Bub1 regulates chromosome segregation in a kinetochore-independent manner

The kinetochore-bound protein kinase Bub1 performs two crucial functions during mitosis: it is essential for spindle checkpoint signaling and for correct chromosome alignment. Interestingly, Bub1 mutations are found in cancer tissues and cancer cell lines. Using an isogenic RNA interference complementation system in transformed HeLa cells and untransformed RPE1 cells, we investigate the effect of structural Bub1 mutants on chromosome segregation. We demonstrate that Bub1 regulates mitosis through the same mechanisms in both cell lines, suggesting a common regulatory network. Surprisingly, Bub1 can regulate chromosome segregation in a kinetochore-independent manner, albeit at lower efficiency. Its kinase activity is crucial for chromosome alignment but plays only a minor role in spindle checkpoint signaling. We also identify a novel conserved motif within Bub1 (amino acids 458–476) that is essential for spindle checkpoint signaling but does not regulate chromosome alignment, and we show that several cancer-related Bub1 mutants impair chromosome segregation, suggesting a possible link to tumorigenesis.     

Implications for molecular mechanisms of glycoprotein hormone receptors using a new sequence-structure-function analysis resource

Comparison between wild-type and mutated glycoprotein hormone receptors (GPHRs), TSH receptor, FSH receptor, and LH-chorionic gonadotropin receptor is established to identify determinants involved in molecular activation mechanism. The basic aims of the current work are 1) the discrimination of receptor phenotypes according to the differences between activity states they represent, 2) the assignment of classified phenotypes to three-dimensional structural positions to reveal 3) functional-structural hot spots and 4) interrelations between determinants that are responsible for corresponding activity states. Because it is hard to survey the vast amount of pathogenic and site-directed mutations at GPHRs and to improve an almost isolated consideration of individual point mutations, we present a system for systematic and diversified sequence-structure-function analysis (http://www.fmp-berlin.de/ssfa). To combine all mutagenesis data into one set, we converted the functional data into unified scaled values. This at least enables their comparison in a rough classification manner. In this study we describe the compiled data set and a wide spectrum of functions for user-driven searches and classification of receptor functionalities such as cell surface expression, maximum of hormone binding capability, and basal as well as hormone-induced Galphas/Galphaq mediated cAMP/inositol phosphate accumulation. Complementary to known databases, our data set and bioinformatics tools allow functional and biochemical specificities to be linked with spatial features to reveal concealed structure-function relationships by a semiquantitative analysis. A comprehensive discrimination of specificities of pathogenic mutations and in vitro mutant phenotypes and their relation to signaling mechanisms of GPHRs demonstrates the utility of sequence-structure-function analysis. Moreover, new interrelations of determinants important for selective G protein-mediated activation of GPHRs are resumed.     

Genome wide association (GWA) study for early onset extreme obesity supports the role of fat mass and obesity associated gene (FTO) variants

Background

Obesity is a major health problem. Although heritability is substantial, genetic mechanisms predisposing to obesity are not very well understood. We have performed a genome wide association study (GWA) for early onset (extreme) obesity.

Methodology/Principal Findings

a) GWA (Genome-Wide Human SNP Array 5.0 comprising 440,794 single nucleotide polymorphisms) for early onset extreme obesity based on 487 extremely obese young German individuals and 442 healthy lean German controls; b) confirmatory analyses on 644 independent families with at least one obese offspring and both parents. We aimed to identify and subsequently confirm the 15 SNPs (minor allele frequency ≥10%) with the lowest p-values of the GWA by four genetic models: additive, recessive, dominant and allelic. Six single nucleotide polymorphisms (SNPs) in FTO (fat mass and obesity associated gene) within one linkage disequilibrium (LD) block including the GWA SNP rendering the lowest p-value (rs1121980; log-additive model: nominal p = 1.13×10⁻⁷, corrected p = 0.0494; odds ratio (OR)_CT 1.67, 95% confidence interval (CI) 1.22–2.27; OR_TT 2.76, 95% CI 1.88–4.03) belonged to the 15 SNPs showing the strongest evidence for association with obesity. For confirmation we genotyped 11 of these in the 644 independent families (of the six FTO SNPs we chose only two representing the LD bock). For both FTO SNPs the initial association was confirmed (both Bonferroni corrected p<0.01). However, none of the nine non-FTO SNPs revealed significant transmission disequilibrium.

Conclusions/Significance

Our GWA for extreme early onset obesity substantiates that variation in FTO strongly contributes to early onset obesity. This is a further proof of concept for GWA to detect genes relevant for highly complex phenotypes. We concurrently show that nine additional SNPs with initially low p-values in the GWA were not confirmed in our family study, thus suggesting that of the best 15 SNPs in the GWA only the FTO SNPs represent true positive findings.     

Accurate quantification of dimethylamine (DMA) in human urine by gas chromatography-mass spectrometry as pentafluorobenzamide derivative: evaluation of the relationship between DMA and its precursor asymmetric dimethylarginine (ADMA) in health and disease

Dimethylamine [DMA, (CH(3))(2)NH)] is abundantly present in human urine. Main sources of urinary DMA have been reported to include trimethylamine N-oxide, a common food component, and asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis. ADMA is excreted in the urine in part unmetabolized and in part after hydrolysis to DMA by dimethylarginine dimethylaminohydrolase (DDAH). Here we describe a GC-MS method for the accurate and rapid quantification of DMA in human urine. The method involves use of (CD(3))(2)NH as internal standard, simultaneous derivatization with pentafluorobenzoyl chloride and extraction in toluene, and selected-ion monitoring of m/z 239 for DMA and m/z 245 for (CD(3))(2)NH in the electron ionization mode. GC-MS analysis of urine samples from 10 healthy volunteers revealed a DMA concentration of 264+/-173 microM equivalent to 10.1+/-1.64 micromol/mmol creatinine. GC-tandem MS analysis of the same urine samples revealed an ADMA concentration of 27.3+/-15.3 microM corresponding to 1.35+/-1.2 micromol/mmol creatinine. In these volunteers, a positive correlation (R=0.83919, P=0.0024) was found between urinary DMA and ADMA, with the DMA/ADMA molar ratio being 10.8+/-6.2. Elevated excretion rates of DMA (52.9+/-18.5 micromol/mmol creatinine) and ADMA (3.85+/-1.65 micromol/mmol creatinine) were found by the method in 49 patients suffering from coronary artery disease, with the DMA/ADMA molar ratio also being elevated (16.8+/-12.8). In 12 patients suffering from end-stage liver disease, excretion rates of DMA (47.8+/-19.7 micromol/mmol creatinine) and ADMA (5.6+/-1.5 micromol/mmol creatinine) were found to be elevated, with the DMA/ADMA molar ratio (9.17+/-4.2) being insignificantly lower (P=0.46). Between urinary DMA and ADMA there was a positive correlation (R=0.6655, P<0.0001) in coronary artery disease, but no correlation (R=0.27339) was found in end-stage liver disease.     

Mirror image duplications of chromosome 21. Three new cases and discussion of the mechanisms of origin

Summary Three new cases of mirror image duplication of a chromosome 21 are studied. In the first case the extra chromosome derived from a single paternal chromosome 21 by intrachromosomal interchange. The analysis of similar published cases suggests that this mechanism may predominate, but interchromosomal interchange, para- and pericentric inversion are likely as well.     

Localization,transmission, spontaneous mutations,and variation of function of the Dpp4 (Dipeptidyl-peptidase IV; CD26) gene in rats

Dipeptidyl-peptidase IV (DPPIV) is involved in endocrine and immune functions via cleavage of regulatory peptides with a N-terminal proline or alanine such as incretins, neuropeptide Y, or several chemokines. So far no systematic investigations on the localization and transmission of the Dpp4 gene or the natural variations of DPPIV-like enzymatic function in different rat strains have been conducted. Here we mapped the Dpp4 gene to rat chromosome 3 and describe a semi-dominant mode of inheritance for Dpp4 in a mutant F344/DuCrj(DPPIV-) rat substrain lacking endogenous DPPIV-like activity. This mutant F344/DuCrj(DPPIV-) rat substrain constantly exhibits a nearly complete lack of DPPIV-like enzymatic activity, while segregation of DPPIV-like enzymatic activity was observed in another DPPIV-negative F344/Crl(Ger/DPPIV-) rat substrain. Screening of 12 different inbred laboratory rat strains revealed dramatic differences in DPPIV-like activity ranging from 11 mU/microl (LEW/Ztm rats) to 40 mU/microl (BN/Ztm and DA/Ztm rats). A lack of DPPIV-like activity in F344 rats was associated with an improved glucose tolerance and blunted natural killer cell function, which indicates the pleiotropic functional role of DPPIV in vivo. Overall, the variations in DPPIV-like enzymatic activity probably represent important confounding factors in studies using rat models for research on regulatory peptides.     

Enhanced cytotoxicity without internuclear spread of adenovirus upon cell fusion by measles virus glycoproteins

The efficiency of viruses in cancer therapy is enhanced by proteins that mediate the fusion of infected cells with their neighbors. It was reported that replication-competent adenovirus particles can spread between nuclei within fusion-generated syncytia. To assess this conjecture, we generated fusogenic adenoviruses that express a balanced ratio of the F and H glycoproteins of measles virus. The viruses displayed enhanced cytotoxicity but largely unchanged replication efficiencies compared to a nonfusogenic virus. Most notably, the virus genomes did not spread through fusion-generated multinuclear cells. Hence, adenovirus replication in syncytia remains largely restricted to initially transduced nuclei.     

Identification of candidate substrates for ectodomain shedding by the metalloprotease-disintegrin ADAM8

ADAM proteases are type I transmembrane proteins with extracellular metalloprotease domains. As for most ADAM family members, ADAM8 (CD156a, MS2) is involved in ectodomain shedding of membrane proteins and is linked to inflammation and neurodegeneration. To identify potential substrates released under these pathologic conditions, we screened 10-mer peptides representing amino acid sequences from extracellular domains of various membrane proteins using the ProteaseSpot system. A soluble ADAM8 protease containing a pro- and metalloprotease domain was expressed in E. coli and purified as active protease owing to autocatalytic prodomain removal. From 34 peptides tested in the peptide cleavage assay, significant cleavage by soluble ADAM8 was observed for 14 peptides representing membrane proteins with functions in inflammation and neurodegeneration, among them the beta-amyloid precursor protein (APP). The in vivo relevance of the ProteaseSpot method was confirmed by cleavage of full-length APP with ADAM8 in human embryonic kidney 293 cells expressing tagged APP. ADAM8 cleaved APP with similar efficiency as ADAM10, whereas the inactive ADAM8 mutant did not. Exchanging amino acids at defined positions in the cleavage sequence of myelin basic protein (MBP) revealed sequence criteria for ADAM8 cleavage. Taken together, the results allowed us to identify novel candidate substrates that could be cleaved by ADAM8 in vivo under pathologic conditions.     

The stability of transmembrane helix interactions measured in a biological membrane

Despite some promising progress in the understanding of membrane protein folding and assembly, there is little experimental information regarding the thermodynamic stability of transmembrane helix interactions and even less on the stability of transmembrane helix-helix interactions in a biological membrane. Here we describe an approach that allows quantitative measurement of transmembrane helix interactions in a biological membrane, and calculation of changes in the interaction free energy resulting from substitution of single amino acids. Dimerization of several variants of the glycophorin A transmembrane domain are characterized and compared to the wild-type (wt) glycophorin A transmembrane helix dimerization. The calculated DeltaDeltaG(app) values are further compared with values found in the literature. In addition, we compare interactions between the wt glycophorin A transmembrane domain and helices in which critical glycine residues are replaced by alanine or serine, respectively. The data demonstrate that replacement of the glycine residues by serine is less destabilizing than replacement by alanine with a DeltaDeltaG(app) value of about 0.4 kcal/mol. Our study comprises the first measurement of a transmembrane helix interaction in a biological membrane, and we are optimistic that it can be further developed and applied.     

Determination of glucose metabolites in stored erythrocytes and in erythrocytes from patients with thalassemia by analytical isotachophoresis

Glycolysis is for some cells, such as erythrocytes, neutrophil granulocytes and many cancer cells, the only or most important source of energy (ATP) production. Based on previous studies we developed an isotachophoretic (ITP) method which allows, in principle, the simultaneous determination of all metabolites of glycolysis. Since glucose metabolites are small anions, mobility of some of them may overlap in isotachophoresis and, therefore, partial mixed zones are generated. By variation of the leading/terminating system, however, it is possible to separate the compounds of interest. In this communication, we describe a method for analysis of glucose metabolites in erythrocytes from healthy donors during storage in blood bags, and from patients with thalassemia, with special respect to intracellular 2,3 bisphosphoglycerate, lactate and ATP/ADP. The well known characteristic changes of glycolysis in erythrocytes during blood storage and in erythrocytes from thalassemia patients, which are often analysed by separate enzymatic assays, could be confirmed with this isotachophoretic procedure. The method is currently adapted for analysis of glycolysis in neutrophil granulocytes and cancer cells which requires some modifications of sample preparation and performance of the isotachophoretic analysis.