Kinetics, Regulation, And Mechanisms Of Biochemical Rxns

自由基和活性氧（ROS）在许多疾病的病因和/或进展、衰老以及信号转导中发挥重要作用。代谢调节组的研究人员进行了研究，阐明自由基和活性氧的产生机制和发挥其生物学效应。在本财政年度，我们的研究重点是：(1)蛋白质summoylation在ros诱导的应激中起重要作用。利用蛋白质组学方法和构建可控的HEK 293 Tet-on稳定细胞系，我们过表达Myc-SUMO-1以扩增SUMO-1 SUMO-1化蛋白的形成，以便鉴定它们。特别是那些通常数量相对较少的。SUMO-1过表达对细胞生长无明显影响，修饰蛋白主要发生在细胞核内，这与对照细胞的观察结果一致。聚合蛋白通过免疫沉淀亲和纯化或分离，并通过质谱方法进行序列分析鉴定。我们的研究结果表明，几种异质核核糖核蛋白（hnRNP）、锌指蛋白和核孔复合物蛋白被转录。由于hnRNPs与RNA剪接、转运、稳定性和翻译有关，我们的观察表明sumo化可能在调节mRNA代谢中起重要作用。这些hnRNPs通过聚合作用的功能影响目前正在研究中。此外，SUMO-2/3的过表达导致生长速度急剧下降，这似乎与p53修饰有关。（ii）之前，我们开发了一种稳定可控的RNAi方法，可以在细胞内生成dsRNA。该方法被扩展到动物实验中，并在细胞中敲除过氧化氢酶、铜/锌超氧化物歧化酶和锰超氧化物歧化酶，以研究这些抗氧化酶如何影响细胞信号传导、凋亡和衰老。为此，我们尝试研究RNA干扰的机制。特别是，发现参与RISC的蛋白质，合成了一个P-32和光亲和片段标记的22 bp dsRNA片段，并用于捞出直接与dsRNA片段相互作用的蛋白质。与dsRNA结合的蛋白被鉴定为TB-RBP/translin。进一步的DNA/ rna结合蛋白分析表明，它同时具有ssRNase和dsRNase活性。由于其对产物的高亲和力，它通过结合dsRNA的开放末端并水解它，以有限的周转率将长dsRNA加工成主要为~ 25bp的片段。ssRNase和dsRNase活性均被常见的RNase抑制剂所抑制。我们目前正在寻找体内控制translin活性的调节单位。（iii）早期我们发现，血清剥夺导致神经母细胞瘤SH-SY5Y细胞中ROS水平升高和凋亡。还观察到刺激作用和抗凋亡作用。这些作用是由环gmp依赖性蛋白激酶途径介导的，该途径导致硫氧还蛋白、mn超氧化物歧化酶和抗凋亡Bcl-2的表达增加。进一步的研究表明，Bcl-2的表达升高，部分是由氧化还原依赖性活化的环amp依赖性蛋白激酶介导的，该激酶磷酸化CREB。（iv）我们之前表明Mn（II）复合物可以作为抗氧化剂发挥作用，高浓度Mn（II）诱导caspase-12介导的细胞凋亡。我们已经克隆并鉴定了小鼠caspase-12基因启动子。为了继续我们对家族性肌萎缩性侧索硬化症的Cu/Zn超氧化物歧化酶突变体的研究，最近的结果表明高尔基体和/或分泌途径可能是FALS突变体毒性的早期靶点。此外，还研究了RNA氧化对翻译效率的影响。(v)以前，我们建立了一个仪器来研究施加外电场对细胞膜完整性的影响，并研究细胞事件。该方法被扩展为使用亚微秒持续时间的更大的外部电脉冲，其主要目的是在含有膜液泡的异质囊泡或细胞中实现选择性渗透。

Free radical and reactive oxygen species (ROS) play important roles in the etiology and/or progression of a number of diseases and in aging as well as in signal transduction. Investigators in the Section on Metabolic Regulation carried out studies to elucidate mechanisms by which free radicals and ROS are generated and exert their biological effects. During this fiscal year, our research was focused on: (i) Protein sumoylation plays an important role in ROS-induced stress. Using proteomic methodology and the construction of controllable HEK 293 Tet-on stable cell lines, we overexpressed Myc-SUMO-1 to amplify the formation of SUMO-1 sumoylated proteins in order to identify them?particularly, those normally existing in relatively low quantities. Overexpression of SUMO-1 does not significantly alter cell growth and the modified proteins mainly occurred in the nucleus, which is consistent with that observed in control cells. The sumoylated proteins were affinity purified or isolated by immunoprecipitation and identified by sequence analysis using mass spectrometric methods. Our results revealed that several heterogeneous nuclear ribonucleoproteins (hnRNP), zinc finger proteins, and nuclear pore complex proteins were sumoylated. Since hnRNPs have been implicated in RNA splicing, transport, stability, and translation, our observation suggests that sumoylation may play an important role in regulating mRNA metabolism. The functional effects of these hnRNPs by sumoylation are currently under investigation. In addition, overexpression of SUMO-2/3 causes a drastic reduction in growth rate that appears to be linked to p53 modification. (ii) Previously, we developed a stable and controllable RNAi method that generated dsRNA intracellularly. This method was extended to animal studies and to knock down catalase, Cu/Zn superoxide dismutase, and Mn-superoxide dismutase in cells to investigate how these anti-oxidant enzymes affect cellular cell signaling, apoptosis, and senescence. To this end, we attempted to study the mechanisms of RNA interference?in particular, finding proteins that are involved in RISC, a P-32- and photoaffinity-moiety-labeled 22 bp dsRNA fragment that was synthesized and used to fish out protein(s) that directly interact with the dsRNA fragment. The protein that binds to the dsRNA was identified as TB-RBP/translin. Further analysis of the DNA/RNA-binding protein revealed that it possesses both ssRNase and dsRNase activities. It processes long dsRNA into mainly ~25 bp fragments by binding to the open ends of dsRNA and hydrolyzing it with limited turnover due to its high affinity towards the products. Both ssRNase and dsRNase activities are inhibited by common RNase inhibitors. We are currently searching for regulatory units that control translin activities in vivo. (iii) Earlier we showed that serum deprivation leads to elevation of ROS levels and apoptosis in neuroblastoma SH-SY5Y cells. Hormesis and anti-apoptotic effects were also observed. These effects are mediated by the cyclic GMP-dependent protein kinase pathway that leads to increased expression of thioredoxin, Mn-superoxide dismutase, and anti-apoptotic Bcl-2. Further studies revealed that the elevated expression of Bcl-2, in part, is mediated by redox-dependent activation of cyclic AMP-dependent protein kinase that phosphorylated CREB. (iv) We previously showed that the Mn(II) complex could function as an anti-oxidant and at high concentrations, Mn(II) induced caspase-12 mediated apoptosis. We have now cloned and characterized the murine caspase-12 gene promoter. To follow up our studies on Cu/Zn superoxide dismutase mutants of familial amyotrophic lateral sclerosis, recent result indicate that Golgi apparatus and/or secretory pathways are likely the early targets of FALS mutant toxicity. In addition, the effect of RNA oxidation on translational efficiency was also investigated. (v) Previously, we built an instrument to study the effects of applying external electric fields on the integrity of the cell membrane and to investigate cellular events. This method was extended using a larger external electric pulse of sub-microsecond duration with a primary aim of achieving selective permeabilization in a hetergenous population of vesicles or cells containing membrane vacuoles.