Bioassays to Monitor Taspase1 Function for the Identification of Pharmacogenetic Inhibitors

Background: Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1's (patho)biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available. Methodology/Findings: Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm, whereas expression of biologically active Taspase1 but not of inactive Taspase1 mutants or of the protease Caspase3 triggers their proteolytic cleavage and nuclear accumulation. Compared to in vitro assays using recombinant components the in vivo assay was highly efficient. Employing an optimized nuclear translocation algorithm, the triple-color assay could be adapted to a high-throughput microscopy platform (Z'factor = 0.63). Automated high-content data analysis was used to screen a focused compound library, selected by an in silico pharmacophor screening approach, as well as a collection of fungal extracts. Screening identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamideand 2-benzyltriazole-4,5-dicarboxylic acid, which partially inhibited Taspase1 cleavage in living cells. Additionally, the assay was exploited to probe endogenous Taspase1 in solid tumor cell models and to identify an improved consensus sequence for efficient Taspase1 cleavage. This allowed the in silico identification of novel putative Taspase1 targets. Those include the FERM Domain-Containing Protein 4B, the Tyrosine-Protein Phosphatase Zeta, and DNA Polymerase Zeta. Cleavage site recognition and proteolytic processing of these substrates were verified in the context of the biosensor. Conclusions: The assay not only allows to genetically probe Taspase1 structure function in vivo, but is also applicable for high-content screening to identify Taspase1 inhibitors. Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance

Populism and Government: Continuity and Paradoxes in the Yellow-Green Experiment

Domestic and comparative constitutional analyses routinely describe the government as the most under-theorised among constitutional organs. This chapter aims to consider whether and how the Italian government has been affected by the rise of populist parties and movements or, more vaguely, by populist arguments and discourse. At first glance, an obvious starting point is the link between the trend towards the presidentialisation of the executives and the institutional programmes of populism. What has been described as the first experiment in purely populist government in Europe seems to contradict such claims and to have revived some long-term characters of the Italian executive. As scholars have put it, electoral laws and European integration are the factors that have most contributed to defining and shaping the role of government over the last three decades: in a nutshell, they have contributed, among other things, to strengthening the institutional role and visibility of the Prime Minister. Still, the Italian government which was formed in 2018 was based on a “contract” signed by the leaders of the Five Star Movement and the League, and to whose drafting Giuseppe Conte, who later went on to be appointed to the premiership, was largely foreign

Are Dynamic Mechanistic Explanations Still Mechanistic?

International audienceA major type of explanation in biology consists of mechanistic explanations (e.g. Machamer et al. 2000, Kaplan and Craver 2011). The explanatory force of mechanisms is apparent in such typical cases as the functioning of an ion channel or the molecular activation of a receptor: it includes the specification of a model of mechanism and the rehearsing of a causal story that tells how the explanandum phenomenon is produced by the mechanism. It is however much less clear how mechanisms explain in the case of complex and non-linear biomolecular networks such as those that underlie the action of hormones and the regulation of genes. While dynamic mechanistic explanations have been proposed as an extension of mechanistic explanations (e.g. Bechtel and Abrahamsen 2010), we argue that the former depart from the latter in that they do not draw their explanatory force from a causal story but from the mathematical warrants they give that the explanandum phenomenon follows from a mathematical model. By analyzing the explanatory force of mechanistic explanation and of dynamic mechanistic explanation, we show that the two types of explanations can be construed as limit cases of a more general pattern of explanation-Causally Interpreted Model Explanations-that draws its explanatory force from a model, a causal interpretation that links the model to biological reality, and a mathematical derivation that links the model to the explanandum phenomenon

Alternative Epigenetic Chromatin States of Polycomb Target Genes

Polycomb (PcG) regulation has been thought to produce stable long-term gene silencing. Genomic analyses in Drosophila and mammals, however, have shown that it targets many genes, which can switch state during development. Genetic evidence indicates that critical for the active state of PcG target genes are the histone methyltransferases Trithorax (TRX) and ASH1. Here we analyze the repertoire of alternative states in which PcG target genes are found in different Drosophila cell lines and the role of PcG proteins TRX and ASH1 in controlling these states. Using extensive genome-wide chromatin immunoprecipitation analysis, RNAi knockdowns, and quantitative RT-PCR, we show that, in addition to the known repressed state, PcG targets can reside in a transcriptionally active state characterized by formation of an extended domain enriched in ASH1, the N-terminal, but not C-terminal moiety of TRX and H3K27ac. ASH1/TRX N-ter domains and transcription are not incompatible with repressive marks, sometimes resulting in a "balanced" state modulated by both repressors and activators. Often however, loss of PcG repression results instead in a "void" state, lacking transcription, H3K27ac, or binding of TRX or ASH1. We conclude that PcG repression is dynamic, not static, and that the propensity of a target gene to switch states depends on relative levels of PcG, TRX, and activators. N-ter TRX plays a remarkable role that antagonizes PcG repression and preempts H3K27 methylation by acetylation. This role is distinct from that usually attributed to TRX/MLL proteins at the promoter. These results have important implications for Polycomb gene regulation, the "bivalent" chromatin state of embryonic stem cells, and gene expression in development