GRP94 Research Articles

Avocado Seeds-Mediated Alleviation of Cyclosporine A-Induced Hepatotoxicity Involves the Inhibition of Oxidative Stress and Proapoptotic Endoplasmic Reticulum Stress.

Previous studies reported disrupted hepatic function and structure following the administration of cyclosporine A (CsA) in humans and animals. Recently, we found that avocado seeds (AvS) ameliorated CsA-induced nephrotoxicity in rats. As a continuation, herein we checked whether AvS could also attenuate CsA-induced hepatotoxicity in rats. Subcutaneous injection of CsA (5 mg/kg) for 7 days triggered hepatotoxicity in rats, as indicated by liver dysfunction, redox imbalance, and histopathological changes. Oral administration of 5% AvS powder for 4 weeks ameliorated CsA-induced hepatotoxicity, as evidenced by (1) decreased levels of liver damage parameters (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and bilirubin), (2) resumed redox balance in the liver (reduced malondialdehyde (MDA) and increased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)), (3) downregulated hepatic expression of endoplasmic reticulum (ER) stress-related genes (X-box binding protein 1 (XBP1), binding immunoglobulin protein (BIP), C/EBP homologous protein (CHOP)), and apoptosis-related genes (Bax and Casp3), (4) upregulated expression of the anti-apoptotic gene Bcl2, (5) reduced DNA damage, and (6) improved liver histology. These results highlight the ability of AvS to ameliorate CsA-induced hepatotoxicity via the inhibition of oxidative stress and proapoptotic ER stress.

Abstract 11616: Obstructive Sleep Apnea Disrupts Endothelial Cellular Cholesterol Trafficking

Introduction: Obstructive sleep apnea (OSA), a highly prevalent disorder, triples cardiovascular risk. Our previous studies showed that intermittent hypoxia (IH), the hallmark of OSA, triggers endothelial cell (EC) inflammation by reducing protection against complement activity, which is cholesterol-dependent. We assessed whether IH promotes accumulation of cellular cholesterol in ECs in OSA patients and investigated the underlying mechanisms. Methods: We used ECs collected from OSA patients and cultured human umbilical vein ECs (HUVECs) exposed to IH (alternating 30 min 21% O 2 for normoxia/30 min 2% O 2 for hypoxia for 8 h). Results: Levels of free, cellular cholesterol were increased and the presence of lipid droplets was reduced in ECs from OSA patients compared with OSA-free controls as well as in HUVECs exposed to IH compared with normoxia. Cholesterol uptake and synthesis were similar in normoxia and IH. In contrast, co-localization of free cholesterol and late endosome/lysosome (LE/LY) was increased in IH compared with normoxia, suggesting impaired trafficking of free cholesterol from LE/LY to endoplasmic reticulum (ER) in IH. Immunoglobulin binding protein (BiP), a marker of ER stress, was upregulated in ECs of OSA patients compared with controls. IH-induced ER stress reduced interaction between LE/LY-bound oxysterol binding protein-related protein-1L (ORP1L) and ER-bound VAMP-associated protein-B (VAPB), which is required for cholesterol trafficking from LE/LY to ER. After screening ER-associated degradation machinery components for potential binding to VAPB in IH, we identified Derlin1 as a ligand that binds increasingly to VAPB in IH thereby potentially reducing interaction between ORP1L and VAPB. In OSA patients, ORP1L-VAPB interaction was reduced whereas Derlin1-VAPB was increased and positive airway pressure, a standard therapy for OSA, reversed these changes. Conclusions: IH induces ER stress that disrupts cholesterol trafficking from LE/LY to ER leading to accumulation of free cholesterol in ECs, which underlies impaired endothelial protection against complement and endothelial inflammation that may increase cardiovascular risk in OSA.

Regular Kratom (Mitragyna speciosa Korth.) Use and Its Association With Endoplasmic Reticulum Stress Response

This study determined the association between expression of the endoplasmic reticulum (ER) stress sensor mRNA in the peripheral leukocytes and the patterns of kratom use and evaluated the correlations between the levels of the ER stress sensor mRNA and the severity of kratom dependence and kratom induced depressive symptoms among people who use kratom (PWUK). A total of 20 PWUK and 20 age matched non-kratom using healthy controls were recruited. Data collected from PWUK included patterns of kratom use, severity of kratom dependence, and severity of depressive symptoms during abstinence from kratom. The mRNA expression of binding immunoglobulin protein ( BiP ), X-box binding protein 1, activating transcription factor 4, and C/-EBP homologous protein ( CHOP ) (major indicators of ER stress response) were analyzed using quantitative reverse transcription polymerase chain reaction in leucocyte-derived total RNA sample of the participants. PWUK regardless of their pattern of kratom use recorded significantly higher expression of BiP mRNA compared with controls. Expression of CHOP mRNA was only significantly higher in those who first consumed kratom at the age of 18 years and above and those who have been using kratom for longer than 6 years, compared with controls. Higher expression of BiP , ATF4 , and CHOP mRNA were significantly positive correlated with greater severity of kratom dependence. Although only higher expression of BiP and CHOP mRNA were significantly positively correlated with greater severity of depressive symptoms. Regular kratom consumption may activate the ER stress pathway and there may be a link between altered ER stress response and kratom dependence and kratom induced depressive symptoms.

20S-ginsenoside Rg3 inhibits the biofilm formation and haemolytic activity of Staphylococcus aureus by inhibiting the SaeR/SaeS two-component system.

Introduction. Staphylococcus aureus is a major cause of chronic diseases and biofilm formation is a contributing factor. 20S-ginsenoside Rg3 (Rg3) is a natural product extracted from the traditional Chinese medicine red ginseng.Gap statement. The effects of Rg3 on biofilm formation and haemolytic activity as well as its antibacterial mechanism against S. aureus have not been reported.Aim. This study aimed to investigate the effects of Rg3 on biofilm formation and haemolytic activity as well as its antibacterial action against clinical S. aureus isolates.Methodology. The effect of Rg3 on biofilm formation of clinical S. aureus isolates was studied by crystal violet staining. Haemolytic activity analysis was carried out. Furthermore, the influence of Rg3 on the proteome profile of S. aureus was studied by quantitative proteomics to clarify the mechanism underlying its antibacterial action and further verified by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR).Results. Rg3 significantly inhibited biofilm formation and haemolytic activity in clinical S. aureus isolates. A total of 63 with >1.5-fold changes in expression were identified, including 34 upregulated proteins and 29 downregulated proteins. Based on bioinformatics analysis, the expression of several virulence factors and biofilm-related proteins, containing CopZ, CspA, SasG, SaeR/SaeS two-component system and SaeR/SaeS-regulated proteins, including leukocidin-like protein 2, immunoglobulin-binding protein G (Sbi) and fibrinogen-binding protein, in the S. aureus of the Rg3-treated group was downregulated. RT-qPCR confirmed that Rg3 inhibited the regulation of SaeR/SaeS and decreased the transcriptional levels of the biofilm-related genes CopZ, CspA and SasG.Conclusions. Rg3 reduces the formation of biofilm by reducing cell adhesion and aggregation. Further, Rg3 can inhibit the SaeR/SaeS two-component system, which acts as a crucial signal transduction system for the anti-virulence activity of Rg3 against clinical S. aureus isolates.

Prodigiosin from Serratia Marcescens in Cockroach Inhibits the Proliferation of Hepatocellular Carcinoma Cells through Endoplasmic Reticulum Stress-Induced Apoptosis.

Hepatocellular carcinoma (HCC) is the most common primary liver malignant tumor, and the targeted therapy for HCC is very limited. Our previous study demonstrated that prodigiosin(PG), a secondary metabolite from Serratia marcescens found in the intestinal flora of cockroaches, inhibits the proliferation of HCC and increases the expression of CHOP, a marker protein for endoplasmic reticulum stress (ERS)-mediated apoptosis, in a dose-dependent manner. However, the mechanisms underlying the activity of PG in vivo and in vitro are unclear. This study explored the molecular mechanisms of PG-induced ERS against liver cancer in vitro and in vivo. The apoptosis of hepatocellular carcinoma cells induced by PG through endoplasmic reticulum stress was observed by flow cytometry, colony formation assay, cell viability assay, immunoblot analysis, and TUNEL assay. The localization of PG in cells was observed using laser confocal fluorescence microscopy. Flow cytometry was used to detect the intracellular Ca2+ concentration after PG treatment. We found that PG could promote apoptosis and inhibit the proliferation of HCC. It was localized in the endoplasmic reticulum of HepG2 cells, where it induces the release of Ca2+. PG also upregulated the expression of key unfolded response proteins, including PERK, IRE1α, Bip, and CHOP, and related apoptotic proteins, including caspase3, caspase9, and Bax, but down-regulated the expression of anti-apoptotic protein Bcl-2 in liver cancer. Alleviating ERS reversed the above phenomenon. PG had no obvious negative effects on the functioning of the liver, kidney, and other main organs in nude mice, but the growth of liver cancer cells was inhibited by inducing ERS in vivo. The findings of this study showed that PG promotes apoptosis of HCC by inducing ERS.

The endoplasmic reticulum (ER) chaperone GRP78 is secreted during ER Stress and alleviates endothelial cell inflammation

Abstract Introduction Glucose-Regulated Protein 78kD (GRP78) is a chaperone and the main regulator of the ER-stress response. Upon ER Stress, GRP78 activates the unfolded protein response (UPR), which aims to clear unfolded proteins and restore ER homeostasis. A prolonged activation of the UPR triggers inflammation, thus contributing to the progression of cardiovascular diseases. Recently, extracellular secretion of GRP78 was described. However, the pathophysiological relevance of secreted GRP78 in atherosclerosis and endothelial cell inflammation remains to be elucidated. Aim Aim of this study is to investigate the role of GRP78 secretion in endothelial cells. Methods and results First, we sought to investigate if vascular cells secrete GRP78 during ER Stress. Human coronary artery endothelial cells (HCAEC) were treated with the ER stress inductor tunicamycin for up to 48h. After ER Stress induction, Western Blot and ELISA experiments detected an increased intracellular GRP78 expression. Intriguingly, prolonged ER Stress also promoted extracellular secretion of GRP78. Proteomic analysis confirmed that after ER-Stress induction, GRP78 is one of the most highly upregulated extracellular proteins (2.43-fold). Co-incubation with Brefeldin A, an inhibitor of ER-Golgi protein transport, abolished extracellular secretion (Fig.1). Hence, ER-Stress-induced GRP78 secretion is an actively regulated process. Next, the effect of GRP78 containing conditioned medium (CM) on HCAEC was analyzed. Treatment with GRP78 containing CM decreased GRP78 mRNA expression in target cells (0.35-fold vs. control [+BFA], p<0.0001). Furthermore, it increased viability (93.0% vs. 79.6%, p=0.017) and decreased formation of reactive oxygen species (0.78-fold). Moreover, expression of markers of vascular inflammation and ER Stress (e.g., NF-κB and CHOP) was decreased when compared to control CM with additional BFA treatment. However, ER Stress induced by tunicamycin exhibits deleterious effects on donor cells and is therefore not feasible for in vivo usage. Thus, we utilized Bip protein inducer x (Bix), a recently described small-molecule activator of GRP78. Treatment with Bix also promoted expression of GRP78 and general UPR activation (e.g., ATF4, XBP1). Moreover, in contrast to tunicamycin, Bix treatment did not impair viability of HCAEC. After treatment with Bix-induced CM, apoptosis (0.77-fold vs. 1.64-fold, p<0.0001) and expression of markers of vascular inflammation (e.g., Il-6) were significantly decreased compared to control CM. Furthermore, presence of GRP78 was able to promote proliferation and viability. Conclusion Endothelial ER Stress promotes GRP78 secretion. Presence of GRP78 in conditioned medium ameliorates subsequent ER Stress and endothelial inflammation, which play a critical role in atherogenesis. Modification of GRP78 secretion by Bix might be a feasible and innovative therapeutic option for vascular inflammation and endothelial regeneration. Funding Acknowledgement Type of funding sources: None.

Stem cells from human exfoliated deciduous teeth attenuate trigeminal neuralgia in rats by inhibiting endoplasmic reticulum stress.

BackgroundThe treatment of trigeminal neuralgia remains a challenging issue. Stem cells from human exfoliated deciduous teeth (SHED) provide optimized therapy for chronic pain. This study aimed to investigate the mechanisms underlying the attenuation of trigeminal neuralgia by SHED.MethodsTrigeminal neuralgia was induced by chronic constriction injury of the infraorbital nerve. The mechanical threshold was assessed after model establishment and local SHED transplantation. Endoplasmic reticulum (ER) morphology and Caspase12 expression in trigeminal ganglion (TG) was evaluated as well. BiP expression was observed in PC12 cells induced by tunicamycin.ResultsThe local transplantation of SHED could relieve trigeminal neuralgia in rats. Further, transmission electron microscopy revealed swelling of the ER in rats with trigeminal neuralgia. Moreover, SHED inhibited the tunicamycin-induced up-regulated expression of BiP mRNA and protein in vitro. Additionally, SHED decreased the up-regulated expression of Caspase12 mRNA and protein in the TG of rats caused by trigeminal neuralgia after chronic constriction injury of the infraorbital nerve mode.ConclusionsThis findings demonstrated that SHED could alleviate pain by relieving ER stress which provide potential basic evidence for clinical pain treatment.

Dysregulation of Long Noncoding RNA NEAT1/miR-199a-5/BiP Axis in Patients with Diabetic Neuropathy.

Diabetic neuropathy (DN) is a type of nerve damage and the most common complication of diabetes. Regarding the association between endoplasmic reticulum (ER) stress with the pathogenesis of neuropathy, this study aims to examine binding immunoglobulin protein (BiP) gene expression and long noncoding RNA nuclear enriched abundant transcript 1 (NEAT1), miR-199a-5 as its regulator in the peripheral blood of DN patients compared to diabetic patients without neuropathy. Peripheral blood samples were obtained from DN (n = 20) patients and diabetic patients without neuropathy (non-DN) (n = 20). After RNA extraction from peripheral blood mononuclear cells, reverse transcription-quantitative polymerase chain reaction was performed to evaluate RNA expression. The results showed that the expression level of NEAT1 and BiP genes in the DN group increased significantly compared to the non-DN group. Also, the expression level of miR-199a-5p in the DN group was significantly downregulated. As a result, the axis of NEAT1, miR-199a-5p, and BiP may have a role in the DN pathogenesis.

Impact of a Decade of Research Into Atopic Dermatitis

The last decade has seen an unprecedented pace of change, particularly of clinical research in atopic dermatitis (AD). This review summarizes some key discoveries. Over the last 10 years, nearly half of all studies investigated the efficacy and safety of novel therapeutic agents, particularly biologics and small molecules. Clear demonstration of benefit in clinical trials with no significant safety concerns provided strong evidence leading to subsequent Food and Drug Administration approval and routine use of the anti-IL-4 receptor alpha antagonist dupilumab in patients 6 months and older, the selective Janus kinase 1 (JAK1) inhibitors upadacitinib for patients 12 years and older and abrocitinib, the IL-13 antagonist tralokinumab, and the JAK1/2 inhibitor baricitinib for adults 18 years and older. Several other drugs are in the pipeline. Other areas under the spotlight have been trials of skin moisturizers and probiotics in the prevention of AD, investigating the role of filaggrin and skin barrier function and the role of skin and gut microbiome, with Staphylococcus aureus second immunoglobulin-binding protein having been found to uniquely trigger allergic skin responses in AD. Skin microbiome, epidermal metabolites/structural components, and local inflammatory biomarkers are now commonly assessed using genomic and proteomic analysis of tape strips rather than more invasive biopsy to identify factors such as C-C motif chemokine ligand-17 that correlate with disease severity and response to therapy. Overall, the last decade has ushered in a new and exciting era in our understanding, diagnosis, and treatment of this common allergic skin disease.

BiP inactivation due to loss of the deAMPylation function of FICD causes a motor neuron disease

The chaperone protein BiP is the master regulator of the unfolded protein response in the endoplasmic reticulum. BiP chaperone activity is regulated by the post-translational modification AMPylation, exclusively provided by FICD. We investigated whether FICD variants identified in patients with motor neuron disease could interfere with BiP activity regulation. Exome sequencing was performed to identify causative pathogenic variants associated with motor neuron diseases. Functional studies were conducted on fibroblasts from patients to explore the molecular mechanism of the disease. We identified biallelic variants in FICD causing a neurodegenerative disease of upper and lower motor neurons. Affected individuals harbor a specific missense variant, Arg374His, positioned in the catalytic motif of the enzyme and important for adenosine triphosphate binding. The mutated residue abolishes intramolecular interaction with the regulatory residue Glu234, essential to inhibit AMPylation and to promote de-AMPylation by FICD. Consequently, fibroblasts from patients with FICD variants have abnormally increased levels of AMPylated and thus inactivated BiP. Loss of BiP chaperone activity in patients likely results in a chronic impairment of the protein quality control system in the endoplasmic reticulum. These findings will guide the development of therapeutic strategies for motoneuron and related diseases linked to proteotoxic stress.

Exercise and Metformin Intervention Prevents Lipotoxicity-Induced Hepatocyte Apoptosis by Alleviating Oxidative and ER Stress and Activating the AMPK/Nrf2/HO-1 Signaling Pathway in db/db Mice.

Objective Nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2DM) commonly coexist and act synergistically to drive adverse clinical outcomes. This study is aimed at investigating the effects of exercise intervention and oral hypoglycaemic drug of metformin (MET) alone or combined on hepatic lipid accumulation. To investigate if oxidative stress and endoplasmic reticulum stress (ERS) are involved in lipotoxicity-induced hepatocyte apoptosis in diabetic mice and whether exercise and/or MET alleviated oxidative stress or ERS-apoptosis by AMPK-Nrf2-HO-1 signaling pathway. Methods Forty db/db mice with diabetes (random blood glucose ≥ 250 mg/dL) were randomly allocated into four groups: control (CON), exercise training alone (EX), metformin treatment alone (MET), and exercise combined with metformin (EM) groups. Hematoxylin-eosin and oil red O staining were carried out to observe hepatic lipid accumulation. Immunohistochemical and TUNEL methods were used to detect the protein expression of the binding immunoglobulin protein (BiP) and superoxide dismutase-1 (SOD1) and the apoptosis level of hepatocytes. ERS-related gene expression and the AMPK-Nrf2-HO-1 signaling pathway were tested by western blotting. Results Our data showed that db/db mice exhibited increased liver lipid accumulation, which induced oxidative and ER stress of the PERK-eIF2α-ATF4 pathway, and hepatocyte apoptosis. MET combined with exercise training significantly alleviated hepatic lipid accumulation by suppressing BiP expression, the central regulator of ER homeostasis, and its downstream PERK-eIF2α-ATF4 pathway, as well as upregulated the AMPK-Nrf2-HO-1 signaling pathway. Moreover, the combination of exercise and MET displayed protective effects on hepatocyte apoptosis by downregulating Bax expression and TUNEL-positive staining, restoring the balance of cleaved-caspase-3 and caspase-3, and improving the antioxidant defense system to prevent oxidative damage in db/db mice. Conclusion Compared to MET or exercise intervention alone, the combined exercise and metformin exhibited significant effect on ameliorating hepatic steatosis, inhibiting oxidative and ER stress-induced hepatocyte apoptosis via improving the capacity of the antioxidant defense system and suppression of the PERK-eIF2α-ATF4 pathway. Furthermore, upregulation of AMPK-Nrf2-HO-1 signaling pathway might be a key crosstalk between MET and exercise, which may have additive effects on alleviating hepatic lipid accumulation.

Oxidative and endoplasmic reticulum stress develop adverse metabolic effects due to the high-fat high-fructose diet consumption from birth to young adulthood

AimsThe early postnatal dietary intake has been considered a crucial factor affecting the offspring later life metabolic status. Consistently, this study investigated the oxidative and endoplasmic reticulum (ER) stress interventions in the induction of adverse metabolic effects due to the high-fat high-fructose diet (HFHFD) consumption from birth to young adulthood in rat offspring. Materials and methodsAfter delivery, the dams with their pups were randomly allocated into the normal diet (ND) and HFHFD groups. At weaning, the male offspring were divided into ND-None, ND-DMSO, ND-4-phenyl butyric acid (4-PBA), HFHFD-None, HFHFD-DMSO, and HFHFD-4-PBA groups and fed on their respected diets for five weeks. Then, the drug was injected for ten days. Subsequently, glucose and lipid metabolism parameters, oxidative and ER stress markers, and Wolfram syndrome1 (Wfs1) expression were assessed. Key findingsIn the HFHFD group, anthropometrical parameters, plasma high-density lipoprotein (HDL), and glucose-stimulated insulin secretion and content were decreased. Whereas, the levels of plasma leptin, low-density lipoprotein (LDL) and glucose, hypothalamic leptin, pancreatic catalase activity and glutathione (GSH), pancreatic and hypothalamic malondialdehyde (MDA), binding immunoglobulin protein (BIP) and C/EBP homologous protein (CHOP), and pancreatic WFS1 protein were increased. 4-PBA administration in the HFHFD group, decreased the hypothalamic and pancreatic MDA, BIP and CHOP levels, while, increased the Insulin mRNA and glucose-stimulated insulin secretion and content. SignificanceHFHFD intake from birth to young adulthood through the development of pancreatic and hypothalamic oxidative and ER stress, increased the pancreatic WFS1 protein and impaired glucose and lipid homeostasis in male rat offspring.

Bioinformatics study of expression from genomes of epidemiologically related MRSA CC398 isolates from human and wild animal samples

One of the most important livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) genetic lineages is the clonal complex (CC) 398, which can cause typical S. aureus-associated infections in people. In this work, whole-genome sequencing, RNA-sequencing, and gel-based comparative proteomics were applied to study the genetic characteristics of three MRSA CC398 isolates recovered from humans (strains C5621 and C9017), and from an animal (strain OR418). Of the three strains, C9017 presented the broadest resistance genotype, including resistance to fluroquinolone, clindamycin, tiamulin, macrolide and aminoglycoside antimicrobial classes. The scn, sak, and chp genes of the immune evasion cluster system were solely detected in OR418. Pangenome analysis showed a total of 288 strain-specific genes, most of which are hypothetical or phage-related proteins. OR418 had the most pronounced genetic differences. RNAIII (δ-hemolysin) gene was clearly the most expressed gene in OR418 and C5621, but it was not detected in C9017. Significant differences in the proteome profiles were found between strains. For example, the immunoglobulin-binding protein Sbi was more abundant in OR418. Considering that Sbi is a multifunctional immune evasion factor in S. aureus, the results point to OR418 strain having high zoonotic potential. Overall, multiomics biomarker signatures can assume an important role to advance precision medicine in the years to come. SignificanceMRSA is one of the most representative drug-resistant pathogens and its dissemination is increasing due to MRSA capability of establishing new reservoirs. LA-MRSA is considered an emerging problem worldwide and CC398 is one of the most important genetic lineages. In this study, three MRSA CC398 isolates recovered from humans and from a wild animal were analyzed through whole-genome sequencing, RNA-sequencing, and gel-based comparative proteomics in order to gather systems-wide omics data and better understand the genetic characteristics of this lineage to identify distinctive markers and genomic features of relevance to public health.

Inflammatory Cytokines Rewire the Proinsulin Interaction Network in Human Islets.

Aberrant biosynthesis and secretion of the insulin precursor proinsulin occurs in both type I and type II diabetes. Inflammatory cytokines are implicated in pancreatic islet stress and dysfunction in both forms of diabetes, but the mechanisms remain unclear. We sought to determine the effect of the diabetes-associated cytokines on proinsulin folding, trafficking, secretion, and β-cell function. Human islets were treated with interleukin-1β and interferon-γ for 48 hours, followed by analysis of interleukin-6, nitrite, proinsulin and insulin release, RNA sequencing, and unbiased profiling of the proinsulin interactome by affinity purification-mass spectrometry. Cytokine treatment induced secretion of interleukin-6, nitrites, and insulin, as well as aberrant release of proinsulin. RNA sequencing showed that cytokines upregulated genes involved in endoplasmic reticulum stress, and, consistent with this, affinity purification-mass spectrometry revealed cytokine induced proinsulin binding to multiple endoplasmic reticulum chaperones and oxidoreductases. Moreover, increased binding to the chaperone immunoglobulin binding protein was required to maintain proper proinsulin folding in the inflammatory environment. Cytokines also regulated novel interactions between proinsulin and type 1 and type 2 diabetes genome-wide association studies candidate proteins not previously known to interact with proinsulin (eg, Ataxin-2). Finally, cytokines induced proinsulin interactions with a cluster of microtubule motor proteins and chemical destabilization of microtubules with Nocodazole exacerbated cytokine induced proinsulin secretion. Together, the data shed new light on mechanisms by which diabetes-associated cytokines dysregulate β-cell function. For the first time, we show that even short-term exposure to an inflammatory environment reshapes proinsulin interactions with critical chaperones and regulators of the secretory pathway.

Ligustilide Improves Cognitive Impairment via Regulating the SIRT1/IRE1α/XBP1s/CHOP Pathway in Vascular Dementia Rats.

Vascular dementia (VaD), the second cause of dementia, is caused by chronic cerebral hypoperfusion, producing progressive damage to cerebral cortex, hippocampus, and white matter. Ligustilide (LIG), one of the main active ingredients of Angelica sinensis, exerts the neuroprotective effect on neurodegenerative diseases. However, the mechanism remains unclear. An in vivo model of bilateral common carotid artery occlusion and in vitro model of oxygen glucose deprivation (OGD) were employed in this study. LIG (20 or 40 mg/kg/day) was intragastrically administered to the VaD rats for four weeks. The results of the Morris water maze test demonstrated that LIG effectively ameliorated learning and memory deficiency in VaD rats. LIG obviously relieved neuronal oxidative stress damage by increasing the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) and decreasing the level of malondialdehyde (MDA) in VaD rats. Nissl staining showed that LIG increased the number of the Nissl body in VaD rats. After LIG administration, the apoptotic-related protein, Bax, was decreased and Bcl-2 was increased in the hippocampus of VaD rats. Moreover, the expressions of sirtuin 1 (SIRT1) and protein disulfide isomerase (PDI) were decreased, binding immunoglobulin protein (BIP) and phospho-inositol-requiring enzyme-1α (P-IRE1α), X-box binding protein 1 (XBP1s), and C/EBP-homologous protein (CHOP) were increased in VaD rats. After LIG treatment, these changes were reversed. The immunofluorescence results further showed that LIG upregulated the expression of SIRT1 and downregulated the expression of P-IRE1α in VaD rats. In addition, in vitro experiment showed that EX-527 (SIRT1 inhibitor) partly abolished the inhibitory effect of LIG on the IRE1α/XBP1s/CHOP pathway. In conclusion, these studies indicated that LIG could improve cognitive impairment by regulating the SIRT1/IRE1α/XBP1s/CHOP pathway in VaD rats.

Evidence From Human Placenta, Endoplasmic Reticulum-Stressed Trophoblasts, and Transgenic Mice Links Transthyretin Proteinopathy to Preeclampsia.

We have demonstrated that protein aggregation plays a pivotal role in the pathophysiology of preeclampsia and identified several aggregated proteins in the circulation of preeclampsia patients, the most prominent of which is the serum protein TTR (transthyretin). However, the mechanisms that underlie protein aggregation remain poorly addressed. We examined TTR aggregates in hypoxia/reoxygenation-exposed primary human trophoblasts (PHTs) and the preeclampsia placenta using complementary approaches, including a novel protein aggregate detection assay. Mechanistic analysis was performed in hypoxia/reoxygenation-exposed PHTs and Ttr transgenic mice overexpressing transgene-encoded wild-type human TTR or Ttr-/- mice. High-resolution ultrasound analysis was used to measure placental blood flow in pregnant mice. TTR aggregation was inducible in PHTs and the TCL-1 trophoblast cell line by endoplasmic reticulum stress inducers or autophagy-lysosomal disruptors. PHTs exposed to hypoxia/reoxygenation showed increased intracellular BiP (binding immunoglobulin protein), phosphorylated IRE1α (inositol-requiring enzyme-1α), PDI (protein disulfide isomerase), and Ero-1, all markers of the unfolded protein response, and the apoptosis mediator caspase-3. Blockade of IRE1α inhibited hypoxia/reoxygenation-induced upregulation of Ero-1 in PHTs. Excessive unfolded protein response activation was observed in the early-onset preeclampsia placenta. Importantly, pregnant human TTR mice displayed aggregated TTR in the junctional zone of the placenta and severe preeclampsia-like features. High-resolution ultrasound analysis revealed low blood flow in uterine and umbilical arteries in human TTR mice compared with control mice. However, Ttr-/- mice did not show any pregnancy-associated abnormalities. These observations in the preeclampsia placenta, cultured trophoblasts, and Ttr transgenic mice indicate that TTR aggregation is an important causal contributor to preeclampsia pathophysiology.

Fic-mediated AMPylation tempers the unfolded protein response during physiological stress

The proper balance of synthesis, folding, modification, and degradation of proteins, also known as protein homeostasis, is vital to cellular health and function. The unfolded protein response (UPR) is activated when the mechanisms maintaining protein homeostasis in the endoplasmic reticulum become overwhelmed. However, prolonged or strong UPR responses can result in elevated inflammation and cellular damage. Previously, we discovered that the enzyme filamentation induced by cyclic-AMP (Fic)can modulate the UPR response via posttranslational modification of binding immunoglobulin protein (BiP) by AMPylation during homeostasis and deAMPylation during stress. Loss of fic in Drosophila leads to vision defects and altered UPR activation in the fly eye. To investigate the importance of Fic-mediated AMPylation in a mammalian system, we generated a conditional null allele of Fic in mice and characterized the effect of Fic loss on the exocrine pancreas. Compared to controls, Fic-/- mice exhibit elevated serum markers for pancreatic dysfunction and display enhanced UPR signaling in the exocrine pancreas in response to physiological and pharmacological stress. In addition, both fic-/- flies and Fic-/- mice show reduced capacity to recover from damage by stress that triggers the UPR. These findings show that Fic-mediated AMPylation acts as a molecular rheostat that is required to temper the UPR response in the mammalian pancreas during physiological stress. Based on these findings, we propose that repeated physiological stress in differentiated tissues requires this rheostat for tissue resilience and continued function over the lifetime of an animal.

Ipatasertib, an oral AKT inhibitor, in combination with paclitaxel exhibits anti-proliferative and anti-tumorigenic effects in preclinical studies for endometrioid endometrial cancer: Endometrial cancer molecularly targeted therapy consortium (227)

<b>Objectives:</b> Ipatasertib (IPAT) is an orally administered selective protein kinase B (AKT) inhibitor that has demonstrated clinical activity in triple-negative breast and metastatic prostate cancer. The PI3K/ AKT pathway is altered in the vast majority of endometrioid endometrial cancers (ECs). A recent clinical trial demonstrated that IPAT combined with paclitaxel showed a significant prolonged overall survival in breast cancer patients. Thus, we aimed to evaluate the antitumorigenic effects of IPAT in combination with paclitaxel in EC cell lines and an EC mouse model. <b>Methods:</b> The human endometrioid EC cell lines Ishikawa <i>(PTEN</i> mutant), and HEC-1A (<i>PTEN</i> wild type, <i>PIK3CA</i> mutant, <i>KRAS</i> mutant) were exposed to varying concentrations and durations of IPAT (Genentech) and paclitaxel. Cell proliferation was assessed by MTT assay. To assess the synergy of IPAT and paclitaxel, the combination index (CI) was calculated by the method of Chou and Talalay. Apoptosis was assessed by cleaved caspase-3 assay. Western immunoblotting determined the effects of IPAT and paclitaxel on apoptosis (MCL, BCL-xl), cellular stress (ATF, PERK, Bip, PDI, IER), and DNA damage (RAD50). LKB1^fl/^flp53^fl/fl mice were used to evaluate the effect of IPAT, paclitaxel, and the combination of these two drugs on EC growth. The mice were treated with placebo, IPAT (50mg/kg daily orally for four weeks), paclitaxel (10 mg/kg IP weekly), or IPAT/paclitaxel starting eight weeks after tumor induction via AdCre injection. <b>Results:</b> IPAT and paclitaxel inhibited cellular proliferation in a dose-dependent fashion in both cell lines after 72 hours of treatment. The median IC50 for IPAT was 0.13µM for Ishikawa and 4.9µM for HEC-1A, respectively. The median IC50 for paclitaxel was 5.6nM for Ishikawa and 1.9nM for HEC-1A, respectively. Simultaneous exposure of cells to various doses of paclitaxel in combination with IPAT resulted in a significant synergistic antiproliferative effect (CI<1). IPAT and paclitaxel induced the activity of cleaved caspase-3 by 3.5-fold in Ishikawa and 1.12-fold in HEC-1A, while simultaneously increasing expression of the apoptotic proteins MCL-1 and BCL-xl. IPAT and paclitaxel also increased Bip, PERK, ATF4, PDI, and IER protein expression. Treatment of cells with IPAT and paclitaxel decreased phosphorylated-S6 (p-S6) expression and induced the expression of p-AKT and RAD50. Compared to control mice, treatment of LKB1^fl/flp53^fl/fl mice with IPAT reduced tumor weight by 62.1%, and treatment with paclitaxel decreased tumor weight by 73.9% (p<0.05). The combination of IPAT and paclitaxel reduced tumor growth by 78.3% and showed improved efficacy over IPAT alone (p<0.05), but not over Taxol alone. <b>Conclusions:</b> The combination of IPAT and paclitaxel demonstrates synergy in human endometrioid EC cell lines and improved efficacy over IPAT alone in the LKB^1fl/fl/p53^fl/fl mouse model of EC. These results suggest that IPAT with paclitaxel is worthy of further exploration in clinical trials for EC.

Ipatasertib, an oral AKT inhibitor, in combination with carboplatin exhibits anti-proliferative effects in serous endometrial cancer: Endometrial Cancer Molecularly Targeted Therapy Consortium (238)

<b>Objectives:</b> Ipatasertib (IPAT) is an investigational, orally-administered, ATP-competitive, highly selective inhibitor of protein kinase B (AKT) that has demonstrated antitumor activity in metastatic prostate cancer and longer progression-free survival in triple-negative breast cancer. Hyperactivation of the PI3K/AKT signaling pathway is frequently seen in endometrial cancer (EC), including serous histology tumors. Thus, we evaluated the antiproliferative efficacy of IPAT in combination with standard-of-care carboplatin in human serous EC cell lines. <b>Methods:</b> The human serous EC cell lines, ARK-1 and SPEC-2, were exposed to varying concentrations of IPAT (Genetech) and carboplatin. Cell proliferation was assessed by MTT assay. To assess the synergy of IPAT and carboplatin, the combination index (CI) was calculated by the method of Chou and Talalay. Apoptosis was assessed by cleaved caspase-3 assay. Cellular stress was measured by DCFDA assay. Western immunoblotting determined the effects of IPAT and carboplatin on apoptotic (MCL-1 and BCL-2) and cellular stress (Bip and PERK) pathways in the ARK-1 and SPEC-2 cell lines. <b>Results:</b> IPAT and carboplatin significantly inhibited cell proliferation in a dose-dependent manner after 72 hours of treatment. The median IC₅₀ for IPAT was 8µM in the ARK-1 and 5µM in SPEC-2. The median IC₅₀ for carboplatin was 50µM in the ARK-1 and 65µM in SPEC-2. Simultaneous exposure of cells to various doses of carboplatin in combination with IPAT resulted in a significant synergistic antiproliferative effect (CI<1). IPAT and carboplatin induced the activity of cleaved-caspase-3 by 2.3-fold in ARK-1 and 3.8-fold in SPEC-2 (p<0.05) while simultaneously reducing expression of the apoptotic proteins MCL-1 and BCL-2. DCFDA assay analysis demonstrated that IPAT and carboplatin induced ROS products by 1.39-fold and 1.17-fold in the ARK-1 and SPEC-2 cell lines (p<0.05), respectively, and increased Bip and PERK protein expression. The effects of IPAT combined with carboplatin on apoptosis and cellular stress were greater when compared to IPAT or carboplatin alone in both cell lines (p<0.05). <b>Conclusions:</b> The combination of IPAT and carboplatin showed synergistic antiproliferative activity in human serous EC cell lines. Thus, IPAT plus carboplatin may be a promising combination in the treatment of serous EC. Studies in transgenic and patient-derived xenograft EC mouse models are underway to assess the in vivo efficacy of IPAT +/- carboplatin.

CdTe quantum dots trigger oxidative stress and endoplasmic reticulum stress-induced apoptosis and autophagy in rat Schwann cell line RSC96.

In the current study, the cytotoxicity and mechanisms of cadmium telluride quantum dots (CdTe QDs) on RSC96 cells were evaluated by exposing different doses of CdTe QDs for 24 h. Two types of cell death, including apoptosis and autophagy, as well as two important organelles, mitochondria and endoplasmic reticulum, were focused after CdTe QDs exposure. The results showed that CdTe QDs induced apoptosis in RSC96 cells in a concentration-dependent manner; promoted the accumulation of intracellular reactive oxygen species; decreased the mitochondrial membrane potential; caused the release of cytochrome c; and also increased the expression of Bcl-2 associated X protein, caspase-3, and cytochrome c proteins and decreased the expression of Bcl-2 protein. Further results also confirmed that CdTe QDs could be internalized by RSC96 cells, and the exposure and internalization of CdTe QDs could induce excessive endoplasmic reticulum stress in the cells, and the expression levels of binding immunoglobulin protein, C/EBP homologous protein, and caspase12 proteins were increased in a concentration-dependent manner. Moreover, autophagy-related proteins LC3II, Beclin1, and P62 all increased after CdTe QDs exposure, suggesting that CdTe QDs exposure both promoted autophagosome formation and inhibited autophagosome degradation, and that CdTe QDs affected the autophagic flow in RSC96 cells. In conclusion, CdTe QDs are able to cause apoptosis and autophagy in RSC96 cells through mitochondrial and endoplasmic reticulum stress pathways, and the possible neurotoxicity of CdTe QDs should be further investigated.