Accelerated disassembly of IgE-receptor complexes by a disruptive macromolecular inhibitor

IgE antibodies bind the high-affinity IgE Fc receptor (FcεRI), found primarily on mast cells and basophils, and trigger inflammatory cascades of the allergic response. Inhibitors of IgE-FcεRI binding have been identified and an anti-IgE therapeutic antibody (omalizumab) is used to treat severe allergic asthma. However, preformed IgE-FcεRI complexes that prime cells before allergen exposure dissociate extremely slowly and cannot be disrupted by strictly competitive inhibitors. IgE-Fc conformational flexibility indicated that inhibition could be mediated by allosteric or other non-classical mechanisms. Here we demonstrate that an engineered protein inhibitor, DARPin E2_79 (refs 9, 10, 11), acts through a non-classical inhibition mechanism, not only blocking IgE-FcεRI interactions, but actively stimulating the dissociation of preformed ligand-receptor complexes. The structure of the E2_79-IgE-Fc(3-4) complex predicts the presence of two non-equivalent E2_79 sites in the asymmetric IgE-FcεRI complex, with site 1 distant from the receptor and site 2 exhibiting partial steric overlap. Although the structure is indicative of an allosteric inhibition mechanism, mutational studies and quantitative kinetic modelling indicate that E2_79 acts through a facilitated dissociation mechanism at site 2 alone. These results demonstrate that high-affinity IgE-FcεRI complexes can be actively dissociated to block the allergic response and suggest that protein-protein complexes may be more generally amenable to active disruption by macromolecular inhibitors.

The molecular role of the common gamma c subunit in signal transduction reveals functional asymmetry within multimeric cytokine receptor complexes.

The specific signal transduction function of the gamma c subunit in the interleukin (IL) 2, IL-4, IL-7, IL-9, and IL-15 receptor complexes remains undefined. The present structure-function analyses demonstrated that the entire cytoplasmic tail of gamma c could be functionally replaced in the IL-2 receptor (IL-2R) signaling complex by a severely truncated erythropoietin receptor cytoplasmic domain lacking tyrosine residues. Heterodimerization of IL-2R beta with either gamma c or the truncated erythropoietin receptor chain led to an array of specific signals normally derived from the native IL-2R despite the substitution of Janus kinase JAK2 for JAK3 in the receptor complex. These findings thus suggest a model in which the gamma c subunit serves as a common and generic "trigger" chain by providing a nonspecific Janus kinase for signaling program initiation, while signal specificity is determined by the unique "driver" subunit in each of the gamma c- containing receptor complexes. Furthermore, these results may have important functional implications for the asymmetric design of many cytokine receptor complexes and the evolutionary design of receptor subfamilies that share common trigger or driver subunits.

Structural characterization of CD81-Claudin-1 hepatitis C virus receptor complexes

Tetraspanins are thought to exert their biological function(s) by co-ordinating the lateral movement and trafficking of associated molecules into tetraspanin-enriched microdomains. A second four-TM (transmembrane) domain protein family, the Claudin superfamily, is the major structural component of cellular TJs (tight junctions). Although the Claudin family displays low sequence homology and appears to be evolutionarily distinct from the tetraspanins, CD81 and Claudin-1 are critical molecules defining HCV (hepatitis C virus) entry; we recently demonstrated that CD81-Claudin-1 complexes have an essential role in this process. To understand the molecular basis of CD81-Claudin-1 complex formation, we produced and purified milligram quantities of full-length CD81 and Claudin-1, alone and in complex, in both detergent and lipid contexts. Structural characterization of these purified proteins will allow us to define the mechanism(s) underlying virus-cell interactions and aid the design of therapeutic agents targeting early steps in the viral life cycle.

Signaling of neuronal cell death by the p75NTR neurotrophin receptor

The neurotrophin receptor (p75NTR) is best known for mediating tropic support by participating in the formation of high-affinity nerve growth factor (NGF) receptor complexes with trkA, however, p75NTR more recently has been shown to act as a bona fide death-signaling receptor, which can signal independently of trkA. This article discusses the evidence for an active role of p75NTR in neuronal cell death and the mechanisms controlling this process, including roles for Bcl-2 family members, the c-jun stress kinase JNK, the transcription factor nuclear factor kappa B (NF kappa B), and caspases.

Immunophilin chaperones in steroid receptor signalling

The immunophilin cochaperones, cyclophilin 40 (CyP40), FKBP51 and FKBP52 and PP5, a serine/threonine protein phosphatase, have been implicated as modulators of steroid receptor function through their association with Hsp90, a molecular chaperone with a key role in steroid hormone signalling. Although progress towards a satisfying definition for the role of these components in steroid receptor complexes has been slow, recent developments arising from novel approaches in both yeast and mammalian systems, together with available crystal structures for Hsp90 and some of these cochaperones, are beginning to provide important clues about their function. Hsp90, recently identified as a member of the GHKL superfamily of ATPases, is the central player in receptor assembly, an energy-driven process that allows receptor and the immunophilins to be proximally located, or to interact directly, on a Hsp90 scaffold. Immunophilin structure, relative abundance, their binding affinity for Hsp90 and their ability to interact with specific receptors may all contribute to a selective preference of the immunophilins for individual receptors. Association of receptors with different immunophilins leads to differential functional consequences for receptor activity. Observations of glucocorticoid resistance in New World primates, attributed to FKBP51 overexpression and incorporation into glucocorticoid receptor complexes, have provided the first evidence that these cochaperones can control hormone-binding affinity. Application of a yeast model to FKBP52 function in the glucocorticoid receptor system has now provided crucial evidence that this immunophilin enhances receptor transcriptional activity by increasing receptor avidity for hormone through PPIase-mediated conformational changes in the ligand-binding domain. A recent novel finding suggests that hormone binding may induce a functional exchange of immunophilins in receptor complexes and that the modified complex directs receptor to the nucleus.

Identificació i caracterització funcional dels complexes proteics dels receptors BRL1 i BRL3 en el teixit vascular d’Arabidopsis Thaliana

Els esteroids juguen papers clau en el creixement I el desenvolupament d’eucariotes multicel•lulars. En plantes, aquestes hormones, anomenades Brassinosteroides (BRs), estan involucrades en una gran varietat de processos biològics essencials per a les plantes. S’han descrit anteriorment dos receptors de BRs del tipus Leucine Rich Repeat Receptor Like Kinase LRR-RLK, BRASSINOSTEROID RECEPTOR LIKE 1 i 3 (BRL1 i BRL3 respectivalemt) que són homòlegs al receptor principal BRI1 i són necessaris pel desenvolupament vascular. Tot i que els principals components de la senyal ja han estat identificats pel seu homòleg més pròxim, el receptor BRI1, els complexes de BRL1 i BRL3 juntament amb els candidats co-receptors així com els components de la ruta de senyalització encara no han sigut identificats. Per tal d’entendre millor la funció molecular d’aquests receptors de BRs en la planta aquesta tesis doctoral planteja dues aproximacions: com a primera aproximació, vaig realitzar un estudi fenotípic del desenvolupament del teixit vascular a la planta model Arabidopsis thaliana (Arabidopsis). Disposant d'una amplia bateria de mutants de síntesis de la hormona i senyalització del receptor BRI1, vam analitzar quantitativament el seu patró vascular a la tija d'Arabidopsis. Vam establir els paràmetres en les plantes silvestres [Col-0 wild type, (WT)] i els vam analitzar a tots i cadascun dels mutants. Això conjuntament amb una col•laboració amb la Dr. Marta Ibañes, física de la Universitat de Barcelona que va construir un model matemàtic per simular la formació del patró vascular ens va permetre el•laborar una hipòtesis que vam demostrar experimentalment i va ser publicada a la revista PNAS. Posteriorment vam observar que les plantes knock-out d'aquests dos receptors BRL1 y BRL3 a diferència de BRI1, no tenien cap fenotip obvi en el teixit vascular de la planta adulta. Així, a continuació, per entendre quina necessitat té la planta de disposar de tres receptors tant altament homòlegs que poden percebre la mateixa hormona, vam utilitzar una aproximació bioquímica en col•laboració amb el Prof. de Vries de la Universitat de Wageningen (Holanda) per tal de purificar els complexes dels receptors in vivo i els seus interactors. Això ens ha permès entendre millor el paper funcional d'aquests receptors en la planta. Els resultats d’aquests experiments estan resumits en un article en preparació que aviat estarà en revisió.

Study of nuclear receptor dynamics by BRET

Les récepteurs nucléaires (RN) sont des facteurs de transcription ligand dépendants qui contrôlent une grande variété de processus biologiques de la physiologie humaine, ce qui a fait d'eux des cibles pharmacologiques privilégiées pour de nombreuses maladies. L'un de ces récepteurs, le récepteur de l’œstrogène alpha (ERα), peut activer la prolifération cellulaire dans certaines sections de l'épithélium mammaire tandis qu’un autre, le récepteur de l'acide rétinoïque alpha (RARα), peut provoquer un arrêt de la croissance et la différenciation cellulaire. La signalisation de ces deux récepteurs peut être altérée dans le cancer du sein, contribuant à la tumorigénèse mammaire. L’activité d’ERα peut être bloquée par les anti-oestrogènes (AE) pour inhiber la prolifération des cellules tumorales mammaires. Par contre, l’activation des voies de RARα avec des rétinoïdes dans un contexte clinique a rencontré peu de succès. Ceci pourrait résulter du manque de spécificité des ligands testés pour RARα et/ou de leur activité seulement dans certains sous-types de tumeurs mammaires. Puisque les récepteurs nucléaires forment des homo- et hétéro-dimères, nous avons cherché à développer de nouveaux essais pharmacologiques pour étudier l'activité de complexes dimériques spécifiques, leur dynamique d’association et la structure quaternaire des récepteurs des œstrogènes. Nous décrivons ici une nouvelle technique FRET, surnommée BRET avec renforcement de fluorescence par transferts combinés (BRETFect), qui permet de détecter la formation de complexes de récepteurs nucléaires ternaires. Le BRETFect peut suivre l'activation des hétérodimères ERα-ERβ et met en évidence un mécanisme allostérique d'activation que chaque récepteur exerce sur son partenaire de dimérisation. L'utilisation de BRETFect en combinaison avec le PCA nous a permis d'observer la formation de multimères d’ERα fonctionnels dans des cellules vivantes pour la première fois. La formation de multimères est favorisée par les AE induisant la dégradation du récepteur des oestrogènes, ce qui pourrait contribuer à leurs propriétés spécifiques. Ces essais de BRET apportent une nette amélioration par rapport aux tests de vecteurs rapporteur luciférase classique, en fournissant des informations spécifiques aux récepteurs en temps réel sans aucune interférence par d'autres processus tels que la transcription et de la traduction. L'utilisation de ces tests nous a permis de caractériser les propriétés de modulation de l’activité des récepteurs nucléaires d’une nouvelle classe de molécules hybrides qui peuvent à la fois lier ERa ou RAR et inhiber les HDACs, conduisant au développement de nouvelles molécules prometteuses bifonctionnelles telles que la molécule hybride RAR-agoniste/HDACi TTNN-HA.

Functional Analysis of saxophone, the Drosophila Gene Encoding the BMP Type I Receptor Ortholog of Human ALK1/ACVRL1 and ACVR1/ALK2

In metazoans, bone morphogenetic proteins (BMPS) direct a myriad of developmental and adult homeostatic evens through their heterotetrameric type I and type II receptor complexes. We examined 3 existing and 12 newly generated mutations in the Drosophila type I receptor gene, saxophone (sax), the ortholog of the human Activin Receptor-Like. Kinasel and -2 (ALK1/ACVR1 and ALK2/ACVR1) genes. Our genetic analyses identified two distinct classes of sax alleles. The first class consists of homozygous viable gain-of-function (GOF) alleles that exhibit (1) synthetic lethality in combination with mutations in BMP pathway components, and (2) significant maternal effect lethality that can be rescued by an increased dosage of the BMP encoding gene, dpp(+). In contrast, the second class consists of alleles that are recessive lethal and do not exhibit lethality in combination with mutations in other BMP pathway components. The alleles in this second class are clearly loss-of-function (LOF) with both complete and partial loss-of-function mutations represented. We find that one allele in the second class of recessive lethals exhibits dominant-negative behavior, albeit distinct from the GOF activity of the first class of viable alleles. On the basis of the fact that the first class of viable alleles can be reverted to lethality and on our ability to independently generate recessive lethal sat mutations, our analysis demonstrates that sax is an essential gene. Consistent with this conclusion, we find that a normal sax transcript is produced by sax(P), a viable allele previously reported to be mill, and that this allele can be reverted to lethality. Interestingly, we determine that two mutations in the first: class of sax alleles show the same amino acid substitutions as mutations in the human receptors ALK1/ACVR1-1 and ACVR1/ALK2, responsible for cases of hereditary hemorrhagic telangiectasia type 2 (HHT2) and fibrodysplasia ossificans progressiva (FOP), respectively. Finally, the data presented here identify different functional requirements for the Sax receptor, support the proposal that Sax participates in a heteromeric receptor complex, and provide a mechanistic framework for future investigations into disease states that arise from defects in BMP/TGF-beta signaling.

Lipopolysaccharides from atherosclerosis-associated bacteria antagonize TLR4, induce formation of TLR2/1/CD36 complexes in lipid rafts and trigger TLR2-induced inflammatory responses in human vascular endothelial cells

Infection with bacteria such as Chlamydia pneumonia, Helicobacter pylori or Porphyromonas gingivalis may be triggering the secretion of inflammatory cytokines that leads to atherogenesis. The mechanisms by which the innate immune recognition of these pathogens could lead to atherosclerosis remain unclear. In this study, using human vascular endothelial cells or HEK-293 cells engineered to express pattern-recognition receptors (PRRs), we set out to determine Toll-like receptors (TLRs) and functionally associated PRRs involved in the innate recognition of and response to lipopolysaccharide (LPS) from H. pylori or P. gingivalis. Using siRNA interference or recombinant expression of cooperating PRRs, we show that H. pylori and P. gingivalis LPS-induced cell activation is mediated through TLR2. Human vascular endothelial cell activation was found to be lipid raft-dependent and to require the formation of heterotypic receptor complexes comprising of TLR2, TLR1, CD36 and CD11b/CD18. In addition, we report that LPS from these bacterial strains are able to antagonize TLR4. This antagonistic activity of H. pylori or P. gingivalis LPS, as well as their TLR2 activation capability may be associated with their ability to contribute to atherosclerosis.

Nck recruitment to Eph receptor, EphB1/ELK, couples ligand activation to c-Jun kinase.

Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis; yet the signaling systems that couple these receptors to targeting and cell-cell assembly responses are incompletely defined. Functional links to regulators of cytoskeletal structure are anticipated based on receptor mediated cell-cell aggregation and migratory responses. We used two-hybrid interaction cloning to identify EphB1-interactive proteins. Six independent cDNAs encoding the SH2 domain of the adapter protein, Nck, were recovered in a screen of a murine embryonic library. We mapped the EphB1 subdomain that binds Nck and its Drosophila homologue, DOCK, to the juxtamembrane region. Within this subdomain, Tyr594 was required for Nck binding. In P19 embryonal carcinoma cells, activation of EphB1 (ELK) by its ligand, ephrin-B1/Fc, recruited Nck to native receptor complexes and activated c-Jun kinase (JNK/SAPK). Transient overexpression of mutant EphB1 receptors (Y594F) blocked Nck recruitment to EphB1, attenuated downstream JNK activation, and blocked cell attachment responses. These findings identify Nck as an important intermediary linking EphB1 signaling to JNK.

SYNTHETIC PROBES FOR STUDYING TUFTSIN-RECEPTOR INTERACTIONS ON HUMAN POLYMORPHONUCLEAR LEUKOCYTES

Tuftsin is an immunopotentiating tetrapeptide of the sequence L-Thr-L-Lys-L-Pro-L-Arg with anti-microbial and anti-tumor enhancing capabilities. These enhancing functions are manifested through the host's granulocytes and monocytes. In delineating tuftsin's mechanism of action, both radiolabeled and fluorescent probes were synthesized. The radiolabeled probe of tuftsin, L-proly-3,4-('3)H(N) -tuftsin, was obtained through the synthesis and subsequent catalytic hydrogenation of L-3,4-dehydroprolyl ('3)-tuftsin using tritium gas. This procedure yielded a probe with a specific activity of 44.9 Ci/mmole. This radiolabeled probe of tuftsin was used in competitive inhibition studies with tuftsin, the tuftsin analogues Lys-Pro-Arg, Thr-Lys-Pro-Arg(NO(,2)) and (DELTA)('3)-pro('3) -tuftsin as well as with the chemotactic peptide f-Met-Leu-Phe. From the competitive binding curves, the K(,D) for tuftsin was estimated to be 80 nM, a value that approaches the concentration of tuftsin that evokes a half maximal biological response. The approximate Ki's for the tuftsin analogues (33 nM) approached that of tuftsin itself (40 nM). On the other hand, approximately a two log difference in the Ki was seen with the chemotactic tripeptide, indicating that tuftsin may indeed be acting through the chemotactic peptide receptor. This conclusion is further strengthened by studies using an N-terminal derivitized mono-fluoresceinated tuftsin probe and image intensification microscopy. These studies showed that like the chemotactic peptide, tuftsin initially binds to diffusely distributed receptors on the surface of human granulocytes. The tuftsin-receptor complexes then rapidly redistribute to form patches (5 min @ 37(DEGREES)C) which are then internalized. Whether redistribution and internalization of tuftsin-receptor complexes is crucial in effecting a biological response, or simply an intermediary point leading ultimately to degradation, is still not clear. This process, however, may provide the target cell with an early time point in modulating the biological effects of tuftsin through down-regulation of cell surface receptor sites. ^

The Kinetics of Mannose 6-Phosphate Receptor Trafficking in the Endocytic Pathway in HEp-2 Cells: The Receptor Enters and Rapidly Leaves Multivesicular Endosomes without Accumulating in a Prelysosomal Compartment

We have previously shown that in HEp-2 cells, multivesicular bodies (MVBs) processing internalized epidermal growth factor–epidermal growth factor receptor complexes mature and fuse directly with lysosomes in which the complexes are degraded. The MVBs do not fuse with a prelysosomal compartment enriched in mannose 6-phosphate receptor (M6PR) as has been described in other cell types. Here we show that the cation-independent M6PR does not become enriched in the endocytic pathway en route to the lysosome, but if a pulse of M6PR or an M6PR ligand, cathepsin D, is followed, a significant fraction of these proteins are routed from the trans-Golgi to MVBs. Accumulation of M6PR does not occur because when the ligand dissociates, the receptor rapidly leaves the MVB. At steady state, most M6PR are distributed within the trans-Golgi and trans-Golgi network and in vacuolar structures distributed in the peripheral cytoplasm. We suggest that these M6PR-rich vacuoles are on the return route from MVBs to the trans-Golgi network and that a separate stable M6PR-rich compartment equivalent to the late endosome/prelysosome stage does not exist on the endosome–lysosome pathway in these cells.

Structural interactions of fibroblast growth factor receptor with its ligands

Fibroblast growth factors (FGFs) effect cellular responses by binding to FGF receptors (FGFRs). FGF bound to extracellular domains on the FGFR in the presence of heparin activates the cytoplasmic receptor tyrosine kinase through autophosphorylation. We have crystallized a complex between human FGF1 and a two-domain extracellular fragment of human FGFR2. The crystal structure, determined by multiwavelength anomalous diffraction analysis of the selenomethionyl protein, is a dimeric assemblage of 1:1 ligand:receptor complexes. FGF is bound at the junction between the two domains of one FGFR, and two such units are associated through receptor:receptor and secondary ligand:receptor interfaces. Sulfate ion positions appear to mark the course of heparin binding between FGF molecules through a basic region on receptor D2 domains. This dimeric assemblage provides a structural mechanism for FGF signal transduction.

Modulation of growth factor receptor function by isoform heterodimerization.

Activation of prolactin (PRL)-dependent signaling occurs as the result of ligand-induced dimerization of receptor (PRLr). Although three PRLr isoforms (short, intermediate, and long) have been characterized and are variably coexpressed in PRL-responsive tissues, the functional effects of ligand-induced PRLr isoform heterodimerization have not been examined. To determine whether heterodimeric PRLr complexes were capable of ligand-induced signaling and cellular proliferation, chimeras consisting of the extracellular domain of either the alpha or beta subunit of human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFr) and the intracellular domain of the rat intermediate or short PRLr isoforms (PRLr-I or PRLr-S) were synthesized. Because high affinity binding of GM-CSF is mediated by the extracellular domain of one alpha and beta GM-CSFr pair, use of GM-CSFr/PRLr chimera specifically directed the dimerization of the PRLr intracellular domains within ligand-receptor complexes. Stable transfection of these constructs into the Ba/F3 line was demonstrated by Northern blot and immunoprecipitation analyses. Flow cytometry revealed specific binding of a phycoerythrin-conjugated human GM-CSF to the transfectants, confirming cell surface expression of the chimeric receptors. When tested for their ability to proliferate in response to GM-CSF, only chimeric transfectants expressing GM-CSFr/PRLr-I homodimers demonstrated significant [3H]thymidine incorporation. GM-CSF stimulation of transfectants expressing either GM-CSFr/PRLr-S homodimers or GM-CSFr/PRLr-S+1 heterodimers failed to induce proliferation. Consistent with these data, the GM-CSF-induced activation of two phosphotyrosine kinases, Jak2 and Fyn, was observed only in homodimeric GM-CSFr/PRLr-I transfectants. These results show that the PRLr-S functions as a dominant negative isoform, down-regulating both signaling and proliferation mediated by the receptor complex. Thus, structural motifs necessary for Jak2 and Fyn activation within the carboxy terminus of the PRLr-I, absent in the PRLr-S, are required in each member of the dimeric PRLr complex.

Cell-surface-expressed T-cell antigen-receptor zeta chain is associated with the cytoskeleton.

The T-cell antigen receptor zeta chain plays an important role in coupling antigen recognition to several intracellular signal-transduction pathways. zeta chain can associate with certain protein tyrosine kinases and retains the capacity to transduce signals independently of the other receptor subunits. Thus, zeta chain could couple cell-surface-expressed T-cell antigen receptors to the intracellular signal-transduction apparatus by its association with various intracellular molecules in addition to tyrosine kinases. In the process of searching for zeta chain-associated molecules we observed that after lysis of resting T cells with Triton X-100, zeta chain is localized in the detergent-insoluble fraction, in addition to its presence in the detergent-soluble fraction. Treatment of T cells with cytochalasin B, an actin-depolymerizing agent, leads to the complete dissociation of zeta chain from the Triton-insoluble fraction, suggesting a linkage between zeta chain and the cytoskeletal matrix. We have also determined that cytoskeletal-associated zeta chain is expressed on the cell surface. Furthermore, a tyrosine-phosphorylated 16-kDa zeta chain was detected only in the Triton-insoluble cytoskeletal fraction of resting T cells. zeta chain also maintains its association with the cytoskeleton when expressed in COS cells, inferring that the cytoskeletal elements involved in this linkage may be ubiquitous. Finally, we have localized a 42-amino acid region in the intracytoplasmic domain of zeta chain, which is crucial for maximal interaction between zeta chain and the cytoskeleton. Anchorage of cell-surface-expressed zeta chain to the cytoskeleton in resting T cells may facilitate recycling of receptor complexes and/or allow the transduction of external stimuli into the cell.