Genetic conflicts and the origin of self/nonself-discrimination in the vertebrate immune system.

Genetic conflicts shape the genomes of prokaryotic and eukaryotic organisms. Here, we argue that some of the key evolutionary novelties of adaptive immune systems of vertebrates are descendants of prokaryotic toxin-antitoxin (TA) systems. Cytidine deaminases and RAG recombinase have evolved from genotoxic enzymes to programmable editors of host genomes, supporting the astounding discriminatory capability of variable lymphocyte receptors of jawless vertebrates, as well as immunoglobulins and T cell receptors of jawed vertebrates. The evolutionarily recent lymphoid lineage is uniquely sensitive to mutations of the DNA maintenance methylase, which is an orphaned distant relative of prokaryotic restriction-modification systems. We discuss how the emergence of adaptive immunity gave rise to higher order genetic conflicts between genetic parasites and their vertebrate host.

Fluorescence protein complementation in microscopy: applications beyond detecting bi-molecular interactions.

Conventional fragments of fluorescent proteins used in bimolecular fluorescence complementation technique (BiFC), form light-emitting species only when they are kept in close proximity by interacting proteins of interest. By contrast, certain fluorescent protein fragments complement spontaneously, namely those corresponding to the 1st to 10th beta-strands (GFP1-10) and the 11th beta-strand of superfolder GFP (GFP11). They were designed as folding reporters for high throughput expression and structure biology. Besides, for light microscopy, self-associating fluorescent protein fragments constitute a valuable and sometimes unique tool. The GFP11 tag is very advantageous when a full-length fluorescent protein cannot be fused to a protein of interest, namely for live imaging of certain pathogens. Self-associating GFP fragments enable live labelling of specific synapses, visualization of proteins topology and their exposure to particular subcellular compartments. Present review aims to attract attention of scientific community to these tools and to inspire their further development and applications.

Robo1 Forms a Compact Dimer-of-Dimers Assembly.

Roundabout (Robo) receptors provide an essential repulsive cue in neuronal development following Slit ligand binding. This important signaling pathway can also be hijacked in numerous cancers, making Slit-Robo an attractive therapeutic target. However, little is known about how Slit binding mediates Robo activation. Here we present the crystal structure of Robo1 Ig1-4 and Robo1 Ig5, together with a negative stain electron microscopy reconstruction of the Robo1 ectodomain. These results show how the Robo1 ectodomain is arranged as compact dimers, mainly mediated by the central Ig domains, which can further interact in a "back-to-back" fashion to generate a tetrameric assembly. We also observed no change in Robo1 oligomerization upon interaction with the dimeric Slit2-N ligand using fluorescent imaging. Taken together with previous studies we propose that Slit2-N binding results in a conformational change of Robo1 to trigger cell signaling.

Structural Characterization of the SMRT Corepressor Interacting with Histone Deacetylase 7.

The 2525 amino acid SMRT corepressor is an intrinsically disordered hub protein responsible for binding and coordinating the activities of multiple transcription factors and chromatin modifying enzymes. Here we have studied its interaction with HDAC7, a class IIa deacetylase that interacts with the corepressor complex together with the highly active class I deacetylase HDAC3. The binding site of class IIa deacetylases was previously mapped to an approximate 500 amino acid region of SMRT, with recent implication of short glycine-serine-isoleucine (GSI) containing motifs. In order to characterize the interaction in detail, we applied a random library screening approach within this region and obtained a range of stable, soluble SMRT fragments. In agreement with an absence of predicted structural domains, these were characterized as intrinsically disordered by NMR spectroscopy. We identified one of them, comprising residues 1255-1452, as interacting with HDAC7 with micromolar affinity. The binding site was mapped in detail by NMR and confirmed by truncation and alanine mutagenesis. Complementing this with mutational analysis of HDAC7, we show that HDAC7, via its surface zinc ion binding site, binds to a 28 residue stretch in SMRT comprising a GSI motif followed by an alpha helix.

Biological and biophysical properties of the histone deacetylase inhibitor suberoylanilide hydroxamic acid are affected by the presence of short alkyl groups on the phenyl ring.

Inhibition of histone deacetylases (HDACs) leads to growth arrest, differentiation, or apoptosis of tumor cell lines, suggesting HDACs as promising targets for cancer therapy. At present, only one HDAC inhibitor (HDACi) is used in therapy: suberoylanilide hydroxamic acid (SAHA). Here, we describe the synthesis and biological evaluation of a new series of compounds derived from SAHA by substituting short alkyl chains at various positions of the phenyl ring. Such modifications induced variable effects ranging from partial loss of activity to increased potency. Through molecular modeling, we describe a possible interaction between HDAC7 proline 809, a residue that is strictly conserved within class 2 enzymes only, and the amide group of HDACi, while nuclear magnetic resonance experiments indicated that dimethyl m-substitution may stabilize the inhibitor in the active site. Our data provide novel information on the structure-activity relationship of HDACi and suggest new ways for developing second generation SAHA-like molecules.