Innate immunity and intestinal microbiota in the development of Type 1 diabetes

Type 1 diabetes (T1D) is a debilitating autoimmune disease that results from T-cell-mediated destruction of insulin-producing beta-cells. Its incidence has increased during the past several decades in developed countries, suggesting that changes in the environment (including the human microbial environment) may influence disease pathogenesis. The incidence of spontaneous T1D in non-obese diabetic (NOD) mice can be affected by the microbial environment in the animal housing facility or by exposure to microbial stimuli, such as injection with mycobacteria or various microbial products. Here we show that specific pathogen-free NOD mice lacking MyD88 protein (an adaptor for multiple innate immune receptors that recognize microbial stimuli) do not develop T1D. The effect is dependent on commensal microbes because germ-free MyD88-negative NOD mice develop robust diabetes, whereas colonization of these germ-free MyD88-negative NOD mice with a defined microbial consortium (representing bacterial phyla normally present in human gut) attenuates T1D. We also find that MyD88 deficiency changes the composition of the distal gut microbiota, and that exposure to the microbiota of specific pathogen-free MyD88-negative NOD donors attenuates T1D in germ-free NOD recipients. Together, these findings indicate that interaction of the intestinal microbes with the innate immune system is a critical epigenetic factor modifying T1D predisposition.     

Structural basis for the selectivity of the external thioesterase of the surfactin synthetase

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) found in bacteria, fungi and plants use two different types of thioesterases for the production of highly active biological compounds. Type I thioesterases (TEI) catalyse the release step from the assembly line of the final product where it is transported from one reaction centre to the next as a thioester linked to a 4'-phosphopantetheine (4'-PP) cofactor that is covalently attached to thiolation (T) domains. The second enzyme involved in the synthesis of these secondary metabolites, the type II thioesterase (TEII), is a crucial repair enzyme for the regeneration of functional 4'-PP cofactors of holo-T domains of NRPS and PKS systems. Mispriming of 4'-PP cofactors by acetyl- and short-chain acyl-residues interrupts the biosynthetic system. This repair reaction is very important, because roughly 80% of CoA, the precursor of the 4'-PP cofactor, is acetylated in bacteria. Here we report the three-dimensional structure of a type II thioesterase from Bacillus subtilis free and in complex with a T domain. Comparison with structures of TEI enzymes shows the basis for substrate selectivity and the different modes of interaction of TEII and TEI enzymes with T domains. Furthermore, we show that the TEII enzyme exists in several conformations of which only one is selected on interaction with its native substrate, a modified holo-T domain.     

Dynamic thiolation-thioesterase structure of a non-ribosomal peptide synthetase

Non-ribosomal peptide synthetases (NRPS) and polyketide synthases (PKS) produce numerous secondary metabolites with various therapeutic/antibiotic properties. Like fatty acid synthases (FAS), these enzymes are organized in modular assembly lines in which each module, made of conserved domains, incorporates a given monomer unit into the growing chain. Knowledge about domain or module interactions may enable reengineering of this assembly line enzymatic organization and open avenues for the design of new bioactive compounds with improved therapeutic properties. So far, little structural information has been available on how the domains interact and communicate. This may be because of inherent interdomain mobility hindering crystallization, or because crystallized molecules may not represent the active domain orientations. In solution, the large size and internal dynamics of multidomain fragments (>35 kilodaltons) make structure determination by nuclear magnetic resonance a challenge and require advanced technologies. Here we present the solution structure of the apo-thiolation-thioesterase (T-TE) di-domain fragment of the Escherichia coli enterobactin synthetase EntF NRPS subunit. In the holoenzyme, the T domain carries the growing chain tethered to a 4'-phosphopantetheine whereas the TE domain catalyses hydrolysis and cyclization of the iron chelator enterobactin. The T-TE di-domain forms a compact but dynamic structure with a well-defined domain interface; the two active sites are at a suitable distance for substrate transfer from T to TE. We observe extensive interdomain and intradomain motions for well-defined regions and show that these are modulated by interactions with proteins that participate in the biosynthesis. The T-TE interaction described here provides a model for NRPS, PKS and FAS function in general as T-TE-like di-domains typically catalyse the last step in numerous assembly-line chain-termination machineries.     

通过镍(II)-硫键和碘离子基元反应有理合成碘-二硫醇盐桥联的双核镍-镍配合物

本文报道由于碘离子对不稳定的Ni(II)-S(芳基硫醇盐)键的亲核作用,使得Fe(CO)412和Ni(SR)2(dppe(SR=芳基硫醇盐)之间的反应生成NiI2(dppe).含碘和二芳基硫醇盐离子桥联的Ni-Ni双核配合物[(dppe)Ni(μ-I)(μ-pdt)Ni(dppe)]I和[(dppe)Ni(μ-I)(μ-edt)Ni(dppe)]I可方便地由[NiI2(dppe)]和[Ni(pdt)(dppe)]或[Ni(edt)(dppe)]在二氯甲烷的溶液中的反应制得;该类反应可认为是由于硫醇盐离子基团中S-供体上的孤对电子对Ni-I键的进攻所致.另一方面,我们观察到[FeCp(CO)2I]和[Ni(pdt)(dppe)]或[Ni(edt)(dppe)]在二氯甲烷中的反应极其缓慢;但当向上述反应体系中加入NH4PF6进行复分解置换后,源于碘离子和Ni(II)-S键的作用同样可得到含碘与二芳基硫醇盐离子桥联的Ni-Ni双核配合物[(dppe)Ni(μ-I)(μ-pdt)Ni(dppe)]PF6和 [(dppe)Ni(μ-I)(μ-edt)Ni(dppe)]PF6.实验结果说明在本文所讨认的镍(II)-硫醇盐离子-膦配合物中,Ni(II)-S键的反应活性随桥联的第二金属离子和不同的碘离子基元而改变.     

An inhibitor of Bcl-2 family proteins induces regression of solid tumours

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.     

Independent recruitment of a conserved developmental mechanism during leaf evolution

Vascular plants evolved in the Middle to Late Silurian period, about 420 million years ago. The fossil record indicates that these primitive plants had branched stems with sporangia but no leaves. Leaf-like lateral outgrowths subsequently evolved on at least two independent occasions. In extant plants, these events are represented by microphyllous leaves in lycophytes (clubmosses, spikemosses and quillworts) and megaphyllous leaves in euphyllophytes (ferns, gymnosperms and angiosperms). Our current understanding of how leaves develop is restricted to processes that operate during megaphyll formation. Because microphylls and megaphylls evolved independently, different mechanisms might be required for leaf formation. Here we show that this is not so. Gene expression data from a microphyllous lycophyte, phylogenetic analyses, and a cross-species complementation experiment all show that a common developmental mechanism can underpin both microphyll and megaphyll formation. We propose that this mechanism might have operated originally in the context of primitive plant apices to facilitate bifurcation. Recruitment of this pathway to form leaves occurred independently and in parallel in different plant lineages.     

Modular epistasis in yeast metabolism

Epistatic interactions, manifested in the effects of mutations on the phenotypes caused by other mutations, may help uncover the functional organization of complex biological networks. Here, we studied system-level epistatic interactions by computing growth phenotypes of all single and double knockouts of 890 metabolic genes in Saccharomyces cerevisiae, using the framework of flux balance analysis. A new scale for epistasis identified a distinctive trimodal distribution of these epistatic effects, allowing gene pairs to be classified as buffering, aggravating or noninteracting. We found that the ensuing epistatic interaction network could be organized hierarchically into function-enriched modules that interact with each other 'monochromatically' (i.e., with purely aggravating or purely buffering epistatic links). This property extends the concept of epistasis from single genes to functional units and provides a new definition of biological modularity, which emphasizes interactions between, rather than within, functional modules. Our approach can be used to infer functional gene modules from purely phenotypic epistasis measurements.