Group A Streptococcus host-pathogen dual crosstalk.

Kennedy disease, a degenerative disorder caused by an expanded glutamine tract, is mediated by misfolding of the mutant androgen receptor (AR) protein, a process that may disrupt proteasome function. We hypothesized that this might lead to endoplasmic reticulum (ER) stress and induction of the unfolded protein response (UPR), a complex physiologic pathway that regulates cell survival. To test this hypothesis, we used aminoterminal fragments of wild type (AR16Q) or mutant (AR112Q) AR that triggered glutamine length-dependent cell death and activated an ER stress-inducible promoter. To evaluate the role of the UPR, we examined the contributions of three proximal sensors of ER stress: activating transcription factor 6 (ATF6), inositol requiring 1 (IRE1), and PKR-like endoplasmic reticulum kinase (PERK). AR112Q toxicity was significantly increased by a dominant negative ATF6 mutant and significantly decreased by a constitutively active ATF6 mutant, indicating that ATF6 promoted cell survival. In contrast, co-transfection with three separate IRE1alpha dominant negative mutants failed to alter glutamine length-dependent toxicity, suggesting that this arm of the UPR did not significantly affect AR112Q induced cell death. Activation of PERK, an ER transmembrane protein that functions as the third proximal UPR sensor, promoted glutamine length-dependent toxicity. Although nuclear localization sequence- and nuclear export sequence-targeted proteins both activated the UPR, this pathway more potently influenced toxicity when proteins were targeted to the cytoplasm. Taken together, our data demonstrate that the UPR is activated in cells expressing long glutamine tracts and that this pathway modulates polyglutamine toxicity.

The efficient functioning of the endoplasmic reticulum (ER) is essential for most cellular activities and survival. Conditions that interfere with ER function lead to the accumulation and aggregation of unfolded proteins. ER transmembrane receptors detect the onset of ER stress and initiate the unfolded protein response (UPR) to restore normal ER function. If the stress is prolonged, or the adaptive response fails, apoptotic cell death ensues. Many studies have focused on how this failure initiates apoptosis, as ER stress-induced apoptosis is implicated in the pathophysiology of several neurodegenerative and cardiovascular diseases. In this review, we examine the role of the molecules that are activated during the UPR in order to identify the molecular switch from the adaptive phase to apoptosis. We discuss how the activation of these molecules leads to the commitment of death and the mechanisms that are responsible for the final demise of the cell.

Endoplasmic reticulum (ER) stress is a causative factor of inflammatory bowel diseases. ER stress mediators, including CCAAT enhancer-binding protein (C/EBP) homologous protein (CHOP), are elevated in intestinal epithelia from patients with inflammatory bowel diseases. The present study arose from the question of how chemical ER stress and CHOP protein were associated with nuclear factor-κB (NF-κB)-mediated epithelial inflammatory response. In a human intestinal epithelial cell culture model, chemical ER stresses induced proinflammatory cytokine interleukin-8 (IL-8) expression and the nuclear translocation of CHOP protein. CHOP was positively involved in ER-activated IL-8 production and was negatively associated with expression of peroxisome proliferator-activated receptor γ (PPARγ). ER stress-induced IL-8 production was enhanced by NF-κB activation that was negatively regulated by PPARγ. Mechanistically, ER stress-induced CHOP suppressed PPARγ transcription by sequestering C/EBPβ and limiting availability of C/EBPβ binding to the PPARγ promoter. Due to the CHOP-mediated regulation of PPARγ action, ER stress can enhance proinflammatory NF-κB activation and maintain an increased level of IL-8 production in human intestinal epithelial cells. In contrast, PPARγ was a counteracting regulator of gut inflammatory response through attenuation of NF-κB activation. The collective results support the view that balances between CHOP and PPARγ are crucial for epithelial homeostasis, and disruption of these balances in mucosal ER stress can etiologically affect the progress of human inflammatory bowel diseases.

Palmitic acid (PA) upregulates oxidized LDL receptor-1 (LOX-1), a scavenger receptor responsible for uptake of oxidized LDL (oxLDL), and enhances oxLDL uptake in macrophages. However, the precise underlying mechanism remains to be elucidated. PA is known to induce endoplasmic reticulum (ER) stress in various cell types. Therefore, we investigated whether ER stress is involved in PA-induced LOX-1 upregulation. PA induced ER stress, as determined by phosphorylation of PERK, eIF2α, and JNK, as well as induction of CHOP in macrophage-like THP-1 cells. Inhibitors [4-phenylbutyric acid (PBA), sodium tauroursodeoxycholate (TUDCA), and salubrinal] and small interfering RNA (siRNA) for the ER stress response decreased PA-induced LOX-1 upregulation. Thapsigargin, an ER stress inducer, upregulated LOX-1, which was decreased by PBA and TUDCA. We next examined whether unsaturated FAs could counteract the effect of PA. Both oleic acid (OA) and linoleic acid (LA) suppressed PA-induced LOX-1. Activation of the ER stress response observed in the PA-treated cells was markedly attenuated when the cells were cotreated with OA or LA. In addition, OA and LA suppressed thapsigargin-induced LOX-1 upregulation with reduced activation of ER stress markers. Our results indicate that activation of ER stress is involved in PA-induced LOX-1 upregulation in macrophages, and that OA and LA inhibit LOX-1 induction through suppression of ER stress.

Endoplasmic reticulum (ER) stress is sensed by cells in different physiopathological conditions in which there is an accumulation of unfolded proteins in the ER. A coordinated adaptive program called the unfolded protein response is triggered and includes translation inhibition, transcriptional activation of a set of genes encoding mostly intracellular proteins, and ultimately apoptosis. Here we show that insulin-like growth factor (IGF)-binding protein-1 (IGFBP-1), a secreted protein that modulates IGF bioavailability and has other IGF-independent effects, is potently induced during ER stress in human hepatocytes. Various ER stress-inducing agents were able to increase IGFBP-1 mRNA levels, as well as cellular and secreted IGFBP-1 protein up to 20-fold. A distal regulatory region of the human IGFBP-1 gene (-6682/-6384) containing an activating transcription factor 4 (ATF4) composite site was required for promoter activation upon ER stress. Mutation of the ATF4 composite site led to the loss of IGFBP-1 regulation. Electrophoretic mobility shift assay revealed an ER stress-inducible complex that was displaced by an ATF4 antibody. Knockdown of ATF4 expression using two specific small interfering RNAs impaired up-regulation of IGFBP-1 mRNA, which highlights the relevance of ATF4 in endogenous IGFBP-1 gene induction. In addition to intracellular proteins involved in secretory and metabolic pathways, we conclude that ER stress induces the synthesis of secreted proteins. Increased secretion of IGFBP-1 during hepatic ER stress may thus constitute a signal to modulate cell growth and metabolism and induce a systemic adaptive response.

ER stress: Can the liver cope?

Objective To investigate the relationship between endoplasmic reticulum stress protein kinase R-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2a (eIF-2α)/activating transcription factor 6 (ATF6) pathway and proliferation and migration of rat hepatoma cells. Methods Rat liver cancer model was established by intermittent administration of diethylnitrosamine (DEN). Morphological changes of rat liver were observed by hematoxylin-eosin staining (HE) staining. The expression of PERK and eIF-2α protein in rat hepatocarcinoma tissues was detected by immunohistochemistry. The endoplasmic reticulum stress cell model was constructed by inducing normal liver cell LO2 and highly differentiated hepatoma cell line CBRH-7919 with absolute ethanol. Cell counting kit-8 (CCK-8) method was used to detect the proliferation of cells; the migration and invasion ability of cells were detected by scratch test; the PERK, eIF-2α, ATF6, and B lymphoma-2 were detected by Western blotting. Expression of bcl-2 and cysteine-containing aspartate proteolytic enzyme-3 (Caspase-3) protein, and statistical calculation. Results Liver cells in liver cancer were found to be irregularly arranged. Hepatocytes showed severe abnormalities, nuclear membrane depression, deep nuclear staining, and abnormal nuclear morphology of liver cells. After ethanol-induced LO2 and CBRH-7919 cells undergo endoplasmic reticulum stress, the cell migration ability of LO2 and CBRH-7919 cells was significantly decreased (tLO2 cells=8.910, P<0.01; tCBRH-7919 cells=30.010, P<0.01). The survival rates of LO2 and CBRH-7919 cells were significantly lower, and the difference was statistically significant (tLO2 cells=11.840, P<0.01; tCBRH-7919 cells=19.080, P<0.01). The expressions of PERK, eIF-2α, ATF6 and Caspase-3 in the liver cancer tissues of the experimental group were significantly increased, and the expression of bcl-2 protein was significantly decreased (tPERK=8.070, P<0.01; teIF-2α=11.790, P<0.01; tATF6= 7.750, P<0.01; tbcl-2=8.670, P<0.01; tCaspase-3=7.720, P<0.01). After ethanol-induced endoplasmic reticulum stress in LO2 cells, the expression levels of PERK, eIF-2α, ATF6, bcl-2 and Caspase-3 in LO2 cells were significantly increased, and bcl-2 protein expression was significantly decreased (tPERK=12.910, P<0.01; teIF-2α= 15.810, P<0.01; tATF6=7.690, P<0.01; tbcl-2=23.260, P<0.01; tCaspase-3=22.280, P<0.01). After the endoplasmic reticulum stress induced by absolute ethanol in CBRH-7919 cells, the expression of PERK, eIF-2α, ATF6, bcl-2 and Caspase-3 protein in CBRH-7919 cells was significantly increased, and the expression of bcl-2 protein was significantly decreased (tPERK= 9.560, P<0.01; teIF-2α= 12.400, P<0.01; tATF6=6.880, P<0.01; tbcl-2=13.840, P<0.01; tCaspase-3=9.950, P<0.01). Conclusion Activation of the PERK/eIF-2α/ATF4 pathway in endoplasmic reticulum stress significantly inhibits the proliferation and migration of rat hepatoma cells. Key words: Liver cancer; Endoplasmic reticulum stress; Protein kinase R-like endoplasmic reticulum kinase; Eukaryotic initiation factor 2α; Transcriptional activator-6

Protein misfolding in the endoplasmic reticulum (ER) triggers a signaling pathway termed the unfolded protein response path-way (UPR). UPR signaling is transduced through the transmembrane ER effectors PKR-like ER kinase (PERK), inositol requiring kinase-1 (IRE-1), and activating transcription factor 6 (ATF6). PERK activation triggers phosphorylation of eIF2alpha leading to repression of protein synthesis, thereby relieving ER protein load and directly inhibiting cyclin D1 translation thereby contributing to cell cycle arrest. However, PERK(-/-) murine embryonic fibroblasts have an attenuated G(1)/S arrest that is not attributable to cyclin D1 loss, suggesting a cyclin D1-independent mechanism. Here we show that the UPR triggers p53 accumulation and activation. UPR induction promotes enhanced interaction between the ribosome proteins (rpL5, rpL11, and rpL23) and Hdm2 in a PERK-dependent manner. Interaction with ribosomal proteins results in inhibition of Hdm2-mediated ubiquitination and degradation of p53. Our data demonstrate that ribosomal subunit:Hdm2 association couples the unfolded protein response to p53-dependent cell cycle arrest.

Objective To investigate the role of endoplasmic reticulum stress (ERS) pathway involving protein kinase R-like ER kinase (PERK)-activating transcription factor 4 (ATF4)-CCAAT/enhancer binding protein homologous protein (CHOP) in apoptosis in lungs of rats with bronchopulmonary dysplasis (BPD). Methods Forty-eight premature SD rats were divided into BPD group and control group according to random number table.Rats in BPD group were continually exposed to O2 with volumetric concentration factor of 850 mL/L, while rats in control group were exposed to air.Lung tissues in each group were obtained in 7, 14 and 21 days respectively.The apoptosis in lung cells was evaluated by terminal dexynucleotifyl transferase-mediated dUTP nick end labeling (TUNEL) assay.The mRNA levels of glucose regulated protein 78 (GRP78), PERK, ATF4 and CHOP were detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The protein levels of GRP78, phosphorylated PERK (pho-PERK), ATF4 and CHOP were detected by using Western blot. Results Compared with control group, the lung cells of the rats in BPD group developed more serious apoptosis.Furthermore, the apoptosis index (AI) in lung cells of the rats increased rapidly with the hyperoxia exposure time.This had been statistically verified by comparison with the control group at different timing(7 d: 15.50±0.58 vs 1.25±0.50, 14 d: 27.75±1.71 vs 3.25±0.96, 21 d: 50.50±3.70 vs 4.00±1.15; t=57.00, 20.58, 25.16, all P<0.01). The mRNA levels of GRP78, PERK, ATF4 and CHOP in BPD group increased significantly compared to the control group [GRP78: 7 d (33.88±3.73 )vs(11.65±1.00), 14 d (54.50±2.18)vs(12.84±1.41), 21 d (95.34±7.61)vs(12.43±0.59); PERK: 7 d (5.23±0.92)vs(1.45±0.46), 14 d (7.60±1.56)vs(2.18±0.97), 21 d (16.55±0.50)vs(2.90±1.18); ATF4: 7 d (23.04±2.45)vs(12.56±2.81), 14 d (28.66±2.66)vs(15.18±2.92), 21 d (36.63±2.99)vs(15.14±2.09); CHOP: 7 d (2.21±0.19)vs(0.81±0.02), 14 d (4.19±0.17)vs(0.90±0.08), 21 d (6.08±0.38)vs(0.88±0.10); all P<0.05]. The protein levels of GRP78, pho-PERK, ATF4 and CHOP in BPD group increased significantly as well [GRP78: 7 d (1.33±0.03 )vs(0.85±0.04), 14 d (1.31±0.02)vs(0.92±0.01), 21 d (1.82±0.28)vs(0.87±0.01); pho-PERK: 7 d (0.68±0.02)vs(0.54±0.01), 14 d (1.04±0.01)vs(0.65±0.01), 21 d (1.29±0.02)vs(0.73±0.01); ATF4: 7 d (1.26±0.01)vs(0.83±0.01), 14 d (1.39±0.02)vs(0.87±0.02), 21 d (1.67±0.02)vs(0.94±0.02); CHOP: 7 d (1.37±0.01)vs(0.47±0.06), 14 d (1.50±0.04)vs(0.74±0.05), 21 d (1.61±0.03)vs(0.55±0.02); all P<0.05]. Positive correlation was demonstrated between the expression levels of CHOP protein and AI, PERK, ATF4 in the BPD group (r=0.87, 0, 92, 0.93 respectively, all P<0.05). Conclusion PERK-ATF4-CHOP mediated ERS may participate in and contribute to the apoptosis mechanism in lungs of rats with BPD. Key words: Endoplasmic reticulum stress; Protein kinase R-like ER kinase; CCAAT/enhancer binding protein homologous protein; Bronchopulmonary dysplasis; Apoptosis

Non-melanoma skin cancer (NMSC) is the most common cancer in the United States. NMSC and other epithelial tumors overexpress cyclooxygenase-2 (COX-2), differentiating them from normal epithelial cells. COX-2 metabolizes arachidonic acid to prostaglandins of the E-, F-, D-, and J-series. While E-series prostaglandins promote tumor cell proliferation, J-series prostaglandins induce apoptosis by various mechanisms including the induction of endoplasmic reticulum (ER) stress. In response to excessive ER stress, sensors such as PKR-like ER kinase (PERK) and activating transcription factor 6 (ATF6) are activated and initiate the apoptotic cascade by increasing the expression of C/EBP homologous protein (CHOP 10). Anandamide (AEA) is an endogenous cannabinoid neurotransmitter which induces apoptotic cell death in a variety of tumor cell types. AEA is metabolized by COX-2 to ethanolamide conjugates of E-, F-, and D-series prostaglandins. Data from our previous study showed that J-series prostaglandins are also metabolic products of AEA however, the identity of the specific J-series prostaglandins produced was unknown. In the current study, we identify the J-series prostaglandins produced by AEA and test the hypothesis that these ethanolamide conjugates of J-series prostaglandins (PGJ2-EA) initiate AEA-induced ER stress in NMSC cells. To determine if AEA induces ER stress in NMSC cells we examined the phosphorylation of PERK and eukaryotic initiation factor 2 alpha (eIF2α), the induction of CHOP 10 expression and the nuclear translocation of ATF 6 by Western blot or immunocytochemical analysis. Our data show that AEA increased PERK and eIF2α phosphorylation, CHOP 10 expression and ATF 6 nuclear localization. To determine whether COX-2 and J-series prostaglandins are necessary for AEA-induced ER stress, non-tumorigenic HaCaT keratinocytes, which express low basal levels of COX-2, were transfected with a plasmid containing human COX-2 cDNA or an empty vector and the cells treated with AEA or drug vehicle. We observed that AEA-induced phosphorylation of PERK and eIF2α occurred only in the presence of COX-2. To identify the specific J-series PGs synthesized from AEA in NMSC cells, JWF2 keratinocytes were treated with AEA, the PGs extracted from the culture media of treated cells, and the samples analyzed using LC/MS. Our data show that ethanolamide conjugates of 15-deoxyΔ 12, 14PGJ2, Δ12PGJ2, and PGJ2 are metabolic products of AEA. These findings suggest that COX-2 mediates the induction of ER stress by AEA. Therefore, the initiation of ER stress may be responsible for the pro-apoptotic activity of AEA in tumor cells. As such, AEA or chemical derivatives of AEA could be ideal topical agents for the eradication of non-melanoma skin tumors that overexpress COX-2. Citation Format: Eman Soliman, Allison Danell, Rukiyah Van Dross. Cyclooxygenase-2 regulates anandamide-induced endoplasmic reticulum stress in tumorigenic keratinocytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2128. doi:10.1158/1538-7445.AM2013-2128

We have previously shown that ASK1-interacting protein 1 (AIP1) transduces tumor necrosis factor-induced ASK1-JNK signaling. Because endoplasmic reticulum (ER) stress activates ASK1-JNK signaling cascade, we investigated the role of AIP1 in ER stress-induced signaling. We created AIP1-deficient mice (AIP1-KO) from which mouse embryonic fibroblasts and vascular endothelial cells were isolated. AIP1-KO cells show dramatic reductions in ER stress-induced, but not oxidative stress-induced, ASK1-JNK activation and cell apoptosis. The ER stress-induced IRE1-JNK/XBP-1 axis, but not the PERK-CHOP1 axis, is blunted in AIP1-KO cells. ER stress induced formation of an AIP1-IRE1 complex, and the PH domain of AIP1 is critical for the IRE1 interaction. Furthermore, reconstitution of AIP1-KO cells with AIP1 wild type, not an AIP1 mutant with a deletion of the PH domain (AIP1-DeltaPH), restores ER stress-induced IRE1-JNK/XBP-1 signaling. AIP1-IRE1 association facilitates IRE1 dimerization, a critical step for activation of IRE1 signaling. More importantly, AIP1-KO mice show impaired ER stress-induced IRE1-dependent signaling in vivo. We conclude that AIP1 is essential for transducing the IRE1-mediated ER stress response.

To explore the protective effect and mechanism of ethyl acetate extract from Bidens bipinnata on hepatocyte damage induced by endoplasmic reticulum stress. Tunicamycin was used to establish the damage model in L02 cells. Methyl thiazolyl tetrazolium(MTT) colorimetric assay was used to investigate the survival rate of ethyl acetate extract from B. bipinnata in L02 cells injury induced by endoplasmic reticulum stress; the protein expressions of endoplasmic reticulum stress-related molecule glucose regulated protein 78(GRP78), PKR-like ER kinase(PERK), eukaryotic initiation factor-2(eIF2α), activating transcription factor 4(ATF4), C/EBP homologous protein(CHOP), B-cell CLL/lymphoma 2(Bcl-2), Bal-2 associated X apoptosis regulator(Bax) were examined by Wes-tern blot. The expressions of the above proteins were also detected after endoplasmic reticulum stress inhibitor(4-phenyl butyric acid) and CHOP shRNA-mediated knockdowns were added. The expressions of GRP78, PERK, CHOP in L02 cells were observed by immunofluorescence method. The results showed that ethyl acetate extract from B. bipinnata could significantly increase the survival rate of L02 cell injury caused by endoplasmic reticulum stress in a dose and time-dependent manner(P&lt;0.05 or P&lt;0.01). The expression levels of GRP78, PERK, eIF2α, ATF4, CHOP and Bax in the drug treatment groups were significantly down-regulated(P&lt;0.05 or P&lt;0.01), while Bcl-2 was significantly up-regulated(P&lt;0.01). After endoplasmic reticulum stress inhibitor and CHOP shRNA-mediated knockdowns were added, the expression levels of GRP78, PERK, eIF2α, ATF4, CHOP, Bax in the drug treatment groups were significantly down-regulated(P&lt;0.01), whereas Bcl-2 was significantly up-regulated(P&lt;0.01). Immunofluorescence results showed that the expressions of GRP78, PERK, CHOP were consistent with the Western blot method. In conclusion, ethyl acetate extract from B. bipinnata has a significant protective effect on the damage of L02 cells caused by endoplasmic reticulum stress. The mechanism may be related to the inhibition of endoplasmic reticulum stress and the down-regulation of apoptosis in cells through the PERK/eIF2α/ATF4/CHOP signaling pathway.

Gene Therapy Strategies to Restore ER Proteostasis in Disease.

Advanced oxidation protein products (AOPPs) accelerate the progression of chronic kidney disease. We previously demonstrated that AOPPs induce hypertrophy and epithelial-to-mesenchymal transition (EMT) in human proximal tubular cells (HK-2 cells) through induction of endoplasmic reticulum (ER) stress. However, which pathway of unfolded protein response (UPR) induced by ER stress plays crucial roles in this process remains unclear. In this study, we investigated the roles of the protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) pathways of UPR in this process in HK-2 cells. AOPP treatment induced the overexpression of cleaved ATF6 and spliced form of X-box binding protein-1, and induced the phosphorylation of PERK, eukaryotic translation initiation factor 2α and IRE1. Furthermore, silencing of ATF6 increased E-cadherin and zonula occludens-1 expression, lowered the expression of vimentin, and downregulated total protein content, whereas knockdown of PERK or IRE1 resulted in no difference compared with the scramble siRNA-transfected cells. AOPP-induced phosphorylation of Src, which was reproduced by thapsigargin, an inducer of ER stress, was partly reversed by salubrinal, an inhibitor of ER stress. Furthermore, the Src inhibitor saracatinib effectively blocked AOPP-induced phosphorylation of Src, activation of ER stress, hypertrophy, and EMT in HK-2 cells. Collectively, our results indicate that AOPPs induce the PERK, ATF6, and IRE1 pathways of UPR, and the ATF6 pathway rather than the other two pathways mediates AOPP-induced HK-2-cell hypertrophy and EMT. We also suggest that the ER stress involved in this process is likely mediated by the activation of Src kinase.

Thiamine responsive megaloblastic anemia (TRMA) is a genetic disease caused by SLC19A2 gene mutation. This study aimed to preliminarily explore the relationship between endoplasmic reticulum stress (ERS)-PERK signaling pathway and the pathogenesis of hyperglycemia induced by TRMA. Islet β (INS.1 and β-TC-6) and HEK293T cell line models with stable overexpression of SLC19A2 and SLC19A2 (c.1409insT) were established. The cells were divided into empty virus group (control), wild-type group (overexpressed SLC19A2), and mutation group (overexpressed SLC19A2 (c.1409insT)). Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and western blotting were used to detect the expression levels of ERS-PERK signaling pathway-related proteins, including glucose-regulated protein 78 (GRP78), protein kinase R-like ER kinase (PERK), and eukaryotic initiation factor 2 (eIF2α), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) in islet β cells. Protein localization was assessed by immunofluorescence staining. Compared with the control group, the mRNA expression levels of SLC19A2 in wild-type and mutant islet β cells (INS.1 and β-TC-6) and HEK293T cells were significantly upregulated (all p < 0.05). Compared with the control group and the wild-type group, the mRNA expression levels of GRP78, PERK, eIF2α, ATF4, and CHOP were increased (all p < 0.05) in the mutant islet β cells; the protein expression levels of PERK, GRP78, and eIF2α were elevated (all p < 0.05). In addition, the results of immunofluorescence staining showed that SLC19A2 (c.1409insT) mutation changed the localization of the proteins in the cells. Thus, they were not located on the cell surface, but in the cytoplasm and nuclei, and protein aggregation occurred in the cytoplasm. 1. Islet β and HEK293Tcell lines, stably overexpressing SLC19A2 and SLC19A2 (c.1409insT) mutations, were successfully constructed. 2. SLC19A2 (c.1409insT) mutation could raise the expression levels of ERS-PERK signaling pathway-related proteins (GRP78, PERK, eIF2α, ATF4, and CHOP), and activate apoptosis pathway. 3. SLC19A2 (c.1409insT) mutation could change the localization of proteins and produce protein aggregation in cells. It could lead to protein misfolding and ERS, which would participate in the pathological mechanism of hyperglycemia induced by TRMA.