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The Phosphatase PP1 Promotes Mitotic Slippage through Mad3 Dephosphorylation

International audienceAccurate chromosome segregation requires bipolar attachment of kinetochores to spindle microtubules. A conserved surveillance mechanism, the spindle assembly checkpoint (SAC), responds to lack of kinetochore-microtubule connections and delays anaphase onset until all chromosomes are bipolarly attached [1]. SAC signaling fires at kinetochores and involves a soluble mitotic checkpoint complex (MCC) that inhibits the anaphase-promoting complex (APC) [2, 3]. The mitotic delay imposed by SAC, however, is not everlasting. If kinetochores fail to establish bipolar connections, cells can escape from the SAC-induced mitotic arrest through a process called mitotic slippage [4]. Mitotic slippage occurs in the presence of SAC signaling at kinetochores [5, 6], but whether and how MCC stability and APC inhibition are actively controlled during slippage is unknown. The PP1 phosphatase has emerged as a key factor in SAC silencing once all kinetochores are bipolarly attached [7, 8]. PP1 turns off SAC signaling through dephosphorylation of the SAC scaffold Knl1/Blinkin at kinetochores [9-11]. Here, we show that, in budding yeast, PP1 is also required for mitotic slippage. However, its involvement in this process is not linked to kinetochores but rather to MCC stability. We identify S268 of Mad3 as a critical target of PP1 in this process. Mad3 S268 dephosphorylation destabilizes the MCC without affecting the initial SAC-induced mitotic arrest. Conversely, it accelerates mitotic slippage and overcomes the slippage defect of PP1 mutants. Thus, slippage is not the mere consequence of incomplete APC inactivation that brings about mitotic exit, as originally proposed, but involves the exertive antagonism between kinases and phosphatases

Chaperone-mediated ordered assembly of the SAGA and NuA4 transcription co-activator complexes in yeast

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Comparison of Hydrophobic, Lipophilic and Immunodepletion Pre- Fractionation Methods for Label-Free LC-MS/MS Identification of Biomarkers in Human Cerebrospinal Fluid

International audienceBackground: Proteomics analysis of human cerebrospinal fluid (CSF) is a major tool for identifying novel biomarkers for neurological diseases. However, the complexity and wide dynamic range of CSF represent a major challenge for detecting specific low-abundance biomarkers. One way to overcome this problem is to rely on different pre-fractionation techniques. However, the most relevant technique remains to be determined. Methods: This study compared three different well-known pre-fractionation methods: immuno-depletion of major proteins (Seppro® IgY14), hydrophobic solid phase extraction (Oasis® HLB), and lipophilic sorbent concentration (Liposorb™). Unfractionated and pre-fractionated CSF was digested with trypsin and analyzed by RP-LC-MS/MS with an OrbitrapTM mass spectrometer. We documented the number of peptides detected and sets of proteins identified. Experiments were repeated to minimize pre-analytical and analytical variability.Results: Compared to unfractionated CSF, the OASIS® HLB fractionated CSF method showed a significant 28% increase in the total number of proteins identified, while the Liposorb™ capture resulted in a significant 46% decrease. Interestingly, results based on the number of peptides detected were different. We also evaluated the capacity of these pre-fractionation methods to detect different proteins in terms of their molecular weight, isoelectrophoretic point (IEP) or nature. Each of these pre-fractionation methods identified a specific subset of proteins, when compared to unfractionated CSF, and/or other methods. This was particularly obvious for the lipophilic sorbent, which allowed the detection of many lipoproteins.Conclusion: Direct analysis of digested CSF led to the identification of several proteins despite matrix complexity. As expected, single pre-fractionation methods that can be included in simple and cost-effective workflows, yielded significant differences in terms of number, or range of proteins identified. This suggests that a single pre-fractionation method cannot cover the full range of protein species present in a complex sampl

Cloning, expression, molecular characterization and preliminary studies on immunomodulating properties of recombinant Trypanosoma congolense calreticulin

Trypanosomes are bloodstream protozoan parasites, which are pathogens of veterinary and medical importance. Several mammalian species, including humans, can be infected by different species of the genus Trypanosoma (T. congolense, T. evansi, T. brucei, T. vivax) exhibiting more or less virulent and pathogenic phenotypes. A previous screening of the excreted-secreted proteins of T. congolense demonstrated an overexpression of several proteins correlated with the virulence and pathogenicity of the strain. Of these proteins, calreticulin (CRT) has shown differential expression between two T. congolense strains with opposite infectious behavior and has been selected as a target molecule based on its immune potential functions in parasitic diseases. In this study, we set out to determine the role of T. congolense calreticulin as an immune target. Immunization of mice with recombinant T. congolense calreticulin induced antibody production, which was associated with delayed parasitemia and increased survival of the challenged animal. These results strongly suggest that some excreted-secreted proteins of T. congolense are a worthwhile target candidate to interfere with the infectious process

Dynamic interactions of the 5-HT 6 receptor with protein partners control dendritic tree morphogenesis

International audienceThe serotonin (5-hydroxytrypatmine) receptor 5-HT6 (5-HT6R) has emerged as a promising target to alleviate the cognitive symptoms of neurodevelopmental diseases. We previously demonstrated that 5-HT6R finely controls key neurodevelopmental steps, including neuronal migration and the initiation of neurite growth, through its interaction with cyclin-dependent kinase 5 (Cdk5). Here, we showed that 5-HT6R recruited G protein-regulated inducer of neurite outgrowth 1 (GPRIN1) through a Gs-dependent mechanism. Interactions between the receptor and either Cdk5 or GPRIN1 occurred sequentially during neuronal differentiation. The 5-HT6R-GPRIN1 interaction enhanced agonist-independent, receptor-stimulated cAMP production without altering the agonist-dependent response in NG108-15 neuroblastoma cells. This interaction also promoted neurite extension and branching in NG108-15 cells and primary mouse striatal neurons through a cAMP-dependent protein kinase A (PKA)-dependent mechanism. This study highlights the complex allosteric modulation of GPCRs by protein partners and demonstrates how dynamic interactions between GPCRs and their protein partners can control the different steps of highly coordinated cellular processes, such as dendritic tree morphogenesis

SNAP23-Kif5 complex controls mGlu1 receptor trafficking

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Depletion of one, six, twelve or twenty major blood proteins before proteomic analysis: the more the better?

International audienceDepletion of major blood proteins is one of the most promising approaches to access low abundant biomarkers using proteomics. Immunocapture columns often used for this purpose exist in different formats depending on the number of major proteins removed. In this article, we compared the relative interest of depleting either one (albumin), six (albumin, IgG, IgA, transferrin, α1-antitrypsin, and haptoglobin), twelve (the previous six and apo A-I and-II, orosomucoid, α2-macroglobulin, fibrinogen, IgM) or twenty blood proteins (the previous twelve and IgD, ceruloplasmin, apo B, complement C1q, C3, C4, plasminogen, and prealbumin). Such study raises interesting issues related to the reproducibility, practicability, specificity of the immunocapture, and to the impact of removing not only the selected molecules, but also associated peptides and proteins. Depleted sera were here analysed using different proteomic approaches, including two dimensional electrophoresis and SELDI–TOF. Altogether, our results clearly confirmed the interest of depleting major blood proteins for the proteomic detection of low abundant components. However, we observed that increasing the number of depleted proteins from twelve to twenty had a limited beneficial impact and might increase drawbacks in removing associated peptides and proteins. This conclusion is however related to the technologies that we have used, and we believe that it is necessary to adapt the immunocapture to the analytical method employed, and to the ratio between wanted and unwanted proteins removed

Trypanosoma cruzi: gene expression surveyed by proteomic analysis reveals interaction between different genotypes in mixed in vitro cultures.

We have analyzed the comportment in in vitro culture of 2 different genotypes of Trypanosoma cruzi, the agent of Chagas disease, pertaining to 2 major genetic subdivisions (near-clades) of this parasite. One of the stocks was a fast-growing one, highly virulent in mice, while the other one was slow-growing, mildly virulent in mice. The working hypothesis was that mixtures of genotypes interact, a pattern that has been observed by us in empirical experimental studies. Genotype mixtures were followed every 7 days and characterized by the DIGE technology of proteomic analysis. Proteic spots of interest were characterized by the SAMESPOT software. Patterns were compared to those of pure genotypes that were also evaluated every 7 days. One hundred and three spots exhibited changes in time by comparison with T = 0. The major part of these spots (58%) exhibited an under-expression pattern by comparison with the pure genotypes. 32% of the spots were over-expressed; 10% of spots were not different from those of pure genotypes. Interestingly, interaction started a few minutes after the mixtures were performed. We have retained 43 different proteins that clearly exhibited either under- or over-expression. Proteins showing interaction were characterized by mass spectrometry (MALDI-TOF). Close to 50% of them were either tubulins or heat shock proteins. This study confirms that mixed genotypes of T. cruzi interact at the molecular level. This is of great interest because mixtures of genotypes are very frequent in Chagas natural cycles, both in insect vectors and in mammalian hosts, and may play an important role in the transmission and severity of Chagas disease. The methodology proposed here is potentially applicable to any micropathogen, including fungi, bacteria and viruses. It should be of great interest in the case of bacteria, for which the epidemiological and clinical consequences of mixed infections could be underestimated