シンポジウム

Additional file 14. A table with description of strains and plasmids generated and used in this study and graphical representation of over-expressed operons/genes

Nuclear RNA foci from C9ORF72 expansion mutation form paraspeckle-like bodies

The GGGGCC (G(4)C(2)) repeat expansion mutation in the C9ORF72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Transcription of the repeat and formation of nuclear RNA foci, which sequester specific RNA-binding proteins, is one of the possible pathological mechanisms. Here, we show that (G(4)C(2))(n) repeat RNA predominantly associates with essential paraspeckle proteins SFPQ, NONO, RBM14, FUS and hnRNPH and colocalizes with known paraspeckle-associated RNA hLinc-p21. As formation of paraspeckles in motor neurons has been associated with early phases of ALS, we investigated the extent of similarity between paraspeckles and (G(4)C(2))(n) RNA foci. Overexpression of (G(4)C(2))(72) RNA results in their increased number and colocalization with SFPQ-stained nuclear bodies. These paraspeckle-like (G(4)C(2))(72) RNA foci form independently of the known paraspeckle scaffold, the long non-coding RNA NEAT1. Moreover, the knockdown of SFPQ protein in C9ORF72 expansion mutation-positive fibroblasts significantly reduces the number of (G(4)C(2))(n) RNA foci. In conclusion, (G(4)C(2))(n) RNA foci have characteristics of paraspeckles, which suggests that both RNA foci and paraspeckles play roles in FTD and ALS, and implies approaches for regulation of their formation

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Additional file 4. A table for validation of microarray data by qPCR

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Additional file 8. Results of the 2-D PAGE experiments: representation of 2-D gels of WT and HP strain in different time points (t1-t4), comparison of 2-D images between HP and WT in t1 and t2

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Additional file 2. An integrated table of all obtained genomic, transcriptomic and proteomic data as well as data obtained and published in previous studies (Peano et al., 2012; Marcellin et al., 2013, Li et al., 2011)

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Additional file 12. Figures representing western blot analyses of constitutively over-expressed ilvB1 gene and mms operon (mmsOp)

A road map for prioritizing warheads for cysteine targeting covalent inhibitors

WOS:000450383500008International audienceTargeted covalent inhibitors have become an integral part of a number of therapeutic protocols and are the subject of intense research. The mechanism of action of these compounds involves the formation of a covalent bond with protein nucleophiles, mostly cysteines. Given the abundance of cysteines in the proteome, the specificity of the covalent inhibitors is of utmost importance and requires careful optimization of the applied warheads. In most of the cysteine targeting covalent inhibitor programs the design strategy involves incorporating Michael acceptors into a ligand that is already known to bind non-covalently. In contrast, we suggest that the reactive warhead itself should be tailored to the reactivity of the specific cysteine being targeted, and we describe a strategy to achieve this goal. Here, we have extended and systematically explored the available organic chemistry toolbox and characterized a large number of warheads representing different chemistries. We demonstrate that in addition to the common Michael addition, there are other nucleophilic addition, addition-elimination, nucleophilic substitution and oxidation reactions suitable for specific covalent protein modification. Importantly, we reveal that warheads for these chemistries impact the reactivity and specificity of covalent fragments at both protein and proteome levels. By integrating surrogate reactivity and selectivity models and subsequent protein assays, we define a road map to help enable new or largely unexplored covalent chemistries for the optimization of cysteine targeting inhibitors. (C) 2018 Elsevier Masson SAS. All rights reserved