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Active Notch signalling is elicited through receptor-ligand interactions that result in release of the Notch intracellular domain (NICD), which translocates into the nucleus. NICD activates transcription at target genes forming a complex with the DNA-binding transcription factor CSL (CBF1/Su(H)/Lag-1) and co-activator Mastermind. Despite this, CSL lacks its own nuclear localisation sequence, and it remains unclear where the tripartite complex is formed. To probe mechanisms involved, we designed an optogenetic approach to control NICD release (OptIC-Notch) and monitored consequences on complex formation and target gene activation. Strikingly we observed that, when uncleaved, OptIC-Notch sequestered CSL in the cytoplasm. Hypothesising that exposure of a juxta membrane ΦWΦP motif is key to sequestration, we masked this motif with a second light sensitive domain in OptIC-Notch{ω}, which was sufficient to prevent CSL sequestration. Furthermore, NICD produced by light-induced cleavage of OptIC-Notch or OptIC-Notch{ω} chaperoned CSL into the nucleus and induced target gene expression, showing efficient light controlled activation. Our results demonstrate that exposure of the ΦWΦP motif leads to CSL recruitment and suggest this can occur in the cytoplasm prior to nuclear entry.Summary statementLight controlled exposure of the Notch ΦWΦP motif leads to CSL sequestration and suggest this interaction could occur prior to nuclear entry during normal signalling.

Tumor necrosis factor (TNF) α-induced nuclear translocation of the NF-κB subunit RELA has been implicated in several pathological conditions. Here we report the discovery of a spirocyclic dimer (SpiD7) that covalently modifies RELA to inhibit TNFα-induced nuclear translocation. This is a previously unexplored strategy to inhibit TNFα-induced NF-κB activation., Discovery of a spirocyclic dimer (SpiD7) that covalently modifies RELA to generate stable high molecular weight complexes. SpiD7 inhibits TNFα-induced nuclear translocation of RELA resulting in the blockade of NF-kB gene transcription, through a previously unexplored modality.

Drought is the leading environmental threat affecting crop productivity, and plants have evolved a series of mechanisms to adapt to drought stress. The FT-interacting proteins (FTIPs) and phosphatidylethanolamine-binding proteins (PEBPs) play key roles in developmental processes, whereas their roles in the regulation of stress response are still largely unknown. Here, we report that OsFTIP1 negatively regulates drought response in rice. We showed that OsFTIP1 interacts with rice MOTHER OF FT AND TFL1 (OsMFT1), a PEBP that promotes rice tolerance to drought treatment. Further studies discovered that OsMFT1 interacts with two key drought-related transcription factors, OsbZIP66 and OsMYB26, regulating their binding capacity on drought-related genes and thereby enhancing drought tolerance in rice. Interestingly, we found that OsFTIP1 impedes the nucleocytoplasmic translocation of OsMFT1, implying that dynamic modulation of drought-responsive genes by the OsMFT1-OsMYB26 and OsMFT1-OsbZIP66 complexes is integral to OsFTIP1-modulated nuclear accumulation of OsMFT1. Our findings also suggest that OsMFT1 might act as a hitherto unknown nucleocytoplasmic trafficking signal that regulates drought tolerance in rice in response to environmental signals.

In our previous study, we demonstrated that norcantharidin (NCTD) is a potential therapeutic agent for renal interstitial fibrosis (RIF). Recently, we found that lncRNA Gm26669 (Gm26669) contributed to the development of RIF and could be regulated by NCTD. However, the upstream mechanisms of Gm26669 and whether the anti-RIF effects of NCTD are related to its regulatory action on Gm26669 remain unclear. Our bioinformatics analysis indicated that special protein1 (Sp1), a transcription factor, may bind to the promoter of Gm26669. In the present study, we observed a significant increase in the nuclear translocation of Sp1 using both in vivo and in vitro models of RIF. Furthermore, the knockdown of Sp1 inhibited the expression of collagen type I (CoL-I) and fibronectin (Fn). Mechanistically, Sp1 promoted the expression levels of CoL-I and Fn by directly binding to the promoter of Gm26669 to elevate its expression level. Moreover, we found that NCTD alleviated RIF by inhibiting Gm26669 and the nuclear translocation of Sp1. Collectively, above results suggested that NCTD might prevent RIF via targeting the Sp1/Gm26669 axis, thus providing a new theoretical basis for the clinical application of NCTD in the treatment of RIF.

Pathogenic helminths have evolved mechanisms to preserve reproductive function while surviving long-term in the host via robust protective responses. A protective role of antioxidant enzymes in preventing DNA degradation has long been proposed, but little evidence has been provided. Here, we show that omega-class glutathione transferases (GSTOs) are critical for maintaining viability by protecting the reproductive cell DNA of the carcinogenic liver fluke, Clonorchis sinensis. Clonorchis sinensis GSTO (CsGSTO) activities modified by changes in the GSH/GSSG and NADPH/NADP+ molar ratios suppressed the overproduction of reactive oxygen species. CsGSTO1 and CsGSTO2 catalyzed deglutathionylation under physiologic and low-stress conditions (GSH/GSSG ratio of 6:1 or higher) but promoted glutathionylation under high-stress conditions (GSH/GSSG ratio of 3:1 or lower). Gliotoxin-induced functional disruption of CsGSTOs in living C. sinensis reduced the GSH/GSSG molar ratio and increased the production of protein glutathionylation (PSSG) under physiologic and low-stress conditions, indicating that suppression of GSTO function did not affect deglutathionylation. However, the perturbation of CsGSTOs decreased the GSH/GSSG ratio but also reduced PSSG production under high oxidative stress, demonstrating that glutathionylation was impeded. In response to oxidative stimuli, C. sinensis decreased GSTO-specific dehydroascorbate reductase and thiol transferase activities and the GSH/GSSG ratio, while it increased the NADPH/NADP+ ratio and PSSG. CsGSTOs utilized GSH to regulate GSH/GSSG and NADPH/NADP+ recycling and triggered a redox signal leading to nuclear translocation. Nuclear-imported CsGSTOs were modified by glutathionylation to prevent DNA damage. Antibodies specific to CsGSTOs dose-dependently inhibited this process. Disruption of CsGSTOs or the depletion of GSH caused glutathionylation defects, leading to DNA degradation. Our results demonstrate that CsGSTOs and the GSH system play a previously unappreciated role in protecting DNA from oxidative stress.

Our study examined for the inhibitory effect of ethanolic extract of Eleagnus Conferta Roxb leaves on TNF –α induced NF- kB nuclear translocation in lung cancer A549 cell line using flow cytometry. Apoptosis also studied to know about the antiproliferative and anticancer effects. However, our results revealed in apoptosis, Elaeagnus conferta Roxb leaves showed (33.54%) increased in proportion of cells. In the study of pre-treatment of A549 cells with Elaeagnus conferta Roxb leaves followed by TNF- α caused the increased proportion of cells (20.78%) at apoptosis induced cell death, which was statistically significant (p< 0.001). The untreated A549 cells had minimal NF-kB expression (0.25± 0.01%). However, the approach of A549 cells with Elaeagnus conferta Roxb had induced NF-kB production many fold (11.50± 1.05%). Therefore, we conclude our study was proved the impact of Elaeagnus conferta Roxb leaves inhibit the cellular growth of NSCLC-A549 cell line and induces apoptosis. Hence, from our findings, we proved this plant has anticancer activity, further feasibly taken for drug formulation.

Humans possess two Sin3 paralogs, SIN3A and SIN3B, that serve as scaffolds within the Sin3 complexes. There is existing evidence that paralog identity influences Sin3 complex composition. Using proteomic profiling, we characterize this influence and highlight heterogeneity within the population of Sin3 complexes. We incorporate chemical cross-linking mass spectrometry into our assays to define domains within SIN3A and SIN3B that mediate protein interactions. These data highlight the modular nature of Sin3 complexes., Graphical Abstract Highlights • Sin3 paralog identity influences Sin3 complex composition. • Chemical cross-linking mass spectrometry identifies domains in SIN3A and SIN3B that mediate complex formation. • Complex subunit homology to yeast Sin3 complex components may assist in defining distinct forms of the Sin3 complex in humans. • A nuclear import signal within SIN3B is identified via chemical cross-linking mass spectrometry., Despite the continued analysis of HDAC inhibitors in clinical trials, the heterogeneous nature of the protein complexes they target limits our understanding of the beneficial and off-target effects associated with their application. Among the many HDAC protein complexes found within the cell, Sin3 complexes are conserved from yeast to humans and likely play important roles as regulators of transcriptional activity. The presence of two Sin3 paralogs in humans, SIN3A and SIN3B, may result in a heterogeneous population of Sin3 complexes and contributes to our poor understanding of the functional attributes of these complexes. Here, we profile the interaction networks of SIN3A and SIN3B to gain insight into complex composition and organization. In accordance with existing data, we show that Sin3 paralog identity influences complex composition. Additionally, chemical cross-linking MS identifies domains that mediate interactions between Sin3 proteins and binding partners. The characterization of rare SIN3B proteoforms provides additional evidence for the existence of conserved and divergent elements within human Sin3 proteins. Together, these findings shed light on both the shared and divergent properties of human Sin3 proteins and highlight the heterogeneous nature of the complexes they organize.

Anthracyclines are a class of conventional and commonly used frontline chemotherapy drugs to treat breast cancer. However, the anthracycline-based regimens can only reduce breast cancer mortality by 20–30%. Furthermore, there is no appropriate biomarker for predicting responses to this kind of chemotherapy currently. Here we report our findings that may fill this gap by showing the AQP1 (Aquaporin1) protein as a potential response predictor in the anthracycline chemotherapy. We showed that breast cancer patients with a high level of AQP1 expression who underwent the anthracycline treatment had a better clinical outcome relative to those with a low level of AQP1 expression. In the exploration of the underlying mechanisms, we found that the AQP1 and glycogen synthase kinase-3β (GSK3β) competitively interacted with the 12 armadillo repeats of β-catenin, followed by the inhibition of the β-catenin degradation that led to β-catenin’s accumulation in the cytoplasm and nuclear translocation. The nuclear β-catenin interacted with TopoIIα and enhanced TopoIIα’s activity, which resulted in a high sensitivity of breast cancer cells to anthracyclines. We also found, the miR-320a-3p can attenuate the anthracycline’s chemosensitivity by inhibiting the AQP1 expression. Taken together, our findings suggest the efficacy of AQP1 as a response predictor in the anthracycline chemotherapy. The application of our study includes, but is not limited to, facilitating screening of the most appropriate breast cancer patients (who have a high AQP1 expression) for better anthracycline chemotherapy and improved prognosis purposes.

Hypoxia-inducible factors (HIFs) are key mediators expressed under hypoxic condition and involved in many kinds of disease such as cancer and abnormal angiogenesis. Thus, development of their inhibitor has been extensively explored. Here, we describe a finding that Remodelin, a specific inhibitor of NAT10, could also inhibit the expression of HIFs. The presence of Remodelin could suppress the elevated level of HIF-1α protein and its nuclear translocation induced by either treatment of cobalt chloride (CoCl2) or hypoxia in dose or time-dependent way. More importantly, Remodelin could also inhibit the constitutional expression of HIF-1α and HIF-2α in VHL mutant 786-0 cells. With using of cells with depletion of NAT10 by shRNA or Crispr-Cas9 edited, we further demonstrated that inhibition of HIFs by Remodelin should need NAT10 activity. In biological analysis, the treatment of cultured HUVECs with Remodelin could inhibit in vitro cell migration and invasion and tube-formation. Our investigation implied that Remodelin could be a new potential inhibitor of HIFs for using in angiogenesis targeting therapy in either cancers or inflammatory diseases.

DNA damage signals transducer RING finger protein 8 (RNF8) is involved in maintaining genomic stability by facilitating the repair of DNA double-strand breaks (DSB) via ubiquitin signaling. By analyzing the TCGA database and colon cancer tissue microarrays, we found that the expression level of RNF8 was positively correlated with that of c-Myc in colon cancer, which were closely associated with poor survival of colon cancer patients. Furthermore, overexpressing and knocking down RNF8 increased and decreased the expression of c-Myc in colon cancer cells, respectively. In addition, RNF8 interacted with β-catenin and facilitated its nuclear translocation by conjugating K63 polyubiquitination on it. These observations suggested a de novo role of RNF8 in promoting the progression of colon cancer by inducing β-catenin-mediated c-Myc expression.

Mechanical stimulation is well known to be important for maintaining tissue and organ homeostasis. Here, we found that hydrostatic pressure induced nuclear translocation of a forkhead box O (FOXO) transcription factor DAF-16, in C. elegans within minutes, whereas the removal of this pressure resulted in immediate export of DAF-16 to the cytoplasm. We also monitored DAF-16-dependent transcriptional changes by exposure to 1 MPa pressure for 5 min, and found significant changes in collagen and other genes in a DAF-16 dependent manner. Lifespan was markedly prolonged with exposure to cyclic pressure treatment (1 MPa once a day for 5 min from L1 larvae until death). Furthermore, age-dependent decline in locomotor activity was suppressed by the treatment. In contrast, the nuclear translocation of the yes-associated protein YAP-1 was not induced under the same pressure conditions. Thus, moderate hydrostatic pressure improves ageing progression through activation of DAF-16/FOXO in C. elegans.

Squamous cell carcinoma of the head and neck (HNSCC) is a common malignant tumor in humans and animals. In humans, papillomavirus (PV)-induced HNSCCs have a better prognosis than papillomavirus-unrelated HNSCCs. The ability of tumor cells to switch from epithelial to mesenchymal, endothelial, or therapy-resistant stem-cell-like phenotypes promotes disease progression and metastasis. In equine HNSCC, PV-association and tumor cell phenotype switching are poorly understood. We screened 49 equine HNSCCs for equine PV (EcPV) type 2, 3 and 5 infection. Subsequently, PV-positive versus -negative lesions were analyzed for expression of selected epithelial (keratins, β-catenin), mesenchymal (vimentin), endothelial (COX-2), and stem-cell markers (CD271, CD44) by immunohistochemistry (IHC) and immunofluorescence (IF; keratins/vimentin, CD44/CD271 double-staining) to address tumor cell plasticity in relation to PV infection. Only EcPV2 PCR scored positive for 11/49 equine HNSCCs. IHC and IF from 11 EcPV2-positive and 11 EcPV2-negative tumors revealed epithelial-to-mesenchymal transition events, with vimentin-positive cells ranging between 50%. CD44- and CD271-staining disclosed the intralesional presence of infiltrative tumor cell fronts and double-positive tumor cell subsets independently of the PV infection status. Our findings are indicative of (partial) epithelial–mesenchymal transition events giving rise to hybrid epithelial/mesenchymal and stem-cell-like tumor cell phenotypes in equine HNSCCs and suggest CD44 and CD271 as potential malignancy markers that merit to be further explored in the horse.

Glutathione S-transferase pi (GSTpi) is an important phase Ⅱ detoxifying enzyme that participates in various physiological processes, such as antioxidant, detoxification, and signal transduction. The high expression level of GSTpi has been reported to be related to drug-resistant and anti-inflammatory and it functioned via its non-catalytic ligandin. However, the previous protection mechanism of GSTpi in DNA damage has not been addressed so far. Nijmegen breakage syndrome 1 (NBS1) is one of the most important sensor proteins to detect damaged DNA. Here, we investigated the interaction between GSTpi and NBS1 in HEK-293T cells and human breast adenocarcinoma cells during DNA damage. Our results showed that overexpression of GSTpi in cells by transfecting DNA vector decreased the DNA damage level after Methyl methanesulfonate (MMS) or Adriamycin (ADR) treatment. We found that cytosolic GSTpi could increase NBS1 ubiquitin-mediated degradation in unstimulated cells, which suggested that GSTpi could maintain the basal level of NBS1 during normal conditions. In response to DNA damage, GSTpi can be phosphorylated in Ser184 and inhibit the ubiquitination degradation of NBS1 mediated by Skp2 to recover NBS1 protein level. Phosphorylated GSTpi can further enhance NBS1 nuclear translocation to activate the ATM-Chk2-p53 signaling pathway. Finally, GSTpi blocked the cell cycle in the G2/M phase to gain more time for cells to repair. Thus, our finding revealed the novel mechanism of GSTpi via its Ser184 phosphorylation to protect cells from cell death during DNA damage and it enriches the function of GSTpi in drug resistance.

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.

Juvenile hormone (JH) plays vital roles in insect reproduction, development, and in many aspects of physiology. JH primarily acts at the gene-regulatory level through interaction with an intracellular receptor (JHR), a ligand-activated complex of transcription factors consisting of the JH-binding protein Methoprene-tolerant (MET) and its partner Taiman (TAI). Initial studies indicated significance of post-transcriptional phosphorylation, subunit assembly, and nucleocytoplasmic transport of JHR in JH signaling. However, our knowledge of JHR regulation at the protein level remains rudimentary, partly due to the difficulty of obtaining purified, functional JHR proteins. Here we present a method for high-yield expression and purification of JHR complexes from two insect species, the beetle Tribolium castaneum and the mosquito Aedes aegypti. Recombinant JHR subunits from each species were co-expressed in an insect cell line using a baculovirus system. MET-TAI complexes were purified through affinity chromatography and anion exchange columns to yield proteins capable of binding both the hormonal ligand (JH III) and DNA bearing cognate JH-response elements. We further examined the beetle JHR complex (TcJHR) in greater detail. Biochemical analyses and mass spectrometry confirmed that TcJHR was a 1:1 heterodimer consisting of TcMET and TcTAI proteins, stabilized by the JHR agonist ligand methoprene. Phosphoproteomics uncovered multiple phosphorylation sites in the TcMET protein, some of which were induced by methoprene treatment. Finally, we report a functional bipartite nuclear localization signal, straddled by phosphorylated residues, within the disordered C-terminal region of TcMET. Our present characterization of the recombinant JHR is an initial step towards understanding JHR structure and function.

Eleven new polycyclic polyprenylated acylphloroglucinols (PPAPs), hyperwilsones A–K (1–11), along with five known PPAPs (12–16), were isolated from Hypericum wilsonii. Their structures were established via spectroscopic methods, the careful analysis of calculated and experimental electronic circular dichroism (ECD) spectra, single-crystal X-ray diffraction, the modified Mosher’s method, and [Rh2(OCOCF3)4]-induced ECD. Hyperwilsone A (1) and hyperwilsone B (2) possessed the unique acetal functionality. Hyperwilsone C (3) was a rare example of [3.3.1]-type PPAP possessing a 3-isopropylfuran moiety. In bioassay, compounds 9 and 10 showed potent anti-inflammatory activity against LPS-induced NO production by inhibiting the nuclear translocation of NF-κB p65 and thus reducing the production of proinflammatory cytokines. Compounds 5, 8, 11, and 14 exhibited moderate inhibitory activity against SUDHL-4 and HL60 cancer cells with IC50 values in the range of 5.74–19.82 μM.

Dorsal-ventral patterning of the Drosophila embryo depends on the NFκB superfamily transcription factor Dorsal (Dl). Toll receptor activation signals for degradation of the IκB inhibitor Cactus (Cact), leading to a ventral-to-dorsal nuclear Dl gradient. Cact is critical for Dl nuclear import, as it binds to and prevents Dl from entering the nuclei. Quantitative analysis of cact mutants revealed an additional Cact function to promote Dl nuclear translocation in ventral regions of the embryo. To investigate this dual Cact role, we developed a predictive model based on a reaction-diffusion regulatory network. This network distinguishes non-uniform Toll-dependent Dl nuclear import and Cact degradation, from the Toll-independent processes of Cact degradation and reversible nuclear-cytoplasmic Dl flow. In addition, it incorporates translational control of Cact levels by Dl. Our model successfully reproduces wild-type data and emulates the Dl nuclear gradient in mutant dl and cact allelic combinations. Our results indicate that the dual role of Cact depends on the dynamics of Dl-Cact trimers along the dorsal-ventral axis: In the absence of Toll activation, free Dl-Cact trimers retain Dl in the cytoplasm, limiting the flow of Dl into the nucleus; in ventral-lateral regions, Dl-Cact trimers are recruited by Toll activation into predominant signaling complexes and promote Dl nuclear translocation. Simulations suggest that the balance between Toll-dependent and Toll-independent processes are key to this dynamics and reproduce the full assortment of Cact effects. Considering the high evolutionary conservation of these pathways, our analysis should contribute to understanding NFκB/c-Rel activation in other contexts such as in the vertebrate immune system and disease., Author summary In Drosophila, Toll pathway establishes spatially distinct gene expression territories that define the embryonic dorsal-ventral axis. Toll activation leads to degradation of the IκB inhibitor Cactus, releasing the NFκB superfamily transcription factor Dorsal for nuclear entry. Recently, quantitative analysis of cact mutants revealed that Cact displays an additional function to promote Dl nuclear translocation in ventral regions of the embryo. To understand this novel activity, we developed a predictive theoretical model that shows that the kinetics of Dorsal-Cactus complex formation prior to their recruitment to Toll-signaling complexes is an essential regulatory hub. Cactus controls the balance between the recruitment of these complexes by active Toll receptor and association-dissociation events that generate free Dorsal for direct nuclear import.

Proteasome inhibition (PSMI) is known to activate macroautophagy (autophagy hereafter), but the underlying mechanisms remain to be fully delineated. Here we discuss our recent work identifying an important PPP3/calcineurin-TFEB-SQSTM1/p62 pathway in mediating activation of autophagy by PSMI, a compensatory process for the heart with proteasome malfunction. Through increasing PPP3/calcineurin activity and inhibiting MTOR signaling, PSMI promotes the dephosphorylation and thereby nuclear translocation of TFEB, resulting in transactivation of genes in the autophagic-lysosomal pathway (ALP) such as Mcoln1 and Sqstm1. We have discovered that SQSTM1 is required for not only induction of autophagy but also cardiac activation of TFEB by PSMI, unveiling a novel feedforward role for SQSTM1 in TFEB activation.

Serine–arginine (SR) proteins are essential splicing factors that promote numerous steps associated with mRNA processing and whose biological function is tightly regulated through multi-site phosphorylation. In the nucleus, the cdc2-like kinases (CLKs) phosphorylate SR proteins on their intrinsically disordered Arg–Ser (RS) domains, mobilizing them from storage speckles to the splicing machinery. The CLKs have disordered N termini that bind tightly to RS domains, enhancing SR protein phosphorylation. The N termini also promote nuclear localization of CLKs, but their transport mechanism is presently unknown. To explore cytoplasmic–nuclear transitions, several classical nuclear localization sequences in the N terminus of the CLK1 isoform were identified, but their mutation had no effect on subcellular localization. Rather, we found that CLK1 amplifies its presence in the nucleus by forming a stable complex with the SR protein substrate and appropriating its NLS for transport. These findings indicate that, along with their well-established roles in mRNA splicing, SR proteins use disordered protein–protein interactions to carry their kinase regulator from the cytoplasm to the nucleus.

The human enhancer of filamentation 1 (HEF1) is a multi-domain docking protein of the p130 Cas family. Research reports on the mechanism of HEF1 in gastric cancer (GC) differentiation are limited. In this study, we reveal that HEF1 plays an essential role in regulating of differentiation in human GC. HEF1 was found to be highly expressed in GC tissues. Besides, In GC tissues or cells, cellular level of HEF1 negatively correlated with tumor differentiation. In addition, we showed that upregulation of HEF1 increased Wnt5a expression and the nuclear translocation of β-catenin, thereby resulting in poor differentiation in GC. Notably, GC patients with a higher expression of HEF1 showed significantly poorer disease-free and overall survival. Thus, our findings suggest that HEF1 reduces differentiation through the Wnt5a/β-catenin signaling pathway and that HEF1 is an independent unfavorable prognostic death factor in GC.

We recently established a new plasma peptidomic technique and comprehensively identified a large number of low-molecular weight and low-abundance native peptides using a single drop of human plasma. To discover a novel polypeptide that potently modulates the cardiovascular system, we performed a bioinformatics analysis of the large-scale identification results, sequentially synthesized the selected peptide sequences, tested their biological activities, and identified a 30-amino-acid proatherogenic peptide, GIP_HUMAN[22–51], as a potent proatherosclerotic peptide hormone. GIP_HUMAN[22–51] has a common precursor with the glucose-dependent insulinotropic polypeptide (GIP) and is located immediately N-terminal to GIP. Chronic infusion of GIP_HUMAN[22–51] into ApoE−/− mice accelerated the development of aortic atherosclerotic lesions, which were inhibited by co-infusions with an anti-GIP_HUMAN[22–51] antibody. GIP_HUMAN[22–51] increased the serum concentrations of many inflammatory and proatherogenic proteins, whereas neutralising antibodies reduced their levels. GIP_HUMAN[22–51] induced IκB-α degradation and nuclear translocation of NF-κB in human vascular endothelial cells and macrophages. Immunoreactive GIP_HUMAN[22–51] was detected in human tissues but there was no colocalization with the GIP. The plasma GIP_HUMAN[22–51] concentration in healthy humans determined using a stable-isotope tagged peptide was approximately 0.6 nM. This study discovered a novel endogenous proatherogenic peptide by using a human plasma native peptidomic resource.

A year ago, when I first contemplated writing this article, my intent was to provide a detailed review of the contributions of the diverse community of talented scientists in my lab to the nuclear receptor research field. In the throes of a deadly pandemic, political turmoil, and Black Lives Matter, however, I found myself compelled to tell a more personal story. While I will still cover milestones in our understanding of the intracellular trafficking of the thyroid hormone receptor, now these will be set against the backdrop of my path as a woman in STEM and on being intentionally inclusive. By sharing reflections on my journey, I hope to encourage young investigators to persist in their pursuit of a career in science.

We have observed overexpression of PACS-1, a cytosolic sorting protein in primary cervical tumors. Absence of exonic mutations and overexpression at the RNA level suggested a transcriptional and/or posttranscriptional regulation. University of California Santa Cruz genome browser analysis of PACS-1 micro RNAs (miR), revealed two 8-base target sequences at the 3′ terminus for hsa-miR-34a and hsa-miR-449a. Quantitative RT-PCR and Northern blotting studies showed reduced or loss of expression of the two microRNAs in cervical cancer cell lines and primary tumors, indicating dysregulation of these two microRNAs in cervical cancer. Loss of PACS-1 with siRNA or exogenous expression of hsa-miR-34a or hsa-miR-449a in HeLa and SiHa cervical cancer cell lines resulted in DNA damage response, S-phase cell cycle arrest, and reduction in cell growth. Furthermore, the siRNA studies showed that loss of PACS-1 expression was accompanied by increased nuclear γH2AX expression, Lys(382)-p53 acetylation, and genomic instability. PACS-1 re-expression through LNA-hsa-anti-miR-34a or -449a or through PACS-1 cDNA transfection led to the reversal of DNA damage response and restoration of cell growth. Release of cells post 24-h serum starvation showed PACS-1 nuclear localization at G(1)-S phase of the cell cycle. Our results therefore indicate that the loss of hsa-miR-34a and hsa-miR-449a expression in cervical cancer leads to overexpression of PACS-1 and suppression of DNA damage response, resulting in the development of chemo-resistant tumors.

The human genome contains vast genetic diversity as naturally occurring coding variants, yet the impact of these variants on protein function and physiology is poorly understood. RGS14 is a multifunctional signaling protein that suppresses synaptic plasticity in dendritic spines of hippocampal neurons. RGS14 also is a nucleocytoplasmic shuttling protein, suggesting that balanced nuclear import/export and dendritic spine localization are essential for RGS14 functions. We identified genetic variants L505R (LR) and R507Q (RQ) located within the nuclear export sequence (NES) of human RGS14. Here we report that RGS14 encoding LR or RQ profoundly impacts protein functions in hippocampal neurons. RGS14 membrane localization is regulated by binding Gαi-GDP, whereas RGS14 nuclear export is regulated by Exportin 1 (XPO1). Remarkably, LR and RQ variants disrupt RGS14 binding to Gαi1-GDP and XPO1, nucleocytoplasmic equilibrium, and capacity to inhibit long-term potentiation (LTP). Variant LR accumulates irreversibly in the nucleus, preventing RGS14 binding to Gαi1, localization to dendritic spines, and inhibitory actions on LTP induction, while variant RQ exhibits a mixed phenotype. When introduced into mice by CRISPR/Cas9, RGS14-LR protein expression was detected predominantly in the nuclei of neurons within hippocampus, central amygdala, piriform cortex, and striatum, brain regions associated with learning and synaptic plasticity. Whereas mice completely lacking RGS14 exhibit enhanced spatial learning, mice carrying variant LR exhibit normal spatial learning, suggesting that RGS14 may have distinct functions in the nucleus independent from those in dendrites and spines. These findings show that naturally occurring genetic variants can profoundly alter normal protein function, impacting physiology in unexpected ways.

We investigated the therapeutic potential and mechanism of chitosan oligosaccharides (COS) for experimental autoimmune uveoretinitis (EAU) in mice. EAU was induced in C57/BL6 mice by injection of human interphotoreceptor retinoid-binding protein (IRBP) peptides. At the same time, a high or low dose (20 or 10 mg/kg) of COS or phosphate-buffered saline (PBS) was given to mice daily after EAU induction. We found that mouse EAU is ameliorated by the high-dose COS treatment when compared with PBS treatment. In the retinas of high-dose COS-treated mice, the nuclear translocation of NF-&kappa, B subunit (p65) was suppressed, and the expression of several key EAU inflammatory mediators, IFN-&gamma, TNF-&alpha, IL-1&alpha, IL-4, IL-5, IL-6, IL-10, IL-17 and MCP-1 was lowered. These results suggest that COS may be a potential treatment for posterior uveitis.

Disorders that disrupt myelin formation during development or in adulthood, such as multiple sclerosis and peripheral neuropathies, lead to severe pathologies, illustrating myelin's crucial role in normal neural functioning. However, although our understanding of glial biology is increasing, the signals that emanate from axons and regulate myelination remain largely unknown. To identify the core components of the myelination process, here we adopted a microarray analysis approach combined with laser-capture microdissection of spinal motoneurons during the myelinogenic phase of development. We identified neuronal genes whose expression was enriched during myelination and further investigated hepatoma-derived growth factor-related protein 3 (HRP3 or HDGFRP3). HRP3 was strongly expressed in the white matter fiber tracts of the peripheral (PNS) and central (CNS) nervous systems during myelination and remyelination in a cuprizone-induced demyelination model. The dynamic localization of HPR3 between axons and nuclei during myelination was consistent with its axonal localization during neuritogenesis. To study this phenomenon, we identified two splice variants encoded by theHRP3gene: the canonical isoform HRP3-I and a newly recognized isoform, HRP3-II. HRP3-I remained solely in the nucleus, whereas HRP3-II displayed distinct axonal localization both before and during myelination. Interestingly, HRP3-II remained in the nuclei of unmyelinated neurons and glial cells, suggesting the existence of a molecular machinery that transfers it to and retains it in the axons of neurons fated for myelination. Overexpression of HRP3-II, but not of HRP3-I, increased Schwann cell numbers and myelination in PNS neuron-glia co-cultures. However, HRP3-II overexpression in CNS co-cultures did not alter myelination. Christopher and Dana Reeve Foundation Roche Foundation Swiss National Science Foundation (SNSF) American Heart Association Paul G. Allen Frontiers Group Grant Leona M. and Harry B. Helmsley Charitable Trust Grant Grace Foundation JPB Foundation Robert and Mary Jane Engman Foundation Ray and Dagmar Dolby Family Fund Crick-Jacob Center for Theoretical and Computational Biology Neurosurgery Neuroscience Consortium Fellowship

The MAP kinase ERK5 contains an N-terminal kinase domain and a unique C-terminal tail including a nuclear localization signal and a transcriptional activation domain. ERK5 is activated in response to growth factors and stresses and regulates transcription at the nucleus by either phosphorylation or interaction with transcription factors. MEK5-ERK5 pathway plays an important role regulating cancer cell proliferation and survival. Therefore, it is important to define the precise molecular mechanisms implicated in ERK5 nucleo-cytoplasmic shuttling. We previously described that the molecular chaperone Hsp90 stabilizes and anchors ERK5 at the cytosol and that ERK5 nuclear shuttling requires Hsp90 dissociation. Here, we show that MEK5 or overexpression of Cdc37&mdash, mechanisms that increase nuclear ERK5&mdash, induced ERK5 Small Ubiquitin-related Modifier (SUMO)-2 modification at residues Lys6/Lys22 in cancer cells. Furthermore, mutation of these SUMO sites abolished the ability of ERK5 to translocate to the nucleus and to promote prostatic cancer PC-3 cell proliferation. We also show that overexpression of the SUMO protease SENP2 completely abolished endogenous ERK5 nuclear localization in response to epidermal growth factor (EGF) stimulation. These results allow us to propose a more precise mechanism: in response to MEK5 activation, ERK5 SUMOylation favors the dissociation of Hsp90 from the complex, allowing ERK5 nuclear shuttling and activation of the transcription.

The characteristic constituent and anti-inflammatory activity of 5- n-alkylresorcinols (ARs) from 21 wheat bran samples in China were investigated in this study. The amount of ARs ranged from 697 to 1732 μg/g in the tested samples, which were composed of five different homologues. Among these homologues, C19:0 and C21:0 were the most abundant, followed by C17:0, C23:0, and C25:0. Moreover, the mRNA expression of IL-1β, IL-6, and TNF-α in LPS-activated RAW264.7 macrophage cells were significantly inhibited by ARs supplementation. The molecular mechanisms behind its anti-inflammatory activity could result from the suppression of nuclear factor-κB (NF-κB) and JNK/MAPK activation. ARs treatment notably decreased NF-κB p65 nuclear translocation and inhibitor κB (IκBα) kinase and JNK phosphorylation. Additionally, ARs homologues C17:0 had been proven to be the main active constituent. The results from this study could be used to promote the comprehensive utilization of wheat and its byproducts in improving human health.

Classical swine fever virus (CSFV) causes a contagious disease of pigs. The virus can break the mucosal barrier to establish its infection. Type III interferons (IFN-&lambda, s) play a crucial role in maintaining the antiviral state in epithelial cells. Limited information is available on whether or how CSFV modulates IFN-&lambda, s production. We found that IFN-&lambda, 3 showed dose-dependent suppression of CSFV replication in IPEC-J2 cells. Npro-deleted CSFV mutant (∆Npro) induced significantly higher IFN-&lambda, s transcription from 24 h post-infection (hpi) than its parental strain (wtCSFV). The strain wtCSFV strongly inhibited IFN-&lambda, s transcription and IFN-&lambda, 3 promoter activity in poly(I:C)-stimulated IPEC-J2 cells, whereas ∆Npro did not show such inhibition. Npro overexpression caused significant reduction of IFN-&lambda, 3 promoter activity. Both wtCSFV and ∆Npro infection induced time-dependent IRF1 expression in IPEC-J2 cells, with &Delta, Npro showing more significant induction, particularly at 24 hpi. However, infection with wtCSFV or Npro overexpression led not only to significant reduction of IRF1 expression and its promoter activity in poly(I:C)-treated IPEC-J2 cells but also to blockage of IRF1 nuclear translocation. This study provides clear evidence that CSFV Npro suppresses IRF1-mediated type III IFNs production by inhibiting IRF1 expression and its nuclear translocation.

// Mihwa Kim 1 , Liza D. Morales 1,2 , Minwoo Baek 1 , Thomas J. Slaga 3 , John DiGiovanni 4 and Dae Joon Kim 1 1 Department of Biomedical Sciences, School of Medicine, University of Texas Rio Grande Valley, Edinburg, TX, USA 2 South Texas Diabetes and Obesity Institute, School of Medicine, University of Texas Rio Grande Valley, Edinburg, TX, USA 3 Department of Pharmacology, School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA 4 Division of Pharmacology & Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, TX, USA Correspondence to: Dae Joon Kim, email: // Keywords : TC-PTP, nuclear translocation, AKT, 14-3-3σ, keratinocytes Received : June 26, 2017 Accepted : September 15, 2017 Published : October 11, 2017 Abstract Understanding protein subcellular localization is important to determining the functional role of specific proteins. T-cell protein tyrosine phosphatase (TC-PTP) contains bipartite nuclear localization signals (NLSI and NLSII) in its C-terminus. We previously have demonstrated that the nuclear form of TC-PTP (TC45) is mainly localized to the cytoplasm in keratinocytes and it is translocated to the nucleus following UVB irradiation. Here, we report that TC45 is translocated by an AKT/14-3-3σ-mediated mechanism in response to UVB exposure, resulting in increased apoptosis and decreased keratinocyte proliferation. We demonstrate that UVB irradiation increased phosphorylation of AKT and induced nuclear translocation of 14-3-3σ and TC45. However, inhibition of AKT blocked nuclear translocation of TC45 and 14-3-3σ. Site-directed mutagenesis of 14-3-3σ binding sites within TC45 showed that a substitution at Threonine 179 (TC45/T179A) effectively blocked UVB-induced nuclear translocation of ectopic TC45 due to the disruption of the direct binding between TC45 and 14-3-3σ. Overexpression of TC45/T179A in keratinocytes resulted in a decrease of UVB-induced apoptosis which corresponded to an increase in nuclear phosphorylated STAT3, and cell proliferation was higher in TC45/T179A-overexpressing keratinocytes compared to control keratinocytes following UVB irradiation. Furthermore, deletion of TC45 NLSII blocked its UVB-induced nuclear translocation, indicating that both T179 and NLSII are required. Taken together, our findings suggest that AKT and 14-3-3σ cooperatively regulate TC45 nuclear translocation in a critical step of an early protective mechanism against UVB exposure that signals the deactivation of STAT3 in order to promote keratinocyte cell death and inhibit keratinocyte proliferation.

The ATR pathway is one of the major DNA damage checkpoints, and Rad17 is a DNA-binding protein that is phosphorylated upon DNA damage by ATR kinase. Rad17 recruits the 9-1-1 complex that mediates the checkpoint activation, and proteasomal degradation of Rad17 is important for recovery from the ATR pathway. Here, we identified several Rad17 mutants deficient in nuclear localization and resistant to proteasomal degradation. The nuclear localization signal was identified in the central basic domain of Rad17. Rad17 Δ230–270 and R240A/L243A mutants that were previously postulated to lack the destruction box, a sequence that is recognized by the ubiquitin ligase/anaphase-promoting complex that mediates degradation of Rad17, also showed cytoplasmic localization. Our data indicate that the nuclear translocation of Rad17 is functionally linked to the proteasomal degradation. The ATP-binding activity of Rad17, but not hydrolysis, is essential for the nuclear translocation, and the ATPase domain orchestrates the nuclear translocation, the proteasomal degradation, as well as the interaction with the 9-1-1 complex. The Rad17 mutant that lacked a nuclear localization signal was proficient in the interaction with the 9-1-1 complex, suggesting cytosolic association of Rad17 and the 9-1-1 complex. Finally, we identified two tandem canonical and noncanonical destruction boxes in the N-terminus of Rad17 as the bona fide destruction box, supporting the role of anaphase-promoting complex in the degradation of Rad17. We propose a model in which Rad17 is activated in the cytoplasm for translocation into the nucleus and continuously degraded in the nucleus even in the absence of exogenous DNA damage.

Morinda (Morinda officinalis) is widely consumed as a health-care herb in Asia and reported to possess various biological activities. In this study, anti-inflammatory phytochemicals were investigated and two pairs of new methyl-2-naphthoate enantiomers (1a/1b, 2a/2b), one new anthraquinone (3), three new natural unknown anthraquinones (5–6, 23), and eighteen known anthraquinones were isolated and elucidated from the roots of morinda. Anti-inflammatory activities of the isolated compounds were assessed in lipopolysaccharide-stimulated RAW 264.7 macrophages. Compounds 2b and 19 significantly inhibited the production of NO with IC50 values of 34.32 ± 4.87 and 17.17 ± 4.13 μM (indomethacin, IC50 26.71 ± 6.32 μM), and they were further corroborated via immunoblotting, quantitative real-time PCR and immunofluorescence staining assays. They could dose-dependent suppress lipopolysaccharide-stimulated pro-inflammatory factors (COX-2 and iNOS) production and block nuclear translocation of NF-κB. The results implied that reasonable consumption of morinda may be beneficial for preventing and reducing the occurrence of inflammatory-associated diseases.

Myoferlin, a member of ferlin family of proteins, was first discovered as a candidate gene for muscular dystrophy and cardiomyopathy. Recently, myoferlin was shown to be also expressed in endothelial and cancer cells where it was shown to modulate vascular endothelial growth factor (VEGFR)-2 and epidermal growth factor receptor (EGFR) signaling by enhancing their stability and recycling. Based on these reports, we hypothesized that myoferlin might be regulating IL-6 signaling by modulating IL-6R stabilization and recycling. However, in our immunoprecipitation (IP) experiments, we did not observe myoferlin binding with IL-6R. Instead, we made a novel discovery that in resting cells myoferlin was bound to EHD2 protein and when cells were treated with IL-6, myoferlin dissociated from EHD2 and binds to activated STAT3. Interestingly, myoferlin depletion did not affect STAT3 phosphorylation, but completely blocked STAT3 translocation to nucleus. In addition, inhibition of STAT3 phosphorylation by phosphorylation-defective STAT3 mutants or JAK inhibitor blocked STAT3 binding to myoferlin and nuclear translocation. Myoferlin knockdown significantly decreased IL-6-mediated tumor cell migration, tumorsphere formation and ALDH-positive cancer stem cell population, in vitro. Furthermore, myoferlin knockdown significantly decreased IL-6-meditated tumor growth and tumor metastasis. Based on these results, we have proposed a novel model for the role of myoferlin in chaperoning phosphorylated STAT3 to the nucleus.

Emerged porcine kobuvirus (PKV) has adversely affected the global swine industry since 2008, but the etiological biology of PKV is unclear. Screening PKV-encoded structural and non-structural proteins with a type I IFN-responsive luciferase reporter showed that PKV VP3 protein inhibited the IFN-β-triggered signaling pathway, resulting in the decrease of VSV-GFP replication. QPCR data showed that IFN-β downstream cytokine genes were suppressed without cell-type specificity as well. The results from biochemical experiments indicated that PKV VP3 associated with STAT2 and IRF9, and interfered with the formation of the STAT2-IRF9 and STAT2-STAT2 complex, impairing nuclear translocation of STAT2 and IRF9. Taken together, these data reveal a new mechanism for immune evasion of PKV.

Huntington's disease (HD) is caused by a genetically mutated huntingtin (mHtt) protein with expanded polyQ stretch, which impairs cytosolic sequestration of the repressor element‐1 silencing transcription factor (REST), resulting in excessive nuclear REST and subsequent repression of neuronal genes. We recently demonstrated that REST undergoes extensive, context‐dependent alternative splicing, of which exon‐3 skipping (∆E3)—a common event in human and nonhuman primates—causes loss of a motif critical for REST nuclear targeting. This study aimed to determine whether ∆E3 can be targeted to reduce nuclear REST and rescue neuronal gene expression in mouse striatal‐derived, mHtt‐expressing STHdhQ111/Q111 cells—a well‐established cellular model of HD. We designed two morpholino antisense oligos (ASOs) targeting the splice sites of Rest E3 and examined their effects on ∆E3, nuclear Rest accumulation and Rest‐controlled gene expression in STHdhQ111/Q111 cells. We found that (1) the ASOs treatment significantly induced ∆E3, reduced nuclear Rest, and rescued transcription and/or mis‐splicing of specific neuronal genes (e.g. Syn1 and Stmn2) in STHdhQ111/Q111 cells; and (2) the ASOs‐induced transcriptional regulation was dependent on ∆E3 induction and mimicked by siRNA‐mediated knock‐down of Rest expression. Our findings demonstrate modulation of nuclear REST by ∆E3 and its potential as a new therapeutic target for HD and provide new insights into environmental regulation of genome function and pathogenesis of HD. As ∆E3 is modulated by cellular signalling and linked to various types of cancer, we anticipate that ∆E3 contributes to environmentally tuned REST function and may have a broad range of clinical implications.

Compression-induced programmed cell death of nucleus pulposus (NP) cells is an important contributor to intervertebral disc degeneration (IDD). Dynamin-related protein 1 (Drp1), a crucial mitochondrial fission protein, triggers programmed necrosis upon cellular injury. However, limited information is available about the role of Drp1 in compression-induced programmed necrosis of NP cells. In the present study, we found that compression resulted in upregulation and mitochondrial translocation of Drp1. Inhibition of Drp1 by siRNA or mitochondrial division inhibitor 1 (mdivi-1) effectively prevented the programmed necrosis of NP cells treated with compression. Furthermore, Drp1 promoted mitochondrial translocation of p53 and nuclear translocation of apoptosis-inducing factor (AIF) in compression-treated NP cells. Inhibition of p53 mitochondrial translocation by pifithrin-μ (PFT-μ) and silencing of AIF expression by siRNA significantly alleviated compression-induced NP cell programmed necrosis. These data indicates that Drp1 mediates compression-induced programmed necrosis of NP cells by promoting mitochondrial translocation of p53 and nuclear translocation of AIF.

In this study, newly identified host interactors of hepatitis C virus (HCV) proteins were assessed for a role in modulating the innate immune response. The analysis revealed enrichment for components of the nuclear transport machinery and the crucial interaction with NS3/4A protein in suppression of interferon‐β (IFNB1) induction. Using a comprehensive microscopy‐based high‐content screening approach combined to the gene silencing of nuclear transport factors, we showed that NS3/4A‐interacting proteins control the nucleocytoplasmic trafficking of IFN regulatory factor 3 (IRF3) and NF‐κB p65 upon Sendai virus (SeV) infection. Notably, importin β1 (IMPβ1) knockdown—a hub protein highly targeted by several viruses—decreases the nuclear translocation of both transcription factors and prevents IFNB1 and IFIT1 induction, correlating with a rapid increased of viral proteins and virus‐mediated apoptosis. Here we show that NS3/4A triggers the cleavage of IMPβ1 and inhibits nuclear transport to disrupt IFNB1 production. Importantly, mutated IMPβ1 resistant to cleavage completely restores signaling, similar to the treatment with BILN 2061 protease inhibitor, correlating with the disappearance of cleavage products. Overall, the data indicate that HCV NS3/4A targeting of IMPβ1 and related modulators of IRF3 and NF‐κB nuclear transport constitute an important innate immune subversion strategy and inspire new avenues for broad‐spectrum antiviral therapies., Hepatitis C virus (HCV) hijacks the cellular host machinery to promote replication and to evade immune response. Using a microscopy‐based High Content Screening (HCS) assay, we demonstrated that HCV‐host interactors involved in nuclear transport are crucial for IRF3 and NFκB‐p65 signaling of IFNB1 response. To subvert innate immunity, NS3/4A interacts and cleaves IMPβ1, a key nucleocytoplasmic transport receptor, to prevent nuclear translocation of IRF3 and NFκB‐p65 transcription factors. Future studies will confirm if IMPβ1 is a prime target for several viruses to evade host defense.

// Rongjie Yu 1, 2 , Hongyu Liu 1, 2 , Xinhe Peng 1, 2 , Yue Cui 1, 2 , Suqin Song 1, 2 , Like Wang 1, 2 , Huahua Zhang 3 , An Hong 1, 2 and Tianhong Zhou 4 1 Institute of Biomedicine, School of Life Science and Technology, Jinan University, Guangzhou, Guangdong, China 2 National Engineering Research Center of Genetic Medicine, Jinan University, Guangzhou, Guangdong, China 3 Department of Medical Genetics, Guangdong Medical University, Dongguan, Guangdong, China 4 Department of Bioengineering, School of Life Science and Technology, Jinan University, Guangzhou, Guangdong, China Correspondence to: Rongjie Yu, email: rongjie_yu1123@163.com Keywords: vasoactive intestinal peptide receptor 1 (VPAC1), cysteine (Cys), palmitoylation, nuclear translocation, anti-apoptotic activity Received: March 06, 2017 Accepted: April 14, 2017 Published: April 27, 2017 ABSTRACT VPAC1 is class B G protein-coupled receptors (GPCR) shared by pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP). The first cysteine (Cys37) in the N-terminal extracellular domain of mature VPAC1 is a free Cys not involved in the formation of conserved intramolecular disulfide bonds. In order to investigate the biological role of this Cys37 in VPAC1, the wild-type VPAC1 and Cys37/Ala mutant (VPAC1-C37/A) were expressed stably as fusion proteins with enhanced yellow fluorescent protein (EYFP) respectively in Chinese hamster ovary (CHO) cells. Both VPAC1-EYFP and VPAC1-C37/A-EYFP trafficked to the plasma membrane normally, and CHO cells expressing VPAC1-EYFP displayed higher anti-apoptotic activity against camptothecin (CPT) induced apoptosis than the cells expressing VPAC1-C37/A-EYFP, while VPAC1-C37/A-CHO cells showed higher proliferative activity than VPAC1-CHO cells. Confocal microscopic analysis, western blotting and fluorescence quantification assay showed VPAC1-EYFP displayed significant nuclear translocation while VPAC1-C37/A-EYFP did not transfer into nucleus under the stimulation of VIP (0.1 nM). Acyl-biotin exchange assay and click chemistry-based palmitoylation assay confirmed for the first time the palmitoylation of Cys37, which has been predicted by bioinformatics analysis. And the palmitoylation inhibitor 2-bromopalmitate significantly inhibited the nuclear translocation of VPAC1-EYFP and its anti-apoptotic activity synchronously. These results indicated the palmitoylation of the Cys37 in the N-terminal extracellular domain of VPAC1 mediates the nuclear translocation of VPAC1 contributing to its anti-apoptotic activity. These findings reveal for the first time the lipidation-mediating nuclear translocation of VPAC1 produces a novel anti-apoptotic signal pathway, which may help to promote new drug development strategy targeting VPAC1.

Parabens owing to their many advantageous properties are widely applied in cosmetics, food products and pharmaceuticals. However, recent research results have shown that they possess the ability to accumulate in the human body and exert many adverse effects. In this study, the impact of methylparaben (MP) as the most frequently used preservative in cosmetics, on human dermal fibroblasts and collagen production was evaluated. In cells treated with 0.01, 0.03 and 0.05% MP a dose-dependent decrease in collagen biosynthesis was revealed, which was positively correlated with the activity of prolidase responsible for the recovery of proline. Consequently, the concentration of total collagen secreted into the medium was markedly diminished. A similar reduction in expression of the major skin collagen type I at both the protein and mRNA level as well as collagen type III and VI at the mRNA level was also detected. The decrease in the collagen level may result not only from the reduced synthesis but also increased degradation owing to MP-induced activation of pro-MMP-2 (72 kDa). The increase in activity of MMP-2 (66 kDa) was accompanied by a reduction in the inhibitory activity of TIMP-2. In addition, an inhibitory effect of MP on cell survival and proliferation was revealed in this study. The increased expression and nuclear translocation of caspase-3 as well as increased Bax and decreased Bcl-2 expression may suggest MP-induced cell apoptosis. In summary, we have provided new data on the adverse effects of methylparaben on human dermal fibroblasts and the main structural protein of the skin. Further studies on the mechanisms responsible for its action are in progress. Copyright © 2017 John Wiley & Sons, Ltd.

Many transcription factors play a key role in cellular differentiation and the delineation of cell phenotype. Transcription factors are regulated by phosphorylation, ubiquitination, acetylation/deacetylation and interactions between two or more proteins controlling multiple signaling pathways. These pathways regulate different physiological processes and pathological events, such as cancer and other diseases. The Forkhead box O (FOXO) is one subfamily of the fork head transcription factor family with important roles in cell fate decisions and this subfamily is also suggested to play a pivotal functional role as a tumor suppressor in a wide range of cancers. During apoptosis, FOXOs are involved in mitochondria-dependent and -independent processes triggering the expression of death receptor ligands like Fas ligand, TNF apoptosis ligand and Bcl‑XL, bNIP3, Bim from Bcl-2 family members. Different types of growth factors like insulin play a vital role in the regulation of FOXOs. The most important pathway interacting with FOXO in different types of cancers is the PI3K/AKT pathway. Some other important pathways such as the Ras-MEK-ERK, IKK and AMPK pathways are also associated with FOXOs in tumorigenesis. Therapeutically targeting the FOXO signaling pathway(s) could lead to the discovery and development of efficacious agents against some cancers, but this requires an enhanced understanding and knowledge of FOXO transcription factors and their regulation and functioning. This review focused on the current understanding of cell biology of FOXO transcription factors which relates to their potential role as targets for the treatment and prevention of human cancers. We also discuss drugs which are currently being used for cancer treatment along with their target pathways and also point out some potential drawbacks of those drugs, which further signifies the need for development of new drug strategies in the field of cancer treatment.

Image-based flow cytometry combines the throughput of traditional flow cytometry with the ability to visually confirm findings and collect novel data that would not be possible otherwise. Since image-based flow cytometry borrows measurement parameters and analysis techniques from microscopy, it is possible to collect unique measures (i.e. nuclear translocation, co-localization, cellular synapse, cellular endocytosis, etc.) that would not be possible with traditional flow cytometry. The ability to collect unique outcomes has led many researchers to develop novel assays for the monitoring and detection of a variety of clinical conditions and diseases. In many cases, investigators have innovated and expanded classical assays to provide new insight regarding clinical conditions and chronic disease. Beyond human clinical applications, image-based flow cytometry has been used to monitor marine biology changes, nano-particles for solar cell production, and particle quality in pharmaceuticals. This review article summarizes work from the major scientists working in the field of image-based flow cytometry.

Deregulation of the TAM (TYRO3, AXL, and MERTK) family of receptor tyrosine kinases (RTKs) has recently been demonstrated to predominately promote survival and chemoresistance of cancer cells. Intramembrane proteolysis mediated by presenilin/gamma-secretase is known to regulate the homeostasis of some RTKs. In the present study, we demonstrate that AXL, but not TYRO3 or MERTK, is efficiently and sequentially cleaved by alpha-and g-secretases in various types of cancer cell lines. Proteolytic processing of AXL redirected signaling toward a secretase-mediated pathway, away from the classic, well-known, ligand-dependent canonical RTK signaling pathway. TheAXL intracellular domain cleavage product, but not full-length AXL, was further shown to translocate into the nucleus via a nuclear localization sequence that harbored a basic HRRKK motif. Of interest, we found that the g-secretase-uncleavable AXL mutant caused an elevated chemoresistance in non-small-cell lung cancer cells. Altogether, our findings suggest thatAXL can undergo sequential processing mediated by various proteases kept in a homeostatic balance. This newly discovered post-translational processing ofAXL may provide an explanation for the diverse functions of AXL, especially in the context of drug resistance in cancer cells.-Lu, Y., Wan, J., Yang, Z., Lei, X., Niu, Q., Jiang, L., Passtoors, W. M., Zang, A., Fraering, P. C., Wu, F. Regulated intramembrane proteolysis of the AXL receptor kinase generates an intracellular domain that localizes in the nucleus of cancer cells. FASEB J. 31, 1382-1397 (2017). www.fasebj.org

The extracellular signal-regulated kinases 1/2 (ERK) are central signaling components that regulate stimulated cellular processes such as proliferation and differentiation. When dysregulated, these kinases participate in the induction and maintenance of various pathologies, primarily cancer. While ERK is localized in the cytoplasm of resting cells, many of its substrates are nuclear, and indeed, extracellular stimulation induces a rapid and robust nuclear translocation of ERK. Similarly to other signaling components that shuttle to the nucleus upon stimulation, ERK does not use the canonical importinmechanism of nuclear translocation. Rather, it has its own unique nuclear translocation signal (NTS) that interacts with importin7 to allow stimulated shuttling via the nuclear pores. Prevention of the nuclear translocation inhibits proliferation of B-Raf- and N/K-Ras-transformed cancers. This effect is distinct from the one achieved by catalytic Raf and MEK inhibitors used clinically, as cells treated with the translocation inhibitors develop resistance much more slowly. In this review, we describe the mechanism of ERK translocation, present all its nuclear substrates, discuss its role in cancer and compare its translocation to the translocation of other signaling components. We also present proof of principle data for the use of nuclear ERK translocation as an anti-cancer target. It is likely that the prevention of nuclear ERK translocation will eventually serve as a way to combat Ras and Raf transformed cancers with less side-effects than the currently used drugs.

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.