Having long dreamt of becoming a mad scientist, Fernando pursued physics at Stanford, and then veered off track at Berkeley for his Ph.D. in Biophysics as a way to understand how things work in living organisms (which turned out to be a much brighter goal than bashing together elementary particles in deep tunnels). Following a biochemistry postdoc at UCSF studying protein structures, Fernando dove into immunology with cytokine and genomic discoveries at DNAX in Palo Alto (CA), and then moved to nearby Genentech to apply protein engineering to drug discovery in oncology and immunology. As the CSO at NeuroScience, Fernando is thrilled to be able to tap into the molecular conversation between neuro and immune systems as a way to diagnose and treat complex human disorders. His move to the Midwest has introduced him to the concept of 'seasons', and the lush green foliage and rolling farmlands remind him of his childhood in Costa Rica.
Abstract: Interleukin-1 (IL-1)-family cytokines are mediators of innate and adaptive immunity. They exert proinflammatory effects by binding a primary receptor that recruits a receptor accessory protein to form a signaling-competent heterotrimeric complex. Here we present the crystal structure of IL-1β bound to its primary receptor IL-1RI and its receptor accessory protein IL-1RAcP, providing insight into how IL-1-type cytokines initiate signaling and revealing an evolutionary relationship with the fibroblast growth factor receptor family.
Abstract: Lacking any discernible sequence similarity, interleukin-34 (IL-34) and colony stimulating factor 1 (CSF-1) signal through a common receptor CSF-1R on cells of mononuclear phagocyte lineage. Here, the crystal structure of dimeric IL-34 reveals a helical cytokine fold homologous to CSF-1, and we further show that the complex architecture of IL-34 bound to the N-terminal immunoglobulin domains of CSF-1R is similar to the CSF-1/CSF-1R assembly. However, unique conformational adaptations in the receptor domain geometry and intermolecular interface explain the cross-reactivity of CSF-1R for two such distantly related ligands. The docking adaptations of the IL-34 and CSF-1 quaternary complexes, when compared to the stem cell factor assembly, draw a common evolutionary theme for transmembrane signaling. In addition, the structure of IL-34 engaged by a Fab fragment reveals the mechanism of a neutralizing antibody that can help deconvolute IL-34 from CSF-1 biology, with implications for therapeutic intervention in diseases with myeloid pathogenic mechanisms.
Abstract: Nectins (nectin1-4) and Necls [nectin-like (Necl1-5)] are Ig superfamily cell adhesion molecules that regulate cell differentiation and tissue morphogenesis. Adherens junction formation and subsequent cell-cell signaling is initiated by the assembly of higher-order receptor clusters of cognate molecules on juxtaposed cells. However, the structural and mechanistic details of signaling cluster formation remain unclear. Here, we report the crystal structure of poliovirus receptor (PVR)/Nectin-like-5/CD155) in complex with its cognate immunoreceptor ligand T-cell-Ig-and-ITIM-domain (TIGIT). The TIGIT/PVR interface reveals a conserved specific "lock-and-key" interaction. Notably, two TIGIT/PVR dimers assemble into a heterotetramer with a core TIGIT/TIGIT cis-homodimer, each TIGIT molecule binding one PVR molecule. Structure-guided mutations that disrupt the TIGIT/TIGIT interface limit both TIGIT/PVR-mediated cell adhesion and TIGIT-induced PVR phosphorylation in primary dendritic cells. Our data suggest a cis-trans receptor clustering mechanism for cell adhesion and signaling by the TIGIT/PVR complex and provide structural insights into how the PVR family of immunoregulators function.
Abstract: Jumping Translocation Breakpoint (JTB) is an orphan receptor that is conserved from nematodes to humans and whose gene expression in humans is strikingly upregulated in diverse types of cancers. Translocations occur frequently at the hJTB genomic locus, leading to multiple copies of a truncated JTB gene, which potentially encodes a soluble secreted ectodomain. In addition, JTB and its orthologs likely represent a unique and ancient protein family since homologs could not be identified by direct sequence comparison. In the present study, we have determined the NMR solution structure of the N-terminal ectodomain of human JTB, showing that its fold architecture is a new variant of a three-β-strand antiparallel β-meander. The JTB structure has a distant relationship to the midkine/pleiotrophin fold, particularly in the conservation of distinctive disulfide bridge patterns. The structure of this newly characterized small cysteine-rich domain suggests potential involvement of JTB in interactions with proteins or extracellular matrix and may help to uncover the elusive biological functions of this protein.
Abstract: Toll like receptors (TLRs) use Toll-IL-1 receptor (TIR) domain-containing adapters, such as myeloid differentiation primary response gene 88 (MyD88) and TIR domain-containing adapter inducing IFN-β (TRIF), to induce activation of transcription factors, including NF-κB, MAP kinases, and IFN regulatory factors. TLR signaling also leads to activation of PI3K, but the molecular mechanism is not understood. Here we have discovered a unique role for B-cell adapter for PI3K (BCAP) in the TLR-signaling pathway. We find that BCAP has a functional N-terminal TIR homology domain and links TLR signaling to activation of PI3K. In addition, BCAP negatively regulates proinflammatory cytokine secretion upon TLR stimulation. In vivo, the absence of BCAP leads to exaggerated recruitment of inflammatory myeloid cells following infections and enhanced susceptibility to dextran sulfate sodium-induced colitis. Our results demonstrate that BCAP is a unique TIR domain-containing TLR signaling adapter crucial for linking TLRs to PI3K activation and regulating the inflammatory response.
Abstract: Cilia are microtubule-based protrusions from the cell surface that are involved in a number of essential signaling pathways, yet little is known about many of the proteins that regulate their structure and function. A number of putative cilia genes have been identified by proteomics and comparative sequence analyses, but functional data are lacking for the vast majority. We therefore monitored the effects in three cell lines of small interfering RNA (siRNA) knockdown of 40 of these genes by high-content analysis. We assayed cilia number, length, and transport of two different cargoes (membranous serotonin receptor 6-green fluorescent protein [HTR6-GFP] and the endogenous Hedgehog [Hh] pathway transcription factor Gli3) by immunofluorescence microscopy; and cilia function using a Gli-luciferase Hh signaling assay. Hh signaling was most sensitive to perturbations, with or without visible structural cilia defects. Validated hits include Ssa2 and mC21orf2 with ciliation defects; Ift46 with short cilia; Ptpdc1 and Iqub with elongated cilia; and Arl3, Nme7, and Ssna1 with distinct ciliary transport but not length defects. Our data confirm various ciliary roles for several ciliome proteins and show it is possible to uncouple ciliary cargo transport from cilia formation in vertebrates.
Abstract: One goal of the CASP community wide experiment on the critical assessment of techniques for protein structure prediction is to identify the current state of the art in protein structure prediction and modeling. A fundamental principle of CASP is blind prediction on a set of relevant protein targets, that is, the participating computational methods are tested on a common set of experimental target proteins, for which the experimental structures are not known at the time of modeling. Therefore, the CASP experiment would not have been possible without broad support of the experimental protein structural biology community. In this article, several experimental groups discuss the structures of the proteins which they provided as prediction targets for CASP9, highlighting structural and functional peculiarities of these structures: the long tail fiber protein gp37 from bacteriophage T4, the cyclic GMP-dependent protein kinase Iβ dimerization/docking domain, the ectodomain of the JTB (jumping translocation breakpoint) transmembrane receptor, Autotaxin in complex with an inhibitor, the DNA-binding J-binding protein 1 domain essential for biosynthesis and maintenance of DNA base-J (β-D-glucosyl-hydroxymethyluracil) in Trypanosoma and Leishmania, an so far uncharacterized 73 residue domain from Ruminococcus gnavus with a fold typical for PDZ-like domains, a domain from the phycobilisome core-membrane linker phycobiliprotein ApcE from Synechocystis, the heat shock protein 90 activators PFC0360w and PFC0270w from Plasmodium falciparum, and 2-oxo-3-deoxygalactonate kinase from Klebsiella pneumoniae.
Abstract: Nephronophthisis (NPHP), Joubert (JBTS), and Meckel-Gruber (MKS) syndromes are autosomal-recessive ciliopathies presenting with cystic kidneys, retinal degeneration, and cerebellar/neural tube malformation. Whether defects in kidney, retinal, or neural disease primarily involve ciliary, Hedgehog, or cell polarity pathways remains unclear. Using high-confidence proteomics, we identified 850 interactors copurifying with nine NPHP/JBTS/MKS proteins and discovered three connected modules: "NPHP1-4-8" functioning at the apical surface, "NPHP5-6" at centrosomes, and "MKS" linked to Hedgehog signaling. Assays for ciliogenesis and epithelial morphogenesis in 3D renal cultures link renal cystic disease to apical organization defects, whereas ciliary and Hedgehog pathway defects lead to retinal or neural deficits. Using 38 interactors as candidates, linkage and sequencing analysis of 250 patients identified ATXN10 and TCTN2 as new NPHP-JBTS genes, and our Tctn2 mouse knockout shows neural tube and Hedgehog signaling defects. Our study further illustrates the power of linking proteomic networks and human genetics to uncover critical disease pathways.
Abstract: Psoriasis is a human skin condition characterized by epidermal hyperproliferation and infiltration of multiple leukocyte populations. In characterizing a novel insulin growth factor (IGF)-like (IGFL) gene in mice (mIGFL), we found transcripts of this gene to be most highly expressed in skin with enhanced expression in models of skin wounding and psoriatic-like inflammation. A possible functional ortholog in humans, IGFL1, was uniquely and significantly induced in psoriatic skin samples. In vitro IGFL1 expression was up-regulated in cultured primary keratinocytes stimulated with tumor necrosis factor α but not by other psoriasis-associated cytokines. Finally, using a secreted and transmembrane protein library, we discovered high affinity interactions between human IGFL1 and mIGFL and the TMEM149 ectodomain. TMEM149 (renamed here as IGFLR1) is an uncharacterized gene with structural similarity to the tumor necrosis factor receptor family. Our studies demonstrate that IGFLR1 is expressed primarily on the surface of mouse T cells. The connection between mIGFL and IGFLR1 receptor suggests mIGFL may influence T cell biology within inflammatory skin conditions.
Abstract: Norbin, a neurite-outgrowth promoting protein, has been found to interact with and regulate several membrane proteins, including metabotropic glutamate receptor 5 (mGluR5). The disruption of both Norbin alleles leads to early embryonic death between 3.5 and 6.5 day post coitus.1 Forebrain specific Norbin knockout (KO) mice are defective in synaptic plasticity,2 an interesting feature considering that Norbin was initially discovered in the context of chemical-induced long term potentiation (LTP),3 a form of synaptic plasticity extensively studied in the context of learning and memory.4 The behavioral phenotypes associated with Norbin conditional KO suggest reduced mGluR5 function. Because of its fundamental functions, Norbin is emerging as a key neuronal regulator. The aim of the present review is to summarize current knowledge about Norbin while emphasizing its role in the nervous system.
Abstract: The BBSome is a complex of Bardet-Biedl Syndrome (BBS) proteins that shares common structural elements with COPI, COPII, and clathrin coats. Here, we show that the BBSome constitutes a coat complex that sorts membrane proteins to primary cilia. The BBSome is the major effector of the Arf-like GTPase Arl6/BBS3, and the BBSome and GTP-bound Arl6 colocalize at ciliary punctae in an interdependent manner. Strikingly, Arl6(GTP)-mediated recruitment of the BBSome to synthetic liposomes produces distinct patches of polymerized coat apposed onto the lipid bilayer. Finally, the ciliary targeting signal of somatostatin receptor 3 needs to be directly recognized by the BBSome in order to mediate targeting of membrane proteins to cilia. Thus, we propose that trafficking of BBSome cargoes to cilia entails the coupling of BBSome coat polymerization to the recognition of sorting signals by the BBSome.
Abstract: MCL1 is essential for the survival of stem and progenitor cells of multiple lineages, and is unique among pro-survival BCL2 family members in that it is rapidly turned over through the action of ubiquitin ligases. B- and mantle-cell lymphomas, chronic myeloid leukaemia, and multiple myeloma, however, express abnormally high levels of MCL1, contributing to chemoresistance and disease relapse. The mechanism of MCL1 overexpression in cancer is not well understood. Here we show that the deubiquitinase USP9X stabilizes MCL1 and thereby promotes cell survival. USP9X binds MCL1 and removes the Lys 48-linked polyubiquitin chains that normally mark MCL1 for proteasomal degradation. Increased USP9X expression correlates with increased MCL1 protein in human follicular lymphomas and diffuse large B-cell lymphomas. Moreover, patients with multiple myeloma overexpressing USP9X have a poor prognosis. Knockdown of USP9X increases MCL1 polyubiquitination, which enhances MCL1 turnover and cell killing by the BH3 mimetic ABT-737. These results identify USP9X as a prognostic and therapeutic target, and they show that deubiquitinases may stabilize labile oncoproteins in human malignancies.
Abstract: Wnt/beta-catenin signaling is initiated at the cell surface by association of secreted Wnt with its receptors Frizzled (Fz) and low density lipoprotein receptor-related protein 5/6 (LRP5/6). The study of these molecular interactions has been a significant technical challenge because the proteins have been inaccessible in sufficient purity and quantity. In this report we describe insect cell expression and purification of soluble mouse Fz8 cysteine-rich domain and human LRP6 extracellular domain and show that they inhibit Wnt/beta-catenin signaling in cellular assays. We determine the binding affinities of Wnts and Dickkopf 1 (Dkk1) to the relevant co-receptors and reconstitute in vitro the Fz8 CRD.Wnt3a.LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth beta-propeller domains of LRP6 (E3E4). Surprisingly, we find that Wnt9b binds to a different part of the LRP6 extracellular domain, E1E2, and we demonstrate that Wnt3a and Wnt9b can bind to LRP6 simultaneously. Dkk1 binds to both E1E2 and E3E4 fragments and competes with both Wnt3a and Wnt9b for binding to LRP6. The existence of multiple, independent Wnt binding sites on the LRP6 co-receptor suggests new possibilities for the architecture of Wnt signaling complexes and a model for broad-spectrum inhibition of Wnt/beta-catenin signaling by Dkk1.
Abstract: The Hedgehog (Hh) signaling pathway is inappropriately activated in certain human cancers, including medulloblastoma, an aggressive brain tumor. GDC-0449, a drug that inhibits Hh signaling by targeting the serpentine receptor Smoothened (SMO), has produced promising anti-tumor responses in early clinical studies of cancers driven by mutations in this pathway. To evaluate the mechanism of resistance in a medulloblastoma patient who had relapsed after an initial response to GDC-0449, we determined the mutational status of Hh signaling genes in the tumor after disease progression. We identified an amino acid substitution at a conserved aspartic acid residue of SMO that had no effect on Hh signaling but disrupted the ability of GDC-0449 to bind SMO and suppress this pathway. A mutation altering the same amino acid also arose in a GDC-0449-resistant mouse model of medulloblastoma. These findings show that acquired mutations in a serpentine receptor with features of a G protein-coupled receptor can serve as a mechanism of drug resistance in human cancer.
Abstract: Hedgehog (Hh) signaling is crucial for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancer. Hedgehog-interacting protein (Hhip) is a surface receptor antagonist that is equipotent against all three mammalian Hh homologs. The crystal structures of human HHIP alone and bound to Sonic hedgehog (SHH) now reveal that HHIP is comprised of two EGF domains and a six-bladed beta-propeller domain. In the complex structure, a critical loop from HHIP binds the pseudo active site groove of SHH and directly coordinates its Zn2+ cation. Notably, sequence comparisons of this SHH binding loop with the Hh receptor Patched (Ptc1) ectodomains and HHIP- and PTC1-peptide binding studies suggest a 'patch for Patched' at the Shh pseudo active site; thus, we propose a role for Hhip as a structural decoy receptor for vertebrate Hh.
Abstract: A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-kappaB (nuclear factor kappaB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-kappaB-inducing kinase) and regulate the non-canonical NF-kappaB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys(48)- and Lys(63)-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain-ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile(44) of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.
Abstract: Members of the interleukin-1 (IL-1) family of cytokines play major roles in host defense and immune system regulation in infectious and inflammatory diseases. IL-1 cytokines trigger a biological response in effector cells by assembling a heterotrimeric signaling complex with two IL-1 receptor chains, a high-affinity primary receptor and a low-affinity coreceptor. To gain insights into the signaling mechanism of the novel IL-1-like cytokine IL-33, we first solved its solution structure and then performed a detailed biochemical and structural characterization of the interaction between IL-33, its primary receptor ST2, and the coreceptor IL-1RAcP. Using nuclear magnetic resonance data, we obtained a model of the IL-33/ST2 complex in solution that is validated by small-angle X-ray scattering (SAXS) data and is similar to the IL-1beta/IL-1R1 complex. We extended our SAXS analysis to the IL-33/ST2/IL-1RAcP and IL-1beta/IL-1R1/IL-1RAcP complexes and propose a general model of the molecular architecture of IL-1 ternary signaling complexes.
Abstract: The crystal structure of the human p300 histone acetyltransferase (HAT) domain reveals a familiar alpha + beta fold with unique structural elaborations that merit its classification as a third divergent HAT branch alongside the GCN5-related N-acetyltransferase (GNAT) and MYST (MOZ, Ybf2/Sas3, Sas2, Tip60) families. Two key departures from the core GNAT/MYST HAT fold--a long unstructured chain (or "flap") overlaying the acetyl-CoA (AcCoA) binding groove, and a four-alpha-helix "tower" excursion from the main beta-sheet--critically contribute to the recognition and presumptive catalytic machinery of p300/CBP HAT enzymes. Kinetic and mutant analysis of this enlarged residue constellation in p300 (which is distinct from functional fingerprints drawn from GNAT or MYST complexes) led Liu et al., to suggest that p300/CBP works with an unorthodox "hit and run" mechanism that enlists Tyr1467 as the critical catalytic residue. In order to extend the evolutionary testbed for this variant HAT mechanism beyond the thin roll of p300/CBP orthologs, I propose that Rtt109, a novel yeast HAT that has so far eluded classification, is the prototype of a fungal clan of p300-related enzymes that preserve the embellished HAT fold, but further diversify its catalytic options.
Abstract: Primary cilium dysfunction affects the development and homeostasis of many organs in Bardet-Biedl syndrome (BBS). We recently showed that seven highly conserved BBS proteins form a stable complex, the BBSome, that functions in membrane trafficking to and inside the primary cilium. We have now discovered a BBSome subunit that we named BBIP10. Similar to other BBSome subunits, BBIP10 localizes to the primary cilium, BBIP10 is present exclusively in ciliated organisms, and depletion of BBIP10 yields characteristic BBS phenotypes in zebrafish. Unexpectedly, BBIP10 is required for cytoplasmic microtubule polymerization and acetylation, two functions not shared with any other BBSome subunits. Strikingly, inhibition of the tubulin deacetylase HDAC6 restores microtubule acetylation in BBIP10-depleted cells, and BBIP10 physically interacts with HDAC6. BBSome-bound BBIP10 may therefore function to couple acetylation of axonemal microtubules and ciliary membrane growth.
Abstract: Production of type I interferon (IFN-I) is a critical host defense triggered by pattern-recognition receptors (PRRs) of the innate immune system. Deubiquitinating enzyme A (DUBA), an ovarian tumor domain-containing deubiquitinating enzyme, was discovered in a small interfering RNA-based screen as a regulator of IFN-I production. Reduction of DUBA augmented the PRR-induced IFN-I response, whereas ectopic expression of DUBA had the converse effect. DUBA bound tumor necrosis factor receptor-associated factor 3 (TRAF3), an adaptor protein essential for the IFN-I response. TRAF3 is an E3 ubiquitin ligase that preferentially assembled lysine-63-linked polyubiquitin chains. DUBA selectively cleaved the lysine-63-linked polyubiquitin chains on TRAF3, resulting in its dissociation from the downstream signaling complex containing TANK-binding kinase 1. A discrete ubiquitin interaction motif within DUBA was required for efficient deubiquitination of TRAF3 and optimal suppression of IFN-I. Our data identify DUBA as a negative regulator of innate immune responses.
Abstract: Neuropilins (Nrps) are co-receptors for class 3 semaphorins and vascular endothelial growth factors and important for the development of the nervous system and the vasculature. The extracellular portion of Nrp is composed of two domains that are essential for semaphorin binding (a1a2), two domains necessary for VEGF binding (b1b2), and one domain critical for receptor dimerization (c). We report several crystal structures of Nrp1 and Nrp2 fragments alone and in complex with antibodies that selectively block either semaphorin or vascular endothelial growth factor (VEGF) binding. In these structures, Nrps adopt an unexpected domain arrangement in which the a2, b1, and b2 domains form a tightly packed core that is only loosely connected to the a1 domain. The locations of the antibody epitopes together with in vitro experiments indicate that VEGF and semaphorin do not directly compete for Nrp binding. Based upon our structural and functional data, we propose possible models for ligand binding to neuropilins.
Abstract: Primary cilium dysfunction underlies the pathogenesis of Bardet-Biedl syndrome (BBS), a genetic disorder whose symptoms include obesity, retinal degeneration, and nephropathy. However, despite the identification of 12 BBS genes, the molecular basis of BBS remains elusive. Here we identify a complex composed of seven highly conserved BBS proteins. This complex, the BBSome, localizes to nonmembranous centriolar satellites in the cytoplasm but also to the membrane of the cilium. Interestingly, the BBSome is required for ciliogenesis but is dispensable for centriolar satellite function. This ciliogenic function is mediated in part by the Rab8 GDP/GTP exchange factor, which localizes to the basal body and contacts the BBSome. Strikingly, Rab8(GTP) enters the primary cilium and promotes extension of the ciliary membrane. Conversely, preventing Rab8(GTP) production blocks ciliation in cells and yields characteristic BBS phenotypes in zebrafish. Our data reveal that BBS may be caused by defects in vesicular transport to the cilium.
Abstract: Missense variants are commonly identified in genomic sequence but only a small fraction directly contribute to oncogenesis. The ability to distinguish those missense changes that contribute to cancer progression from those that do not is a difficult problem usually only accomplished through functional in vivo analyses. Using two computational algorithms, Sorting Intolerant from Tolerant (SIFT) and the Pfam-based LogR.E-value method, we have identified features that distinguish cancer-associated missense mutations from other classes of missense change. Our data reveal that cancer mutants behave similarly to Mendelian disease mutations, but are clearly distinct from either complex disease mutations or common single-nucleotide polymorphisms. We show that both activating and inactivating oncogenic mutations are predicted to be deleterious, although activating changes are likely to increase protein activity. Using the Gene Ontology and data from the SIFT and LogR.E-value metrics, a classifier was built that predicts cancer-associated missense mutations with a very low false-positive rate. The classifier does remarkably well in a number of different experiments designed to distinguish polymorphisms from true cancer-associated mutations. We also show that recurrently observed mutations are much more likely to be predicted to be cancer-associated than rare mutations, suggesting that our classifier will be useful in distinguishing causal from passenger mutations. In addition, from an expressed sequence tag-based screen, we identified a previously unknown germ line change (P1104A) in tumor tissues that is predicted to disrupt the function of the TYK2 protein. The data presented here show that this novel bioinformatics approach to classifying cancer-associated variants is robust and can be used for large-scale analyses.
Abstract: Signal transduction pathways are frequently found to repress transcription of target genes in the absence of stimulation and, conversely, to upregulate transcription in the presence of a signal. Transcription factors are central in this dual regulatory mechanism and widely use a generalized mechanism to repress transcription through recruitment of a Sin3-histone deacetylase (HDAC) complex to their binding sites on DNA. The protein SAP18 (Sin3-associated polypeptide of 18 kDa) has been shown to play a key role in gene-specific recruitment of the HDAC complex by a number of transcription factors including Gli, GAGA, and Bicoid. The solution structure of SAP18 reveals a ubiquitin-like fold with several large loop insertions relative to other family members. This fold supports the functional role of SAP18 as a protein-protein adapter module and provides insight for how SAP18 may bridge the Sin3-HDAC complex to transcription factors.
Abstract: ADP-ribosylation is an enzyme-catalyzed posttranslational protein modification in which mono(ADP-ribosyl)transferases (mARTs) and poly(ADP-ribosyl)transferases (pARTs) transfer the ADP-ribose moiety from NAD onto specific amino acid side chains and/or ADP-ribose units on target proteins.
Abstract: Toll-like receptors (TLRs) are the archetypal pattern recognition receptors (PRRs) envisioned by as innate sensors of pathogen attack and host triggers of an adaptive immune response. Two recent papers (Choe et al., 2005; Bell et al., 2005) reveal the distinctive architecture of a TLR sensor domain and hint at how this structural design facilitates the recognition of a wide array of pathogen molecules.
Abstract: Cytokines of the interleukin-1 (IL-1) family, such as IL-1 alpha/beta and IL-18, have important functions in host defense, immune regulation, and inflammation. Insight into their biological functions has led to novel therapeutic approaches to treat human inflammatory diseases. Within the IL-1 family, IL-1 alpha/beta, IL-1Ra, and IL-18 have been matched to their respective receptor complexes and have been shown to have distinct biological functions. The most prominent orphan IL-1 receptor is ST 2. This receptor has been described as a negative regulator of Toll-like receptor-IL-1 receptor signaling, but it also functions as an important effector molecule of T helper type 2 responses. We report a member of the IL-1 family, IL-33, which mediates its biological effects via IL-1 receptor ST 2, activates NF-kappaB and MAP kinases, and drives production of T(H)2-associated cytokines from in vitro polarized T(H)2 cells. In vivo, IL-33 induces the expression of IL-4, IL-5, and IL-13 and leads to severe pathological changes in mucosal organs.
Abstract: The recently discovered cytokine IL-27 belongs to the IL-6/IL-12 family of cytokines and induced proliferation of naive CD4(+) T cells and the generation of a Th1-type adaptive immune response. Although binding of IL-27 to the cytokine receptor WSX-1 was demonstrated, this interaction proved insufficient to mediate cellular effects. Hence, IL-27 was believed to form a heteromeric signaling receptor complex with WSX-1 and another, yet to be identified, cytokine receptor subunit. In this study, we describe that WSX-1 together with gp130 constitutes a functional signal-transducing receptor for IL-27. We show that neither of the two subunits itself is sufficient to mediate IL-27-induced signal transduction, but that the combination of both is required for this event. Expression analysis of WSX-1 and gp130 by quantitative PCR suggests that IL-27 might have a variety of cellular targets besides naive CD4(+) T cells: we demonstrate gene induction of a subset of inflammatory cytokines in primary human mast cells and monocytes in response to IL-27 stimulation. Thus, IL-27 not only contributes to the development of an adaptive immune response through its action on CD4(+) T cells, it also directly acts on cells of the innate immune system.
Abstract: Failure of axon regeneration in the adult mammalian central nervous system (CNS) is at least partly due to inhibitory molecules associated with myelin. Recent studies suggest that an axon surface protein, the Nogo receptor (NgR), may play a role in this process through an unprecedented degree of crossreactivity with myelin-associated inhibitory ligands. Here, we report the 1.5 A crystal structure and functional characterization of a soluble extracellular domain of the human Nogo receptor. Nogo receptor adopts a leucine-rich repeat (LRR) module whose concave exterior surface contains a broad region of evolutionarily conserved patches of aromatic residues, possibly suggestive of degenerate ligand binding sites. A deep cleft at the C-terminal base of the LRR may play a role in NgR association with the p75 coreceptor. These results now provide a detailed framework for focused structure-function studies aimed at assessing the physiological relevance of NgR-mediated protein-protein interactions to axon regeneration inhibition.
Abstract: Whether epithelial cells play a role in triggering the immune cascade leading to T helper 2 (T(H)2)-type allergic inflammation is not known. We show here that human thymic stromal lymphopoietin (TSLP) potently activated CD11c(+) dendritic cells (DCs) and induced production of the T(H)2-attracting chemokines TARC (thymus and activation-regulated chemokine; also known as CCL17) and MDC (macrophage-derived chemokine; CCL22). TSLP-activated DCs primed naïve T(H) cells to produce the proallergic cytokines interleukin 4 (IL-4), IL-5, IL-13 and tumor necrosis factor-alpha, while down-regulating IL-10 and interferon-gamma. TSLP was highly expressed by epithelial cells, especially keratinocytes from patients with atopic dermatitis. TSLP expression was associated with Langerhans cell migration and activation in situ. These findings shed new light on the function of human TSLP and the role played by epithelial cells and DCs in initiating allergic inflammation.
Abstract: ADP-ribosyltransferases including toxins secreted by Vibrio cholera, Pseudomonas aerurginosa, and other pathogenic bacteria inactivate the function of human target proteins by attaching ADP-ribose onto a critical amino acid residue. Cross-species polymerase chain reaction (PCR) and database mining identified the orthologs of these ADP-ribosylating toxins in humans and the mouse. The human genome contains four functional toxin-related ADP-ribosyltransferase genes (ARTs) and two related intron-containing pseudogenes; the mouse has six functional orthologs. The human and mouse ART genes map to chromosomal regions with conserved linkage synteny. The individual ART genes reveal highly restricted expression patterns, which are largely conserved in humans and the mouse. We confirmed the predicted extracellular location of the ART proteins by expressing recombinant ARTs in insect cells. Two human and four mouse ARTs contain the active site motif (R-S-EXE) typical of arginine-specific ADP-ribosyltransferases and exhibit the predicted enzyme activities. Two other human ARTs and their murine orthologues deviate in the active site motif and lack detectable enzyme activity. Conceivably, these ARTs may have acquired a new specificity or function. The position-sensitive iterative database search program PSI-BLAST connected the mammalian ARTs with most known bacterial ADP-ribosylating toxins. In contrast, no related open reading frames occur in the four completed genomes of lower eucaryotes (yeast, worm, fly, and mustard weed). Interestingly, these organisms also lack genes for ADP-ribosylhydrolases, the enzymes that reverse protein ADP-ribosylation. This suggests that the two enzyme families that catalyze reversible mono-ADP-ribosylation either were lost from the genomes of these nonchordata eucaryotes or were subject to horizontal gene transfer between kingdoms.
Abstract: An efficient Th1-driven adaptive immune response requires activation of the T cell receptor and secretion of the T cell stimulatory cytokine IL-12 by activated antigen-presenting cells. IL-12 triggers Th1 polarization of naive CD4(+) T cells and secretion of IFN-gamma. We describe a new heterodimeric cytokine termed IL-27 that consists of EBI3, an IL-12p40-related protein, and p28, a newly discovered IL-12p35-related polypeptide. IL-27 is an early product of activated antigen-presenting cells and drives rapid clonal expansion of naive but not memory CD4(+) T cells. It also strongly synergizes with IL-12 to trigger IFN-gamma production of naive CD4(+) T cells. IL-27 mediates its biologic effects through the orphan cytokine receptor WSX-1/TCCR.
Abstract: The stimulation of interferon (IFN)-gamma by interleukin (IL)-12 has been shown to provide protection from intracellular pathogens such as Listeria monocytogenes. Tumor necrosis factor (TNF) is also a major player in the resolution of Listeria infections and is suggested to have more global effects than can be explained by the induction of IFN-gamma alone. Since IL-18 synergizes with IL-12 to induce IFN-gamma production by natural killer and T helper (Th)1 cells, we determined its role in responses to Listeria. IL-18 appeared to be even more potent than either IL-12 or IFN-gamma for protection against this pathogen and IL-18 enhanced bacterial clearance in the complete absence of IFN-gamma. Indeed IL-18 was comparable to TNF in its ability to resolve the infection and showed a lowered protective capacity in the absence of TNF. Moreover, IL-18 induced macrophages to secrete both TNF and nitric oxide after a Listeria infection. IL-18 was also essential for optimal IFN-gamma production by antigen-specific T cells. Therefore, IL-18 operates via its effects on both the innate immune response, including macrophages, as well as on Th1 cells, to protect against Listeria.
Abstract: IL-1 is of utmost importance in the host response to immunological challenges. We identified and functionally characterized two novel IL-1 ligands termed IL-1delta and IL-1epsilon. Northern blot analyses show that these IL-1s are highly abundant in embryonic tissue and tissues containing epithelial cells (i.e., skin, lung, and stomach). In extension, quantitative real-time PCR revealed that of human skin-derived cells, only keratinocytes but not fibroblasts, endothelial cells, or melanocytes express IL-1delta and epsilon. Levels of keratinocyte IL-1delta are approximately 10-fold higher than those of IL-1epsilon. In vitro stimulation of keratinocytes with IL-1beta/TNF-alpha significantly up-regulates the expression of IL-1epsilon mRNA, and to a lesser extent of IL-1delta mRNA. In NF-kappaB-luciferase reporter assays, we demonstrated that IL-1delta and epsilon proteins do not initiate a functional response via classical IL-1R pairs, which confer responsiveness to IL-1alpha and beta or IL-18. However, IL-1epsilon activates NF-kappaB through the orphan IL-1R-related protein 2 (IL-1Rrp2), whereas IL-1delta, which shows striking homology to IL-1 receptor antagonist, specifically and potently inhibits this IL-1epsilon response. In lesional psoriasis skin, characterized by chronic cutaneous inflammation, the mRNA expression of both IL-1 ligands as well as IL-1Rrp2 are increased relative to normal healthy skin. In total, IL-1delta and epsilon and IL-1Rrp2 may constitute an independent signaling system, analogous to IL-1alphabeta/receptor agonist and IL-1R1, that is present in epithelial barriers of our body and takes part in local inflammatory responses.
Abstract: Toll-like receptors (TLRs) are ancient microbial pattern recognition receptors highly conserved from Drosophila to humans. To investigate if subsets of human dendritic cell precursors (pre-DC), including monocytes (pre-DC1), plasmacytoid DC precursors (pre-DC2), and CD11c(+) immature DCs (imDCs) are developed to recognize different microbes or microbial antigens, we studied their TLR expression and responses to microbial antigens. We demonstrate that whereas monocytes preferentially express TLR 1, 2, 4, 5, and 8, plasmacytoid pre-DC strongly express TLR 7 and 9. In accordance with these TLR expression profiles, monocytes respond to the known microbial ligands for TLR2 (peptidoglycan [PGN], lipoteichoic acid) and TLR4 (lipopolysaccharide), by producing tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. In contrast, plasmacytoid pre-DCs only respond to the microbial TLR9-ligand, CpG-ODNs (oligodeoxynucleotides [ODNs] containing unmethylated CpG motifs), by producing IFN-alpha. CD11c(+) imDCs preferentially express TLR 1, 2, and 3 and respond to TLR 2-ligand PGN by producing large amounts of TNF-alpha, and to viral double-stranded RNA-like molecule poly I:C, by producing IFN-alpha and IL-12. The expression of distinct sets of TLRs and the corresponding difference in reactivity to microbial molecules among subsets of pre-DCs and imDCs support the concept that they have developed through distinct evolutionary pathways to recognize different microbial antigens.
Abstract: ADP-ribosyltransferases (ADPRTs) form an interesting class of enzymes with well-established roles as potent bacterial toxins and metabolic regulators. ADPRTs catalyze the transfer of the ADP-ribose moiety from NAD(+) onto specific substrates including proteins. ADP-ribosylation usually inactivates the function of the target. ADPRTs have become adapted to function in extra- and intracellular settings. Regulation of ADPRT activity can be mediated by ligand binding to associated regulatory domains, proteolytic cleavage, disulphide bond reduction, and association with other proteins. Crystallisation has revealed a conserved core set of elements that define an unusual minimal scaffold of the catalytic domain with remarkably plastic sequence requirements--only a single glutamic acid residue critical to catalytic activity is invariant. These inherent properties of ADPRTs suggest that the ADPRT catalytic fold is an attractive, malleable subject for protein design.
Abstract: Melanoma inhibitory activity (MIA) is a 12-kDa protein that is secreted from both chondrocytes and malignant melanoma cells. MIA has been reported to have effects on cell growth and adhesion, and it may play a role in melanoma metastasis and cartilage development. We report the 1.4-A crystal structure of human MIA, which consists of an Src homology 3 (SH3)-like domain with N- and C-terminal extensions of about 20 aa. each. The N- and C-terminal extensions add additional structural elements to the SH3 domain, forming a previously undescribed fold. MIA is a representative of a recently identified family of proteins and is the first structure of a secreted protein with an SH3 subdomain. The structure also suggests a likely protein interaction site and suggests that, unlike conventional SH3 domains, MIA does not recognize polyproline helices.
Abstract: p19, a molecule structurally related to IL-6, G-CSF, and the p35 subunit of IL-12, is a subunit of the recently discovered cytokine IL-23. Here we show that expression of p19 in multiple tissues of transgenic mice induced a striking phenotype characterized by runting, systemic inflammation, infertility, and death before 3 mo of age. Founder animals had infiltrates of lymphocytes and macrophages in skin, lung, liver, pancreas, and the digestive tract and were anemic. The serum concentrations of the proinflammatory cytokines TNF-alpha and IL-1 were elevated, and the number of circulating neutrophils was increased. In addition, ubiquitous expression of p19 resulted in constitutive expression of acute phase proteins in the liver. Surprisingly, liver-specific expression of p19 failed to reproduce any of these abnormalities, suggesting specific requirements for production of biologically active p19. Bone marrow transfer experiments showed that expression of p19 by hemopoietic cells alone recapitulated the phenotype induced by its widespread expression, pointing to hemopoietic cells as the source of biologically active p19. These findings indicate that p19 shares biological properties with IL-6, IL-12, and G-CSF and that cell-specific expression is required for its biological activity.
Abstract: The sequence of a novel hemopoietic cytokine was discovered in a computational screen of genomic databases, and its homology to mouse thymic stromal lymphopoietin (TSLP) suggests that it is the human orthologue. Human TSLP is proposed to signal through a heterodimeric receptor complex that consists of a new member of the hemopoietin family termed human TSLP receptor and the IL-7R alpha-chain. Cells transfected with both receptor subunits proliferated in response to purified, recombinant human TSLP, with induced phosphorylation of Stat3 and Stat5. Human TSLPR and IL-7Ralpha are principally coexpressed on monocytes and dendritic cell populations and to a much lesser extent on various lymphoid cells. In accord, we find that human TSLP functions mainly on myeloid cells; it induces the release of T cell-attracting chemokines from monocytes and, in particular, enhances the maturation of CD11c(+) dendritic cells, as evidenced by the strong induction of the costimulatory molecules CD40 and CD80 and the enhanced capacity to elicit proliferation of naive T cells.
Abstract: The outstanding problems facing immunology are whole system issues: curing allergic and autoimmune disease and developing vaccines to stimulate stronger immune responses against pathogenic organisms and cancer. We hope that the human genome sequence will reveal the molecular checks and balances that ensure both an effective immunogenic response against pathogenic microorganisms and a suitably tolerogenic response to self antigens and innocuous environmental antigens. Three synergistic approaches--sequence homology searches, messenger RNA expression profiling on microarrays, and mutagenesis in mice--provide the best opportunities to reveal, in the genome sequence, key proteins and pathways for targeting by new immunomodulatory treatments.
Abstract: Nuclear Factor of Activated T cell (NFAT) is a family of transcription factors that are important for the coordinate expression of various cytokines and immunoregulatory cell surface molecules in T cells and other types of cells in the immune system. In addition, analysis of gene disrupted mice revealed that some members of NFAT family are important for the development of myocardium, myocardial hypertrophy, and mesenchymal stem cells. NFAT family proteins have two conserved domains, the NFAT Homology Domain (NHD) and the Rel Similarity Domain (RSD). The RSD is DNA binding and AP-1 interacting domain which has structural similarity to the Rel Homology Region, the DNA binding domain of Rel family proteins. The NHD is a regulatory domain required for the Ca regulated translocation of NFAT. We report here the isolation and initial characterization of a novel RSD containing protein designated NFATz. NFATz has a RSD but no NHD. NFATz protein is localized in the nucleus without Ca signal. There is no detectable binding to a typical NFAT site even in the presence of AP-1, and it is not capable of activating transcription through the NFAT site. The chromosomal location determined by FISH revealed that NFATz and NFATx genes are in the same region.
Abstract: The Interleukin-1 receptor (IL-1R) and Toll signaling pathways share the evolutionarily conserved Toll homology domain (THD), which is a critical component in the signaling cascade of the host defense responses to infection and inflammation. Our initial genomic database searches uncovered a novel THD signature sequence between DNA markers DXS87 and DXS366. The feasibility of subsequently applying a coordinated computational approach, including various exon-finding programs, homology-based searches, and receptor profile searches, in revealing the exons encoding this novel IL-1R family member is described. IL-1R9 shows restricted expression in fetal brain and is highly homologous to IL1RAPL (A. Carrie et al., 1999 Nat. Genet. 23: 25-31), which is reportedly involved in nonsyndromic X-linked mental retardation. These genes are scattered over separate genomic intervals in excess of 1.0 Mb and encode receptors with extended C-terminal tails. In our functional NF-kappaB reporter assays, IL1RAPL, IL-1R9, or versions lacking the extended C-terminal sequences failed in responding either to IL-1 directly or to IL-18 when various permutations of IL-18R ectodomain chimeras were fused to their cytoplasmic domains. Evolutionary sequence analyses reinforce our conclusion that these novel orphan receptors probably form a functionally distinct subset of the IL-1R superfamily.
Abstract: IL-18 is critical in eliciting IFN-gamma production from Th1 cells both in vitro and in vivo. Th1 cells have been implicated in the pathogenesis of autoimmune disorders, making antagonists of IL-18 promising therapeutics. However, specificity and binding characteristics of IL-18R components have only been superficially explored. In this study, we show that IL-1R related protein 1 (IL-1Rrp1) and IL-1R accessory protein-like (IL-1RAcPL) confer responsiveness to IL-18 in a highly specific (no response to other IL-1 ligands) and unique manner (no functional pairing with other IL-1Rs and IL-1R-like molecules). Cotransfection with both receptor components resulted in expression of both low and high affinity binding sites for IL-18 (K:(d) of 11 and 0.4 nM, respectively). We prepared anti-IL-1RAcPL mAb TC30-28E3, which, in contrast to soluble R proteins, effectively inhibited the IL-18-induced activation of NF-kappaB. Quantitative PCR showed that Th1 but not Th2 cells are unique in that they coexpress IL-1Rrp1 and IL-1RAcPL. mAb TC30-28E3 inhibited IL-18-induced production of IFN-gamma by Th1 cells, being at least 10-fold more potent than anti-IL-18 ligand mAb. This study shows that IL-1RAcPL is highly specific to IL-18, is required for high affinity binding of IL-18, and that the anti-IL-1RAcPL mAb TC30-28E3 potently antagonizes IL-18 responses in vitro, providing a rationale for the use of anti-IL-1RAcPL Abs to inhibit Th1-mediated inflammatory pathologies.
Abstract: Doublecortin (DCX) missense mutations are found in two clusters in patients with defective cortical neuronal migration. Although DCX can function as a microtubule-associated protein (MAP), the potential relationship between its MAP activity and neuronal migration is not understood. Here we show that the two clusters of patient mutations precisely define an internal tandem repeat. Each repeat alone binds tubulin, whereas neither repeat is sufficient for co-assembly with microtubules. The two tandem repeats are sufficient to mediate microtubule polymerization, and representative patient missense mutations lead to impaired polymerization both in vitro and in vivo as well as impaired microtubule stabilization. Furthermore, each repeat is predicted to have the secondary structure of a beta-grasp superfold motif, a motif not found in other MAPs. The patient mutations are predicted to disrupt the structure of the motif, suggesting that the motif may be critical for the DCX-tubulin interaction. These data provide both genetic and biochemical evidence that the interaction of DCX with microtubules is dependent upon this novel repeated tubulin-binding motif.
Abstract: A novel sequence discovered in a computational screen appears distantly related to the p35 subunit of IL-12. This factor, which we term p19, shows no biological activity by itself; instead, it combines with the p40 subunit of IL-12 to form a novel, biologically active, composite cytokine, which we term IL-23. Activated dendritic cells secrete detectable levels of this complex. IL-23 binds to IL-12R beta 1 but fails to engage IL-12R beta 2; nonetheless, IL-23 activates Stat4 in PHA blast T cells. IL-23 induces strong proliferation of mouse memory (CD4(+)CD45Rb(low)) T cells, a unique activity of IL-23 as IL-12 has no effect on this cell population. Similar to IL-12, human IL-23 stimulates IFN-gamma production and proliferation in PHA blast T cells, as well as in CD45RO (memory) T cells.
Abstract: In this study we describe the isolation and characterization of a new chicken (Gallus gallus) chemokine. This molecule belongs to the C or gamma-chemokine family and is related to the mouse and human lymphotactin (Lptn). Mouse and human Lptn are distinguished from alpha and beta chemokines by the absence of two cysteines (Cys 1 and 3) that form a disulfide bridge; the novel chicken chemokine shows the same cysteine pattern, but replaces a long carboxy-terminal tail found in the other Lptn proteins with a short extension rich in Arg residues. The 1-kb mRNA is mainly expressed in spleen, although weaker signals have been detected in liver and colon. It is interesting to note that the chicken chemokine seems to preferentially induce the migration of spleen B cells over T cells or B cells from the bursa of Fabricius.
Abstract: Fractalkine, a novel CX3C chemokine, is unusual because of both its membrane-associated structure and its direct role in cell adhesion. We have solved the solution structure of the chemokine domain of fractalkine (residues 1-76) by heteronuclear NMR methods. The 20 lowest energy structures in the ensemble have an average backbone rmsd of 0.43 A, excluding the termini. In contrast to many other chemokines which form homodimers, fractalkine's chemokine module is monomeric. Comparison of the structure to CC and CXC chemokines reveals interesting differences which are likely to be relevant to receptor binding. These include a bulge formed by the CX3C motif, the relative orientation of the N-terminus and 30's loop (residues 30-38), and the conformation of the N-loop (residues 9-19). 15N backbone relaxation experiments indicate that these same regions of the protein are dynamic. We also titrated 15N-labeled protein with a peptide from the N-terminus of the receptor CX3CR1 and confirmed that this region of the receptor contacts the fractalkine chemokine domain. Interestingly, the binding site maps roughly to the regions of greatest flexibility and structural variability. Together, these data provide a first glimpse of how fractalkine interacts with its receptor and should help guide mutagenesis studies to further elucidate the molecular details of binding and signaling through CX3CR1.
Abstract: We report the expression cloning of a novel leptin-binding protein of the immunoglobulin superfamily (OB-BP1) and a cross-hybridizing clone (OB-BP2) that is identical to a recently described sialic acid-binding I-type lectin called Siglec-5. Comparisons to other known Siglec family members (CD22, CD33, myelin-associated glycoprotein, and sialoadhesin) show that OB-BP1, OB-BP2/Siglec-5, and CD33/Siglec-3 constitute a unique related subgroup with a high level of overall amino acid identity: OB-BP1 versus Siglec-5 (59%), OB-BP1 versus CD33 (63%), and OB-BP2/Siglec-5 versus CD33 (56%). The cytoplasmic domains are not as highly conserved, but display novel motifs which are putative sites of tyrosine phosphorylation, including an immunoreceptor tyrosine kinase inhibitory motif and a motif found in SLAM and SLAM-like proteins. Human tissues showed high levels of OB-BP1 mRNA in placenta and moderate expression in spleen, peripheral blood leukocytes, and small intestine. OB-BP2/Siglec-5 mRNA was detected in peripheral blood leukocytes, lung, spleen, and placenta. A monoclonal antibody specific for OB-BP1 confirmed high expression in the cyto- and syncytiotrophoblasts of the placenta. Using this antibody on peripheral blood leukocytes showed an almost exclusive expression pattern on B cells. Recombinant forms of the extracellular domains of OB-BP1, OB-BP2/Siglec-5, and CD33/Siglec-3 were assayed for specific binding of leptin. While OB-BP1 exhibited tight binding (K(d) 91 nM), the other two showed weak binding with K(d) values in the 1-2 microM range. Studies with sialylated ligands indicated that OB-BP1 selectively bound Neu5Acalpha2-6GalNAcalpha (sialyl-Tn) allowing its formal designation as Siglec-6. The identification of OB-BP1/Siglec-6 as a Siglec family member, coupled with its restricted expression pattern, suggests that it may mediate cell-cell recognition events by interacting with sialylated glycoprotein ligands expressed on specific cell populations. We also propose a role for OB-BP1 in leptin physiology, as a molecular sink to regulate leptin serum levels.
Abstract: Signaling through the hematopoietic receptors requires activation of receptor-associated Janus (Jak) kinases. For example, Jak1 and Jak3 bind specifically to the IL-2 receptor beta (IL-2Rbeta) and common gamma (gammac) chains, respectively, and initiate biochemical signals critical in controlling immune responses. The region of Jak responsible for receptor interactions, however, is not well characterized. Here we describe a naturally occurring Jak3 mutation from a patient with autosomal severe combined immunodeficiency (SCID), where a single amino acid substitution, Y100C, in Janus homology domain 7 (JH7) prevents kinase-receptor interaction. This mutation also results in a loss of IL-2-induced signaling in a B-cell line derived from this patient. Using mutational analysis we have identified a region of Jak3, including portions of JH6 and JH7, that is sufficient for kinase-receptor contact and show that this segment interacts with the proline-rich Box1 region of the receptor. Furthermore, a Jak3-Jak1 chimera containing only the JH6 and JH7 domains of Jak3 interacts with gammac and can reconstitute IL-2-dependent responses, including receptor phosphorylation and activation of signal transducer and activator of transcription (STAT) 5b. Our results suggest that the N-terminus of Jak kinases is critical for receptor binding, and is therefore likely to determine specificity of Jak kinase-receptor interactions.
Abstract: We report here the identification and characterization of the mouse homologue of a human CX3C chemokine described by F. Bazan et al. (1997, Nature 385, 640-644). Termed fractalkine, this molecule constitutes a fourth or delta chemokine structural type that displays a novel CX3C sequence fingerprint. Distinct from the alpha, beta, or gamma chemokine families, the polypeptide chain of CX3C predicts a 373-amino-acid type I transmembrane glycoprotein with the chemokine domain resting on top of an extended mucin-like stalk. Comparison of the mouse and human protein chains shows a high degree of conservation in all the globular segments with the exception of the stalk portion. The striking identity of an amino acid stretch encompassing a putative juxtamembrane cleavage site suggests the evolutionary conservation of both membrane-bound and processed CX3C forms. Northern analysis reveals the presence of mouse CX3C mRNA in heart, brain, lung, kidney, skeletal muscle, and testis tissues. The mouse CX3C gene was further localized to the central region of chromosome 8 by interspecific backcross mapping; a related locus was detected on chromosome 11. The novel location of this gene from other chemokine gene clusters adds to the notion that CX3C is a fundamentally new class of chemokine.
Abstract: The discovery of sequence homology between the cytoplasmic domains of Drosophila Toll and human interleukin 1 receptors has sown the conviction that both molecules trigger related signaling pathways tied to the nuclear translocation of Rel-type transcription factors. This conserved signaling scheme governs an evolutionarily ancient immune response in both insects and vertebrates. We report the molecular cloning of a class of putative human receptors with a protein architecture that is similar to Drosophila Toll in both intra- and extracellular segments. Five human Toll-like receptors--named TLRs 1-5--are probably the direct homologs of the fly molecule and, as such, could constitute an important and unrecognized component of innate immunity in humans. Intriguingly, the evolutionary retention of TLRs in vertebrates may indicate another role--akin to Toll in the dorsoventralization of the Drosophila embryo--as regulators of early morphogenetic patterning. Multiple tissue mRNA blots indicate markedly different patterns of expression for the human TLRs. By using fluorescence in situ hybridization and sequence-tagged site database analyses, we also show that the cognate Tlr genes reside on chromosomes 4 (TLRs 1, 2, and 3), 9 (TLR4), and 1 (TLR5). Structure prediction of the aligned Toll-homology domains from varied insect and human TLRs, vertebrate interleukin 1 receptors and MyD88 factors, and plant disease-resistance proteins recognizes a parallel beta/alpha fold with an acidic active site; a similar structure notably recurs in a class of response regulators broadly involved in transducing sensory information in bacteria.
Abstract: The myeloid differentiation (MyD) marker MyD88 was initially characterized as a primary response gene, upregulated in mouse M1 myeloleukemic cells in response to differentiation induced by interleukin-6. Subsequent analysis revealed that MyD88 possesses a unique modular structure, which consists of an N-terminal "death domain," similar to the intracellular segments of TNF receptor 1 and Fas, and a C-terminal region related to the cytoplasmic domains of the Drosophila morphogen Toll and vertebrate interleukin-1 receptors. In this report we describe the cloning and gene structure of mouse MyD88. The complete coding sequence of mouse MyD88 spans five exons, with the first exon encoding the complete death domain. Zooblot analysis revealed that MyD88 is an evolutionarily conserved gene. MyD88 was localized to the distal region of mouse chromosome 9 by interspecific backcross mapping. The human homolog (hMyD88) was mapped to chromosome 3p22-p21.3 by PCR analysis of a human chromosome 3 somatic cell hybrid mapping panel. Northern blot analysis revealed widespread expression of MyD88 in many adult mouse tissues, and RT-PCR studies detected MyD88 mRNA in T and B cell lines and differentiating embryonic stem cells. The broad expression pattern demonstrates that mouse MyD88 expression is not restricted to cells of myeloid lineage as was originally believed.
Abstract: Interferon-gamma inducing factor (IGIF) is a recently identified cytokine which stimulates the production of interferon-gamma (IFN-gamma) by T cells and enhances natural killer (NK) cell cytolytic activity. Protein fold recognition, structure prediction and comparative modeling have revealed that IGIF is a member of the interleukin (IL)-1 cytokine family and has prompted the designation IL-1 gamma. Here we report functional similarities between members of the IL-1 family by comparing the effects of IL-1 alpha, IL-1 beta and IGIF on NK cell production of IFN-gamma. All three IL-1 types enhanced NK cell production of IFN-gamma when induced by IL-2 or IL-12, although at high concentrations (> 10 ng/ml), IGIF was five- to tenfold more potent than IL-1 alpha or IL-1 beta. This effect correlated with enhanced levels of mRNA for IFN-gamma when NK cells were stimulated with IGIF plus IL-12. In contrast to IL-12 and IL-2, the ability of IGIF to stimulate NK cell production of IFN-gamma was not increased by IL-1 alpha or IL-1 beta. The ability of IGIF to enhance IFN-gamma production was independent of the type I and type II IL-1 receptors or the IL-1R accessory protein. Together, these results identify IGIF as a potent stimulator of NK cell production of IFN-gamma and demonstrate that the effect of IGIF on NK cell production of IFN-gamma is similar to that of IL-1 alpha and IL-1 beta but distinct from that of IL-12.
Abstract: Chemokines direct the trafficking of white blood cells in immune surveillance, playing a key role in inflammatory and infectious diseases such as AIDS. All chemokines studied so far are secreted proteins of relative molecular mass approximately 7K-15K and fall into three families that are defined by a cysteine signature motif: CXC, CC and C (refs 3, 6, 7), where C is a cysteine and X any amino-acid residue. We report here the identification and characterization of a fourth human chemokine type, derived from non-haemopoietic cells and bearing a new CX3C fingerprint. Unlike other chemokine types, the polypeptide chain of the human CX3C chemokine is predicted to be part of a 373-amino-acid protein that carries the chemokine domain on top of an extended mucin-like stalk. This molecule can exist in two forms: either membrane-anchored or as a shed 95K glycoprotein. The soluble CX3C chemokine has potent chemoattractant activity for T cells and monocytes, and the cell-surface-bound protein, which is induced on activated primary endothelial cells, promotes strong adhesion of those leukocytes. The structure, biochemical features, tissue distribution and chromosomal localization of CX3C chemokine all indicate that it represents a unique class of chemokine that may constitute part of the molecular control of leukocyte traffic at the endothelium.
Abstract: A novel gene detected in mouse embryonic sites of hematopoiesis was cloned and shown to be a eukaryotic analog of the Escherichia coli selenophosphate synthetase gene. Unlike the E. coli enzyme, which is not a selenoprotein, the presence of selenocysteine in the mouse enzyme is indicated by a TGA codon in the open reading frame of the gene in a position corresponding to the essential cysteine of the E. coli enzyme. An ionized selenol group in place of a cysteine sulfhydryl group could render this mammalian selenocysteine-containing enzyme a more active catalyst. The native cDNA clone and also a mutant form containing a TGC (cysteine) codon in place of TGA were expressed in a baculovirus-insect cell system. Based on recovery of purified proteins, expression of the mutant enzyme was about 40 times higher than wild-type enzyme. The cysteine mutant enzyme exhibited selenophosphate synthetase activity in the assay that measures selenide-dependent AMP formation from ATP. Although expression of wild-type enzyme has not been optimized, the mutant form of the fetal mouse enzyme can be produced in amounts sufficient for isolation in homogeneous form and precise physicochemical and mechanistic studies allowing direct comparison with the analogous cysteine-containing prokaryotic enzyme.
Abstract: In these studies, IFN gamma-inducing factor (IGIF), unlike IL-12, did not drive Th1 development in BALB/c or C57BL/6 mice, but like IL-1alpha, potentiated IL-12-driven Th1 development in BALB/c mice. IGIF and IL-12 synergized for IFN gamma production from Th1 cells. Unlike IL-1alpha, IGIF had no effect on Th2 cells. IGIF signaled through IRAK, IL-1 receptor-associated kinase, to induce nuclear translocation of p65/p50 NFkappaB in Th1 cells. IL-1alpha had no effect on proliferation, cytokine production, or NFkappaB activation in Th1 cells but activated NFkappaB and proliferation in Th2 cells. Thus, Th1 and Th2 cells may differ in responsiveness and receptor expression for IL-1 family molecules. IGIF and IL-1alpha may differentially amplify Th1 and Th2 effector responses, respectively.
Abstract: Bacteriophage T4 codes at least for two ADP-ribosylating activities, the 76 kDa Alt and the 24 kDa Mod gene products. The main target for both enzymes is the host RNA polymerase. We cloned and sequenced the alt gene and overexpressed the corresponding enzyme. The recombinant protein shows ADP-ribosylating activities in vitro, as had been described earlier for the native enzyme isolated from phage heads. The native as well as the recombinant protein ADP-ribosylate the alpha-subunit of RNA polymerase, but also subunits beta, beta' and sigma 70 and perform an autoribosylation reaction. Taking advantage of the pKWIII test system, constructed to measure promoter strengths in vivo, it was found that ADP-ribosylation of RNA polymerase leads to an increase of transcription from T4 early promoters up to a factor of two. In an infected host cell this should cause an enhanced expression of T4 genes. Depending on whether RNA polymerase was ADP-ribosylated or not, it initiated transcription at T4 promoters with different sequence characteristics: unribosylated RNA polymerase recognizes the early T4 promoters by an extended -10 region, whereas the ribosylated enzyme selects for T4 early promoters with an extended T4-specific and highly conserved -35 region. These results may reflect how the virus, step by step imposes its genetic program on the host cell, and in part they give a rationale for the extension of the consensus sequence observed with these promoters. We also sequenced the genomic region of the T4 mod gene and found two open reading frames coding both for proteins of approximately 24 kDa. Up to now none of the reading frames could be cloned into E. coli in an active form, making it highly probable that the ADP-ribosylation pattern inflicted by gene product Mod on host RNA polymerase is deleterious to these bacteria. Comparisons of the amino acid sequences showed significant homologies among the two reading frames. Computer analysis reveals that both Mod sequences and also the sequence of the Alt protein exhibit a structural concordance with the catalytic domains of other prokaryotic ADP-mono-ribosyltransferases such as the Pseudomonas aeruginosa exotoxin A, the cholera labile enterotoxin, the diphteria toxin, the heat labile enterotoxin A of E. coli, and pertussis toxin. We present a detailed model for T4 transcription regulation.
Abstract: The Wnt family constitutes a set of structurally related glycoproteins that have been implicated in oncogenesis and developmental processes. The vertebrate Wnt10 family includes mouse, Xenopus and salamander proteins. We undertook a bioinformatics based approach to characterize a novel human Wnt gene. The gene (WNT10B) encodes a 389-amino acid protein with 96.6% sequence identity to mouse Wnt10b. The expression pattern of WNT10B reveals that it is synthesized in many adult tissues with the highest levels found in heart and skeletal muscle. WNT10B expression was also detected in several human cancer cell lines with elevated mRNA levels observed in HeLa (cervical cancer) cells. WNT10B was localized to 12q13.1 by PCR typing of a human/rodent monochromosomal panel and fluorescent in situ hybridization.
Abstract: We searched the database of expressed sequence tags (dbEST) for relatives of the known human and murine mono(ADP-ribosyl)transferases (mADPRT), poly(ADP-ribosyl)polymerases (PARP), ADP-ribosyl cyclases, and ADP-ribosylarginine hydrolases (ARH). By May 31, 1996, all of the known enzymes except for RT6 were represented in dbEST by exact sequence matches from mouse and/or human tissues. Several ESTs show significant sequence similarity but not identity to known mADPRTs. We isolated, cloned, and sequenced the corresponding genes. Our results show that seven human ESTs stem from a novel gene, provisionally designated LART, which is specifically expressed in lymphatic tissues. Five human ESTs stem from a novel gene, here designated TART1, which is specifically expressed in testis. This gene is also represented by a single mouse EST. One other mouse EST stems from a distinct gene, here designated TART2, which is also expressed in testis. These genes have similar exon/intron structures. The predicted LART and TART1 gene products contain hydrophobic N- and C-terminal signal peptides characteristic for GPI-anchored surface proteins, TART2 lacks the GPI-anchor signal peptide. The predicted native proteins show 28-42% sequence identity to one another. They each contain four cysteine residues that probably form conserved disulfide bonds. They each also contain a conserved glutamic acid residue within the proposed active site motif LART and TART1 show interesting deviations from the surrounding consensus sequence.
Abstract: Mono-ADP-ribosylation is one of the posttranslational protein modifications regulating cellular metabolism, e.g., nitrogen fixation, in prokaryotes. Several bacterial toxins mono-ADP-ribosylate and inactivate specific proteins in their animal hosts. Recently, two mammalian GPI-anchored cell surface enzymes with similar activities were cloned (designated ART1 and ART2). We have now identified six related expressed sequence tags (ESTs) in the public database and cloned the two novel human genes from which these are derived (designated ART3 and ART4). The deduced amino acid sequences of the predicted gene products show 28% sequence identity to one another and 32-41% identity vs the muscle and T cell enzymes. They contain signal peptide sequences characteristic of GPI anchorage. Southern Zoo blot analyses suggest the presence of related genes in other mammalian species. By PCR screening of somatic cell hybrids and by in situ hybridization, we have mapped the two genes to human chromosomes 4p14-p15.1 and 12q13.2-q13.3. Northern blot analyses show that these genes are specifically expressed in testis and spleen, respectively. Comparison of genomic and cDNA sequences reveals a conserved exon/intron structure, with an unusually large exon encoding the predicted mature membrane proteins. Secondary structure prediction analyses indicate conserved motifs and amino acid residues consistent with a common ancestry of this emerging mammalian enzyme family and bacterial mono(ADP-ribosyl)transferases. It is possible that the four human gene family members identified so far represent the "tip of an iceberg," i.e., a larger family of enzymes that influences the function of target proteins via mono-ADP-ribosylation.
Abstract: The low resolution structure of the Pseudomonas aeroginosa exotoxin A (ETA) presented in 1986 provided the first tantalizing three-dimensional view of an ADP-ribosyl-transferase (ADPRT) catalytic domain. The major features of this protein fold have recurred in the more recently solved crystal structures of the cholera toxin-related heat-labile enterotoxin (LT), diphtheria toxin (DT) and pertussis toxin (PT). A core set of alpha + beta elements define a minimal, conserved scaffold with remarkably plastic sequence requirements-only a single glutamic acid residue critical to catalytic activity is invariant. Other interchangeable residues in locations important for catalysis and binding are suggested by the cocrystal structures of DT with the inhibitor ApUp, ETA with bound AMP and nicotinamide, and DT with substrate NAD-in close accord with labeling and mutagenic data. Faint sequence resemblances that were earlier noticed among prokaryotic ADPRTs have now been securely extended by the structural concordance between toxin folds; more recently, eukaryotic ADPRTs have surfaced and their sequences can be reliably threaded into the conserved core fold. We will briefly summarize efforts in Palo Alto and Hamburg to explore these latter relationships, and to mount a rigorous search for new ADPRT families in the growing sequence databases.
Abstract: Mono ADP-ribosylation is a posttranslational protein modification that has been implicated in the regulation of key biological functions in bacteria as well as in animals. Recently, the first cDNAs for eucaryotic mono(ADPribosyl)transferases were cloned and found to exhibit significant sequence similarity only to one other known protein, the T cell differentiation antigen Rt6. In this paper we describe secondary structure analyses of Rt6 and related proteins and show conserved structure motifs and amino acid residues consistent with a common ancestry of these eucaryotic proteins and bacterial ADP-ribosyltransferases. Moreover, we have expressed soluble mouse Rt6-1 and Rt6-2 gene products in which C-terminal tags (FLAG-His6) replace the native glycosylphosphatidylinositol anchor signal sequences. Purified recombinant Rt6-2, but not Rt6-1, shows NAD+ glycohydrolase activity, which is inhibited by the arginine analogue agmatine. Immunoprecipitation of recombinant Rt6-1 and Rt6-2 with anti-FLAG M2 antibody followed by incubation with [32P]NAD+ leads to rapid and covalent incorporation of radioactivity into the light chain of the M2 antibody. The bound label is resistant to treatment with HgCl2 but sensitive to NH2OH, characteristic of arginine-linked ADP-ribosylation. These results demonstrate that Rt6-1 and RT6-2 possess the enzymatic activities typical for NAD+-dependent arginine/protein mono(ADPribosyl)transferases (EC 2.4.2.31). They are the first such enzymes to be molecularly characterized in the immune system.
Abstract: The smooth progression of the eukaryotic cell cycle relies on the periodic activation of members of a family of cell cycle kinases by regulatory proteins called cyclins. Outside of the cell cycle, cyclin homologs play important roles in regulating the assembly of transcription complexes; distant structural relatives of the conserved cyclin core or "box" can also function as general transcription factors (like TFIIB) or survive embedded in the chain of the tumor suppressor, retinoblastoma protein. The present work attempts the prediction of the canonical secondary, supersecondary, and tertiary fold of the minimal cyclin box domain using a combination of techniques that make use of the evolutionary information captured in a multiple alignment of homolog sequences. A tandem set of closely packed, helical modules are predicted to form the cyclin box domain.
Abstract: O-Acetylation and de-O-acetylation of sialic acids have been implicated in the regulation of a variety of biological phenomena, including endogenous lectin recognition, tumor antigenicity, virus binding, and complement activation. Applying a strategy designed to identify genes preferentially expressed in active sites of embryonic hematopoiesis, we isolated a novel cDNA from the pluripotent hematopoietic cell line FDCPmixA4 whose open reading frame contained sequences homologous to peptide fragments of a lysosomal sialic acid O-acetylesterase (Lse) previously purified from rat liver, but with no evident similarity to endoplasmic reticulum-derived acetylesterases. The expressed Lse protein exhibits sialic-acid O-acetylesterase activity that is not attributable to a typical serine esterase active site. lse expression is spatially and temporally restricted during embryogenesis, and its mRNA levels correlate with differences in O-acetylesterase activity described in adult tissues and blood cell types. Using interspecific backcross analysis, we further mapped the lse gene to the central region of mouse chromosome 9. This constitutes the first report on the molecular cloning of a sialic acid-specific O-acetylesterase in vertebrates and suggests novel roles for the 9-O-acetyl modification of sialic acids during the development and differentiation of mammalian organisms.
Abstract: Structure prediction algorithms have tagged leptin as the newest member of the haemopoietic cytokine family, a diverse class of secreted hormone-like factors with pleiotropic effects in immunity and haemopoietic development. While haemopoietic cytokines typically lack sequence similarity, they conserve a distinctive three-dimensional fold, a four-alpha-helix bundle structure that is recognized by the cognate family of haemopoietic cellular receptors. We have constructed a detailed molecular model of the human leptin helical fold that places the two cysteine residues of the leptin chain, Cys96 and Cys146, in close spatial proximity to each other. In this report, we present evidence that these cysteines are involved in an intrachain disulfide bridge that is critical for the structural integrity and stability of leptin. A leptin variant that is unable to form the disulfide link shows a reduced biological response when administered to leptin-deficient, ob/ob mice.
Abstract: MyD88 was first characterized as a myeloid differentiation primary response gene in mice, activated in M1 myeloleukemic cells following interleukin-6 (IL-6) induced growth arrest and terminal differentiation. Analysis of expressed sequence tags (ESTs) from activated dendritic cell libraries led to the indentification of cDNAs encoding the human homolog (hMyD88). The original description of MyD88 as a 243 aa protein may reflect a truncated mouse cDNA since the 2682 nt hMyD88 cDNA predicts a 296 aa cytoplasmic protein. Consistent with this proposal is the detection of a 33 kDa protein in human heart, kidney and liver tissue. The expression pattern of MyD88 is also more widespread than originally believed: a 2.6 kb hMyD88 mRNA species was found to be constitutively expressed in many adult human tissues; in addition MyD88 expression was observed in monocyte, T, B, NK and dendritic cells. The MyD88 protein has a modular structure composed of an N-terminal 'death domain' (DD) similar to the intracellular segments of TNF receptor 1 (TNFR1) and FAS and a C-terminal region related to the signaling domains of vertebrate interleukin-1 receptors (IL-1R) and the Drosophila morphogen Toll. This intriguing structural framework may endow MyD88 with unique signaling capabilities.
Abstract: Escherichia coli selenophosphate synthetase (SPS, the selD gene product) catalyzes the production of monoselenophosphate, the selenium donor compound required for synthesis of selenocysteine (Sec) and seleno-tRNAs. We report the molecular cloning of human and mouse homologs of the selD gene, designated Sps2, which contains an in-frame TGA codon at a site corresponding to the enzyme's putative active site. These sequences allow the identification of selD gene homologs in the genomes of the bacterium Haemophilus influenzae and the archaeon Methanococcus jannaschii, which had been previously misinterpreted due to their in-frame TGA codon. Sps2 mRNA levels are elevated in organs previously implicated in the synthesis of selenoproteins and in active sites of blood cell development. In addition, we show that Sps2 mRNA is up-regulated upon activation of T lymphocytes and have mapped the Sps2 gene to mouse chromosome 7. Using the mouse gene isolated from the hematopoietic cell line FDCPmixA4, we devised a construct for protein expression that results in the insertion of a FLAG tag sequence at the N terminus of the SPS2 protein. This strategy allowed us to document the readthrough of the in-frame TGA codon and the incorporation of 75Se into SPS2. These results suggest the existence of an autoregulatory mechanism involving the incorporation of Sec into SPS2 that might be relevant to blood cell biology. This mechanism is likely to have been present in ancient life forms and conserved in a variety of living organisms from all domains of life.
Abstract: The lymphocyte cell-surface Ag CD38 catabolizes NAD to adenosine 5' diphosphoribose (ADPR) and cyclic ADPR (cADPR). We show here that the soluble extracellular domain of CD38 (sCD38) mediates ADP ribosylation of several proteins. This was demonstrated by mass spectrometric analyses which revealed the addition of mass in units of 541.1 Da to these proteins, presumably corresponding to the covalent attachment of one or more ADPR moieties. Separate experiments showed that the same proteins became specifically radiolabeled following incubation with [32P]NAD plus sCD38. Additionally, it is shown that sCD38 can autoribosylate. Moreover, sCD38-mediated protein ribosylation was found to occur specifically at cysteine residues, since it was effectively blocked by addition of L-cysteine but not by other amino acids, and CD38-mediated protein ribosylation could be reversed by the addition of HgCl2, which specifically cleaves thiol-glycosidic bonds. ADPR purified from the reaction of sCD38 with NAD could itself be covalently transferred to target proteins at rates similar to the sCD38-mediated reaction, indicating that the ribosylation proceeds via the generation of this reactive intermediate. In vitro mutagenesis of a catalytic Glu residue that is conserved in numerous ADP-ribosyl transferases revealed that this amino acid is also important for catalysis in CD38. These data suggest that CD38 has the potential to cause ribosylation of experimental proteins, and raises the possibility that its specific ribosylation of a currently unidentified lymphocyte protein may contribute to its array of immunoregulatory activities.
Abstract: Dimeric proteins can arise from monomers by the simple exchange of secondary structural elements or a wholesale swapping of domains. These results have implications for the construction of novel oligomeric molecules and illuminate how existing structures may have evolved.
Abstract: To understand the mechanisms that control the differentiation of uncommitted mesoderm precursors into haematopoietic stem cells (HSCs) and the activation of haematopoiesis, we conducted a study to identify genes expressed at the earliest stages of both in vivo and in vitro haematopoietic development. Our strategy was to utilize Differential Display by means of the Polymerase Chain Reaction (DD-PCR) to compare patterns of gene expression between mRNA populations representing different levels of haematopoietic activity obtained from the mouse embryo, embryoid bodies (EBs) and mouse cell lines. We report the molecular cloning of two groups of genes expressed in the yolk sac: a group of genes expressed in the day-8.5 yolk sac at higher levels than in the day-8.5 embryo proper and up-regulated during EB development, and another group of day-8.5 yolk sac genes not expressed in the day-8.5 embryo proper or in EBs. Specifically, we describe the molecular cloning of the first nucleobase permease gene to be found in vertebrates, yolk sac permease-like molecule 1 (Ysp11). The Ysp11 gene has the unique property of encoding both intracellular, transmembrane and extracellular protein forms, revealing novel aspects of nucleotide metabolism that may be relevant during mammalian development.
Abstract: Human obese (hOB) protein was recently identified as a secreted hormone-like factor that is exclusively produced by adipose tissue and appears to regulate the size of the body's fat stores. We describe a rapid and efficient repetitive extension PCR method for the construction of a 453-bp synthetic hOB gene with high-frequency codons to optimize expression in Escherichia coli. The use of a bacterial signal sequence fused to hOB together with expression of bacteriocin release protein resulted in efficient release of hOB into the culture medium. The protein was readily purified to homogeneity from the culture medium using a two-step procedure.
Abstract: Amino acid sequence comparison suggests that the structure of Escherichia coli methionine aminopeptidase (EC 3.4.11.18) and the C-terminal domain of Pseudomonas putida creatinase (EC 3.5.3.3) are related. A detailed comparison of the three-dimensional folds of the two enzymes confirms this homology: with an approximately 260-residue chain segment, 218 C alpha atoms of the structures superimpose within 2.5 A; only 41 of these overlapping positions (i.e., 19%) feature identical amino acids in the two protein chains. Notwithstanding this striking correspondence in structure, methionine aminopeptidase binds and is stimulated by Co2+, while creatinase is not a metal-dependent enzyme. Searches of protein data banks using sequence and structure-based profiles reveal other enzymes, including aminopeptidase P (EC 3.4.11.9), prolidase (EC 3.4.13.9), and agropine synthase, that likely share the same "pita-bread" fold common to creatinase and methionine aminopeptidase.
Abstract: Cytokines and growth factors are soluble proteins that regulate the development and activities of many cell types. One group of these proteins have structures based on a four-helix bundle, though this similarity is not apparent from amino acid sequence comparisons. An understanding of how diverse sequences can adopt the same fold would be useful for recognizing and aligning distant homologs and for applying structural information gained from one protein to other sequences.
Abstract: The FLT3/FLK2 receptor tyrosine kinase is closely related to two receptors, c-Kit and c-Fms, which function with their respective ligands, Kit ligand and macrophage colony-stimulating factor to control differentiation of haematopoietic and non-haematopoietic cells. FLT3/FLK2 is thought to be present on haematopoietic stem cells and found in brain, placenta and testis. We have purified to homogeneity and partially sequenced a soluble form of the FLT3/FLK2 ligand produced by mouse thymic stromal cells. We isolated several mouse and human complementary DNAs that encode polypeptides with identical N termini and different C termini. Some variants contain hydrophobic transmembrane segments, suggesting that processing may be required to release soluble ligand. The purified ligand enhances the response of mouse stem cells and a primitive human progenitor cell population to other growth factors such as interleukins IL-3 and IL-6 and to granulocyte-macrophage colony-stimulating factor, and also stimulates fetal thymocytes.
Abstract: In this study, the cytokine-producing profile of progenitor T cells (pro-T cells) was determined. During screening of a complementary DNA library generated from activated mouse pro-T cells, a cytokine designated lymphotactin was discovered. Lymphotactin is similar to members of both the Cys-Cys and Cys-X-Cys chemokine families but lacks two of the four cysteine residues that are characteristic of the chemokines. Lymphotactin is also expressed in activated CD8+ T cells and CD4-CD8- T cell receptor alpha beta + thymocytes. It has chemotactic activity for lymphocytes but not for monocytes or neutrophils. The gene encoding lymphotactin maps to chromosome one. Taken together, these observations suggest that lymphotactin represents a novel addition to the chemokine superfamily.
Abstract: CD40 is an integral membrane protein found on the surface of B lymphocytes, dendritic cells, follicular dendritic cells, hematopoietic progenitor cells, epithelial cells, and carcinomas. It is a 45-50 kDa glycoprotein of 277 aa, which is a member of the tumor necrosis factor receptor superfamily. The CD40 gene maps to human chromosome 20q11-2-q13-2. CD40 binds to a ligand (CD40-L) which is an approximately 35 kDa glycoprotein of 261 aa, a member of the tumor necrosis factor superfamily. The CD40-L gene maps to human chromosome Xq24. This CD40-L is expressed on activated T cells, mostly CD4+ but also some CD8+ as well as basophils/mast cells. The CD40-L is defective in the X-linked hyper-IgM syndrome. Cross-linking of CD40 with immobilized anti-CD40 or cells expressing CD40-L induces B cells to proliferate strongly, and addition of IL-4 or IL-13 allows the generation of factor-dependent long-term normal human B cell lines and the secretion of IgE following isotype switching. Addition of IL-10 results in very high immunoglobulin production with limited cell proliferation. IL-10 induces naive B cells to produce IgG3, IgG1, and IgA1, and further addition of TGF beta permits the secretion of IgA2. Several evidences suggest that CD40-dependent activation of B cells is important for the generation of memory B cells within the germinal centers: (i) CD40 activated germinal center B cells cultured in the presence of IL-4 acquire a memory B cell phenotype, (ii) CD40 activated B cells can undergo isotype switching, (iii) the deficit of CD40-L results in the hyper-IgM syndrome characterized by lack of germinal centers in secondary lymphoid organ follicles and lack of IgG, IgA, and IgE, and (iv) CD40-L positive T cells are present in secondary follicles. Thymic epithelial cells, activated monocytes, and dendritic cells express CD40 antigen which may be involved in an enhanced cytokine production by these cells, allowing an amplification of T cell proliferation. Finally, as other members of the tumor necrosis factor receptor family have been shown to bind several ligands, it is possible that CD40 may bind other ligands that may trigger CD40 on different cell types such as hematopoietic cells or epithelial cells.
Abstract: We isolated cDNAs encoding a mouse interleukin 10 receptor (mIL-10R) from mouse mast cell and macrophage cell lines. The two cDNAs are substantially identical and express an approximately 110-kDa polypeptide in COS7 cells, which binds mIL-10 specifically. A mouse pro-B-cell line (Ba/F3) expressing transfected recombinant mIL-10R binds IL-10 with high affinity (approximately 70 pM) and proliferates in response to mIL-10. mIL-10R is structurally related to interferon receptors (IFNRs). Since IL-10 inhibits macrophage activation by IFN-gamma, a possible implication of this relationship interaction of IL-10R and IFN-gamma R or their signaling pathways.
Abstract: PH-30, a sperm surface protein involved in sperm-egg fusion, is composed of two subunits, alpha and beta, which are synthesized as precursors and processed, during sperm development, to yield the mature forms. The mature PH-30 alpha/beta complex resembles certain viral fusion proteins in membrane topology and predicted binding and fusion functions. Furthermore, the mature subunits are similar in sequence to each other and to a family of disintegrin domain-containing snake venom proteins. We report here the sequences of the PH-30 alpha and beta precursor regions. Their domain organizations are similar to each other and to precursors of snake venom metalloproteases and disintegrins. The alpha precursor region contains, from amino to carboxyl terminus, pro, metalloprotease, and disintegrin domains. The beta precursor region contains pro and metalloprotease domains. Residues diagnostic of a catalytically active metalloprotease are present in the alpha, but not the beta, precursor region. We propose that the active sites of the PH-30 alpha and snake venom metalloproteases are structurally similar to that of astacin. PH-30, acting through its metalloprotease and/or disintegrin domains, could be involved in sperm development as well as sperm-egg binding and fusion. Phylogenetic analysis indicates that PH-30 stems from a multidomain ancestral protein.
Abstract: CD38 is a 42-kilodalton glycoprotein expressed extensively on B and T lymphocytes. CD38 exhibits a structural homology to Aplysia adenosine diphosphate (ADP)-ribosyl cyclase. This enzyme catalyzes the synthesis of cyclic ADP-ribose (cADPR), a metabolite of nicotinamide adenine dinucleotide (NAD+) with calcium-mobilizing activity. A complementary DNA encoding the extracellular domain of murine CD38 was constructed and expressed, and the resultant recombinant soluble CD38 was purified to homogeneity. Soluble CD38 catalyzed the formation and hydrolysis of cADPR when added to NAD+. Purified cADPR augmented the proliferative response of activated murine B cells, potentially implicating the enzymatic activity of CD38 in lymphocyte function.
Abstract: The cytoplasmic tyrosine kinase, Bruton's tyrosine kinase (Btk, formerly bpk or atk), is crucial for B cell development. Loss of kinase activity results in the human immunodeficiency, X-linked agammaglobulinemia, characterized by a failure to produce B cells. In the murine X-linked immunodeficiency (XID), B cells are present but respond abnormally to activating signals. The Btk gene, btk, was mapped to the xid region of the mouse X chromosome by interspecific backcross analysis. A single conserved residue within the amino terminal unique region of Btk was mutated in XID mice. This change in xid probably interferes with normal B cell signaling mediated by Btk protein interactions.
Abstract: What is the current state of the art in protein design? This question was approached in a recent two-week protein design workshop sponsored by EMBO and held at the EMBL in Heidelberg. The goals were to test available design tools and to explore new design strategies. Five novel proteins were designed: Shpilka, a sandwich of two four-stranded beta-sheets, a scaffold on which to explore variations in loop topology; Grendel, a four-helical membrane anchor, ready for fusion to water-soluble functional domains; Finger-clasp, a dimer of interdigitating beta-beta-alpha units, the simplest variant of the "handshake" structural class; Aida, an antibody binding surface intended to be specific for flavodoxin; Leather--a minimal NAD binding domain, extracted from a larger protein. Each design is available as a set of three-dimensional coordinates, the corresponding amino acid sequence and a set of analytical results. The designs are placed in the public domain for scrutiny, improvement, and possible experimental verification.
Abstract: Among the molecules that determine the developmental fate of sympathetic neurons from noradrenergic to cholinergic function are two apparently unrelated proteins, cholinergic differentiation factor and ciliary neurotrophic factor (CDF and CNTF, respectively). The present work suggests a structural basis for their functional overlap: sequence pattern-matching and predictive structure analysis contends that CDF and CNTF are homologous and share a common helical framework. An integrated CDF/CNTF profile also reveals similar sequence/structure motifs in a group of hematopoietic cytokines composed of granulocyte colony-stimulating factor, interleukin-6, and a novel factor called oncostatin M; a more distant relationship is indicated with interleukin-3 and interferons-alpha/beta. Evolutionary ties between neuropoietic and hematopoietic cytokines predict a distinctive tertiary architecture for the uncharacterized CDF and CNTF receptors. The intertwined cytokine/receptor networks signal a closer relationship between the molecular mechanisms underlying neuro- and hematopoiesis.
Abstract: cdc25 controls the activity of the cyclin-p34cdc2 complex by regulating the state of tyrosine phosphorylation of p34cdc2. Drosophila cdc25 protein from two different expression systems activates inactive cyclin-p34cdc2 and induces M phase in Xenopus oocytes and egg extracts. We find that the cdc25 sequence shows weak but significant homology to a phylogenetically diverse group of protein tyrosine phosphatases. cdc25 itself is a very specific protein tyrosine phosphatase. Bacterially expressed cdc25 directly dephosphorylates bacterially expressed p34cdc2 on Tyr-15 in a minimal system devoid of eukaryotic cell components, but does not dephosphorylate other tyrosine-phosphorylated proteins at appreciable rates. In addition, mutations in the putative catalytic site abolish the in vivo activity of cdc25 and its phosphatase activity in vitro. Therefore, cdc25 is a specific protein phosphatase that dephosphorylates tyrosine and possibly threonine residues on p34cdc2 and regulates MPF activation.
Abstract: A bewitching interplay of proteins, variously clothed as chemical messengers and cellular receptors, control the pace of growth and the course of progressive differentiation in blood cell types. The messengers are lymphokines, interleukins, colony-stimulating factors, growth hormones and interferons: generically, the cytokines. The second components of the regulatory pairs are membrane-spanning receptor proteins: these molecules transduce the specific binding of cognate cytokines into a mitogenic cellular response. In this article, Fernando Bazan discusses a provocative structural model for cytokine-receptor interactions which, if correct, will alter perceptions of the evolutionary design of the haemopoietic system.
Abstract: Sequence homology and molecular modeling studies have suggested that the N-terminal one-third of the flavirvirus nonstructural protein NS3 functions as a trypsin-like serine protease. To examine the putative proteolytic activity of NS3, segments of the yellow fever virus genome were subcloned into plasmid transcription/translation vectors and cell-free translation products were characterized. The results suggest that a protease activity encoded within NS2B and the N-terminal one-third of yellow fever virus NS3 is capable of cis-acting site-specific proteolysis at the NS2B-NS3 cleavage site and dilution-insensitive cleavage of the NS2A-NS2B site. Site-directed mutagenesis of the His-53, Asp-77, and Ser-138 residues of NS3 that compose the proposed catalytic triad implicates this domain as a serine protease. Infectious virus was not recovered from mammalian cells transfected with RNAs transcribed from full-length yellow fever virus cDNA templates containing mutations at Ser-138 (which abolish or dramatically reduce protease activity in vitro), suggesting that the protease is required for viral replication.
Abstract: A family of cytokine receptors comprising molecules specific for a diverse group of hematopoietic factors and growth hormones has been principally defined by a striking homology of binding domains. This work proposes that the approximately 200-residue binding segment of the canonical cytokine receptor is composed of two discrete folding domains that share a significant sequence and structural resemblance. Analogous motifs are found in tandem approximately 100-amino acid domains in the extracellular segments of a receptor family formed by the interferon-alpha/beta and -gamma receptors and tissue factor, a membrane tether for a coagulation protease. Domains from the receptor supergroup reveal clear evolutionary links to fibronectin type III structures, approximately 90-amino acid modules that are typically found in cell surface molecules with adhesive functions. Predictive structural analysis of the shared receptor and fibronectin domains locates seven beta-strands in conserved regions of the chain; these strands are modeled to fold into antiparallel beta-sandwiches with a topology that is similar to immunoglobulin constant domains. These findings have strong implications for understanding the evolutionary emergence of an important class of regulatory molecules from primitive adhesive modules. In addition, the resulting double-barrel design of the receptors and the spatial clustering of conserved residues suggest a likely binding site for cytokine ligands.
Abstract: The bifunctional rat liver enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (ATP:D-fructose-6-phosphate 2-phosphotransferase/D-fructose-2,6-bisphosphate 2-phosphohydrolase, EC 2.7.1.105/EC 3.1.3.46) is constructed of two independent catalytic domains. We present evidence that the kinase and bisphosphatase halves of the bifunctional enzyme are, respectively, structurally similar to the glycolytic enzymes 6-phosphofructo-1-kinase and phosphoglycerate mutase. Computer-assisted modeling of the C-terminal bisphosphatase domain reveals a hydrophobic core and active site residue constellation equivalent to the yeast mutase structure; structural differences map to length-variable, surface-located loops. Sequence patterns derived from the structural alignment of mutases and the bisphosphatase further detect a significant similarity to a family of acid phosphatases. The N-terminal kinase domain, in turn, is predicted to form a nucleotide-binding fold that is analogous to a segment of 6-phosphofructo-1-kinase, suggesting that these unrelated enzymes bind fructose 6-phosphate and ATP substrates in a similar geometry. This analysis indicates that the bifunctional enzyme is the likely product of gene fusion of kinase and mutase/phosphatase catalytic units.
Abstract: We propose through a sequence and structural-pattern analysis that a protein domain of undefined function encoded by the enveloped RNA flavi- and pestivruses is a Ser active-center enzyme related to the cellular trypsin family. A further homology is emphasized with the group of (Cys active-center) viral proteases encoded by nonenveloped RNA viruses of the picorna-, como-, nepo-, and potyvirus classes. Structural modeling of the putative flaviviral protease domain suggests amino acids that are crucial for catalytic activity and substrate binding.
Abstract: Lymphokine and hematopoietic growth factors control the differentiation and proliferation of diverse cell types by binding to specific cell-surface receptors. Strikingly, the recently elucidated sequences of the interleukin-6 and erythropoietin receptors, and the interleukin-2 receptor beta-chain (p75), display a significant evolutionary resemblance of their extracellular domains. This homology extends to the binding domains of the growth hormone/prolactin class of receptors. Alternatively, little similarity exists between the cytoplasmic extensions of these diverse receptors. I discuss the evolutionary and functional implications of this broad, mosaic receptor relationship, with particular reference to possible structural resemblances between the cognate growth factors.
Abstract: The 49-kDa proteinase of tobacco etch virus (TEV) cleaves the polyprotein derived from the TEV genomic RNA at five locations. Molecular genetic and biochemical analyses of the 49-kDa TEV proteinase were performed to test its homology to the cellular trypsin-like serine proteases. A cDNA fragment, containing the TEV 49-kDa proteinase gene and flanking sequences, was expressed in a cell-free transcription/translation system and resulted in the formation of a polyprotein precursor that underwent rapid self-processing. Site-directed mutagenesis was used to test the effect of altering individual 49-kDa amino acid residues on proteolysis. The data suggest that the catalytic triad of the TEV 49-kDa proteinase could be composed of the His234, Asp269, and Cys339. These findings are consistent with the hypothesis that the TEV 49-kDa proteinase is structurally similar to the trypsin-like family of serine proteinases with the substitution of Cys339 as the active site nucleophile. A structural model of the TEV 49-kDa proteinase proposes other virus-specific differences in the vicinity of the active site triad and substrate-binding pocket. The structure may explain the observed negligible effect of most cellular proteinase inhibitors on the activity of this viral proteinase.
Abstract: Proteases that are encoded by animal picornaviruses and plant como- and potyviruses form a related group of cysteine-active-center enzymes that are essential for virus maturation. We show that these proteins are homologous to the family of trypsin-like serine proteases. In our model, the active-site nucleophile of the trypsin catalytic triad, Ser-195, is changed to a Cys residue in these viral proteases. The other two residues of the triad, His-57 and Asp-102, are otherwise absolutely conserved in all the viral protease sequences. Secondary structure analysis of aligned sequences suggests the location of the component strands of the twin beta-barrel trypsin fold in the viral proteases. Unexpectedly, the 2a and 3c subclasses of viral cysteine proteases are, respectively, homologous to the small and large structural subclasses of trypsin-like serine proteases. This classification allows the molecular mapping of residues from viral sequences onto related tertiary structures; we precisely identify amino acids that are strong determinants of specificity for both small and large viral cysteine proteases.
Abstract: The integral membrane sialoglycoprotein PrPSc is the only identifiable component of the scrapie prion. Scrapie in animals and Creutzfeldt-Jakob disease in humans are transmissible, degenerative neurological diseases caused by prions. Standard predictive strategies have been used to analyze the secondary structure of the prion protein in conjunction with Fourier analysis of the primary sequence hydrophobicities to detect potential amphipathic regions. Several hydrophobic segments, a proline- and glycine-rich repeat region and putative glycosylation sites are incorporated into a model for the integral membrane topology of PrP. The complete amino acid sequences of the hamster, human and mouse prion proteins are compared and the effects of residue substitutions upon the predicted conformation of the polypeptide chain are discussed. While PrP has a unique primary structure, its predicted secondary structure shares some interesting features with the serum amyloid A proteins. These proteins undergo a post-translational modification to yield amyloid A, molecules that share with PrP the ability to polymerize into birefringent filaments. Our analyses may explain some experimental observations on PrP, and suggest further studies on the properties of the scrapie and cellular PrP isoforms.
Abstract: Sterol carrier protein 2 (SCP2) is involved in the later steps of cholesterol biosynthesis and in the intracellular transport of cholesterol. In the present investigation, the amino acid sequence of SCP2 from rat liver has been determined. It is a single polypeptide chain with 122 amino acid residues. Secondary structure prediction indicates an amphipathic alpha-helix region for residues 21-34 and antiparallel beta-sheet structure for residues 35-95. A major finding is the significant homology which exists over approximately 80 residues between SCP2 and the variable domains of the heavy chain of immunoglobulin G.
