Polarization of various cell types depends on a complex containing the PDZ domain-containing scaffolding proteins Par-3 and Par-6 and atypical protein kinase C. Noting that phosphoinositides (PIPs) have also been implicated in cell polarization and that PDZ domains--although better known for mediating protein-protein interactions--can bind phosphatidylinositol (PI) lipids, Wu et al . investigated interactions between Par protein PDZ domains and PI lipids. The second Par-3 PDZ domain (PDZ2) bound to brain-derived liposomes with an affinity comparable to that of the PLCδ pleckstrin homology (PH) domain (a PI lipid-binding domain); strip-based assays and experiments with defined liposomes indicated that it bound directly to PIPs. Lipid-binding assays of 74 human PDZ domains and 14 PDZ tandems revealed that a subset bound PI lipids. Structural and biochemical analysis indicated that lipid binding by the Par-3 PDZ2 depended on a cluster of positively charged residues and that PDZ2 contained a PIP head group-binding pocket that overlapped its peptide ligand-binding site. In MDCK epithelial cells, Par-3 primarily localized to the cell membrane; mutational analysis indicated that membrane localization depended on PI lipid binding by PDZ2. Par-3 knockdown disrupted tight junction formation and reestablishment of polarization in MDCK cells. Epithelial repolarization was rescued by wild-type Par-3 or by a mutant in which the PLCδ PH domain replaced PDZ2 but not by mutants defective in lipid binding. The Par-3 PDZ3 domain bound to the lipid phosphatase PTEN. The authors conclude that lipid binding by the Par-3 PDZ2 domain is critical to its role in epithelial polarization and that the combination of PIP-binding by PDZ2 and PTEN-binding by PDZ3 could enable Par-3 to integrate PIP signaling. H. Wu, W. Feng, J. Chen, L.-N. Chan, S. Huang, M. Zhang, PDZ domains of Par-3 as potential phosphoinositide signaling integrators. Mol. Cell 28 , 886-898 (2007). [PubMed]
Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases that can initiate a variety of signaling events. Many human cancers involve mutations that activate PI3Kα, a heterodimer composed of a catalytic subunit, p110α, and a regulatory subunit, p85α, both of which contain multiple domains. Huang et al. describe the crystal structure of a complex between the full-length human p110α catalytic subunit and the binding and activation domains of the p85α regulatory subunit. The structure provides insight into how oncogenic mutations affect enzyme activity and could assist in the future design of isoform- or mutation-specific inhibitors. C.-H. Huang, D. Mandelker, O. Schmidt-Kittler, Y. Samuels, V. E. Velculescu, K. W. Kinzler, B. Vogelstein, S. B. Gabelli, L. M. Amzel, The structure of a human p110α/p85α complex elucidates the effects of oncogenic PI3Kα mutations. Science 318 , 1744-1748 (2007). [Abstract] [Full Text]
This Teaching Resource describes a course designed to expose graduate students to various hurdles they will encounter during their development as scientists. This course aims to train students in such elements of scientific life as working within a research group, presenting research, writing proposals, and publishing peer-reviewed manuscripts and to provide them with the tools necessary to accomplish these tasks. A critical element of this course is that it is designed to provide students with valuable "real world" experiences. A course description and a syllabus are provided.
A number of thiol-dependent oxidoreductases are released from cells and act on the cell surface. Correspondingly, several cell-surface processes appear to depend on catalyzed thiol-disulfide exchange, including integrin activation and the fusion of viral particles with the host membrane. Tumor cells frequently increase the abundance of secreted and cell-surface forms of particular oxidoreductases, and evidence suggests that oxidoreductases released from tumor cells promote growth and contribute to the remodeling of the cellular microenvironment. Few cell-surface or membrane proteins that are targeted by extracellular redox enzymes have been identified. One major reason for this slow progress is the highly transient nature of thiol-disulfide exchange, making its detection by conventional techniques difficult or impossible. Here we describe the application of an activity-based proteomics approach, also known as "mechanism-based kinetic trapping," to identify individual cell-surface target proteins that engage in disulfide exchange with thiol-dependent oxidoreductases. Although we have applied this approach to thioredoxin-1, it should also be applicable to other members of the thioredoxin superfamily whose activity is based on the CXXC active-site motif.
Dendritic cells have a key role in protecting organisms from infections. These cells detect antigens derived from invading bacteria or viruses and present them to T cells. Zaru et al. explored the roles of protein kinases activated in response to mitogen-activated protein kinases (MAPKs) and not only demonstrate the importance of various MAPK-activated protein kinases (MKs)--the MKs known as MK2 and MK3 and the p90 ribosomal S6 kinases (Rsks)--but also reveal a previously unrecognized regulatory network for control of downstream kinases in a MAPK system. The authors monitored Toll-like receptor-activated endocytosis by detecting uptake of fluorescently labeled dextran and observed that dendritic cells lacking both MK2 and MK3 had a reduced response. They also observed that pharmacological inhibition of Rsk family members with two structurally distinct inhibitors inhibited Toll-like receptor-induced endocytosis. The surprise was that inhibition of ERK1 and ERK2, the MAPKs known to activate Rsks, could not block receptor-induced Rsk activation in this system. Inhibition of ERKs only blocked Rsk activation in cells lacking MK2 and MK3, kinases that are activated by the MAPK p38 rather then ERKs. Rsks have two kinase domains, and the C-terminal kinase domain of Rsk proteins is phosphorylated by ERKs and autophosphorylates a serine residue located between the two kinase domains [thus recruiting yet another kinase (PDK1) to complete the activation]. Zaru et al . realized, however, that this critical serine residue in Rsk fits the consensus for recognition by MK2 and MK3 and showed that MK2 could phosphorylate Rsk2 at this site in vitro. Thus, the p38 MAPK provides an alternative mechanism for activating Rsks that bypasses the mechanism used for their activation by the ERK MAPKs, a mechanism that appears to be restricted to dendritic cells. The authors and Karin (in associated commentary) speculate on ways in which this modified regulatory scheme might be advantageous in these particular cells. R. Zaru, N. Ronkina, M. Gaestel, J. S. C. Arthur, C. Watts, The MAPK-activated kinase Rsk controls an acute Toll-like receptor signaling response in dendritic cells and is activated through two distinct pathways. Nat. Immunol. 8 , 1227-1235 (2007). [PubMed] M. Karin, Rsk Tolls the bell for endocytosis in DCs. Nat. Immunol. 8 , 1197-1199 (2007). [PubMed]
Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) are transmembrane proteins that respond to diverse extracellular stimuli to trigger many different cellular responses. They are important pharmacological targets, but drug design has been hampered because structural information is only available for the rather distinctive member of this family, rhodopsin (see the Perspective by Ranganathan). Three studies now provide structural insights into another GPCR, the human β 2 -adrenergic receptor. All three groups crystallized the protein in the presence of an inverse agonist that stabilizes the inactive conformation of the receptor. Rosenbaum et al . stabilized the protein for crystallization by protein engineering and analyzed mutagenesis data in the context of the structure to provide insight into how ligand binding is coupled. Cherezov et al . describe in detail the 2.4 Å structure of the fusion protein and how it compares to the rhodopsin structure. Rasmussen et al . crystallized the protein in a lipid environment in the presence of a monoclonal antibody fragment to obtain a 3.4 Å structure. It appears that conserved helices provide a common core in class A GPCRs, whereas variable helices provide binding-site plasticity. V. Cherezov, D. M. Rosenbaum, M. A. Hanson, S. G. F. Rasmussen, F. S. Thian, T. S. Kobilka, H.-J. Choi, P. Kuhn, W. I. Weis, B. K. Kobilka, R. C. Stevens, High-resolution crystal structure of an engineered human β 2 -adrenergic G protein-coupled receptor. Science 318 , 1258-1265 (2007). [Abstract] [Full Text] D. M. Rosenbaum, V. Cherezov, M. A. Hanson, S. G. F. Rasmussen, F. S. Thian, T. S. Kobilka, H.-J. Choi, X.-J. Yao, W. I. Weis, R. C. Stevens, B. K. Kobilka, GPCR engineering yields high-resolution structural insights into β 2 -adrenergic receptor function. Science 318 , 1266-1273 (2007). [Abstract] [Full Text] S. G. F. Rasmussen, H.-J. Choi, D. M. Rosenbaum, T. S. Kobilka, F. S. Thian, P. C. Edwards, M. Burghammer, V. R. P. Ratnala, R. Sanishvili, R. F. Fischetti, G. F. X. Schertler, W. I. Weis, B. K. Kobilka, Crystal structure of the human β 2 adrenergic G-protein-coupled receptor. Nature 450 , 383-387 (2007). [PubMed] R. Ranganathan, Signaling across the cell membrane. Science 318 , 1253-1254 (2007). [Abstract] [Full Text]
The PDZ domain is a frequently occurring interaction domain in eukaryotic proteins that binds to the C termini of target proteins. PDZ domains have been thought to cluster into functional classes, each with distinct sequence binding preferences. Using a combination of experimental data and modeling, Stiffler et al . predict PDZ domain-peptide interactions across the mouse proteome. Instead of observing clustering, they find that PDZ domains are evenly distributed throughout selectivity space likely to minimize cross-reactivity. The study highlights focusing on families of interaction domains as a productive approach to gain insight into protein function. M. A. Stiffler, J. R. Chen, V. P. Grantcharova, Y. Lei, D. Fuchs, J. E. Allen, L. A. Zaslavskaia, G. MacBeath, PDZ domain binding selectivity is optimized across the mouse proteome. Science 317 , 364-369 (2007). [Abstract] [Full Text]
Hirayama et al. provide new understanding of the signaling feedback loops that control the mammalian circadian clock. CLOCK and BMAL1 are clock proteins that form a complex and promote clock-controlled transcription of target genes. CLOCK is a histone acetyltransferase that acts to regulate gene expression by initiating remodeling of chromatin. The new work showed that CLOCK acetylated its binding partner BMAL1 when transfected, tagged proteins were expressed in cultured cells or when purified proteins were mixed in vitro. Reconstitution of mouse embryo fibroblasts lacking BMAL1 with a BMAL mutant that lacked the lysine residue acetylated by CLOCK failed to restore circadian gene regulation. Acetylation of BMAL1 appeared to increase association of the CLOCK-BMAL1 complex with cryptochrome 1 (Cry1), which acts as a repressor of CLOCK-BMAL1-induced transcription. Cry1-mediated repression was lacking in cells expressing the acetylation-deficient mutant of BMAL1, and interaction of Cry1 with the CLOCK-BMAL1 complex was lost in a two-hybrid assay with the mutant BMAL1. Acetylation of BMAL1 in vivo also occurs at a time in the circadian cycle when transcription of clock-controlled genes is diminished. Thus, the authors conclude that acetylation of BMAL1 by its binding partner CLOCK contributes to a negative feedback loop that decreases clock-regulated transcription. The positive and negative feedback loops thus appear to be tightly associated because CLOCK’s effect on BMAL contrasts with its role in acetylation of histones, which stimulates transcription of clock-dependent genes. J. Hirayama, S. Sahar, B. Grimaldi, T. Tamaru, K. Takamatsu, Y. Nakahata, P. Sassone-Corsi, CLOCK-mediated acetylation of BMAL1 controls circadian function. Nature, 450, 1086-1090 (2007). [PubMed]
Despite substantial effort, it has remained relatively mysterious how the protein known as Hedgehog (Hh) activates signaling pathways that regulate various biological processes, including stem cell function, development, and cancer. Rohatgi et al . (see the Perspective by Christensen and Ott) show that mammalian cells use their primary cilium as an antenna that samples the surrounding environment for the presence of Hh. When Hh bound to its receptor Patched 1 (Ptc1), the receptor left the cilia, where (in the absence of stimulation) it acts to restrain Hh signaling by preventing accumulation of the signaling protein Smoothened (Smo). Accumulation of Smo in the cilia of stimulated cells corresponded to activation of Hh signaling. Further understanding the molecular mechanisms that influence cellular localization of Ptc1 and Smo will improve understanding of the signaling pathway and may lead to new therapeutic targets. R. Rohatgi, L. Milenkovic, M. P. Scott, Patched1 regulates Hedgehog signaling at the primary cilium. Science 317 , 372-376 (2007). [Abstract] [Full Text] S. T. Christensen, C. M. Ott, A ciliary signaling switch. Science 317 , 330-331 (2007). [Summary] [Full Text]
Chronic lung disease, including cystic fibrosis, bronchiectasis, and chronic obstructive pulmonary disease (COPD), is characterized by high concentrations of interleukin-8 (IL-8) in cells around the airway and accumulation of neutrophils, yet the lungs of these patients are frequently colonized by bacteria, such as Pseudomonas aeruginosa . Hartl et al . show that IL-8 acts through the CXCR1 receptor, not the CXCR2 receptor, to promote the bactericidal activity of neutrophils. By comparing neutrophils from the peripheral blood and airway (bronchial alveolar lavage and sputum) of healthy individuals with those from patients with cystic fibrosis, bronchiectasis, or COPD, Hartl et al . found that the airway neutrophils from the diseased lungs had decreased CXCR1 at the surface and exhibited impaired bactericidal activity. The protease elastase is increased in abundance in airway fluids from patients with cystic fibrosis. Further statistical analysis of various factors associated with cystic fibrosis, including infection with P. aeruginosa , suggested that the concentration of free elastase correlated with the loss of CXCR1 on the airway neutrophils. Addition of airway fluids from individuals with cystic fibrosis to peripheral neutrophils caused loss of CXCR1 and impaired bacterial killing, and these effects were prevented if the elastase activity was inhibited pharmacologically in the airway fluid prior to addition to the isolated neutrophils. Cleavage of CXCR1 by elastase produced two soluble CXCR1 fragments that were also detected in the airway fluids from patients with cystic fibrosis, bronchiectasis, or COPD. Application of purified soluble CXCR1 fragments stimulated bronchial epithelial cells to produce IL-8 through a mechanism requiring the Toll-like receptor TLR2. Cystic fibrosis patients who inhaled α1 antitrypsin to inhibit elastase activity for 4 weeks showed increased CXCR1 at the surface of airway neutrophils, decreased abundance of soluble CXCR1 fragments in the airway fluid, and decreased P. aeruginosa in the sputum. In commentary, Sabroe and Whyte describe how failure of mucosal immunity contributes to disease pathogenesis, especially in cystic fibrosis. D. Hartl, P. Latzin, P. Hordijk, V. Marcos, C. Rudolph, M. Woischnik, S. Krauss-Etschmann, B. Koller, D. Reinhardt, A. A. Roscher, D. Roos, M. Griese, Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease. Nat. Med. 13 , 1423-1430 (2007). [PubMed] I. Sabroe, M. K. B. Whyte, Incapacitating the immune system in cystic fibrosis. Nat. Med. 13 , 1417-1418 (2007). [PubMed]
Adams et al . identified the V-ATPase H + pump in a pharmacological screen for ion transporters involved in Xenopus larval tail regeneration. When the pumping activity of the V-ATPase was inhibited with concanamycin, tail regeneration was inhibited, whereas primary tail development and wound healing were unaffected. Using a dominant-negative V-ATPase, the same inhibition of regeneration was observed, confirming V-ATPase as the target. Expression of a yeast P-type H + pump, which is insensitive to concanamycin, was localized to the plasma membrane and also prevented concanamycin inhibition of regeneration, confirming that it was transport of H + efflux at the plasma membrane that was important for regeneration. Six hours after amputation, the abundance of the mRNA for the c subunit of the V-ATPase had increased, and by 24 hours after amputation the H + pump was abundant in the plasma membrane of the cells of the regeneration bud. The cells of the regeneration bud were depolarized six hours after amputation; however, by 24 hours after amputation, the cells were repolarized, which is consistent with the hyperpolarizing activity of the H + pump. Furthermore, depolarization of the tails using a drug that converts the Na + ,K + ATPase into a Na + /K + channel also inhibited regeneration, supporting the concept that membrane voltage is a critical regulator of regeneration. Inhibition of the H + pump decreased the number of proliferating cells in the regeneration bud and inhibited the expression of the KCNK1 gene, which encodes a K + channel that is expressed at early stages of regeneration (12 hours after amputation). Axon patterning in the regeneration bud was also aberrant when the H + pump activity was inhibited. Thus, H + pump activity contributes to changes in membrane potential that are important for regulation of gene expression, cell proliferation, and proper patterning of the new tissues. D. S. Adams. A. Masi, M. Levin, H + pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regeneration. Development 134 , 1323-1335 (2007). [Abstract] [Full Text]
Cell behaviors are regulated by signaling pathways triggered by the activation of cell surface receptors. A key aspect of receptor signaling is the location of these receptors relative to their ligands and to other receptors, particularly in epithelia, whose cells are polarized by tight junction barriers into apical and basolateral membrane regions. In polarized epithelia, the co-receptor ErbB2 is often localized to the apical surface by its intramembrane ligand Muc4, thus segregating it from its partner ErbB3, which is sequestered at the lateral surface, co-localized with cadherin junctions. The ErbB2-ErbB3 receptor heterodimer, when activated, is a potent stimulator of cell proliferation; thus, the segregation mechanism helps maintain these cells in a differentiated state. Similarly, epidermal growth factor, the ligand for ErbB1, which is present in the apical fluid of some epithelia, is segregated from its receptor by the tight junction barrier. Loss of cell polarity and the tight junction barrier facilitates the interaction of ErbB2 with the hemidesmosome integrin α 6 β 4 . This integrin acts as a docking site for signaling pathways to promote cell proliferation and further disruption of cell junctions. The ultimate dissolution of tight junctions may result from activation of transforming growth factor–β receptors, one subunit of which is directly associated with the junction. This activation triggers degradation of critical tight junction components. These sequestration and segregation phenomena provide a model by which overexpression of the ErbB2 receptor kinase may trigger oncogenesis by initiating junction breakdown. Equally important, these mechanisms may act as a sensor for epithelial damage that can activate repair mechanisms.
The (E3) ubiquitin ligase, GRAIL (gene related to anergy in lymphocytes), is associated with CD4 + T cell anergy, a state of T cell nonresponsiveness to antigen. MacKenzie et al . found that amounts of GRAIL mRNA and protein were greater in mouse CD4 + CD25 + Tregs than in CD4 + CD25 – naïve cells. Transfer of naïve CD4 + T cells from TCR-DO11.10/Rag2 –/– (DO11) mice to BALB/c mice, followed by injection of the mice with OVA peptide, induced tolerized CD4 + CD25 + cells detected with the DO11-specific antibody KJ1.26. This is a routinely used method for studying tolerance, because the donor cells express a T cell receptor (TCR) that responds only to OVA peptide, and the donor mice cannot produce any mature T cells because of the lack of the Rag2 gene (recombinase activating gene 2). KJ1.26 + CD25 + cells had larger amounts of GRAIL mRNA and Foxp3 mRNA and protein than did the KJ1.26 + CD25 – subset, as measured by real-time reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. A retroviral system was used to generate cell lines producing large amounts of GRAIL (DO11 GRAIL) or GFP (DO11 GFP). Co-culture of naïve CD4 + CD25 – T cells with antigen-presenting cells, OVA peptide, and DO GFP cells resulted in proliferation of naïve cells. However, when DO11 GRAIL cells were used instead of DO11 GFP cells, proliferation of naïve CD4 + CD25 – T cells was suppressed. DO11 GRAIL cells produced more transforming growth factor-β (TGF-β), a cytokine associated with Treg function, than did DO11 cells. DO11 GRAIL cells also had larger amounts of the surface Treg markers, CD25, GITR, and CTLA4. Surprisingly, DO11 GRAIL cells did not produce the cytokine, IL-10, or the transcription factor, Foxp3, both of which are associated with Treg function. However, this study demonstrates the presence of GRAIL in CD4 + CD25 + Tregs and suggests a role for GRAIL in suppressor function. D. A. MacKenzie, J. Schartner, J. Lin, A. Timmel, M. Jennens-Clough, C. G. Fathman, C. M. Seroogy, GRAIL is up-regulated in CD4 + CD25 + T regulatory cells and is sufficient for conversion of T cells to a regulatory phenotype. J. Biol. Chem. 282 , 9696-9702 (2007). [Abstract] [Full Text]
The cytoplasmic CARD-containing DExD/H box RNA helicases RIG-I and MDA5 act as sensors of viral infections through recognition of viral double-stranded (ds) RNAs. They both associate with the mitochondrial adaptor IPS-1 (also referred to as MAVS, VISA, and CARDIF) through homotypic CARD-CARD interactions. IPS-1, in turn, triggers signaling pathways, including activation of the protein kinases TBK1 and IKKε, responsible for the phosphorylation of IRF3, a key transcription factor involved in interferon (IFN) synthesis, one essential element of the innate immune response. RIG-I remains in an autoinhibited state in the absence of dsRNA, through an internal repressor domain (RD) that binds within both its CARD and its RNA helicase domains and therefore acts in cis to control its multimerization and interaction with IPS-1. Ectopic expression of the RD prevents signaling and increases cell permissiveness to viruses, including hepatitis C virus. LGP2, which is another DExD/H RNA helicase of the RIG-I and MDA5 family and which is devoid of CARD domain, negatively controls IFN induction at different levels: by sequestering dsRNA, by blocking RIG-I’s multimerization in trans through a domain analogous to the RIG-I RD, and by competing with the protein kinase IKKε for a common interaction site on IPS-1. The ability of RIG-I and LGP2 to exert such a feedback control at the earliest steps of IFN synthesis allows the cells to exert a tight regulation of the induction of the innate immune response.
The nodulation of roots in legumes is a key factor in nitrogen fixation. Working in the leguminous plant Lotus japonica , Tirichine et al . and Murray et al . have identified how the hormone cytokinin fits into the signaling cascade by which leguminous plants establish nitrogen-fixation nodules filled with symbiotic bacteria. Gain-of-function mutation in a cytokinin receptor results in spontaneous formation of bacteria-free nodules, whereas loss of function results in too few nodules despite aggressive formation of bacterial infection threads. L. Tirichine, N. Sandal, L. H. Madsen, S. Radutoiu, A. S. Albrektsen, S. Sato, E. Asamizu, S. Tabata, J. Stougaard, A gain-of-function mutation in a cytokinin receptor triggers spontaneous root nodule organogenesis. Science 315 , 104-107 (2007). [Abstract] [Full Text] J. D. Murray, B. J. Karas, S. Sato, S. Tabata, L. Amyot, K. Szczyglowski, A cytokinin perception mutant colonized by Rhizobium in the absence of nodule organogenesis. Science 315 , 101-104 (2007). [Abstract] [Full Text] G. E. D. Oldroyd, Nodules and hormones. Science 315 , 52-53 (2007). [Summary] [Full Text]
In the immune system, T cells respond to fragments of antigenic proteins presented at the cell surface by molecules encoded by genes of the major histocompatibility complex. This process is vital not only for recognition of antigens carried by pathogens and tumors but also in the selection of T cells as they develop in the thymus. A large multisubunit complex called the proteasome, which exists in a variety of forms containing different catalytic subunits, generates these peptides. Murata et al. (see the Perspective by Bevan) now identify a proteasome subunit, β5t, that was found exclusively in the thymic epithelial cells and directs positive selection of T cells. Indeed, mice lacking β5t showed significant disruption of T cell development. S. Murata, K. Sasaki, T. Kishimoto, S.-i. Niwa, H. Hayashi, Y. Takahama, K. Tanaka, Regulation of CD8 + T cell development by thymus-specific proteasomes. Science 316 , 1349-1353 (2007). [Abstract] [Full Text] M. J. Bevan, The cutting edge of T cell selection. Science 316 , 1291-1292 (2007). [Summary] [Full Text]
For mammals, inflammatory responses of the innate immune system provide an indispensable protective mechanism against infection. But these signaling mechanisms may also contribute to certain diseases, including cancer. O’Connell et al . investigated the role of microRNAs (noncoding RNAs that regulate gene expression through interaction with cellular mRNAs) in the inflammatory response to viral infection and may have unearthed a molecular link between inflammation and cancer. The authors used microarrays to monitor expression of a panel of 200 microRNAs in response to polyriboinosinic:polyribocytidylic acid [poly(I:C)], a synthetic double-stranded RNA used to mimic viral infection, or the antiviral cytokine interferon-β (IFN-β). They identified one microRNA, miR-155, that showed increased expression in macrophages in response to both stimuli. Expression of miR-155 was regulated through multiple signaling pathways. The response to poly(I:C) required signaling through Toll receptors and the MyD88 or TRIF adaptor proteins. Interferons, on the other hand, stimulated expression of miR-155 through a slower mechanism that required autocrine signals mediated by tumor necrosis factor α. Both pathways appeared to converge on regulation of an AP-1 transcription factor binding site, and an inhibitor of the JNK mitogen-activated protein kinase inhibited the response to poly(I:C) or interferon. The miR-155 microRNA is also an oncogene, and its overexpression can cause B cell lymphoma. Thus, the authors note that the finding of miR-155 as a common target of pathways activated by viral infection presents a potential link between inflammatory responses and cancer. R. M. O’Connell, K. D. Taganov, M. P. Boldin, G. Cheng, D. Baltimore, MicroRNA-155 is induced during the macrophage inflammatory response. Proc. Natl. Acad. Sci. U.S.A. 104 , 1604-1609 (2007). [Abstract] [Full Text]
The cytoplasmic contents of the cell are targeted to lysosomes for degradation or recycling by a process known as autophagy. Jounai et al . investigated the role of autophagy in the response to infection by vesicular stomatitis virus (VSV). Whereas mouse embryonic fibroblasts (MEFs) from wild-type mice were efficiently infected with VSV, as assessed by measurement of virus in infected cell cultures, MEFs from mice deficient in Atg5 (Atg5 KO), a critical component of the autophagic process, produced much lower amounts of virus. Real-time reverse transcription polymerase chain reaction and Western blotting analyses demonstrated that VSV infection of Atg5 KO MEFs resulted in the higher abundance of interferon-β (IFN-β) mRNA and increased phosphorylation and activation of interferon regulatory factor 3 (IRF-3), a transcription factor that activates IFN gene expression, than did infection of wild-type MEFs. Viral RNA is recognized by DExD/H box RNA helicases, such as retinoic acid-inducible gene I (RIG-I), which then associates with and activates the adaptor molecule IFN-β promoter stimulator 1 (IPS-1). This association is mediated by interactions between the caspase recruitment domains (CARDs) of RIG-I and IPS-1 and leads to IRF-3 activation. Reporter assays in 293 cells demonstrated that RIG-1-induced activation of the IFN promoter was inhibited by both Agt5 and a conjugate of Agt5 and Agt12 (the physiologically relevant form of Atg5). Atg5-Atg12 coimmunoprecipitated with RIG-I and IPS-1 in 293 cells, and VSV infection enhanced the association between Atg5-Atg12 and IPS-1. Experiments with mutant forms of IPS-1 and RIG-I showed that the CARD domains were the targets of Atg5-Atg12. This study suggests that Atg5-Atg12 blocks the interaction between RIG-I and IPS-1 and thus inhibits the innate immune response to viral infection. N. Jounai, F. Takeshita, K. Kobiyama, A. Sawano, A. Miyawaki, K.-Q. Xin, K. J. Ishii, T. Kawai, S. Akira, K. Suzuki, K. Okuda, The Atg5-Atg12 conjugate associates with innate antiviral immune responses. Proc. Natl. Acad. Sci. U.S.A. 104 , 14050-14055 (2007). [Abstract] [Full Text]
Nerve growth factor (NGF) binds to two structurally unrelated cell surface receptors, the p75 neurotrophin receptor (p75) and the receptor tyrosine kinase TrkA. p75 increases TrkA’s responsiveness to NGF, and coexpression of the two receptors appears to lead to the formation of binding sites with higher affinity for NGF than that of either receptor alone. This suggests that the high-affinity NGF-binding site may involve a complex containing both p75 and TrkA. Wehrmann et al . determined the crystal structure of the complete TrkA extracellular domain in complex with NGF and found that this structure was not incompatible with the existence of a 1:2:1 TrkA-NGF-p75 ternary complex. To investigate this possibility in a cellular context, the authors determined interactions between proteins (p75 and a truncated form of TrkA) fused to fragments of β-galactosidase (which become functional when brought together) that were expressed in C2C12 myoblasts. NGF promoted formation of TrkA homodimers but not that of TrkA-p75 heterodimers (which also did not exist in untreated cells); p75 formed oligomers in the absence of ligand, and NGF did not affect this oligomerization. When corrected for background binding, Scatchard analysis of 125 I-NGF binding in both PC12 cells and C2C12 cells expressing TrkA and p75 constructs failed to reveal the higher-affinity NGF binding site. Thus, the authors conclude that functional interactions between p75 and TrkA likely depend on downstream signaling rather than physical complexes involving the two receptors. In discussing this research, Barker suggests that p75 could pass NGF to TrkA in a ternary complex that exists too briefly to be detected by the β-galactosidase complementation assay. T. Wehrman, X. He, B. Raab, A. Dukipatti, H. Blau, K. C. Garcia, Structural and mechanistic insights into nerve growth factor interactions with the TrkA and p75 receptors. Neuron 53 , 25-38 (2007). [PubMed] P. A. Barker, High affinity not in the vicinity? Neuron 53 , 1-4 (2007). [PubMed]
The effector molecules in RNA interference (RNAi) are small interfering RNAs (siRNAs). The initial population of "primary" siRNAs, ~22 nucleotides in length with 5′-monophosphates groups, is generated by the Dicer nuclease. Amplification and "spreading" of the initial trigger population are thought to contribute to strength of the RNAi response in a number of systems and involve an RNA-dependent RNA polymerase (RDRP) (see the Perspective by Baulcombe). To investigate the nature of this secondary response, Pak and Fire and Sijen et al . analyzed the course of an experimentally induced RNAi reaction in the nematode worm Caenorhabditis elegans and also examined endogenous small RNAs. They found distinct populations of "secondary" siRNAs that are antisense to the messenger RNA target, that have a di- or triphosphate moiety at their 5′ ends, and that may map both upstream and downstream of the original dsRNA trigger. Primary siRNAs do not appear to act as primers for RdRP but rather guide RdRP to targeted messages for the de novo synthesis of secondary siRNAs that further boost the RNAi response. J. Pak, A. Fire, Distinct populations of primary and secondary effectors during RNAi in C. elegans . Science 315 , 241-244 (2007). [Abstract] [Full Text] T. Sijen, F. A. Steiner, K. L. Thijssen, R. H. A. Plasterk, Secondary siRNAs result from unprimed RNA synthesis and form a distinct class. Science 315 , 244-247 (2007). [Abstract] [Full Text] D. C. Baulcombe, Amplified silencing. Science 315 , 199-200 (2007). [Summary] [Full Text]
