Yaeta Endo

Novel protein fold discovered in the PabI family of restriction enzymes

Although structures of many DNA-binding proteins have been solved, they fall into a limited number of folds. Here, we describe an approach that led to the finding of a novel DNA-binding fold. Based on the behavior of Type II restriction–modification gene complexes as mobile elements, our earlier work identified a restriction enzyme, R.PabI, and its cognate modification enzyme in Pyrococcus abyssi through comparison of closely related genomes. While the modification methyltransferase was easily recognized, R.PabI was predicted to have a novel 3D structure. We expressed cytotoxic R.PabI in a wheat-germ-based cell-free translation system and determined its crystal structure. R.PabI turned out to adopt a novel protein fold. Homodimeric R.PabI has a curved anti-parallel β-sheet that forms a ‘half pipe’. Mutational and in silico DNA-binding analyses have assigned it as the double-strand DNA-binding site. Unlike most restriction enzymes analyzed, R.PabI is able to cleave DNA in the absence of Mg2+. These results demonstrate the value of genome comparison and the wheat-germ-based system in finding a novel DNA-binding motif in mobile DNases and, in general, a novel protein fold in horizontally transferred genes.

Anti-interleukin-5 and multiple autoantibodies are associated with human atherosclerotic diseases and serum interleukin-5 levels

Atherosclerotic diseases, such as coronary artery disease and peripheral artery disease, are systemic disorders and among the leading causes of mortality and morbidity throughout the world. However, the exact pathophysiological mechanisms underlying the development of atherosclerosis remain unknown; currently, atherosclerosis is thought to involve an inflammatory process. Systemic inflammatory reactions and accumulation of immune cells in atherosclerotic lesions in situ are considered essential. We have comprehensively analyzed autoantibodies in patients with atherosclerosis by means of a newly developed high‐throughput autoantibody analysis system. A wide range of autoantibodies was found in sera from patients with atherosclerosis. After we statistically analyzed the titers of each autoantibody with conventional techniques, the results underwent text‐mining analyses based on natural language processing. Combinatory analysis revealed a close association between anti‐interleukin (IL)‐5 antibody and atherosclerosis. Titers of anti‐IL‐5 antibodies and serum IL‐5 concentrations were also closely associated with other risk factors, such as low‐density lipoprotein cholesterol, serum creatinine, fasting plasma glucose, gender, and age, suggesting that suppressed IL‐5 function mediated by autoantibodies in patients with atherosclerosis plays an important role in the disease process. To validate the clinical significance of these findings, we computed the specificity and sensitivity of titers of anti‐IL‐5 autoantibodies for human atherosclerosis. When antibody titers of 1.49 were assumed to predict the presence of atherosclerosis, the sensitivity was 95.0% and the specificity 91.0%, with an area under the curve of 0.940. Our results provide important clues to understanding the role of autoantibody‐mediated immune reactions in human atherosclerosis and suggest novel therapeutic opportunities for management of the disease.—Ishigami, T., Abe, K., Aoki, I., Minegishi, S., Ryo, A., Matsunaga, S., Matsuoka, K., Takeda, H., Sawasaki, T., Umemura, S., and Endo, Y., Anti‐interleukin‐5 and multiple autoantibodies are associated with human atherosclerotic diseases and serum interleukin‐5 levels. FASEB J. 27, 3437–3445 (2013). www.fasebj.org

The Solanum chacoense ovary receptor kinase 11 (ScORK11) undergoes tissue-dependent transcriptional, translational and post-translational regulation

Using a subtraction screen to isolate weakly expressed transcripts from ovule and ovary libraries, we uncovered 30 receptor-like kinases that were predominantly expressed in ovary and fruit tissues following fertilization [1]. Here we describe the analysis of Solanum chacoense ovule receptor kinase 11 (ScORK11), a member of the large LRR III receptor kinase subfamily that localizes to the plasma membrane. In situ analyses demonstrated that ScORK11 gene expression was mainly restricted to the ovule integument, the embryo sac and the pericarp of the fruit. Tight regulation of ScORK11 expression at the mRNA level was also accompanied by both translational and post-translational regulation of protein levels.

Protein Synthesis in Test Tube

細胞を使わずにタンパク質をつくるタンパク質の無細胞合成法とは,生体の遺伝情報発現系を人工容器内に取りそろえ,高品質タンパク質を試験管内で鋳型合成しようとするものである。この方法では,生命体の生理学的制約を受けないので,遺伝子からのタンパク質合成のハイスループット化が可能となり,さらに合成可能なアミノ酸配列種を劇的に拡大できるものと期待できる。

Highly Productive Plant Continuous Cell-Free System

The development of a system capable of synthesizing any desired protein on a preparative scale is one of the most important endeavors in biotechnology today. Three strategies are currently being used: chemical synthesis, in vivo expression, and cell-free protein synthesis. The first two methods have severe limitations: chemical synthesis is not feasible for the synthesis of long peptides because of low yield, and in vivo expression can produce only those proteins that do not affect the physiology of the host cell [1–3]. Cell-free translation systems, in contrast, can synthesize proteins with high speed and accuracy, approaching in vivo rates [4–5], and they can express proteins that would interfere with cell physiology. However, they are relatively inefficient because of their instability[6].