的。内质网(Endoplasmic reticulum,ER)是蛋白质合成、折叠和修饰的细胞器,也是贮存钙的主要场所之一。酵母细胞内质网钙平衡与UPR的作用是相互的;两个MAPK途径——HOG途径和CWI途径都是细胞应答内质网应激压力时生存所必需的;重金属镉离子能够激活UPR途径,它通过激活钙离子通道Cch1/Mid1进入细胞影响钙离子的功能。本文结合最新研究进展对酿酒酵母细胞中的两个MAPK途径、镉离子和钙离子稳态与内质网应激激活的UPR途径之间相互关系进行综述。     

内质网应激与疾病

内质网是蛋白质合成与折叠、维持Ca²⁺动态平衡及合成脂类和固醇的场所。遗传或环境损伤引起内质网功能紊乱导致内质网应激,激活未折叠蛋白反应。未折叠蛋白反应是一种细胞自我保护性措施,但是内质网应激过强或持续时间过久可引起细胞凋亡。因此,内质网应激与众多人类疾病的发生发展密切相关。最近研究证明,癌症、炎症性疾病、代谢性疾病、骨质疏松症及神经退行性疾病等有内质网应激信号传递参与。然而内质网应激作为一个有效靶点参与各种疾病发挥作用的功能和机制仍然有待进一步研究。在近年来发表的文献基础上对内质网应激与疾病的关系,以及其可能的作用机制进行综述。     

Cross‐compartment proteostasis regulation during redox imbalance induced ER stress

Imbalance in protein homeostasis in specific subcellular organelles is alleviated through organelle‐specific stress response pathways. As a canonical example of stress activated pathway, accumulation of misfolded proteins in ER activates unfolded protein response (UPR) in almost all eukaryotic organisms. However, very little is known about the involvement of proteins of other organelles that help to maintain the cellular protein homeostasis during ER stress. In this study, using iTRAQ‐based LC–MS approach, we identified organelle enriched proteins that are differentially expressed in yeast (Saccharomyces cerevisiae) during ER stress in the absence of UPR sensor Ire1p. We have identified about 750 proteins from enriched organelle fraction in three independent iTRAQ experiments. Induction of ER stress resulted in the differential expression of 93 proteins in WT strains, 40 of which were found to be dependent on IRE1. Our study reveals a cross‐talk between ER‐ and mitochondrial proteostasis exemplified by an Ire1p‐dependent induction of Hsp60p, a mitochondrial chaperone. Thus, in this study, we show changes in protein levels in various organelles in response to ER stress. A large fraction of these changes were dependent on canonical UPR signalling through Ire1, highlighting the importance of interorganellar cross‐talk during stress.