GSH-dependent regulation of Fas-mediated caspase-8 activation by acrolein

Activation of the cysteine protease caspase-8 by the death receptor Fas (CD95/APO-1) in B lymphoblastoid SKW6.4 cells or Jurkat T cells is associated with GSH depletion. Conversely, GSH depletion by the aldehyde acrolein (3-30 microM) was associated with inhibition of Fas-induced caspase-8 activation, although GSH depletion by buthionine sulfoximine (BSO) did not affect caspase-8 activation. In contrast to BSO, acrolein caused a loss of caspase-8 cysteine content in association with direct alkylation of caspase-8. Our findings indicate that inhibition of caspase-8 by thiol-reactive agents such as acrolein is not due to GSH depletion but caused by direct protein thiol modifications.     

CSF-1 and TPA stimulate independent pathways leading to lysosomal degradation or regulated intramembrane proteolysis of the CSF-1 receptor

The CSF-1 receptor is a protein-tyrosine kinase that has been shown to undergo regulated intramembrane proteolysis, or RIPping. Here, we have compared receptor downregulation and RIPping in response to CSF-1 and TPA. Our studies show that CSF-1 is a relatively poor inducer of RIPping and that CSF-1-induced receptor downregulation is largely independent of RIPping. TPA is a strong inducer of RIPping and TPA-induced receptor downregulation is mediated by RIPping. We further found that RIPping is dependent on TACE or a TACE-like protease, that CSF-1 and TPA use independent pathways to initiate RIPping, and that the intracellular domain is targeted for degradation through ubiquitination.     

Structural characterization and oligomerization of PB1-F2, a proapoptotic influenza A virus protein

Recently, a novel 87-amino acid influenza A virus protein with proapoptotic properties, PB1-F2, has been reported that originates from an alternative reading frame in the PB1 polymerase gene and is encoded in most known human influenza A virus isolates. Here we characterize the molecular structure of a biologically active synthetic version of the protein (sPB1-F2). Western blot analysis, chemical cross-linking, and NMR spectroscopy afforded direct evidence of the inherent tendency of sPB1-F2 to undergo oligomerization mediated by two distinct domains located in the N and C termini, respectively. CD and (1)H NMR spectroscopic analyses indicate that the stability of structured regions in the molecule clearly depends upon the hydrophobicity of the solvent. In aqueous solutions, the behavior of sPB1-F2 is typical of a largely random coil peptide that, however, adopts alpha-helical structure upon the addition of membrane mimetics. (1)H NMR analysis of three overlapping peptides afforded, for the first time, direct experimental evidence of the presence of a C-terminal region with strong alpha-helical propensity comprising amino acid residues Ile(55)-Lys(85) connected via an essentially random coil structure to a much weaker helix-like region, located in the N terminus between residues Trp(9) and Lys(20). The C-terminal helix is not a true amphipathic helix and is more compact than previously predicted. It corresponds to a positively charged region previously shown to include the mitochondrial targeting sequence of PB1-F2. The consequences of the strong oligomerization and helical propensities of the molecule are discussed and used to formulate a hypothetical model of its interaction with the mitochondrial membrane.     

Structural and functional fingerprint of the mitochondrial ATP-binding cassette transporter Mdl1 from Saccharomyces cerevisiae

The ATP-binding cassette half-transporter Mdl1 from Saccharomyces cerevisiae has been proposed to be involved in the quality control of misassembled respiratory chain complexes by exporting degradation products generated by the m-AAA proteases from the matrix. Direct functional or structural data of the transport complex are, however, not known so far. After screening expression in various hosts, Mdl1 was overexpressed 100-fold to 1% of total mitochondrial membrane protein in S. cerevisiae. Based on detergent screens, Mdl1 was solubilized and purified to homogeneity. Mdl1 showed a high binding affinity for MgATP (Kd = 0.26 microm) and an ATPase activity with a Km of 0.86 mm (Hill coefficient of 0.98) and a turnover rate of 2.6 ATP/s. Mutagenesis of the conserved glutamate downstream of the Walker B motif (E599Q) or the conserved histidine of the H-loop (H631A) abolished ATP hydrolysis, whereas ATP binding was not affected. Mdl1 reconstituted into liposomes showed an ATPase activity similar to the solubilized complex. By single particle electron microscopy, a first three-dimensional structure of the mitochondrial ATP-binding cassette transporter was derived at 2.3-nm resolution, revealing a homodimeric complex in an open conformation.     

The contribution of conformational adjustments and long-range electrostatic forces to the CD2/CD58 interaction

CD2 is a T cell surface molecule that enhances T and natural killer cell function by binding its ligands CD58 (humans) and CD48 (rodents) on antigen-presenting or target cells. Here we show that the CD2/CD58 interaction is enthalpically driven and accompanied by unfavorable entropic changes. Taken together with structural studies, this indicates that binding is accompanied by energetically significant conformational adjustments. Despite having a highly charged binding interface, neither the affinity nor the rate constants of the CD2/CD58 interaction were affected by changes in ionic strength, indicating that long-range electrostatic forces make no net contribution to binding.     

Mad3 KEN boxes mediate both Cdc20 and Mad3 turnover, and are critical for the spindle checkpoint

Mitotic progression is controlled by proteolytic destruction of securin and cyclin. The mitotic E3 ubiquitin ligase, known as the anaphase promoting complex or cyclosome (APC/C), in partnership with its activators Cdc20p and Cdh1p, targets these proteins for degradation. In the presence of defective kinetochore-microtubule interactions, APC/C(Cdc20) is inhibited by the spindle checkpoint, thereby delaying anaphase onset and providing more time for spindle assembly. Cdc20p interacts directly with Mad2p, and its levels are subject to careful regulation, but the precise mode(s) of APC/C( Cdc20) inhibition remain unclear. The mitotic checkpoint complex (MCC, consisting of Mad3p, Mad2p, Bub3p and Cdc20p in budding yeast) is a potent APC/C inhibitor. Here we focus on Mad3p and how it acts, in concert with Mad2p, to efficiently inhibit Cdc20p. We identify and analyse the function of two motifs in Mad3p, KEN30 and KEN296, which are conserved from yeast Mad3p to human BubR1. These KEN amino acid sequences resemble 'degron' signals that confer interaction with APC/C activators and target proteins for degradation. We show that both Mad3p KEN boxes are necessary for spindle checkpoint function. Mutation of KEN30 abolished MCC formation and stabilised Cdc20p in mitosis. In addition, mutation of Mad3-KEN30, APC/C subunits, or Cdh1p, stabilised Mad3p in G1, indicating that the N-terminal KEN box could be a Mad3p degron. To determine the significance of Mad3p turnover, we analysed the consequences of MAD3 overexpression and found that four-fold overproduction of Mad3p led to chromosome bi-orientation defects and significant chromosome loss during recovery from anti-microtubule drug induced checkpoint arrest. In conclusion, Mad3p KEN30 mediates interactions that regulate the proteolytic turnover of Cdc20p and Mad3p, and the levels of both of these proteins are critical for spindle checkpoint signaling and high fidelity chromosome segregation.     

A prospective study of breakfast consumption and weight gain among U.S. men

Objective: The aim was to investigate the association between breakfast consumption and long‐term weight gain in an adult male population. Research Methods and Procedures: We evaluated prospective data on 20,064 U.S men, 46 to 81 years of age, who participated in the Health Professionals Follow‐up Study. Data on body weight, dietary factors, and lifestyle variables were obtained by validated questionnaires. We examined weight gain during 10 years of follow‐up. Results: Overall, 5857 men had a weight gain of 5 kg or greater during 10 years of follow‐up. Breakfast consumption was inversely associated with the risk of 5‐kg weight gain after adjustment for age [hazard ratio (HR) = 0.77 (95% confidence interval [CI], 0.72 to 0.82)], and this association was independent of lifestyle and BMI at baseline [HR = 0.87 (95% CI, 0.82 to 0.93)]. Fiber and nutrient intakes partially explained the association between breakfast consumption and weight gain. The inverse association between breakfast consumption and weight gain was more pronounced in men with a baseline BMI of 25 kg/m² or lower [multivariate HR = 0.78 (95% CI, 0.70 to 0.87)] than in men who were overweight at baseline [HR = 0.92 (95% CI, 0.85 to 1.00)]. Furthermore, we observed that an increasing number of eating occasions in addition to three standard meals was associated with a higher risk of 5‐kg weight gain [HR = 1.15 (95% CI, 1.06 to 1.25, for ≥2 vs. 0 additional eating occasions)]. Discussion: These findings suggest that the consumption of breakfast may modestly contribute to the prevention of weight gain as compared with skipping breakfast in middle‐aged and older men.     

Secular trends of obesity and cardiovascular risk factors in a Mediterranean population

Objective: To evaluate time trends of obesity, abdominal obesity, and cardiovascular risk factors (CRFs) according to BMI and waist circumference (WC) categories in a Mediterranean population. Research Methods and Procedures: Subjects were Spanish men (n = 2383) and women (n = 2525) 25 to 74 years old, examined in 1994 to 1995 and 1999 to 2000 in two independent population‐based cross‐sectional surveys in the northeast of Spain. Lifestyle measures, CRFs, and anthropometric variables were analyzed. Results: Over the 5 years of the study, mean age‐standardized BMI increased by 1.0 units in men and by 0.8 units in women. At the same time the prevalence of obesity increased from 15.4% to 21.9% in men and from 15.4% to 21.4% in women. An upward trend was observed for WC and abdominal obesity (WC > 102 cm in men and WC > 88 cm in women) only in men. The proportion of men and women with hypercholesterolemia, diabetes, and low high‐density lipoprotein‐cholesterol plasma concentration remained stable within BMI and WC categories. The proportion of hypertension and smoking in obese men significantly increased from 1995 to 2000. Discussion: The 5‐year increase in BMI and WC is of considerable magnitude in the present population, although several CRFs remained stable within BMI and WC categories.     

Applications of a new subspace clustering algorithm (COSA) in medical systems biology

A novel clustering approach named Clustering Objects on Subsets of Attributes (COSA) has been proposed (Friedman and Meulman, (2004). Clustering objects on subsets of attributes. J. R. Statist. Soc. B 66, 1–25.) for unsupervised analysis of complex data sets. We demonstrate its usefulness in medical systems biology studies. Examples of metabolomics analyses are described as well as the unsupervised clustering based on the study of disease pathology and intervention effects in rats and humans. In comparison to principal components analysis and hierarchical clustering based on Euclidean distance, COSA shows an enhanced capability to trace partial similarities in groups of objects enabling a new discovery approach in systems biology as well as offering a unique approach to reveal common denominators of complex multi-factorial diseases in animal and human studies. Doris Damian, Matej Orešič, and Elwin Verheij contributed equally to this work.