Kinase-like Endoplasmic Reticulum Research Articles

Natural compound Alternol actives multiple endoplasmic reticulum stress-responding pathways contributing to cell death

Background: Alternol is a small molecular compound isolated from the fermentation of a mutant fungus obtained from Taxus brevifolia bark. Our previous studies showed that Alternol treatment induced reactive oxygen species (ROS)-dependent immunogenic cell death. This study conducted a comprehensive investigation to explore the mechanisms involved in Alternol-induced immunogenic cell death.Methods: Prostate cancer PC-3, C4-2, and 22RV1 were used in this study. Alternol interaction with heat shock proteins (HSP) was determined using CETSA assay. Alternol-regulated ER stress proteins were assessed with Western blot assay. Extracellular adenosine triphosphate (ATP) was measured using ATPlite Luminescence Assay System.Results: Our results showed that Alternol interacted with multiple cellular chaperone proteins and increased their expression levels, including endoplasmic reticulum (ER) chaperone hypoxia up-regulated 1 (HYOU1) and heat shock protein 90 alpha family class B member 1 (HSP90AB1), as well as cytosolic chaperone heat shock protein family A member 8 (HSPA8). These data represented a potential cause of unfolded protein response (UPR) after Alternol treatment. Further investigation revealed that Alternol treatment triggered ROS-dependent (ER) stress responses via R-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α). The double-stranded RNA-dependent protein kinase (PKR) but not activating transcription factor 6 (ATF6) cascades, leading to ATF-3/ATF-4 activation, C/EBP-homologous protein (CHOP) overexpression, and X-box binding protein XBP1 splicing induction. In addition, inhibition of these ER stress responses cascades blunted Alternol-induced extracellular adenosine triphosphate (ATP) release, one of the classical hallmarks of immunogenic cell death.Conclusion: Taken together, our data demonstrate that Alternol treatment triggered multiple ER stress cascades, leading to immunogenic cell death.

Cyanidin-3-O-glucoside protects Zearalenone-induced in vitro maturation disorders of porcine oocytes by alleviating NOX4-dependent oxidative stress and endoplasmic reticulum stress in cumulus cells

Zearalenone (ZEN) is widely found in foodstuffs and has serious harmful effects on female fertility, especially in pigs. Cyanidin-3-O-glucoside (C3G), a type of anthocyanin, exists in most dark fruits and vegetables; it has many positive dietary effects including as an antioxidant, anti-inflammatory, or anti-apoptotic agent. However, the beneficial effects of C3G alongside ZEN-induced damage in porcine oocytes and the underlying molecular mechanism have not been investigated. In this work, porcine cumulus-oocyte complexes (COCs) were divided into Control (Ctrl), ZEN, ZEN + C3G (Z + C), and C3G, and treated for 44–46 h in vitro. The results showed that C3G could alleviate ZEN-induced disorders of first polar body (PBI) extrusion, abnormalities of spindle assembly, cortical granule distribution, and mitochondrial distribution; these results were produced via restoring transzonal projections (TZPs), and inhibiting nicotinamide adenine dinucleotide phosphate oxidase (NOX4)-dependent oxidative stress and ‘glucose regulatory protein 78/protein kinase-like endoplasmic reticulum kinase/α subunit of eukaryotic initiation factor 2α/activating transcription factor 4/C/EBP-homologous protein’ (GRP78/PERK/eIF2α/ATF4/CHOP)-mediated endoplasmic reticulum stress (ERS) during oocyte maturation. Moreover, the over-expression of NOX4 in cumulus cells could result in a significant increase in ROS levels and ER fluorescence intensity in oocytes. In conclusion, C3G promoted in vitro maturation of porcine oocytes exposed to ZEN via mitigating NOX4-dependent oxidative stress and ERS in cumulus cells. These results contribute to our comprehension of the molecular mechanisms underlying the protective effects of C3G against ZEN toxicity in porcine oocytes, and they provide a novel theoretical foundation and strategy for future applications of C3G in the improvement of female reproduction.

Allosteric Inhibition of c-Abl to Induce Unfolded Protein Response and Cell Death in Multiple Myeloma.

Endoplasmic reticulum stress activates inositol-requiring enzyme 1α (IRE1α) and protein kinase, R-like endoplasmic reticulum kinase (PERK), the two principal regulators of the unfolded protein response (UPR). In multiple myeloma, adaptive IRE1α signaling is predominantly activated and regulates cell fate along with PERK. Recently, we demonstrated that GNF-2, an allosteric c-Abl inhibitor, rheostatically enhanced IRE1α activity and induced apoptosis through c-Abl conformational changes in pancreatic β cells. Herein, we analyzed whether the pharmacological modulation of c-Abl conformation resulted in anti-myeloma effects. First, we investigated the effects of GNF-2 on IRE1α activity and cell fate, followed by an investigation of the anti-myeloma effects of asciminib, a new allosteric c-Abl inhibitor. Finally, we performed RNA sequencing to characterize the signaling profiles of asciminib. We observed that both GNF-2 and asciminib decreased cell viability and induced XBP1 mRNA splicing in primary human myeloma cells and myeloma cell lines. RNA sequencing identified the induction of UPR- and apoptosis-related genes by asciminib. Asciminib re-localized c-Abl to the endoplasmic reticulum, and its combination with a specific IRE1α inhibitor, KIRA8, enhanced cell death with the reciprocal induction of CHOP mRNA expression. Together, the allosteric inhibition of c-Abl-activated UPR with anti-myeloma effects; this could be a novel therapeutic target for multiple myeloma.

Study of calcitriol anti-aging effects on human natural killer cells in vitro

ABSTRACT Vitamin D is widely considered to have a regulatory effect on the immune system. Some clinical investigations have shown that the demand for vitamin D increases with age. Calcitriol is the biologically active form of vitamin D. However, its effect on human natural killer (NK) cells remains unclear. Therefore, in this study, we investigated the anti-aging and immunomodulatory effects of calcitriol on NK cells using a series of immunological methods to explore its important role in innate immunity. We found that calcitriol reversed the expression of aging-related biomarkers in NK cells and inhibited their expansion by maintaining these cells in the G1 phase, without any apoptosis and exhaustion. Calcitriol repressed the release of inflammation-related cytokines, such as interleukin-5 (IL-5), interleukin-13 (IL-13), interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). The degranulation of NK cells was downregulated by calcitriol when these cells were co-cultured with K562 tumor cells. We also found that calcitriol upregulated the aging-related sirtuin 1- protein/kinase R-like endoplasmic reticulum kinase (SIRT1/pERK) pathway and SIRT1-deltaExon8 (SIRT1-∆Exon8) expression by activating the vitamin D receptor (VDR). Moreover, calcitriol could be a potential negative regulator of NK cell apoptosis and mitochondrial inactivation which caused by oxidative stress. Thus, calcitriol exhibits anti-aging effects on human NK cells in vitro by activating the SIRT1-PERK axis and resisting oxidative senescence.

Gut Microbe-Derived Metabolite Trimethylamine N-Oxide Activates PERK to Drive Mesenchymal Differentiation and Fibrosis

Intestinal dysbiosis is prominent in systemic sclerosis (SSc), but it remains unknown if and how it contributes to synchronous microvascular injury and fibrosis that characterize this disease. Trimethylamine (TMA) is generated in the gut and converted by flavin-containing monooxygenase (FMO3) to trimethylamine N-oxide (TMAO), which has been implicated in chronic cardiovascular diseases. We now show that TMAO reprograms skin fibroblasts, vascular endothelial cells and adipocytic progenitor cells to myofibroblasts in a process mediated via R-like endoplasmic reticulum kinase (PERK), a TMAO-binding protein. Remarkably, FMO3, principally expressed in the liver, was found also to be present in both isolated skin fibroblasts and in skin explants, and its expression was elevated in pericytes and fibroblasts in SSc skin biopsies and explanted dermal fibroblasts. A TMAO meta-organismal pathway in SSc thus potentially links the gut microbiome to vasculopathy and fibrosis via stromal cell reprogramming, implicating this pathway in pathogenesis, and as a target for therapy.

Similarities and Distinctions in the Effects of Metformin and Carbon Monoxide in Immunometabolism.

Immunometabolism, defined as the interaction of metabolic pathways with the immune system, influences the pathogenesis of metabolic diseases. Metformin and carbon monoxide (CO) are two pharmacological agents known to ameliorate metabolic disorders. There are notable similarities and differences in the reported effects of metformin and CO on immunometabolism. Metformin, an anti-diabetes drug, has positive effects on metabolism and can exert anti-inflammatory and anti-cancer effects via adenosine monophosphate-activated protein kinase (AMPK)-dependent and AMPK-independent mechanisms. CO, an endogenous product of heme oxygenase-1 (HO-1), can exert anti-inflammatory and antioxidant effects at low concentration. CO can confer cytoprotection in metabolic disorders and cancer via selective activation of the protein kinase R-like endoplasmic reticulum (ER) kinase (PERK) pathway. Both metformin and CO can induce mitochondrial stress to produce a mild elevation of mitochondrial ROS (mtROS) by distinct mechanisms. Metformin inhibits complex I of the mitochondrial electron transport chain (ETC), while CO inhibits ETC complex IV. Both metformin and CO can differentially induce several protein factors, including fibroblast growth factor 21 (FGF21) and sestrin2 (SESN2), which maintain metabolic homeostasis; nuclear factor erythroid 2-related factor 2 (Nrf2), a master regulator of the antioxidant response; and REDD1, which exhibits an anticancer effect. However, metformin and CO regulate these effects via different pathways. Metformin stimulates p53- and AMPK-dependent pathways whereas CO can selectively trigger the PERK-dependent signaling pathway. Although further studies are needed to identify the mechanistic differences between metformin and CO, pharmacological application of these agents may represent useful strategies to ameliorate metabolic diseases associated with altered immunometabolism.