Michal Dadlez

CENP-C Is a Structural Platform for Kinetochore Assembly

Centromeres provide a region of chromatin upon which kinetochores are assembled in mitosis. Centromeric protein C (CENP-C) is a core component of this centromeric chromatin that, when depleted, prevents the proper formation of both centromeres and kinetochores. CENP-C localizes to centromeres throughout the cell cycle via its C-terminal part, whereas its N-terminal part appears necessary for recruitment of some but not all components of the Mis12 complex of the kinetochore. We now find that all kinetochore proteins belonging to the KMN (KNL1/Spc105, the Mis12 complex, and the Ndc80 complex) network bind to the N-terminal part of Drosophila CENP-C. Moreover, we show that the Mis12 complex component Nnf1 interacts directly with CENP-C in vitro. To test whether CENP-Cs N-terminal part was sufficient to recruit KMN proteins, we targeted it to the centrosome by fusing it to a domain of Plk4 kinase. The Mis12 and Ndc80 complexes and Spc105 protein were then all recruited to centrosomes at the expense of centromeres, leading to mitotic abnormalities typical of cells with defective kinetochores. Thus, the N-terminal part of Drosophila CENP-C is sufficient to recruit core kinetochore components and acts as the principal linkage between centromere and kinetochore during mitosis.

Regulation of Nicotiana tabacum osmotic stress-activated protein kinase and its cellular partner GAPDH by nitric oxide in response to salinity

Several studies focusing on elucidating the mechanism of NO (nitric oxide) signalling in plant cells have highlighted that its biological effects are partly mediated by protein kinases. The identity of these kinases and details of how NO modulates their activities, however, remain poorly investigated. In the present study, we have attempted to clarify the mechanisms underlying NO action in the regulation of NtOSAK (Nicotiana tabacum osmotic stress-activated protein kinase), a member of the SNF1 (sucrose non-fermenting 1)-related protein kinase 2 family. We found that in tobacco BY-2 (bright-yellow 2) cells exposed to salt stress, NtOSAK is rapidly activated, partly through a NO-dependent process. This activation, as well as the one observed following treatment of BY-2 cells with the NO donor DEA/NO (diethylamine-NONOate), involved the phosphorylation of two residues located in the kinase activation loop, one being identified as Ser158. Our results indicate that NtOSAK does not undergo the direct chemical modifications of its cysteine residues by S-nitrosylation. Using a co-immunoprecipitation-based strategy, we identified several proteins present in immunocomplex with NtOSAK in salt-treated cells including the glycolytic enzyme GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Our results indicate that NtOSAK directly interacts with GAPDH in planta. Furthermore, in response to salt, GAPDH showed a transient increase in its S-nitrosylation level which was correlated with the time course of NtOSAK activation. However, GADPH S-nitrosylation did not influence its interaction with NtOSAK and did not have an impact on the activity of the protein kinase. Taken together, the results support the hypothesis that NtOSAK and GAPDH form a cellular complex and that both proteins are regulated directly or indirectly by NO.

Cildb: a knowledgebase for centrosomes and cilia

Ciliopathies, pleiotropic diseases provoked by defects in the structure or function of cilia or flagella, reflect the multiple roles of cilia during development, in stem cells, in somatic organs and germ cells. High throughput studies have revealed several hundred proteins that are involved in the composition, function or biogenesis of cilia. The corresponding genes are potential candidates for orphan ciliopathies. To study ciliary genes, model organisms are used in which particular questions on motility, sensory or developmental functions can be approached by genetics. In the course of high throughput studies of cilia in Paramecium tetraurelia, we were confronted with the problem of comparing our results with those obtained in other model organisms. We therefore developed a novel knowledgebase, Cildb, that integrates ciliary data from heterogeneous sources. Cildb links orthology relationships among 18 species to high throughput ciliary studies, and to OMIM data on human hereditary diseases. The web interface of Cildb comprises three tools, BioMart for complex queries, BLAST for sequence homology searches and GBrowse for browsing the human genome in relation to OMIM information for human diseases. Cildb can be used for interspecies comparisons, building candidate ciliary proteomes in any species, or identifying candidate ciliopathy genes. Database URL: http://cildb.cgm.cnrs-gif.fr

Plk4 Phosphorylates Ana2 to Trigger Sas6 Recruitment and Procentriole Formation

Summary Centrioles are 9-fold symmetrical structures at the core of centrosomes and base of cilia whose dysfunction has been linked to a wide range of inherited diseases and cancer [1]. Their duplication is regulated by a protein kinase of conserved structure, the C. elegans ZYG-1 or its Polo-like kinase 4 (Plk4) counterpart in other organisms [2–4]. Although Plk4’s centriolar partners and mechanisms that regulate its stability are known, its crucial substrates for centriole duplication have never been identified. Here we show that Drosophila Plk4 phosphorylates four conserved serines in the STAN motif of the core centriole protein Ana2 to enable it to bind and recruit its Sas6 partner. Ana2 and Sas6 normally load onto both mother and daughter centrioles immediately after their disengagement toward the end of mitosis to seed procentriole formation. Nonphosphorylatable Ana2 still localizes to the centriole but can no longer recruit Sas6 and centriole duplication fails. Thus, following centriole disengagement, recruitment of Ana2 and its phosphorylation by Plk4 are the earliest known events in centriole duplication to recruit Sas6 and thereby establish the architecture of the new procentriole engaged with its parent.

Ion mobility separation coupled with MS detects two structural states of Alzheimer's disease Aβ1-40 peptide oligomers.

Mounting evidence points to the soluble oligomers of amyloid β (Aβ) peptide as important neurotoxic species in Alzheimers disease, causing synaptic dysfunction and neuronal injury, and finally leading to neuronal death. The mechanism of the Aβ peptide self-assembly is still under debate. Here, Aβ1-40 peptide oligomers were studied using mass spectrometry combined with ion mobility spectrometry, which allowed separation of the signals of numerous oligomers and measurement of their collisional cross-section values (Ω). For several oligomers, at least two different species of different Ω values were detected, indicating the presence of at least two families of conformers: compact and extended. The obtained results are rationalized by a set of molecular models of Aβ1-40 oligomer structure that provided a very good correlation between the experimental and theoretical Ω values, both for the compact and the extended forms. Our results indicate that mass spectrometry detects oligomeric species that are on-pathway in the process of fibril formation or decay, but also alternative structures which may represent off-pathway evolution of oligomers.

Integrating proteomic and transcriptomic high-throughput surveys for search of new biomarkers of colon tumors

To the search of new colon tumor biomarkers in the transition from normal colon (NC) mucosa to adenoma (AD) and adenocarcinoma (AC), we integrated microarray data with the results of a high-throughput proteomic workflow. In proteomic study, we used a modified isoelectric focusing protocol on strips with an immobilized pH gradient to separate peptides labeled with iTRAQ (isobaric tags for relative and absolute quantitation) tags followed by liquid chromatography–tandem mass spectrometry analysis. Gene expression measurements were done using Affymetrix GeneChip HG-U133plus2 microarrays and quantitative reverse transcriptase PCR (q-RT-PCR). We identified 3,886 proteins with at least two peptides. Of them, 1,061 proteins were differentially expressed [FC ≥ 1.5; FDR ≤ 0.01] in two pair-wise comparisons: AD vs. NC and AC vs. AD while 15 and 23 proteins were progressively up-regulated and down-regulated in the NC/AD/AC sequence, respectively. The quantitative proteomic information was subsequently correlated with microarray data. For a collection of genes with the same direction of changes of both mRNA and protein levels, we obtained 785/853/795 genes in AD vs. NC/AC vs. NC/AC vs. AD comparison, respectively. Further evaluation of sequentially altered gene expression by q-RT-PCR on individual samples of 24 NCs, 42 ADs, and 26 ACs confirmed progressive expression of six genes: biglycan, calumenin, collagen type XII, alpha 1 (COL12A1), monoamine oxidase A (MAOA), ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5), and MOCO sulphurase C-terminal domain-containing 2 (MOSC2). Among them, three continuously down-regulated (MAOA, ENTPD5, and MOSC2) and one continuously overexpressed (COL12A1) are reported, to our best knowledge, for the first time in a connection to colon cancer onset.

Trimethylamine-N-oxide: a carnitine-derived metabolite that prolongs the hypertensive effect of angiotensin II in rats.

BACKGROUND Recent evidence suggests that an elevated plasma trimethylamine N-oxide (TMAO) level is associated with an increased risk of adverse cardiovascular events in humans; however, the mechanism is not clear. The aims of this study were to establish the plasma TMAO level in rats and to evaluate the effect of TMAO on arterial blood pressure (BP) and the hemodynamic effects of angiotensin II (Ang II). METHODS Twelve-week-old, Sprague-Dawley rats were implanted with telemetric transmitters, and continuous recordings of heart rate, systolic BP (SBP), and diastolic BP (DBP) were made for 7 days before and 14 days during osmotic minipump-driven subcutaneous infusion of saline (controls), TMAO, low-dose Ang II, or Ang II + TMAO. Plasma TMAO concentration was evaluated using liquid chromatography coupled with triple-quadrupole mass spectrometry. RESULTS The plasma TMAO concentration in controls was 0.57 μmol/L, whereas in TMAO-infused rats it was 58 μmol/L. Neither saline nor TMAO infusion affected SBP and DBP. Infusion of Ang II significantly increased SBP and DBP for the first 5 days of infusion only. In contrast, infusion of Ang II + TMAO produced a hypertensive response that lasted until the end of the experiment. TMAO infusions did not affect body weight and motor activity. CONCLUSIONS We showed that physiological plasma TMAO concentration in rats was approximately 10 times lower than that reported in humans. Furthermore, the new finding of the study is that TMAO does not affect BP in normotensive animals. However, it prolongs the hypertensive effect of Ang II.

Discovery of novel potent ΔF508-CFTR correctors that target the nucleotide binding domain

The deletion of Phe508 (ΔF508) in the first nucleotide binding domain (NBD1) of CFTR is the most common mutation associated with cystic fibrosis. The ΔF508‐CFTR mutant is recognized as improperly folded and targeted for proteasomal degradation. Based on molecular dynamics simulation results, we hypothesized that interaction between ΔF508‐NBD1 and housekeeping proteins prevents ΔF508‐CFTR delivery to the plasma membrane. Based on this assumption we applied structure‐based virtual screening to identify new low‐molecular‐weight compounds that should bind to ΔF508‐NBD1 and act as protein–protein interaction inhibitors. Using different functional assays for CFTR activity, we demonstrated that in silico‐selected compounds induced functional expression of ΔF508‐CFTR in transfected HeLa cells, human bronchial CF cells in primary culture, and in the nasal epithelium of homozygous ΔF508‐CFTR mice. The proposed compounds disrupt keratin8‐ΔF508‐CFTR interaction in ΔF508‐CFTR HeLa cells. Structural analysis of ΔF508‐NBD1 in the presence of these compounds suggests their binding to NBD1. We conclude that our strategy leads to the discovery of new compounds that are among the most potent correctors of ΔF508‐CFTR trafficking defect known to date.

A Direct Determination of the Dissociation Constant for the Cu(II) Complex of Amyloid β 1-40 Peptide

Interactions of amyloid beta (Abeta) peptides with Cu(II) are believed to play a crucial role in the molecular mechanisms of neurotoxicity of Alzheimers disease. There is, however, a serious disagreement regarding the strength of Cu(II) binding to these peptides. We used recombinant amyloid beta peptide 1-40 (Abeta40) to determine the stoichiometry and dissociation constants of Cu(II)-Abeta40 complexes using fluorescence spectroscopy. A single Cu(Abeta40) complex, characterized with the conditional dissociation constant K(d)(cond) = 57 +/- 5 nM was identified. This complex does not bind Hepes buffer molecules, as indicated by the total lack of relationship between K(d)(cond) values and Hepes concentration. The differences between this and other determinations of this constant and its relevance for the understanding of Cu(II) interaction with Abeta peptides are discussed.

Suppression of Scant Identifies Endos as a Substrate of Greatwall Kinase and a Negative Regulator of Protein Phosphatase 2A in Mitosis

Protein phosphatase 2A (PP2A) plays a major role in dephosphorylating the targets of the major mitotic kinase Cdk1 at mitotic exit, yet how it is regulated in mitotic progression is poorly understood. Here we show that mutations in either the catalytic or regulatory twins/B55 subunit of PP2A act as enhancers of gwlScant, a gain-of-function allele of the Greatwall kinase gene that leads to embryonic lethality in Drosophila when the maternal dosage of the mitotic kinase Polo is reduced. We also show that heterozygous mutant endos alleles suppress heterozygous gwlScant; many more embryos survive. Furthermore, heterozygous PP2A mutations make females heterozygous for the strong mutation polo11 partially sterile, even in the absence of gwlScant. Heterozygosity for an endos mutation suppresses this PP2A/polo11 sterility. Homozygous mutation or knockdown of endos leads to phenotypes suggestive of defects in maintaining the mitotic state. In accord with the genetic interactions shown by the gwlScant dominant mutant, the mitotic defects of Endos knockdown in cultured cells can be suppressed by knockdown of either the catalytic or the Twins/B55 regulatory subunits of PP2A but not by the other three regulatory B subunits of Drosophila PP2A. Greatwall phosphorylates Endos at a single site, Ser68, and this is essential for Endos function. Together these interactions suggest that Greatwall and Endos act to promote the inactivation of PP2A-Twins/B55 in Drosophila. We discuss the involvement of Polo kinase in such a regulatory loop.