HSP105 interacts with GRP78 and GSK3 and promotes ER stress-induced caspase-3 activation

Stress of the endoplasmic reticulum (ER stress) is caused by the accumulation of misfolded proteins, which occurs in many neurodegenerative diseases. ER stress can lead to adaptive responses or apoptosis, both of which follow activation of the unfolded protein response (UPR). Heat shock proteins (HSP) support the folding and function of many proteins, and are important components of the ER stress response, but little is known about the role of one of the major large HSPs, HSP105. We identified several new partners of HSP105, including glycogen synthase kinase-3 (GSK3), a promoter of ER stress-induced apoptosis, and GRP78, a key component of the UPR. Knockdown of HSP105 did not alter UPR signaling after ER stress, but blocked caspase-3 activation after ER stress. In contrast, caspase-3 activation induced by genotoxic stress was unaffected by knockdown of HSP105, suggesting ER stress-specificity in the apoptotic action of HSP105. However, knockdown of HSP105 did not alter cell survival after ER stress, but instead diverted signaling to a caspase-3-independent cell death pathway, indicating that HSP105 is necessary for apoptotic signaling after UPR activation by ER stress. Thus, HSP105 appears to chaperone the responses to ER stress through its interactions with GRP78 and GSK3, and without HSP105 cell death following ER stress proceeds by a non-caspase-3-dependent process.     

The Endoplasmic Reticulum-Resident Chaperone Heat Shock Protein 47 Protects the Golgi Apparatus from the Effects of O-Glycosylation Inhibition

The Golgi apparatus is important for the transport of secretory cargo. Glycosylation is a major post-translational event. Recognition of O-glycans on proteins is necessary for glycoprotein trafficking. In this study, specific inhibition of O-glycosylation (Golgi stress) induced the expression of endoplasmic reticulum (ER)-resident heat shock protein (HSP) 47 in NIH3T3 cells, although cell death was not induced by Golgi stress alone. When HSP47 expression was downregulated by siRNA, inhibition of O-glycosylation caused cell death. Three days after the induction of Golgi stress, the Golgi apparatus was disassembled, many vacuoles appeared near the Golgi apparatus and extended into the cytoplasm, the nuclei had split, and cell death assay-positive cells appeared. Six hours after the induction of Golgi stress, HSP47-knockdown cells exhibited increased cleavage of Golgi-resident caspase-2. Furthermore, activation of mitochondrial caspase-9 and ER-resident unfolded protein response (UPR)-related molecules and efflux of cytochrome c from the mitochondria to the cytoplasm was observed in HSP47-knockdown cells 24 h after the induction of Golgi stress. These findings indicate that (i) the ER-resident chaperon HSP47 protected cells from Golgi stress, and (ii) Golgi stress-induced cell death caused by the inhibition of HSP47 expression resulted from caspase-2 activation in the Golgi apparatus, extending to the ER and mitochondria.     

ERS和microRNA间的相互作用与肿瘤

内质网是细胞蛋白质翻译后修饰和折叠的重要场所. 多种外界因素诸如热激、病毒感染和低氧等均可导致内质网功能受损,表现为细胞中未折叠或者发生错误折叠的蛋白质在内质网腔内大量聚集,这种聚集将会引发细胞产生应激反应,称为内质网应激. MicroRNAs 是一类内源性非编码 RNAs,通过调控基因的表达来发挥重要的功能. 越来越多的研究表明,内质网应激和microRNAs之间通过相互作用参与诸多重要的生理过程. 本文综述了内质网应激和microRNAs两者的相互作用与肿瘤发生发展的关系,以期对肿瘤发生发展的过程调控机制有更为深入的理解,为发展新的肿瘤治疗方案提供思路.     

Structural organization of the spinach endoplasmic reticulum-luminal 70-kilodalton heat-shock cognate gene and expression of 70-kilodalton heat-shock genes during cold acclimation.

The 70-kD heat-shock proteins (HSP70s) are encoded by a multigene family in eukaryotes. In plants, the 70-kD heat-shock cognate (HSC70) proteins are located in organellar and cytosolic compartments of cells in most tissues. Previous work has indicated that HSC70 proteins of spinach (Spinacia oleracea) are actively synthesized during cold-acclimating conditions. We have isolated, sequenced, and characterized cDNA and genomic clones for the endoplasmic reticulum (ER) luminal HSC70 protein (immunoglobulin heavy chain-binding protein; BiP) of spinach. The spinach ER-luminal HSC70 is a constitutively expressed gene consisting of eight exons. Spinach BiP mRNA appears to be up-regulated during cold acclimation but is not expressed during water stress or heat shock. In contrast to the differential regulation of mRNA, the ER-luminal HSC70 protein levels remain constant in response to various environmental stresses. Two other members of the spinach 70-kD heat-shock (HS70) multigene family also show differential expression in response to a variety of environmental stresses. A constitutively expressed cytosolic HSC70 protein in spinach appears also to be up-regulated in response to both cold-acclimating and heat-shock treatments. Spinach also contains a cold-shock-induced HS70 gene that is not expressed during heat shock or water stress. Since HSP70s are considered to be involved with the chaperoning and folding of proteins, the data further support the concept that they may be important for maintaining cellular homeostasis and proper protein biogenesis during cold acclimation of spinach.     

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.     

植物内质网胁迫研究进展

内质网是蛋白质折叠和蛋白质糖基化修饰的重要场所。在内质网中存在多种调控机制来确保其中的蛋白质被正确地折叠、修饰和组装,以维持内质网稳态,这对于细胞正常的生理活动十分重要。然而,多种物理、化学因素均可使内质网稳态失衡,即在应激条件下,错误折叠和未折叠蛋白质的大量积累将导致内质网胁迫(endoplasmic reticulum stress, ERS),进而会引起未折叠蛋白质响应(unfolded protein response, UPR),极端情况下还会启动细胞程序性死亡(program cell death, PCD)。目前,植物内质网胁迫方面的研究较酵母和动物滞后,因此,从内质网质量控制系统和未折叠蛋白质响应2个方面对植物内质网胁迫现有研究进行了综述,以期为进一步理解内质网胁迫与植物逆境胁迫的关系提供参考。     

The Emp24 complex recruits a specific cargo molecule into endoplasmic reticulum-derived vesicles

Members of the yeast p24 family, including Emp24p and Erv25p, form a heteromeric complex required for the efficient transport of selected proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. The specific functions and sites of action of this complex are unknown. We show that Emp24p is directly required for efficient packaging of a lumenal cargo protein, Gas1p, into ER-derived vesicles. Emp24p and Erv25p can be directly cross-linked to Gas1p in ER-derived vesicles. Gap1p, which was not affected by emp24 mutation, was not cross-linked. These results suggest that the Emp24 complex acts as a cargo receptor in vesicle biogenesis from the ER.     

Effects of calpain inhibitor on the apoptosis of hepatic stellate cells induced by calcium ionophore A23187

We previously showed that changes in calcium concentrations were related to cell apoptosis in vitro. The endoplasmic reticulum (ER) is the main component of calcium storage and signal transduction, and disrupting the balance of intracellular Ca²⁺ can cause endoplasmic reticulum stress (ERS). In this process, the ER releases stored Ca ²⁺ into the cytoplasm and activates calpain-2. To further investigate the effect of calpain in hepatic stellate cells (HSCs), in the current study, we examine the effect of N-acetyl-leu-leu-norleucinal (ALLN) on apoptosis resulting from calcium ionophore A23187–induced ERS. Our findings indicate that calpain inhibition reduces calcium ionophore A23187–induced apoptosis of HSCs and decreases the expression of ER stress proteins that may be related to the calpain/caspase signaling pathway.     

The absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of selected proteins to the Golgi.

Emp24p is a type I transmembrane protein that is involved in secretory protein transport from the endoplasmic reticulum (ER) to the Golgi complex. A yeast mutant that lacks Emp24p (emp24 delta) is viable, but periplasmic invertase and the glycosylphosphatidyl-inositol-anchored plasma membrane protein Gas1p are delivered to the Golgi apparatus with reduced kinetics, whereas transport of alpha-factor, acid phosphatase and two vacuolar proteins is unaffected. Oligomerization and protease digestion studies of invertase suggest that the selective transport phenotype observed in the emp24 delta mutant is not due to a defect in protein folding or oligomerization. Consistent with a role in ER to Golgi transport, Emp24p is a component of COPII-coated, ER-derived transport vesicles that are isolated from a reconstituted in vitro budding reaction. We propose that Emp24p is involved in the sorting and/or concentration of a subset of secretory proteins into ER-derived transport vesicles.     

LAG1 puts the focus on ceramide signaling

Longevity-assurance gene 1 (LAG 1) is a yeast longevity gene. Homologues of the Lag 1 protein can be found throughout phylogeny, although sequence similarity is very limited. The Lag 1 protein is located in the endoplasmic reticulum (ER), where it helps to accelerate the transport of glycosylphosphatidylinositol (GPI)-anchored proteins to the Golgi. This function of Lag 1 p results from its participation in ceramide synthesis. Thus, Lag 1 p and its homologues are likely to play a role in ceramide signaling, which affects growth, proliferation, stress resistance, and apoptosis. This provides a wide range of physiologic processes through which Lag 1 p may impinge upon life span.     

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。     

X-box binding protein 1 (XBP1) function in diseases

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) causes endoplasmic reticulum stress (ERS), which is characteristic of cells with high levels of secretory activity and is involved in a variety of diseases. In response to ERS, cells initiate an adaptive process named the unfolding protein response (UPR) to maintain intracellular homeostasis and survival. However, long term and unresolved ERS can also induce apoptosis. As the most conserved signaling branch of UPR, the IRE1-XBP1 pathway plays an important role in both physiological and pathological states, and its activity has a profound impact on disease progression and prognosis. Here, the latest research progress of IRE1-XBP1 pathway in cancer, metabolic diseases, and other diseases was briefly introduced, and the relationship between several diseases and this pathway was analyzed. Besides, the new understanding and prospect of IRE1-XBP1 pathway regulating male reproduction were reviewed.     

Depletion of oxidative and endoplasmic reticulum stress regulators in Pick disease

Both oxidative and endoplasmic reticulum (ER) stress is associated with multiple neurodegenerative, age-related diseases. The rare disorder Pick disease (PiD) shares some pathological hallmarks of other neurodegenerative diseases that may be related to oxidative stress. Importantly, activation of an ER stress response, which is also involved in aging, has not yet been investigated in PiD. In this study, we assessed the implication of ER stress associated with oxidative stress in PiD as a potential mechanism involved in its pathogenesis. Samples from morphologically affected frontal cortex and apparently pathologically preserved occipital cortex showed region-dependent increases in different protein oxidative damage pathways. The oxidative modifications targeted antioxidant enzymes, proteases, heat shock proteins, and synaptic proteins. These effects were associated with compromised proteasomal function and ER stress in frontal cortex samples. In addition, we observed a depletion in ER chaperones (glucose-regulated proteins Grp78/BiP and glucose-regulated protein 94) and differences in tissue content and distribution of nuclear factor-erythroid 2 p45-related respiratory 2, required for cell survival during the unfolded protein response. These results demonstrate increased region-specific protein oxidative damage in PiD, with proteasomal alteration and dysfunctional ER stress response. We suggest this was caused by complete and specific depletion of Grp78/BiP, contributing to the pathophysiology of this neurodegenerative disease.     

Yeast genes controlling responses to topogenic signals in a model transmembrane protein

Yeast protein insertion orientation (PIO) mutants were isolated by selecting for growth on sucrose in cells in which the only source of invertase is a C-terminal fusion to a transmembrane protein. Only the fraction with an exocellular C terminus can be processed to secreted invertase and this fraction is constrained to 2-3% by a strong charge difference signal. Identified pio mutants increased this to 9-12%. PIO1 is SPF1, encoding a P-type ATPase located in the endoplasmic reticulum (ER) or Golgi. spf1-null mutants are modestly sensitive to EGTA. Sensitivity is considerably greater in an spf1 pmr1 double mutant, although PIO is not further disturbed. Pmr1p is the Golgi Ca(2+) ATPase and Spf1p may be the equivalent ER pump. PIO2 is STE24, a metalloprotease anchored in the ER membrane. Like Spf1p, Ste24p is expressed in all yeast cell types and belongs to a highly conserved protein family. The effects of ste24- and spf1-null mutations on invertase secretion are additive, cell generation time is increased 60%, and cells become sensitive to cold and to heat shock. Ste24p and Rce1p cleave the C-AAX bond of farnesylated CAAX box proteins. The closest paralog of SPF1 is YOR291w. Neither rce1-null nor yor291w-null mutations affected PIO or the phenotype of spf1- or ste24-null mutants. Mutations in PIO3 (unidentified) cause a weaker Pio phenotype, enhanced by a null mutation in BMH1, one of two yeast 14-3-3 proteins.     

ER-golgi traffic is a prerequisite for efficient ER degradation

Protein quality control is an essential function of the endoplasmic reticulum. Misfolded proteins unable to acquire their native conformation are retained in the endoplasmic reticulum, retro-translocated back into the cytosol, and degraded via the ubiquitin-proteasome system. We show that efficient degradation of soluble malfolded proteins in yeast requires a fully competent early secretory pathway. Mutations in proteins essential for ER-Golgi protein traffic severely inhibit ER degradation of the model substrate CPY*. We found ER localization of CPY* in WT cells, but no other specific organelle for ER degradation could be identified by electron microscopy studies. Because CPY* is degraded in COPI coat mutants, only a minor fraction of CPY* or of a proteinaceous factor required for degradation seems to enter the recycling pathway between ER and Golgi. Therefore, we propose that the disorganized structure of the ER and/or the mislocalization of Kar2p, observed in early secretory mutants, is responsible for the reduction in CPY* degradation. Further, we observed that mutations in proteins directly involved in degradation of malfolded proteins (Der1p, Der3/Hrd1p, and Hrd3p) lead to morphological changes of the endoplasmic reticulum and the Golgi, escape of CPY* into the secretory pathway and a slower maturation rate of wild-type CPY.     

内质网应激反应分子机理研究进展

内质网应激是导致心脑组织缺血梗塞、神经退行性疾病等发生的重要环节 .目前发现同型半胱氨酸、氧化应激、钙代谢紊乱等都能引起内质网应激级联反应 ,表现为蛋白质合成暂停、内质网应激蛋白表达和细胞凋亡等 .这些表现包括在未折叠蛋白反应 (UPR)、整合应激反应 (ISR)和内质网相关性死亡 (ERAD)三个相互关联的动态过程中 ,每一过程的分子机理现已逐步被揭示 .作为细胞保护性应对机制的内质网应激体系一旦遭到破坏 ,细胞将不能合成应有的蛋白质 ,亦不能发挥正常的生理功能 ,甚至会出现细胞凋亡 .掌握内质网应激过程对进一步理解多种疾病的发生机理有十分重要的理论意义     

Dexmedetomidine attenuates neuronal injury after spinal cord ischaemia‐reperfusion injury by targeting the CNPY2‐endoplasmic reticulum stress signalling

Dexmedetomidine (Dex) has been proven to exert protective effects on multiple organs in response to ischaemia‐reperfusion injury, but the specific mechanism by which this occurs has not been fully elucidated. The purpose of this study was to investigate whether Dex attenuates spinal cord ischaemia‐reperfusion injury (SCIRI) by inhibiting endoplasmic reticulum stress (ERS). Our team established a model of SCIRI and utilized the endoplasmic reticulum agonist thapsigargin. Dex (25 g/kg) was intraperitoneally injected 30 minutes before spinal cord ischaemia. After 45 minutes of ischaemia, the spinal cord was reperfused for 24 hours. To evaluate the neuroprotective effect of Dex on SCIRI, neurological function scores were assessed in rats and apoptosis of spinal cord cells was determined by TUNEL staining. To determine whether the endoplasmic reticulum apoptosis pathway CNPY2‐PERK was involved in the neuroprotective mechanism of Dex, the expression levels of related proteins (CNPY2, GRP78, PERK, CHOP, caspase‐12, caspase‐9 and caspase‐3) were detected by western blot analysis and RT‐PCR. We observed that Dex significantly increased the neurological function scores after SCIRI and decreased apoptosis of spinal cord cells. The expression of ERS‐related apoptosis proteins was significantly increased by SCIRI but was significantly decreased in response to Dex administration. Taken together, the results of this study indicate that Dex may attenuate SCIRI by inhibiting the CNPY2‐ERS apoptotic pathway.     

The ADP-ribosylation factor GTPase-activating protein Glo3p is involved in ER retrieval.

Retrograde transport of proteins from the Golgi to the endoplasmic reticulum (ER) has been the subject of some interest in the recent past. Here a new thermosensitive yeast mutant defective in retrieval of dilysine-tagged proteins from the Golgi back to the endoplasmic reticulum was characterized. The ret4-1 mutant also exhibited a selective defect in forward ER-to-Golgi transport of some secreted proteins at the non-permissive temperature. The corresponding RET4 gene was found to encode Glo3p, a GTPase-activating protein (GAP) specific for ADP-ribosylation factor (ARF). In vitro, the Glo3 thermosensitive mutant showed a reduced ARF1-GAP activity. The Glo3 protein belongs to a family of zinc finger proteins that may include additional ARF-GAPs. Gene deletion experiments of other family members showed that only GLO3 deletion resulted in impaired retrieval of dilysine-tagged proteins back to the ER. These results demonstrate that Glo3p is the main ARF-GAP specifically involved in ER retrieval.     

The yeast p24 complex regulates GPI-anchored protein transport and quality control by monitoring anchor remodeling

Glycosylphosphatidylinositol (GPI)-anchored proteins are secretory proteins that are attached to the cell surface of eukaryotic cells by a glycolipid moiety. Once GPI anchoring has occurred in the lumen of the endoplasmic reticulum (ER), the structure of the lipid part on the GPI anchor undergoes a remodeling process prior to ER exit. In this study, we provide evidence suggesting that the yeast p24 complex, through binding specifically to GPI-anchored proteins in an anchor-dependent manner, plays a dual role in their selective trafficking. First, the p24 complex promotes efficient ER exit of remodeled GPI-anchored proteins after concentration by connecting them with the COPII coat and thus facilitates their incorporation into vesicles. Second, it retrieves escaped, unremodeled GPI-anchored proteins from the Golgi to the ER in COPI vesicles. Therefore the p24 complex, by sensing the status of the GPI anchor, regulates GPI-anchored protein intracellular transport and coordinates this with correct anchor remodeling.