Endoplasmic Reticulum
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Biology Open ◽  
2021 ◽  
Jennifer Y. Liu ◽  
Yu-Hsiu Tony Lin ◽  
Andrew M. Leidal ◽  
Hector H. Huang ◽  
Jordan Ye ◽  

There is great interest in understanding the cellular mechanisms controlling autophagy, a tightly regulated catabolic and stress response pathway. Prior work has uncovered links between autophagy and the Golgi reassembly stacking protein of 55 kDa (GRASP55), but their precise interrelationship remains unclear. Intriguingly, both autophagy and GRASP55 have been functionally and spatially linked to the endoplasmic reticulum (ER)-Golgi interface, broaching this compartment as a site where GRASP55 and autophagy may intersect. Here, we uncover that loss of GRASP55 enhances LC3 puncta formation, indicating that GRASP55 restricts autophagosome formation. Additionally, using proximity-dependent biotinylation, we identify a GRASP55 proximal interactome highly associated with the ER-Golgi interface. Both nutrient starvation and loss of GRASP55 are associated with coalescence of early secretory pathway markers. In light of these findings, we propose that GRASP55 regulates spatial organization of the ER-Golgi interface, which suppresses early autophagosome formation.

Yuxiang Zhou ◽  
Xueping Wan ◽  
Kerstin Seidel ◽  
Mo Zhang ◽  
Jena B. Goodman ◽  

Background Persistent activation of endoplasmic reticulum stress and the unfolded protein response (UPR) induces vascular cell apoptosis, contributing to atherogenesis. Aging and hypercholesterolemia are 2 independent proatherogenic factors. How they affect vascular UPR signaling remains unclear. Methods and Results Transcriptome analysis of aortic tissues from high fat diet–fed and aged ApoE −/− mice revealed 50 overlapping genes enriched for endoplasmic reticulum stress‐ and UPR‐related pathways. Aortae from control, Western diet (WD)–fed, and aged ApoE −/− mice were assayed for (1) 3 branches of UPR signaling (pancreatic ER eIF2‐alpha kinase /alpha subunit of the eukaryotic translation initiation factor 1/activating transcription factor 4, inositol‐requiring enzyme 1 alpha/XBP1s, activating transcription factor 6); (2) UPR‐mediated protective adaptation (upregulation of immunoglobulin heavy chain‐binding protein and protein disulfide isomerase); and (3) UPR‐mediated apoptosis (induction of C/EBP homologous transcription factor, p‐JNK, and cleaved caspase‐3). Aortic UPR signaling was differentially regulated in the aged and WD‐fed groups. Consumption of WD activated all 3 UPR branches; in the aged aorta, only the ATF6α arm was activated, but it was 10 times higher than that in the WD group. BiP and protein disulfide isomerase protein levels were significantly decreased only in the aged aorta despite a 5‐fold increase in their mRNA levels. Importantly, the aortae of aged mice exhibited a substantially enhanced proapoptotic UPR compared with that of WD‐fed mice. In lung tissues, UPR activation and the resultant adaptive/apoptotic responses were not significantly different between the 2 groups. Conclusions Using a mouse model of atherosclerosis, this study provides the first in vivo evidence that aging and an atherogenic diet activate differential aortic UPR pathways, leading to distinct vascular responses. Compared with dietary intervention, aging is associated with impaired endoplasmic reticulum protein folding and increased aortic apoptosis.

Haiying Liu ◽  
Linyu Dai ◽  
Ming Wang ◽  
Fumin Feng ◽  
Yonghong Xiao

It has been reported that calpain/caspase-mediated apoptosis induced by endoplasmic reticulum stress (ERS) in hepatic stellate cells (HSCs) by previous studies. At present, the activation of HSC is an important cause of liver fibrosis, and the induction of HSC apoptosis plays an irreplaceable role in reversing liver fibrosis. Therefore, it is of great significance to explore mechanisms of action that can induce HSC apoptosis for the reversal of hepatic fibrosis and the clinical prevention and treatment of hepatic-fibrosis-related diseases such as hepatitis, cirrhosis, and liver cancer. In the current study, we demonstrated that tunicamycin (a novel ERS inducer) can induce the apoptosis of HSCs and increase the concentration of intracellular Ca2+ and the expression of ERS protein GRP78, apoptosis protein caspase-12, and Bax, while it can decrease the antiapoptosis protein expression of Bcl-2. Our findings indicate that tunicamycin can induce HSCs apoptosis through calpain-2/Ca2+-dependent ERS pathway.

2021 ◽  
Vol 18 (1) ◽  
Tongtong Liu ◽  
Ting Li ◽  
Xuhui Chen ◽  
Zuofan Li ◽  
Miaomiao Feng ◽  

Abstract Background Central post-stroke pain (CPSP) is a chronic and intolerable neuropathic pain syndrome following a cerebral vascular insult, which negatively impacts the quality of life of stroke survivors but currently lacks efficacious treatments. Though its underlying mechanism remains unclear, clinical features of hyperalgesia and allodynia indicate central sensitization due to excessive neuroinflammation. Recently, the crosslink between neuroinflammation and endoplasmic reticulum (ER) stress has been identified in diverse types of diseases. Nevertheless, whether this interaction contributes to pain development remains unanswered. Epoxyeicosatrienoic acids (EETs)/soluble epoxy hydrolase inhibitors (sEHi) are emerging targets that play a significant role in pain and neuroinflammatory regulation. Moreover, recent studies have revealed that EETs are effective in attenuating ER stress. In this study, we hypothesized that ER stress around the stroke site may activate glial cells and lead to further inflammatory cascades, which constitute a positive feedback loop resulting in central sensitization and CPSP. Additionally, we tested whether EETs/sEHi could attenuate CPSP by suppressing ER stress and neuroinflammation, as well as their vicious cycle, in a rat model of CPSP. Methods Young male SD rats were used to induce CPSP using a model of thalamic hemorrhage and were then treated with TPPU (sEHi) alone or in combination with 14,15-EET or 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, the EET antagonist), tunicamycin (Tm, ER stress inducer), or 4-PBA (ER stress inhibitor). Nociceptive behaviors, ER stress markers, JNK and p38 (two well-recognized inflammatory kinases of mitogen-activated protein kinase (MAPK) signaling) expression, and glial cell activation were assessed. In addition, some healthy rats were intrathalamically microinjected with Tm or lipopolysaccharide (LPS) to test the interaction between ER stress and neuroinflammation in central pain. Results Analysis of the perithalamic lesion tissue from the brain of CPSP rats demonstrated decreased soluble epoxy hydrolase (sEH) expression, which was accompanied by increased expression of ER stress markers, including BIP, p-IRE, p-PERK, and ATF6. In addition, inflammatory kinases (p-p38 and p-JNK) were upregulated and glial cells were activated. Intrathalamic injection of sEHi (TPPU) increased the paw withdrawal mechanical threshold (PWMT), reduced hallmarks of ER stress and MAPK signaling, and restrained the activation of microglia and astrocytes around the lesion site. However, the analgesic effect of TPPU was completely abolished by 14,15-EEZE. Moreover, microinjection of Tm into the thalamic ventral posterior lateral (VPL) nucleus of healthy rats induced mechanical allodynia and activated MAPK-mediated neuroinflammatory signaling; lipopolysaccharide (LPS) administration led to activation of ER stress along the injected site in healthy rats. Conclusions The present study provides evidence that the interaction between ER stress and neuroinflammation is involved in the mechanism of CPSP. Combined with the previously reported EET/sEHi effects on antinociception and neuroprotection, therapy with agents that target EET signaling may serve as a multi-functional approach in central neuropathic pain by attenuating ER stress, excessive neuroinflammation, and subsequent central sensitization. The use of these agents within a proper time window could not only curtail further nerve injury but also produce an analgesic effect.

2021 ◽  
Vol 18 (1) ◽  
Hanhai Zeng ◽  
Huaijun Chen ◽  
Min Li ◽  
Jianfeng Zhuang ◽  
Yucong Peng ◽  

Abstract Background Neuroinflammation and oxidative stress plays an important role in the pathogenesis of early brain injury (EBI) after subarachnoid hemorrhage (SAH). This study is the first to show that activation of autophagy protein nuclear receptor binding factor 2 (NRBF2) could reduce endoplasmic reticulum stress (ERS)-associated inflammation and oxidative stress after SAH. Methods Male C57BL/6J mice were subjected to endovascular perforation to establish a model of SAH. NRBF2 overexpression adeno-associated virus (AAV), NRBF2 small interfering RNAs (siRNA), lysosomal inhibitor-chloroquine (CQ), and late endosome GTPase Rab7 receptor antagonist-CID1067700 (CID) were used to investigate the role of NRBF2 in EBI after SAH. Neurological tests, brain water content, western blotting and immunofluorescence staining were evaluated. Results Our study found that the level of NRBF2 was increased after SAH and peaked at 24 h after SAH. In addition, we found that the overexpression of NRBF2 significantly improved neurobehavioral scores and reduced ERS, oxidative stress, and neuroinflammation in SAH, whereas the inhibition of NRBF2 exacerbated these phenotypes. In terms of mechanism, NRBF2 overexpression significantly promoted autophagosome maturation, with the downregulation of CHOP, Romo-1, TXNIP, NLRP3, TNF-α, and IL-1β expression through interaction with Rab7. The protective effect of NRBF2 on ERS-associated neuroinflammation and oxidative stress after SAH was eliminated by treatment with CQ. Meanwhile, it was also reversed by intraperitoneal injection of CID. Moreover, the MIT domain of NRBF2 was identified as a critical binding site that interacts with Rab7 and thereby promotes autophagosome maturation. Conclusion Our data provide evidence that the autophagy protein NRBF2 has a protective effect on endoplasmic reticulum stress-associated neuroinflammation and oxidative stress by promoting autophagosome maturation through interactions with Rab7 after SAH.

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257435
Miyako Nakano ◽  
Susana Sabido-Bozo ◽  
Kouta Okazaki ◽  
Auxiliadora Aguilera-Romero ◽  
Sofia Rodriguez-Gallardo ◽  

Glycosylphosphatidylinositol (GPI) anchoring of proteins is an essential post-translational modification in all eukaryotes that occurs at the endoplasmic reticulum (ER) and serves to deliver GPI-anchored proteins (GPI-APs) to the cell surface where they play a wide variety of vital physiological roles. This paper describes a specialized method for purification and structural analysis of the GPI glycan of individual GPI-APs in yeast. The protocol involves the expression of a specific GPI-AP tagged with GFP, enzymatic release from the cellular membrane fraction, immunopurification, separation by electrophoresis and analysis of the peptides bearing GPI glycans by mass spectrometry after trypsin digestion. We used specifically this protocol to address the structural remodeling that undergoes the GPI glycan of a specific GPI-AP during its transport to the cell surface. This method can be also applied to investigate the GPI-AP biosynthetic pathway and to directly confirm predicted GPI-anchoring of individual proteins.

2021 ◽  
Gurur Garip ◽  
Berrin Ozdil ◽  
Duygu Calik-Kocaturk ◽  
Fatih Oltulu ◽  
Fatma Zuhal Eroglu ◽  

Although in vitro endoplasmic reticulum (ER) stress studies have been carried out using Tunicamycin in human trophoblast cell lines in recent years, the effect of calcium homeostasis impaired by the effect of Thapsigargin on cell survival - death pathways have not been clearly demonstrated. Here, the effects of ER stress and impaired calcium homeostasis on cell death pathways such as apoptosis and autophagy in 2-dimensional and 3-dimensional cell cultures were investigated using the HTR8 / SVneo cell line representing human trophoectoderm cells and the ER stressor Thapsigargin. By using Real Time PCR, gene and immunofluorescence analyzes were studied at the protein level. In this study, it has been established that the Thapsigargin creates ER stress by increasing the level of GRP78 gene and protein in 2 and 3 dimensions of human trophoectoderm cells and that cells show different characterization properties in 2 and 3 dimensions. It has been determined that while it moves in the direction of EIF2A and IRE1A mechanisms in 2 dimensions, it proceeds in the direction of EIF2A and ATF6 mechanisms in 3 dimensions and creates different responses in survival and programmed cell death mechanisms such as apoptosis and autophagy. With forthcoming studies, it is thought that the effects of Thapsigargin on the intrinsic pathway of apoptosis and the linkage of the autophagy mechanism, the examination of the survival-death pathways in the co-culture model with endometrial cells, therapeutic target molecules that will contribute to the elucidation of intracellular cell dynamics may increase the success of implantation.

2021 ◽  
pp. MOLPHARM-AR-2021-000269
Etta Y.L. Liu ◽  
Shinghung Mak ◽  
Xiangpeng Kong ◽  
Yingjie Xia ◽  
Kenneth K.L. Kwan ◽  

2021 ◽  
Gerd Jürgens ◽  
Sabine Brumm ◽  
Hauke Beckmann ◽  
Sandra Richter ◽  
Manoj K Singh ◽  

Functionally divergent paralogs of homomeric proteins do not form potentially deleterious heteromers, which requires distinction between self and non-self (Hochberg et al., 2018; Marchant et al, 2019; Marsh and Teichmann, 2015). In Arabidopsis, two ARF guanine-nucleotide exchange factors (ARF-GEFs) related to mammalian GBF1, named GNOM and GNL1, can mediate coatomer complex (COPI)-coated vesicle formation in retrograde Golgi-endoplasmic reticulum (ER) traffic (Geldner et al., 2003; Richter et al., 2007; Teh and Moore, 2007). Unlike GNL1, however, GNOM is also required for polar recycling of endocytosed auxin efflux regulator PIN1 from endosomes to the plasma membrane. Here we show that these paralogues form homodimers constitutively but no heterodimers. We also address why and how GNOM and GNL1 might be kept separate. These paralogues share a common domain organisation and each N-terminal dimerisation (DCB) domain can interact with the complementary fragment (DDCB) of its own and the other protein. However, unlike self-interacting DCBGNOM (Grebe et al., 2000; Anders et al., 2008), DCBGNL1 did not interact with itself nor DCBGNOM. DCBGNOM removal or replacement with DCBGNL1, but not disruption of cysteine bridges that stabilise DCB-DCB interaction, resulted in GNOM-GNL1 heterodimers which impaired developmental processes such as lateral root formation. We propose precocious self-interaction of the DCBGNOM domain as a mechanism to preclude formation of fitness-reducing GNOM-GNL1 heterodimers.

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