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

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

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 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 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

ADP‐heptose is a newly identified pathogen‐associated molecular pattern of Shigella flexneri

International audienceDuring an infection, the detection of pathogens is mediated through the interactions between pathogen-associated molecular patterns (PAMPs) and pathogen recognition receptors. β-Heptose 1,7-bisphosphate (βHBP), an intermediate of the lipopolysaccharide (LPS) biosynthesis pathway, was recently identified as a bacterial PAMP. It was reported that βHBP sensing leads to oligomerization of TIFA proteins, a mechanism controlling NF-κB activation and pro-inflammatory gene expression. Here, we compare the ability of chemically synthesized βHBP and Shigella flexneri lysate to induce TIFA oligomerization in epithelial cells. We find that, unlike bacterial lysate, βHBP fails to initiate rapid TIFA oligomerization. It only induces delayed signaling, suggesting that βHBP must be processed intracellularly to trigger inflammation. Gene deletion and complementation analysis of the LPS biosynthesis pathway revealed that ADP-heptose is the bacterial metabolite responsible for rapid TIFA oligomerization. ADP-heptose sensing occurs down to 10-10 M. During S. flexneri infection, it results in cytokine production, a process dependent on the kinase ALPK1. Altogether, our results rule out a major role of βHBP in S. flexneri infection and identify ADP-heptose as a new bacterial PAMP

Conformationally constrained Lipid A mimetics for exploration of structural basis of TLR4/MD-2 activation by lipopolysaccharide

Recognition of the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, by the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD-2) complex is essential for the control of bacterial infection. A pro-inflammatory signaling cascade is initiated upon binding of membrane-associated portion of LPS, a glycophospholipid Lipid A, by a co-receptor protein MD-2, which results in a protective host innate immune response. However, activation of TLR4 signaling by LPS may lead to the dysregulated immune response resulting in a variety of inflammatory conditions including sepsis syndrome. Understanding of structural requirements for Lipid A endotoxicity would ensure the development of effective anti-inflammatory medications. Herein we report on design, synthesis and biological activities of a series of conformationally confined Lipid A mimetics based on β,α-trehalose-type scaffold. Replacement of the flexible three-bond β(1→6) linkage in diglucosamine backbone of Lipid A by a two-bond β,α(1↔1) glycosidic linkage afforded novel potent TLR4 antagonists. Synthetic tetraacylated bisphosphorylated Lipid A mimetics based on a β-GlcN(1↔1)α-GlcN scaffold selectively block the LPS binding site on both human and murine MD-2 and completely abolish lipopolysaccharide-induced pro-inflammatory signaling, thereby serving as antisepsis drug candidates. In contrast to their natural counterpart lipid IVa, conformationally constrained Lipid A mimetics do not activate mouse TLR4. The structural basis for high antagonistic activity of novel Lipid A mimetics was confirmed by molecular dynamics simulation. Our findings suggest that besides the chemical structure, also the three-dimensional arrangement of the diglucosamine backbone of MD-2-bound Lipid A determines endotoxic effects on TLR4