Jan W. Thuring

PtdIns(3)P Regulates the Neutrophil Oxidase Complex by Binding to the PX Domain of p40phox

The production of reactive oxygen species (ROS) by neutrophils has a vital role in defence against a range of infectious agents, and is driven by the assembly of a multi-protein complex containing a minimal core of five proteins: the two membrane-bound subunits of cytochrome b558 (gp91phox and p22phox) and three soluble factors (GTP–Rac, p47phox and p67phox (refs 1, 2). This minimal complex can reconstitute ROS formation in vitro in the presence of non-physiological amphiphiles such as SDS. p40phox has subsequently been discovered as a binding partner for p67phox (ref. 3), but its role in ROS formation is unclear. Phosphoinositide-3-OH kinases (PI(3)Ks) have been implicated in the intracellular signalling pathways coordinating ROS formation but through an unknown mechanism. We show that the addition of p40phox to the minimal core complex allows a lipid product of PI(3)Ks, phosphatidylinositol 3-phosphate (PtdIns(3)P), to stimulate specifically the formation of ROS. This effect was mediated by binding of PtdIns(3)P to the PX domain of p40phox. These results offer new insights into the roles for PI(3)Ks and p40phox in ROS formation and define a cellular ligand for the orphan PX domain.

Identification of ARAP3, a novel PI3K effector regulating both Arf and Rho GTPases, by selective capture on phosphoinositide affinity matrices

We show that matrices carrying the tethered homologs of natural phosphoinositides can be used to capture and display multiple phosphoinositide binding proteins in cell and tissue extracts. We present the mass spectrometric identification of over 20 proteins isolated by this method, mostly from leukocyte extracts: they include known and novel proteins with established phosphoinositide binding domains and also known proteins with surprising and unusual phosphoinositide binding properties. One of the novel PtdIns(3,4,5)P3 binding proteins, ARAP3, has an unusual domain structure, including five predicted PH domains. We show that it is a specific PtdIns(3,4,5)P3/PtdIns(3,4)P2-stimulated Arf6 GAP both in vitro and in vivo, and both its Arf GAP and Rho GAP domains cooperate in mediating PI3K-dependent rearrangements in the cell cytoskeleton and cell shape.

Svp1p defines a family of phosphatidylinositol 3,5‐bisphosphate effectors

Phosphatidylinositol 3,5‐bisphosphate (PtdIns(3,5)P2), made by Fab1p, is essential for vesicle recycling from vacuole/lysosomal compartments and for protein sorting into multivesicular bodies. To isolate PtdIns(3,5)P2 effectors, we identified Saccharomyces cerevisiae mutants that display fab1Δ‐like vacuole enlargement, one of which lacked the SVP1/YFR021w/ATG18 gene. Expressed Svp1p displays PtdIns(3,5)P2 binding of exquisite specificity, GFP‐Svp1p localises to the vacuole membrane in a Fab1p‐dependent manner, and svp1Δ cells fail to recycle a marker protein from the vacuole to the Golgi. Cells lacking Svp1p accumulate abnormally large amounts of PtdIns(3,5)P2. These observations identify Svp1p as a PtdIns(3,5)P2 effector required for PtdIns(3,5)P2‐dependent membrane recycling from the vacuole. Other Svp1p‐related proteins, including human and Drosophila homologues, bind PtdIns(3,5)P2 similarly. Svp1p and related proteins almost certainly fold as β‐propellers, and the PtdIns(3,5)P2‐binding site is on the β‐propeller. It is likely that many of the Svp1p‐related proteins that are ubiquitous throughout the eukaryotes are PtdIns(3,5)P2 effectors. Svp1p is not involved in the contributions of FAB1/PtdIns(3,5)P2 to MVB sorting or to vacuole acidification and so additional PtdIns(3,5)P2 effectors must exist.

The role of inositol 1,4,5-trisphosphate receptors in Ca2+ signalling and the generation of arrhythmias in rat atrial myocytes

Various cardio‐active stimuli, including endothelin‐1 (ET‐1), exhibit potent arrhythmogenicity, but the underlying cellular mechanisms of their actions are largely unclear. We used isolated rat atrial myocytes and related changes in their subcellular Ca2+ signalling to the ability of various stimuli to induce diastolic, premature extra Ca2+ transients (ECTs). For this, we recorded global and spatially resolved Ca2+ signals in indo‐1‐ and fluo‐4‐loaded atrial myocytes during electrical pacing. ET‐1 exhibited a higher arrhythmogenicity (arrhythmogenic index; ratio of number of ECTs over fold‐increase in Ca2+ response, 8.60; n= 8 cells) when compared with concentrations of cardiac glycosides (arrhythmogenic index, 4.10; n= 8 cells) or the β‐adrenergic agonist isoproterenol (arrhythmogenic index, 0.11; n= 6 cells) that gave similar increases in the global Ca2+ responses. Seventy‐five percent of the ET‐1‐induced arrhythmogenic Ca2+ transients were accompanied by premature action potentials, while for digoxin this proportion was 25 %. The β‐adrenergic agonist failed to elicit a significant number of ECTs. Direct activation of inositol 1,4,5‐trisphosphate (InsP3) receptors with a membrane‐permeable InsP3 ester (InsP3 BM) mimicked the effect of ET‐1 (arrhythmogenic index, 14.70; n= 6 cells). Inhibition of InsP3 receptors using 2 μM 2‐aminoethoxydiphenyl borate, which did not display any effects on Ca2+ signalling under control conditions, specifically suppressed the arrhythmogenic action of ET‐1 and InsP3 BM. Immunocytochemistry indicated a co‐localisation of peripheral, junctional ryanodine receptors with InsP3Rs. Thus, the pronounced arrhythmogenic potency of ET‐1 is due to the spatially specific recruitment of Ca2+ sparks by subsarcolemmal InsP3Rs. Summation of such sparks efficiently generates delayed afterdepolarisations that trigger premature action potentials. We conclude that the particular spatial profile of cellular Ca2+ signals is a major, previously unrecognised, determinant for arrhythmogenic potency and that the InsP3 signalling cassette might therefore be a promising new target for understanding and managing atrial arrhythmia.

Production and characterization of reduced NAADP (nicotinic acid-adenine dinucleotide phosphate).

The pyridine nucleotide NAADP (nicotinic acid-adenine dinucleotide phosphate) has been shown to act as a Ca2+-releasing intracellular messenger in a wide variety of systems from invertebrates to mammals and has been implicated in a number of cellular processes. NAADP is structurally very similar to its precursor, the endogenous coenzyme NADP and while much is known about the reduced form of NADP, NADPH, it is not known whether NAADP can also exist in a reduced state. Here we report that NAADP can be reduced to NAADPH by endogenous cellular enzymes and that NAADPH is functionally inert at the NAADP receptor. These data suggest that NAADPH could represent a mechanism for rapidly inactivating NAADP in cells.

Synthesis and biological evaluation of a PtdIns(4,5)P2 and a phosphatidic acid affinity matrix

Analogues of dipalmitoyl phosphatidic acid (PA), dilauroyl PA and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] were synthesised and immobilised onto a solid support, Affi-Gel 10. Using them as affinity matrices, a number of known proteins as well as a set of novel proteins were found to bind specifically to PA.

The synthesis of membrane permeant derivatives of myo-inositol 1,4,5-trisphosphate

In order to enable the study of the intracellular second messenger d-myo-inositol 1,4,5-trisphosphate (InsP3) and its receptors (InsP3Rs), it has been desirable to develop protected derivatives of InsP3 that are able to enter the cell, upon extracellular application. The subsequent removal of the lipophilic protecting groups, by intracellular enzymes, releases InsP3 and leads to the activation of InsP3Rs. Two syntheses of d-myo-inositol 1,4,5-trisphosphate hexakis(butyryloxymethyl) ester (d-InsP3/BM) and one of l-InsP3/BM are reported. It is demonstrated that extracellular application of the d-enantiomer results in Ca2+ release, which is thought to occur via InsP3Rs. Application of the l-enantiomer resulted in little Ca2+ release.

Synthesis and biological evaluation of aPtdIns(3,4,5)P3 affinity matrix

New PtdIns(3,4,5)P3 binding proteins have been nidentified utilising PtdIns(3,4,5)P3 modified affinity matrix 1 nwhich was synthesised from myo-inositol derivative 2, nphosphoramidite 9 and an agarose based solid support.