Direct duplication 12p11.21–p13.31 mediated by segmental duplications: a new recurrent rearrangement?

We describe the characterization of an interstitial duplication of 12p, dup(12)(p11.21p13.31), by array-CGH and FISH in a patient with mental retardation and dysmorphic features. The sequence analysis of the breakpoints revealed the presence of homologous low copy repeats (LCRs) flanking the duplication region, thus suggesting that they have mediated the rearrangement. Pip-maker analysis showed that a third cluster of homologous LCRs lie distally to the two mediating the 12p duplication. We hypothesize that this duplication might be a new recurrent rearrangement and that, thanks to the different orientations of the homologous regions lying within each cluster, the three clusters are responsible for at least some of the several 12p aneuploidies reported in the literature such as direct and inverted duplications, deletions and supernumerary analphoid chromosomes. Moreover, we excluded that polymorphic inversions between these three clusters are present in the normal population.Manuela De Gregori, Tiziano Pramparo contributed equally to this paper.     

Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance

The RecQ helicase Sgs1p forms a complex with the type 1 DNA topoisomerase Top3p that resolves double Holliday junctions resulting from Rad51-mediated exchange. We find, however, that Sgs1p functions independently of both Top3p and Rad51p to stimulate the checkpoint kinase Rad53p when replication forks stall due to dNTP depletion on hydroxyurea. Checkpoint activation does not require Sgs1p function as a helicase, and correlates with its ability to bind the Rad53p kinase FHA1 motif directly. On the other hand, Sgs1p's helicase activity is required together with Top3p and the strand-exchange factor Rad51p, to help stabilise DNA polymerase epsilon at stalled replication forks. In this function, the Sgs1p/Top3p complex acts in parallel to the Claspin-related adaptor, Mrc1p, although the sgs1 and mrc1 mutations are epistatic for Rad53p activation. We thus identify two distinct pathways through which Sgs1p contributes to genomic integrity: checkpoint kinase activation requires Sgs1p as a noncatalytic Rad53p-binding site, while the combined Top3p/Sgs1p resolvase activity contributes to replisome stability and recovery from arrested replication forks.     

Beta1 integrins regulate mammary gland proliferation and maintain the integrity of mammary alveoli

Integrin-extracellular matrix interactions play important roles in the coordinated integration of external and internal cues that are essential for proper development. To study the role of beta1 integrin in the mammary gland, Itgbeta1(flox/flox) mice were crossed with WAPiCre transgenic mice, which led to specific ablation of beta1 integrin in luminal alveolar epithelial cells. In the beta1 integrin mutant mammary gland, individual alveoli were disorganized resulting from alterations in cell-basement membrane associations. Activity of focal adhesion kinase (FAK) was also decreased in mutant mammary glands. Luminal cell proliferation was strongly inhibited in beta1 integrin mutant glands, which correlated with a specific increase of p21 Cip1 expression. In a p21 Cip1 null background, there was a partial rescue of BrdU incorporation, providing in vivo evidence linking p21 Cip1 to the proliferative defect observed in beta1 integrin mutant glands. A connection between p21 Cip1 and beta1 integrin as well as FAK was also established in primary mammary cells. These results point to the essential role of beta1 integrin signaling in mammary epithelial cell proliferation.     

5-Lipoxygenase regulates senescence-like growth arrest by promoting ROS-dependent p53 activation

5-Lipoxygenase (5LO) is involved in the production of leukotrienes and reactive oxygen species (ROS) from arachidonic acid. Its strong activation has been associated with several diseases like cancer and neurodegeneration. Here we show that 5LO activity increases during senescence-like growth arrest induced by oncogenic ras or culture history in both human and mouse embryo fibroblasts. Overexpression of 5LO promotes senescence-like growth arrest via a p53/p21-dependent pathway, and this occurs independently of telomerase activity. 5LO stabilizes p53 through phosphorylation at Ser15 and increases expression of the p53-transcriptional target p21. This is achieved by regulating ROS production. Indeed, ROS are increased in 5LO-arrested cells. Antioxidants and a low oxygen environment prevent 5LO-induced growth arrest. Finally, 5LO inhibition reduces the growth arrest induced by oncogenic ras or culture history and these effects are neutralized by the addition of exogenous ROS. These data link the 5LO pathway to oxidative crises of primary fibroblast and suggest that the ability of 5LO to induce senescence-like growth arrest may be important in the pathogenesis of 5LO-associated disorders.     

Degradation of wild-type alpha-synuclein by a molecular chaperone leads to reduced aggregate formation

Alpha-synuclein, a presynaptic protein, was found to be the major component in the Lewy bodies (LB) in an age-related neurodegenerative disease, Parkinson's disease (PD). Even though the function of alpha-synuclein is not completely understood, it has been demonstrated to spontaneously aggregate into amyloid fibrils. With the aim of inhibiting aggregate formation, a molecular chaperone protein, Hsp104p, was investigated since it rescues cells from stress by resolubilizing denatured proteins from insoluble aggregates, in vivo as well as in vitro. Here, in order to examine whether Hsp104p functions as a regulator of aggregate formation for alpha-synuclein, we expressed the His-tagged wild-type (wt) synuclein and the glutathione-S-transferase (GST)-tagged Hsp104p in bacterial systems. Using thioflavin-T fluorescence assays, significant protection against fibril formation was observed with wt Hsp104p regardless of the presence of ATP, but not with mutant Hsp104p. To a lesser extent, the dissociation effect of wild-type Hsp104p was observed only in the presence of ATP. Interaction between Hsp104p and synuclein was also investigated using a GST pull-down experiment. Interestingly, Hsp104p degraded alpha-synuclein in a concentration-dependent manner with the synergistic assistance of ATP. These results suggest that Hsp104p could be developed as a therapeutic candidate in the treatment of protein aggregation-related neurodegenerative disease.     

Dimerization of the p53 oligomerization domain: identification of a folding nucleus by molecular dynamics simulations

Dimerization of the p53 oligomerization domain involves coupled folding and binding of monomers. To examine the dimerization, we have performed molecular dynamics (MD) simulations of dimer folding from the rate-limiting transition state ensemble (TSE). Among 799 putative transition state structures that were selected from a large ensemble of high-temperature unfolding trajectories, 129 were identified as members of the TSE via calculation of a 50% transmission coefficient from at least 20 room-temperature simulations. This study is the first to examine the refolding of a protein dimer using MD simulations in explicit water, revealing a folding nucleus for dimerization. Our atomistic simulations are consistent with experiment and offer insight that was previously unobtainable.     

Structural determinants of substrate access to the disulfide oxidase Erv2p

Erv2p is a small, dimeric FAD-dependent sulfhydryl oxidase that generates disulfide bonds in the lumen of the endoplasmic reticulum. Mutagenic and structural studies suggest that Erv2p uses an internal thiol-transfer relay between the FAD-proximal active site cysteine pair (Cys121-Cys124) and a second cysteine pair (Cys176-Cys178) located in a flexible, substrate-accessible C-terminal tail of the adjacent dimer subunit. Here, we demonstrate that Cys176 and Cys178 are the only amino acids in the tail region required for disulfide transfer and that their relative positioning within the tail peptide is important for activity. However, intragenic suppressor mutations could be isolated that bypass the requirement for Cys176 and Cys178. These mutants were found to disrupt Erv2p dimerization and to increase the activity of Erv2p for thiol substrates such as glutathione. We propose that the two Erv2p subunits act together to direct the disulfide transfer to specific substrates. One subunit provides the catalytic domain composed of the active site cysteine residues and the FAD cofactor, while the second subunit appears to have two functions: it facilitates disulfide transfer to substrates via the tail cysteine residues, while simultaneously shielding the active site cysteine residues from non-specific reactions.     

Characterisation of the Rac/PAK pathway in Amoeba proteus

Summary. Molecular mechanisms underlying the unique locomotion of the highly motile Amoeba proteus still remain poorly understood. Recently, we have shown that blocking the endogenous amoebal Rac-like protein(s) leads to distinct and irreversible changes in the appearance of these large migrating cells as well as to a significant inhibition of their locomotion. To elucidate the mechanism of the Rac pathway in Amoeba proteus, we tested the effects of blocking the endogenous myosin I heavy chain kinase (MIHCK), one of the Rac effectors in Acanthamoeba castellanii and Dictyostelium discoideum, with anti-MIHCK antibodies in migrating amoebae, as well as the effect of inhibiting Rac and MIHCK on the actin polymerisation process. Antibodies against A. castellanii MIHCK detected an A. proteus protein with a molecular mass (ca. 95 kDa) similar to the A. castellanii kinase. The cellular distribution of MIHCK in A. proteus was very similar to those of Rac-like protein in amoebae and MIHCK in A. castellanii. Amoebae microinjected with anti-MIHCK antibodies moved slower and protruded fewer wide pseudopodia (5–6) than the control cells (9–10), resembling to some extent the phenotype of cells microinjected with anti-Rac antibodies. The in vitro studies indicate that the A. proteus Rac-like protein, but not the MIHCK isoform, is engaged in the regulation of the nucleation step of the actin polymerisation process. These observations suggest that MIHCK may be one of the effectors for Rac in these extremely large cells. Correspondence and reprints: Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur ulica, 02-093 Warsaw, Poland.     

Evidence of a dominant negative mutant of yeast methionine aminopeptidase type 2 in Saccharomyces cerevisiae

Eukaryotic methionine aminopeptidase type 2 (MetAP2, MetAP2 gene (MAP2)), together with eukaryotic MetAP1, cotranslationally hydrolyzes initiator methionine from nascent polypeptides when the side chain of the second residue is small and uncharged. In this report, we took advantage of the yeast (Saccharomyces cerevisiae) map1 null strain's reliance on MetAP2 activity for the growth and viability to provide evidence of the first dominant negative mutant of eukaryotic MetAP2. Replacement of the conserved His(174) with alanine within the C-terminal catalytic domain of yeast MetAP2 eliminated detectable catalytic activity against a peptide substrate in vitro. Overexpression of MetAP2 (H174A) under the strong GPD promoter in a yeast map1 null strain was lethal, whereas overexpression under the weaker GAL1 promoter slightly inhibited map1 null growth. Deletion mutants further revealed that the N-terminal region of MetAP2 (residues 2-57) is essential but not sufficient for MetAP2 (H174A) to fully interfere with map1 null growth. Together, these results indicate that catalytically inactive MetAP2 is a dominant negative mutant that requires its N-terminal region to interfere with wild-type MetAP2 function.     

Dexamethasone upregulates the expression of the nuclear cofactor p300 and its interaction with C/EBPbeta in cultured myotubes

Muscle wasting during sepsis and other catabolic conditions is, at least in part, mediated by glucocorticoids and is associated with upregulated transcription of multiple genes in the ubiquitin-proteasome proteolytic pathway. In addition to transcription factors, nuclear cofactors, including p300, regulate gene transcription. We tested the hypothesis that glucocorticoids upregulate the expression of p300 in muscle cells. Treatment of cultured L6 myotubes, a rat skeletal muscle cell line, with dexamethasone resulted in a dose- and time-dependent increase in p300 protein and mRNA levels. Surprisingly, the effect of dexamethasone on p300 levels was not inhibited by the glucocorticoid receptor (GR) antagonist RU38486 and RU38486 exerted an agonist effect on p300, increasing its expression. Co-immunoprecipitation showed that treatment of the myotubes with dexamethasone resulted in protein-protein interaction between p300 and C/EBPbeta, but not C/EBPdelta. The present results suggest that glucocorticoids upregulate the expression of p300 and its interaction with C/EBPbeta in skeletal muscle. Increased expression and activity of p300 may be involved in the regulation of gene transcription in glucocorticoid-dependent muscle wasting.