Withaferin A alleviates inflammation and joint injury in arthritic rats via elevating microRNA-1297 to target karyopherin alpha2.

Withaferin A (WFA) is a natural compound separated from the medicinal plant Withania somnifera. As reported, it has the potential to safely cure rheumatoid arthritis (RA) in animal models. Nevertheless, the action mechanism of WFA in treating RA has not been completely illuminated. The study was to explore the action and mechanism of WFA on arthritic rats. First, a collagen-induced arthritis rat model was established. WFA administration alleviated inflammation and injury in arthritic rats. Subsequently, fibroblast synovial cells (FLS) of arthritic rats were separated and cell proliferation and apoptosis abilities were tested. It was found that WFA was available to repress FLS cell proliferation and accelerate apoptosis. MicroRNA-1297 was downregulated in RA patients. Clinical correlation analysis suggested that miR-1297 in the serum of RA patients was negatively associated with pro-inflammatory factors interleukin (IL)-6, IL-17, tumor necrosis factor (TNF)-α, and RA diagnostic indexes (RF, DAS28). In the meantime, miR-1297 had superior diagnostic value in differentiating RA patients from healthy people. Karyopherin α2 (KPNA2) was the downstream target of miR-1297, while miR-1297 negatively modulated KPNA2 expression. Importantly, WFA further restrained KPNA2 expression via elevating miR-1297 in functional rescue experiments, thereby treating inflammation and injury in arthritic rats and repressing FLS cell proliferation and activation. In short, WFA alleviated inflammation and joint damage in arthritic rats via elevating miR-1297 to target KPNA2.

Structural basis for activation of fungal sterol receptor Upc2 and azole resistance.

Fungal transcription factor Upc2 senses ergosterol levels and regulates sterol biosynthesis and uptake. Constitutive activation of Upc2 causes azole resistance in Candida species. We determined the structure of ergosterol-bound Upc2, revealing the ligand specificity and transcriptional regulation. Ergosterol binding involves conformational changes of the ligand-binding domain, creating a shape-complementary hydrophobic pocket. The conserved helix α12 and glycine-rich loop are critical for sterol recognition by forming the pocket wall. The mutations of the glycine-rich loop inhibit ligand binding by steric clashes and constitutively activate Upc2. The translocation of Upc2 is regulated by Hsp90 chaperone in a sterol-dependent manner. Ergosterol-bound Upc2 associates with Hsp90 using the C-terminal tail, which retains the inactive Upc2 in the cytosol. Ergosterol dissociation induces a conformational change of the C-terminal tail, releasing Upc2 from Hsp90 for nuclear transport by importin α. The understanding of the regulatory mechanism provides an antifungal target for the treatment of azole-resistant Candida infections.

Natural lactucopicrin alleviates importin-α3-mediated NF-κB activation in inflammated endothelial cells and improves sepsis in mice.

Lactucopicrin, a bitter sesquiterpene lactone of leafy vegetables, such as chicory, curly escarole, and lettuce, possesses anti-malarial, anti-cancer and analgesic properties. However, it remains unknown whether lactucopicrin could inhibit vascular endothelial nuclear factor-κB (NF-κB) activation, a hallmark of vascular inflammatory diseases including sepsis. In tumor necrosis factor-α-stimulated human or mouse aortic endothelial cells, lactucopicrin dose-dependently inhibited NF-κB activation, and concomitantly repressed both vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1)-mediated monocyte adhesion. The lactucopicrin effect was not due to modulation of inhibitor of NF-κB kinases (IKK) α/ß/γ, inhibitor of NF-κB alpha (IκBα), and NF-κB/p65 DNA binding activity. Instead, lactucopicrin inhibited importin-α3 expression by destabilization of its mRNA, an effect mediating the lactucopicrin effect on NF-κB activity. More importantly, in lipopolysaccharide (LPS)-elicited septic mice, oral gavage with lactucopicrin decreased mortality by 30.5% as compared with the control treatment. This effect was associated with inhibited importin-α3 expression, suppressed NF-κB activation and VCAM-1/ICAM-1 expression, and inhibited leukocyte influx in the vascular endothelium of both lung and aorta. Collectively, our novel data suggest that dietary supplementation with lactucopicrin inhibits endothelial NF-κB activation by down-regulation of importin-α3 and thereby improves sepsis.

Pharmacoinformatics based elucidation and designing of potential inhibitors against Plasmodium falciparum to target importin α/ß mediated nuclear importation.

Plasmodium falciparum, the prime causative agent of malaria, is responsible for 4, 05,000 deaths per year and fatality rates are higher among the children aged below 5 years. The emerging distribution of the multi-drug resistant P. falciparum becomes a worldwide concern, so the identification of unique targets and novel inhibitors is a prime need now. In the present study, we have employed pharmacoinformatics approaches to analyze 265 lead-like compounds from PubChem databases for virtual screening. Thereafter, 15 lead-like compounds were docked within the active side pocket of importin alpha. Comparative ligand properties and absorption, distribution, metabolism, excretion, and toxicity (ADMET) profile were also assessed. Finally, a novel inhibitor was designed and assessed computationally for its efficacy. From the comparative analysis we have found that our screened compounds possess better results than the existing lead ivermectin; having the highest binding energy of -15.6 kcal/mol, whereas ivermectin has -12.4 kcal/mol. The novel lead compound possessed more fascinating output without deviating any of the rules of Lipinski. It also possessed higher bioavailability and the drug-likeness score of 0.55 and 0.71, respectively compared to ivermectin. Furthermore, the binding study was confirmed by molecular dynamics simulation over 25 ns by evaluating the stability of the complex. Finally, all the screened compounds and the novel compound showed promising ADMET properties likewise. To end, we hope that our proposed screened compounds, as well as the novel compound, might give some advances to treat malaria efficiently in vitro and in vivo.

YY1-mediated overexpression of long noncoding RNA MCM3AP-AS1 accelerates angiogenesis and progression in lung cancer by targeting miR-340-5p/KPNA4 axis.

Lung cancer is famous as an aggressive malignant tumor and is the main cause of cancer-associated mortality globally. Tumor angiogenesis is a vital part in cancer, which influences cell proliferation and metastasis. Increasing studies have claimed that long noncoding RNAs (lncRNAs) were involved in the progression of several cancers. Based on previous studies, this study focused on the role and mechanism of lncRNA MCM3AP antisense RNA 1 (MCM3AP-AS1) in lung cancer. At first, MCM3AP-AS1 expression was found to be elevated in lung cancer cells. Depletion of MCM3AP-AS1 repressed cell proliferation, migration, and angiogenesis in lung cancer cells. YY1 was confirmed to mediate MCM3AP-AS1 transcription in lung cancer cells. Moreover, the molecular mechanism investigation revealed that MCM3AP-AS1 could sponge miR-340-5p and elevate KPNA4 expression. On the basis of rescue assays, we identified that the overexpression of KPNA4 partly counteracted the suppressed effect of MCM3AP-AS1 knockdown on angiogenesis and progression in lung cancer cells. Conclusively, the YY1-mediated overexpression of MCM3AP-AS1 accelerated angiogenesis and progression in lung cancer by targeting miR-340-5p/KPNA4 axis, which highlighted the possibility of MCM3AP-AS1 as a promising therapeutic target for lung cancer.

Vitamin D Deficiency Accelerates Coronary Artery Disease Progression in Swine.

OBJECTIVE: The role of vitamin D deficiency in coronary artery disease (CAD) progression is uncertain. Chronic inflammation in epicardial adipose tissue (EAT) has been implicated in the pathogenesis of CAD. However, the molecular mechanism underlying vitamin D deficiency-enhanced inflammation in the EAT of diseased coronary arteries remains unknown. We examined a mechanistic link between 1,25-dihydroxyvitamin D-mediated suppression of nuclear factor-κB (NF-κB) transporter, karyopherin α4 (KPNA4) expression and NF-κB activation in preadipocytes. Furthermore, we determined whether vitamin D deficiency accelerates CAD progression by increasing KPNA4 and nuclear NF-κB levels in EAT. APPROACH AND RESULTS: Nuclear protein levels were detected by immunofluorescence and Western blot. Exogenous KPNA4 was transported into cells by a transfection approach and constituted lentiviral vector. Swine were administered vitamin D-deficient or vitamin D-sufficient hypercholesterolemic diet. After 1 year, the histopathology of coronary arteries and nuclear protein expression of EAT were assessed. 1,25-dihydroxyvitamin D inhibited NF-κB activation and reduced KPNA4 levels through increased vitamin D receptor expression. Exogenous KPNA4 rescued 1,25-dihydroxyvitamin D-dependent suppression of NF-κB nuclear translocation and activation. Vitamin D deficiency caused extensive CAD progression and advanced atherosclerotic plaques, which are linked to increased KPNA4 and nuclear NF-κB levels in the EAT. CONCLUSIONS: 1,25-dihydroxyvitamin D attenuates NF-κB activation by targeting KPNA4. Vitamin D deficiency accelerates CAD progression at least, in part, through enhanced chronic inflammation of EAT by upregulation of KPNA4, which enhances NF-κB activation. These novel findings provide mechanistic evidence that vitamin D supplementation could be beneficial for the prevention and treatment of CAD.

KPNA2 over-expression is a potential marker of prognosis and therapeutic sensitivity in colorectal cancer patients.

BACKGROUND: Karyopherin α 2 (KPNA2) is a member of the Karyopherin α family and has recently been reported to play an important role in tumor progression. The aim of the current study was to elucidate the clinicopathological significance of KPNA2 over-expression in colorectal cancer (CRC). PATIENTS AND METHODS: KPNA2 expression was evaluated by immunohistochemistry in 122 surgically resected CRC and 13 biopsy specimens obtained at colonoscopy during screening for preoperative hyperthermochemoradiation therapy (HCRT). The association between KPNA2 expression and clinicopathological features and preoperative HCRT efficacy were examined. RESULTS: The high and low KNPA2 expression groups were comprised of 91 (74.6%) and 31 CRC patients, respectively. A significant association was observed between high expression and lymphatic invasion (P = 0.0245). KPNA2 high expression group had decreased overall survival (P = 0.00374). Multivariate analysis demonstrated high KPNA2 expression was independently associated with poor prognosis. Histological examinations revealed 11 (84.6%) and 2 (15.4%) of cases were KPNA2 positive and negative, respectively. Pathological complete response (pCR) was observed in 9.1% of KPNA2-positive cases and 100% of KPNA2-negative cases. CONCLUSION: High KPNA2 expression was found to be associated with poor prognosis and resistance to HCRT.

Hirsutinolide Series Inhibit Stat3 Activity, Alter GCN1, MAP1B, Hsp105, G6PD, Vimentin, TrxR1, and Importin α-2 Expression, and Induce Antitumor Effects against Human Glioma.

We report that hirsutinolide series, 6, 7, 10, 11, 20, and 22, and the semisynthetic analogues, 30, 31, 33, and 36, inhibit constitutively active signal transducer and activator of transcription (Stat)3 and malignant glioma phenotype. A position 13 lipophilic ester group is required for activity. Molecular modeling and nuclear magnetic resonance structural analyses reveal direct hirsutinolide:Stat3 binding. One-hour treatment of cells with 6 and 22 also upregulated importin subunit α-2 levels and repressed translational activator GCN1, microtubule-associated protein (MAP)1B, thioredoxin reductase (TrxR)1 cytoplasmic isoform 3, glucose-6-phosphate 1-dehydrogenase isoform a, Hsp105, vimentin, and tumor necrosis factor α-induced protein (TNAP)2 expression. Active hirsutinolides inhibited anchorage-dependent and three-dimensional spheroid growth, survival, and migration of human glioma lines and glioma patients' tumor-derived xenograft cells harboring constitutively active Stat3. Oral gavage delivery of 6 or 22 inhibited human glioma tumor growth in subcutaneous mouse xenografts. The inhibition of Stat3 signaling represents part of the hirsutinolide-mediated mechanisms to induce antitumor effects.

Vitamin D supplementation reduces airway hyperresponsiveness and allergic airway inflammation in a murine model.

BACKGROUND: Asthma is a chronic disease associated with airway hyperresponsiveness (AHR), airway obstruction and airway remodelling. NF-κB is a transcriptional factor that regulates and co-ordinates the expression of various inflammatory genes. The NF-κB subunits, p50 and Rel-A, are translocated to the nucleus by importin α3 and importin α4. There is growing evidence that vitamin D is a potent immunomodulator. However, the evidence for beneficial or adverse effects of vitamin D in asthma is still unclear. OBJECTIVE: In this study, we examined the effect of vitamin D status on AHR, airway inflammation and cytokines in the bronchoalveolar lavage fluid (BALF) in a murine model of allergic asthma. METHODS: Female BALB/c mice were fed with special vitamin D-deficient or vitamin D-sufficient (2000 IU/kg) or vitamin D-supplemented (10,000 IU/kg) diet for 13 weeks. Mice were sensitized and challenged with ovalbumin (OVA). The effect of vitamin D on lung histology, AHR, T regulatory cells (Tregs) and BALF cytokines was examined. The expression of importin-α3 and Rel-A in the lung of OVA-sensitized mice was analysed using immunofluorescence. RESULTS: Vitamin D deficiency was associated with higher AHR in OVA-sensitized and challenged mice than those in vitamin D-sufficient mice. This was accompanied with marked signs of airway remodelling, high BALF eosinophilia, increased BALF pro-inflammatory cytokines, reduced BALF IL-10 levels, reduced blood Tregs, increased expression of importin-α3 and Rel-A in the lung tissue. Vitamin D supplementation attenuated the pro-inflammatory effects, but did not completely reverse the features of allergic airway inflammation. CONCLUSION AND CLINICAL RELEVANCE: Vitamin D could be beneficial as an adjunct therapy in the treatment of allergic asthma.

Nuclear localization of Beet black scorch virus capsid protein and its interaction with importin α.

Beet black scorch virus (BBSV) is a positive-sense, single-stranded RNA virus belonging to Necrovirus genus. In order to better understand the life cycle of BBSV, we have investigated the subcellular localization of BBSV capsid protein (CP) by its fusion with green fluorescent protein (GFP) agroinfiltrated into Nicotiana benthamiana leaves and by particle bombardment into onion (Allium cepa) epidermal cells. Confocal laser scanning microscopy (CLSM) showed that BBSV CP fused to GFP displayed enhanced fluorescence in nuclei and nuclear import of the CP was confirmed in BBSV-infected N. benthamiana leaves. Mutational analysis revealed that the N-terminal basic amino acid cluster (4)KRNKGGKKSR(13) of the CP is essential for nuclear localization. Bimolecular fluorescence complementation (BiFC) assays indicated that the CP could interact with the nuclear import factor importin α, suggesting that the CP is possibly imported into the nucleus via an importin α-dependent pathway. This is the first report of the nuclear localization of the CP encoded by a necrovirus.

Phospholipid scramblase 1 contains a nonclassical nuclear localization signal with unique binding site in importin alpha.

Nuclear import of proteins containing a classical nuclear localization signal (NLS) is an energy-dependent process that requires the heterodimer importin alpha/beta. Three to six basic contiguous arginine/lysine residues characterize a classical NLS and are thought to form a basic patch on the surface of the import cargo. In this study, we have characterized the NLS of phospholipid scramblase 1 (PLSCR1), a lipid-binding protein that enters the nucleus via the nonclassical NLS (257)GKISKHWTGI(266). This import sequence lacks a contiguous stretch of positively charged residues, and it is enriched in hydrophobic residues. We have determined the 2.2 A crystal structure of a complex between the PLSCR1 NLS and the armadillo repeat core of vertebrate importin alpha. Our crystallographic analysis reveals that PLSCR1 NLS binds to armadillo repeats 1-4 of importin alpha, but its interaction partially overlaps the classical NLS binding site. Two PLSCR1 lysines occupy the canonical positions indicated as P2 and P5. Moreover, we present in vivo evidence that the critical lysine at position P2, which is essential in other known NLS sequences, is dispensable in PLSCR1 NLS. Taken together, these data provide insight into a novel nuclear localization signal that presents a distinct motif for binding to importin alpha.

Isolation of a protein that is essential for the first step of nuclear protein import.

We have purified a cytosolic protein from Xenopus eggs that is essential for selective protein import into the cell nucleus. The purified protein, named importin, promotes signal-dependent binding of karyophilic proteins to the nuclear envelope. We have cloned, sequenced, and expressed a corresponding cDNA. Importin shows 44% sequence identity with SRP1p, a protein associated with the yeast nuclear pore complex. Complete, signal-dependent import into HeLa nuclei can be reconstituted by combining importin purified from Xenopus eggs or expressed in E. coli with Ran/TC4. Evidence for additional stimulatory factors is provided.