The ansamycin antibiotic, rifamycin SV, inhibits BCL6 transcriptional repression and forms a complex with the BCL6-BTB/POZ domain

BCL6 is a transcriptional repressor that is over-expressed due to chromosomal translocations, or other abnormalities, in ~40% of diffuse large B-cell lymphoma. BCL6 interacts with co-repressor, SMRT, and this is essential for its role in lymphomas. Peptide or small molecule inhibitors, which prevent the association of SMRT with BCL6, inhibit transcriptional repression and cause apoptosis of lymphoma cells in vitro and in vivo. In order to discover compounds, which have the potential to be developed into BCL6 inhibitors, we screened a natural product library. The ansamycin antibiotic, rifamycin SV, inhibited BCL6 transcriptional repression and NMR spectroscopy confirmed a direct interaction between rifamycin SV and BCL6. To further determine the characteristics of compounds binding to BCL6-POZ we analyzed four other members of this family and showed that rifabutin, bound most strongly. An X-ray crystal structure of the rifabutin-BCL6 complex revealed that rifabutin occupies a partly non-polar pocket making interactions with tyrosine58, asparagine21 and arginine24 of the BCL6-POZ domain. Importantly these residues are also important for the interaction of BLC6 with SMRT. This work demonstrates a unique approach to developing a structure activity relationship for a compound that will form the basis of a therapeutically useful BCL6 inhibitor

Lectin-like bacteriocins from pseudomonas spp. utilise D-rhamnose containing lipopolysaccharide as a cellular receptor

Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of d-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing d-rhamnose and not d-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins

Unraveling the Phylogenetic Relationships of the Eccoptochilinae, an Enigmatic Array of Ordovician Cheirurid Trilobites

The Cheiruridae are a diverse group of trilobites and several subfamilies within the clade have been the focus of recent phylogenetic studies. This paper focuses on the relationships of one of those subfamilies, the Ordovician Eccoptochilinae. We analyze sixteen species from six genera within the traditionally defined group, using the pilekiid Anacheirurus frederici as an outgroup. To assess the monophyly of the Eccoptochilinae seven sphaerexochine species, Kawina arnoldi, Sphaerexochus arenosus, S. atacius, S. latifrons, S. mirus, S. parvus, and S. scabridus were included in the analysis as well. The results of this analysis show that the genus Eccoptochile represents a paraphyletic grade and species traditionally assigned to Parasphaerexochus and Skelipyx plot within Pseudosphaerexochus. Also, representative species of Sphaerexochinae plot within the traditionally defined Eccoptochilinae, suggesting Eccoptochilinae itself is paraphyletic. To resolve this, we propose all species of Pseudosphaerexochus be placed within Sphaerexochinae and Eccoptochilinae be restricted to a monotypic Eccoptochile clavigera.This research was supported by NSF DEB-0716162

A unique population of effector memory lymphocytes identified by CD146 having a distinct immunophenotypic and genomic profile

<p>Abstract</p> <p>Background</p> <p>CD146 is a well described homotypic adhesion molecule found on endothelial cells and a limited number of other cell types. In cells from the peripheral circulation, CD146 has also been reported to be on activated lymphocytes <it>in vitro </it>and <it>in vivo</it>. The function associated with CD146 expression on lymphoid cells is unknown and very little information is available concerning the nature of CD146+ lymphocytes. In the current study, lymphocytes from healthy donors were characterized based upon the presence or absence of CD146 expression.</p> <p>Results</p> <p>CD146 was expressed on a low percentage of circulating T lymphocytes, B lymphocytes, and NK cells in healthy individuals. CD146 expression can be induced and upregulated <it>in vitro </it>on both B cells and T cells, but does not correlate with the expression of other markers of T cell activation. CD146 positive T cells do not represent clonal expansions as determined with the use of anti Vβ reagents. Data suggest that CD146 positive cells have enhanced adherence to endothelial monolayers in vitro. Gene profiling and immunophenotyping studies between CD146+ and CD146- T cells revealed several striking genotypic distinctions such as the upregulation of IL-8 and phenotypic differences including the paucity of CCR7 and CD45RA among CD146 positive T cells, consistent with effector memory function. A number of genes involved in cell adhesion, signal transduction, and cell communication are dramatically upregulated in CD146+ T cells compared to CD146- T cells.</p> <p>Conclusion</p> <p>CD146 appears to identify small, unique populations of T as well as B lymphocytes in the circulation. The T cells have immunophenotypic characteristics of effector memory lymphocytes. The characteristics of these CD146+ lymphocytes in the circulation, together with the known functions in cell adhesion of CD146 on endothelial cells, suggests that these lymphocytes may represent a small subpopulation of cells primed to adhere to the endothelium and possibly extravasate to sites of inflammation.</p

Equal fitness paradigm explained by a trade-off between generation time and energy production rate

Most plant, animal and microbial species of widely varying body size and lifestyle are nearly equally fit as evidenced by their coexistence and persistence through millions of years. All organisms compete for a limited supply of organic chemical energy, derived mostly from photosynthesis, to invest in the two components of fitness: survival and production. All organisms are mortal because molecular and cellular damage accumulates over the lifetime; life persists only because parents produce offspring. We call this the equal fitness paradigm. The equal fitness paradigm occurs because: (1) there is a trade-off between generation time and productive power, which have equal-but-opposite scalings with body size and temperature; smaller and warmer organisms have shorter lifespans but produce biomass at higher rates than larger and colder organisms; (2) the energy content of biomass is essentially constant, ~22.4 kJ g−1 dry body weight; and (3) the fraction of biomass production incorporated into surviving offspring is also roughly constant, ~10–50%. As organisms transmit approximately the same quantity of energy per gram to offspring in the next generation, no species has an inherent lasting advantage in the struggle for existence. The equal fitness paradigm emphasizes the central importance of energy, biological scaling relations and power–time trade-offs in life history, ecology and evolution

Dynamically-Driven Inactivation of the Catalytic Machinery of the SARS 3C-Like Protease by the N214A Mutation on the Extra Domain

Despite utilizing the same chymotrypsin fold to host the catalytic machinery, coronavirus 3C-like proteases (3CLpro) noticeably differ from picornavirus 3C proteases in acquiring an extra helical domain in evolution. Previously, the extra domain was demonstrated to regulate the catalysis of the SARS-CoV 3CLpro by controlling its dimerization. Here, we studied N214A, another mutant with only a doubled dissociation constant but significantly abolished activity. Unexpectedly, N214A still adopts the dimeric structure almost identical to that of the wild-type (WT) enzyme. Thus, we conducted 30-ns molecular dynamics (MD) simulations for N214A, WT, and R298A which we previously characterized to be a monomer with the collapsed catalytic machinery. Remarkably, three proteases display distinctive dynamical behaviors. While in WT, the catalytic machinery stably retains in the activated state; in R298A it remains largely collapsed in the inactivated state, thus implying that two states are not only structurally very distinguishable but also dynamically well separated. Surprisingly, in N214A the catalytic dyad becomes dynamically unstable and many residues constituting the catalytic machinery jump to sample the conformations highly resembling those of R298A. Therefore, the N214A mutation appears to trigger the dramatic change of the enzyme dynamics in the context of the dimeric form which ultimately inactivates the catalytic machinery. The present MD simulations represent the longest reported so far for the SARS-CoV 3CLpro, unveiling that its catalysis is critically dependent on the dynamics, which can be amazingly modulated by the extra domain. Consequently, mediating the dynamics may offer a potential avenue to inhibit the SARS-CoV 3CLpro

SAD phasing using iodide ions in a high-throughput structural genomics environment

The Seattle Structural Genomics Center for Infectious Disease (SSGCID) focuses on the structure elucidation of potential drug targets from class A, B, and C infectious disease organisms. Many SSGCID targets are selected because they have homologs in other organisms that are validated drug targets with known structures. Thus, many SSGCID targets are expected to be solved by molecular replacement (MR), and reflective of this, all proteins are expressed in native form. However, many community request targets do not have homologs with known structures and not all internally selected targets readily solve by MR, necessitating experimental phase determination. We have adopted the use of iodide ion soaks and single wavelength anomalous dispersion (SAD) experiments as our primary method for de novo phasing. This method uses existing native crystals and in house data collection, resulting in rapid, low cost structure determination. Iodide ions are non-toxic and soluble at molar concentrations, facilitating binding at numerous hydrophobic or positively charged sites. We have used this technique across a wide range of crystallization conditions with successful structure determination in 16 of 17 cases within the first year of use (94% success rate). Here we present a general overview of this method as well as several examples including SAD phasing of proteins with novel folds and the combined use of SAD and MR for targets with weak MR solutions. These cases highlight the straightforward and powerful method of iodide ion SAD phasing in a high-throughput structural genomics environment

Insights into the Mechanism of Bovine CD38/NAD+Glycohydrolase from the X-Ray Structures of Its Michaelis Complex and Covalently-Trapped Intermediates

Bovine CD38/NAD+glycohydrolase (bCD38) catalyses the hydrolysis of NAD+ into nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose (cADPR). We solved the crystal structures of the mono N-glycosylated forms of the ecto-domain of bCD38 or the catalytic residue mutant Glu218Gln in their apo state or bound to aFNAD or rFNAD, two 2′-fluorinated analogs of NAD+. Both compounds behave as mechanism-based inhibitors, allowing the trapping of a reaction intermediate covalently linked to Glu218. Compared to the non-covalent (Michaelis) complex, the ligands adopt a more folded conformation in the covalent complexes. Altogether these crystallographic snapshots along the reaction pathway reveal the drastic conformational rearrangements undergone by the ligand during catalysis with the repositioning of its adenine ring from a solvent-exposed position stacked against Trp168 to a more buried position stacked against Trp181. This adenine flipping between conserved tryptophans is a prerequisite for the proper positioning of the N1 of the adenine ring to perform the nucleophilic attack on the C1′ of the ribofuranoside ring ultimately yielding cADPR. In all structures, however, the adenine ring adopts the most thermodynamically favorable anti conformation, explaining why cyclization, which requires a syn conformation, remains a rare alternate event in the reactions catalyzed by bCD38 (cADPR represents only 1% of the reaction products). In the Michaelis complex, the substrate is bound in a constrained conformation; the enzyme uses this ground-state destabilization, in addition to a hydrophobic environment and desolvation of the nicotinamide-ribosyl bond, to destabilize the scissile bond leading to the formation of a ribooxocarbenium ion intermediate. The Glu218 side chain stabilizes this reaction intermediate and plays another important role during catalysis by polarizing the 2′-OH of the substrate NAD+. Based on our structural analysis and data on active site mutants, we propose a detailed analysis of the catalytic mechanism

Microglial activation and chronic neurodegeneration

Microglia, the resident innate immune cells in the brain, have long been implicated in the pathology of neurode-generative diseases. Accumulating evidence points to activated microglia as a chronic source of multiple neurotoxic factors, including tumor necrosis factor-α, nitric oxide, interleukin-1β, and reactive oxygen species (ROS), driving progressive neuron damage. Microglia can become chronically activated by either a single stimulus (e.g., lipopolysaccharide or neuron damage) or multiple stimuli exposures to result in cumulative neuronal loss with time. Although the mechanisms driving these phenomena are just beginning to be understood, reactive microgliosis (the microglial response to neuron damage) and ROS have been implicated as key mechanisms of chronic and neurotoxic microglial activation, particularly in the case of Parkinson’s disease. We review the mechanisms of neurotoxicity associated with chronic microglial activation and discuss the role of neuronal death and microglial ROS driving the chronic and toxic microglial phenotype