Nonsteroidal anti-inflammatory drugs alter the spatiotemporal organization of Ras proteins on the plasma membrane

Ras proteins on the inner leaflet of the plasma membrane signal from transient nanoscale proteolipid assemblies called nanoclusters. Interactions between the Ras lipid anchors and plasma membrane phospholipids, cholesterol, and actin cytoskeleton contribute to the formation, stability, and dynamics of Ras nanoclusters. Many small biological molecules are amphiphilic and capable of intercalating into membranes and altering lipid immiscibility. In this study we systematically examined whether amphiphiles such as indomethacin influence Ras protein nanoclustering in intact plasma membrane. We found that indomethacin, a nonsteroidal anti-inflammatory drug, induced profound and complex effects on Ras spatial organization, all likely related to liquid-ordered domain stabilization. Indomethacin enhanced the clustering of H-Ras.GDP and N-Ras.GTP in cholesterol-dependent nanoclusters. Indomethacin also abrogated efficient GTP-dependent lateral segregation of H- and N-Ras between cholesterol-dependent and cholesterol-independent clusters, resulting in mixed heterotypic clusters of Ras proteins that normally are separated spatially. These heterotypic Ras nanoclusters showed impaired Raf recruitment and kinase activation resulting in significantly compromised MAPK signaling. All of the amphiphilic anti-inflammatory agents we tested had similar effects on Ras nanoclustering and signaling. The potency of these effects correlated with the membrane partition coefficients of the individual agents and was independent of COX inhibition. This study shows that biological amphiphiles have wide-ranging effects on plasma membrane heterogeneity and protein nanoclustering, revealing a novel mechanism of drug action that has important consequences for cell signaling.     

Structure-based virtual screening and identification of a novel androgen receptor antagonist

Hormonal therapies, mainly combinations of anti-androgens and androgen deprivation, have been the mainstay treatment for advanced prostate cancer because the androgen-androgen receptor (AR) system plays a pivotal role in the development and progression of prostate cancers. However, the emergence of androgen resistance, largely due to inefficient anti-hormone action, limits the therapeutic usefulness of these therapies. Here, we report that 6-(3,4-dihydro-1H-isoquinolin-2-yl)-N-(6-methylpyridin-2-yl)nicotinamide (DIMN) acts as a novel anti-androgenic compound that may be effective in the treatment of both androgen-dependent and androgen-independent prostate cancers. Through AR structure-based virtual screening using the FlexX docking model, fifty-four compounds were selected and further screened for AR antagonism via cell-based tests. One compound, DIMN, showed an antagonistic effect specific to AR with comparable potency to that of the classical AR antagonists, hydroxyflutamide and bicalutamide. Consistent with their anti-androgenic activity, DIMN inhibited the growth of androgen-dependent LNCaP prostate cancer cells. Interestingly, the compound also suppressed the growth of androgen-independent C4-2 and CWR22rv prostate cancer cells, which express a functional AR, but did not suppress the growth of the AR-negative prostate cancer cells PPC-1, DU145, and R3327-AT3.1. Taken together, the results suggest that the synthetic compound DIMN is a novel anti-androgen and strong candidate for useful therapeutic agent against early stage to advanced prostate cancer.     

Selective Gαi subunits as novel direct activators of transient receptor potential canonical (TRPC)4 and TRPC5 channels

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca(2+)-permeable channels and mediate numerous cellular functions. It is commonly assumed that TRPC channels are activated by stimulation of Gα(q)-PLC-coupled receptors. However, whether the Gα(q)-PLC pathway is the main regulator of TRPC4/5 channels and how other Gα proteins may regulate these channels are poorly understood. We previously reported that TRPC4/TRPC5 can be activated by Gα(i). In the current work, we found that Gα(i) subunits, rather than Gα(q), are the primary and direct activators of TRPC4 and TRPC5. We report a novel molecular mechanism in which TRPC4 is activated by several Gα(i) subunits, most prominently by Gα(i2), and TRPC5 is activated primarily by Gα(i3). Activation of Gα(i) by the muscarinic M2 receptors or expression of the constitutively active Gα(i) mutants equally and fully activates the channels. Moreover, both TRPC4 and TRPC5 are activated by direct interaction of their conserved C-terminal SESTD (SEC14-like and spectrin-type domains) with the Gα(i) subunits. Two amino acids (lysine 715 and arginine 716) of the TRPC4 C terminus were identified by structural modeling as mediating the interaction with Gα(i2). These findings indicate an essential role of Gα(i) proteins as novel activators for TRPC4/5 and reveal the molecular mechanism by which G-proteins activate the channels.     

Validation of trans-acting elements that promote exon 7 skipping of SMN2 in SMN2-GFP stable cell line

Spinal muscular atrophy is a genetic disease in which the SMN1 gene is deleted. The SMN2 gene exists in all of the patients. Alternative splicing of these two genes are different. More than 90% of exon 7 included form is produced from SMN1 pre-mRNA, whereas only ～20% of exon 7 included form is produced from SMN2 pre-mRNA. Only exon 7 inclusion form produces functional protein. Exon 7 skipped SMN isoform is unstable. Here we constructed a GFP reporter system that recapitulates the alternative splicing of SMN1 and SMN2 pre-mRNA. We designed a system in which GFP protein is expressed only when exon 7 of is included in alternative splicing. The stable cell that expresses SMN1-GFP produces ～4 times more GFP protein than the stable cell line that expresses SMN2-GFP; as demonstrated by microscopy, FACS analysis and immunoblotting. In addition the ratio of exon 7 inclusion and skipping of SMN1-GFP and SMN2-GFP pre-mRNA was similar to endogenous SMN1 and SMN2 pre-mRNA as shown in RT-PCR. Furthermore the knockdown with hnRNP A1 shRNA, a known protein which promotes exon 7 skipping of SMN2, induces exon 7 inclusion of exon 7 in SMN2-GFP pre-mRNA in SMN2-GFP cell line. We conclude that we have established the stable cell lines that recapitulate alternative splicing of the SMN1 and SMN2 genes. The stable cell line can be used to identify the trans-acting elements with siRNA.     

Erythropoietin promotes oligodendrogenesis and myelin repair following lysolecithin-induced injury in spinal cord slice culture

Here, we sought to delineate the effect of EPO on the remyelination processes using an in vitro model of demyelination. We report that lysolecithin-induced demyelination elevated EPO receptor (EpoR) expression in oligodendrocyte progenitor cells (OPCs), facilitating the beneficial effect of EPO on the formation of oligodendrocytes (oligodendrogenesis). In the absence of EPO, the resultant remyelination was insufficient, possibly due to a limiting number of oligodendrocytes rather than their progenitors, which proliferate in response to lysolecithin-induced injury. By EPO treatment, lysolecithin-induced proliferation of OPCs was accelerated and the number of myelinating oligodendrocytes and myelin recovery was increased. EPO also enhanced the differentiation of neural progenitor cells expressing EpoR at high level toward the oligodendrocyte-lineage cells through activation of cyclin E and Janus kinase 2 pathways. Induction of myelin-forming oligodendrocytes by high dose of EPO implies that EPO might be the key factor influencing the final differentiation of OPCs. Taken together, our data suggest that EPO treatment could be an effective way to enhance remyelination by promoting oligodendrogenesis in association with elevated EpoR expression in spinal cord slice culture after lysolecithin-induced demyelination.     

Antiangiogenic properties of cafestol, a coffee diterpene, in human umbilical vein endothelial cells

As angiogenesis plays important roles in tumor growth and metastasis, searching for antiangiogenic compounds is a promising tactic for treating cancers. Cafestol, a diterpene found mainly in unfiltered coffee, provides benefit through varied biological activity, including antitumorigenic, antioxidative, and anti-inflammatory effects. This study aimed to investigate the effects of cafestol on angiogenesis and to uncover the associated mechanism. We show that cafestol inhibits angiogenesis of human umbilical vascular endothelial cells. This inhibition affects the following specific steps of the angiogenic process: proliferation, migration, and tube formation. The inhibitory effects of cafestol are accompanied by decreasing phosphorylation of FAK and Akt and by a decrease in nitric oxide production. Overall, cafestol inhibits angiogenesis by affecting the angiogenic signaling pathway.     

Replacement of microglial cells using Clodronate liposome and bone marrow transplantation in the central nervous system of SOD1(G93A) transgenic mice as an in vivo model of amyotrophic lateral sclerosis

Disease progression of amyotrophic lateral sclerosis (ALS) is partially mediated by the toxic microenvironment established by microglia. In the present study, we used SOD1G93A transgenic mice as an in vivo ALS model and replaced microglia expressing mutant SOD1 (mSOD1) with microglia expressing wild-type SOD1 (w/tSOD1) to modulate the toxic microenvironment. Stereotactic injection of Clodronate liposome, a selective toxin against the monocyte/macrophage system, into the fourth ventricle of the brains of 12-week-old asymptomatic ALS mice reduced the number of microglia effectively in the central nervous system. Subsequent bone marrow transplantation (BMT) with bone marrow cells (BMCs) expressing w/tSOD1 and GFP leads to replacement of the endogenous microglia of the ALS mice with microglia expressing w/tSOD1 and GFP. The expression of mSOD1 in the other neural cells was not influenced by the replacement procedures, and immunological side effects were not observed. The replacement of microglia significantly slowed disease progression and prolonged survival of the ALS mice compared with the ALS mice treated by stereotactic injection of PBS-liposome and BMT with BMCs expressing mSOD1 or w/tSOD1. These results suggest that replacement of microglia would improve the neural cell microenvironment, thereby slowing disease progression. The mechanisms and functional implications of this replacement require further elucidation.     

Identification of IRF8, TMEM39A, and IKZF3-ZPBP2 as susceptibility loci for systemic lupus erythematosus in a large-scale multiracial replication study

Systemic lupus erythematosus (SLE) is a chronic heterogeneous autoimmune disorder characterized by the loss of tolerance to self-antigens and dysregulated interferon responses. The etiology of SLE is complex, involving both heritable and environmental factors. Candidate-gene studies and genome-wide association (GWA) scans have been successful in identifying new loci that contribute to disease susceptibility; however, much of the heritable risk has yet to be identified. In this study, we sought to replicate 1,580 variants showing suggestive association with SLE in a previously published GWA scan of European Americans; we tested a multiethnic population consisting of 7,998 SLE cases and 7,492 controls of European, African American, Asian, Hispanic, Gullah, and Amerindian ancestry to find association with the disease. Several genes relevant to immunological pathways showed association with SLE. Three loci exceeded the genome-wide significance threshold: interferon regulatory factor 8 (IRF8; rs11644034; p_meta-Euro = 2.08 × 10⁻¹⁰), transmembrane protein 39A (TMEM39A; rs1132200; p_meta-all = 8.62 × 10⁻⁹), and 17q21 (rs1453560; p_meta-all = 3.48 × 10⁻¹⁰) between IKAROS family of zinc finger 3 (AIOLOS; IKZF3) and zona pellucida binding protein 2 (ZPBP2). Fine mapping, resequencing, imputation, and haplotype analysis of IRF8 indicated that three independent effects tagged by rs8046526, rs450443, and rs4843869, respectively, were required for risk in individuals of European ancestry. Eleven additional replicated effects (5 × 10⁻⁸ < p_meta-Euro < 9.99 × 10⁻⁵) were observed with CFHR1, CADM2, LOC730109/IL12A, LPP, LOC63920, SLU7, ADAMTSL1, C10orf64, OR8D4, FAM19A2, and STXBP6. The results of this study increase the number of confirmed SLE risk loci and identify others warranting further investigation.     

Crystal structure of Pyrococcus furiosus PF2050, a member of the DUF2666 protein family

Pyrococcus furiosus PF2050 is an uncharacterized putative protein that contains two DUF2666 domains. Functional and structural studies of PF2050 have not previously been performed. In this study, we determined the crystal structure of PF2050. The structure of PF2050 showed that the two DUF2666 domains interact tightly, forming a globular structure. Each DUF2666 domain comprises an antiparallel β-sheet and an α-helical bundle. One side of the PF2050 structure has a positively charged basic cleft, which may have a DNA-binding function. Furthermore, we confirmed that PF2050 interacts with circular and linear dsDNA.