Structural Insights into Methylated DNA Recognition by the Methyl-CpG Binding Domain of MBD6 from Arabidopsis thaliana

Cytosine methylation is an epigenetic modification essential for formation of mature heterochromatin, gene silencing, and genomic stability. In plants, methylation occurs not only at cytosine bases in CpG but also in CpHpG and CpHpH contexts, where H denotes A, T, or C. Methyl-CpG binding domain (MBD) proteins, which recognize symmetrical methyl-CpG dinucleotides and act as gene repressors in mammalian cells, are also present in plant cells, although their structural and functional properties still remain poorly understood. To fill this gap, in this study, we determined the solution structure of the MBD domain of the MBD6 protein from Arabidopsis thaliana and investigated its binding properties to methylated DNA by binding assays and an in-depth NMR spectroscopic analysis. The AtMBD6 MBD domain folds into a canonical MBD structure in line with its binding specificity toward methyl-CpG and possesses a DNA binding interface similar to mammalian MBD domains. Intriguingly, however, the binding affinity of the AtMBD6 MBD domain toward methyl-CpG-containing DNA was found to be much lower than that of known mammalian MBD domains. The main difference arises from the absence of positively charged residues in AtMBD6 that supposedly interact with the DNA backbone as seen in mammalian MBD/methyl-CpG-containing DNA complexes. Taken together, we have established a structural basis for methyl-CpG recognition by AtMBD6 to develop a deeper understanding how MBD proteins work as mediators of epigenetic signals in plant cells

The crystal structure of the plant small GTPase OsRac1 reveals its mode of binding to NADPH oxidase

This research was originally published in Journal of Biological Chemistry. Ken-ichi Kosami, Izuru Ohki, Minoru Nagano, Kyoko Furuita, Toshihiko Sugiki, Yoji Kawano, Tsutomu Kawasaki, Toshimichi Fujiwara, Atsushi Nakagawa, Ko Shimamoto and Chojiro Kojima. The crystal structure of the plant small GTPase OsRac1 reveals its mode of binding to NADPH oxidase. Journal of Biological Chemistry. 2014; 289, 28569-28578. © the American Society for Biochemistry and Molecular Biology

ヒト メチルカ トクイテキ リプレッサー MBD1 ノ メチルカ CpG ケツゴウ ドメイン ノ NMR ニヨル ケンキュウ

https://library.naist.jp/mylimedio/dllimedio/show.cgi?bookid=100051047&oldid=95317博士 (Doctor)バイオサイエンス (Bioscience)博第154号甲第154号博士(バイオサイエンス)奈良先端科学技術大学院大

MtnBD Is a Multifunctional Fusion Enzyme in the Methionine Salvage Pathway of <i>Tetrahymena thermophila</i>

<div><p>To recycle reduced sulfur to methionine in the methionine salvage pathway (MSP), 5-methylthioribulose-1-phosphate is converted to 2-keto-4-methylthiobutyrate, the methionine precursor, by four steps; dehydratase, enolase, phosphatase, and dioxygenase reactions (catalyzed by MtnB, MtnW, MtnX and MtnD, respectively, in <i>Bacillus subtilis</i>). It has been proposed that the MtnBD fusion enzyme in <i>Tetrahymena thermophila</i> catalyzes four sequential reactions from the dehydratase to dioxygenase steps, based on the results of molecular biological analyses of mutant yeast strains with knocked-out MSP genes, suggesting that new catalytic function can be acquired by fusion of enzymes. This result raises the question of how the MtnBD fusion enzyme can catalyze four very different reactions, especially since there are no homologous domains for enolase and phosphatase (MtnW and MtnX, respectively, in <i>B. subtilis</i>) in the peptide. Here, we tried to identify the domains responsible for catalyzing the four reactions using recombinant proteins of full-length MtnBD and each domain alone. UV-visible and ¹H-NMR spectral analyses of reaction products revealed that the MtnB domain catalyzes dehydration and enolization and the MtnD domain catalyzes dioxygenation. Contrary to a previous report, conversion of 5-methylthioribulose-1-phosphate to 2-keto-4-methylthiobutyrate was dependent on addition of an exogenous phosphatase from <i>B. subtilis</i>. This was observed for both the MtnB domain and full-length MtnBD, suggesting that MtnBD does not catalyze the phosphatase reaction. Our results suggest that the MtnB domain of <i>T. thermophila</i> MtnBD acquired the new function to catalyze both the dehydratase and enolase reactions through evolutionary gene mutations, rather than fusion of MSP genes.</p></div

The gateway reflex regulates tissue-specific autoimmune diseases

Abstract The dynamic interaction and movement of substances and cells between the central nervous system (CNS) and peripheral organs are meticulously controlled by a specialized vascular structure, the blood–brain barrier (BBB). Experimental and clinical research has shown that disruptions in the BBB are characteristic of various neuroinflammatory disorders, including multiple sclerosis. We have been elucidating a mechanism termed the “gateway reflex” that details the entry of immune cells, notably autoreactive T cells, into the CNS at the onset of such diseases. This process is initiated through local neural responses to a range of environmental stimuli, such as gravity, electricity, pain, stress, light, and joint inflammation. These stimuli specifically activate neural pathways to open gateways at targeted blood vessels for blood immune cell entry. The gateway reflex is pivotal in managing tissue-specific inflammatory diseases, and its improper activation is linked to disease progression. In this review, we present a comprehensive examination of the gateway reflex mechanism

UV-visible and ¹H-NMR spectra of metabolites synthesized by <i>Tetrahymena</i> MtnBD.

<p>(A) Conversion of MTRu-1-P into HK-MTPenyl-1-P in D₂O phosphate buffer. Dehydration of MTRu-1-P (upper part) may introduce a proton from solvent into C4 of HK-MTPenyl-1-P (lower part). (B) UV-visible spectral changes after adding MtnBD into reaction mixture containing MTRu-1-P. Spectra are shown in different colors (0, 40, 80, 120, 160, 200, 240, 300, and 420 s). (C) Spectral changes before and after adding 0.14 µg <i>B. subtilis</i> MtnX to reaction product in Fig. 2B. Spectra are shown in different colors (0, 10, 20, 30, 40, 50, 60, 80, 100, and 220 s). In (B) and (C), spectra of products were measured at 35°C in 100 µL 50 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 100 µM MTR-1-P, and 9 µg <i>Bacillus</i> MtnA. MTR-1-P was converted into MTRu-1-P by MtnA before assay. (D) Time course of HK-MTPenyl-1-P production with different amounts of MtnBD protein. The reaction was initiated by adding 0.7, 1.5 or 2.2 µg MtnBD proteins to 100 µL 50 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 100 µM MTR-1-P, and 9 µg MtnA at 35°C. MTR-1-P was converted into MTRu-1-P by MtnA before assay. Concentration of HK-MTPenyl-1-P was estimated using the molecular extinction coefficient (9,500 M⁻¹ cm⁻¹ at 280 nm). (E) ¹H-NMR spectrum of product (MTRu-1-P) from <i>B. subtilis</i> MtnA. Isomerization reaction in 250 µL 25 mM sodium phosphate (pD 7.5), 0.1 mM MgCl₂, 1 mM MTR-1-P, and 20 µg <i>B. subtilis</i> MtnA at 37°C for 30 min. (F to H) ¹H-NMR spectra after adding 10 µg MtnBD into reaction mixture containing MTRu-1-P. * indicates chemical shift associated with degraded compound (elimination of methyl group at C1). Reaction products generated in magnesium phosphate buffer at 25°C and pD 7.5 at 1 h (F), 2 h (G) and 6 h (H). ¹H peak at 4.74 ppm represents proton from residual H₂O.</p

HPLC analyses of reaction products of dioxygenase reaction.

<p>(A) Authentic KMTB (1 mM). (B) Chromatogram of products generated by MtnBD and <i>B. subtilis</i> MtnX. The triangle represents KMTB. * indicates an unidentified peak at 11.8 min.</p