p47 phox is a key cytosolic subunit required for activation of phagocyte NADPH oxidase. The X-ray structure of the p47 phox PX domain revealed two distinct basic pockets on the membrane-binding surface, each occupied by a sulfate. These two pockets have different speci®cities: one preferentially binds phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P 2 ] and is analogous to the phophatidylinositol 3-phosphate (PtdIns3P)-binding pocket of p40 phox , while the other binds anionic phospholipids such as phosphatidic acid (PtdOH) or phosphatidylserine. The preference of this second site for PtdOH may be related to previously observed activation of NADPH oxidase by PtdOH. Simultaneous occupancy of the two phospholipidbinding pockets radically increases membrane af®n-ity. Strikingly, measurements for full-length p47 phox show that membrane interaction by the PX domain is masked by an intramolecular association with the C-terminal SH3 domain (C-SH3). Either a site-speci®c mutation in C-SH3 (W263R) or a mimic of the phosphorylated form of p47 phox [Ser(303, 304, 328, 359, 370)Glu] cause a transition from a closed to an open conformation that binds membranes with a greater af®nity than the isolated PX domain.

More than 50 human proteins with a wide range of functions have a 120 residue phosphoinositide binding module known as the PX domain. The 1.7 A X-ray crystal structure of the PX domain from the p40(phox) subunit of NADPH oxidase bound to PtdIns(3)P shows that the PX domain embraces the 3-phosphate on one side of a water-filled, positively charged pocket and reveals how 3-phosphoinositide specificity is achieved. A chronic granulomatous disease (CGD)-associated mutation in the p47(phox) PX domain that abrogates PtdIns(3)P binding maps to a conserved Arg that does not directly interact with the phosphoinositide but instead appears to stabilize a critical lipid binding loop. The SH3 domain present in the full-length protein does not affect soluble PtdIns(3)P binding to the p40(phox) PX domain.

Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low. Therefore, the design of artificial endonucleases with chosen specificities is under intense investigation. In this report, we describe the first artificial HEs whose specificity has been entirely redesigned to cleave a naturally occurring sequence. First, hundreds of novel endonucleases with locally altered substrate specificity were derived from I-CreI, a Chlamydomonas reinhardti protein belonging to the LAGLIDADG family of HEs. Second, distinct DNA-binding subdomains were identified within the protein. Third, we used these findings to assemble four sets of mutations into heterodimeric endonucleases cleaving a model target or a sequence from the human RAG1 gene. These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities.

Tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3) functions downstream of multiple receptors that induce interferon-α (IFN-α), IFN–β and IFN-λ production, including Toll-like receptor 3 (TLR3), which is deficient in some patients with herpes simplex virus-1 encephalitis (HSE). Mice lacking TRAF3 die in the neonatal period, preventing direct investigation of the role of TRAF3 in immune responses and host defenses in vivo. Here we reported the autosomal dominant, human TRAF3 deficiency in a young adult with a history of HSE in childhood. The TRAF3 mutant allele was a loss-of-expression, loss-of-function, dominant-negative phenotype, and was associated with impaired, but not abolished TRAF3-dependent responses upon stimulation of both TNF receptors and receptors that induce IFN production. TRAF3 deficiency was associated with a clinical phenotype limited to HSE resulting from the impairment of TLR3-dependent induction of IFN. Thus, TLR3-mediated immunity against primary infection by HSV-1 in the central nervous system is critically dependent on TRAF3. Highlight sentence Autosomal dominant TRAF3 deficiency is a genetic etiology of herpes simplex encephalitis. Highlight sentence R118W TRAF3 allele is loss-of-function, loss-of-expression, and dominant-negative. Highlight sentence Human TRAF3 deficiency impairs the TLR3-dependent induction of anti-viral interferons.

No abstract

Cytokines of the gp130 family exert their diverse biological effects by formation of stable high af®nity transmembrane receptor complexes that are characterized by the presence of the shared transmembrane signalling receptor gp130. Different gp130 ligands form signalling complexes that vary in both composition and stoichiometry. Analysis of the threedimensional structure of selected ligands and receptor elements indicates that ligands display three topologically conserved receptor recognition epitopes that interact with complementary ligand recognition elements. The composition of the signalling complex and downstream biological responses is de®ned by the relative af®nity of different receptor components for these epitopes. The detailed structure of receptor recognition epitopes indicates that the generation of small molecule cytokine mimetics may be a feasible objective.

Phospholipase C␥ (PLC␥) isoforms are regulated through activation of tyrosine kinase-linked receptors. The importance of growth factor-stimulated phosphorylation of specific tyrosine residues has been documented for PLC␥1; however, despite the critical importance of PLC␥2 in B-cell signal transduction, neither the tyrosine kinase(s) that directly phosphorylate PLC␥2 nor the sites in PLC␥2 that become phosphorylated after stimulation are known. By measuring the ability of human PLC␥2 to restore calcium responses to the B-cell receptor stimulation or oxidative stress in a B-cell line (DT40) deficient in PLC␥2, we have demonstrated that two tyrosine residues, Tyr 753 and Tyr 759 , were important for the PLC␥2 signaling function. Furthermore, the double mutation Y753F/Y759F in PLC␥2 resulted in a loss of tyrosine phosphorylation in stimulated DT40 cells. Of the two kinases that previously have been proposed to phosphorylate PLC␥2, Btk, and Syk, purified Btk had much greater ability to phosphorylate recombinant PLC␥2 in vitro, whereas Syk efficiently phosphorylated adapter protein BLNK. Using purified proteins to analyze the formation of complexes, we suggest that function of Syk is to phosphorylate BLNK, providing binding sites for PLC␥2. Further analysis of PLC␥2 tyrosine residues phosphorylated by Btk and several kinases from the Src family has suggested multiple sites of phosphorylation and, in the context of a peptide incorporating residues Tyr 753 and Tyr 759 , shown preferential phosphorylation of Tyr 753 .

Despite differences in size and chemical properties, which were suggestive of a unique catalase, the deduced structure of HPII is related to the structure of catalase from Penicillium vitale, whose sequence is not yet known. In particular, both molecules have an additional C-terminal domain that is absent in the bovine catalase. This extra domain contains a Rossmann fold but no bound nucleotides have been detected, and its physiological role is unknown. In HPII, the heme group is modified to a heme d and inverted with respect to the orientation determined in all previously reported heme catalases. HPII is the largest catalase for which the structure has been determined to almost atomic resolution.