5-羟甲基胞嘧啶及其TET氧合酶在神经系统发育和相关疾病中的研究进展

神经系统的正常发育是多种因素相互协调作用的结果,一旦特定因素失衡将引起相关疾病的发生。近年来不断有研究发现,DNA去甲基化过程的一类中间产物5-羟甲基胞嘧啶(5-hydroxymethylcytosine, 5hmC)作为一种新的表观遗传标记,在神经系统中高水平分布,并参与认知、记忆等重要的神经功能。5hmC的形成由氧合酶家族分子(ten-eleven translocation protein, TET)催化,在多种神经系统相关疾病中,5hmC水平和TETs分子的表达都发生改变,提示TET-5hmC表观遗传机制在复杂的神经系统发生发展过程中发挥了重要的调控作用。此外,作为基因表达调控的DNA标记物,5hmC的基因定位与基因表达水平的关系也是重要的研究方向。本文就近年来5hmC和TET家族蛋白分子在神经系统发育和相关疾病方面的重要研究发现进行了综述总结,希望为相关领域研究人员深入开展研究提供重要的思路,并为相关疾病设计治疗策略提供理论支持。     

Rate enhancement of cytokinin oxidase/dehydrogenase using 2,6-dichloroindophenol as an electron acceptor

Cytokinin oxidase/dehydrogenase degrades cytokinins by dehydrogenating the N6-C1 bond of cytokinins. The resulting imine is then hydrolyzed. For example, isopentenyl-adenine is cleaved into 3-methyl-2-butenal (isopentenyl-aldehyde) and adenine . The reducing equivalents from dehydrogenation are transferred to an unknown sink, in vivo. It has been hypothesized that the enzyme requires oxygen , possibly resulting in the formation of hydrogen peroxide. 2,6-dichloroindophenol (DCPIP) can function as an acceptor of reducing equivalents for in vitro cytokinin oxidase/dehydrogenase reactions. For the predominant cytokinin oxidase/dehydrogenase in maize, ZmCKX1, the addition of DCPIP to in vitro reactions increases the reaction rate to nearly 4000-fold faster than the oxygen-dependent rate. Further, the change in absorbance of DCPIP at 600 nm, as it is reduced, forms the basis for an assay suitable for following biochemical purification of cytokinin oxidase/dehydrogenases , detailed kinetic studies , and rapid measurement of cytokinin oxidase/dehydrogenase activity in large numbers of samples.     

Targeting the Ca2+/Calmodulin-dependent protein kinase II by Tetrandrine in human liver cancer cells

Hepatocellular carcinoma (HCC) is the most prevalent malignancy in liver and a leading cause of cancer-related deaths. Despite the pressing need for treatment options, patients with HCC develop significant resistance and adverse side effects to current approved drugs that becomes a major barrier to effective treatment. A natural product Tetrandrine (TET) is a potential alternative treatment option for HCC, with demonstrated effectiveness and low toxicity. However, the mechanisms by which Tetrandrine inhibits HCC are unclear. In the current study, we identify Ca²+/calmodulin-dependent protein kinase II δ (CaMKIIδ) as a potential TET drug target through structural modeling. Screening of a panel of HCC cell lines reveal differential sensitivities toward TET treatment. Interestingly, IC₅₀ of TET inhibition of HCC cell proliferation is positively correlated with CaMKIIδ expression level in these distinct HCC cells. Furthermore, TET treatment resulted in a marked reduction of CaMKIIδ phosphorylation level, and knockdown of CaMKIIδ reduced the sensitivity of HCC cells to TET. Most importantly, CaMKIIδ protein levels in high-grade human HCC samples were significantly elevated as compared to normal liver tissues. Taken together, our studies demonstrate that the natural compound TET targets CaMKIIδ in HCC cells, and that CaMKIIδ level is a potential biomarker to identify HCC patient populations sensitive to Tetrandrine treatment.     

Crystal structures of E. coli laccase CueO at different copper concentrations

CueO protein is a hypothetical bacterial laccase and a good laccase candidate for large scale industrial application. Four CueO crystal structures were determined at different copper concentrations. Low copper occupancy in apo-CueO and slow copper reconstitution process in CueO with exogenous copper were demonstrated. These observations well explain the copper dependence of CueO oxidase activity. Structural comparison between CueO and other three fungal laccase proteins indicates that Glu106 in CueO constitutes the primary counter-work for reconstitution of the trinuclear copper site. Mutation of Glu106 to a Phe enhanced CueO oxidation activity and supported this hypothesis. In addition, an extra alpha-helix from Leu351 to Gly378 covers substrate biding pocket of CueO and might compromises the electron transfer from substrate to type I copper.     

Phosphorylation of TET Proteins Is Regulated via O-GlcNAcylation by the O-Linked N-Acetylglucosamine Transferase (OGT)

TET proteins oxidize 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine and thus provide a possible means for active DNA demethylation in mammals. Although their catalytic mechanism is well characterized and the catalytic dioxygenase domain is highly conserved, the function of the regulatory regions (the N terminus and the low-complexity insert between the two parts of the dioxygenase domains) is only poorly understood. Here, we demonstrate that TET proteins are subject to a variety of post-translational modifications that mostly occur at these regulatory regions. We mapped TET modification sites at amino acid resolution and show for the first time that TET1, TET2, and TET3 are highly phosphorylated. The O-linked GlcNAc transferase, which we identified as a strong interactor with all three TET proteins, catalyzes the addition of a GlcNAc group to serine and threonine residues of TET proteins and thereby decreases both the number of phosphorylation sites and site occupancy. Interestingly, the different TET proteins display unique post-translational modification patterns, and some modifications occur in distinct combinations. In summary, our results provide a novel potential mechanism for TET protein regulation based on a dynamic interplay of phosphorylation and O-GlcNAcylation at the N terminus and the low-complexity insert region. Our data suggest strong cross-talk between the modification sites that could allow rapid adaption of TET protein localization, activity, or targeting due to changing environmental conditions as well as in response to external stimuli.     

Increase in the toxic effects of Tl+ on isolated rat liver mitochondria in the presence of nonactin

The effects of Tl(+) ions on isolated rat liver mitochondria were studied in the presence of nonactin, a cyclic ionophore. Nonenergized rat liver mitochondria were increasingly swollen at an elevated concentration of Tl(+) in the 160 mOsm medium containing 0-150 mM sucrose and 0-75 mM TlNO(3) or 0-50 mM Tl acetate. On the contrary, mitochondria in experiments with nonactin were contracted in the medium with 5-25 mM Tl(+) and were swollen only in the medium with 50-75 mM TlNO(3) or 50 mM Tl acetate. State 4 respiration along with swelling of succinate-energized mitochondria followed contraction after their deenergization was further enhanced at increasing concentration of Tl acetate in a medium containing nonactin. Regardless of the presence of nonactin, State 3 and 2,4-dinitrophenol (DNP)-stimulated respiration and the monoamine oxidase (MAO) activity were not affected in the medium with 0-25 mM Tl acetate and sucrose. DNP-stimulated respiration decreased and the MAO activity somewhat increased in the medium containing 50 mM Tl acetate and nonactin. Uptake of (86)Rb(+) by energized mitochondria in the presence of valinomycin was considerably decreased when Tl(+) and nonactin were simultaneously present in the medium. An increase of the toxic effect of Tl(+) on rat liver mitochondria in the presence of nonactin is accounted for by disruption of mitochondria due to their more extensive swelling and uncoupling of mitochondria, resulting in the stimulation of State 4 and depletion of their energy store.     

几种蛇毒抑菌作用的比较

本文通过平板扩散实验观察了蛇岛蝮蛇毒、长白山白眉蝮蛇毒、江浙蝮蛇毒及中华眼镜蛇毒对革兰氏阳性菌和革兰氏阴性菌的抑菌作用。结果表明四种蛇毒对试验菌株均有不同程度的抑菌作用;其中白眉蝮蛇毒对金黄色葡萄球菌的最小抑菌浓度为０．６３ｍｇ／ｍｌ,对大肠杆菌为５．０ｍｇ／ｍｌ。抑菌作用与蛇毒中Ｌ—氨基酸氧化酶的活力有相关性。利用ＣＭ—ＳｅｐｈａｄｅｘＣ－２５ＳｅｐｈｄｅｘＧ—７５一对江浙蝮蛇毒的抑菌成份进行初步的柱层析分离,从纯化的各组份看,Ｌ—氨基酸氧化酶活力高的其抑菌活性也高。     

TET蛋白的去甲基化机制及其在调控小鼠发育过程中的作用

TET(Ten-eleven translocation)蛋白家族共有3个成员,分别为TET1、TET2和TET3,均属于α-酮戊二酸(α-KG)和Fe²⁺依赖的双加氧酶,可以将5-甲基胞嘧啶(5-methylcytosine, 5 mC)氧化为5-羟甲基胞嘧啶(5-hydroxymethylcytosine, 5 hmC)、5-甲酰基胞嘧啶(5-formylcytosine, 5 fC)及5-羧基胞嘧啶(5-carboxylcytosine, 5 caC)。研究表明,TET蛋白通过不同机制以主动或被动的方式调控DNA去甲基化,且去甲基化的活性可能受其他因子的调控。TET蛋白广泛参与哺乳动物发育过程的调节,其中在原始生殖细胞的形成、胚胎发育、干细胞多能性及神经和脑发育等方面发挥了重要作用。TET蛋白生物功能的发现为表观遗传学研究开辟了全新的研究领域,而且相关研究结果对拓展生命科学研究具有重要意义。文章综述了TET蛋白家族的结构、去甲基化分子机制及在小鼠发育过程中的作用,为深入了解TET蛋白的功能提供理论基础。