Identification of critical residues in the bifunctional phosphoenolpyruvate synthetase kinase/phosphotransferase of Escherichia coli

In bacteria, phosphoenolpyruvate synthetase (EC 2.7.9.2) catalyses the conversion of pyruvate to phosphoenolpyruvate during gluconeogenesis. The enzyme is regulated by an unusual bifunctional serine/threonine kinase-phosphotransferase (PEP synthetase regulatory protein) that involves an ADP-dependent phosphorylation and a Pi-dependent dephosphorylation mechanism. Site-directed mutagenesis studies have revealed that two separate regions of Escherichia coli PEP synthetase regulatory protein are involved in catalysis; a central P-loop that is probably critical for the binding of the protein substrate (PEP synthetase) and a C-terminal region that interacts with the P-loop and is required to bind ADP and Pi. In addition, our findings are consistent with the P-loop and the C-terminal region responsible for ADP and Pi binding being juxtaposed in the functioning enzyme.\ud Two closely associated active sites are present in E. coli PEP synthetase regulatory protein. Given the high degree of sequence similarity between bacterial PEP synthetase regulatory protein and plant pyruvate, orthophosphate dikinase regulatory protein, it is highly likely that there are two active sites involved in the ADP-dependent inactivation and the Pi-dependent activation of both PEP synthetase and pyruvate, orthophosphate dikinase and they are very close together. \u

Choline-releasing glycerophosphodiesterase EDI3 links the tumor metabolome to signaling network activities.

Recently, EDI3 was identified as a key factor for choline metabolism that controls tumor cell migration and is associated with metastasis in endometrial carcinomas. EDI3 cleaves glycerophosphocholine (GPC) to form choline and glycerol-3-phosphate (G3P). Choline is then further metabolized to phosphatidylcholine (PtdC), the major lipid in membranes and a key player in membrane-mediated cell signaling. The second product, G3P, is a precursor molecule for several lipids with central roles in signaling, for example lysophosphatidic acid (LPA), phosphatidic acid (PA) and diacylglycerol (DAG). LPA activates intracellular signaling pathways by binding to specific LPA receptors, including membrane-bound G protein-coupled receptors and the intracellular nuclear receptor, PPAR?. Conversely, PA and DAG mediate signaling by acting as lipid anchors that bind and activate several signaling proteins. For example, binding of GTPases and PKC to PA and DAG, respectively, increases the activation of signaling networks, mediating processes such as migration, adhesion, proliferation or anti-apoptosis-all relevant for tumor development. We present a concept by which EDI3 either directly generates signaling molecules or provides "membrane anchors" for downstream signaling factors. As a result, EDI3 links choline metabolism to signaling activities resulting in a more malignant phenotype

Macromolecular Crowding as a Suppressor of Human IAPP Fibril Formation and Cytotoxicity

<div><p>The biological cell is known to exhibit a highly crowded milieu, which significantly influences protein aggregation and association processes. As several cell degenerative diseases are related to the self-association and fibrillation of amyloidogenic peptides, understanding of the impact of macromolecular crowding on these processes is of high biomedical importance. It is further of particular relevance as most <i>in vitro</i> studies on amyloid aggregation have been performed in diluted solution which does not reflect the complexity of their cellular surrounding. The study presented here focuses on the self-association of the type-2 diabetes mellitus related human islet amyloid polypeptide (hIAPP) in various crowded environments including network-forming macromolecular crowding reagents and protein crowders. It was possible to identify two competing processes: a crowder concentration and type dependent stabilization of globular off-pathway species and a – consequently - retarded or even inhibited hIAPP fibrillation reaction. The cause of these crowding effects was revealed to be mainly excluded volume in the polymeric crowders, whereas non-specific interactions seem to be most dominant in protein crowded environments. Specific hIAPP cytotoxicity assays on pancreatic β-cells reveal non-toxicity for the stabilized globular species, in contrast to the high cytotoxicity imposed by the normal fibrillation pathway. From these findings it can be concluded that cellular crowding is able to effectively stabilize the monomeric conformation of hIAPP, hence enabling the conduction of its normal physiological function and prevent this highly amyloidogenic peptide from cytotoxic aggregation and fibrillation.</p></div

Schematic illustration of the crowder systems' molecular characteristics.

<p><b>A</b> High concentrations of Ficoll and dextran exhibit a network-like structure. <b>B</b>, <b>C</b> The proteins BSA and lysozyme can be described as hard sphere systems with BSA (<b>B</b>) having a larger hydrodynamic radius than lysozyme (<b>C</b>), leading to differences in the dimensions of void volume.</p

Monitoring crowder binding effects on hIAPP aggregation.

<p>Time dependent ThT fluorescence spectroscopic measurements of equimolar solutions of hIAPP and crowding reagents (10 µM each). The left panel shows absolute ThT fluorescence intensities, whereas the corresponding intensity normalized data are presented in the right panel. Data points are mean values (errors bars indicate standard deviations) of ≥6 experiments.</p

Structural differences of hIAPP aggregates upon incubation in crowded environments as detected by AFM.

<p>AFM images showing the structures of 50 µM hIAPP after ∼15 h of incubation in solutions with and without crowding reagents. <b>A</b> hIAPP only. <b>B</b>–<b>E</b> Incubation of hIAPP in 20% or 40% (w/v) of Ficoll, dextran, BSA and lysozyme. For an easier comparison of the hIAPP fibril sizes, insets in the left images of <b>D</b> and <b>E</b> show the same magnification as the other images displaying fibrils. All scale bars correspond to 1 µm. The color scale represents a height of 30 nm for images <b>A</b>–<b>C</b> and 5 nm for images <b>D</b> and <b>E</b>.</p

Influence of the crowder type on IAPP restriction and binding.

<p><b>A</b> Crowder concentration-dependent lateral diffusion constant, <i>D</i>, of 10 µM fluorescent labeled rIAPP (rIAPP-K-Bodipy FL) as monitored by FCS. The polymer-like network forming molecules Ficoll and dextran as well as the proteins BSA and lysozyme were used as crowding reagents at concentrations ranging from 10 µM to 40% (w/v). <b>B</b> Content of rIAPP bound to the different crowding agents as analyzed from the FCS results of the equimolar rIAPP-K-Bodipy FL to crowder mixture (10 µM each). Data points are mean values (errors bars indicate standard deviations) of ≥3 individual measurements.</p