QC in proteomics: lessons from proteored multicentric experiments

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Myogenin protein stability is decreased by BMP-2 through a mechanism implicating Id1

Bone morphogenetic protein-2 (BMP-2) induces a switch in differentiation of mesenchymal cells from the myogenic to the osteogenic lineage. Here we describe that in C2C12 cells, BMP-2 decreases myogenin expression induced by des-(1,3) insulin-like growth factor-1 (des-(1,3)IGF-1) or ectopically expressed from a constitutive promoter, even in conditions where myogenin mRNA levels were unaffected. Addition of BMP-2 decreases myogenin protein half-life to 50%, whereas proteasome inhibitors abolish these effects. Forced expression of Id1, either by transient transfection or under the control of an inducible system, causes degradation of myogenin in the absence of BMP-2. In contrast, E47 overexpression blocks the inhibitory effect of BMP-2 on myogenin levels. Finally, expression of E47 in 293 cells stabilizes myogenin, an effect that is dependent on the heterodimerization mediated by their helix-loop-helix. Our findings indicate that induction of Id1 not only blocks transcriptional activity but also induces myogenin degradation by blocking formation of myogenin-E47 protein complexes

p70 S6 kinase-mediated protein synthesis is a critical step for vascular endothelial cell proliferation

In this work, we analyzed the role of the PI3K-p70 S6 kinase (S6K) signaling cascade in the stimulation of endothelial cell proliferation. We found that inhibitors of the p42/p44 MAPK pathway (PD98059) and the PI3K-p70 S6K pathway (wortmannin, Ly294002, and rapamycin) all block thymidine incorporation stimulated by fetal calf serum in the resting mouse endothelial cell line 1G11. The action of rapamycin can be generalized, since it completely inhibits the mitogenic effect of fetal calf serum in primary endothelial cell cultures (human umbilical vein endothelial cells) and another established capillary endothelial cell line (LIBE cells). The inhibitory effect of rapamycin is only observed when the inhibitor is added at the early stages of G(0)-G(1) progression, suggesting an inhibitory action early in G(1). Rapamycin completely inhibits growth factor stimulation of protein synthesis, which perfectly correlates with the inhibition of cell proliferation. In accordance with its inhibitory action on protein synthesis, activation of cyclin D1 and p21 proteins by growth factors is also blocked by preincubation with rapamycin. Expression of a p70 S6K mutant partially resistant to rapamycin reverses the inhibitory effect of the drug on DNA synthesis, indicating that rapamycin action is via p70 S6K. Thus, in vascular endothelial cells, activation of protein synthesis via p70 S6K is an essential step for cell cycle progression in response to growth factors

Legislación de la cetrería en España y Cataluña

Descripció del recurs: 13 abril 2011.Treball presentat a la Facultat de Veterinària de la Universitat Autònoma de Barcelona.Treball presentat a l'assignatura de Deontologia i Veterinària Legal (21223

p42/p44 MAP kinase module plays a key role in the transcriptional regulation of the vascular endothelial growth factor gene in fibroblasts

Vascular Endothelial Growth Factor (VEGF) is a potent mitogen for vascular endothelial cells that has been implicated in tumor neovascularization. We show that, in hamster fibroblasts (CCL39 cells), VEGF mRNAs are expressed at low levels in serum-deprived or exponentially growing cells, whereas it is rapidly induced after stimulation of quiescent cells with serum. CCL39 derivatives, transformed with Polyoma virus or with active members of the p42/p44 mitogen-activated protein (MAP) kinase pathway, Gly/Val point mutant of Ras at position 12 (Ras-Val12), MKK1 in which Ser218 and Ser222 were mutated to Asp (MKK1-SS/DD)), express very high levels of VEGF mRNA. To analyze the contribution of the p42/p44MAP kinase in this induction, we used the CCL39-derived cell line (Raf-1:ER) expressing an estradiol-activable Raf-1. We show a time and an estradiol dose-dependent up-regulation of VEGF mRNA clearly detectable after 2 h of stimulation. The induction of VEGF mRNA in response to conditioned activation of Raf-1 is reverted by an inhibitor of MKK1, PD 098059, highlighting a specific role for the p42/p44 MAP kinase pathway in VEGF expression. Interestingly, hypoxia has an additive effect on VEGF induction in CCL39 cells stimulated by serum or in Raf-1:ER cells stimulated by estradiol. In contrast to VEGF, the isoforms VEGF-B and VEGF-C are poorly regulated by growth and oncogenic factors. We have identified a GC-rich region of the VEGF promoter between -88 and -66 base pairs which contains all the elements responsible of its up-regulation by constitutive active Ras or MKK1-SS/DD. By mutation of the putative binding sites and electrophoretic mobility supershift experiments, we showed that the GC-rich region constitutively binds Sp1 and AP-2 transcription factors. Furthermore, following activation of the p42/p44 MAP kinase module, the binding of Sp1 and AP-2 is increased in the complexes formed in this region of the promoter. Altogether, these data suggest that hypoxia and p42/p44 MAP kinase independently play a key role in the regulation of the VEGF expression

A comparison of quantitative proteomics methodologies on a differential experiment on test samples

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Caracterización de un proteoma mínimo: Mycoplasma genitalium

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Funastrain c II: A Cysteine Endopeptidase Purified from the Latex of <i>Funastrum clausum</i>

A cysteine endopeptidase, named funastrain c II, was isolated and characterized from the latex of Funastrum clausum (Asclepiadaceae). The molecular mass (mass spectrometry) of the protease was 23.636 kDa. The analysis of funastrain c II by SDS-PAGE revealed a single polypeptide chain. The enzyme showed a remarkable stability of its caseinolytic activity after incubation at temperatures as high as 70°C. Inhibition and activation assays indicated the cysteinic nature of the funastrain c II catalytic site. The optimum pH of funastrain c II enzymatic activity varied according to the substrate used (9.0–10.0 for casein and 6.2–6.8 for PFLNA). Kinetic parameters were determined for N-α-CBZ-Ala p-nitrophenyl ester (Km = 0.0243 mM, kcat = 1.5 s–1) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA; KM = 0.1011 mM, kcat = 0.9 s–1). The N-terminal sequence of funastrain c II showed considerable similarity to other proteases isolated from latex of different Asclepiadaceae species as well as to other cysteine proteinases belonging to the papain family.Centro de Investigación de Proteínas Vegetale

Funastrain c II: A Cysteine Endopeptidase Purified from the Latex of <i>Funastrum clausum</i>

A cysteine endopeptidase, named funastrain c II, was isolated and characterized from the latex of Funastrum clausum (Asclepiadaceae). The molecular mass (mass spectrometry) of the protease was 23.636 kDa. The analysis of funastrain c II by SDS-PAGE revealed a single polypeptide chain. The enzyme showed a remarkable stability of its caseinolytic activity after incubation at temperatures as high as 70°C. Inhibition and activation assays indicated the cysteinic nature of the funastrain c II catalytic site. The optimum pH of funastrain c II enzymatic activity varied according to the substrate used (9.0–10.0 for casein and 6.2–6.8 for PFLNA). Kinetic parameters were determined for N-α-CBZ-Ala p-nitrophenyl ester (Km = 0.0243 mM, kcat = 1.5 s–1) and L-pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA; KM = 0.1011 mM, kcat = 0.9 s–1). The N-terminal sequence of funastrain c II showed considerable similarity to other proteases isolated from latex of different Asclepiadaceae species as well as to other cysteine proteinases belonging to the papain family.Centro de Investigación de Proteínas Vegetale