The catalytic core of a superoxide-producing NADPH oxidase (Nox) in phagocytes is gp91 phox /Nox2, a membrane-integrated protein that forms a heterodimer with p22 phox to constitute flavocytochrome b 558 . The cytochrome becomes activated by interacting with the adaptor proteins p47 phox and p67 phox as well as the small GTPase Rac. Here we describe the cloning of human cDNAs for novel proteins homologous to p47 phox and p67 phox , designated p41 nox and p51 nox , respectively; the former is encoded by NOXO1 (Nox organizer 1), and the latter is encoded by NOXA1 (Nox activator 1). The novel homologue p41 nox interacts with p22 phox via the two tandem SH3 domains, as does p47 phox . The protein p51 nox as well as p67 phox can form a complex with p47 phox and with p41 nox via the C-terminal SH3 domain and binds to GTPbound Rac via the N-terminal domain containing four tetratricopeptide repeat motifs. These bindings seem to play important roles, since p47 phox and p67 phox activate the phagocyte oxidase via the same interactions. Indeed, p41 nox and p51 nox are capable of replacing the corresponding classical homologue in activation of gp91 phox . Nox1, a homologue of gp91 phox , also can be activated in cells, when it is coexpressed with p41 nox and p51 nox , with p41 nox and p67 phox , or with p47 phox and p51 nox ; in the former two cases, Nox1 is partially activated without any stimulants added, suggesting that p41 nox is normally in an active state. Thus, the novel homologues p41 nox and p51 nox likely function together or in combination with a classical one, thereby activating the two Nox family oxidases.

The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole in phagocytes by means of the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system and translocated effector proteins, some of which subvert host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC anchors to the membrane of Legionella-containing vacuoles (LCVs) by specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a nonbiased screen for novel L. pneumophila PI-binding proteins, we identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the predominant PtdIns(4)P-binding protein. Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding domain of SidM was mapped to the 12-kDa C-terminal sequence, termed "P4M" (PtdIns4P binding of SidM/DrrA). The isolated P4M domain is largely helical and displayed higher PtdIns(4)P binding activity in the context of the ␣-helical, monomeric full-length protein. SidM constructs containing P4M were translocated by Icm/Dot-proficient L. pneumophila and localized to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via its P4M domain. An L. pneumophila ⌬sidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally, RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by host PtdIns 4-kinase III␤. Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase III␤ to anchor the effectors SidC and SidM to LCVs.

The basic function of the Src homology 3 (SH3) domain is considered to be binding to proline-rich sequences containing a PxxP motif. Recently, many SH3 domains, including those from Grb2 and Pex13p, were reported to bind sequences lacking a PxxP motif. We report here that the 22 residue peptide lacking a PxxP motif, derived from p47(phox), binds to the C-terminal SH3 domain from p67(phox). We applied the NMR cross-saturation method to locate the interaction sites for the non-PxxP peptides on their cognate SH3 domains from p67(phox), Grb2 and Pex13p. The binding site of the Grb2 SH3 partially overlapped the conventional PxxP-binding site, whereas those of p67(phox) and Pex13p SH3s are located in different surface regions. The non-PxxP peptide from p47(phox) binds to the p67(phox) SH3 more tightly when it extends to the N-terminus to include a typical PxxP motif, which enabled the structure determination of the complex, to reveal that the non-PxxP peptide segment interacted with the p67(phox) SH3 in a compact helix-turn-helix structure (PDB entry 1K4U).

Manual handling of organs during gastrectomy is an important contributor to the molecular and humoral inflammatory response to surgery, supporting the use of minimally invasive techniques in gastrointestinal surgery.

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Identification of the interfaces of large (Mr > 50,000) protein-protein complexes in solution by high resolution NMR has typically been achieved using experiments involving chemical shift perturbation and/or hydrogen-deuterium exchange of the main chain amide groups of the proteins. Interfaces identified using these techniques, however, are not always identical to those revealed using X-ray crystallography. In order to identify the contact residues in a large protein-protein complex more accurately, we developed a novel NMR method that uses cross-saturation phenomena in combination with TROSY detection in an optimally deuterium labeled system.

PAGE 25238:The image shown in the lower panel on the left of Fig. 3A was not correct. The lower panel on the left (the first from the left) incorrectly duplicated the lower panel on the right (the third from the left). In addition, the panels in Fig. 3A were not oriented correctly. The revised figure includes the correct images in the proper orientation. This correction does not affect the interpretation of the results or the conclusions of this work.