Photoreceptor Proteins Initiate Microglial Activation via Toll-like Receptor 4 in Retinal Degeneration Mediated by All-trans-retinal

Although several genetic and biochemical factors are associated with the pathogenesis of retinal degeneration, it has yet to be determined how these different impairments can cause similar degenerative phenotypes. Here, we report microglial/macrophage activation in both a Stargardt disease and age-related macular degeneration mouse model caused by delayed clearance of all-trans-retinal from the retina, and in a retinitis pigmentosa mouse model with impaired retinal pigment epithelium (RPE) phagocytosis. Mouse microglia displayed RPE cytotoxicity and increased production of inflammatory chemokines/cytokines, Ccl2, Il1b, and Tnf, after coincubation with ligands that activate innate immunity. Notably, phagocytosis of photoreceptor proteins increased the activation of microglia/macrophages and RPE cells isolated from model mice as well as wild-type mice. The mRNA levels of Tlr2 and Tlr4, which can recognize proteins as their ligands, were elevated in mice with retinal degeneration. Bone marrow-derived macrophages from Tlr4-deficient mice did not increase Ccl2 after coincubation with photoreceptor proteins. Tlr4^−/−Abca4^−/−Rdh8^−/− mice displayed milder retinal degenerative phenotypes than Abca4^−/−Rdh8^−/− mice. Additionally, inactivation of microglia/macrophages by pharmacological approaches attenuated mouse retinal degeneration. This study demonstrates an important contribution of TLR4-mediated microglial activation by endogenous photoreceptor proteins in retinal inflammation that aggravates retinal cell death. This pathway is likely to represent an underlying common pathology in degenerative retinal disorders.     

The First Identification of Carbohydrate Binding Modules Specific to Chitosan

Two carbohydrate binding modules (DD1 and DD2) belonging to CBM32 are located at the C terminus of a chitosanase from Paenibacillus sp. IK-5. We produced three proteins, DD1, DD2, and tandem DD1/DD2 (DD1+DD2), and characterized their binding ability. Transition temperature of thermal unfolding (T_m) of each protein was elevated by the addition of cello-, laminari-, chitin-, or chitosan-hexamer (GlcN)₆. The T_m elevation (ΔT_m) in DD1 was the highest (10.3 °C) upon the addition of (GlcN)₆ and was markedly higher than that in DD2 (1.0 °C). A synergistic effect was observed (ΔT_m = 13.6 °C), when (GlcN)₆ was added to DD1+DD2. From isothermal titration calorimetry experiments, affinities to DD1 were not clearly dependent upon chain length of (GlcN)_n; ΔG_r° values were −7.8 (n = 6), −7.6 (n = 5), −7.6 (n = 4), −7.6 (n = 3), and −7.1 (n = 2) kcal/mol, and the value was not obtained for GlcN due to the lowest affinity. DD2 bound (GlcN)_n with the lower affinities (ΔG_r° = −5.0 (n = 3) ∼ −5.2 (n = 6) kcal/mol). Isothermal titration calorimetry profiles obtained for DD1+DD2 exhibited a better fit when the two-site model was used for analysis and provided greater affinities to (GlcN)₆ for individual DD1 and DD2 sites (ΔG_r° = −8.6 and −6.4 kcal/mol, respectively). From NMR titration experiments, (GlcN)_n (n = 2∼6) were found to bind to loops extruded from the core β-sandwich of individual DD1 and DD2, and the interaction sites were similar to each other. Taken together, DD1+DD2 is specific to chitosan, and individual modules synergistically interact with at least two GlcN units, facilitating chitosan hydrolysis.     

Structural Basis for Complement Evasion by Lyme Disease Pathogen Borrelia burgdorferi

Borrelia burgdorferi spirochetes that cause Lyme borreliosis survive for a long time in human serum because they successfully evade the complement system, an important arm of innate immunity. The outer surface protein E (OspE) of B. burgdorferi is needed for this because it recruits complement regulator factor H (FH) onto the bacterial surface to evade complement-mediated cell lysis. To understand this process at the molecular level, we used a structural approach. First, we solved the solution structure of OspE by NMR, revealing a fold that has not been seen before in proteins involved in complement regulation. Next, we solved the x-ray structure of the complex between OspE and the FH C-terminal domains 19 and 20 (FH19-20) at 2.83 Å resolution. The structure shows that OspE binds FH19-20 in a way similar to, but not identical with, that used by endothelial cells to bind FH via glycosaminoglycans. The observed interaction of OspE with FH19-20 allows the full function of FH in down-regulation of complement activation on the bacteria. This reveals the molecular basis for how B. burgdorferi evades innate immunity and suggests how OspE could be used as a potential vaccine antigen.     

TG1 integrase-based system for site-specific gene integration into bacterial genomes

Serine-type phage integrases catalyze unidirectional site-specific recombination between the attachment sites, attP and attB, in the phage and host bacterial genomes, respectively; these integrases and DNA target sites function efficiently when transferred into heterologous cells. We previously developed an in vivo site-specific genomic integration system based on actinophage TG1 integrase that introduces ～2-kbp DNA into an att site inserted into a heterologous Escherichia coli genome. Here, we analyzed the TG1 integrase-mediated integrations of att site-containing ～10-kbp DNA into the corresponding att site pre-inserted into various genomic locations; moreover, we developed a system that introduces ～10-kbp DNA into the genome with an efficiency of ～10⁴ transformants/μg DNA. Integrations of attB-containing DNA into an attP-containing genome were more efficient than integrations of attP-containing DNA into an attB-containing genome, and integrations targeting attP inserted near the replication origin, oriC, and the E. coli “centromere” analogue, migS, were more efficient than those targeting attP within other regions of the genome. Because the genomic region proximal to the oriC and migS sites is located at the extreme poles of the cell during chromosomal segregation, the oriC–migS region may be more exposed to the cytosol than are other regions of the E. coli chromosome. Thus, accessibility of pre-inserted attP to attB-containing incoming DNA may be crucial for the integration efficiency by serine-type integrases in heterologous cells. These results may be beneficial to the development of serine-type integrases-based genomic integration systems for various bacterial species.     

Circadian Yin-Yang Regulation and Its Manipulation to Globally Reprogram Gene Expression

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Identification of a novel benzimidazole derivative as a highly potent NPY Y5 receptor antagonist with an anti-obesity profile

Optimization of HTS hit 1 for NPY Y5 receptor binding affinity, CYP450 inhibition, solubility and metabolic stability led to the identification of some orally available oxygen-linker derivatives for in vivo study. Among them, derivative 4i inhibited food intake induced by the NPY Y5 selective agonist, and chronic oral administration of 4i in DIO mice caused a dose-dependent reduction of body weight gain.     

Structure–activity-relationship studies on dihydrofuran-fused perhydrophenanthrenes as an anti-Alzheimer’s disease agent

As an extended study on development of anti-Alzheimer’s disease agent, we newly synthesized various dihydrofuran-fused perhydrophenanthrenes via o-quinodimethane chemistry. This study revealed that the introduction of carbon side-chain on 8-position or removal of the acetal moiety on 3-position arose a cytotoxicity on rat cortical neurons. On the other hand, the ethereal or thio-ethereal substituent on 8-position enhanced the elongation effect on Aβ-damaged neurons. The necessity of the cyano group on 10b position was also proved in this structure–activity-relationship study.     

A sphingosine 1-phosphate receptor 2 selective allosteric agonist

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound.     

Glutathione S‐transferase π complexes with and stimulates Na+,K+‐ATPase

Glutathione S‐transferase (GST) was found to complex with the Na⁺,K⁺‐ATPase as shown by binding assay using quartz crystal microbalance. The complexation was obstructed by the addition of antiserum to the α‐subunit of the Na⁺,K⁺‐ATPase, suggesting the specificity of complexation between GST and the Na⁺,K⁺‐ATPase. Co‐immunoprecipitation experiments, using the anti‐α‐subunit antiserum to precipitate the GST‐Na⁺,K⁺‐ATPase complex and then using antibodies specific to an isoform of GST to identify the co‐precipitated proteins, revealed that GSTπ was complexed with the Na⁺,K⁺‐ATPase. GST stimulated the Na⁺,K⁺‐ATPase activity up to 1.4‐fold. The level of stimulation exhibited a saturable dose–response relationship with the amount of GST added, although the level of stimulation varied depending on the content of GSTπ in the lots of GST received from supplier. The stimulation was also obtained when recombinant GSTπ was used, confirming the results. When GST was treated with reduced glutathione, GST activity was greatly stimulated, whereas the level of stimulation of the Na⁺,K⁺‐ATPase activity was similar to that when untreated GST was added. When GST was treated with H₂O₂, GST activity was greatly diminished while the stimulation of the Na⁺,K⁺‐ATPase activity was preserved. The results suggest that GSTπ complexes with the Na⁺,K⁺‐ATPase and stimulates the latter independent of its GST activity. Copyright © 2012 John Wiley & Sons, Ltd.     

Microbiological Studies of Coli-aerogenes Bacteria

The presence of glucose-6-phosphate markedly stimulated the anaerobic utilization of glyoxylate by either cell-free extracts or partially purified enzyme preparations of coli-aerogenes bacteria. The enzymic reduction of glyoxylate to glycollate was found to occur in the presence of TPN with the following substrates; glucose-6-phosphate, glucose plus ATP, gluconate plus ATP, glucose-1-phosphate or malate. The data indicated that the reduction of glyoxylate to glycollate was coupled to the oxidation of glucose-6-phosphate via the hexose monophosphate shunt pathway. It was propounded that the operation of the hexose monophosphate oxidative pathway might be controlled by TPN-linked glyoxylic reductase, and the mechanisms of enzymic regulation in microbial respiration were also discussed.