Structural basis for activation of the thiamin diphosphate-dependent enzyme oxalyl-CoA decarboxylase by adenosine diphosphate

Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate-dependent enzyme that plays an important role in the catabolism of the highly toxic compound oxalate. We have determined the crystal structure of the enzyme from Oxalobacter formigenes from a hemihedrally twinned crystal to 1.73 A resolution and characterized the steady-state kinetic behavior of the decarboxylase. The monomer of the tetrameric enzyme consists of three alpha/beta-type domains, commonly seen in this class of enzymes, and the thiamin diphosphate-binding site is located at the expected subunit-subunit interface between two of the domains with the cofactor bound in the conserved V-conformation. Although oxalyl-CoA decarboxylase is structurally homologous to acetohydroxyacid synthase, a molecule of ADP is bound in a region that is cognate to the FAD-binding site observed in acetohydroxyacid synthase and presumably fulfils a similar role in stabilizing the protein structure. This difference between the two enzymes may have physiological importance since oxalyl-CoA decarboxylation is an essential step in ATP generation in O. formigenes, and the decarboxylase activity is stimulated by exogenous ADP. Despite the significant degree of structural conservation between the two homologous enzymes and the similarity in catalytic mechanism to other thiamin diphosphate-dependent enzymes, the active site residues of oxalyl-CoA decarboxylase are unique. A suggestion for the reaction mechanism of the enzyme is presented.     

Proteome‐wide analysis of phospho‐regulated PDZ domain interactions

A key function of reversible protein phosphorylation is to regulate protein–protein interactions, many of which involve short linear motifs (3–12 amino acids). Motif‐based interactions are difficult to capture because of their often low‐to‐moderate affinities. Here, we describe phosphomimetic proteomic peptide‐phage display, a powerful method for simultaneously finding motif‐based interaction and pinpointing phosphorylation switches. We computationally designed an oligonucleotide library encoding human C‐terminal peptides containing known or predicted Ser/Thr phosphosites and phosphomimetic variants thereof. We incorporated these oligonucleotides into a phage library and screened the PDZ (PSD‐95/Dlg/ZO‐1) domains of Scribble and DLG1 for interactions potentially enabled or disabled by ligand phosphorylation. We identified known and novel binders and characterized selected interactions through microscale thermophoresis, isothermal titration calorimetry, and NMR. We uncover site‐specific phospho‐regulation of PDZ domain interactions, provide a structural framework for how PDZ domains accomplish phosphopeptide binding, and discuss ligand phosphorylation as a switching mechanism of PDZ domain interactions. The approach is readily scalable and can be used to explore the potential phospho‐regulation of motif‐based interactions on a large scale.     

Bartonella Infections in Deer Keds (Lipoptena cervi) and Moose (Alces alces) in Norway

Infections with Bartonella spp. have been recognized as emerging zoonotic diseases in humans. Large knowledge gaps exist, however, relating to reservoirs, vectors, and transmission of these bacteria. We describe identification by culture, PCR, and housekeeping gene sequencing of Bartonella spp. in fed, wingless deer keds (Lipoptena cervi), deer ked pupae, and blood samples collected from moose, Alces alces, sampled within the deer ked distribution range in Norway. Direct sequencing from moose blood sampled in a deer ked-free area also indicated Bartonella infection but at a much lower prevalence. The sequencing data suggested the presence of mixed infections involving two species of Bartonella within the deer ked range, while moose outside the range appeared to be infected with a single species. Bartonella were not detected or cultured from unfed winged deer keds. The results may indicate that long-term bacteremia in the moose represents a reservoir of infection and that L. cervi acts as a vector for the spread of infection of Bartonella spp. Further research is needed to evaluate the role of L. cervi in the transmission of Bartonella to animals and humans and the possible pathogenicity of these bacteria for humans and animals.     

A ZO-1/α5β1-Integrin Complex Regulates Cytokinesis Downstream of PKCε in NCI-H460 Cells Plated on Fibronectin

Recently, we demonstrated that integrin adhesion to the extracellular matrix at the cleavage furrow is essential for cytokinesis of adherent cells. Here, we report that tight junction protein ZO-1 (Zonula Occludens-1) is required for successful cytokinesis in NCI-H460 cells plated on fibronectin. This function of ZO-1 involves interaction with the cytoplasmic domain of α5-integrin to facilitate recruitment of active fibronectin-binding integrins to the base of the cleavage furrow. In the absence of ZO-1, or a functional ZO-1/α5β1-integrin complex, proper actin-dependent constriction between daughter cells is impaired and cells fail cytokinesis. Super-resolution microscopy reveals that in ZO-1 depleted cells the furrow becomes delocalized from the matrix. We also show that PKCε-dependent phosphorylation at Serine168 is required for ZO-1 localization to the furrow and successful cell division. Altogether, our results identify a novel regulatory pathway involving the interplay between ZO-1, α5-integrin and PKCε in the late stages of mammalian cell division.     

In vivo interconversion of 7beta-hydroxycholesterol and 7-ketocholesterol, potential surrogate markers for oxidative stress

The oxysterols 7beta-hydroxycholesterol and 7-ketocholesterol are cholesterol autoxidation products. These two oxysterols are formed as a result of low density lipoprotein oxidation and in a study on biomarkers for oxidative stress in patients with atherosclerosis, 7beta-hydroxycholesterol was found to be the strongest predictor of progression of carotid atherosclerosis. Interconversion of 7beta-hydroxycholesterol and 7-ketocholesterol in vitro has been reported recently, using recombinant 11beta-hydroxysteroid dehydrogenase or rodent liver microsomes. In this study deuterium-labeled 7beta-hydroxycholesterol or 7-ketocholesterol was administered intravenously to two healthy volunteers and blood samples were collected at different time points. The mean half-life for elimination of 7beta-hydroxycholesterol from the circulation was estimated to be 1.9 h. The corresponding half-life for 7-ketocholesterol was estimated to be 1.5 h. Infusion of deuterium-labeled 7-ketocholesterol resulted in labeling of 7beta-hydroxycholesterol and vice versa. In addition, the biological within-day and between-day variations of the two oxysterols were determined. In summary, the present investigation clearly shows an interconversion of 7beta-hydroxycholesterol and 7-ketocholesterol in humans.     

Antioxidant supplementation enhances the exercise-induced increase in mitochondrial uncoupling protein 3 and endothelial nitric oxide synthase mRNA content in human skeletal muscle

The effects of acute exercise on the mRNA content of selected genes were examined during control conditions and after oral intake of antioxidants. In addition, to provide evidence for formation of reactive oxygen species (ROS) in human skeletal muscle during exercise, cytochrome c reduction was measured in microdialysate from the muscle. For the study on the effects of antioxidants on mRNA content, seven healthy, habitually active, male subjects participated in a double-blinded experimental design in which they, on one occasion, received a placebo and, on another, a mixture of antioxidants containing 1500 mg vitamin C, 120 mg coenzyme Q, and 345 mg alpha-tocopherol every day for 7 days before the experiment. On the experimental day the subjects cycled for 90 min and muscle biopsies were taken preexercise and at 1, 3, and 5 h after exercise. Exercise induced an increase in the eNOS, UCP3, PGC-1alpha, VEGF, Hsp72, and HO-1 mRNA content (p < 0.001), whereas there was no change in the Hsc70 mRNA level. Prior antioxidant treatment further enhanced (p < 0.05) the eNOS and UCP3 mRNA content after exercise. Moreover, the overall level of Hsc70 mRNA tended (p = 0.07) to be higher after antioxidant treatment. In another group of healthy male subjects, cytochrome c reduction was determined in microdialysate from the thigh muscle at rest and during knee extensor exercise to determine ROS formation. There was a significant increase in cytochrome c reduction with exercise both at 14 ( approximately 25%) and at 30 W ( approximately 50%). The data show that ROS are formed within skeletal muscle during exercise and that oral intake of antioxidants can enhance the exercise-induced adaptive mRNA responses of eNOS and UCP3.     

Characterization of Stem-Like Cells in Mucoepidermoid Tracheal Paediatric Tumor

Stem cells contribute to regeneration of tissues and organs. Cells with stem cell-like properties have been identified in tumors from a variety of origins, but to our knowledge there are yet no reports on tumor-related stem cells in the human upper respiratory tract. In the present study, we show that a tracheal mucoepidermoid tumor biopsy obtained from a 6 year-old patient contained a subpopulation of cells with morphology, clonogenicity and surface markers that overlapped with bone marrow mesenchymal stromal cells (BM-MSCs). These cells, designated as MEi (mesenchymal stem cell-like mucoepidermoid tumor) cells, could be differentiated towards mesenchymal lineages both with and without induction, and formed spheroids in vitro. The MEi cells shared several multipotent characteristics with BM-MSCs. However, they displayed differences to BM-MSCs in growth kinectics and gene expression profiles relating to cancer pathways and tube development. Despite this, the MEi cells did not possess in vivo tumor-initiating capacity, as proven by the absence of growth in situ after localized injection in immunocompromised mice. Our results provide an initial characterization of benign tracheal cancer-derived niche cells. We believe that this report could be of importance to further understand tracheal cancer initiation and progression as well as therapeutic development.     

Detection and characterization of merohedral twinning in crystals of oxalyl-coenzyme A decarboxylase from Oxalobacter formigenes

Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate dependent enzyme active in the catabolism of the highly toxic compound oxalate. The enzyme from Oxalobacter formigenes has been expressed as a recombinant protein in Escherichia coli, purified to homogeneity and crystallized. Two crystal forms were obtained, one showing poor diffraction and the other merohedral twinning. Crystals in the former category belong to the tetragonal space group P4(2)2(1)2. Data to 4.1 A resolution were collected from these crystals and an incomplete low resolution structure was initially determined by molecular replacement. Crystals in the latter category were obtained by co-crystallizing the protein with coenzyme A, thiamin diphosphate and Mg(2+)-ions. Data to 1.73 A were collected from one of these crystals with apparent point group 622. The crystal was found to be heavily twinned, and a twin ratio of 0.43 was estimated consistently by different established methods. The true space group P3(1)21 was deduced, and a molecular replacement solution was obtained using the low resolution structure as template when searching in detwinned data.