Recording of elapsed time and temporal information about biological events using Cas9

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Structural basis for the N‐degron specificity of ClpS1 from Arabidopsis thaliana

The N‐degron pathway determines the half‐life of proteins in both prokaryotes and eukaryotes by precisely recognizing the N‐terminal residue (N‐degron) of substrates. ClpS proteins from bacteria bind to substrates containing hydrophobic N‐degrons (Leu, Phe, Tyr, and Trp) and deliver them to the caseinolytic protease system ClpAP. This mechanism is preserved in organelles such as mitochondria and chloroplasts. Bacterial ClpS adaptors bind preferentially to Leu and Phe N‐degrons; however, ClpS1 from Arabidopsis thaliana (AtClpS1) shows a difference in that it binds strongly to Phe and Trp N‐degrons and only weakly to Leu. This difference in behavior cannot be explained without structural information due to the high sequence homology between bacterial and plant ClpS proteins. Here, we report the structure of AtClpS1 at 2.0 Å resolution in the presence of a bound N‐degron. The key determinants for α‐amino group recognition are conserved among all ClpS proteins, but the α3‐helix of eukaryotic AtClpS1 is significantly shortened, and consequently, a loop forming a pocket for the N‐degron is moved slightly outward to enlarge the pocket. In addition, amino acid replacement from Val to Ala causes a reduction in hydrophobic interactions with Leu N‐degron. A combination of the fine‐tuned hydrophobic residues in the pocket and the basic gatekeeper at the entrance of the pocket controls the N‐degron selectivity of the plant ClpS protein.     

Algivirga pacifica gen. nov., sp. nov., a Novel Agar-Degrading Marine Bacterium of the Family Flammeovirgaceae Isolated from Micronesia

An aerobic, Gram-negative, coccoid to short rod-shaped and non-flagellated marine bacterial strain S354^T was isolated from seawater of Micronesia. The strain was capable to degrade agar-forming slight depression into agar plate. Growth occurred at a temperature range of 12–44 °C, a pH range of 5–9, and a salinity range of 1–7 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences suggested that S354^T belongs to the family Flammeovirgaceae. The novel strain was most closely related to Limibacter armeniacum YM 11-185^T with similarity of 92.5 %. The DNA G+C content was 43.8 mol%. The major fatty acids (>10 %) were iso-C_15:0 and C_16:1 ω5c. The predominant isoprenoid quinone was determined to be MK-7. Polar lipid profile of S354^T consisted of phosphatidylethanolamine, unknown polar lipid, and unknown glycolipids. Based on the phenotypic, phylogenetic, biochemical, and physiological tests conducted in this study, S354^T is proposed to represent a type strain of a novel genus and species. The 16S rRNA gene sequence of S354^T is registered in GenBank under the accession number JQ639084. The type of strain Algivirga pacifica gen. nov., sp. nov. is S354^T (=KCCM 90107^T=JCM 18326^T).     

Structural and Functional Analysis of the Natural JNK1 Inhibitor Quercetagetin

c-Jun NH2-terminal kinases (JNKs) and phosphatidylinositol 3-kinase (PI3-K) play critical roles in chronic diseases such as cancer, type II diabetes, and obesity. We describe here the binding of quercetagetin (3,3′,4′,5,6,7-hydroxyflavone), related flavonoids, and SP600125 to JNK1 and PI3-K by ATP-competitive and immobilized metal ion affinity-based fluorescence polarization assays and measure the effect of quercetagetin on JNK1 and PI3-K activities. Quercetagetin attenuated the phosphorylation of c-Jun and AKT, suppressed AP-1 and NF-κB promoter activities, and also reduced cell transformation. It attenuated tumor incidence and reduced tumor volumes in a two-stage skin carcinogenesis mouse model.Our crystallographic structure determination data show that quercetagetin binds to the ATP-binding site of JNK1. Notably, the interaction between Lys55, Asp169, and Glu73 of JNK1 and the catechol moiety of quercetagetin reorients the N-terminal lobe of JNK1, thereby improving compatibility of the ligand with its binding site. The results of a theoretical docking study suggest a binding mode of PI3-K with the hydroxyl groups of the catechol moiety forming hydrogen bonds with the side chains of Asp964 and Asp841 in the p110γ catalytic subunit. These interactions could contribute to the high inhibitory activity of quercetagetin against PI3-K. Our study suggests the potential use of quercetagetin in the prevention or therapy of cancer and other chronic diseases.     

Attenuation by a Vigna nakashimae extract of nonalcoholic fatty liver disease in high-fat diet-fed mice

A Vigna nakashimae (VN) extract has been shown to have antidiabetic and anti-obesity effects. However, the mechanism underlying the effect of a VN extract on hepatic inflammation and endoplasmic reticulum (ER) stress remains unclear. In the present study, we investigated how a VN extract protects against the development of non-alcoholic fatty liver disease (NAFLD). A VN extract for 12 weeks reduced the body weight, serum metabolic parameters, cytokines, and hepatic steatosis in high-fat diet (HFD)-fed mice. A VN extract decreased HFD-induced hepatic acetyl CoA carboxylase and glucose transporter 4 expressions. In addition to the levels of high-mobility group box 1 and receptor for advanced glycation, the hepatic expression of ATF4 and caspase-3 was also reduced by a VN extract. Thus, these data indicate that a chronic VN extract prevented NAFLD through multiple mechanisms, including inflammation, ER stress, and apoptosis in the liver.