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The innate immune response is an important line of defense against invading pathogens in invertebrates. Signaling pathways, including the IMD pathway, play critical roles in the production of antimicrobial peptides (AMPs), which induce the transcription of immune effectors that protect against bacterial invasion. In the present study, the cDNA of IMD from Eriocheir sinensis was cloned (designated Es IMD) and shown to be significantly upregulated following Gram-positive and Gram-negative bacterial infection. In vivo and in vitro studies collectively suggested that both the Gram-negative bacterium Vibrio parahemolyticus and the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis elicit the translocation of Relish. Moreover, Es IMD positively regulated Es Relish translocation from the cytoplasm to the nucleus following stimulation with both Gram-positive and Gram-negative bacteria. Es Relish knockdown in hemocytes significantly suppressed AMPs' expression. Furthermore, both Lys-type and DAP-type peptidoglycan-containing bacteria activated the IMD pathway and elicited antibacterial responses in crab. Conclusively, these findings demonstrate that both Gram-positive and Gram-negative bacteria activate IMD signaling, via a mechanism that is distinct with that by which Gram-negative bacteria activate IMD signaling in Drosophila. These findings might pave the way for a better understanding of the innate immune system and the fundamental network of the IMD signaling pathway in crustacean. • Eriocheir sinensis IMD (Es IMD) is upregulated by bacterial infection. • Bacteria (Gram+/-) induce nuclear translocation of Es Relish. • Nuclear translocation of Es Relish is regulated by Es IMD. • Bacteria (Gram+/-) activate IMD signaling in E. sinensis. [ABSTRACT FROM AUTHOR]

Pharmacoresistance in epilepsy is a major challenge to successful clinical therapy. Glucocorticoid receptor (GR) dysregulation can affect the underlying disease pathogenesis. We recently reported that local drug biotransformation at the blood-brain barrier is upregulated by GR, which controls drug-metabolizing enzymes (e.g., cytochrome P450s, CYPs) and efflux drug transporters (MDR1) in human epileptic brain endothelial cells (EPI-ECs). Here, we establish that this mechanism is influenced upstream by GR and its association with heat shock proteins/co-chaperones (Hsps) during maturation, which differentially affect human epileptic (EPI) tissue and brain endothelial cells. Overexpressed GR, Hsp90, Hsp70, and Hsp40 were found in EPI vs. NON-EPI brain regions. Elevated neurovascular GR expression and co-localization with Hsps was evident in the EPI regions with cortical dysplasia, predominantly in the brain micro-capillaries and neurons. A corresponding increase in ATPase activity (*p < 0.05) was found in the EPI regions. The GR-Hsp90/Hsp70 binding patterns indicated a faster chaperone-promoted maturation of GR, leading to its overactivation in both the tissue and EPI-ECs derived from EPI/focal regions and GR silencing in EPI-ECs slowed such GR-Hsp interactions. Significantly accelerated GR nuclear translocation was determined in EPI-ECs following treatment with GR modulators/ligands dexamethasone, rifampicin, or phenytoin. Our findings reveal that overexpressed GR co-localizes with Hsps in the neurovasculature of EPI brain, increased GR maturation by Hsps accelerates EPI GR machinery, and furthermore this change in EPI and NON-EPI GR-Hsp interaction alters with the age of seizure onset in epileptic patients, together affecting the pathophysiology and drug regulation in the epileptic brain endothelium. [ABSTRACT FROM AUTHOR]

Lactic acid (LA) is a byproduct of glycolysis resulting from intense exercise or a metabolic defect in aerobic processes. LA metabolism is essential to prevent lactic acidosis, but the mechanism through which LA regulates its own metabolism is largely unknown. Here, we identified a LA‐responsive protein, named LRPGC1, which has a distinct role from PGC1α, a key metabolic regulator, and report that LRPGC1 particularly mediates LA response to activate liver LA metabolism. Following LA stimulation, LRPGC1, but not PGC1α, translocates from the cytoplasm to the nucleus through deactivation of nuclear export signals, interacts with the nuclear receptor ERRγ, and upregulates TFAM, which ensures mitochondrial biogenesis. Knockout of PGC1 gene in HepG2 hepatocarcinoma cells decreased the LA consumption and TFAM expression, which were rescued by LRPGC1 expression, but not by PGC1α. These LRPGC1‐induced effects were mediated by ERRγ, concomitantly with mitochondrial activation. The response element for LRPGC1/ERRγ signaling pathway was identified in TFAM promoter. Notably, the survival rate of a mouse model of lactic acidosis was reduced by the liver‐targeted silencing of Lrpgc1, while it was significantly ameliorated by the pharmacological activation of ERRγ. These findings demonstrate LA‐responsive transactivation via LRPGC1 that highlight an intrinsic molecular mechanism for LA homeostasis. [ABSTRACT FROM AUTHOR]

The regulation of hepatic gluconeogenesis is of great significance to improve insulin resistance and benefit diabetes therapy. cAMP-Regulated Transcriptional Co-activator 2 (CRTC2) plays a key role in regulating hepatic gluconeogenesis through controlling the expression of gluconeogenic rate-limiting enzymes such as glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK). Recently, salt-inducible kinase 1 (SIK1) has been identified to play an important role in glucose metabolism disorders, but whether and how SIK1 regulates the CTRC2 signaling in liver cells under high glucose conditions has rarely been intensively elucidated. Here, we show that high glucose stimulation resulted in time-dependent down-regulated expression of SIK1, phosphorylated SIK1 at T182 site, and phosphorylated CRTC2 at S171 site, as well as upregulated expression of total CRTC2 and its downstream targets G6Pase and PEPCK in the human liver cell line HepG2. The nuclear expression levels of SIK1 and CRTC2 were time-dependently upregulated upon high glucose challenge, which was accompanied by enhanced cytoplasm-to-nucleus translocation of SIK1. Manipulation of SIK1 activity using plasmid-mediated SIK1 over-expression and the use of the SIKs inhibitor HG-9-91-01 confirmed that SIK1 regulated the CRTC2 signaling pathway in HepG2 cells. Furthermore, in mouse primary hepatocytes, high glucose exposure down-regulated SIK1 expression, and promoted SIK1 nuclear accumulation. While HG-9-91-01 treatment suppressed SIK1 expression and released the inhibitory effects of SIK1 on the expressions of key molecules involved in the CRTC2 signaling pathway, additional ectopic expression of SIK1 using adenovirus infection reversed the impacts of HG-9-91-01 on the expressions of these molecules in mouse hepatocytes. Therefore, SIK1 regulates CRTC2-mediated gluconeogenesis signaling pathway under both physiological and high glucose-induced pathological conditions. The modulation of the SIK1-CRTC2 signaling axis could provide an attractive means for treating diabetes. [ABSTRACT FROM AUTHOR]

The nuclear receptor peroxisome proliferator-activated receptor (PPAR)γ has been implicated in the pathogenesis of various human diseases including fatty liver. Although nuclear translocation of PPARγ plays an important role in PPARγ signaling, details of the translocation mechanisms have not been elucidated. Here we demonstrate that PPARγ2 translocates to the nucleus and activates signal transduction through H 2 O 2 -dependent formation of a PPARγ2 and transportin (Tnpo)1 complex via redox-sensitive disulfide bonds between cysteine (Cys)176 and Cys180 of the former and Cys512 of the latter. Using hepatocyte cultures and mouse models, we show that cytosolic H 2 O 2 /Tnpo1-dependent nuclear translocation enhances the amount of DNA-bound PPARγ and downstream signaling, leading to triglyceride accumulation in hepatocytes and liver. These findings expand our understanding of the mechanism underlying the nuclear translocation of PPARγ, and suggest that the PPARγ and Tnpo1 complex and surrounding redox environment are potential therapeutic targets in the treatment of PPARγ-related diseases. Image 1 • PPARγ2 forms disulfide bonds with Tnpo1. • Both Cys176 and Cys180 of PPARγ2 form disulfide bonds with Cys512 of Tnpo1. • H 2 O 2 promotes PPARγ2/Tnpo1 nuclear entry. • Redox-dependent PPARγ2/Tnpo1 nuclear entry promotes hepatic triglyceride accumulation. [ABSTRACT FROM AUTHOR]

Heart failure (HF) is a leading cause of morbidity and mortality worldwide. Patients with HF exhibit a loss of junctophilin-2 (JPH2), a structural protein critical in forming junctional membrane complexes in which excitation–contraction takes place. Several mechanisms have been proposed to mediate the loss of JPH2, one being cleavage by the calcium-dependent protease calpain. The downstream mechanisms underlying HF progression after JPH2 cleavage are presently poorly understood. In this study, we used Labcas to bioinformatically predict putative calpain cleavage sites on JPH2. We identified a cleavage site that produces a novel C-terminal JPH2 peptide (JPH2-CTP) using several domain-specific antibodies. Western blotting revealed elevated JPH2-CTP levels in hearts of patients and mice with HF, corresponding to increased levels of calpain-2. Moreover, immunocytochemistry demonstrated nuclear localization of JPH2-CTP within ventricular myocytes isolated from a murine model of pressure overload-induced HF as well as rat ventricular myocytes treated with isoproterenol. Nuclear localization of JPH2-CTP and cellular remodeling were abrogated by a genetic mutation of the nuclear localization sequence within JPH2-CTP. Taken together, our studies identified a novel C-terminal fragment of JPH2 (JPH2-CTP) generated by calpain-2 mediated cleavage which localizes within the cardiomyocyte nucleus during HF. Blocking nuclear localization of JPH2-CTP protects cardiomyocytes from isoproterenol-induced hypertrophy in vitro. Future in vivo studies of the nuclear role of JPH2-CTP may reveal a causal association with adverse remodeling during HF and establish CTP as a therapeutic target. [ABSTRACT FROM AUTHOR]

Polyglutamine (polyQ) spinocerebellar ataxias (SCAs) are the most prevalent subset of SCAs and share the aberrant expansion of Q-encoding CAG repeats within the coding sequences of disease-responsible genes as their common genetic cause. These polyQ SCAs (SCA1, SCA2, SCA3, SCA6, SCA7, and SCA17) are inherited neurodegenerative diseases characterized by the progressive atrophy of the cerebellum and connected regions of the nervous system, which leads to loss of fine muscle movement coordination. Upon the expansion of polyQ repeats, the mutated proteins typically accumulate disproportionately in the neuronal nucleus, where they sequester various target molecules, including transcription factors and other nuclear proteins. However, it is not yet clearly understood how CAG repeat expansion takes place or how expanded polyQ proteins accumulate in the nucleus. In this article, we review the current knowledge on the molecular and cellular bases of nuclear proteotoxicity of polyQ proteins in SCAs and present our perspectives on the remaining issues surrounding these diseases. [ABSTRACT FROM AUTHOR]

The nuclear translocation of insulin‐like growth factor receptor type 1 (IGF‐1R) has been documented in a variety of previous studies. The exact mechanism of this translocation, however, is still poorly understood. Furthermore, the functional role of IGF‐1R in the nucleus shows promise of transcriptional control. This function is particularly important in cancer cells. Understanding this role may also give insights into cancer biology and treatment methods. Processes including SUMOylation and clathrin‐mediated endocytosis are necessary for IGF‐1R nuclear translocation to occur. The antiapoptotic qualities of IGF‐1R likely contribute to its function in cancer cells. This review aims to synthesize the work on IGF‐1R in order to propose a mechanism of translocation. Using this mechanism, new therapeutic targets can be proposed that hinder the role of IGF‐1R in cancer metastasis. [ABSTRACT FROM AUTHOR]

Autosomal dominant hyper IgE syndrome (AD‐HIES) caused by STAT3 gene mutation is a rare primary immunodeficiency disease. To better understand the disease, we described the clinical characteristics of 20 AD‐HIES patients in Chongqing, China and explored the effect of mutations in different domains of STAT3 gene on the function of STAT3 protein by Western blot and confocal microscopy. The mean age at onset was 0.12 years. The mean age at diagnosis was 5.31 years. The most common presentation was eczema, pneumonia, skin abscesses and chronic mucocutaneous candidiasis. Seven patients suffered from BCG complications. R382W/Q were identified in 12 patients, V637M mutation in three patients. Three patients have died. The phosphorylated STAT3 was expressed more in wild‐type(WT) and R382W mutant STAT3 in the cytoplasm of COS7 cells with epidermal growth factor(EGF) stimulation, less in the V637M mutation and T620S mutation. Dynamic observation showed that STAT3 cytoplasmic accumulation and nuclear translocation occurred rapidly after EGF stimulation in WT‐STAT3‐GFP, the time of accumulation and nuclear translocation was later and the expression was less in R382W‐STAT3‐GFP compared with WT‐STAT3‐GFP, followed by V637M and T620S mutation. These results suggested that our patients had earlier onset, diagnostic age and higher rate of BCG complications. However, our patients had higher incidence of mortality though the earlier diagnostic age. We did not find a significant genotype/phenotype correlation, but Src homology 2 domain mutations (V637M and T620S) had a greater effect on STAT3 phosphorylation and nuclear translocation than DNA‐binding domain mutation (R382W) in vitro. [ABSTRACT FROM AUTHOR]

The activity-regulated cytoskeleton-associated protein (Arc) gene is a neural immediate early gene that is involved in synaptic down scaling and is robustly induced by prolonged wakefulness in rodent brains. Converging evidence has led to the hypothesis that wakefulness potentiates, and sleep reduces, synaptic strengthening. This suggests a potential role for Arc in these and other sleep-related processes. However, the role of Arc in sleep remains unknown. Here, we demonstrated that Arc is important for the induction of multiple behavioral and molecular responses associated with sleep homeostasis. Arc knockout (KO) mice displayed increased time spent in rapid eye movement (REM) sleep under baseline conditions and marked attenuation of sleep rebound to both 4 h of total sleep deprivation (SD)and selective REM deprivation. At the molecular level, the following homeostatic sleep responses to 4-h SD were all blunted in Arc KO mice: increase ofα-amino-3-hydroxy-5-methyl-4-isoxazolepropionicacid (AMPA) receptor GluA1 and its phosphorylation in synaptoneurosomes; induction of a subset of SD-response genes; and suppression of the GluA1 messenger RNA in the cortex. In wild-type brains, SD increased Arc protein expression in multiple subcellular locations, including the nucleus, cytoplasm, and synapse, which is reversed in part by recovery sleep. Arc is critical for these behavioral and multiple molecular responses to SD, thus providing a multifunctional role for Arc in the maintenance of sleep homeostasis, which may be attributed by the sleep/wake-associated changes in subcellular location of Arc. [ABSTRACT FROM AUTHOR]

As a ubiquitously expressed protein, cyclophilin A (CypA) is involved in a variety of pathological process, including immune suppression, inflammation, cell apoptosis, viral infection and stress response. However, the functional roles of CypA were largely unknown in economic marine animals. In this report, a novel CypA gene from sea cucumber Apostichopus japonicus (designated as AjCypA) was cloned and its function roles in immune responses were explored. The full-length cDNA of AjCypA was 1297 bp containing an open reading frame of 489 bp encoding a putative protein of 162 amino acids (aa). A conserved cyclophilin-like domain (CLD) with PPIase signature was located from 5 to 155 aa sequences in AjCypA, in which five necessary aa residues was totally conserved. In healthy sea cucumbers, AjCypA was expressed in all detected tissues, with highly expressed in muscles and weakly expressed in coelomocytes. AjCypA transcripts was significantly induced 8.08-fold and 5.65-fold in coelomocytes when sea cucumbers challenged with Vibrio splendidus in vivo and LPS in vitro , respectively. The expression pattern is similar with the expression of AjRel in the same condition. Moreover, GST pull-down and immunofluorescence analysis both revealed that AjCypA might be interacted with AjRel. Furthermore, AjCypA knockdown not only inhibited the expression of inflammation cytokines, but also suppressed the translocation of AjRel in nucleus induced by LPS. Taken together, our results suggested that AjCypA play key roles in V. splendidus mediated immune responses via suppressing the nuclear translocation of AjRel activity in sea cucumber. • AjCypA homologue was cloned and characterized in sea cucumbers. • AjCypA was expressed in all detected tissues, with highly expressed in muscles. • AjCypA transcripts showed similar expression profiles to that of AjRel in Vibrio splendidus challenged sea cucumber and LPS-exposed coelomocytes. • GST pull-down and immunofluorescence analysis both revealed that AjCypA might be interacted with AjRel. • AjCypA knockdown suppressed the translocation of AjRel in nucleus induced by LPS. [ABSTRACT FROM AUTHOR]

Nuclear factor of activated T cells (NFAT) leads to the transcription of diverse inducible genes involved in many biological processes; therefore, aberrant NFAT expression is responsible for the development and exacerbation of various disorders. Since five isoforms of NFAT (NFATc1‐c4, NFAT5) exhibit distinct and overlapping functions, selective control of a part, but not all, of NFAT family members is desirable. By comparing the binding activity of each NFATc1‐c4 with its regulatory enzyme, calcineurin (CN), using a quantitative immunoprecipitation assay, we found a new CN‐binding region (CNBR) selectively functioning in NFATc1 and NFATc4. This region, termed CNBR3, is located between two preexisting CNBR1 and CNBR2, within the Ca2+ regulatory domain. The nuclear translocation of NFATc1 but not NFATc2 in T cells was suppressed by ectopic expression of CNBR3 and, accordingly, NFATc1‐dependent cytokine expression was downregulated. Through competition assays using NFATc1‐derived partial peptides and mass spectrometry with photoaffinity technology, we identified 18 amino acids in NFATc1 (Arg258 to Pro275) and 13 amino acids in CN catalytic subunit (CNA) (Asn77 to Gly89) responsible for CNA/CNBR3 binding in which Cys263 and Asp82, respectively, played crucial roles. The possible selective regulation of NFAT‐mediated biological processes by targeting this new CN/NFAT‐binding region is suggested. [ABSTRACT FROM AUTHOR]

CD44 is a transmembrane receptor that acts as adhesion protein, fundamentally recognizing hyaluronan, an essential component of the extracellular matrix. It has a well-established functional association with cancer metastasis, particularly the CD44 variant forms which are considered essential markers of cancer stem cells. CD44 itself lacks intrinsic kinase activity but rather engages in signalling through specific interactions with kinases and other signalling components. Proteolysis within its transmembrane region also leads to release of the CD44 cytoplasmic domain, which can translocate to the nucleus and regulate transcription. A third signalling modality has been reported where the intact CD44 receptor translocates to the nucleus. Here, we investigated the latter using imaging techniques together with biochemical analyses. Our findings support observations where CD44 is cleaved prior to nuclear translocation and challenges the evidence for the presence of intact CD44 receptors in the cell nucleus. Conclusions regarding the presence of intact CD44 in the cell nucleus as a signalling modality, therefore, require re-evaluation. We highlight artefacts and common technical issues associated with these experiments that can lead to misinterpretation. [ABSTRACT FROM AUTHOR]

Optical manipulations are widely used to analyze neuronal functions in vivo. Blue light is frequently used to activate channelrhodopsins or LOV domains, although the degrees of its absorption and scattering are higher than those of longer wavelength light. High spatial resolution of optical manipulation is easily achieved in vitro , while the light is unevenly scattered and absorbed in tissues due to many factors. It is difficult to spatially measure a blue light transmission area in vivo. Here, we propose a genetic method to visualize blue light transmission in the brain and other organs using light-induced nuclear translocation of fluorescent proteins with a LOV domain. A light-inducible nuclear localization signal (LINuS) consists of a LOV2 domain fused with a nuclear localization signal (NLS). We confirmed that blue light illumination induced reversible translocation of NES-tdTomato-LINuS from the cytosol to the nucleus within 30 min in HEK293 cells. By employing a PHP.eb capsid that can penetrate the blood-brain barrier, retro-orbital sinus injection of adeno-associated virus (AAV) vectors induced scattered expression of nuclear export signal (NES)-tdTomato-LINuS in the brain. We confirmed that 30-min transcranial blue light illumination induced nuclear translocation of NES-tdTomato-LINuS in the cortex, the hippocampus, and even the paraventricular nucleus of the thalamus. We also found that mice exposed to blue light in a shaved abdominal area exhibited a substantial increase in nuclear translocation in the ventral surface lobe of the liver. These results provide a simple way to obtain useful information on light transmission in tissues without any transgenic animals or skillful procedures. • We achieved light-dependent nuclear translocation of tdTomato by employing a light-inducible nuclear localization signal. • We achieved scattered expression of NES-tdTomato-LINuS in the brain by simple intravenous injection of AAV vectors. • We achieved visualization of blue light transmission areas by quantifying nuclear translocation of NES-tdTomato-LINuS. [ABSTRACT FROM AUTHOR]

Transforming growth factor (TGF)-β signalling pathways are intensively investigated because of their diverse association with physiological and pathophysiological states. Smad transcription factors are the key mediators of TGF-β signalling. Smads can be directly phosphorylated in the carboxy terminal by the TGF-β receptor or in the linker region via multiple intermediate serine/threonine kinases. Growth factors in addition to hormones and TGF-β can activate many of the same kinases which can phosphorylate the Smad linker region. Historically, Smad linker region phosphorylation was shown to prevent nuclear translocation of Smads and inhibit TGF-β signalling pathways; however, it was subsequently shown that Smad linker region phosphorylation can be a driver of gene expression. This review will cover the signalling pathways of Smad linker region phosphorylation that drive the expression of genes involved in pathology and pathophysiology. The role of Smad signalling in cell biology is expanding rapidly beyond its role in TGF-β signalling and many signalling paradigms need to be re-evaluated in terms of Smad involvement. [ABSTRACT FROM AUTHOR]

The Y-box binding protein 1 (YB-1) is an RNA/DNA-binding protein regulating gene expression in the cytoplasm and the nucleus. Although mostly cytoplasmic, YB-1 accumulates in the nucleus under stress conditions. Its nuclear localization is associated with aggressiveness and multidrug resistance of cancer cells, which makes the understanding of the regulatory mechanisms of YB-1 subcellular distribution essential. Here, we report that inhibition of RNA polymerase II (RNAPII) activity results in the nuclear accumulation of YB-1 accompanied by its phosphorylation at Ser102. The inhibition of kinase activity reduces YB-1 phosphorylation and its accumulation in the nucleus. The presence of RNA in the nucleus is shown to be required for the nuclear retention of YB-1. Thus, the subcellular localization of YB-1 depends on its post-translational modifications (PTMs) and intracellular RNA distribution. [ABSTRACT FROM AUTHOR]

• Hepatic ischemia–reperfusion injury damages hepatocytes through promoting inflammation induced by activating the cGAS-STING pathway. • Hepatic ischemia–reperfusion injury inhibits Sirt3 expression, resulting in the activation of cGAS-STING pathway and enhanced inflammation. • Sirt3 inhibition aggravates hepatocyte damage by promoting nuclear translocation of p65 and facilitating cGAS transcription. Hepatic ischemia–reperfusion injury (IRI) typically manifests during subtotal hepatectomy and inflicts substantial damage to liver function in the perioperative period. Although the central role of cGAS-STING-mediated immune inflammation in hepatocyte damage during hepatic IRI is acknowledged, the precise regulatory mechanisms remain elusive. The current study aims to elucidate how Sirt3 inhibition activates the cGAS-STING pathway and exacerbates hepatocyte damage in hepatic IRI. We established both in vivo and in vitro models by creating hepatic IRI mice model and subjecting AML-12 hepatocyte cell lines to oxygen-glucose deprivation/reperfusion (OGD/R). Hepatic IRI compromised liver and mitochondrial function while elevating cytosolic mitochondrial DNA (mtDNA) levels in hepatocytes. Additionally, both in vivo hepatic IRI and in vitro OGD/R induced increased phosphorylation and activation of cGAS, STING, and IRF3, accompanied by heightened levels of pro-inflammatory factors, including TNF-α, IL-1β, and type I interferon (IFN-β). Importantly, knockdown of cGAS or STING through siRNA effectively attenuated hepatic IRI-induced inflammation and ameliorated liver function in both experimental settings, underscoring the dynamic involvement of the cGAS-STING pathway in hepatic IRI-induced inflammation. Furthermore, we observed a significant reduction in Sirt3 expression following hepatic IRI, both in vivo and in vitro. Then we generated Sirt3-deficient mice and applied Sirt3 knockdown in AML-12 hepatocytes. Notably, Sirt3 deficiency led to increased phosphorylation and activation of cGAS, STING, and IRF3, coupled with elevated TNF-α, IL-1β, and IFN-β levels in both in vivo and in vitro conditions. Moreover, upon silencing various downstream targets of Sirt3, such as transcription factors Sp1, Pu1, and p65, we observed that specifically knocking down p65 in AML-12 hepatocytes reduced cGAS mRNA levels. Co-immunoprecipitation assays confirmed a direct interaction between Sirt3 and p65. The absence of Sirt3 significantly increased nuclear translocation of p65 in mice, whereas Sirt3 knockdown in AML-12 hepatocytes heightened nuclear translocation of p65. ChIP-PCR assays demonstrated that Sirt3 deficiency notably enhanced the binding of p65 to two cGAS promoters, ultimately promoting cGAS transcription. Collectively, our results underscored that inhibition of Sirt3 activates the cGAS-STING pathway to aggravate hepatocyte damage by increasing cytosolic mtDNA and promoting nuclear translocation of p65 to promote cGAS transcription in hepatic IRI. These findings hold promise for potential therapeutic interventions in hepatic IRI by targeting the Sirt3-cGAS-STING axis, offering new avenues for the development of clinical strategies to mitigate liver damage during the perioperative period. [ABSTRACT FROM AUTHOR]

Potential genotoxicity and carcinogenicity of carbon nanotubes (CNT), as well as the underlying mechanisms, remains a pressing topic. The study aimed to evaluate and compare the genotoxic effect and mechanisms of DNA damage under exposure to different types of CNT. Immortalized human cell lines of respiratory origin BEAS-2B, A549, MRC5-SV40 were exposed to three types of CNT: MWCNT Taunit-M, pristine and purified SWCNT TUBALL™ at concentrations in the range of 0.0006–200 μg/ml. Data on the CNT content in the workplace air were used to calculate the lower concentration limit. The genotoxic potential of CNTs was investigated at non-cytotoxic concentrations using a DNA comet assay. We explored reactive oxygen species (ROS) formation, direct genetic material damage, and expression of a profibrotic factor TGFB1 as mechanisms related to genotoxicity upon CNT exposure. An increase in the number of unstable DNA regions was observed at a subtoxic concentration of CNT (20 μg/ml), with no genotoxic effects at concentrations corresponding to industrial exposures being found. While the three test articles of CNTs exhibited comparable genotoxic potential, their mechanisms appeared to differ. MWCNTs were found to penetrate the nucleus of respiratory cells, potentially interacting directly with genetic material, as well as to enhance ROS production and TGFB1 gene expression. For A549 and MRC5-SV40, genotoxicity depended mainly on MWCNT concentration, while for BEAS-2B – on ROS production. Mechanisms of SWCNT genotoxicity were not so obvious. Oxidative stress and increased expression of profibrotic factors could not fully explain DNA damage under SWCNT exposure, and other mechanisms might be involved. • DNA damage was observed at CNT concentration exceeding industrial exposures. • MWCNT interact with cell nucleus, enhance ROS production and TGFB1 gene expression. • In BEAS-2B cells exposed to MWCNT, DNA damage was quantitatively related to ROS. • ROS and increased TGFB1 expression could not explain DNA damage under SWCNT exposure. • CNT genotoxicity in various cell cultures exerted through distinct pathways. [ABSTRACT FROM AUTHOR]

Valosin-containing protein (VCP) was found to play a vital protective role against cardiac stresses. Genetic mutations of VCP are associated with human dilated cardiomyopathy. However, the essential role of VCP in the heart during the physiological condition remains unknown since the VCP knockout in mice is embryonically lethal. We generated a cardiac-specific dominant-negative VCP transgenic (DN-VCP TG) mouse to determine the effects of impaired VCP activity on the heart. Using echocardiography, we showed that cardiac-specific overexpression of DN-VCP induced a remarkable cardiac dilation and progressively declined cardiac function during the aging transition. Mechanistically, DN-VCP did not affect the endogenous VCP (EN-VCP) expression but significantly reduced cardiac ATPase activity in the DN-VCP TG mouse hearts, indicating a functional inhibition. DN-VCP significantly impaired the aging-related cytoplasmic/nuclear shuffling of EN-VCP and its co-factors in the heart tissues and interrupted the balance of the VCP-cofactors interaction between the activating co-factors, ubiquitin fusion degradation protein 1 (UFD-1)/nuclear protein localization protein 4 (NPL-4) complex, and its inhibiting co-factor P47, leading to the binding preference with the inhibitory co-factor, resulting in functional repression of VCP. This DN-VCP TG mouse provides a unique functional-inactivation model for investigating VCP in the heart in physiological and pathological conditions.

Macrophage stimulation by pathogen-associated molecular patterns (PAMPs) like lipopolysaccharide (LPS) or lipoteichoic acid (LTA) drives a proinflammatory phenotype and induces a metabolic reprogramming to sustain the cell’s function. Nevertheless, the relationship between metabolic shifts and gene expression remains poorly explored. In this context, the metabolic enzyme ATP citrate lyase (ACLY), the producer of citrate-derived acetyl-coenzyme A (CoA), plays a critical role in supporting a proinflammatory response. Through immunocytochemistry and cytosol–nucleus fractionation, we found a short-term ACLY nuclear translocation. Protein immunoprecipitation unveiled the role of nuclear ACLY in NF-κB acetylation and in turn its full activation in human PBMC-derived macrophages. Notably, sepsis in the early hyperinflammatory phase triggers ACLY-mediated NF-κB acetylation. The ACLY/NF-κB axis increases the expression levels of proinflammatory genes, including SLC25A1—which encodes the mitochondrial citrate carrier—and ACLY, thus promoting the existence of a proinflammatory loop involving SLC25A1 and ACLY genes.

In Arabidopsis, the cytosolic redox protein thioredoxin h2 (Trx-h2) is anchored to the cytoplasmic endomembrane through the myristoylated second glycine residue (Gly2). However, under cold stress, the cytosolic Trx-h2 is rapidly translocated to the nucleus, where it interacts with and reduces the cold-responsive C-repeat-binding factors (CBFs), thus activating cold-responsive (COR) genes. In this study, we investigated the significance of fatty acid modification of Trx-h2 under cold conditions by generating transgenic Arabidopsis lines in the trx-h2 mutant background, overexpressing Trx-h2 (Trx-h2OE/trx-h2) and its point mutation variant Trx-h2(G/A) [Trx-h2(G/A)OE/trx-h2], in which the Gly2 was replaced by alanine (Ala). Due to the lack of Gly2, Trx-h2(G/A) was incapable of myristoylation, and a part of Trx-h2(G/A) localized to the nucleus even under warm temperature. As no time is spent on the demyristoylation and subsequent nuclear translocation of Trx-h2(G/A) under a cold snap, the ability of Trx-h2(G/A) to protect plants from cold stress was greater than that of Trx-h2. Additionally, COR genes were up-regulated earlier in Trx-h2(G/A)2OE/trx-h2 plants than in Trx-h2OE/trx-h2 plants under cold stress. Consequently, Trx-h2(G/A)2OE/trx-h2 plants showed greater cold tolerance than Col-0 (wild type) and Trx-h2OE/trx-h2 plants. Overall, our results clearly demonstrate the significance of the demyristoylation of Trx-h2 in enhancing plant cold/freezing tolerance.

Tumor cells undergoing epithelial-mesenchymal transition (EMT) lose cell surface adhesion molecules and gain invasive and metastatic properties. EMT is a plastic process and tumor cells may shift between different epithelial-mesenchymal states during metastasis. However, how this is regulated is not fully understood. Syndecan-1 (SDC1) is the major cell surface proteoglycan in epithelial cells and has been shown to regulate carcinoma progression and EMT. Recently, it was discovered that SDC1 translocates into the cell nucleus in certain tumor cells. Nuclear SDC1 inhibits cell proliferation, but whether nuclear SDC1 contributes to the regulation of EMT is not clear. Here, we report that loss of nuclear SDC1 is associated with cellular elongation and an E-cadherin-to-N-cadherin switch during TGF-β1-induced EMT in human A549 lung adenocarcinoma cells. Further studies showed that nuclear translocation of SDC1 contributed to the repression of mesenchymal and invasive properties of human B6FS fibrosarcoma cells. The results demonstrate that nuclear translocation contributes to the capacity of SDC1 to regulate epithelial-mesenchymal plasticity in human tumor cells and opens up to mechanistic studies to elucidate the mechanisms involved.

Infection with the influenza A (H1N1) virus is a major challenge for public health because it can cause severe morbidity and even mortality in humans. The over-secretion of inflammatory cytokines (cytokine storm) is considered to be a key contributor to the severe pneumonia caused by H1N1 infection. It has been reported that hypoxia-inducible factor 1-alpha (HIF-1α) is associated with the production of proinflammatory molecules, but whether HIF-1α participates in the acute inflammatory responses against H1N1 infection is still unclear. To investigate the role of HIF-1α in H1N1 infection, the expression and nuclear translocation of HIF-1α in A549 and THP-1 cell lines infected with H1N1 virus were observed. The results showed that without altering the intracellular mRNA or protein expression of HIF-1α, H1N1 infection only induced nuclear translocation of HIF-1α under normal oxygen concentrations. The use of 2-methoxyestradiol (2ME2), a HIF-1α inhibitor that blocks HIF-1α nuclear accumulation, in H1N1-infected cells decreased the mRNA and protein expression of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-6 and increased the levels of IL-10. In contrast, H1N1-infected cells under hypoxic conditions had increased HIF-1α nuclear accumulation, increased expression of TNF-α and IL-6 and decreased levels of IL-10. In conclusion, our data implied that in vitro H1N1 infection induced nuclear translocation of HIF-1α without altering the expression of HIF-1α, which may promote the secretion of proinflammatory cytokines during H1N1 infection.Emerging Microbes & Infections (2017) 6, e39; doi:10.1038/emi.2017.21; published online 24 May 2017

5-lipoxygenase (5-LOX) is a non-heme iron-containing dioxygenase expressed in immune cells that catalyzes the two initial steps in the biosynthesis of leukotrienes. It is well known that 5-LOX activation in innate immunity cells is related to different iron-associated pro-inflammatory disorders, including cancer, neurodegenerative diseases, and atherosclerosis. However, the molecular and cellular mechanism(s) underlying the interplay between iron and 5-LOX activation are largely unexplored. In this study, we investigated whether iron (in the form of Fe3+ and hemin) might modulate 5-LOX influencing its membrane binding, subcellular distribution, and functional activity. We proved by fluorescence resonance energy transfer approach that metal removal from the recombinant human 5-LOX, not only altered the catalytic activity of the enzyme, but also impaired its membrane-binding. To ascertain whether iron can modulate the subcellular distribution of 5-LOX in immune cells, we exposed THP-1 macrophages and human primary macrophages to exogenous iron. Cells exposed to increasing amounts of Fe3+ showed a redistribution (ranging from ~45 to 75%) of the cytosolic 5-LOX to the nuclear fraction. Accordingly, confocal microscopy revealed that acute exposure to extracellular Fe3+, as well as hemin, caused an overt increase in the nuclear fluorescence of 5-LOX, accompanied by a co-localization with the 5-LOX activating protein (FLAP) both in THP-1 macrophages and human macrophages. The functional relevance of iron overloading was demonstrated by a marked induction of the expression of interleukin-6 in iron-treated macrophages. Importantly, pre-treatment of cells with the iron-chelating agent deferoxamine completely abolished the hemin-dependent translocation of 5-LOX to the nuclear fraction, and significantly reverted its effect on interleukin-6 overexpression. These results suggest that exogenous iron modulates the biological activity of 5-LOX in macrophages by increasing its ability to bind to nuclear membranes, further supporting a role for iron in inflammation-based diseases where its homeostasis is altered and suggesting further evidence of risks related to iron overload.

Abstract Introduction Mutations in the angiogenin (ANG) gene are known to be associated with both familial and sporadic amyotrophic lateral sclerosis (ALS). The majority of disease‐causing mutations of ANG result in loss of either ribonucleolytic activity, nuclear translocation activity or both. Methods We sequenced ANG gene from a total of 136 sporadic ALS patients and 112 healthy controls of Hungarian origin. To elucidate the role of the R33W mutation in the disease mechanism, computational, and functional analyses were performed. Results Mutation screening revealed a mutation located in the signal peptide (M‐24I) and two mutations that affect the mature protein (R33W, V103I). The R33W mutation, which has not been previously detected in ALS patients, affects the key amino acid of the nuclear translocation signal of the ANG protein. Molecular dynamics simulations suggested that the R33W mutation results in partial loss of ribonucleolytic activity and reduced nuclear translocation activity. The ribonucleolytic assay and nuclear translocation assay of the R33W ANG protein confirmed the molecular dynamics results. Conclusions In the Hungarian ALS population, the observed frequency of ANG mutations was 2.9%, which is higher than previously reported for sporadic cohorts. The evidence from computational and functional analyses support the deleterious effect of the novel R33W variant detected in this study.

The constitutive androstane receptor (CAR) plays an important role in hepatic drug metabolism and detoxification but has recently been projected as a potential drug target for metabolic disorders due to its repression of lipogenesis and gluconeogenesis. Thus, identification of physiologically-relevant CAR modulators has garnered significant interest. Here, we adapted the previously characterized human CAR (hCAR) nuclear translocation assay in human primary hepatocytes (HPH) to a high-content format and screened an FDA-approved drug library containing 978 compounds. Comparison of hCAR nuclear translocation results with the Tox21 hCAR luciferase reporter assay database in 643 shared compounds revealed significant overlap between these two assays, with approximately half of hCAR agonists also mediating nuclear translocation. Further validation of these compounds in HPH and/or using published data from literature demonstrated that hCAR translocation exhibits a higher correlation with the induction of hCAR target genes, such as CYP2B6, than the luciferase assay. In addition, some CAR antagonists which repress CYP2B6 mRNA expression in HPH, such as sorafenib, rimonabant, and CINPA1, were found to translocate hCAR to the nucleus of HPH. Notably, both the translocation assay and the luciferase assay identified mosapride citrate (MOS), a gastroprokinetic agent that is known to reduce fasting blood glucose levels in humans, as a novel hCAR activator. Further studies with MOS in HPH uncovered that MOS can repress the expression of gluconeogenic genes and decrease glucose output from hepatocytes, providing a previously unidentified liver-specific mechanism by which MOS modulates blood glucose levels. [ABSTRACT FROM AUTHOR]

Constitutive androstane receptor (CAR), a member of the nuclear receptor superfamily, is retained as an inactive form phosphorylated at threonine in the cytoplasm of hepatocytes. Upon activation, CAR is dephosphorylated to move into the nucleus and induces the transcription of genes. Thus, nuclear translocation is a key step for CAR activation in hepatocytes. However, this nuclear translocation has not been demonstrated in conventional two-dimensionally-cultured immortalized cell lines such as HepG2, in which CAR spontaneously accumulates in the nucleus. In this study, we showed that treatment with the indirect CAR activator phenobarbital activated transcription of the CYP3A4 gene in three-dimensionally (3D)-cultured HepG2 cells. CAR was retained as its phosphorylated form in the cytoplasm and was translocated to the nucleus in 3D-cultured HepG2 cells in response to treatment with phenobarbital. Moreover, okadaic acid and epidermal growth factor, were found to repress phenobarbital-induced CAR nuclear translocation and subsequent activation of the CYP3A4 gene promoter. These results suggested that 3D-cultured HepG2 cells properly regulated CAR activation as has been observed in hepatocytes. [ABSTRACT FROM AUTHOR]

Rare missense variants play a crucial role in amyotrophic lateral sclerosis (ALS) pathophysiology. We report rare/novel missense variants from 154 Indian ALS patients, identified through targeted sequencing of 25 ALS-associated genes. As pathogenic variants could explain only a small percentage of ALS pathophysiology in our cohort, we investigated the frequency of tolerated and benign novel/rare variants, which could be potentially ALS susceptible. These variants were identified in 5.36% (8/149) of sporadic ALS (sALS) cases; with one novel variant each in ERBB4, SETX, DCTN1, and MATR3; four rare variants, one each in PON2 and ANG and two different rare variants in SETX. Identified variants were either absent or present at extremely rare frequencies (MAF < 0.01) in large population databases and were absent in 50 healthy controls sequenced through Sanger method. Furthermore, an oligogenic basis of ALS was observed in three sALS, with co-occurrence of intermediate-length repeat expansions in ATXN2 and a rare/novel variant in DCTN1 and SETX genes. Additionally, molecular dynamics and biochemical functional analysis of an angiogenin variant (R21G) identified from our cohort demonstrated loss of ribonucleolytic and nuclear translocation activities. Our findings suggest that rare variants could be potentially pathogenic and functional studies are warranted to decisively establish the pathogenic mechanisms associated with them. [ABSTRACT FROM AUTHOR]

Mutations of PTEN-induced kinase 1 (PINK1) cause recessive familial Parkinson's disease. Cells lacking PINK1 display mitochondrial deficits and increased sensitivity to oxidative and proteasomal stress. It has been shown that the 52-kDa (mature) form of PINK1 in the cytoplasm mitigates proteasomal stress-induced cell death by enhancing aggresomes formation and autophagy. Here we newly demonstrate that proteasome dysfunction triggers mono-ubiquitination and nuclear translocation of mature PINK1. Enhancing PINK1 mono-ubiquitination by two different means increased nuclear accumulation of PINK1 independent of proteasome inhibition. Moreover, we show that PINK1 harbors a hitherto unknown nuclear export sequence (NES) in its C-terminus. Blocking CRM1-dependent nuclear export with leptomycin B augmented PINK1 levels in the nucleus of MG132-treated cells but not in normal cells. Overall, these results show that proteasomal stress-induced mono-ubiquitination of PINK1 mediates PINK1 nuclear translocation, while PINK1 is excluded from the nucleus of healthy cells via its NES. Therefore, mature PINK1 may have a nuclear function in cells under proteasomal stress. • Mature PINK1 harbors a CRM1-dependent nuclear export signal. • Proteasome inhibition results in mono-ubiquitination of mature PINK1. • Mono-ubiquitination promotes PINK1 nuclear translocation. • PINK1 may have a proteasomal stress-dependent function in the nucleus. [ABSTRACT FROM AUTHOR]

Signaling pathways that originate at the plasma membrane, including regulated intramembrane proteolysis (RIP), enable extracellular cues to control transcription. We modified the yeast Gal4 transcription system to study the nuclear translocation of transcriptionally active complexes using the fluorescent protein citrine (Cit) as a reporter. This enabled highly sensitive quantitative analysis of transcription in situ at the single cell level. The Gal4/UAS-Cit transcription assay displayed a sigmoidal response limited by the number of integrated reporter cassettes. We validated the assay by analyzing nuclear translocation of the amyloid precursor protein (APP) intracellular domain (AICD) and confirmed the requirement of Fe65 for nuclear translocation of AICD. In addition to the strong on-off effects on transcriptional activity, the results of this assay establish that phosphorylation modifies nuclear signaling. The Y682F mutation in APP showed the strongest increase in Cit expression, underscoring its role in regulating Fe65 binding. Together, we established a highly sensitive fluorescent protein-based assay that can monitor transcriptional activity at the single cell level and demonstrate that AICD phosphorylation affects Fe65 nuclear activity. This assay also introduces a platform for future single cellbased drug screening methods for nuclear translocation. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy (DN) is a major cause of end-stage renal disease (ESRD). Vitamin D receptor (VDR) belongs to the nuclear receptor superfamily and exerts a renoprotective effect through inhibiting fibrosis. Microtubule-associated protein 1 light chain 3 (LC3), a key regulator of autophagy, is abundant in the nucleus, although its primary function is in the cytoplasm. The role of nuclear LC3 and the mechanism by which LC3 shuttles between the cytoplasm and nucleoplasm has not been fully elucidated. We found that LC3 binds to VDR in an LC3-interacting region (LIR)-independent manner and promotes the nuclear translocation of VDR. Further study indicated that LC3 promotes the formation of the VDR:retinoid X receptor (RXR) heterodimer and inhibits fibrogenic genes expression in HK-2 cells induced by high glucose. Our result demonstrates that LC3 is a negative regulator of high glucose-induced fibrogenic genes expression through its ability to promote VDR signaling. • LC3 promotes the nuclear translocation of the VDR. • LC3 binds the VDR in an LC3-interacting region (LIR)-independent manner. • LC3 promotes the formation of the VDR:retinoid X receptor (RXR) heterodimer. • LC3 inhibits fibrogenic genes expression in HK-2 cells induced by high glucose. [ABSTRACT FROM AUTHOR]

Intermediate filament protein keratin (K) 8 binds to Hsp70 and p38 MAPK, and is phosphorylated at Ser74 by p38. However, a p38 binding site on K8 and the molecular mechanism of K8-p38 interaction related to Hsp70 are unknown. Here, we identify a p38 docking site on K8 (Arg148/149 and Leu159/161) that is highly conserved in other intermediate filaments. The docking-deficient K8 mutation causes increased p38-Hsp70 interaction and enhanced p38 nuclear localization, indicating that the p38 dissociated from mutant K8 makes a complex with Hsp70, known as a potential chaperone for p38 nuclear translocation. Comparison of p38 MAPK binding with liver disease-associated keratin variants shows that K18-I150V variant dramatically reduces binding with p38, which is similar to the effect of the p38 docking-deficient mutation on K8. Since the p38 docking site on K8 (Arg148/149 and Leu159/161) and the K18-Ile150 residue are closely localized in parallel K8/K18 heterodimer, the K18-I150V mutation may interfere K8-p38 interaction. These findings show that keratins modulate the p38 nuclear localization, functioning as a cytoplasmic anchor for p38, which might affect a number of p38-mediated signal transduction pathways. [ABSTRACT FROM AUTHOR]

Peste des petits ruminants virus (PPRV) is the etiological agent of peste des petits ruminants, causing acute immunosuppression in its natural hosts. However, the molecular mechanisms by which PPRV antagonizes the host immune responses have not been fully characterized. In particular, how PPRV suppresses the activation of the host RIG-I-like receptor (RLR) pathway has yet to be clarified. In this study, we demonstrated that PPRV infection significantly suppresses RLR pathway activation and type I interferon (IFN) production and identified PPRV N protein as an extremely important antagonistic viral factor that suppresses beta interferon (IFN-β) and IFN-stimulated gene (ISG) expression. A detailed analysis showed that PPRV N protein inhibited type I IFN production by targeting interferon regulatory factor 3 (IRF3), a key molecule in the RLR pathway required for type I IFN induction. PPRV N protein interacted with IRF3 (but not with other components of the RLR pathway, including MDA5, RIG-I, VISA, TBK1, and MITA) and abrogated the phosphorylation of IRF3. As expected, PPRV N protein also considerably impaired the nuclear translocation of IRF3. The TBK1-IRF3 interaction was involved significantly in IRF3 phosphorylation, and we showed that PPRV N protein inhibits the association between TBK1 and IRF3, which in turn inhibits IRF3 phosphorylation. The amino acid region 106 to 210 of PPRV N protein was determined to be essential for suppressing the nuclear translocation of IRF3 and IFN-β production, and the 140 to 400 region of IRF3 was identified as the crucial region for the N-IRF3 interaction. Together, our findings demonstrate a new mechanism evolved by PPRV to inhibit type I IFN production and provide structural insights into the immunosuppression caused by PPRV. IMPORTANCE Peste des petits ruminants is a highly contagious animal disease affecting small ruminants, which threatens both small livestock and endangered susceptible wildlife populations in many countries. The causative agent, peste des petits ruminants virus (PPRV), often causes acute immunosuppression in its natural hosts during infection. Here, for the first time, we demonstrate that N protein, the most abundant protein of PPRV, plays an extremely important role in suppression of interferon regulatory factor 3 (IRF3) function and type I interferon (IFN) production by interfering with the formation of the TBK1-IRF3 complex. This study explored a novel antagonistic mechanism of PPRV. [ABSTRACT FROM AUTHOR]

The availability of intracellular, stabilized β-catenin, a transcription factor coactivator, is tightly regulated; β-catenin is translocated into the nucleus in response to Wnt ligand binding to its cell membrane receptors. Here we show that Wnt signal activation in mammalian cells activates intracellular mobilization of connexin 43 (Cx43), which belongs to a gap junction protein family, a new target protein in response to extracellular Wnt signal activation. Transmission electron microscopy showed that the nuclear localization of Cx43 was increased by 8- to 10-fold in Wnt5A- and 9B-treated cells compared with controls; this Wnt-induced increase was negated in the cells where Cx43 and β-catenin were knocked down using shRNA. There was a significant (P < 0.001) and concomitant depletion of the cell membrane and cytosolic signal of Cx43 in Wnt-treated cells with an increase in the nuclear signal for Cx43; this was more obvious in cells where β-catenin was knocked down using shRNA. Conversely, Cx43 knockdown resulted in increased β-catenin in the nucleus in the absence of Wnt activation. Coimmunoprecipitation of Cx43 and β-catenin proteins with a casein kinase (CKIδ) antibody showed that Cx43 interacts with β-catenin and may form part of the so-called destruction complex. Functionally, Wnt activation increased the rate of wound reepithelization in rat skin in vivo. [ABSTRACT FROM AUTHOR]

Background/Aims: The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase (MAPK) family. Upon stimulation, these kinases translocate from the cytoplasm to the nucleus, where they induce physiological processes such as proliferation and differentiation. The mechanism of translocation of this kinase involves phosphorylation of two Ser residues within a nuclear translocation signal (NTS), which allows binding to importin7 and a subsequent penetration via nuclear pores. However, the regulation of this process and the protein kinases involved are not yet clear. Methods: To answer this point we developed specific anti phospho-SPS antibody, used this and other antibodies in Western blots and crystalized the phospho-mimetic mutated ERK. Results: Here we show that the phosphorylation of both Ser residues is mediated mainly by casein kinase 2 (CK2) and that active ERK may assist in the phosphorylation of the N-terminal Ser. We also demonstrate that the phosphorylation is dependent on the release of ERK from cytoplasmic anchoring proteins. Crystal structure of the phosphomimetic ERK revealed that the NTS phosphorylation creates an acidic patch in ERK. Our model is that in resting cells ERK is bound to cytoplasmic anchors, which prevent its NTS phosphorylation. Upon stimulation, phosphorylation of the ERK TEY domain releases ERK and allows phosphorylation of its NTS by CK2 and active ERK to generate a negatively charged patch in ERK, binding to importin 7 and nuclear translocation. Conclusion: These results provide an important role of CK2 in regulating nuclear ERK activities. [ABSTRACT FROM AUTHOR]

Aberrantly elevated expression levels of histone deacetylase 1 (HDAC1) and vimentin are closely associated with disease progression in hepatocellular carcinoma (HCC). It was previously demonstrated that knocking down expression of HDAC1 resulted in a concurrent decrease in the expression levels of vimentin. However, a causal link between these two proteins has not yet been demonstrated, to the best of our knowledge. In the present study, the association between HDAC1 and vimentin was investigated using an HDAC1 overexpression platform. HDAC1 and vimentin were significantly increased in HCC cells, and HDAC1 overexpression enhanced vimentin mRNA and protein expression levels in an HDAC1 dose-dependent manner. Subsequently, truncation and mutation of a vimentin promoter demonstrated that HDAC1-induced vimentin expression was dependent on a nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) binding site in the vimentin promoter sequence. Furthermore, HDAC1 induced vimentin expression by promoting NF-κB translocation between the cytoplasm and the nucleus, as opposed to modulating the total expression level of vimentin directly. The data in the present study demonstrated that HDAC1 is overexpressed in HCC and that HDAC1 may upregulate vimentin expression through the NF-κB signaling pathway, thus demonstrating a causal link between HDAC1 and vimentin in HCC, and may provide valuable information in understanding the pathogenesis of HCC. [ABSTRACT FROM AUTHOR]

Disturbed neuronal cholesterol homeostasis has been observed in Alzheimer disease (AD) and contributes to the pathogenesis of AD. As the master switch of cholesterol biosynthesis, the sterol regulatory element‐binding protein 2 (SREBP‐2) translocates to the nucleus after cleavage/activation, but its expression and activation have not been studied in AD which is the focus of the current study. We found both a significant decrease in the nuclear translocation of N‐terminal SREBP‐2 accompanied by a significant accumulation of C‐terminal SREBP‐2 in NFT‐containing pyramidal neurons in AD. N‐terminal‐ SREBP‐2 is also found in dystrophic neurites around plaques in AD brain. Western blot confirmed a significantly reduced nuclear translocation of mature SREBP‐2 (mSREBP‐2) in AD brain. Interestingly, reduced nuclear mSREBP‐2 was only found in animal models of tauopathies such as 3XTg AD mice and P301L Tau Tg mice but not in CRND8 APP transgenic mice, suggesting that tau alterations likely are involved in the changes of mSREBP‐2 distribution and activation in AD. Altogether, our study demonstrated disturbed SREBP‐2 signaling in AD and related models, and proved for the first time that tau alterations contribute to disturbed cholesterol homeostasis in AD likely through modulation of nuclear mSREBP‐2 translocation. [ABSTRACT FROM AUTHOR]

5-lipoxygenase (5-LOX) is a non-heme iron-containing dioxygenase expressed in immune cells that catalyzes the two initial steps in the biosynthesis of leukotrienes. It is well known that 5-LOX activation in innate immunity cells is related to different iron-associated pro-inflammatory disorders, including cancer, neurodegenerative diseases, and atherosclerosis. However, the molecular and cellular mechanism(s) underlying the interplay between iron and 5-LOX activation are largely unexplored. In this study, we investigated whether iron (in the form of Fe3+ and hemin) might modulate 5-LOX influencing its membrane binding, subcellular distribution, and functional activity. We proved by fluorescence resonance energy transfer approach that metal removal from the recombinant human 5-LOX, not only altered the catalytic activity of the enzyme, but also impaired its membrane-binding. To ascertain whether iron can modulate the subcellular distribution of 5-LOX in immune cells, we exposed THP-1 macrophages and human primary macrophages to exogenous iron. Cells exposed to increasing amounts of Fe3+ showed a redistribution (ranging from ~45 to 75%) of the cytosolic 5-LOX to the nuclear fraction. Accordingly, confocal microscopy revealed that acute exposure to extracellular Fe3+, as well as hemin, caused an overt increase in the nuclear fluorescence of 5-LOX, accompanied by a co-localization with the 5-LOX activating protein (FLAP) both in THP-1 macrophages and human macrophages. The functional relevance of iron overloading was demonstrated by a marked induction of the expression of interleukin-6 in iron-treated macrophages. Importantly, pre-treatment of cells with the iron-chelating agent deferoxamine completely abolished the hemin-dependent translocation of 5-LOX to the nuclear fraction, and significantly reverted its effect on interleukin-6 overexpression. These results suggest that exogenous iron modulates the biological activity of 5-LOX in macrophages by increasing its ability to bind to nuclear membranes, further supporting a role for iron in inflammation-based diseases where its homeostasis is altered and suggesting further evidence of risks related to iron overload. [ABSTRACT FROM AUTHOR]

Abstract Multiple lines of evidence have indicated that pyruvate kinase M2 (PKM2) is upregulated in most cancer cells and it is increasingly recognized as a potential therapeutic target in oncology. In a continuation of our discovery of lead compound 5 and SAR study, the 7-azaindole moiety in compound 5 was systematically optimized. The results showed that compound 6f , which has a difluoroethyl substitution on the 7-azaindole ring, exhibited high PKM2 activation potency and anti-proliferation activities on A375 cell lines. In a xenograft mouse model, oral administration of compound 6f led to significant tumor regression without obvious toxicity. Further mechanistic studies revealed that 6f could influence the translocation of PKM2 into nucleus, as well as induction of apoptosis and autophagy of A375 cells. More importantly, compound 6f significantly inhibited migration of A375 cells in a concentration-dependent manner. Collectively, 6f may serve as a lead compound in the development of potent PKM2 activators for cancer therapy. Graphical abstract Image 1 Highlights • 7-Azaindole derivatives containing pyridin-3-ylmethyl dithiocarbamate moiety were optimized as potent PKM2 activators. • Compound 6f has great anti-proliferation activities on A375 cell lines both in vitro and in vivo. • Compound 6f could influence the translocation of PKM2 into nucleus on A375 cells. • Compound 6f could efficiently exhibit antimetastatic potential against cancer cells. [ABSTRACT FROM AUTHOR]

The aryl hydrocarbon receptor (AHR) plays a major role in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced toxicity phenotypes. TCDD bound to AHR elicits both genomic action in which target genes are transcriptionally upregulated and nongenomic action in which cytosolic phospholipase A2α (cPLA2α) is rapidly activated. However, how either of these actions, separately or in combination, induces toxicity phenotypes is largely unknown. In this study, we used AHRnls/nls mice as a model in which AHR was mutated to lack nuclear translocation sequence (NLS), and AHRd/− mice as the corresponding control. Using this model, we studied TCDD-induced alterations in cPLA2α activation and related factors because of the pivotal roles of cPLA2α both in AHR's nongenomic action and in regulation of causative genes of TCDD-induced hydronephrosis. Dams were orally administered TCDD at a dose of 300 µg/kg body weight on postnatal day 1, and pups subsequently exposed to TCDD via milk were examined for gene expression on PND 7 and for histological changes on PND 14. The activation of the AHR genomic action and hydronephrosis onset were observed in the control group but not in the AHRnls/nls group. An ex vivo experiment using peritoneal macrophages exposed to 100 nM TCDD resulted in rapid activation of cPLA2α, an indicator of the nongenomic action, only in the control group but not in the AHRnls/nls group. These results indicated that an NLS is required for the AHR's genomic and nongenomic actions. [ABSTRACT FROM AUTHOR]

Key message: Overexpression of VaWRKY12, whose nuclear translocation increased under low temperature, enhanced the cold tolerance of Arabidopsis and grapevine calli and significantly increased the expression of antioxidant-related genes. Low temperature causes injuries to buds during winter and to young shoots during early spring, thereby affecting grapevine quality and yield. Understanding the regulatory mechanisms of cold stress responses is essential for the breeding of new grapevine cultivars with excellent cold tolerance. Previous studies indicated that WRKY family genes are induced by low temperature in grapevine, but their function in cold stress responses was not clear. Here, a cold-induced WRKY gene, named VaWRKY12, was cloned from Vitis amurensis, which displays remarkable cold tolerance. An atypical transmembrane (TM) region was found in its C-terminal region. Transient expression assays showed that VaWRKY12 was localized in the nucleus and cytoplasm at normal temperature but only in the nucleus after cold treatment. By contrast, a truncated version of VaWRKY12 without the TM region was found specifically in the nucleus at normal temperature, and its binding activity to tandem W-box elements in yeast was stronger than that of VaWRKY12, indicating that the TM region might affect the location and function of VaWRKY12. Overexpression of VaWRKY12 enhanced the cold tolerance of transformed Arabidopsis and grapevine calli. Transcriptome data revealed that the expression of genes encoding antioxidant enzymes, including peroxidases and glutathione S-transferases, was upregulated after cold treatment in VaWRKY12-overexpressing grapevine calli compared to the control calli. This study identifies candidate target genes as a basis for further studies on the roles of VaWRKY12 in cold stress responses in grapevine. [ABSTRACT FROM AUTHOR]

Main conclusion: The physiological importance of MpUVR8 in UV-B resistance and translocation in a UV-B-dependent manner from the cytosol into the nucleus is characterized in Marchantia polymorpha. UV RESISTANCE LOCUS 8 (UVR8) is an ultraviolet-B (UV-B) light receptor functioning for UV-B sensing and tolerance in Arabidopsis thaliana and other species. It is unclear whether UVR8 physiologically functions in UV-B-induced defense responses in Marchantia polymorpha, which belongs to the earliest diverging group of embryophyte lineages. Here, we demonstrate that UVR8 has a physiological function in UV-B tolerance and that there is a UVR8-dependent pathway involved. In addition, a UVR8-independent pathway is revealed. We examine the tissue-specific expression pattern of M. polymorpha UVR8 (MpUVR8), showing that it is highly expressed in the apical notch in thalli and gametangiophores, as well as in antheridial and archegonial heads. Furthermore, Mpuvr8KO plant transformants, in which the MpUVR8 locus was disrupted, were produced and analyzed to understand the physiological and molecular function of MpUVR8. Analysis using these plants indicates the important roles of MpUVR8 and MpUVR8-regulated genes, and of MpUVR8-independent pathways in UV-B tolerance. Subcellular localization of Citrine-fused MpUVR8 in M. polymorpha cells was also investigated. It was found to translocate from the cytosol into the nucleus in response to UV-B irradiation. Our findings indicate strong conservation of the physiological function of UVR8 and the molecular mechanisms for UVR8-dependent signal transduction through regulation of gene expression in embryophytes. [ABSTRACT FROM AUTHOR]

Background/Aims: The rapid nuclear translocation of signaling proteins upon stimulation is important for the regulation of de-novo gene expression. However, the molecular mechanisms of this translocation is not well understood, although some studies suggest that much of this translocation may be mediated by beta-like importins (Imps). Here we undertook to study the stimulated nuclear shuttling of JNK and p38 MAPKs. Methods: For this purpose, we used coimmunoprecipitation, proximity ligation assay, gel filtration and immunostaining to examine the mechanism of nuclear translocation of these proteins. Results: We found that JNK and p38 MAPKs translocate into the nucleus in a Ran dependent, but NLS- or NTS-independent manner, unrelated to their catalytic activity. We show that this translocation involves three β-like Imps, 3, 7 and 9. Knockdown of these Imps inhibits the nuclear translocation of the MAPKs, and thereby, phosphorylation of their transcription factor targets. We further demonstrate that the translocation requires the stimulated formation of heterotrimers composed of Imp3/ Imp7/MAPK or Imp3/Imp9/MAPK. JNK1/2 and p38α/β bind to either Imp7 or Imp9 upon stimulated post-translational modifications of the two Imps, while Imp3 joins the complex after its stimulation-induced phosphorylation. Once formed, these heterotrimers move to the nuclear envelope where Imp3 remains, while Imp7 or Imp9 escort the MAPKs into the nucleus. Conclusion: These results suggest that β-like Imps are central mediators of stimulated nuclear translocation of signaling proteins, providing a central level of regulation of the induction of cellular processes such as transcription upon stimulation. [ABSTRACT FROM AUTHOR]

Rewiring glucose metabolism, termed as Warburg effect or aerobic glycolysis, is a common signature of cancer cells to meet their high energetic and biosynthetic demands of rapid growth and proliferation. Pyruvate kinase M2 isoform (PKM2) is a key player in such metabolic reshuffle, which functions as a rate-limiting glycolytic enzyme in the cytosol of highly-proliferative cancer cells. During the recent decades, PKM2 has been extensively studied in non-canonical localizations such as nucleus, mitochondria, and extracellular secretion, and pertained to novel biological functions in tumor progression. Such functions of PKM2 open a new avenue for cancer researchers. This review summarizes up-to-date functions of PKM2 at various subcellular localizations of cancer cells and draws attention to the translocation of PKM2 from cytosol into the nucleus induced by posttranslational modifications. Moreover, PKM2 in tumor cells could have an important role in resistance acquisition processes against various chemotherapeutic drugs, which have raised a concern on PKM2 as a potential therapeutic target. Finally, we summarize the current status and future perspectives to improve the potential of PKM2 as a therapeutic target for the development of anticancer therapeutic strategies. [ABSTRACT FROM AUTHOR]

Abstract Aims To investigate whether mucin 1 (MUC1) downregulation reduced the sensitivity of tumor necrosis factor-alpha (TNF-α)-induced bronchial epithelial cells to glucocorticoid-mediated necroptosis and explore the underlying mechanisms. Main methods The human lung bronchial epithelial cell line (16HBE) was transfected with small interfering RNA (siRNA) against MUC1 and then stimulated by TNF-α, where some cells were pretreated with dexamethasone. Flow cytometry was performed to analyze necroptosis in 16HBE cells, and western blot analysis was used to detect protein expression levels of MUC1, glucocorticoid receptor (GR)α, GRβ, NF-κB p65, phospho-p65 (p-p65), and histone deacetylase-2 (HDAC2). Additionally, nuclear translocation of MUC1 and GRα was assessed by immunofluorescence. Key findings We observed that MUC1 downregulation by siRNA significantly augmented TNF-α-induced necroptosis in 16HBE cells, and that dexamethasone showed impaired anti-necroptotic effects of MUC1 downregulation. Furthermore, we found that GRα nuclear translocation was inhibited in 16HBE cells with MUC1 downregulation, and that dexamethasone-mediated inhibition of p65 phosphorylation was lower in cells transfected with MUC1-siRNA compared to those transfected with negative control siRNA. Significance Impaired GRα nuclear translocation and inhibited p-p65 expression might contribute to glucocorticoid resistance caused by MUC1 deficiency in TNF-α-induced necroptosis in 16HBE cells, and should be considered as a potential target for the development of novel therapeutics for asthma. [ABSTRACT FROM AUTHOR]

Hepatocyte cell adhesion molecule (HepaCAM) is a tumour suppressor. However, the mechanism of HepaCAM function in prostate cancer (PCa) remains unknown. In the present study, HepaCAM, androgen receptor (AR) and Ran were analysed in 46 PCa tissue samples using immunohistochemistry. Subsequently, the influence of HepaCAM and its cytoplasmic domain on cell proliferation, migration, and invasion, and associated proteins was examined using MTT, wound healing, Transwell and western blotting assays, respectively. Furthermore, nuclear translocation of AR and Ran was analysed using immunofluorescence and Western blot assays. The results demonstrated that HepaCAM expression was reduced in PCa, and there was an association between downregulation of HepaCAM and changes in the distribution of AR and Ran. Furthermore, HepaCAM, specifically the cytoplasmic domain, was involved in cell proliferation, migration and invasion. Nuclear translocation of AR was dependent on HepaCAM and its cytoplasmic domain. Additionally, HepaCAM suppression of the nuclear translocation of AR occurred via Ran. The results suggest that HepaCAM and its cytoplasmic domain suppress the nuclear translocation of AR via Ran in PCa. The cytoplasmic domain of HepaCAM may serve as a novel target for therapy in PCa. [ABSTRACT FROM AUTHOR]

Abstract The insulin-like growth factor-1 receptor (IGF1R) mediates the biological actions of both IGF1 and IGF2. In recent years, evidence has accumulated showing that, in addition to its classical cell-surface distribution, IGF1R translocates to cell nucleus via an apparently SUMO-1-dependent mechanism. While the role of IGF1R in nucleus has not yet been settled, available information suggests that the nuclear receptor displays activities usually linked to transcription factors, including DNA binding and transcription regulation. To gain insight into the biological pathways associated with nuclear IGF1R action we conducted a mass spectrometry-based proteomic analysis aimed at identifying interactors of IGF1R in nucleus of both benign and malignant breast cells. The nucleolar NOM1 molecule belongs to a family of proteins that contain the middle domain of eukaryotic initiation factor 4G (MIF4G) and/or interaction module (MA3), and functions in translation, cell growth and proliferation. Using a combination of co-immunoprecipitation and silencing assays we provide evidence of a complex, bi-directional interplay between nuclear IGF1R and nucleolar protein NOM1. Inhibition of nuclear IGF1R translocation by dansylcadaverine reduced NOM1 levels in nuclei of MCF7 cells. On the other hand, IGF1R overexpression enhanced NOM1 levels in the nuclear fraction. Of interest, NOM1 silencing led to a major increase in IGF1R biosynthesis. In summary, results are consistent with a physiologically-relevant interplay between the nuclear IGF1 signaling pathway and nucleolar protein NOM1. [ABSTRACT FROM AUTHOR]

The mitogen-activated protein kinase (MAPK) cascades are central signaling pathways that play a central role in the regulation of most stimulated cellular processes including proliferation, differentiation, stress response and apoptosis. Currently 4 such cascades are known, each termed by its downstream MAPK components: the extracellular signal-regulated kinase 1/2 (ERK1/2), cJun-N-terminal kinase (JNK), p38 and ERK5. One of the hallmarks of these cascades is the stimulated nuclear translocation of their MAPK components using distinct mechanisms. ERK1/2 are shuttled into the nucleus by importin7, JNK and p38 by a dimer of importin3 with either importin9 or importin7, and ERK5 by importin-α/β. Dysregulation of these cascades often results in diseases, including cancer and inflammation, as well as developmental and neurological disorders. Much effort has been invested over the years in developing inhibitors to the MAPK cascades to combat these diseases. Although some inhibitors are already in clinical use or clinical trials, their effects are hampered by development of resistance or adverse side-effects. Recently, our group developed 2 myristoylated peptides: EPE peptide, which inhibits the interaction of ERK1/2 with importin7, and PERY peptide, which prevents JNK/p38 interaction with either importin7 or importin9. These peptides block the nuclear translocation of their corresponding kinases, resulting in prevention of several cancers, while the PERY peptide also inhibits inflammation-induced diseases. These peptides provide a proof of concept for the use of the nuclear translocation of MAPKs as therapeutic targets for cancer and/or inflammation. [ABSTRACT FROM AUTHOR]

Highlights • Membrane-initiated glucocorticoid signaling induces GR nuclear translocation. • Nuclear translocation of an unliganded glucocorticoid receptor. • Distinct transcriptional regulation by the unliganded glucocorticoid receptor. Abstract Glucocorticoid binding to the intracellular glucocorticoid receptor (GR) stimulates the translocation of the GR from the cytosol to the nucleus, which leads to the transactivation or transrepression of gene transcription. However, multiple lines of evidence suggest that glucocorticoid signaling can also be initiated from the plasma membrane. Here, we provide evidence for membrane-initiated glucocorticoid signaling by a membrane-impermeant dexamethasone-bovine serum albumin (Dex-BSA) conjugate, which induced GR nuclear trafficking in hypothalamic neurons in vitro and in vivo. The GR nuclear translocation induced by a membrane-impermeant glucocorticoid suggests trafficking of an unliganded GR. The membrane-initiated GR trafficking was not blocked by inhibiting ERK MAPK, p38 MAPK, PKA, Akt, Src kinase, or calcium signaling, but was inhibited by Akt activation. Short-term exposure of hypothalamic neurons to dexamethasone (Dex) activated the glucocorticoid response element (GRE), suggesting transcriptional transactivation, whereas exposure to the Dex-BSA conjugate failed to activate the GRE, suggesting differential transcriptional activity of the liganded compared to the unliganded GR. Microarray analysis revealed divergent transcriptional regulation by Dex-BSA compared to Dex. Together, our data suggest that signaling from a putative membrane glucocorticoid receptor induces the trafficking of unliganded GR to the nucleus, which elicits a pattern of gene transcription that differs from that of the liganded receptor. The differential transcriptional signaling by liganded and unliganded receptors may contribute to the broad range of genetic regulation by glucocorticoids, and may help explain some of the different off-target actions of glucocorticoid drugs. [ABSTRACT FROM AUTHOR]