Crystal structure of human cytosolic aspartyl‐tRNA synthetase,a component of multi‐tRNA synthetase complex

Human cytosolic aspartyl‐tRNA synthetase (DRS) catalyzes the attachment of the amino acid aspartic acid to its cognate tRNA and it is a component of the multi‐tRNA synthetase complex (MSC) which has been known to be involved in unexpected signaling pathways. Here, we report the crystal structure of DRS at a resolution of 2.25 Å. DRS is a homodimer with a dimer interface of 3750.5 Ų which comprises 16.6% of the monomeric surface area. Our structure reveals the C‐terminal end of the N‐helix which is considered as a unique addition in DRS, and its conformation further supports the switching model of the N‐helix for the transfer of tRNA^Asp to elongation factor 1α. From our analyses of the crystal structure and post‐translational modification of DRS, we suggest that the phosphorylation of Ser146 provokes the separation of DRS from the MSC and provides the binding site for an interaction partner with unforeseen functions.Proteins 2013; 81:1840–1846. © 2013 Wiley Periodicals, Inc.     

The structure of a shellfish specific GST class glutathione S‐transferase from antarctic bivalve Laternula elliptica reveals novel active site architecture

Glutathione‐S‐transferases have been identified in all the living species examined so far, yet little is known about their function in marine organisms. In a previous report, the recently identified GST from Antarctic bivalve Laternula elliptica (LeGST) was classified into the rho class GST, but there are several unique features of LeGST that may justify reclassification, which could represent specific shellfish GSTs. Here, we determined the crystal structure of LeGST, which is a shellfish specific class of GST. The structural analysis showed that the relatively open and wide hydrophobic H‐site of the LeGST allows this GST to accommodate various substrates. These results suggest that the H‐site of LeGST may be the result of adaptation to their environments as sedentary organisms. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Cyclooxygenase-2 dependent metabolism of 20-HETE increases adiposity and adipocyte enlargement in mesenchymal stem cell-derived adipocytes

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE), a product of the cytochrome P450 (CYP)-catalyzed ω-hydroxylation of arachidonic acid, induces oxidative stress and, in clinical studies, is associated with increased body mass index (BMI) and the metabolic syndrome. This study was designed to examine the effects of exogenous 20-HETE on mesenchymal stem cell (MSC)-derived adipocytes. The expression levels of CYP4A11 and CYP4F2 (major 20-HETE synthases in humans) in MSCs decreased during adipocyte differentiation; however, exogenous administration of 20-HETE (0.1–1 μM) increased adipogenesis in a dose-dependent manner in these cells (P < 0.05). The inability of a 20-HETE analog to reproduce these effects suggested the involvement of a metabolic product of 20-HETE in mediating its pro-adipogenic effects. A cyclooxygenase (COX)-1 selective inhibitor enhanced, whereas a COX-2 selective or a dual COX-1/2 inhibitor attenuated adipogenesis induced by 20-HETE. The COX-derived metabolite of 20-HETE, 20-OH-PGE₂, enhanced adipogenesis and lipid accumulation in MSCs. The pro-adipogenic effects of 20-HETE and 20-OH-PGE₂ resulted in the increased expression of the adipogenic regulators PPARγ and β-catenin in MSC-derived adipocytes. Taken together we show for the first time that 20-HETE-derived COX-2-dependent 20-OH-PGE₂ enhances mature inflamed adipocyte hypertrophy in MSC undergoing adipogenic differentiation.     

Inactivation of Max-interacting Protein 1 Induces Renal Cilia Disassembly through Reduction in Levels of Intraflagellar Transport 20 in Polycystic Kidney

Cilia in ciliated cells consist of protruding structures that sense mechanical and chemical signals from the extracellular environment. Cilia are assembled with variety molecules via a process known as intraflagellar transport (IFT). What controls the length of cilia in ciliated cells is critical to understand ciliary disease such as autosomal dominant polycystic kidney disease, which involves abnormally short cilia. But this control mechanism is not well understood. Previously, multiple tubular cysts have been observed in the kidneys of max-interacting protein 1 (Mxi1)-deficient mice aged 6 months or more. Here, we clarified the relationship between Mxi1 inactivation and cilia disassembly. Cilia phenotypes were observed in kidneys of Mxi1-deficient mice using scanning electron microscopy to elucidate the effect of Mxi1 on renal cilia phenotype, and cilia disassembly was observed in Mxi1-deficient kidney. In addition, genes related to cilia were validated in vitro and in vivo using quantitative PCR, and Ift20 was selected as a candidate gene in this study. The length of cilium decreased, and p-ERK level induced by a cilia defect increased in kidneys of Mxi1-deficient mice. Ciliogenesis of Mxi1-deficient mouse embryonic fibroblasts (MEFs) decreased, and this abnormality was restored by Mxi1 transfection in Mxi1-deficient MEFs. We confirmed that ciliogenesis and Ift20 expression were regulated by Mxi1 in vitro. We also determined that Mxi1 regulates Ift20 promoter activity via Ets-1 binding to the Ift20 promoter. These results indicate that inactivating Mxi1 induces ciliary defects in polycystic kidney.     

Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2 Sequestration

Carbonic anhydrase is an enzyme that reversibly catalyzes the hydration of carbon dioxide (CO₂). It has been suggested recently that this remarkably fast enzyme can be used for sequestration of CO₂, a major greenhouse gas, making this a promising alternative for chemical CO₂ mitigation. To promote the economical use of enzymes, we engineered the carbonic anhydrase from Neisseria gonorrhoeae (ngCA) in the periplasm of Escherichia coli, thereby creating a bacterial whole-cell catalyst. We then investigated the application of this system to CO₂ sequestration by mineral carbonation, a process with the potential to store large quantities of CO₂. ngCA was highly expressed in the periplasm of E. coli in a soluble form, and the recombinant bacterial cell displayed the distinct ability to hydrate CO₂ compared with its cytoplasmic ngCA counterpart and previously reported whole-cell CA systems. The expression of ngCA in the periplasm of E. coli greatly accelerated the rate of calcium carbonate (CaCO₃) formation and exerted a striking impact on the maximal amount of CaCO₃ produced under conditions of relatively low pH. It was also shown that the thermal stability of the periplasmic enzyme was significantly improved. These results demonstrate that the engineered bacterial cell with periplasmic ngCA can successfully serve as an efficient biocatalyst for CO₂ sequestration.     

Binding characteristics of a bacterial expansin (BsEXLX1) for various types of pretreated lignocellulose

BsEXLX1 from Bacillus subtilis is the first discovered bacterial expansin as a structural homolog of a plant expansin, and it exhibited synergism with cellulase on the cellulose hydrolysis in a previous study. In this study, binding characteristics of BsEXLX1 were investigated using pretreated and untreated Miscanthus x giganteus in comparison with those of CtCBD3, a cellulose-binding domain from Clostridium thermocellum. The amounts of BsEXLX1 bound to cellulose-rich substrates were significantly lower than those of CtCBD3. However, the amounts of BsEXLX1 bound to lignin-rich substrates were much higher than those of CtCBD3. A binding competition assay between BsEXLX1 and CtCBD3 revealed that binding of BsEXLX1 to alkali lignin was not affected by the presence of CtCBD3. This preferential binding of BsEXLX1 to lignin could be related to root colonization in plants by bacteria, and the bacterial expansin could be used as a lignin blocker in the enzymatic hydrolysis of lignocellulose.