ER Stress and Unfolded Protein Response in Amyotrophic Lateral Sclerosis

Several theories on the pathomechanism of amyotrophic lateral sclerosis (ALS) have been proposed: misfolded protein aggregates, mitochondrial dysfunction, increased glutamate toxicity, increased oxidative stress, disturbance of intracellular trafficking, and so on. In parallel, a number of drugs that have been developed to alleviate the putative key pathomechanism of ALS have been under clinical trials. Unfortunately, however, almost all studies have finished unsuccessfully. This fact indicates that the key ALS pathomechanism still remains a tough enigma. Recent studies with autopsied ALS patients and studies using mutant SOD1 (mSOD1) transgenic mice have suggested that endoplasmic reticulum (ER) stress-related toxicity may be a relevant ALS pathomechanism. Levels of ER stress-related proteins were upregulated in motor neurons in the spinal cords of ALS patients. It was also shown that mSOD1, translocated to the ER, caused ER stress in neurons in the spinal cord of mSOD1 transgenic mice. We recently reported that the newly identified ALS-causative gene, vesicle-associated membrane protein-associated protein B (VAPB), plays a pivotal role in unfolded protein response (UPR), a physiological reaction against ER stress. The ALS-linked P56S mutation in VAPB nullifies the function of VAPB, resulting in motoneuronal vulnerability to ER stress. In this review, we summarize recent advances in research on the ALS pathomechanism especially addressing the putative involvement of ER stress and UPR dysfunction.     

内质网应激诱导自噬的分子机制

在真核细胞中,内质网对蛋白质的折叠和运输至关重要,多种病理因素对内质网稳态的扰乱,可导致内质网腔中未折叠或错误折叠蛋白蓄积,即内质网应激(ERS)。细胞为此通过激活一种叫做未折叠蛋白反应(UPR)的防御反应来恢复内质网稳态。自噬是一种被描述为\"自我吞食\"的细胞代谢过程,其通过批量清除和降解未折叠蛋白以及破损细胞器在ERS时作为一种重要的保护机制。近年的研究显示这两个系统动态互联,且ERS可以通过多种方式诱导自噬的发生。在本文中,我们将总结目前关于ERS尤其是UPR诱导自噬的分子机制的相关知识,以进一步指导关于ERS与自噬关系的的研究。     

Activation of mammalian IRE1α upon ER stress depends on dissociation of BiP rather than on direct interaction with unfolded proteins

IRE1, an ER-localized transmembrane protein, plays a central role in the unfolded protein response. Upon ER stress, IRE1 senses the accumulation of unfolded proteins in the ER, and transfers signal from the ER to the cytosol. Recently, it was reported that the luminal domain of yeast Ire1 senses the unfolded proteins via a two-step mechanism, namely dissociation of BiP and direct interaction with unfolded proteins. However, it has been unclear whether a similar mechanism is applicable to mammalian IRE1α. To address this point, we analyzed luminal-domain mutants of mammalian IRE1α in cells, and evaluated the anti-aggregation activity of the luminal fragment of IRE1α in vitro. We generated a mutant that has low affinity for BiP, and this mutant was significantly activated even under normal conditions. Moreover, the luminal fragments of mammalian IRE1α did not exhibit anti-aggregation activity. These results suggest that in contrast to yeast Ire1, the regulation of mammalian IRE1α strongly depends on the dissociation of BiP.     

Synthetic embryonic lethality upon deletion of the ER cochaperone p58 and the ER stress sensor ATF6α

The unfolded protein response (UPR) is activated as a consequence of alterations to ER homeostasis. It upregulates a group of ER chaperones and cochaperones, as well as other genes that improve protein processing within the secretory pathway. The UPR effector ATF6α augments—but is not essential for—maximal induction of ER chaperones during stress, yet its role, if any, in protecting cellular function during normal development and physiology is unknown. A systematic analysis of multiple tissues from Atf6α−/− mice revealed that all tissues examined were grossly insensitive to loss of ATF6α. However, combined deletion of ATF6α and the ER cochaperone p58^IPK resulted in synthetic embryonic lethality. These findings reveal for the first time that an intact UPR can compensate for the genetic impairment of protein folding in the ER in vivo. The also expose a role for p58^IPK in normal embryonic development.     

酵母PMT1基因参与内质网应激的调控机制

【目的】内质网应激(Endoplasmic reticulum stress,ERS)可激活细胞保护性信号级联反应——未折叠蛋白质反应(Unfolded protein response,UPR)。研究表明,酵母细胞中的UPR信号通路由转录因子Hac1p和ERS感应因子Ire1p共同介导。前期研究发现:蛋白质-O-甘露糖转移酶1(Protein-O-mannosyltransferase 1,PMT1)基因缺失能延长酵母细胞的复制性寿命,其机制与上调UPR通路活性相关。本文进一步探讨PMT1基因缺失在酵母ERS反应中的作用。【方法】观察PMT1基因与IRE1或HAC1基因双缺失酵母菌株(pmt1?hac1?和pmt1?ire1?)在ERS反应条件下的克隆形成能力;通过比色法检测各菌株的细胞增殖活性;RT-PCR检测各菌株UPR通路下游部分靶基因的转录水平。【结果】与对照菌株比较,PMT1基因缺失菌株(pmt1?)在ERS反应条件下生长较慢,而HAC1和IRE1单基因缺失菌株(hac1?和ire1?)在ERS反应条件下无法存活;在hac1?或ire1?菌株的基础上进一步缺失PMT1基因,可以改善hac1?菌株在ERS反应条件下的生长状态;但缺失PMT1基因没有上调hac1?菌株UPR通路靶基因的转录水平。【结论】缺失PMT1基因可增强hac1?菌株对ERS诱导剂衣霉素的抗性,机制与已知的UPR通路不相关,提示可能存在其它途径参与ERS反应的调控。     

The unfolded protein response as a target for cancer therapy

Various physiological and pathological conditions generate an accumulation of misfolded proteins in the endoplasmic reticulum (ER). This results in ER stress followed by a cellular response to cope with this stress and restore homeostasis: the unfolded protein response (UPR). Overall, the UPR leads to general translational arrest and the induction of specific factors to ensure cell survival or to mediate cell death if the stress is too severe. In multiple cancers, components of the UPR are overexpressed, indicating increased dependence on the UPR. In addition, the UPR can confer resistance to anti-cancer treatment. Therefore, modification of the UPR should be explored for its anti-cancer properties. This review discusses factors associated with the UPR that represent potential therapeutic targets.     

Rottlerin induces pro-apoptotic endoplasmic reticulum stress through the protein kinase C-δ-independent pathway in human colon cancer cells

Rottlerin, a compound reported to be a PKC δ-selective inhibitor, has been shown to induce growth arrest or apoptosis of human cancer cell lines. In our study, rottlerin dose-dependently induced apoptotic cell death in colon carcinoma cells. Treatment of HT29 human colon carcinoma cells with rottlerin was found to induce a number of signature ER stress markers; phosphorylation of eukaryotic initiation factor-2α (eIF-2α), ER stress-specific XBP1 splicing, and up-regulation of glucose-regulated protein (GRP)-78 and CCAAT/enhancer-binding protein-homologous protein (CHOP). However, suppression of PKC δ expression by siRNA or overexpression of WT-PKC δ and DN-PKC δ did not abrogate the rottlerin-mediated induction of CHOP. These results suggest that rottlerin induces up-regulation of CHOP via PKC δ-independent pathway. Furthermore, down-regulation of CHOP expression using CHOP siRNA attenuated rottlerin-induced apoptosis. Taken together, the present study thus provides strong evidence to support an important role of ER stress response in mediating the rottlerin-induced apoptosis.     

Curcumin induces pro-apoptotic endoplasmic reticulum stress in human leukemia HL-60 cells

Curcumin has been shown to induce apoptosis in many cancer cells. However, the molecular mechanism(s) responsible for curcumin-induced apoptosis is not well understood and most probably involves several pathways. In HL-60 cells, curcumin induced apoptosis and endoplasmic reticulum (ER) stress as evidenced by the survival molecules such as phosphorylated protein kinase-like ER-resident kinase, phosphorylated eukaryotic initiation factor-2alpha, glucose-regulated protein-78, and the apoptotic molecules such as caspase-4 and CAAT/enhancer binding protein homologous protein (CHOP). Inhibition of caspase-4 activity by z-LEVD-FMK, blockage of CHOP expression by small interfering RNA, and treatment with salubrinal, an ER inhibitor, significantly reduced curcumin-induced apoptosis. Removing two double bonds in curcumin, which was speculated to form Michael adducts with thiols in secretory proteins, resulted in a loss of the ability of curcumin to induce apoptosis as well as ER stress. Thus, the present study shows that curcumin-induced apoptosis is associated with its ability to cause ER stress.     

The unfolded protein response in human corneal endothelial cells following hypothermic storage: implications of a novel stress pathway

Human corneal endothelial cells (HCEC) have become increasingly important for a range of eye disease treatment therapies. Accordingly, a more detailed understanding of the processing and preservation associated stresses experienced by corneal cells might contribute to improved therapeutic outcomes. To this end, the unfolded protein response (UPR) pathway was investigated as a potential mediator of corneal cell death in response to hypothermic storage. Once preservation-induced failure had begun in HCECs stored at 4 °C, it was noted that necrosis accounted for the majority of cell death but with significant apoptotic involvement, peaking at several hours post-storage (4–8 h). Western blot analysis demonstrated changes associated with apoptotic activation (caspase 9, caspase 3, and PARP cleavage). Further, the activation of the UPR pathway was observed through increased and sustained levels of ER folding and chaperone proteins (Bip, PDI, and ERO1-Lα) in samples experiencing significant cell death. Modulation of the UPR pathway using the specific inhibitor, salubrinal, resulted in a 2-fold increase in cell survival in samples experiencing profound cold-induced failure. Furthermore, this increased cell survival was associated with increased membrane integrity, cell attachment, and decreased necrotic cell death populations. Conversely, addition of the UPR inducer, tunicamycin, during cold exposure resulted in a significant decrease in HCEC survival during the recovery period. These data implicate for the first time that this novel cell stress pathway may be activated in HCEC as a result of the complex stresses associated with hypothermic exposure. The data suggest that the targeted control of the UPR pathway during both processing and preservation protocols may improve cell survival and function of HCEC thus improving the clinical utility of these cells as well as whole human corneas.     

Mutant proinsulin proteins associated with neonatal diabetes are retained in the endoplasmic reticulum and not efficiently secreted

Mutations in the preproinsulin protein that affect processing of preproinsulin to proinsulin or lead to misfolding of proinsulin are associated with diabetes. We examined the subcellular localization and secretion of 13 neonatal diabetes-associated human proinsulin proteins (A24D, G32R, G32S, L35P, C43G, G47V, F48C, G84R, R89C, G90C, C96Y, S101C and Y108C) in rat INS-1 insulinoma cells. These mutant proinsulin proteins accumulate in the endoplasmic reticulum (ER) and are poorly secreted except for G84R and in contrast to wild-type and hyperproinsulinemia-associated mutant proteins (H34D and R89H) which were sorted to secretory granules and efficiently secreted. We also examined the effect of C96Y mutant proinsulin on the synthesis and secretion of wild-type insulin and observed a dominant-negative effect of the mutant proinsulin on the synthesis and secretion of wild-type insulin due to induction of the unfolded protein response and resulting attenuation of overall translation.