SIRT1 and SIRT3 deacetylate homologous substrates: AceCS1,2 and HMGCS1,2.

SIRT1 and SIRT3 are NAD+-dependent protein deacetylases that are evolutionarily conserved across mammals. These proteins are located in the cytoplasm/nucleus and mitochondria, respectively. Previous reports demonstrated that human SIRT1 deacetylates Acetyl-CoA Synthase 1 (AceCS1) in the cytoplasm, whereas SIRT3 deacetylates the homologous Acetyl-CoA Synthase 2 (AceCS2) in the mitochondria. We recently showed that 3-hydroxy-3-methylglutaryl CoA synthase 2 (HMGCS2) is deacetylated by SIRT3 in mitochondria, and we demonstrate here that SIRT1 deacetylates the homologous 3-hydroxy-3-methylglutaryl CoA synthase 1 (HMGCS1) in the cytoplasm. This novel pattern of substrate homology between cytoplasmic SIRT1 and mitochondrial SIRT3 suggests that considering evolutionary relationships between the sirtuins and their substrates may help to identify and understand the functions and interactions of this gene family. In this perspective, we take a first step by characterizing the evolutionary history of the sirtuins and these substrate families

The Japan Public Health Center-based Prospective Study for the Next Generation (JPHC-NEXT): Study Design and Participants

Background: Lifestyle and life-environment factors have undergone drastic changes in Japan over the last few decades. Further, many molecular epidemiologic studies have reported that genetic, epigenetic, and other biomarker information may be useful in predicting individual disease risk.Methods: The Japan Public Health Center-based Prospective Study for the Next Generation (JPHC-NEXT) was launched in 2011 to identify risk factors for lifestyle-related disease, elucidate factors that extend healthy life expectancy, and contribute toward personalized healthcare based on our more than 20 years’ experience with the JPHC Study. From 2011 through 2016, a baseline survey was conducted at 16 municipalities in seven prefectures across the country. A self-administered questionnaire was distributed to all registered residents aged 40–74, which mainly asked about lifestyle factors, such as socio-demographic situation, personal medical history, smoking, alcohol and dietary habits. We obtained informed consent from each participant to participate in this long follow-up study of at least 20 years, including consent to the potential use of their residence registry, medical records, medical fee receipts, care insurance etc., and to the provision of biospecimens (blood and urine), including genomic analysis.Results: As of December 31, 2016, we have established a population-based cohort of 115,385 persons (Response rate 44.1%), among whom 55,278 (47.9% of participants) have provided blood and urine samples. The participation rate was slightly higher among females and in the older age group.Conclusion: We have established a large-scale population-based cohort for next-generation epidemiological study in Japan

Selenium-based S-adenosylmethionine analog reveals the mammalian seven-beta-strand methyltransferase METTL10 to be an EF1A1 lysine methyltransferase.

Lysine methylation has been extensively studied in histones, where it has been shown to provide specific epigenetic marks for the regulation of gene expression; however, the molecular mechanism and physiological function of lysine methylation in proteins other than histones remains to be fully addressed. To better understand the substrate diversity of lysine methylation, S-adenosylmethionine (SAM) derivatives with alkyne-moieties have been synthesized. A selenium-based SAM analog, propargylic Se-adenosyl-l-selenomethionine (ProSeAM), has a wide spectrum of reactivity against various lysine methyltransferases (KMTs) with sufficient stability to support enzymatic reactions in vitro. By using ProSeAM as a chemical probe for lysine methylation, we identified substrates for two seven-beta-strand KMTs, METTL21A and METTL10, on a proteomic scale in mammalian cells. METTL21A has been characterized as a heat shock protein (HSP)-70 methyltransferase. Mammalian METTL10 remains functionally uncharacterized, although its ortholog in yeast, See1, has been shown to methylate the translation elongation factor eEF1A. By using ProSeAM-mediated alkylation followed by purification and quantitative MS analysis, we confirmed that METTL21A labels HSP70 family proteins. Furthermore, we demonstrated that METTL10 also methylates the eukaryotic elongation factor EF1A1 in mammalian cells. Subsequent biochemical characterization revealed that METTL10 specifically trimethylates EF1A1 at lysine 318 and that siRNA-mediated knockdown of METTL10 decreases EF1A1 methylation levels in vivo. Thus, our study emphasizes the utility of the synthetic cofactor ProSeAM as a chemical probe for the identification of non-histone substrates of KMTs

世界文化遺産登録に向けた鹿児島市の観光まちづくり : 鹿児島駅を起点とする交通ターミナルの課題と提言

Ⅰ.はじめに Ⅱ.「明治日本の産業革命遺産」の構成資産 (1)構成資産候補の新規性：シリアルノミネーションと稼働資産 (2)鹿児島県における構成資産候補の歴史的意義 Ⅲ.世界遺産登録に向けた鹿児島観光の課題 (1)ドレスデンの教訓と「かごしま観光まちづくり研究会」の設立 (2)構成資産候補の地理的分布と磯地区の交通渋滞問題 (3)磯地区にかかる観光圧力の試算 (4)「周遊型観光」から「滞在型観光」へ Ⅳ.解決策の提示 (1)JR鹿児島駅に対する有効利用性の注目 (2)鹿児島駅と西鹿児島駅の推移 (3)観光まちづくり課題解決の実践的活動と鹿児島駅の役割 Ⅴ.おわり

METTL10 knockdown reduces the level of lysine 318 methylation in EF1A1 <i>in vivo</i>.

<p>A, HEK293T cells were transfected with either scramble siRNA (Scr) or siRNA against METTL10 (siMETTL10). Twenty-four hours after the transfection, the plasmid for FLAG-EF1A1 was transfected and cultured for an additional 48 hours. Cells were harvested and the METTL10 expression level was analyzed with RT-PCR. n = 3, mean ± SD, **p<0.01. B, FLAG-EF1A1 was purified with anti-FLAG agarose beads from siRNA treated cells. EF1A1 lysine 318 mono-methylation (K318-Me1), di-methylation (K318-Me2), tri-methylation (K318-Me3), and unmethylation (K318-Me0) were analyzed by using a triple stage quadrupole mass spectrometer. The intensity of each peptide was normalized to the quantity of each sample's EF1A1 as determined by the presence of five EF1A1 peptide fragments, and the relative intensity was normalized to Scr of K318-Me0; n = 3, mean ± SD, *p<0.05, **p<0.01. C, <i>In vivo</i> METTL10 knockdown is required for <i>in vitro</i> robust methylation by METTL10. FLAG-EF1A1 purified from Scr or siMETTL10 treated cells was incubated with or without His-METTL10 in the presence of ¹⁴C-labeled SAM. Proteins were separated with SDS-PAGE and stained with Coomassie blue (bottom), the autoradiography was performed using a BAS-5000 image analyzer (top).</p

ProSeAM, a synthetic SAM analog, has a wide spectrum of reactivity for histones and non-histone substrates.

<p>A, Schematic overview for analyzing lysine methylation. A synthetic cofactor was used to transfer an alkyne moiety to the ε-amino group of lysine by KMTs (1). The modified proteins were tagged with biotin via CuAAC reaction (2). Tagged-proteins in the crude lysates were pulled down with affinity beads (3), and the precipitants were further analyzed with a LC-MS apparatus (4). B, Chemical structure of SAM (1), propargylated SAM (2) and ProSeAM (3). C, H3 peptide (1-21 a.a.) and ProSeAM was incubated with or without GST-G9a at 20°C for 2 h, then the peptide was analyzed by MALDI-TOF MS. D, full-length Histone H3 (1 µg) and ProSeAM (500 µM) were incubated with indicated KMTs (0.5 µg) for 2 h at 20°C. The histones were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with streptavidin-HRP (top) or anti-Histone H3 antibody (bottom). E, The non-histone substrates His-HSP90 and His-HSP70 (1 µg) were incubated with His-SMYD2 and His-METTL21A (1 µg), respectively. After the reaction, proteins were separated by SDS-PAGE (right). Their modifications were detected by western blotting with streptavidin-HRP as in Fig. 1D. *and ** showed automodification of SMYD2 and METTL21A, respectively (left). F, His-HSP70 (WT and K561R) were incubated with or without His-METTL21A in the presence of ProSeAM for 2 h at 20°C. Modified proteins were biotinylated and detected with streptavidin-HRP (top) or anti-HSP70 antibody for the loading control (bottom).</p