Distinct genes encode 6 human receptors for IgG (hFc␥Rs), 3 of which have 2 or 3 polymorphic variants. The specificity and affinity of individual hFc␥Rs for the 4 human IgG subclasses is unknown. This information is critical for antibodybased immunotherapy which has been increasingly used in the clinics. We investigated the binding of polyclonal and monoclonal IgG1, IgG2, IgG3, and IgG4 to Fc␥RI; Fc␥RIIA, IIB, and IIC; Fc␥RIIIA and IIIB; and all known polymorphic variants. Wild-type and low-fucosylated IgG1 anti-CD20 and anti-RhD mAbs were also examined. We found that (1) IgG1 and IgG3 bind to all hFc␥Rs; (2) IgG2 bind not only to Fc␥RIIA H131 , but also, with a lower affinity, to Fc␥RIIA R131 4 Other FcRs are inserted in the outer layer of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor and contain no signaling motif. 5 FcRs have been associated with many antibodydependent diseases 6 and are key molecules in antibody-based immunotherapy. These include the treatment, for instance, of non-Hodgkin lymphomas by mouse/human chimeric IgG1 anti-CD20 antibodies 7 and the prevention of hemolytic disease of the newborn by a mixture of polyclonal IgG1 and IgG3 anti-RhD antibodies (eg, Rophylac). Therapeutic antibodies are, however, potentially harmful, as exemplified by a recent clinical trial using IgG4 anti-CD28 antibodies.Four human subclasses of IgG are produced in different amounts in response to various antigens. T-dependent protein antigens elicit primarily IgG1 and IgG3 antibodies, whereas T-independent carbohydrate antigens elicit primarily IgG2 antibodies. Chronic antigen stimulation, as in allergic desensitization, elicits IgG4 antibodies. The biological activities of each subclass of IgG are poorly known. IgG receptors (Fc␥Rs) are strikingly numerous in humans. They comprise high-affinity and low-affinity receptors. 8 Both high-affinity and low-affinity Fc␥Rs bind IgGimmune complexes with a high avidity, but only high-affinity Fc␥Rs bind monomeric IgG. There is one high-affinity IgG receptor in humans, hFc␥RI (CD64), and 2 families of low-affinity IgG receptors, hFc␥RIIA, IIB, and IIC (CD32), and hFc␥RIIIA and IIIB (CD16). hFc␥RI and hFc␥RIIIA are FcR␥-associated activating receptors, hFc␥RIIA and hFc␥RIIC are single-chain activating receptors, hFc␥RIIB are single-chain inhibitory receptors, and hFc␥RIIIB are GPI-anchored receptors whose function is uncertain. 1 The multiplicity of hFc␥Rs is further increased by a series of polymorphisms in their extracellular domains (reviewed in van Sorge et al 9 ). Two alleles of the gene encoding hFc␥RIIA generate 2 variants differing at position 131, named low-responder (H 131 ) and high-responder (R 131 ). 10 The H 131 and R 131 alleles are differentially distributed in whites, Japanese, and Chinese. 11 Two alleles of the gene-encoding hFc␥RIIIA generate 2 variants differing at 23 and hFc␥RIIIB NA2 to SLE in Japanese people. 24 The subclass specificity of hFc␥Rs has been investigated since the 1980s, that is, at a time when the complexity of hFc␥R...

Zika virus (ZIKV) is an arthropod-borne enveloped virus belonging to the Flavivirus genus in the family Flaviviridae, which also includes the human pathogenic yellow fever, dengue, West Nile and tick-borne encephalitis viruses 1 . Flaviviruses have two structural glycoproteins, prM and E (for precursor membrane and envelope proteins, respectively), which form a heterodimer in the endoplasmic reticulum (ER) of the infected cell and drive the budding of spiky immature virions into the ER lumen. These particles transit through the cellular secretory pathway, during which the trans-Golgi-resident protease furin cleaves prM. This processing is required for infectivity, and results in the loss of a large fragment of prM and reorganization of E on the virion surface. The mature particles have a smooth aspect, with 90 E dimers organized with icosahedral symmetry in a 'herringbone' pattern 2,3 .Three-dimensional cryo-electron microscopy (cryo-EM) structures of the mature ZIKV particles have recently been reported to near atomic resolution (3.8 Å) 4,5 , showing that the virus has essentially the same organization as the other flaviviruses of known structure, such as dengue virus (DENV) 3 and West Nile virus 6,7 . The E protein is about 500 amino acids long, with the 400 N-terminal residues forming the ectodomain essentially folded as β-sheets with three domains, named I, II and III, aligned in a row with domain I at the centre. The conserved fusion loop is at the distal end of the rod in domain II, buried at the E dimer interface. At the C terminus, the E ectodomain is followed by the 'stem' , featuring two α-helices lying flat on the viral membrane (the stem helices), which link to two C-terminal transmembrane α-helices. The main distinguishing feature of the ZIKV virion is an insertion within a glycosylated loop of E (the '150' loop), which protrudes from the mature virion surface 4,5 .Flaviviruses have been grouped into serocomplexes based on cross-neutralization studies with polyclonal immune sera 8 . The E protein is the main target of neutralizing antibodies, and is also the viral fusogen; cleavage of prM allows E to respond to the endosomal pH by undergoing a large-scale conformational change that catalyses membrane fusion and releases the viral genome into the cyotosol. Loss of the precursor fragment of prM lets the E protein fluctuate from its tight packing at the surface of the virion, transiently exposing otherwise buried surfaces. One surface exposed by this 'breathing' is the fusionloop epitope (FLE), which is a dominant cross-reactive antigenic site 9 . Although antibodies to this site can protect by complement-mediated mechanisms, as shown in a mouse model for West Nile virus 10 , they are poorly neutralizing and lead to antibody-dependent enhancement 11-15 , thereby aggravating Flavivirus pathogenesis and complicating the development of safe and effective vaccines.We recently reported the functional and structural characterization of a panel of antibodies isolated from patients with dengue disease 13,16 . ...

Post-translational modification with ubiquitin is one of the most important mechanisms in the regulation of protein stability and function. However, the high reversibility of this modification is the main obstacle for the isolation and characterization of ubiquitylated proteins. To overcome this problem, we have developed tandem-repeated ubiquitin-binding entities (TUBEs) based on ubiquitin-associated (UBA) domains. TUBEs recognize tetra-ubiquitin with a markedly higher affinity than single UBA domains, allowing poly-ubiquitylated proteins to be efficiently purified from cell extracts in native conditions. More significant is the fact that TUBEs protect poly-ubiquitin-conjugated proteins, such as p53 and IjBa, both from proteasomal degradation and de-ubiquitylating activity present in cell extracts, as well as from existing proteasome and cysteine protease inhibitors. Therefore, these new 'molecular traps' should become valuable tools for purifying endogenous poly-ubiquitylated proteins, thus contributing to a better characterization of many essential functions regulated by these post-translational modifications.

The 6-kDa early secreted antigenic target ESAT-6 and the 10-kDa culture filtrate protein CFP-10 of Mycobacterium tuberculosis are secreted by the ESX-1 system into the host cell and thereby contribute to pathogenicity. Although different studies performed at the organismal and cellular levels have helped to explain ESX-1-associated phenomena, not much is known about how ESAT-6 and CFP-10 contribute to pathogenesis at the molecular level. In this study we describe the interaction of both proteins with lipid bilayers, using biologically relevant liposomal preparations containing dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol, and cholesterol. Using floatation gradient centrifugation, we demonstrate that ESAT-6 showed strong association with liposomes, and in particular with preparations containing DMPC and cholesterol, whereas the interaction of CFP-10 with membranes appeared to be weaker and less specific. Most importantly, binding to the biomembranes no longer occurred when the proteins were present as a 1:1 ESAT-6 ⅐ CFP-10 complex. However, lowering of the pH resulted in dissociation of the protein complex and subsequent protein-liposome interaction. Finally, cryoelectron microscopy revealed that ESAT-6 destabilized and lysed liposomes, whereas CFP-10 did not. In conclusion, we propose that one of the main features of ESAT-6 in the infection process of M. tuberculosis is the interaction with biomembranes that occurs after dissociation from its putative chaperone CFP-10 under acidic conditions typically encountered in the phagosome.Mycobacterium tuberculosis is one of the most successful human pathogens, infecting nearly one-third of the world's population. Among the various factors that contribute, it is certainly the bacterium's ability to multiply and persist within professional phagocytic cells that is of primary importance (30).The extended RD1 region of M. tuberculosis encodes ESX-1, a novel protein secretion system involved in the immunogenicity and pathogenicity. The system, which is absent from the attenuated vaccines Mycobacterium bovis BCG and Mycobacterium microti (4, 6, 24), is responsible for the export of the 6-kDa early secreted antigenic target ESAT-6 and the 10-kDa culture filtrate protein CFP-10. ESX-1 is present in the saprophyte Mycobacterium smegmatis (9), where it acts in DNA uptake (11), which suggests that pathogenic mycobacteria might have adapted an ancestral conjugation system for protein secretion, which is required for survival and multiplication in the host cell.The importance of ESX-1 proteins for pathogenicity was shown by reintroduction of the extended RD1 region into BCG (34), deletion of RD1 from M. tuberculosis (23), signaturetagged and insertional mutagenesis (8,40,46), and targeted gene deletions (5,15,18). Several effects related to pathogenicity have been found to be associated with the expression of ESX-1 in M. tuberculosis and/or Mycobacterium marinum, a fish pathogen that harbors an ESX-1 system similar to that of M. tuberculosis (14,27). These ...

Hepatitis C virus (HCV), a major cause of chronic liver disease in humans, is the focus of intense research efforts worldwide. Yet structural data on the viral envelope glycoproteins E1 and E2 are scarce, in spite of their essential role in the viral life cycle. To obtain more information, we developed an efficient production system of recombinant E2 ectodomain (E2e), truncated immediately upstream its trans-membrane (TM) region, using Drosophila melanogaster cells. This system yields a majority of monomeric protein, which can be readily separated chromatographically from contaminating disulfide-linked aggregates. The isolated monomeric E2e reacts with a number of conformation-sensitive monoclonal antibodies, binds the soluble CD81 large external loop and efficiently inhibits infection of Huh7.5 cells by infectious HCV particles (HCVcc) in a dose-dependent manner, suggesting that it adopts a native conformation. These properties of E2e led us to experimentally determine the connectivity of its 9 disulfide bonds, which are strictly conserved across HCV genotypes. Furthermore, circular dichroism combined with infrared spectroscopy analyses revealed the secondary structure contents of E2e, indicating in particular about 28% β-sheet, in agreement with the consensus secondary structure predictions. The disulfide connectivity pattern, together with data on the CD81 binding site and reported E2 deletion mutants, enabled the threading of the E2e polypeptide chain onto the structural template of class II fusion proteins of related flavi- and alphaviruses. The resulting model of the tertiary organization of E2 gives key information on the antigenicity determinants of the virus, maps the receptor binding site to the interface of domains I and III, and provides insight into the nature of a putative fusogenic conformational change.

Studies of the dynamics of the antibody-mediated immune response have been hampered by the absence of quantitative, high-throughput systems to analyze individual antibody-secreting cells. Here we describe a simple microfluidic system, DropMap, in which single cells are compartmentalized in tens of thousands of 40-pL droplets and analyzed in two-dimensional droplet arrays using a fluorescence relocation-based immunoassay. Using DropMap, we characterized antibody-secreting cells in mice immunized with tetanus toxoid (TT) over a 7-week protocol, simultaneously analyzing the secretion rate and affinity of IgG from over 0.5 million individual cells enriched from spleen and bone marrow. Immunization resulted in dramatic increases in the range of both single-cell secretion rates and affinities, which spanned at maximum 3 and 4 logs, respectively. We observed differences over time in dynamics of secretion rate and affinity within and between anatomical compartments. This system will not only enable immune monitoring and optimization of immunization and vaccination protocols but also potentiate antibody screening.

Programmed necrosis induced by DNA alkylating agents, such as MNNG, is a caspase-independent mode of cell death mediated by apoptosis-inducing factor (AIF). After poly(ADP-ribose) polymerase 1, calpain, and Bax activation, AIF moves from the mitochondria to the nucleus where it induces chromatinolysis and cell death. The mechanisms underlying the nuclear action of AIF are, however, largely unknown. We show here that, through its C-terminal proline-rich binding domain (PBD, residues 543-559), AIF associates in the nucleus with histone H2AX. This interaction regulates chromatinolysis and programmed necrosis by generating an active DNA-degrading complex with cyclophilin A (CypA). Deletion or directed mutagenesis in the AIF C-terminal PBD abolishes AIF/H2AX interaction and AIF-mediated chromatinolysis. H2AX genetic ablation or CypA downregulation confers resistance to programmed necrosis. AIF fails to induce chromatinolysis in H2AX or CypA-deficient nuclei. We also establish that H2AX is phosphorylated at Ser139 after MNNG treatment and that this phosphorylation is critical for caspase-independent programmed necrosis. Overall, our data shed new light in the mechanisms regulating programmed necrosis, elucidate a key nuclear partner of AIF, and uncover an AIF apoptogenic motif.

No abstract