Over the past 15 years, investigators have shown that T lymphocytes can recognize not only peptides in the context of MHC class I and class II molecules but also foreign and self-lipids in ...association with the nonclassical MHC class I-like molecules, CD1 proteins. In this review, we describe the most recent events in the field, with particular emphasis on (a) structural and functional aspects of lipid presentation by CD1 molecules, (b) the development of CD1d-restricted invariant natural killer T (iNKT) cells and transcription factors required for their differentiation, (c) the ability of iNKT cells to modulate innate and adaptive immune responses through their cross talk with lymphoid and myeloid cells, and (d) MR1-restricted and group I (CD1a, CD1b, and CD1c)-restricted T cells.
The relationship between the presence of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the risk of subsequent reinfection remains unclear.
We investigated the ...incidence of SARS-CoV-2 infection confirmed by polymerase chain reaction (PCR) in seropositive and seronegative health care workers attending testing of asymptomatic and symptomatic staff at Oxford University Hospitals in the United Kingdom. Baseline antibody status was determined by anti-spike (primary analysis) and anti-nucleocapsid IgG assays, and staff members were followed for up to 31 weeks. We estimated the relative incidence of PCR-positive test results and new symptomatic infection according to antibody status, adjusting for age, participant-reported gender, and changes in incidence over time.
A total of 12,541 health care workers participated and had anti-spike IgG measured; 11,364 were followed up after negative antibody results and 1265 after positive results, including 88 in whom seroconversion occurred during follow-up. A total of 223 anti-spike-seronegative health care workers had a positive PCR test (1.09 per 10,000 days at risk), 100 during screening while they were asymptomatic and 123 while symptomatic, whereas 2 anti-spike-seropositive health care workers had a positive PCR test (0.13 per 10,000 days at risk), and both workers were asymptomatic when tested (adjusted incidence rate ratio, 0.11; 95% confidence interval, 0.03 to 0.44; P = 0.002). There were no symptomatic infections in workers with anti-spike antibodies. Rate ratios were similar when the anti-nucleocapsid IgG assay was used alone or in combination with the anti-spike IgG assay to determine baseline status.
The presence of anti-spike or anti-nucleocapsid IgG antibodies was associated with a substantially reduced risk of SARS-CoV-2 reinfection in the ensuing 6 months. (Funded by the U.K. Government Department of Health and Social Care and others.).
FLRTs are broadly expressed proteins with the unique property of acting as homophilic cell adhesion molecules and as heterophilic repulsive ligands of Unc5/Netrin receptors. How these functions ...direct cell behavior and the molecular mechanisms involved remain largely unclear. Here we use X-ray crystallography to reveal the distinct structural bases for FLRT-mediated cell adhesion and repulsion in neurons. We apply this knowledge to elucidate FLRT functions during cortical development. We show that FLRTs regulate both the radial migration of pyramidal neurons, as well as their tangential spread. Mechanistically, radial migration is controlled by repulsive FLRT2-Unc5D interactions, while spatial organization in the tangential axis involves adhesive FLRT-FLRT interactions. Further, we show that the fundamental mechanisms of FLRT adhesion and repulsion are conserved between neurons and vascular endothelial cells. Our results reveal FLRTs as powerful guidance factors with structurally encoded repulsive and adhesive surfaces.
•Distinct FLRT surfaces mediate homophilic adhesion and Unc5-dependent repulsion•Neurons expressing Unc5 and FLRT integrate adhesive and repulsive FLRT signals•FLRTs direct radial migration and tangential spread of cortical neurons•FLRT3 controls retinal vascularization
Cortical development depends on a balance of adhesive and repulsive interactions between cells. Seiradake et al. show how FLRT proteins fine-tune adhesion and repulsion between cells migrating through the neocortex. Vascular cells are also guided by FLRT using structurally conserved mechanisms.
Extracellular modulators of Wnt signalling Malinauskas, Tomas; Jones, E Yvonne
Current opinion in structural biology,
December 2014, 2014-Dec, 2014-12-00, 20141201, Volume:
29
Journal Article
Peer reviewed
Open access
•Structure and binding of lipid-modified Wnt8 with Frizzled8 cysteine-rich domain.•Modularity of Wnt inhibitory factor 1 combining Wnt-binding and heparan sulphate-binding.•Integration of multiple ...interaction sites in LRP5/6 ectodomain architecture.•Ternary complex assembly of RNF43/ZNRF3 and LGR4/5/6 bridged by R-spondin.•Ectodomain dimerisation of the Frizzled-specific E3 ligase ZNRF3.
Wnt morphogens are secreted signalling proteins that play leading roles in embryogenesis and tissue homeostasis throughout life. Wnt signalling is controlled by multiple mechanisms, including posttranslational modification of Wnts, antagonist binding (to Wnts or their receptors), and regulation of the availability of Wnt receptors. Recent crystallographic, structure-guided biophysical and cell-based studies have advanced our understanding of how Wnt signalling is regulated at the cell surface. Structures include Wnt in complex with the cysteine-rich domain (CRD) of Frizzled, extracellular fragments of Wnt co-receptor LRP6, LRP6-binding antagonists Dickkopf and Sclerostin, antagonists 5T4/WAIF1 and Wnt inhibitory factor 1 (WIF-1), as well as Frizzled–ubiquitin ligases ZNRF3/RNF43 (in isolation and in complexes with Wnt signalling promoters R-spondins and LGR5). We review recent discoveries and remaining questions.
Structural Perspectives on Axon Guidance Seiradake, Elena; Jones, E. Yvonne; Klein, Rüdiger
Annual review of cell and developmental biology,
10/2016, Volume:
32, Issue:
1
Journal Article
Peer reviewed
Open access
Axon guidance relies on a combinatorial code of receptor and ligand interactions that direct adhesive attractive and repulsive cellular responses. Recent structural data have revealed many of the ...molecular mechanisms that govern these interactions and enabled the design of sophisticated mutant tools to dissect their biological functions. Here, we discuss the structure function relationships of four major classes of guidance cues (ephrins, semaphorins, slits, netrins) and examples of morphogens (Wnt, Shh) and of cell adhesion molecules (FLRT). These cell signaling systems rely on specific modes of receptor-ligand binding that are determined by selective binding sites; however, defined structure-encoded receptor promiscuity also enables cross talk between different receptor ligand families and can also involve extracellular matrix components. A picture emerges in which a multitude of highly context-dependent structural assemblies determines the finely tuned cellular behavior required for nervous system development.
Most proteins for structural biology studies are produced by high‐level expression in Escherichia coli. However, prokaryotic based expression systems fail to generate correctly folded functional ...forms of many proteins and hence a variety of eukaryotic based expression systems have been developed. Of these, yeast and baculovirus‐infected insect cells currently represent the expression systems of choice for structural biologists. Here, protocols for a simple, fast and affordable method for transient protein expression in mammalian cells are reported. The results demonstrate that it combines several features necessary for the production of suitable samples for structural biology, in particular protein crystallography, namely high protein yield, straightforward purification, selenomethionine incorporation and control of N‐linked glycosylation. The system is suitable for use in conventional laboratories or can be implemented in a medium‐ or high‐throughput pipeline.
Coxsackievirus A10 (CV-A10) is responsible for an escalating number of severe infections in children, but no prophylactics or therapeutics are currently available. KREMEN1 (KRM1) is the entry ...receptor for the largest receptor-group of hand-foot-and-mouth disease causing viruses, which includes CV-A10. We report here structures of CV-A10 mature virus alone and in complex with KRM1 as well as of the CV-A10 A-particle. The receptor spans the viral canyon with a large footprint on the virus surface. The footprint has some overlap with that seen for the neonatal Fc receptor complexed with enterovirus E6 but is larger and distinct from that of another enterovirus receptor SCARB2. Reduced occupancy of a particle-stabilising pocket factor in the complexed virus and the presence of both unbound and expanded virus particles suggests receptor binding initiates a cascade of conformational changes that produces expanded particles primed for viral uncoating.
Structural, biochemical and biophysical studies of eukaryotic soluble and membrane proteins require their production in milligram quantities. Although large-scale protein expression strategies based ...on transient or stable transfection of mammalian cells are well established, they are associated with high consumable costs, limited transfection efficiency or long and tedious selection of clonal cell lines. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines. In this protocol, we describe the design and step-by-step application of a lentiviral plasmid suite, termed pHR-CMV-TetO
, for the constitutive or inducible large-scale production of soluble and membrane proteins in HEK293 cell lines. Optional features include bicistronic co-expression of fluorescent marker proteins for enrichment of co-transduced cells using cell sorting and of biotin ligase for in vivo biotinylation. We demonstrate the efficacy of the method for a set of soluble proteins and for the G-protein-coupled receptor (GPCR) Smoothened (SMO). We further compare this method with baculovirus transduction of mammalian cells (BacMam), using the type-A γ-aminobutyric acid receptor (GABA
R) β3 homopentamer as a test case. The protocols described here are optimized for simplicity, speed and affordability; lead to a stable polyclonal cell line and milligram-scale amounts of protein in 3-4 weeks; and routinely achieve an approximately three- to tenfold improvement in protein production yield per cell as compared to transient transduction or transfection.
Secreted Wnt morphogens are essential for embryogenesis and homeostasis and require a lipid/palmitoleoylate modification for receptor binding and activity. Notum is a secreted Wnt antagonist that ...belongs to the α/β hydrolase superfamily, but its mechanism of action and roles in vertebrate embryogenesis are not fully understood. Here, we report that Notum hydrolyzes the Wnt palmitoleoylate adduct extracellularly, resulting in inactivated Wnt proteins that form oxidized oligomers incapable of receptor binding. Thus, Notum is a Wnt deacylase, and palmitoleoylation is obligatory for the Wnt structure that maintains its active monomeric conformation. Notum is expressed in naive ectoderm and neural plate in Xenopus and is required for neural and head induction. These findings suggest that Notum is a prerequisite for the “default” neural fate and that distinct mechanisms of Wnt inactivation by the Tiki protease in the Organizer and the Notum deacylase in presumptive neuroectoderm orchestrate vertebrate brain development.
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•Notum is an extracellular Wnt deacylase•Acylation maintains Wnt folding and its active monomeric conformation•Notum in neuroectoderm is required for vertebrate neural and head induction•Distinct Wnt inactivation mechanisms by Notum and Tiki orchestrate brain development
Wnt proteins require a lipid modification for receptor binding and activity. Zhang and Cheong et al. report that Notum, a secreted Wnt antagonist, is a Wnt deacylase that inactivates Wnt proteins and is required for vertebrate brain development. Deacylated Wnt forms oxidized-oligomers, suggesting that acylation is essential for Wnt structure.
A computational strategy has delivered a redesigned, more stable version of a cytokine protein that mimics the natural protein's interactions with receptors, opening the way for designer ...cytokine-based therapeutics.
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