Sensory circuitry controls cytosolic calcium-mediated phytochrome B phototransduction.

Although Ca2+ has long been recognized as an obligatory intermediate in visual transduction, its role in plant phototransduction remains elusive. Here, we report a Ca2+ signaling that controls photoreceptor phyB nuclear translocation in etiolated seedlings during dark-to-light transition. Red light stimulates acute cytosolic Ca2+ increases via phyB, which are sensed by Ca2+-binding protein kinases, CPK6 and CPK12 (CPK6/12). Upon Ca2+ activation, CPK6/12 in turn directly interact with and phosphorylate photo-activated phyB at Ser80/Ser106 to initiate phyB nuclear import. Non-phosphorylatable mutation, phyBS80A/S106A, abolishes nuclear translocation and fails to complement phyB mutant, which is fully restored by combining phyBS80A/S106A with a nuclear localization signal. We further show that CPK6/12 function specifically in the early phyB-mediated cotyledon expansion, while Ser80/Ser106 phosphorylation generally governs phyB nuclear translocation. Our results uncover a biochemical regulatory loop centered in phyB phototransduction and provide a paradigm for linking ubiquitous Ca2+ increases to specific responses in sensory stimulus processing.

OptIC-Notch reveals mechanism that regulates receptor interactions with CSL.

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. However, CSL lacks its own nuclear localisation sequence, and it remains unclear where the tripartite complex is formed. To probe the mechanisms involved, we designed an optogenetic approach to control NICD release (OptIC-Notch) and monitored the subsequent 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 (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.

ER chaperone GRP78/BiP translocates to the nucleus under stress and acts as a transcriptional regulator.

Cancer cells are commonly subjected to endoplasmic reticulum (ER) stress. To gain survival advantage, cancer cells exploit the adaptive aspects of the unfolded protein response such as upregulation of the ER luminal chaperone GRP78. The finding that when overexpressed, GRP78 can escape to other cellular compartments to gain new functions regulating homeostasis and tumorigenesis represents a paradigm shift. Here, toward deciphering the mechanisms whereby GRP78 knockdown suppresses EGFR transcription, we find that nuclear GRP78 is prominent in cancer and stressed cells and uncover a nuclear localization signal critical for its translocation and nuclear activity. Furthermore, nuclear GRP78 can regulate expression of genes and pathways, notably those important for cell migration and invasion, by interacting with and inhibiting the activity of the transcriptional repressor ID2. Our study reveals a mechanism for cancer cells to respond to ER stress via transcriptional regulation mediated by nuclear GRP78 to adopt an invasive phenotype.

Fluorescent tagging of endogenous IRS2 with an auxin-dependent degron to assess dynamic intracellular localization and function.

Insulin Receptor Substrate 2 (IRS2) is a signaling adaptor protein for the insulin (IR) and Insulin-like Growth Factor-1 (IGF-1R) receptors. In breast cancer, IRS2 contributes to both the initiation of primary tumor growth and the establishment of secondary metastases through regulation of cancer stem cell (CSC) function and invasion. However, how IRS2 mediates its diverse functions is not well understood. We used CRISPR/Cas9-mediated gene editing to modify endogenous IRS2 to study the expression, localization, and function of this adaptor protein. A cassette containing an auxin-inducible degradation (AID) sequence, 3x-FLAG tag, and mNeon-green was introduced at the N-terminus of the IRS2 protein to provide rapid and reversible control of IRS2 protein degradation and analysis of endogenous IRS2 expression and localization. Live fluorescence imaging of these cells revealed that IRS2 shuttles between the cytoplasm and nucleus in response to growth regulatory signals in a PI3K-dependent manner. Inhibition of nuclear export or deletion of a putative nuclear export sequence in the C-terminal tail promotes nuclear retention of IRS2, implicating nuclear export in the mechanism by which IRS2 intracellular localization is regulated. Moreover, the acute induction of IRS2 degradation reduces tumor cell invasion, demonstrating the potential for therapeutic targeting of this adaptor protein. Our data highlight the value of our model of endogenously tagged IRS2 as a tool to study IRS2 localization and function.

MERS-CoV-nsp5 expression in human epithelial BEAS 2b cells attenuates type I interferon production by inhibiting IRF3 nuclear translocation.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an enveloped, positive-sense RNA virus that emerged in 2012, causing sporadic cases and localized outbreaks of severe respiratory illness with high fatality rates. A characteristic feature of the immune response to MERS-CoV infection is low type I IFN induction, despite its importance in viral clearance. The non-structural proteins (nsps) of other coronaviruses have been shown to block IFN production. However, the role of nsp5 from MERS-CoV in IFN induction of human respiratory cells is unclear. In this study, we elucidated the role of MERS-CoV-nsp5, the viral main protease, in modulating the host's antiviral responses in human bronchial epithelial BEAS 2b cells. We found that overexpression of MERS-CoV-nsp5 had a dose-dependent inhibitory effect on IFN-ß promoter activation and cytokine production induced by HMW-poly(I:C). It also suppressed IFN-ß promoter activation triggered by overexpression of key components in the RIG-I-like receptor (RLR) pathway, including RIG-I, MAVS, IKK-ε and IRF3. Moreover, the overexpression of MERS-CoV-nsp5 did not impair expression or phosphorylation of IRF3, but suppressed the nuclear translocation of IRF3. Further investigation revealed that MERS-CoV-nsp5 specifically interacted with IRF3. Using docking and molecular dynamic (MD) simulations, we also found that amino acids on MERS-CoV-nsp5, IRF3, and KPNA4 may participate in protein-protein interactions. Additionally, we uncovered protein conformations that mask the nuclear localization signal (NLS) regions of IRF3 and KPNA4 when interacting with MERS-CoV-nsp5, suggesting a mechanism by which this viral protein blocks IRF3 nuclear translocation. Of note, the IFN-ß expression was restored after administration of protease inhibitors targeting nsp5, indicating this suppression of IFN-ß production was dependent on the enzyme activity of nsp5. Collectively, our findings elucidate a mechanism by which MERS-CoV-nsp5 disrupts the host's innate antiviral immunity and thus provides insights into viral pathogenesis.

RKIP localizes to the nucleus through a bipartite nuclear localization signal and interaction with importin α to regulate mitotic progression.

Raf kinase inhibitor protein (RKIP) is a multifunctional modulator of intracellular signal transduction. Although most of its functions have been considered cytosolic, we show here that the localization of RKIP is primarily nuclear in both growing and quiescent Madin-Darby canine kidney epithelial cells and in Cal-51 and BT-20 human breast cancer cells. We have identified a putative bipartite nuclear localization signal (NLS) in RKIP that maps to the surface of the protein surrounding a known regulatory region. Like classical NLS sequences, the putative NLS of RKIP is rich in arginine and lysine residues. Deletion of and point mutations in the putative NLS lead to decreased nuclear localization. Point mutation of all the basic residues in the putative NLS of RKIP particularly strongly reduces nuclear localization. We found consistent results in reexpression experiments with wildtype or mutant RKIP in RKIP-silenced cells. A fusion construct of the putative NLS of RKIP alone to a heterologous reporter protein leads to nuclear localization of the fusion protein, demonstrating that this sequence alone is sufficient for import into the nucleus. We found that RKIP interacts with the nuclear transport factor importin α in BT-20 and MDA-MB-231 human breast cancer cells, suggesting importin-mediated active nuclear translocation. Evaluating the biological function of nuclear localization of RKIP, we found that the presence of the putative NLS is important for the role of RKIP in mitotic checkpoint regulation in MCF-7 human breast cancer cells. Taken together, these findings suggest that a bipartite NLS in RKIP interacts with importin α for active transport of RKIP into the nucleus and that this process may be involved in the regulation of mitotic progression.

Identification and functional characterization of the nuclear and nucleolar localization signals in the intrinsically disordered region of nucleomethylin.

The nucleolar localization of proteins is regulated by specific signals directing their trafficking to nucleus and nucleolus. Here, we elucidate the mechanism underlying the nuclear and nucleolar localization of the nucleomethylin (NML) protein, focusing on its nuclear localization signals (NLSs) and nucleolar localization signal (NoLS). Using a combination of bioinformatic analysis and experimental validation, we identified two monopartite and one bipartite NLS motifs within NML. The combined presence of both monopartite NLSs significantly enhances nuclear localization of the protein, while specific basic amino acid clusters within the bipartite NLS are crucial for their functionality. We also reveal the functional role of the NLS-coupled NoLS motif in driving nucleolar localization of NML, which contains an arginine-rich motif essential for its function. The basic residues of the arginine-rich motif of NoLS of NML interacts with nucleophosmin 1 (NPM1), allowing the possible liquid-liquid phase separation and retention of NML in the nucleolus. Remarkably, the strong NoLS of NML can direct the nucleolar localization of a cytosolic protein, aldolase, emphasizing its potency. Overall, our findings provide insights into the combinatorial functioning of NLSs and NoLS in regulating the subcellular localization of NML, highlighting the intricate regulatory mechanisms governing its localization within the nucleus and nucleolus.

The RNA-binding protein Cpeb4 regulates splicing of the Id2 gene in osteoclast differentiation.

Cytoplasmic polyadenylation element-binding protein 4 (Cpeb4) is an RNA-binding protein that regulates posttranscriptional regulation, such as regulation of messenger RNA stability and translation. In the previous study, we reported that Cpeb4 localizes to nuclear bodies upon induction of osteoclast differentiation by RANKL. However, the mechanisms of the localization of Cpeb4 and osteoclastogenesis by Cpeb4 remain unknown. Here, we show that Cpeb4 localizes to the nuclear bodies by its RNA-binding ability and partially regulates normal splicing during osteoclast differentiation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with Phos-tag® revealed that the phosphorylation levels of Cpeb4 were already high in the RAW264.7 cells and were not altered by RANKL treatment. Immunofluorescence showed that exogenous Cpeb4 in HEK293T cells without RANKL stimulation localized to the same foci as shown in RANKL-stimulated RAW264.7 cells. Furthermore, when nuclear export was inhibited by leptomycin B treatment, Cpeb4 accumulated throughout the nucleus. Importantly, RNA recognition motif (RRM) 7 of Cpeb4 was essential for the localization. In contrast, the intrinsically disordered region, RRM1, and zinc finger domain CEBP_ZZ were not necessary for the localization. The mechanistic study showed that Cpeb4 co-localized and interacted with the splicing factors serine/arginine-rich splicing factor 5 (SRSF5) and SRSF6, suggesting that Cpeb4 may be involved in the splicing reaction. RNA-sequencing analysis revealed that the expression of genes related to cell proliferation processes, such as mitotic cell cycle and regulation of cell cycle processes, was elevated in osteoclasts depleted of Cpeb4. Interestingly, the splicing pattern of the inhibitor of DNA binding 2 (Id2) gene, which suppresses osteoclast differentiation, was altered by the depletion of Cpeb4. These results provide new insight into the role of Cpeb4 as a player of normal splicing of Id2 in osteoclast differentiation.

The Prognostic Biomarker RAB7A Promotes Growth and Metastasis of Liver Cancer Cells by Regulating Glycolysis and YAP1 Activation.

Activating transcription factor 6 (ATF6) and its downstream genes are involved in progression of hepatocellular carcinoma (HCC). Herein, we demonstrated that sulfhydration of Ras-related protein Rab-7a (RAB7A) was regulated by ATF6. High expression of RAB7A indicated poor prognosis of HCC patients. RAB7A overexpression contributed to proliferation, colony formation, migration, and invasion of HepG2 and Hep3B cells. Furthermore, we found that RAB7A enhanced aerobic glycolysis in HepG2 cells, indicating a higher degree of tumor malignancy. Mechanistically, RAB7A suppressed Yes-associated protein 1 (YAP1) binding to 14-3-3 and conduced to YAP1 nuclear translocation and activation, promoting its downstream gene expression, thereby promoting growth and metastasis of liver cancer cells. In addition, knocking down RAB7A attenuated the progression of orthotopic liver tumors in mice. These findings illustrate the important role of RAB7A in regulating HCC progression. Thus, RAB7A may be a potential innovative target for HCC treatment.

CircPPAP2B controls metastasis of clear cell renal cell carcinoma via HNRNPC-dependent alternative splicing and targeting the miR-182-5p/CYP1B1 axis.

BACKGROUND: Renal cell carcinoma (RCC) is one of the most common malignant tumor worldwide. Metastasis is a leading case of cancer-related deaths of RCC. Circular RNAs (circRNAs), a class of noncoding RNAs, have emerged as important regulators in cancer metastasis. However, the functional effects and regulatory mechanisms of circRNAs on RCC metastasis remain largely unknown. METHODS: High-throughput RNA sequencing techniques were performed to analyze the expression profiles of circRNAs and mRNAs in highly and poorly invasive clear cell renal cell carcinoma (ccRCC) cell lines. Functional experiments were performed to unveil the regulatory role of circPPAP2B in the proliferation and metastatic capabilities of ccRCC cells. RNA pulldown, Mass spectrometry analysis, RNA methylation immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), co-immunoprecipitation (CoIP), next-generation RNA-sequencing and double luciferase experiments were employed to clarify the molecular mechanisms by which circPPAP2B promotes ccRCC metastasis. RESULTS: In this study, we describe a newly identified circular RNA called circPPAP2B, which is overexpressed in highly invasive ccRCC cells, as determined through advanced high-throughput RNA sequencing techniques. Furthermore, we observed elevated circPPAP2B in ccRCC tissues, particularly in metastatic ccRCC tissues, and found it to be associated with poor prognosis. Functional experiments unveiled that circPPAP2B actively stimulates the proliferation and metastatic capabilities of ccRCC cells. Mechanistically, circPPAP2B interacts with HNRNPC in a m6A-dependent manner to facilitate HNRNPC nuclear translocation. Subcellular relocalization was dependent upon nondegradable ubiquitination of HNRNPC and stabilization of an HNRNPC/Vimentin/Importin α7 ternary complex. Moreover, we found that circPPAP2B modulates the interaction between HNRNPC and splicing factors, PTBP1 and HNPNPK, and regulates pre-mRNA alternative splicing. Finally, our studies demonstrate that circPPAP2B functions as a miRNA sponge to directly bind to miR-182-5p and increase CYP1B1 expression in ccRCC. CONCLUSIONS: Collectively, our study provides comprehensive evidence that circPPAP2B promotes proliferation and metastasis of ccRCC via HNRNPC-dependent alternative splicing and miR-182-5p/CYP1B1 axis and highlights circPPAP2B as a potential therapeutic target for ccRCC intervention.

The pyruvate dehydrogenase complex at the epigenetic crossroads of acetylation and lactylation.

The pyruvate dehydrogenase complex (PDC) is remarkable for its size and structure as well as for its physiological and pathological importance. Its canonical location is in the mitochondrial matrix, where it primes the tricarboxylic acid (TCA) cycle by decarboxylating glycolytically-derived pyruvate to acetyl-CoA. Less well appreciated is its role in helping to shape the epigenetic landscape, from early development throughout mammalian life by its ability to "moonlight" in the nucleus, with major repercussions for human healthspan and lifespan. The PDC's influence on two crucial modifiers of the epigenome, acetylation and lactylation, is the focus of this brief review.

Mechanisms related to carbon nanotubes genotoxicity in human cell lines of respiratory origin.

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.

Functional regulation of the protein phosphatase PPM1M by phosphorylation at multiple sites with Ser/Thr-Pro motifs.

The imbalance in the phosphorylation and the dephosphorylation of proteins leads to various diseases. Therefore, in vivo, the functions of protein kinases and protein phosphatases are strictly regulated. Mg2+/Mn2+-dependent protein phosphatase PPM1M has been implicated in immunity and cancer; however, the regulation mechanism remains unknown. In this study, we show that PPM1M is regulated in different ways by multiple phosphorylation. PPM1M has four Ser/Thr-Pro motifs (Ser27, Ser43, Ser60, and Thr254) that are recognized by proline-directed kinases, and Ser60 was found to be phosphorylated by cyclin-dependent kinase 5 (CDK5) in the cell. The phospho-mimetic mutation of Ser27 and Ser43 in the N-terminal domain suppresses the nuclear localization of PPM1M and promotes its accumulation in the cytoplasm. The phospho-mimetic mutation of Ser60 decreases PPM1M activity; conversely, the phospho-mimetic mutation of Thr254 increases PPM1M activity. These results suggest that the subcellular localization and phosphatase activity of PPM1M are regulated by protein kinases, including CDK5, via phosphorylation at multiple sites. Thus, PPM1M is differentially regulated by proline-directed kinases, including CDK5.

Chitinase 3-like 1 overexpression aggravates hypoxia-reoxygenation injury in IEC-6 cells by inhibiting the PI3K/AKT signalling pathway.

Intestinal ischaemia-reperfusion (I/R) is a common clinical pathology with high incidence and mortality rates. However, the mechanisms underlying intestinal I/R injury remain unclear. In this study, we investigated the role and mechanism of chitinase 3-like 1 (CHI3L1) during intestinal I/R injury. Therefore, we analysed the expression levels of CHI3L1 in the intestinal tissue of an intestinal I/R rat model and explored its effects and mechanism in a hypoxia-reoxygenation (H/R) IEC-6 cell model. We found that intestinal I/R injury elevated CHI3L1 levels in the serum, ileum and duodenum, whereas H/R enhanced CHI3L1 expression in IEC-6 cells. The H/R-induced inhibition of proliferation and apoptosis was alleviated by CHI3L1 knockdown and aggravated by CHI3L1 overexpression. In addition, CHI3L1 knockdown alleviated, and CHI3L1 overexpression aggravated, the H/R-induced inflammatory response and oxidative stress. Mechanistically, CHI3L1 overexpression weakened the activation of the phosphoinositide 3-kinase (PI3K)/AKT pathway, suppressed the nuclear translocation of Nrf2, and promoted the nuclear translocation of nuclear factor κB (NF-κB). Moreover, CHI3L1 knockdown had the opposite effect on the PI3K/AKT pathway, Nrf2, and NF-κB. Moreover, the PI3K inhibitor LY294002 blocked the effect of CHI3L1 knockdown on the H/R-induced inhibition of proliferation, apoptosis, inflammatory response and oxidative stress. In conclusion, CHI3L1 expression was induced during intestinal I/R and H/R injury in IEC-6 cells, and CHI3L1 overexpression aggravated H/R injury in IEC-6 cells by inhibiting the PI3K/AKT signalling pathway. Therefore, CHI3L1 may be an effective target for controlling intestinal I/R injury.

Diarylpropionitrile-stimulated ERß nuclear accumulation promotes MyoD-induced muscle regeneration in mdx mice by interacting with FOXO3A.

Duchenne muscular dystrophy (DMD) is an X-linked recessive progressive degenerative disease of skeletal muscle, characterized by intramuscular inflammation, muscle regeneration disorder and replacement of muscle with fibroadipose tissue. DMD is caused by the absence of normal dystrophy. Impaired self-renew ability and limited differentiation capacity of satellite cells are proved as main reasons for muscle regeneration failure. The deficiency of estrogen impedes the process of muscle regeneration. However, the role of estrogen receptor ß (ERß) in muscle regeneration is still unclear. This study aims to investigate the role and the pharmacological effect of ERß activation on muscle regeneration in mdx mice. This study showed that mRNA levels of ERß and myogenic-related genes both witnessed increasing trends in dystrophic context. Our results revealed that treatment with selective ERß agonist (DPN, diarylpropionitrile) significantly increased myogenic differentiation 1 (MyoD-1) level and promoted muscle regeneration in mdx mice. Similarly, in mdx mice with muscle-specific estrogen receptor α (ERα) ablation, DPN treatment still promoted muscle regeneration. Moreover, we demonstrated that myoblasts differentiation was accompanied by raised nuclear accumulation of ERß. DPN treatment augmented the nuclear accumulation of ERß and, thus, contributed to myotubes formation. One important finding was that forkhead box O3A (FOXO3A), as a pivotal transcription factor in Myod-1 transcription, participated in the ERß-promoted muscle regeneration. Overall, we offered an interesting explanation about the crucial role of ERß during myogenesis.

Apoptosis-inducing factor 1 mediates Vibrio splendidus-induced coelomocyte apoptosis via importin ß dependent nuclear translocation in Apostichopus japonicus.

As is well known, apoptosis is an important form of immune response and immune regulation, particularly playing a crucial role in combating microbial infections. Apoptosis-inducing factor 1 (AIF-1) is essential for apoptosis to induce chromatin condensation and DNA fragmentation via a caspase-independent pathway. The nuclear translocation of AIF-1 is a key step in apoptosis but the molecular mechanism is still unclear. In this study, the homologous gene of AIF-1, named AjAIF-1, was cloned and identified in Apostichopus japonicus. The mRNA expression of AjAIF-1 was significantly increased by 46.63-fold after Vibrio splendidus challenge. Silencing of AjAIF-1 was found to significantly inhibit coelomocyte apoptosis because the apoptosis rate of coelomocyte decreased by 0.62-fold lower compared with the control group. AjAIF-1 was able to promote coelomocyte apoptosis through nuclear translocation under the V. splendidus challenge. Moreover, AjAIF-1 and Ajimportin ß were mainly co-localized around the nucleus in vivo and silencing Ajimportin ß significantly inhibited the nuclear translocation of AjAIF-1 and suppressed coelomocyte apoptosis by 0.64-fold compared with control. In summary, nuclear translocation of AjAIF-1 will likely mediate coelomocyte apoptosis through an importin ß-dependent pathway in sea cucumber.

Toxicological assessment of the Achyrocline satureioides aqueous extract in the Caenorhabditis elegans alternative model.

Achyrocline satureioides, popularly called "marcela" in Brazil, is used in traditional medicine in South America. A. satureioides, inflorescences are used for many conditions, including to minimize the Sars-Cov-2 symptoms. Therefore, the aim of this study was to determine the toxicity profile of A. satureioides aqueous extract (ASAE), using the Caenorhabditis elegans (C. elegans) alternative model. Survival, reproduction, development, and transgenerational assays were performed. The effects of ASAE were investigated under conditions of thermal stress and presence of oxidant hydrogen peroxide (H2O2). In addition, C. elegans strains containing high antioxidant enzyme levels and elevated lineages of daf-16, skn-1 and daf-2 regulatory pathways were examined. The ASAE LC50 value was found to be 77.3 ± 4 mg/ml. The concentration of ASAE 10 mg/ml (frequently used in humans) did not exhibit a significant reduction in worm survival at either the L1 or L4 stage, after 24 or 72 hr treatment. ASAE did not markedly alter the body area. In N2 strain, ASAE (10 or 25 mg/ml) reversed the damage initiated by H2O2. In addition, ASAE protected the damage produced by H2O2 in strains containing significant levels of sod-3, gst-4 and ctl - 1,2,3, suggesting modulation in these antioxidant systems by this plant extract. ASAE exposure activated daf-16 and skn-1 stress response transcriptional pathways independently of daf-2, even under extreme stress. Data suggest that ASAE, at the concentrations tested in C. elegans, exhibits a reliable toxicity profile, which may contribute to consideration for safe use in humans.

Lyn-mediated glycolysis enhancement of microglia contributes to neuropathic pain through facilitating IRF5 nuclear translocation in spinal dorsal horn.

The pro-inflammatory phenotype of microglia usually induces neuroinflammatory reactions in neuropathic pain. Glycometabolism shift to glycolysis can promote the pro-inflammatory phenotype transition of microglia. The omics data analysis suggest a critical role for Lyn dysregulation in neuropathic pain. The present study aimed at exploring the mechanism of Lyn-mediated glycolysis enhancement of microglia in neuropathic pain. Neuropathic pain model was established by chronic constriction injury (CCI), then pain thresholds and Lyn expression were measured. Lyn inhibitor Bafetinib and siRNA-lyn knockdown were administrated intrathecally to evaluate the effects of Lyn on pain thresholds, glycolysis and interferon regulatory factor 5 (IRF5) nuclear translocation of microglia in vivo and in vitro. ChIP was carried out to observe the binding of transcription factors SP1, PU.1 to glycolytic gene promoters by IRF5 knockdown. Finally, the relationship between glycolysis and pro-inflammatory phenotype transition of microglia was evaluated. CCI led to the upregulation of Lyn expression and glycolysis enhancement in microglia of spinal dorsal horn. Bafetinib or siRNA-lyn knockdown intrathecally alleviated pain hyperalgesia, suppressed glycolysis enhancement and inhibited nuclear translocation of IRF5 in CCI mice. Also, IRF5 promoted the binding of transcription factors SP1, PU.1 to glycolytic gene promoters, and then the enhanced glycolysis facilitated the proliferation and pro-inflammatory phenotype transition of microglia and contributed to neuropathic pain. Lyn-mediated glycolysis enhancement of microglia contributes to neuropathic pain through facilitating IRF5 nuclear translocation in spinal dorsal horn.

The Ragulator complex serves as a substrate-specific mTORC1 scaffold in regulating the nuclear translocation of transcription factor EB.

The mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is activated by intracellular nutritional sufficiency and extracellular growth signals. It has been reported that mTORC1 acts as a hub that integrates these inputs to orchestrate a number of cellular responses, including translation, nucleotide synthesis, lipid synthesis, and lysosome biogenesis. However, little is known about specific control of mTORC1 signaling downstream of this complex. Here, we demonstrate that Ragulator, a heteropentameric protein complex required for mTORC1 activation in response to amino acids, is critical for inhibiting the nuclear translocation of transcription factor EB (TFEB). We established a unique RAW264.7 clone that lacked Ragulator but retained total mTORC1 activity. In a nutrition-sufficient state, the nuclear translocation of TFEB was markedly enhanced in the clone despite total mTORC1 kinase activity. In addition, as a cellular phenotype, the number of lysosomes was increased by tenfold in the Ragulator-deficient clone compared with that of control cells. These findings indicate that mTORC1 essentially requires the Ragulator complex for regulating the subcellular distribution of TFEB. Our findings also suggest that other scaffold proteins may be associated with mTORC1 for the specific regulation of downstream signaling.

Phospholipase Dδ and phosphatidic acid mediate heat-induced nuclear localization of glyceraldehyde-3-phosphate dehydrogenase in Arabidopsis.

Cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) is a glycolytic enzyme, but undergoes stress-induced nuclear translocation for moonlighting. We previously reported that in response to heat stress, GAPC accumulated in the nucleus to modulate transcription and thermotolerance. Here we show a cellular and molecular mechanism that mediates heat-induced nuclear translocation of cytosolic GAPC in Arabidopsis thaliana. Heat-induced GAPC nuclear accumulation and plant heat tolerance were reduced in Arabidopsis phospholipase D (PLD) knockout mutants of pldδ and pldα1pldδ, but not of pldα1. These changes were restored to wild type by genetic complementation with active PLDδ, but not with catalytically inactive PLDδ. GAPC overexpression enhanced the seedling thermotolerance and the expression of heat-inducible genes, but this effect was abolished in the pldδ background. Heat stress elevated the levels of the PLD product phosphatidic acid (PA) in the nucleus in wild type, but not in pldδ plants. Lipid labeling demonstrated the heat-induced nuclear co-localization of PA and GAPC, which was impaired by zinc, which inhibited the PA-GAPC interaction, and by the membrane trafficking inhibitor brefeldin A (BFA). The GAPC nuclear accumulation and seedling thermotolerance were also decreased by treatment with zinc or BFA. Our data suggest that PLDδ and PA are critical for the heat-induced nuclear translocation of GAPC. We propose that PLDδ-produced PA mediates the process via lipid-protein interaction and that the lipid mediation acts as a cellular conduit linking stress perturbations at cell membranes to nuclear functions in plants coping with heat stress.