Monolysocardiolipins accumulate in Barth syndrome but do not lead to enhanced apoptosis

Barth syndrome (BTHS) is an X-linked recessive disorder that is biochemically characterized by low cellular levels of the mitochondrial phospholipid cardiolipin (CL). Previously, we discovered that the yeast disruptant of the TAZ ortholog in Saccharomyces cerevisiae not only displays CL deficiency but also accumulates monolysocardiolipins (MLCLs), which are intermediates in CL remodeling. Therefore, we set out to investigate whether MLCL accumulation also occurs in BTHS. Indeed, we observed MLCL accumulation in heart, muscle, lymphocytes, and cultured lymphoblasts of BTHS patients; however, only very low levels of these lysophospholipids were found in platelets and fibroblasts of these patients. Although the fatty acid composition of the MLCLs was different depending on the tissue source, it did parallel the fatty acid composition of the (remaining) CLs. The possible implications of these findings for the two reported CL remodeling mechanisms, transacylation and deacylation/reacylation, are discussed. Because MLCLs have been proposed to be involved in the initiation of apoptosome-mediated cell death by the sequestration of the proapoptotic protein (t)BH3-interacting domain death agonist (Bid) to the mitochondrial membrane, we used control and BTHS lymphoblasts to investigate whether the accumulation of MLCLs results in higher levels of apoptosis. We found no differences in susceptibility to death receptor-mediated apoptosis or in cellular distribution of Bid, cytochrome c, and other parameters, implying that MLCL accumulation does not lead to enhanced apoptosis in cultured BTHS lymphoblasts.     

Database-derived potentials dependent on protein size for in silico folding and design

Knowledge-based potentials are widely used in simulations of protein folding, structure prediction, and protein design. Their advantages include limited computational requirements and the ability to deal with low-resolution protein models compatible with long-scale simulations. Their drawbacks comprehend their dependence on specific features of the dataset from which they are derived, such as the size of the proteins it contains, and their physical meaning is still a subject of debate. We address these issues by probing the theoretical validity of these potentials as mean-force potentials that take the solvent implicitly into account and involve entropic contributions due to atomic degrees of freedom and solvation. The dependence on the size of the system is checked on distance-dependent amino acid pair potentials, derived from six protein structure sets containing proteins of increasing length N. For large inter-residue distances, they are found to display the theoretically predicted 1/N behavior weighted by a factor depending on the boundaries and the compressibility of the system. For short distances, different trends are observed according to the nature of the residue pairs and their ability to form, for example, electrostatic, cation-pi or pi-pi interactions, or hydrophobic packing. The results of this analysis are used to devise a novel protein size-dependent distance potential, which displays an improved performance in discriminating native sequence-structure matches among decoy models.     

Apolipoprotein A-I-stimulated apolipoprotein E secretion from human macrophages is independent of cholesterol efflux

Apolipoprotein A-I (apoA-I)-mediated cholesterol efflux involves the binding of apoA-I to the plasma membrane via its C terminus and requires cellular ATP-binding cassette transporter (ABCA1) activity. ApoA-I also stimulates secretion of apolipoprotein E (apoE) from macrophage foam cells, although the mechanism of this process is not understood. In this study, we demonstrate that apoA-I stimulates secretion of apoE independently of both ABCA1-mediated cholesterol efflux and of lipid binding by its C terminus. Pulse-chase experiments using (35)S-labeled cellular apoE demonstrate that macrophage apoE exists in both relatively mobile (E(m)) and stable (E(s)) pools, that apoA-I diverts apoE from degradation to secretion, and that only a small proportion of apoA-I-mobilized apoE is derived from the cell surface. The structural requirements for induction of apoE secretion and cholesterol efflux are clearly dissociated, as C-terminal deletions in recombinant apoA-I reduce cholesterol efflux but increase apoE secretion, and deletion of central helices 5 and 6 decreases apoE secretion without perturbing cholesterol efflux. Moreover, a range of 11- and 22-mer alpha-helical peptides representing amphipathic alpha-helical segments of apoA-I stimulate apoE secretion whereas only the C-terminal alpha-helix (domains 220-241) stimulates cholesterol efflux. Other alpha-helix-containing apolipoproteins (apoA-II, apoA-IV, apoE2, apoE3, apoE4) also stimulate apoE secretion, implying a positive feedback autocrine loop for apoE secretion, although apoE4 is less effective. Finally, apoA-I stimulates apoE secretion normally from macrophages of two unrelated subjects with genetically confirmed Tangier Disease (mutations C733R and c.5220-5222delTCT; and mutations A1046D and c.4629-4630insA), despite severely inhibited cholesterol efflux. We conclude that apoA-I stimulates secretion of apoE independently of cholesterol efflux, and that this represents a novel, ABCA-1-independent, positive feedback pathway for stimulation of potentially anti-atherogenic apoE secretion by alpha-helix-containing molecules including apoA-I and apoE.     

Chondrocyte protein with a poly-proline region (CHPPR) is a novel mitochondrial protein and promotes mitochondrial fission

We have recently identified a chondrocyte protein with a poly-proline region, referred to as CHPPR, and showed that this protein is expressed intracellularly in chick embryo chondrocytes. Conventional fluorescence and confocal localization of CHPPR shows that CHPPR is sorted to mitochondria. Furthermore, immunoelectron microscopy of CHPPR transfected cells demonstrates that this protein is mostly associated with the mitochondrial inner membranes. Careful analysis of CHPPR expressing cells reveals, instead of the regular mitochondrial tubular network, the presence of a number of small spheroid mitochondria. Here we show that the domain responsible for network-spheroid transition spans amino acid residues 182-309 including the poly-proline region. Functional analyses of mitochondrial activity rule out the possibility of mitochondrial damage in CHPPR transfected cells. Since cartilage expresses high levels of CHPPR mRNA when compared to other tissues and because CHPPR is associated with late stages of chondrocyte differentiation, we have investigated mitochondrial morphology in hypertrophic chondrocytes by MitoTracker Orange labeling. Confocal microscopy shows that these cells have spheroid mitochondria. Our data demonstrate that CHPPR is able to promote mitochondrial fission with a sequence specific mechanism suggesting that this event may be relevant to late stage of chondrocyte differentiation.     

Patency Outcomes of Aortic Connectors

OBJECTIVE:: Controlled outcome analysis of mechanical aortic connectors for proximal saphenous vein bypass graft anastomosis is lacking. We report the clinical and angiographic outcome of patients receiving the Symmetry aortic connector (St. Jude Medical, Inc St. Paul, MN, US) within a multicenter, prospective, randomized study. METHODS:: Twenty-five patients at 3 study sites received aortic connectors at the time of coronary artery bypass surgery. Protocol-defined angiographic follow-up was completed in 19 of 25 patients (76%) at time-points up to 14 months postoperatively; 32 connector anastomoses were evaluated in these 19 patients. Beating heart surgery was performed in 17 patients, and 2 were performed with cardiopulmonary bypass. Age was 69.7 ± 8.1 year; all patients were males. RESULTS:: The connector anastomosis patency rate was 15.6% (5/32). There were no deaths during the follow-up period. Four patients (21%) suffered myocardial infarction and 2 additional patients (10.5%) required percutaneous coronary interventions; one of who required 3 percutaneous coronary interventions, the other received one percutaneous coronary intervention. CONCLUSIONS:: In this nonrandomized cohort of patients, occlusion rate with Symmetry connectors was significantly greater than anticipated. Patients who have received these connectors during coronary artery bypass surgery may require closer follow-up and evaluation. While the manufacturer has stopped producing this device, there has been no recall of the product, clinical support remains ongoing, and next generation connectors have now been marketed. Consideration should be given to discontinuation of the clinical use of Symmetry connectors.     

Rapid alteration of the phosphoproteome in the moss Physcomitrella patens after cytokinin treatment

Cytokinin hormones are crucial regulators of a large number of processes in plant development. Recently, significant progress has been made toward the elucidation of the molecular details of cytokinin that has led to a model for signal transduction involving a phosphorylation cascade. However, the current knowledge of cytokinin action remains largely unknown and does not explain the different roles of this hormone. To gain further insights into this aspect of cytokinin action and the inducible phosphorelay, we have produced the first large-scale map of a phosphoproteome in the moss Physcomitrella patens. Using a protocol that we recently published (Heintz, D.; et al. Electrophoresis 2004, 25, 1149-1159) that combines IMAC, MALDI-TOF-MS, and LC-MS/MS, a total of 172 phosphopeptide sequences were obtained by a peptide de novo sequencing strategy. Specific P. patens EST and raw genomic databases were interrogated, and protein homology searches resulted in the identification of 112 proteins that were then classified into functional categories. In addition, the temporal dynamics of the phosphoproteome in response to cytokinin stimulation was studied at 2, 4, 6, and 15 min after hormone addition. We identified 13 proteins that were not previously known targets of cytokinin action. Among the responsive proteins, some were involved in metabolism, and several proteins of unknown function were also identified. We have mapped the time course of their activation in response to cytokinin and discussed their hypothetical biological significance. Deciphering these early induced phosphorylation events has shown that the cytokinin effect can be rapid (few minutes), and the duration of this effect can be variable. Also phosphorylation events can be differentially regulated. Taken together our proteomic study provides an enriched look of the multistep phosphorelay system mediating cytokinin response and suggests the existence of a multidirectional interaction between cytokinin and numerous other pathways.     

Cell type-dependent, infection-induced, aberrant DNA methylation in gastric cancer

Epigenetic changes correspond to heritable modifications of the chromatin structure, which do not involve any alteration of the DNA sequence but nonetheless affect gene expression. These mechanisms play an important role in cell differentiation, but aberrant occurrences are also associated with a number of diseases, including cancer and neural development disorders.In particular, aberrant DNA methylation induced by H. Pylori has been found to be a significant risk factor in gastric cancer. To investigate the sensitivity of different genes and cell types to this infection, a computational model of methylation in gastric crypts is developed.In this article, we review existing results from physical experiments and outline their limitations, before presenting the computational model and investigating the influence of its parameters.     

Biochemical and Spectroscopic Characterization of the Human Mitochondrial Amidoxime Reducing Components hmARC-1 and hmARC-2 Suggests the Existence of a New Molybdenum Enzyme Family in Eukaryotes

The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that is presumed to form the catalytical part of a three-component enzyme system, consisting of mARC, heme/cytochrome b₅, and NADH/FAD-dependent cytochrome b₅ reductase. mARC proteins share a significant degree of homology to the molybdenum cofactor-binding domain of eukaryotic molybdenum cofactor sulfurase proteins, the latter catalyzing the post-translational activation of aldehyde oxidase and xanthine oxidoreductase. The human genome harbors two mARC genes, referred to as hmARC-1/MOSC-1 and hmARC-2/MOSC-2, which are organized in a tandem arrangement on chromosome 1. Recombinant expression of hmARC-1 and hmARC-2 proteins in Escherichia coli reveals that both proteins are monomeric in their active forms, which is in contrast to all other eukaryotic molybdenum enzymes that act as homo- or heterodimers. Both hmARC-1 and hmARC-2 catalyze the N-reduction of a variety of N-hydroxylated substrates such as N-hydroxy-cytosine, albeit with different specificities. Reconstitution of active molybdenum cofactor onto recombinant hmARC-1 and hmARC-2 proteins in the absence of sulfur indicates that mARC proteins do not belong to the xanthine oxidase family of molybdenum enzymes. Moreover, they also appear to be different from the sulfite oxidase family, because no cysteine residue could be identified as a putative ligand of the molybdenum atom. This suggests that the hmARC proteins and sulfurase represent members of a new family of molybdenum enzymes.     

Structural Basis for the Activity and Substrate Specificity of Fluoroacetyl-CoA Thioesterase FlK

The thioesterase FlK from the fluoroacetate-producing Streptomyces cattleya catalyzes the hydrolysis of fluoroacetyl-coenzyme A. This provides an effective self-defense mechanism, preventing any fluoroacetyl-coenzyme A formed from being further metabolized to 4-hydroxy-trans-aconitate, a lethal inhibitor of the tricarboxylic acid cycle. Remarkably, FlK does not accept acetyl-coenzyme A as a substrate. Crystal structure analysis shows that FlK forms a dimer, in which each subunit adopts a hot dog fold as observed for type II thioesterases. Unlike other type II thioesterases, which invariably utilize either an aspartate or a glutamate as catalytic base, we show by site-directed mutagenesis and crystallography that FlK employs a catalytic triad composed of Thr⁴², His⁷⁶, and a water molecule, analogous to the Ser/Cys-His-acid triad of type I thioesterases. Structural comparison of FlK complexed with various substrate analogues suggests that the interaction between the fluorine of the substrate and the side chain of Arg¹²⁰ located opposite to the catalytic triad is essential for correct coordination of the substrate at the active site and therefore accounts for the substrate specificity.