Proteomic Profiling of the TRAF3 Interactome Network Reveals a New Role for the ER-to-Golgi Transport Compartments in Innate Immunity

Tumor Necrosis Factor receptor-associated factor-3 (TRAF3) is a central mediator important for inducing type I interferon (IFN) production in response to intracellular double-stranded RNA (dsRNA). Here, we report the identification of Sec16A and p115, two proteins of the ER-to-Golgi vesicular transport system, as novel components of the TRAF3 interactome network. Notably, in non-infected cells, TRAF3 was found associated with markers of the ER-Exit-Sites (ERES), ER-to-Golgi intermediate compartment (ERGIC) and the cis-Golgi apparatus. Upon dsRNA and dsDNA sensing however, the Golgi apparatus fragmented into cytoplasmic punctated structures containing TRAF3 allowing its colocalization and interaction with Mitochondrial AntiViral Signaling (MAVS), the essential mitochondria-bound RIG-I-like Helicase (RLH) adaptor. In contrast, retention of TRAF3 at the ER-to-Golgi vesicular transport system blunted the ability of TRAF3 to interact with MAVS upon viral infection and consequently decreased type I IFN response. Moreover, depletion of Sec16A and p115 led to a drastic disorganization of the Golgi paralleled by the relocalization of TRAF3, which under these conditions was unable to associate with MAVS. Consequently, upon dsRNA and dsDNA sensing, ablation of Sec16A and p115 was found to inhibit IRF3 activation and anti-viral gene expression. Reciprocally, mild overexpression of Sec16A or p115 in Hec1B cells increased the activation of IFNβ, ISG56 and NF-κB -dependent promoters following viral infection and ectopic expression of MAVS and Tank-binding kinase-1 (TBK1). In line with these results, TRAF3 was found enriched in immunocomplexes composed of p115, Sec16A and TBK1 upon infection. Hence, we propose a model where dsDNA and dsRNA sensing induces the formation of membrane-bound compartments originating from the Golgi, which mediate the dynamic association of TRAF3 with MAVS leading to an optimal induction of innate immune responses.     

Structural Basis for Auto-Inhibition of the NDR1 Kinase Domain by an Atypically Long Activation Segment

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High-Density Proximity Mapping Reveals the Subcellular Organization of mRNA-Associated Granules and Bodies

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The insulin-induced signalling pathway leading to S6 and initiation factor 4E binding protein 1 phosphorylation bifurcates at a rapamycin-sensitive point immediately upstream of p70s6k.

Employing specific inhibitors and docking-site mutants of growth factor receptors, recent studies have indicated that the insulin-induced increase in 40S ribosomal protein S6 and initiation factor 4E binding protein 1 (4E-BP1) phosphorylation is mediated by the mTOR/FRAP-p70s6k signal transduction pathway. However, it has not been resolved whether the phosphorylation of both proteins is mediated by p70s6k or whether they reside on parallel pathways which bifurcate upstream of p70s6k. Here we have used either rapamycin-resistant, kinase-dead, or wild-type p70s6k variants to distinguish between these possibilities. The rapamycin-resistant p70s6k, which has high constitutive activity, was able to signal to S6 in the absence of insulin and to prevent the rapamycin-induced block of S6 phosphorylation. This same construct did not increase the basal state of 4E-BP1 phosphorylation or protect it from the rapamycin-induced block in phosphorylation. Unexpectedly, the rapamycin-resistant p70s6k inhibited insulin-induced 4E-BP1 phosphorylation in a dose-dependent manner. This effect was mimicked by the kinase-dead and wild-type p70s6k constructs, which also blocked insulin-induced dissociation of 4E-BP1 from initiation factor 4E. Both the kinase-dead and wild-type constructs also blocked reporter p70s6k activation, although only the kinase-dead p70s6k had a dominant-interfering effect on S6 phosphorylation. Analysis of phosphopeptides from reporter 4E-BP1 and p70s6k revealed that the kinase-dead p70s6k affected the same subset of sites as rapamycin in both proteins. The results demonstrate, for the first time, that activated p70s6k mediates increased S6 phosphorylation in vivo. Furthermore, they show that increased 4E-BP1 phosphorylation is controlled by a parallel signalling pathway that bifurcates immediately upstream of p70s6k, with the two pathways sharing a common rapamycin-sensitive activator.     

Amiloride does not alter NaCl avoidance in Fischer-344 rats

Fischer-344 (F-344) rats differ from other common rat strains in that they fail to show any preference for NaCl at any concentration in two- bottle preference tests. Because 100 microM amiloride partially blocks the NaCl-evoked chorda tympani (CT) response in electrophysiological studies, we tested NaCl preference (0.068-0.273 M) in F-344 rats with and without 100 microM amiloride solution as the solvent. A third group was tested with unadulterated NaCl solutions following CT transection. Amiloride had no significant effect on the NaCl preference-aversion function, whereas CT transection significantly reduced NaCl avoidance. These results suggest that the amiloride-sensitive component of the NaCl response is not necessary for F-344 rats to display avoidance of NaCl, but the entire CT input is.      

Comparison between personal exposure to 60 Hz magnetic fields and stationary home measurements for people living near and away from a 735 kV power line.

This study compares stationary home measurements with a personal exposure monitor of 60 Hz magnetic fields in a group of 18 people living near a 735 kV line and 17 people living far away from the line. Most of them were white collar workers who worked during the day. They wore a personal Positron meter for 24 h, while a similar meter was left in their home, away from any appliances. For people living away from the line, the impact of residential activities appeared rather weak when considering the average intensity of the field during the awake period (at home): 0.22 microT for personal exposure versus 0.18 microT for stationary measurements (P = 0.09). The impact of residential activities during the awake period was more detectable when using the percentage of time with exposure above 0.78 microT: median 0.4 for personal vs. 0.0 for stationary measurements (P =.01). The temporal variability of the exposure during the awake period was also significantly higher for personal exposure than for stationary measurements. For people living near the line, the intensity of the magnetic field from the line dominated the personal exposure when considering the mean of measurements and the percentage of time above a threshold. However, the temporal variability was greater for the personal exposure during the awake period. Although limited due to its small sample size, the present study seems to demonstrate the usefulness of considering different indexes of exposure when assessing residential exposure to 60 Hz magnetic fields.     

Human eukaryotic translation initiation factor 4G (eIF4G) recruits mnk1 to phosphorylate eIF4E.

Human eukaryotic translation initiation factor 4E (eIF4E) binds to the mRNA cap structure and interacts with eIF4G, which serves as a scaffold protein for the assembly of eIF4E and eIF4A to form the eIF4F complex. eIF4E is an important modulator of cell growth and proliferation. It is the least abundant component of the translation initiation machinery and its activity is modulated by phosphorylation and interaction with eIF4E-binding proteins (4E-BPs). One strong candidate for the eIF4E kinase is the recently cloned MAPK-activated protein kinase, Mnk1, which phosphorylates eIF4E on its physiological site Ser209 in vitro. Here we report that Mnk1 is associated with the eIF4F complex via its interaction with the C-terminal region of eIF4G. Moreover, the phosphorylation of an eIF4E mutant lacking eIF4G-binding capability is severely impaired in cells. We propose a model whereby, in addition to its role in eIF4F assembly, eIF4G provides a docking site for Mnk1 to phosphorylate eIF4E. We also show that Mnk1 interacts with the C-terminal region of the translational inhibitor p97, an eIF4G-related protein that does not bind eIF4E, raising the possibility that p97 can block phosphorylation of eIF4E by sequestering Mnk1.     

Diaphragm adaptations in patients with COPD

Inspiratory muscle weakness in patients with COPD is of major clinical relevance. For instance, maximum inspiratory pressure generation is an independent determinant of survival in severe COPD. Traditionally, inspiratory muscle weakness has been ascribed to hyperinflation-induced diaphragm shortening. However, more recently, invasive evaluation of diaphragm contractile function, structure, and biochemistry demonstrated that cellular and molecular alterations occur, of which several can be considered pathologic of nature. Whereas the fiber type shift towards oxidative type I fibers in COPD diaphragm is regarded beneficial, rendering the overloaded diaphragm more resistant to fatigue, the reduction of diaphragm fiber force generation in vitro likely contributes to diaphragm weakness. The reduced diaphragm force generation at single fiber level is associated with loss of myosin content in these fibers. Moreover, the diaphragm in COPD is exposed to oxidative stress and sarcomeric injury. This review postulates that the oxidative stress and sarcomeric injury activate proteolytic machinery, leading to contractile protein wasting and, consequently, loss of force generating capacity of diaphragm fibers in patients with COPD. Interestingly, several of these presumed pathologic alterations are already present early in the course of the disease (GOLD I/II), although these patients appear not limited in their daily life activities. Treatment of diaphragm dysfunction in COPD is complex since its etiology is unclear, but recent findings indicate the ubiquitin-proteasome pathway as a prime target to attenuate diaphragm wasting in COPD.     

The Structure of an Interdomain Complex That Regulates Talin Activity

Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. It exists in both globular and extended conformations, and an intramolecular interaction between the N-terminal F3 FERM subdomain and the C-terminal part of the talin rod contributes to an autoinhibited form of the molecule. Here, we report the solution structure of the primary F3 binding domain within the C-terminal region of the talin rod and use intermolecular nuclear Overhauser effects to determine the structure of the complex. The rod domain (residues 1655–1822) is an amphipathic five-helix bundle; Tyr-377 of F3 docks into a hydrophobic pocket at one end of the bundle, whereas a basic loop in F3 (residues 316–326) interacts with a cluster of acidic residues in the middle of helix 4. Mutation of Glu-1770 abolishes binding. The rod domain competes with β3-integrin tails for binding to F3, and the structure of the complex suggests that the rod is also likely to sterically inhibit binding of the FERM domain to the membrane.The cytoskeletal protein talin has emerged as a key player, both in regulating the affinity of the integrin family of cell adhesion molecules for ligand (1) and in coupling integrins to the actin cytoskeleton (2). Thus, depletion of talin results in defects in integrin activation (3), integrin signaling through focal adhesion kinase, the maintenance of cell spreading, and the assembly of focal adhesions in cultured cells (4). In the whole organism, studies on the single talin gene in worms (5) and flies (6) show that talin is essential for a variety of integrin-mediated events that are crucial for normal embryonic development. In vertebrates, there are two talin genes, and mice carrying a talin1 null allele fail to complete gastrulation (7). Tissue-specific inactivation of talin1 results in an inability to activate integrins in platelets (8, 9), defects in the membrane-cytoskeletal interface in megakaryocytes (10), and disruption of the myotendinous junction in skeletal muscle (11). In contrast, mice homozygous for a talin2 gene trap allele have no phenotype, although the allele may be hypomorphic (12).Recent structural studies have provided substantial insights into the molecular basis of talin action. Talin is composed of an N-terminal globular head (∼50 kDa) linked to an extended flexible rod (∼220 kDa). The talin head contains a FERM² domain (made up of F1, F2, and F3 subdomains) preceded by a domain referred to here as F0 (2). Studies by Wegener et al. (30) have shown how the F3 FERM subdomain, which has a phosphotyrosine binding domain fold, interacts with both the canonical NPXY motif and the membrane-proximal helical region of the cytoplasmic tails of integrin β-subunits (13). The latter interaction apparently activates the integrin by disrupting the salt bridge between the integrin α- and β-subunit tails that normally keeps integrins locked in a low affinity state. The observation that the F0 region is also important in integrin activation (14) may be explained by our recent finding that F0 binds, albeit with low affinity, Rap1-GTP,³ a known activator of integrins (15, 16). The talin rod is made up of a series of amphipathic α-helical bundles (17–20) and contains a second integrin binding site (IBS2) (21), numerous binding sites for the cytoskeletal protein vinculin (22), at least two actin binding sites (23), and a C-terminal helix that is required for assembly of talin dimers (20, 24).Both biochemical (25) and cellular studies (16) suggest that the integrin binding sites in full-length talin are masked, and both phosphatidylinositol 4,5-bisphosphate (PIP2) and Rap1 have been implicated in exposing these sites. It is well established that some members of the FERM domain family of proteins are regulated by a head-tail interaction (26); gel filtration, sedimentation velocity, and electron microscopy studies all show that talin is globular in low salt buffers, although it is more elongated (∼60 nm in length) in high salt (27). By contrast, the talin rod liberated from full-length talin by calpain-II cleavage is elongated in both buffers, indicating that the head is required for talin to adopt a more compact state. Direct evidence for an interaction between the talin head and rod has recently emerged from NMR studies by Goksoy et al. (28), who demonstrated binding of ¹⁵N-labeled talin F3 to a talin rod fragment spanning residues 1654–2344, an interaction that was confirmed by surface plasmon resonance (K_d = 0.57 μm) (28). Chemical shift data also showed that this segment of the talin rod partially masked the binding site in F3 for the membraneproximal helix of the β3-integrin tail (28), directly implicating the talin head-rod interaction in regulating the integrin binding activity of talin. Goksoy et al. (28) subdivided the F3 binding site in this rod fragment into two sites with higher affinity (K_d ∼3.6 μm; residues 1654–1848) and lower affinity (K_d ∼78 μm; residues 1984–2344). Here, we define the rod domain boundaries and determine the NMR structure of residues 1655–1822, a five-helix bundle. We further show that this domain binds F3 predominantly via surface-exposed residues on helix 4, with an affinity similar to the high affinity site reported by Goksoy et al. (28). We also report the structure of the complex between F3 and the rod domain and show that the latter masks the known binding site in F3 for the β3-integrin tail and is expected to inhibit the association of the talin FERM domain with the membrane.