Salicylic acid- and ethylene-dependent effects of the ER stress-inducer tunicamycin on the photosynthetic light reactions in tomato plants.

Plant hormones such as ethylene (ET) and salicylic acid (SA) have an elementary role in the regulation of ER stress and unfolded protein response (UPR) in plants via modulating defence responses or inducing oxidative stress. Chloroplasts can be sources and targets of reactive oxygen species (ROS) that affect photosynthetic efficiency, which has not been investigated under tunicamycin (Tm)-induced ER stress. In this study, the direct and indirect effects of Tm on chloroplastic ROS production were first investigated in leaves of wild-type tomato (Solanum lycopersicum L.) plants. Secondly changes in activities of photosystem II and I were analysed under Tm exposure and after application of the chemical chaperone 4-phenylbutyrate (PBA) in different genotypes, focusing on the regulatory role of SA and ET Tm treatments significantly but indirectly induced ROS production in tomato leaves and in parallel it decreased the effective quantum yield of PSII [Y(II)] and PSI [Y(I)], as well as the photochemical quenching coefficient (qP) and the quantum yield of non-photochemical energy dissipation in PSI due to acceptor-side limitation [Y(NA)]. At the same time, Tm increased non-photochemical quenching (NPQ) and cyclic electron flow (CEF) in tomato leaves after 24 h. However, the photosynthetic activity of the SA hydroxylase-overexpressing NahG tomato plants was more severely affected by Tm as compared to wild-type and ET-insensitive Never ripe (Nr) plants. These results suggest the protective role of SA in the regulation of photosynthetic activity contributing to UPR and the survival of plants under ER stress. Interestingly, the activation of photoprotective mechanisms by NPQ was independent of SA but dependent on active ET signalling under ER stress, whereas CEF was reduced by ET due to its higher ratio in Nr plants.

The Effects of N-Glycosylation on the Expression and Transport Activity of OATP1A2 and OATP2B1.

Organic anion transporting polypeptide (OATP)1A2 and OATP2B1 have potential N-glycosylation sites, but their influence remains unclear. This study aimed to identify the N-glycosylation sites of OATP1A2/2B1 and investigate their impact on the expression and function of OATP1A2/2B1. Human embryonic kidney cells expressing OATP1A2 or OATP2B1 (HEK293-OATP1A2/2B1) were exposed to tunicamycin, an N-glycosylation inhibitor, and a plasma membrane fraction (PMF) Western blot assay and an estrone 3-sulfate (E3S) uptake study were conducted. HEK293-OATP1A2/OATP2B1 cell lines with mutation(s) at potential N-glycosylation sites were established, and the Western blotting and uptake study were repeated. Tunicamycin reduced the PMF levels and E3S uptake of OATP1A2/OATP2B1. The Asn124Gln, Asn135Gln, and Asn492Gln mutations in OATP1A2 and Asn176Gln and Asn538Gln mutations in OATP2B1 reduced the molecular weights of the OATP molecules and their PMF levels. The PMF levels of OATP1A2 Asn124/135Gln, OATP1A2 Asn124/135/492Gln, and OATP2B1 Asn176/538Gln were further reduced. The maximum transport velocities of OATP1A2 Asn124Gln, OATP1A2 Asn135Gln, and OATP2B1 Asn176/538Gln were markedly reduced to 10 %, 4 %, and 10 % of the wild-type level, respectively. In conclusion, the N-glycans at Asn124 and Asn135 of OATP1A2 and those at Asn176 and Asn538 of OATP2B1 are essential for the plasma membrane expression of these molecules and also affect their transport function.

An EMC-Hpo-Yki axis maintains intestinal homeostasis under physiological and pathological conditions.

Balanced control of stem cell proliferation and differentiation underlines tissue homeostasis. Disruption of tissue homeostasis often results in many diseases. However, how endogenous factors influence the proliferation and differentiation of intestinal stem cells (ISCs) under physiological and pathological conditions remains poorly understood. Here, we find that the evolutionarily conserved endoplasmic reticulum membrane protein complex (EMC) negatively regulates ISC proliferation and intestinal homeostasis. Compromising EMC function in progenitors leads to excessive ISC proliferation and intestinal homeostasis disruption. Mechanistically, the EMC associates with and stabilizes Hippo (Hpo) protein, the key component of the Hpo signaling pathway. In the absence of EMC, Yorkie (Yki) is activated to promote ISC proliferation due to Hpo destruction. The EMC-Hpo-Yki axis also functions in enterocytes to maintain intestinal homeostasis. Importantly, the levels of the EMC are dramatically diminished in tunicamycin-treated animals, leading to Hpo destruction, thereby resulting in intestinal homeostasis disruption due to Yki activation. Thus, our study uncovers the molecular mechanism underlying the action of the EMC in intestinal homeostasis maintenance under physiological and pathological conditions and provides new insight into the pathogenesis of tunicamycin-induced tumorigenesis.

Combination Organelle Mitochondrial Endoplasmic Reticulum Therapy (COMET) for Multidrug Resistant Breast Cancer.

It is time for the story of mitochondria and intracellular communication in multidrug resistant cancer to be rewritten. Herein we characterize the extent and cellular advantages of mitochondrial network fusion in multidrug resistant (MDR) breast cancer and have designed a novel nanomedicine that disrupts mitochondrial network fusion and systematically manipulates organelle fusion and function. Combination Organelle Mitochondrial Endoplasmic reticulum Therapy (COMET) is an innovative translational nanomedicine for treating MDR triple negative breast cancer (TNBC) that has superior safety and equivalent efficacy to the current standard of care (paclitaxel). Our study has demonstrated that the increased mitochondrial networks in MDR TNBC contribute to apoptotic resistance and network fusion is mediated by mitofusin2 (MFN2) on the outer mitochondrial membrane. COMET consists of three components; Mitochondrial Network Disrupting (MiND) nanoparticles (NPs) that are loaded with an anti-MFN2 peptide, tunicamycin, and Bam7. The therapeutic rationale of COMET is to reduce the apoptotic threshold in MDR cells with MiND NPs, followed by inducing the endoplasmic reticulum mediated unfolded protein response (UPR) by stressing MDR cells with tunicamycin, and finally, directly inducing mitochondrial apoptosis with Bam7 which is a specific bcl-2 Bax activator. MiND NPs are PEGylated liposomes with the 21 amino acid (2577.98 MW) anti-MFN2 peptide compartmentalized in the aqueous core. Hypoxia (0.5% oxygen) was used to create MDR derivatives of MDA-MB-231 cells and BT-549 cells. Mitochondrial networks were quantified using 3D analysis of 60× live cell images acquired with a Keyence BZ-X710 microscope and MiND NPs effectively fragmented mitochondrial networks in drug sensitive and MDR TNBC cells. The IC50 values, combination index, and dose reduction index derived from dose response studies demonstrate that MiND NPs decrease the apoptotic threshold of both drug sensitive and MDR TNBC cells and COMET is a synergistic drug combination. Complex V (ATP synthase) extracted from bovine cardiac mitochondria was used to assess the effect of MiND NPs on OXPHOS; both MiND NPs and anti-MFN2 peptide solution significantly decrease the activity of mitochondrial complex V and decrease the capacity of OXPHOS. A BacMam viral vector based fluorescent biosensor was used to quantify the unfolded protein response (UPR) at the level of the endoplasmic reticulum and tunicamycin specifically induces the UPR in drug sensitive and MDR TNBC cells. A caspase 3 colorimetric assay demonstrated that the synergistic triple drug combination of COMET increases the ability of Bam7 to specifically induce apoptosis. Dose limiting toxicity and off target effects are a significant challenge for current chemotherapy regimens including paclitaxel. COMET has significantly lower cytotoxicity than paclitaxel in human embryonic kidney epithelial cells and has the potential to fulfill the clinical need for safer cancer therapeutics. COMET is a promising early stage translational nanomedicine for treating MDR TNBC. Manipulating intracellular communication and organelle fusion is a novel approach to treating MDR cancer. The data from this study has rewritten the story of mitochondria, organelle fusion, and intracellular communication and by targeting this intersection, COMET is an exciting new chapter in cancer therapeutics that could transform the clinical outcome of MDR TNBC.

Krüppel-like factor 15 counteracts endoplasmic reticulum stress and suppresses lung fibroblast proliferation and extracellular matrix accumulation.

The incidence of pulmonary fibrosis is on the rise, and existing treatments have limited efficacy in improving patient survival. The purpose of this study was to reveal the potential of Krüppel-like factor (KLF)15 activation in alleviating pulmonary fibrosis. Transforming growth factor beta (TGF-ß) was utilized to induce lung fibroblasts to establish an in vitro model of pulmonary fibrosis. The impacts of TGF-ß and KLF15 level on cell proliferation, migration, extracellular matrix (ECM) accumulation, and endoplasmic reticulum stress (ERS) were assessed. Additionally, tunicamycin, an ERS agonist, was used to investigate the role of ERS in KLF15 regulation. The results showed that KLF15 was dropped in response to TGF-ß treatment. However, KLF15 overexpression reduced cell proliferation, migration, ECM accumulation, and ERS, alleviating the effects of TGF-ß stimulation. Subsequent treatment with tunicamycin diminished the effects of KLF15 overexpression, demonstrating that ERS mediated the modulation of KLF15. KLF15 acts against ERS and suppresses excessive proliferation and ECM accumulation in lung fibroblast. These findings suggest that activating KLF15 is a promising strategy for alleviating pulmonary fibrosis.

Involvement of IGF1 in endoplasmic reticulum stress contributes to cataract formation through regulating Nrf2/NF-κB signaling.

Endoplasmic reticulum (ER) stress is reportedly involved in the development of ophthalmic diseases. This study aimed to investigate the role and potential mechanism of insulin-like growth factor 1 (IGF1) in ER stress. A mouse cataract model was constructed by subcutaneous injection of sodium selenite, and sh-IGF1 was used to evaluate the effect of silencing IGF1 on cataract progression. Slit-lamp and histological examination of the lens were performed to examine lens damage. The regulatory effects of IGF1 on inflammatory responses, oxidative stress, and ER stress were evaluated using ELISA, reverse transcription-quantitative PCR (RT-qPCR), and immunoblotting analysis. Tunicamycin was used to induce ER stress in the lens of epithelial cells. The NF-E2 related factor-2 (Nrf2) inhibitor ML385 and nuclear factor-κB (NF-κB) agonist diprovocim were used to confirm whether IGF1 regulates inflammation and ER stress through Nrf2/NF-κB signaling. Silencing IGF1 alleviated lens damage and reduced lens turbidity in the cataract mice. Silencing IGF1 inhibited inflammatory response, oxidative stress and ER stress response. Meanwhile, IGF1 was highly expressed in sodium selenite-treated lens epithelial cells. The ER stress agonist tunicamycin suppressed cell viability as well as induced ER stress, oxidative stress and inflammation. Silencing IGF1 increased cell viability, EdU-positive rate and migration. Also, silencing of IGF1 reduced inflammation and ER stress via regulating Nrf2/NF-κB pathway. This study reveals silencing IGF1 attenuated cataract through regulating Nrf2/NF-κB signaling, which shares novel insights into the underlying mechanism of cataract and provides potential therapeutic target for cataract.

Knockdown of Rab7B, But Not of Rab7A, Which Antagonistically Regulates Oligodendroglial Cell Morphological Differentiation, Recovers Tunicamycin-Induced Defective Differentiation in FBD-102b Cells.

In the central nervous system (CNS), insulative myelin sheaths are generated from the differentiated plasma membranes of oligodendrocytes (oligodendroglial cells) and surround neuronal axons to achieve saltatory conduction. Despite the functional involvement of myelin sheaths in the CNS, the molecular mechanism by which oligodendroglial cells themselves undergo differentiation of plasma membranes remains unclear. It also remains to be explored whether their signaling mechanisms can be applied to treating diseases of the oligodendroglial cells. Here, we describe that Rab7B of Rab7 subfamily small GTPases negatively regulates oligodendroglial cell morphological differentiation using FBD-102b cells, which are model cells undergoing differentiation of oligodendroglial precursors. Knockdown of Rab7B or Rab7A by the respective specific siRNAs in cells positively or negatively regulated morphological differentiation, respectively. Consistently, these changes were supported by changes on differentiation- and myelination-related structural protein and protein kinase markers. We also found that knockdown of Rab7B has the ability to recover inhibition of morphological differentiation following tunicamycin-induced endoplasmic reticulum (ER) stress, which mimics one of the major molecular pathological causes of hereditary hypomyelinating disorders in oligodendroglial cells, such as Pelizaeus-Merzbacher disease (PMD). These results suggest that the respective molecules among very close Rab7 homologues exhibit differential roles in morphological differentiation and that knocking down Rab7B can recover defective differentiating phenotypes under ER stress, thereby adding Rab7B to the list of molecular therapeutic cues taking advantage of signaling mechanisms for oligodendroglial diseases like PMD.

Excessive ER-phagy contributes to ochratoxin A-induced apoptosis.

The nephrotoxic secondary fungal metabolite ochratoxin A (OTA) is ubiquitously existed in foodstuffs and feeds. Although our earlier research provided preliminary evidence that endoplasmic reticulum (ER) was crucial in OTA-induced nephrotoxicity, more research is necessary to understand the fine-tune mechanisms involving ER stress (ERS), ER-phagy, and apoptosis. In the present study, the cell viability and protein expressions of human proximal tubule epithelial (HK-2) cells in response to OTA and/or chloroquine/rapamycin/sodium phenylbutyrate/tunicamycin were determined via cell viability assay, apoptosis analysis, and Western blot analysis. The findings showed that a 24 h-treatment of 0.25-4 µM OTA could significantly reduced the cell viability (P < 0.05), which notably increased with the addition of chloroquine and sodium phenylbutyrate, while decreased with the addition of rapamycin and tunicamycin as compared to group OTA (P < 0.05). A 24 h-treatment of 1-4 µM OTA could markedly induce apoptosis via increasing the protein expressions of GRP78, p-eIF2α, Chop, LC3B-II, Bak, and Bax, and inhibiting the protein expressions of DDRGK1, UBA5, Lonp1, Tex264, FAM134B, p-mTOR, p62, and Bcl-2 in HK-2 cells (P < 0.05). In conclusion, OTA activated ERS, unfolded protein response, and subsequent excessive ER-phagy, thus inducing apoptosis, and the vicious cycle between excessive ER-phagy and ERS could further promote apoptosis in vitro.

ATF4-mediated CD36 upregulation contributes to palmitate-induced lipotoxicity in hepatocytes.

Hepatic lipotoxicity plays a central role in the pathogenesis of nonalcoholic fatty liver disease; however, the underlying mechanisms remain elusive. Here, using both cultured hepatocytes (AML-12 cells and primary mouse hepatocytes) and the liver-specific gene knockout mice, we investigated the mechanisms underlying palmitate-elicited upregulation of CD36, a class B scavenger receptor mediating long-chain fatty acids uptake, and its role in palmitate-induced hepatolipotoxicity. We found that palmitate upregulates hepatic CD36 expression. Despite being a well-established target gene of PPARγ transactivation, our data demonstrated that the palmitate-induced CD36 upregulation in hepatocytes is in fact PPARγ-independent. We previously reported that the activation of ATF4, one of three canonical pathways activated upon endoplasmic reticulum (ER) stress induction, contributes to palmitate-triggered lipotoxicity in hepatocytes. In this study, our data revealed for the first time that ATF4 plays a critical role in mediating hepatic CD36 expression. Genetic inhibition of ATF4 attenuated CD36 upregulation induced by either palmitate or ER stress inducer tunicamycin in hepatocytes. In mice, tunicamycin upregulates liver CD36 expression, whereas hepatocyte-specific ATF4 knockout mice manifest lower hepatic CD36 expression when compared with control animals. Furthermore, we demonstrated that CD36 upregulation upon palmitate exposure represents a feedforward mechanism in that siRNA knockdown of CD36 in hepatocytes blunted ATF4 activation induced by both palmitate and tunicamycin. Finally, we confirmed that the ATF4-CD36 pathway activation contributes to palmitate-induced hepatolipotoxicity as genetic inhibition of either ATF4 or CD36 alleviated cell death and intracellular triacylglycerol accumulation. Collectively, our data demonstrate that CD36 upregulation by ATF4 activation contributes to palmitate-induced hepatic lipotoxicity.NEW & NOTEWORTHY We provided the initial evidence that ATF4 is a principal transcription factor mediating hepatic CD36 expression in that both palmitate- and ER stress-elicited CD36 upregulation was blunted by ATF4 gene knockdown in hepatocytes, and hepatocyte-specific ATF4 knockout mice manifested lower hepatic CD36 expression. We further confirmed that the ATF4-CD36 pathway activation contributes to palmitate-induced hepatolipotoxicity as genetic inhibition of either ATF4 or CD36 alleviated cell death and intracellular triacylglycerol accumulation in response to exogenous palmitate exposure.

Tunicamycin induces depression-like behaviors in male rats, accompanied by initiated chaperon-mediated autophagy and decreased synaptic protein expression in the hippocampus.

BACKGROUND AND AIM: Endoplasmic reticulum (ER) stress participates in the occurrence and development of depression, but the underlying mechanism is not fully understood. This study aimed to investigate the behavioral performance and intracerebral molecular changes in an ER stress model of male rats. METHODS: Intrahippocampal injection of tunicamycin (TM) was performed on male rats as a model of ER stress. The body weight was determined, and behavioral tests, including sucrose preference test (SPT), open field test (OFT), and forced swimming test (FST), were performed to evaluate depressive and anxiety-like phenotypes within 8 days after injection. The levels of chaperone-mediated autophagy (CMA), synaptic proteins, and neuroinflammation related factors in this model were measured via real-time quantitative PCR and Western blot analysis. RESULTS: Intrahippocampal injection of TM (2 or 1 µg) induced depression-like behaviors in rats, as indicated by the reduced body weight, sucrose preference in SPT, central time in OFT, and increased immobility time in FST. The mRNA and protein levels of GRP78, ATF4, CHOP, LAMP2A, IL-1ß, IL-6, and TNF-α were significantly increased, while the expressions of MEF2D, PSD95, SYN, p-CREB (Ser133), and BDNF were significantly decreased in the hippocampus in the model group compared with the sham group. CONCLUSIONS: These results confirmed that intrahippocampal injection of TM was a valid method to induce an ER stress rat model with depression-like behaviors accompanied by decreased synaptic protein expression and neuroinflammation. The alteration in CMA-related proteins in this ER stress depression model indicated the involvement of CMA in the development of depression.

Deletion induced splicing in RIC3 drives nicotinic acetylcholine receptor regulation with implications for endoplasmic reticulum stress in human astrocytes.

Nicotinic acetylcholine receptor (nAChR) dysregulation in astrocytes is reported in neurodegenerative disorders. Modulation of nAChRs through agonists confers protection to astrocytes from stress but regulation of chaperones involved in proteostasis with pathological implications is unclear. Resistance to inhibitors of cholinesterase 3 (RIC3), a potential chaperone of nAChRs is poorly studied in humans. We characterized RIC3 in astrocytes derived from an isogenic wild-type and Cas9 edited "del" human iPSC line harboring a 25 bp homozygous deletion in exon2. Altered RIC3 transcript ratio due to deletion induced splicing and an unexpected gain of α7nAChR expression were observed in "del" astrocytes. Transcriptome analysis showed higher expression of neurotransmitter/G-protein coupled receptors mediated by cAMP and calcium/calmodulin-dependent kinase signaling with increased cytokines/glutamate secretion. Functional implications examined using tunicamycin induced ER stress in wild-type astrocyte stress model showed cell cycle arrest, RIC3 upregulation, reduction in α7nAChR membrane levels but increased α4nAChR membrane expression. Conversely, tunicamycin-treated "del" astrocytes showed a comparatively higher α4nAChR membrane expression and upsurged cAMP signaling. Furthermore, reduced expression of stress markers CHOP, phospho-PERK and lowered XBP1 splicing in western blot and qPCR, validated by proteome-based pathway analysis indicated lowered disease severity. Findings indicate (i) a complex RNA regulatory mechanism via exonic deletion induced splicing; (ii) RIC-3 as a disordered protein having contrasting effects on co-expressed nAChR subtypes under basal/stress conditions; and (iii) RIC3 as a potential drug target against ER stress in astrocytes for neurodegenerative/nicotine-related brain disorders. Cellular rescue mechanism through deletion induced exon skipping may encourage ASO-based therapies for tauopathies.

Unfolded protein response is involved in the metabolic and apoptotic regulation of oral squamous cell carcinoma.

Endoplasmic reticulum (ER) stress and unfolded protein response (UPR) have been shown to be crucial in the pathogenesis and response to treatment in various cancers. However, such response has not been profiled in oral squamous cell carcinoma (OSCC), the most frequent form of cancer in the head and neck region. Cell lines derived from OSCC (SCC4, SCC15 and SCC25) and normal oral mucosa (OKF4, OKF6 and OKP7) were subjected to tunicamycin-induced ER stress (2.5 µg/mL for 24 h) after which the differential regulation of 84 key UPR/ER stress genes were assessed using Quantitative real-time reverse transcription polymerase chain reaction. The expression of the transcription factors SREBP1 and CREB3L3, and the activation of SREBP1, were examined using ELISA and a transcription factor assay. The expression of DDIT3 was immunohistochemically verified in OSCC tissue samples. SREBP1 and CREB3L3 were significantly up-regulated in OSCC with and without tunicamycin-induced ER stress. A significantly higher level of SREBP1 transcriptional activation was observed in OSCC. Apoptosis-associated genes (DDIT3, HTRA4 and HSPA1L) were also significantly up-regulated in OSCC upon ER stress induction. The findings demonstrated the involvement of UPR and ER stress in the pathogenesis of OSCC through the identification of apoptosis-associated genes (DDIT3, HSPA1L and HTRA4) and regulators of metabolism (SREBP1 and CREB3L3) as the key factors differentiating between normal and malignant oral keratinocytes.

[IRE1α deficiency impairs autophagy in chondrocytes by upregulating calcium homeostasis endoplasmic reticulum protein].

OBJECTIVE: To explore the mechanism by which inositol-requiring enzyme-1α (IRE1α) regulates autophagy function of chondrocytes through calcium homeostasis endoplasmic reticulum protein (CHERP). METHODS: Cultured human chondrocytes (C28/I2 cells) were treated with tunicamycin, 4µ8c, rapamycin, or both 4µ8c and rapamycin, and the expressions of endoplasmic reticulum (ER) stress- and autophagy-related proteins were detected with Western blotting. Primary chondrocytes from ERN1 knockout (ERN1 CKO) mice and wild-type mice were examined for ATG5 and ATG7 mRNA expressions, IRE1α and p-IRE1α protein expressions, and intracellular calcium ion content using qPCR, Western blotting and flow cytometry. The effect of bafilomycin A1 treatment on LC3 Ⅱ/LC3 Ⅰ ratio in the isolated chondrocytes was assessed with Western blotting. Changes in autophagic flux of the chondrocytes in response to rapamycin treatment were detected using autophagy dual fluorescent virus. The changes in autophagy level in C28/I2 cells overexpressing CHERP and IRE1α were detected using immunofluorescence assay. RESULTS: Tunicamycin treatment significantly up-regulated ER stress-related proteins and LC3 Ⅱ/LC3 Ⅰ ratio and down-regulated the expression of p62 in C28/I2 cells (P < 0.05). Rapamycin obviously up-regulated LC3 Ⅱ/LC3 Ⅰ ratio (P < 0.001) in C28/I2 cells, but this effect was significantly attenuated by co-treatment with 4µ8c (P < 0.05). Compared with the cells from the wild-type mice, the primary chondrocytes from ERN1 knockout mice showed significantly down-regulated mRNA levels of ERN1 (P < 0.01), ATG5 (P < 0.001) and ATG7 (P < 0.001), lowered or even lost expressions of IRE1α and p-IRE1α proteins (PP < 0.01), and increased expression of CHERP (P < 0.05) and intracellular calcium ion content (P < 0.001). Bafilomycin A1 treatment obviously increased LC3 Ⅱ/ LC3 Ⅰ ratio in the chondrocytes from both wild-type and ERN1 knockout mice (P < 0.01 or 0.05), but the increment was more obvious in the wild-type chondrocytes (P < 0.05). Treatment with autophagy dual-fluorescence virus resulted in a significantly greater fluorescence intensity of LC3-GFP in rapamycin-treated ERN1 CKO chondrocytes than in wild-type chondrocytes (P < 0.05). In C28/I2 cells, overexpression of CHERP obviously decreased the fluorescence intensity of LC3, and overexpression of IRE1α enhanced the fluorescence intensity and partially rescued the fluorescence reduction of LC3 caused by CHERP. CONCLUSION: IRE1α deficiency impairs autophagy in chondrocytes by upregulating CHERP and increasing intracellular calcium ion content.

Evaluation of Cell Models to Study Monocyte Functions in PMM2 Congenital Disorders of Glycosylation.

Congenital disorders of glycosylation (CDG) are inherited metabolic diseases characterized by mutations in enzymes involved in different steps of protein glycosylation, leading to aberrant synthesis, attachment or processing of glycans. Recently, immunological dysfunctions in several CDG types have been increasingly documented. Despite these observations, detailed studies on immune cell dysfunction in PMM2-CDG and other CDG types are still scarce. Studying PMM2-CDG patient immune cells is challenging due to limited availability of patient material, which is a result of the low incidence of the disease and the often young age of the subjects. Dedicated immune cell models, mimicking PMM2-CDG, could circumvent many of these problems and facilitate research into the mechanisms of immune dysfunction. Here we provide initial observations about the immunophenotype and the phagocytic function of primary PMM2-CDG monocytes. Furthermore, we assessed the suitability of two different glycosylation-impaired human monocyte models: tunicamycin-treated THP-1 monocytes and PMM2 knockdown THP-1 monocytes induced by shRNAs. We found no significant differences in primary monocyte subpopulations of PMM2-CDG patients as compared to healthy individuals but we did observe anomalous surface glycosylation patterns in PMM2-CDG patient monocytes as determined using fluorescent lectin binding. We also looked at the capacity of monocytes to bind and internalize fungal particles and found a slightly increased uptake of C. albicans by PMM2-CDG monocytes as compared to healthy monocytes. Tunicamycin-treated THP-1 monocytes showed a highly decreased uptake of fungal particles, accompanied by a strong decrease in glycosylation levels and a high induction of ER stress. In contrast and despite a drastic reduction of the PMM2 enzyme activity, PMM2 knockdown THP-1 monocytes showed no changes in global surface glycosylation levels, levels of fungal particle uptake similar to control monocytes, and no ER stress induction. Collectively, these initial observations suggest that the absence of ER stress in PMM2 knockdown THP-1 cells make this model superior over tunicamycin-treated THP-1 cells and more comparable to primary PMM2-CDG monocytes. Further development and exploitation of CDG monocyte models will be essential for future in-depth studies to ultimately unravel the mechanisms of immune dysfunction in CDG.

The yeast two-component SLN1 branch of the HOG pathway and the scaffolding activity of Pbs2 modulate the response to endoplasmic reticulum stress induced by tunicamycin.

In addition to the UPR pathway, yeast cells require components of the HOG pathway to respond to ER stress. In this work, we found that unphosphorylated Sln1 and Ssk1 are required to mount an appropriate response to Tn. We also found that the MAPKKKs Ssk2 participates in the Tn response, but its osmo-redundant protein Ssk22 does not. We also found that the Pbs2 docking sites for Ssk2 (RDS-I and KD) are partially dispensable when mutated separately; however, the prevention of Ssk2 binding to Pbs2, by the simultaneous mutation of RDS-I and KD, caused strong sensitivity to Tn. In agreement with the lack of Hog1 phosphorylation during Tn treatment, a moderate resistance to Tn is obtained when a Pbs2 version lacking its kinase activity is expressed; however, the presence of mutual Pbs2-Hog1 docking sites is essential for the Tn response. Finally, we detected that Tn induced a transcriptional activation of some components of the SLN1 branch. These results indicate that the Tn response requires a complex formed by the MAPK module and components of the SLN1 branch but not their canonical osmoregulatory activities.

[Bridge between Total Synthesis of Bioactive Natural Products and Development of Drug Leads].

Although natural products are rich sources for drug discovery, only a small percentage of natural products themselves have been approved for clinical use, thus it is necessary to modulate various properties, such as efficacy, toxicity, and metabolic stability. A question in natural product drug discovery is how to logically design natural product derivatives with desired biological properties. This review describes our recent studies regarding the medicinal chemistry of tunicamycin. Tunicamycin inhibits bacterial phospho-N-acetylmuramic acid (MurNAc)-pentapeptide translocase (MraY), which is an essential enzyme in bacteria and a good target for antibacterial drug discovery. The usefulness of tunicamycin as antibacterial agents is limited by off-target inhibition of human UDP-N-acetylglucosamine (GlcNAc): polyprenol phosphate translocase (GPT). We positioned the total synthesis of tunicamycin as a starting point for the research and have accomplished the synthesis of tunicamycin V by using the Achmatowicz reaction, [3,3] sigmatropic rearrangement of allyl cyanate, and stereoselective glycosylation as key reactions. Next, the minimum structural requirements for tunicamycin V for MraY inhibition were established by systematic structure-activity relationship studies with truncated analogs of tunicamycin V. Our collaborative study elucidated a crystal structure of human GPT in complex with tunicamycin. This structural information was then exploited to rationally design an MraY-specific inhibitor of tunicamycin V in which the GlcNAc moiety was modified to a MurNAc amide. The analog was identified as a highly selective MraYAA inhibitor.

Endoplasmic reticulum stress downregulates PGC-1α in skeletal muscle through ATF4 and an mTOR-mediated reduction of CRTC2.

BACKGROUND: Peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1α (PGC-1α) downregulation in skeletal muscle contributes to insulin resistance and type 2 diabetes mellitus. Here, we examined the effects of endoplasmic reticulum (ER) stress on PGC-1α levels in muscle and the potential mechanisms involved. METHODS: The human skeletal muscle cell line LHCN-M2 and mice exposed to different inducers of ER stress were used. RESULTS: Palmitate- or tunicamycin-induced ER stress resulted in PGC-1α downregulation and enhanced expression of activating transcription factor 4 (ATF4) in human myotubes and mouse skeletal muscle. Overexpression of ATF4 decreased basal PCG-1α expression, whereas ATF4 knockdown abrogated the reduction of PCG-1α caused by tunicamycin in myotubes. ER stress induction also activated mammalian target of rapamycin (mTOR) in myotubes and reduced the nuclear levels of cAMP response element-binding protein (CREB)-regulated transcription co-activator 2 (CRTC2), a positive modulator of PGC-1α transcription. The mTOR inhibitor torin 1 restored PCG-1α and CRTC2 protein levels. Moreover, siRNA against S6 kinase, an mTORC1 downstream target, prevented the reduction in the expression of CRTC2 and PGC-1α caused by the ER stressor tunicamycin. CONCLUSIONS: Collectively, these findings demonstrate that ATF4 and the mTOR-CRTC2 axis regulates PGC-1α transcription under ER stress conditions in skeletal muscle, suggesting that its inhibition might be a therapeutic target for insulin resistant states. Video Abstract.

ATF6-Mediated Unfolded Protein Response Facilitates Adeno-associated Virus 2 (AAV2) Transduction by Releasing the Suppression of the AAV Receptor on Endoplasmic Reticulum Stress.

Adeno-associated virus (AAV) is extensively used as a viral vector to deliver therapeutic genes during human gene therapy. A high-affinity cellular receptor (AAVR) for most serotypes was recently identified; however, its biological function as a gene product remains unclear. In this study, we used AAVR knockdown cell models to show that AAVR depletion significantly attenuated cells to activate unfolded protein response (UPR) pathways when exposed to the endoplasmic reticulum (ER) stress inducer, tunicamycin. By analyzing three major UPR pathways, we found that ATF6 signaling was most affected in an AAVR-dependent fashion, distinct from CHOP and XBP1 branches. AAVR capacity in UPR regulation required the full native AAVR protein, and AAV2 capsid binding to the receptor altered ATF6 dynamics. Conversely, the transduction efficiency of AAV2 was associated with changes in ATF6 signaling in host cells following treatment with different small molecules. Thus, AAVR served as an inhibitory molecule to repress UPR responses via a specificity for ATF6 signaling, and the AAV2 infection route involved the release from AAVR-mediated ATF6 repression, thereby facilitating viral intracellular trafficking and transduction. IMPORTANCE The native function of the AAVR as an ER-Golgi localized protein is largely unknown. We showed that AAVR acted as a functional molecule to regulate UPR signaling under induced ER stress. AAVR inhibited the activation of the transcription factor, ATF6, whereas receptor binding to AAV2 released the suppression effects. This finding has expanded our understanding of AAV infection biology in terms of the physiological properties of AAVR in host cells. Importantly, our research provides a possible strategy which may improve the efficiency of AAV-mediated gene delivery during gene therapy.

NHR-8 and P-glycoproteins uncouple xenobiotic resistance from longevity in chemosensory C. elegans mutants.

Longevity is often associated with stress resistance, but whether they are causally linked is incompletely understood. Here we investigate chemosensory-defective Caenorhabditis elegans mutants that are long-lived and stress resistant. We find that mutants in the intraflagellar transport protein gene osm-3 were significantly protected from tunicamycin-induced ER stress. While osm-3 lifespan extension is dependent on the key longevity factor DAF-16/FOXO, tunicamycin resistance was not. osm-3 mutants are protected from bacterial pathogens, which is pmk-1 p38 MAP kinase dependent, while TM resistance was pmk-1 independent. Expression of P-glycoprotein (PGP) xenobiotic detoxification genes was elevated in osm-3 mutants and their knockdown or inhibition with verapamil suppressed tunicamycin resistance. The nuclear hormone receptor nhr-8 was necessary to regulate a subset of PGPs. We thus identify a cell-nonautonomous regulation of xenobiotic detoxification and show that separate pathways are engaged to mediate longevity, pathogen resistance, and xenobiotic detoxification in osm-3 mutants.

Imoxin inhibits tunicamycin-induced endoplasmic reticulum stress and restores insulin signaling in C2C12 myotubes.

Prolonged endoplasmic reticulum (ER) stress can mediate inflammatory myopathies and insulin signaling pathways. The double-stranded RNA (dsRNA)-activated protein kinase R (PKR) has been implicated in skeletal muscle dysfunction. However, pathological roles of PKR in ER stress in muscle are not fully understood. The current study aimed to investigate the effect of imoxin (IMX), a selective PKR inhibitor, on tunicamycin (TN)-induced promotion of ER stress and suppression of insulin signaling in C2C12 myotubes. Cells were pretreated with 5 µM IMX for 1 h and exposed to 0.5 µg/mL TN for 23 h. A subset of cells was stimulated with 100 nM insulin for the last 15 min. mRNA expression and protein levels involved in ER stress were measured by RT-PCR and Western blotting, respectively. TN significantly augmented PKR phosphorylation by 231%, which was prevented by IMX. In addition, IMX reduced mRNA and protein levels of ER stress-related markers, including CCAAT-enhancer-binding protein homologous protein (CHOP, mRNA: 95% decrease; protein: 98% decrease), activating transcription factor 4 (ATF4, mRNA: 69% decrease; protein: 99% decrease), cleavage of ATF6, and spliced X-box-binding protein 1 (XBP-1s, mRNA: 88% decrease; protein: 79% decrease), which were induced by TN. Furthermore, IMX ameliorated TN-induced suppression of phospho-insulin receptor ß (317% increase) and Akt phosphorylation (by 36% at Ser473 and 30% at Thr308) in myotubes, while augmenting insulin-stimulated AS160 phosphorylation and glucose uptake (by ∼30%). These findings suggest that IMX may protect against TN-induced skeletal muscle ER stress and insulin resistance, which are potentially mediated by PKR.