S6K1 Negatively Regulates TAK1 Activity in the Toll-Like Receptor Signaling Pathway

Transforming growth factor β (TGF-β)-activated kinase 1 (TAK1) is a key regulator in the signals transduced by proinflammatory cytokines and Toll-like receptors (TLRs). The regulatory mechanism of TAK1 in response to various tissue types and stimuli remains incompletely understood. Here, we show that ribosomal S6 kinase 1 (S6K1) negatively regulates TLR-mediated signals by inhibiting TAK1 activity. S6K1 overexpression causes a marked reduction in NF-κB and AP-1 activity induced by stimulation of TLR2 or TLR4. In contrast, S6K1^−/− and S6K1 knockdown cells display enhanced production of inflammatory cytokines. Moreover, S6K1^−/− mice exhibit decreased survival in response to challenge with lipopolysaccharide (LPS). We found that S6K1 inhibits TAK1 kinase activity by interfering with the interaction between TAK1 and TAB1, which is a key regulator protein for TAK1 catalytic function. Upon stimulation with TLR ligands, S6K1 deficiency causes a marked increase in TAK1 kinase activity that in turn induces a substantial enhancement of NF-κB-dependent gene expression, indicating that S6K1 is negatively involved in the TLR signaling pathway by the inhibition of TAK1 activity. Our findings contribute to understanding the molecular pathogenesis of the impaired immune responses seen in type 2 diabetes, where S6K1 plays a key role both in driving insulin resistance and modulating TLR signaling.     

Genome Sequence of the Fleming Strain of Micrococcus luteus,a Simple Free-Living Actinobacterium

Micrococcus luteus (NCTC2665, “Fleming strain”) has one of the smallest genomes of free-living actinobacteria sequenced to date, comprising a single circular chromosome of 2,501,097 bp (G+C content, 73%) predicted to encode 2,403 proteins. The genome shows extensive synteny with that of the closely related organism, Kocuria rhizophila, from which it was taxonomically separated relatively recently. Despite its small size, the genome harbors 73 insertion sequence (IS) elements, almost all of which are closely related to elements found in other actinobacteria. An IS element is inserted into the rrs gene of one of only two rrn operons found in M. luteus. The genome encodes only four sigma factors and 14 response regulators, a finding indicative of adaptation to a rather strict ecological niche (mammalian skin). The high sensitivity of M. luteus to β-lactam antibiotics may result from the presence of a reduced set of penicillin-binding proteins and the absence of a wblC gene, which plays an important role in the antibiotic resistance in other actinobacteria. Consistent with the restricted range of compounds it can use as a sole source of carbon for energy and growth, M. luteus has a minimal complement of genes concerned with carbohydrate transport and metabolism and its inability to utilize glucose as a sole carbon source may be due to the apparent absence of a gene encoding glucokinase. Uniquely among characterized bacteria, M. luteus appears to be able to metabolize glycogen only via trehalose and to make trehalose only via glycogen. It has very few genes associated with secondary metabolism. In contrast to most other actinobacteria, M. luteus encodes only one resuscitation-promoting factor (Rpf) required for emergence from dormancy, and its complement of other dormancy-related proteins is also much reduced. M. luteus is capable of long-chain alkene biosynthesis, which is of interest for advanced biofuel production; a three-gene cluster essential for this metabolism has been identified in the genome.Micrococcus luteus, the type species of the genus Micrococcus (family Micrococcaceae, order Actinomycetales) (117), is an obligate aerobe. Three biovars have been distinguished (138). Its simple, coccoid morphology delayed the recognition of its relationship to actinomycetes, which are typically morphologically more complex. In the currently accepted phylogenetic tree of the actinobacteria, Micrococcus clusters with Arthrobacter and Renibacterium. Some other coccoid actinobacteria originally also called Micrococcus, but reclassified into four new genera (Kocuria, Nesterenkonia, Kytococcus, and Dermacoccus), are more distant relatives (121). The genus Micrococcus now includes only five species: M. luteus, M. lylae, M. antarcticus, M. endophyticus, and M. flavus (20, 69, 70, 121).We report here the genome sequence of Micrococcus luteus NCTC2665 (DSM 20030^T), a strain of historical interest, since Fleming used it to demonstrate bacteriolytic activity (due to lysozyme) in a variety of body tissues and secretions (29, 30), leading to its designation as Micrococcus lysodeikticus until its taxonomic status was clarified in 1972 (59). M. luteus has been used in a number of scientific contexts. The ease with which its cell wall could be removed made it a favored source of bacterial cell membranes and protoplasts for investigations in bioenergetics (28, 34, 89, 93). Because of the exceptionally high GC content of its DNA, M. luteus was used to investigate the relationship between codon usage and tRNA representation in bacterial genomes (51, 52, 61). Although it does not form endospores, M. luteus can enter a profoundly dormant state, which could explain why it may routinely be isolated from amber (39). Dormancy has been convincingly demonstrated under laboratory conditions (53-55, 83), and a secreted protein (Rpf) with muralytic activity is involved in the process of resuscitation (81, 82, 84, 85, 87, 125, 133).Micrococci are also of biotechnological interest. In addition to the extensive exploitation of these and related organisms by the pharmaceutical industry for testing and assaying compounds for antibacterial activity, micrococci can synthesize long-chain alkenes (1, 2, 127). They are also potentially useful for ore dressing and bioremediation applications, since they are able to concentrate heavy metals from low-grade ores (26, 66, 67, 116).Given its intrinsic historical and biological importance, and its biotechnological potential, it is perhaps surprising that the genome sequence of M. luteus was not determined previously (130). We consider here the strikingly small genome sequence in these contexts and also in relation to the morphological simplicity of M. luteus compared to many of its actinobacterial relatives, which include important pathogens as well as developmentally complex, antibiotic-producing bacteria with some of the largest bacterial genomes.     

Mechanisms of haplotype divergence at the <Emphasis Type="Italic">RGA08</Emphasis> nucleotide-binding leucine-rich repeat gene locus in wild banana <Emphasis Type="Italic">(Musa balbisiana)</Emphasis>

Background  

Comparative sequence analysis of complex loci such as resistance gene analog clusters allows estimating the degree of sequence conservation and mechanisms of divergence at the intraspecies level. In banana (Musa sp.), two diploid wild species Musa acuminata (A genome) and Musa balbisiana (B genome) contribute to the polyploid genome of many cultivars. The M. balbisiana species is associated with vigour and tolerance to pests and disease and little is known on the genome structure and haplotype diversity within this species. Here, we compare two genomic sequences of 253 and 223 kb corresponding to two haplotypes of the RGA08 resistance gene analog locus in M. balbisiana "Pisang Klutuk Wulung" (PKW).     

Interactions of the Escherichia coli hydrogenase biosynthetic proteins: HybG complex formation

Assembly of the active site of the [NiFe]-hydrogenase enzymes involves a multi-step pathway and the coordinated activity of many accessory proteins. To analyze complex formation between these factors in Escherichia coli, they were genomically tagged and native multi-protein complexes were isolated. This method validated multiple interactions reported in separate studies from several organisms and defined a new complex containing the putative chaperone HybG and the large subunit of hydrogenase 1 or 2. The complex also includes HypE and HypD, which interact with each other before joining the larger complex.     

Structural insights into substrate traffic and inhibition in acetylcholinesterase

Acetylcholinesterase (AChE) terminates nerve-impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a substrate molecule bound at the peripheral site. At the higher concentration, substrate inhibition arises from prevention of exit of acetate due to binding of two substrate molecules within the active-site gorge.     

The bestrophin- and TMEM16A-associated Ca2+-activated Cl– channels in vascular smooth muscles

The presence of Ca²⁺-activated Cl^– currents (I_Cl(Ca)) in vascular smooth muscle cells (VSMCs) is well established. I_Cl(Ca) are supposedly important for arterial contraction by linking changes in [Ca²⁺]_i and membrane depolarization. Bestrophins and some members of the TMEM16 protein family were recently associated with I_Cl(Ca). Two distinct I_Cl(Ca) are characterized in VSMCs; the cGMP-dependent I_Cl(Ca) dependent upon bestrophin expression and the ‘classical’ Ca²⁺-activated Cl^– current, which is bestrophin-independent. Interestingly, TMEM16A is essential for both the cGMP-dependent and the classical I_Cl(Ca). Furthermore, TMEM16A has a role in arterial contraction while bestrophins do not. TMEM16A’s role in the contractile response cannot be explained however only by a simple suppression of the depolarization by Cl^– channels. It is suggested that TMEM16A expression modulates voltage-gated Ca²⁺ influx in a voltage-independent manner and recent studies also demonstrate a complex role of TMEM16A in modulating other membrane proteins.     

Automated microinjection of recombinant BCL-X into mouse zygotes enhances embryo development

Progression of fertilized mammalian oocytes through cleavage, blastocyst formation and implantation depends on successful implementation of the developmental program, which becomes established during oogenesis. The identification of ooplasmic factors, which are responsible for successful embryo development, is thus crucial in designing possible molecular therapies for infertility intervention. However, systematic evaluation of molecular targets has been hampered by the lack of techniques for efficient delivery of molecules into embryos. We have developed an automated robotic microinjection system for delivering cell impermeable compounds into preimplantation embryos with a high post-injection survival rate. In this paper, we report the performance of the system on microinjection of mouse embryos. Furthermore, using this system we provide the first evidence that recombinant BCL-XL (recBCL-XL) protein is effective in preventing early embryo arrest imposed by suboptimal culture environment. We demonstrate that microinjection of recBCL-XL protein into early-stage embryos repairs mitochondrial bioenergetics, prevents reactive oxygen species (ROS) accumulation, and enhances preimplantation embryo development. This approach may lead to a possible treatment option for patients with repeated in vitro fertilization (IVF) failure due to poor embryo quality.     

Ribosome-dependent ATPase interacts with conserved membrane protein in Escherichia coli to modulate protein synthesis and oxidative phosphorylation

Elongation factor RbbA is required for ATP-dependent deacyl-tRNA release presumably after each peptide bond formation; however, there is no information about the cellular role. Proteomic analysis in Escherichia coli revealed that RbbA reciprocally co-purified with a conserved inner membrane protein of unknown function, YhjD. Both proteins are also physically associated with the 30S ribosome and with members of the lipopolysaccharide transport machinery. Genome-wide genetic screens of rbbA and yhjD deletion mutants revealed aggravating genetic interactions with mutants deficient in the electron transport chain. Cells lacking both rbbA and yhjD exhibited reduced cell division, respiration and global protein synthesis as well as increased sensitivity to antibiotics targeting the ETC and the accuracy of protein synthesis. Our results suggest that RbbA appears to function together with YhjD as part of a regulatory network that impacts bacterial oxidative phosphorylation and translation efficiency.