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International audience; Here, we enhanced the popular yeast display method by multiple rounds of DNA and protein engineering. We introduced surface exposure-tailored reporters, eUnaG2 and DnbALFA, creating a new platform of C and N terminal fusion vectors. The optimization of eUnaG2 resulted in five times brighter fluorescence and 10°C increased thermostability than UnaG. The optimized DnbALFA has 10-fold the level of expression of the starting protein. Following this, different plasmids were developed to create a complex platform allowing a broad range of protein expression organizations and labeling strategies. Our platform showed up to five times better separation between nonexpressing and expressing cells compared with traditional pCTcon2 and c-myc labeling, allowing for fewer rounds of selection and achieving higher binding affinities. Testing 16 different proteins, the enhanced system showed consistently stronger expression signals over c-myc labeling. In addition to gains in simplicity, speed, and cost-effectiveness, new applications were introduced to monitor protein surface exposure and protein retention in the secretion pathway that enabled successful protein engineering of hard-to-express proteins. As an example, we show how we optimized the WD40 domain of the ATG16L1 protein for yeast surface and soluble bacterial expression, starting from a nonexpressing protein. As a second example, we show how using the here-presented enhanced yeast display method we rapidly selected high-affinity binders toward two protein targets, demonstrating the simplicity of generating new protein−protein interactions. While the methodological changes are incremental, it results in a qualitative enhancement in the applicability of yeast display for many applications.

Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic conditions, protein and lipid compositions, membrane curvature) and the different partners associated with specific membrane reshaping processes, we undertake in vitro bottom-up approaches. In recent years, there has been keen interest in revealing the role of septin proteins associated with major diseases. Septins are essential and ubiquitous cytoskeletal proteins that interact with the plasma membrane. They are implicated in cell division, cell motility, neuro-morphogenesis, and spermiogenesis, among other functions. It is, therefore, important to understand how septins interact and organize at membranes to subsequently induce membrane deformations and how they can be sensitive to specific membrane curvatures. This article aims to decipher the interplay between the ultra-structure of septins at a molecular level and the membrane remodeling occurring at a micron scale. To this end, budding yeast, and mammalian septin complexes were recombinantly expressed and purified. A combination of in vitro assays was then used to analyze the self-assembly of septins at the membrane. Supported lipid bilayers (SLBs), giant unilamellar vesicles (GUVs), large unilamellar vesicles (LUVs), and wavy substrates were used to study the interplay between septin self-assembly, membrane reshaping, and membrane curvature.

Class I histone deacetylases (HDACs) are key regulators of cell proliferation and they are frequently dysregulated in cancer cells. We report here the synthesis of a novel series of class-I selective HDAC inhibitors (HDACi) containing a 2-aminobenzamide moiety as a zinc-binding group connected with a central (piperazin-1-yl)pyrazine or (piperazin-1-yl)pyrimidine moiety. Some of the compounds were additionally substituted with an aromatic capping group. Compounds were tested in vitro against human HDAC1, 2, 3, and 8 enzymes and compared to reference class I HDACi (Entinostat (MS-275), Mocetinostat, CI994 and RGFP-966). The most promising compounds were found to be highly selective against HDAC1, 2 and 3 over the remaining HDAC subtypes from other classes. Molecular docking studies and MD simulations were performed to rationalize the in vitro data and to deduce a complete structure activity relationship (SAR) analysis of this novel series of class-I HDACi. The most potent compounds, including 19f, which blocks HDAC1, HDAC2, and HDAC3, as well as the selective HDAC1/HDAC2 inhibitors 21a and 29b, were selected for further cellular testing against human acute myeloid leukemia (AML) and erythroleukemic cancer (HEL) cells, taking into consideration their low toxicity against human embryonic HEK293 cells. We found that 19f is superior to the clinically tested class-I HDACi Entinostat (MS-275). Thus, 19f is a new and specific HDACi with the potential to eliminate blood cancer cells of various origins.

Structural bioinformatics provides the scientific methods and tools to analyse, archive, validate, and present the biomolecular structure data generated by the structural biology community. It also provides an important link with the genomics community, as structural bioinformaticians also use the extensive sequence data to predict protein structures and their functional sites. A very broad and active community of structural bioinformaticians exists across Europe, and 3D-Bioinfo will establish formal platforms to address their needs and better integrate their activities and initiatives. Our mission will be to strengthen the ties with the structural biology research communities in Europe covering life sciences, as well as chemistry and physics and to bridge the gap between these researchers in order to fully realize the potential of structural bioinformatics. Our Community will also undertake dedicated educational, training and outreach efforts to facilitate this, bringing new insights and thus facilitating the development of much needed innovative applications e.g. for human health, drug and protein design. Our combined efforts will be of critical importance to keep the European research efforts competitive in this respect. Here we highlight the major European contributions to the field of structural bioinformatics, the most pressing challenges remaining and how Europe-wide interactions, enabled by ELIXIR and its platforms, will help in addressing these challenges and in coordinating structural bioinformatics resources across Europe. In particular, we present recent activities and future plans to consolidate an ELIXIR 3D-Bioinfo Community in structural bioinformatics and propose means to develop better links across the community. These include building new consortia, organising workshops to establish data standards and seeking community agreement on benchmark data sets and strategies. We also highlight existing and planned collaborations with other ELIXIR Communities and other European infrastructures, such as the structural biology community supported by Instruct-ERIC, with whom we have synergies and overlapping common interests.

International audience; Electrostatic interactions play important roles in the functional mechanisms exploited by intrinsically disordered proteins (IDPs). The atomic resolution description of long-range and local structural propensities that can both be crucial for the function of highly charged IDPs presents significant experimental challenges. Here, we investigate the conformational behavior of the δ subunit of RNA polymerase from Bacillus subtilis whose unfolded domain is highly charged, with 7 positively charged amino acids followed by 51 acidic amino acids. Using a specifically designed analytical strategy, we identify transient contacts between the two regions using a combination of NMR paramagnetic relaxation enhancements, residual dipolar couplings (RDCs), chemical shifts, and small-angle scattering. This strategy allows the resolution of long-range and local ensemble averaged structural contributions to the experimental RDCs, and reveals that the negatively charged segment folds back onto the positively charged strand, compacting the conformational sampling of the protein while remaining highly flexible in solution. Mutation of the positively charged region abrogates the long-range contact, leaving the disordered domain in an extended conformation, possibly due to local repulsion of like-charges along the chain. Remarkably, in vitro studies show that this mutation also has a significant effect on transcription activity, and results in diminished cell fitness of the mutated bacteria in vivo. This study highlights the importance of accurately describing electrostatic interactions for understanding the functional mechanisms of IDPs.

Contains fulltext : 202265.pdf (Publisher’s version ) (Closed access) Cancer cells without mitochondrial DNA (mtDNA) do not form tumors unless they reconstitute oxidative phosphorylation (OXPHOS) by mitochondria acquired from host stroma. To understand why functional respiration is crucial for tumorigenesis, we used time-resolved analysis of tumor formation by mtDNA-depleted cells and genetic manipulations of OXPHOS. We show that pyrimidine biosynthesis dependent on respiration-linked dihydroorotate dehydrogenase (DHODH) is required to overcome cell-cycle arrest, while mitochondrial ATP generation is dispensable for tumorigenesis. Latent DHODH in mtDNA-deficient cells is fully activated with restoration of complex III/IV activity and coenzyme Q redox-cycling after mitochondrial transfer, or by introduction of an alternative oxidase. Further, deletion of DHODH interferes with tumor formation in cells with fully functional OXPHOS, while disruption of mitochondrial ATP synthase has little effect. Our results show that DHODH-driven pyrimidine biosynthesis is an essential pathway linking respiration to tumorigenesis, pointing to inhibitors of DHODH as potential anti-cancer agents.

19 p.-6 fig., In mammals, germ cell differentiation is initiated in the Primordial Germ Cells (PGCs) during fetal development. Prenatal exposure to environmental toxicants such as endocrine disruptors may alter PGC differentiation, development of the male germline and induce transgenerational epigenetic disorders. The anti-androgenic compound vinclozolin represents a paradigmatic example of molecule causing transgenerational effects on germ cells. We performed prenatal exposure to vinclozolin in mice and analyzed the phenotypic and molecular changes in three successive generations. A reduction in the number of embryonic PGCs and increased rate of apoptotic cells along with decrease of fertility rate in adult males were observed in F1 to F3 generations. Blimp1 is a crucial regulator of PGC differentiation. We show that prenatal exposure to vinclozolin deregulates specific microRNAs in PGCs, such as miR-23b and miR-21, inducing disequilibrium in the Lin28/let-7/Blimp1 pathway in three successive generations of males. As determined by global maps of cytosine methylation, we found no evidence for prominent changes in DNA methylation in PGCs or mature sperm. Our data suggest that embryonic exposure to environmental endocrine disruptors induces transgenerational epigenetic deregulation of expression of microRNAs affecting key regulatory pathways of germ cells differentiation., We thank M. Quesada, O. Barcón and M. Moreno for caring for the animals. We also acknowledged the support from "EpiGeneSys European Network of Excellence".