The proton-pumping NADH:ubiquinone oxidoreductase (complex I) of Aquifex aeolicus

The proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, is the first energy-transducing complex of many respiratory chains. Homologues of complex I are present in the three domains of life. Here, we report the properties of complex I in membranes of the hyperthermophilic bacterium Aquifex aeolicus. The complex reacted with NADH but not with NADPH and F(420)H(2) as electron donors. Short-chain analogues of ubiquinone like decyl-ubiquinone and ubiquinone-2 were suitable electron acceptors. The affinities towards NADH and ubiquinone-2 were comparable to the ones obtained with the Escherichia coli complex I. The reaction was inhibited by piericidin A at the same concentration as in E. coli. The complex showed an unusual pH optimum at pH 9 and a maximal rate at 80 degrees C. We found no evidence for the presence of an alternative, single subunit NADH dehydrogenase in A. aeolicus membranes. The NADH:ferricyanide reductase activity of detergent extracts of A. aeolicus membranes sedimented as a protein with a molecular mass of approximately 550 kDa. From the data we concluded that A. aeolicus contains a NADH:ubiquinone oxidoreductase resembling complex I of mesophilic bacteria.     

PPAR activators inhibit endothelial cell migration by targeting Akt

Peroxisome proliferator-activated receptors (PPARs) regulate lipid and glucose metabolism and exert several vascular effects that may provide a dual benefit of these receptors on metabolic disorders and atherosclerotic vascular disease. Endothelial cell migration is a key event in the pathogenesis of atherosclerosis. We therefore investigated the effects of lipid-lowering PPARalpha-activators (fenofibrate, WY14643) and antidiabetic PPARgamma-activators (troglitazone, ciglitazone) on this endothelial cell function. Both PPARalpha- and PPARgamma-activators significantly inhibited VEGF-induced migration of human umbilical vein endothelial cells (EC) in a concentration-dependent manner. Chemotactic signaling in EC is known to require activation of two signaling pathways: the phosphatidylinositol-3-kinase (PI3K)-->Akt- and the ERK1/2 mitogen-activated protein kinase (ERK MAPK) pathway. Using the pharmacological PI3K-inhibitor wortmannin and the ERK MAPK-pathway inhibitor PD98059, we observed a complete inhibition of VEGF-induced EC migration. VEGF-induced Akt phosphorylation was significantly inhibited by both PPARalpha- and gamma-activators. In contrast, VEGF-stimulated ERK MAPK-activation was not affected by any of the PPAR-activators, indicating that they inhibit migration either downstream of ERK MAPK or independent from this pathway. These results provide first evidence for the antimigratory effects of PPAR-activators in EC. By inhibiting EC migration PPAR-activators may protect the vasculature from pathological alterations associated with metabolic disorders.     

The phosphorylation state of threonine-220, a uniquely phosphatase-sensitive protein kinase A site in microtubule-associated protein MAP2c,regulates microtubule binding and stability

Phosphorylation of microtubule-associated protein 2 (MAP2) has a profound effect on microtubule stability and organization. In this work a consensus protein kinase A (PKA) phosphorylation site, T(220), of juvenile MAP2c is characterized. As confirmed by mass spectrometry, this site can be phosphorylated by PKA but shows less than average reactivity among the 3.5 +/- 0.5 phosphate residues incorporated into the protein. In contrast, T(220) is uniquely sensitive to dephosphorylation: three major Ser/Thr protein phosphatases, in the order of efficiency PP2B > PP2A(c) > PP1(c), remove this phosphate group first. MAP2c specifically dephosphorylated at this site binds and stabilizes microtubules stronger than either fully phosphorylated or nonphosphorylated MAP2c. Phosphorylation of this site also affects proteolytic sensitivity of MAP2c, which might represent a further level of control in this system. Thus, the phosphorylation state of T(220) may be a primary determinant of microtubule function.     

Conformation and stability of alpha-helical membrane proteins. 1. Influence of salts on conformational equilibria between active and Inactive states of rhodopsin

We studied the influence of salts on the pH-dependent conformational equilibria between the active and the inactive photoproduct states of rhodopsin, Meta II and Meta I, respectively, and between the active and inactive conformations of the apoprotein opsin. In both equilibria, the active species is favored in the presence of medium to high concentration of salt. The ion selectivity for the Meta I/Meta II equilibrium is particularly pronounced for the anions and follows the series trichloroacetate > thiocyanate > iodide > bromide > sulfate > chloride > acetate. The Hill coefficient of this salt-induced transition is close to 2.0. Both ion selectivity and Hill coefficient suggest that the transition is mainly regulated by ion binding to two specific charged binding sites in the protein with smaller contributions being due to the Hofmeister effect. We propose that these putative ion binding sites are identical to those sites that are titrated in the corresponding pH-dependent conformational transition. They presumably function as ionic locks, which keep the receptor in an inactive conformation, and which may be disrupted either by pH-dependent protonation or by salt-dependent ion binding.     

Enzymatic synthesis of D-glucosone 6-phosphate (D-arabino-hexos-2-ulose 6-(dihydrogen phosphate)) and NMR analysis of its isomeric forms

D-Glucosone 6-phosphate (D-arabino-hexos-2-ulose 6-(dihydrogen phosphate)) was prepared from D-glucosone (D-arabino-hexos-2-ulose) by enzymatic conversion with hexokinase. The isomeric composition of D-glucosone 6-phosphate in aqueous solution was quantitatively determined by NMR spectroscopy and compared to D-glucosone. The main isomers are the alpha-anomer (58%) and the beta-anomer (28%) of the hydrated pyranose form, and the beta-D-fructofuranose form (14%).     

Voltage-dependent anion-selective channel (VDAC) interacts with the dynein light chain Tctex1 and the heat-shock protein PBP74

The voltage-dependent anion-selective channel 1 (VDAC1), i.e. eukaryotic porin, functions as a channel in membranous structures as described for the outer mitochondrial membrane, the cell membrane, endosomes, caveolae, the sarcoplasmatic reticulum, synaptosomes, and post-synaptic density fraction.The identification of VDAC1 interacting proteins may be a promising approach for better understanding the biological context and function of the channel protein. In this study human VDAC1 was used as a bait protein in a two-hybrid screening, which is based on the Sos recruitment system (SRS). hVDAC1 interacts with the dynein light chain Tctex-1 and the heat-shock protein peptide-binding protein 74 (PBP74)/mitochondrial heat-shock protein 70 (mtHSP70)/glucose-regulated protein 75 (GRP75)/mortalin in vivo. Both interactions were confirmed by overlay-assays using recombinant partner proteins and purified hVDAC1. Indirect immunofluorescence on HeLa cells indicates a co-localisation of hVDAC1 with the dynein light chain and the PBP74. In addition, HeLa cells were transfected transiently with enhanced green fluorescent protein (EGFP)-hVDAC1 fusion proteins, which also clearly co-localise with both proteins. The functional relevance of the identified protein interactions was analysed in planar lipid bilayer (PLB) experiments. In these experiments both recombinant binding partners altered the electrophysiological properties of hVDAC1. While rTctex-1 increases the voltage-dependence of hVDAC1 slightly, the rPBP74 drastically minimises the voltage-dependence, indicating a modulation of channel properties in each case. Since the identified proteins are known to be involved in the transport or processing of proteins, the results of this study represent additional evidence of membrane-associated trafficking of the voltage-dependent anion-selective channel 1.     

A new allele of Gli3 and a new mutation,circletail (Crc), resulting from a single transgenic experiment

While generating transgenic lines, transgene-linked mutations can occur, which are caused by an insertional mutation at a given locus. More rarely, mutations unlinked to the transgene insertion site are observed. In the process of generating a mouse overexpressing the enzyme tyrosinase, we have obtained one transgenic line that appears to carry a semidominant insertional mutation at the Gli3 (extra toes) locus, characterized by polydactyly and skeletal malformations. Additionally, the transgenic line contained a second mutation, Crc (circletail), which appears to be unlinked to the transgene insertion site. Heterozygous Crc mice are incompletely penetrant for a circled-tail phenotype, while all homozygous Crc/Crc mice die at birth of a severe neural tube defect (craniorachischisis). Anatomical evidence from a Crc/Crc; Gli3/+ fetus indicates that these two genes may interact.