siRNA screen of the human signaling proteome identifies the PtdIns(3,4,5)P3-mTOR signaling pathway as a primary regulator of transferrin uptake

A survey of 1,804 human dicer-generated signaling siRNAs using automated quantitative imaging identified the phosphatidylinositol-3,4,5-trisphosphate-mTOR signaling pathway as a primary regulator of iron-transferrin uptake

Pathogen-induced binding of the soybean zinc finger homeodomain proteins GmZF-HD1 and GmZF-HD2 to two repeats of ATTA homeodomain binding site in the calmodulin isoform 4 (GmCaM4) promoter

Calmodulin (CaM) is involved in defense responses in plants. In soybean (Glycine max), transcription of calmodulin isoform 4 (GmCaM4) is rapidly induced within 30 min after pathogen stimulation, but regulation of the GmCaM4 gene in response to pathogen is poorly understood. Here, we used the yeast one-hybrid system to isolate two cDNA clones encoding proteins that bind to a 30-nt A/T-rich sequence in the GmCaM4 promoter, a region that contains two repeats of a conserved homeodomain binding site, ATTA. The two proteins, GmZF-HD1 and GmZF-HD2, belong to the zinc finger homeodomain (ZF-HD) transcription factor family. Domain deletion analysis showed that a homeodomain motif can bind to the 30-nt GmCaM4 promoter sequence, whereas the two zinc finger domains cannot. Critically, the formation of super-shifted complexes by an anti-GmZF-HD1 antibody incubated with nuclear extracts from pathogen-treated cells suggests that the interaction between GmZF-HD1 and two homeodomain binding site repeats is regulated by pathogen stimulation. Finally, a transient expression assay with Arabidopsis protoplasts confirmed that GmZF-HD1 can activate the expression of GmCaM4 by specifically interacting with the two repeats. These results suggest that the GmZF-HD1 and –2 proteins function as ZF-HD transcription factors to activate GmCaM4 gene expression in response to pathogen

PRAS40 and PRR5-Like Protein Are New mTOR Interactors that Regulate Apoptosis

TOR (Target of Rapamycin) is a highly conserved protein kinase and a central controller of cell growth. TOR is found in two functionally and structurally distinct multiprotein complexes termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). In the present study, we developed a two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) based proteomic strategy to identify new mammalian TOR (mTOR) binding proteins. We report the identification of Proline-rich Akt substrate (PRAS40) and the hypothetical protein Q6MZQ0/FLJ14213/CAE45978 as new mTOR binding proteins. PRAS40 binds mTORC1 via Raptor, and is an mTOR phosphorylation substrate. PRAS40 inhibits mTORC1 autophosphorylation and mTORC1 kinase activity toward eIF-4E binding protein (4E-BP) and PRAS40 itself. HeLa cells in which PRAS40 was knocked down were protected against induction of apoptosis by TNFα and cycloheximide. Rapamycin failed to mimic the pro-apoptotic effect of PRAS40, suggesting that PRAS40 mediates apoptosis independently of its inhibitory effect on mTORC1. Q6MZQ0 is structurally similar to proline rich protein 5 (PRR5) and was therefore named PRR5-Like (PRR5L). PRR5L binds specifically to mTORC2, via Rictor and/or SIN1. Unlike other mTORC2 members, PRR5L is not required for mTORC2 integrity or kinase activity, but dissociates from mTORC2 upon knock down of tuberous sclerosis complex 1 (TSC1) and TSC2. Hyperactivation of mTOR by TSC1/2 knock down enhanced apoptosis whereas PRR5L knock down reduced apoptosis. PRR5L knock down reduced apoptosis also in mTORC2 deficient cells. The above suggests that mTORC2-dissociated PRR5L may promote apoptosis when mTOR is hyperactive. Thus, PRAS40 and PRR5L are novel mTOR-associated proteins that control the balance between cell growth and cell death

Aberrant actin depolymerization triggers the pyrin inflammasome and autoinflammatory disease that is dependent on IL-18, not IL-1beta

Gain-of-function mutations that activate the innate immune system can cause systemic autoinflammatory diseases associated with increased IL-1β production. This cytokine is activated identically to IL-18 by an intracellular protein complex known as the inflammasome; however, IL-18 has not yet been specifically implicated in the pathogenesis of hereditary autoinflammatory disorders. We have now identified an autoinflammatory disease in mice driven by IL-18, but not IL-1β, resulting from an inactivating mutation of the actin-depolymerizing cofactor Wdr1. This perturbation of actin polymerization leads to systemic autoinflammation that is reduced when IL-18 is deleted but not when IL-1 signaling is removed. Remarkably, inflammasome activation in mature macrophages is unaltered, but IL-18 production from monocytes is greatly exaggerated, and depletion of monocytes in vivo prevents the disease. Small-molecule inhibition of actin polymerization can remove potential danger signals from the system and prevents monocyte IL-18 production. Finally, we show that the inflammasome sensor of actin dynamics in this system requires caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain, and the innate immune receptor pyrin. Previously, perturbation of actin polymerization by pathogens was shown to activate the pyrin inflammasome, so our data now extend this guard hypothesis to host-regulated actin-dependent processes and autoinflammatory disease.Man Lyang Kim, Jae Jin Chae, Yong Hwan Park, Dominic De Nardo, Roslynn A. Stirzaker ... Benjamin T Kile ... et al

IKKα regulation of canonical NF-κB activation downstream of Nod1-mediated peptidoglycan recognition : "and" Endocytosis-independent function of clathrin heavy chain in the control of basal NF-κB activation

NF-κB is a transcription factor involved in the regulation of inflammation and innate immunity. The IκB kinase (IKK) complex contains two catalytic subunits, IKKα and IKKβ, and plays an essential role in the activation of NF-κB through the phosphorylation and degradation of the NF-κB inhibitor IκBα, thereby allowing translocation of NF-κB into the nucleus. Numerous evidences indicate that IKKβ mediates NF-κB activation in response to pro-inflammatory cytokines and microbial products, but the role of IKKα in inflammation and innate immunity is unknown. In the first part of dissertation, we focus on understanding the previously unknown function of IKKα in the canonical NF-κB pathway, associated with inflammation and innate immunity. We show that silencing of IKKα by RNA interference (RNAi) significantly reduced phosphorylation and degradation of IκBα, and nuclear translocation of NF-κB, and secretion of the pro-inflammatory chemokine interleukin-8 (IL-8) during Shigella flexneri infection of human epithelial HeLa cells. This suggests that IKKα like IKKβ plays a pivotal role in inflammation and innate immunity by mediating NF-κB activation in response to microbial infection. Proper control of NF-κB activation is essential for inflammation and innate immunity triggered by microbial infection, but the dysregulation of NF-κB is associated with various diseases such as chronic inflammatory diseases and cancers. Thus, the NF-κB pathway has been a target of therapeutic drug development. Although constitutive and excessive NF-κB activation has been detected in many inflammation-related diseases, the cause of the constitutive NF-κB activation in non-stimulated cells is largely unknown. In the second part of dissertation, we focus on clathrin heavy chain (CHC), a well-known regulator of endocytosis that plays a novel endocytosis-independent function as an inhibitor of basal NF-κB activation. We show that silencing of CHC induced constitutive NF-κB nuclear translocation and high level of IL-8 secretion in resting cells. We revealed that constitutive NF-κB nuclear translocation was mediated through the constant IκBα degradation in an IKKα-dependent mechanism. We further showed that CHC depletion-induced constitutive IκBα degradation and high level of IL-8 secretion in resting cells was independent of the inhibition of clathrin-mediated endocytosis (CME) as silencing of μ2 subunit of AP2 complex (AP2M1), an adaptor protein essential for CME failed to induce the constitutive IκBα degradation and high level of IL-8 secretion. Therefore, the results presented may suggest a potential link between a defect in CHC expression and chronic inflammatory disorders and cancers

CHC prevents constitutive degradation and phosphorylation of IκBα by an IKKα-dependent mechanism.

<p>(<b>A</b>) Reduced level of IκBα after CHC knockdown in HeLa cells. Cell lysates from control or CHC siRNA transfected cells were analyzed by western immunoblotting using indicated antibodies. Actin is shown as a loading control (representative of 3 independent experiments). (<b>B</b>) Quantification of the level of <i>IκBα</i> mRNA by quantitative RT-PCR in control or CHC-depleted HeLa cells. <i>GAPDH</i> mRNA was used as an internal control for normalization (results are expressed as the mean ± SD of 3 independent experiments). (<b>C</b>) IKKα-depletion abolishes the constitutive degradation of IκBα induced by CHC knockdown. Lysates from cells transfected with different combinations of IKKα and CHC siRNAs were analyzed by immunoblotting using indicated antibodies. Total siRNA concentration was kept constant by adding appropriate amounts of control siRNAs. Actin is shown as a loading control (data representative of 2 independent experiments). (<b>D</b>) CHC prevents enhanced basal phosphorylation of IκBα at position serine 32. Lysates from control or CHC-depleted HeLa cells were analyzed by immunoblotting using the indicated antibodies. Actin is shown as a loading control. (<b>E</b>) Densitometric quantification of the p-IκBα/IκBα ratio (results are expressed as the mean ± SD of 3 independent experiments).</p

CHC regulates NF-κB activation independently of endocytosis and CLCa.

<p>(<b>A</b>) Uptake of Alexa 594-transferrin (Alexa 594-Tf) in cells transfected with control (left panels), AP2M1 (middle panels) or CHC (right panels) siRNAs; Scale bars, 10 µm. (<b>B</b>) Quantification of transferrin uptake by automated image analysis (results are expressed as the mean ± SD of 12 images; graph representative of 2 independent experiments). (<b>C</b>) AP2M1 knockdown fails to enhance IκBα degradation. Cell lysates from control, AP2M1 or CHC siRNA-transfected cells were analyzed by immunoblotting using indicated antibodies. Actin is shown as a loading control. (<b>D</b>) Densitometric quantification of the levels of IκBα shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017158#pone-0017158-g004" target="_blank">Figure 4C</a> (graph representative of 2 independent experiments). (<b>E</b>) AP2M1 knockdown fails to induce constitutive IL-8 expression. HeLa cells were transfected with control, AP2M1 or CHC siRNAs for 72 hours. Supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments). (<b>F</b>) Inhibition of transferrin uptake after dynasore and PAO treatment. HeLa cells were left untreated (Ctrl) or treated with dynasore (80 µM) (Dyn) or PAO (5 µM) 10 minutes before and during the transferrin uptake assay (results are expressed as the mean ± SD of 18 images; graph representative of 2 independent experiments). (<b>G</b>) Dynasore and PAO fail to enhance basal degradation of IκBα. HeLa cells were pretreated for 10 minutes with dynasore (80 µM) or PAO (5 µM) and analyzed by western immunoblotting using an IκBα antibody. Actin is shown as a loading control (results representative of 2 independent experiments). (<b>H</b>) Long-term inhibition of endocytosis in dynasore-treated HeLa cells. Transferrin uptake in HeLa cells left untreated or treated with dynasore (80 µM) for 48 hours (results are expressed as the mean ± SD of 18 images; graph representative of 2 independent experiments). (<b>I</b>) Long-term inhibition of endocytosis fails to enhance the basal degradation of IκBα. Basal degradation of IκBα in HeLa cells left untreated or treated with dynasore (80 µM) for 48 hours. As positive control of the degradation of IκBα, cells were stimulated for 20 minutes with TNFα (results representative of 2 independent experiments). (<b>J</b>) CLCa knockdown fails to enhance IκBα degradation. Cell lysates from control, CLCa or CHC siRNA transfected cells were analyzed by immunoblotting using indicated antibodies. Actin is shown as loading control. (<b>K</b>) Densitometric quantification of IκBα levels shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017158#pone-0017158-g004" target="_blank">Figure 4F</a> (Graph representative of 2 independent experiments). (<b>L</b>) CLCa knockdown fails to induce constitutive IL-8 expression. HeLa cells were transfected with control, CLCa or CHC siRNAs for 72 hours and supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments).</p

CHC prevents constitutive IL-8 expression in unstimulated epithelial cells.

<p>(<b>A</b>) Constitutive IL-8 expression after knockdown of CHC in HeLa cells. Cells were transfected with control or CHC siRNAs. After 72 hours, supernatants were collected and analyzed for their content in IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments). (<b>B</b>) Constitutive IL-8 expression after knockdown of CHC in MCF-7 cells. MCF-7 cells were treated as described in (A) (results are expressed as the mean ± SD of 3 independent experiments). (<b>C</b>) IKKα-depletion abolishes the constitutive secretion of IL-8 induced by CHC knockdown. HeLa cells were transfected with different combinations of IKKα and CHC siRNAs for 72 hours. Total siRNA concentration was kept constant by adding appropriate amounts of control siRNAs. Supernatants were collected to measure the concentration of IL-8 by ELISA (results are expressed as the mean ± SD of 3 independent experiments).</p

CHC prevents constitutive NF-κB p65 nuclear localization in unstimulated epithelial cells.

<p>(<b>A</b>) Effective knockdown of CHC after siRNA transfection. Lysates from HeLa cells transfected with control (Ctrl) or CHC siRNAs for 72 hours were analyzed by western immunoblotting using indicated antibodies. Actin is shown as a loading control. (<b>B</b>) Constitutive nuclear localization of p65 after CHC knockdown. HeLa cells were transfected with either control or CHC siRNA and p65 localization was visualized by immunofluorescence microscopy. White arrows indicate cells showing a clear nuclear localization of p65. Scale bars, 10 µm. (<b>C</b>) Quantification of the nuclear/cytosolic p65 intensity ratio in control and CHC siRNA transfected HeLa cells (results are expressed as the mean ± SD of 12 images; *p = 3.14E-07, graph representative of 3 independent experiments).</p