Kinetics, Regulation, And Mechanisms Of Biochemical Reac

自由基和活性氧（ROS）与许多疾病的病因学和/或进展、衰老以及正常生物功能有关。代谢调节部分的研究人员进行了研究，以阐明产生自由基和ROS并发挥其生物学效应的机制。于本财政年度，我们的研究集中于：（i）蛋白质类小泛素化已被证明在氧化应激中发挥作用。使用蛋白质组学和分子生物学方法，我们已经表明，SUMO-1过表达并没有显着改变HEK 293细胞的生长，像对照细胞，修饰的蛋白质主要发生在细胞核中。为了研究SUMO家族成员的整体修饰模式并鉴定SUMO-2和SUMO-3的未知修饰蛋白，将编码Myc-His标记的SUMO-1、SUMO-2和SUMO-3及其非缀合形式SUMO-1/2/3 DeltaGG的质粒构建在pTRE 2 hyg 2-Myc载体中并转染到HEK 293 Tet-on细胞系中。使用潮霉素选择获得稳定转染的细胞系。当这些细胞系被强力霉素诱导时，基于使用抗Myc抗体的Western印迹分析，发现Myc-His标记的SUMO-1/2/3和SUMO-1/2/3 DeltaGG被高度表达。我们观察到，SUMO-2和SUMO-3的整体修饰模式非常相似，但它们与SUMO-1有显著差异。利用这种方法，14种蛋白被鉴定为SUMO-2/3靶标，包括肿瘤抑制蛋白、p53（也是SUMO-1的靶标）和pRb。此外，过表达SUMO-2或SUMO-3的细胞表现出早衰表型。这与p21的升高有关。敲低eitehr p53或pRb显著降低过表达SUMO-2/3的早衰表型，表明过表达SUMO-2/3诱导的衰老是p53和pRb依赖性的。类似的方法被用于鉴定另一种泛素样蛋白NEDD 8的靶蛋白。(ii)我们已经建立了一种稳定和可控的RNAi技术，在细胞过程或动物生命的期望阶段敲低蛋白质。该方法已被证明在细胞水平上是成功的。我们目前正在进行小鼠模型研究。我们正处于筛选同时携带Tet-on控制元件和靶蛋白的小鼠的最后阶段。此外，利用RNAi技术研究了超氧阴离子自由基和过氧化氢在EGF诱导的细胞生长中的作用。(iii)SOD 1基因编码区的错义突变与家族性肌萎缩性侧索硬化症（FALS）有关。人们普遍认为，FALSSOD 1突变体通过获得细胞毒性功能发挥作用，但其确切性质仍有争议。目前的数据表明，由FALS突变体引起的过程导致含SOD 1的聚集体的形成。我们以前表明，两个FALS突变体增强催化活性，产生自由基，这可能有助于反应，导致蛋白质聚集。我们目前正在研究纯化的野生型和FALS突变体在体外形成蛋白质聚集体的影响。为此，在杆状病毒中正确折叠的突变蛋白的产生受到SOD（CCS）的铜分子伴侣的相对低表达的阻碍。我们改进了过量表达方法，并对感染的昆虫细胞进行了大量培养。与纯化的突变酶，我们计划研究的性质获得的功能和聚集体形成在明确定义的条件。(iv)我们的自由基介导的兴奋效应对神经母细胞瘤SH-SY 5 Y细胞凋亡的研究表明，启动的过程中ROS诱导的NO的升高，这反过来又导致激活PKG介导的硫氧还蛋白，MnSOD和Bcl-2的表达。进一步的研究表明，Bcl-2的表达增加是由硫氧还蛋白的水平调节，并在一定程度上，与cAMP依赖性蛋白激酶（PKA）催化的磷酸化的CREB（环AMP反应元件结合蛋白）诱导的硫氧还蛋白处理。PKA活性通过PKA催化亚基在其低pKa半胱氨酸C199处的谷氧还蛋白催化的脱谷胱甘肽化而升高。这与以下观点一致，即该催化亚基的谷胱甘肽化形式已显示出促进其苏氨酸197的去磷酸化并将其转化为无活性的酶。(v)外部电场以多种方式影响细胞系统，从与信号传导、伤口愈合、生长和运输相关的低场效应到导致膜透化或通过凋亡途径导致细胞死亡的相对大的脉冲场。使用相对较大但较短的脉冲场，我们展示了一种实现选择性膜透化的方法。我们的研究结果显示，在具有相似大小分布但具有不同内部渗透性的混合囊泡群体中的选择性群体的透化，以及COS-7细胞中直径为2-3微米的内吞膜空泡的透化，其对外部细胞膜的完整性具有限制性影响。

Free radical and reactive oxygen species (ROS) have been implicated in the etiology and/or progression of a number of diseases and in aging as well as in normal biological functions. 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 focused on: (i) Protein sumoylation has been shown to play a role in oxidative stress. Using proteomic and molecular biological methods, we have shown that SUMO-1 overexpression did not significantly alter HEK 293 cell growth and, like the control cells, the modified proteins mainly occurred in the nucleus. To investigate the global modification patterns of SUMO family members and to identify unknown modified proteins by SUMO-2 and SUMO-3, plasmids encoding Myc-His-tagged SUMO-1, SUMO-2, and SUMO-3 and their nonconjugatable forms, SUMO-1/2/3 DeltaGG, were constructed in pTRE2hyg2-Myc vector and transfected into HEK 293 Tet-on cell lines. The stable transfected cell lines were obtained using hygromycin selection. When these cell lines were induced by doxycycline, Myc-His-tagged SUMO-1/2/3 and SUMO-1/2/3 DeltaGG were found to be highly expressed based on Western blot analyses using anti-Myc antibody. We observed that the global modification patterns of SUMO-2 and SUMO-3 are very similar, but they are significantly different from that of SUMO-1. With this method, 14 proteins were identified as SUMO-2/3 targets, including tumor suppressor proteins, p53 (also a target for SUMO-1), and pRb. Furthermore, cells overexpressing SUMO-2 or SUMO-3 showed premature senescence phenotype. This correlates with the elevation of p21. Knockdown of eitehr p53 or pRb significantly reduces the premature senescence phenotype of overexpressing SUMO-2/3 indicating that overexpressing SUMO-2/3 induced senescence is p53 and pRb dependent. Similar methods were used to identify the target proteins for another ubiquitin-like protein, NEDD8. (ii) We have established a stable and controllable RNAi technique to knockdown proteins at desired stages of cellular processes or animal life. The method has proven to be successful at cellular levels. We are currently involved in mouse model studies. We are in the final stage of screening mice that harbor both the Tet-on controlling element and the target protein for knockdown. Furthermore, the RNAi technique is used to investigate the roles of superoxide anion radicals and hydrogen peroxide in EGF-induced cell growth. (iii) Missense mutations in the coding regions of the SOD1 gene have been linked to familial amyotrophic lateral sclerosis (FALS). It is widely accepted that FALS SOD1 mutants act through gain of cytotoxic function(s), whose exact nature is under debate. Current data suggest that the processes caused by FALS mutants lead to the formation of SOD1-containing aggregates. We previously showed that two FALS mutants enhance catalytic activity for generating free radicals, which could facilitate reactions leading to protein aggregation. We are currently investigating the effects of purified wild-type and FALS mutants on the formation of protein aggregates in vitro. To this end, the production of properly folded mutant proteins in Bacculovirus have been hampered by the relatively low expression of the copper chaperon for SOD (CCS). We have improved overexpression methods and carried out mass-culture of the infected insect cells. With the purified mutant enzymes, we plan to study the nature of the gain-of-function and aggregate formation in well-defined conditions. (iv) Our studies on free radical-mediated hormesis against apoptosis in neuroblastoma SH-SY5Y cells revealed that the process initiated by ROS-induced elevation of NO, which in turn leads to activation of PKG-mediated expression of thioredoxin, MnSOD, and Bcl-2. Further studies showed that increased expression of Bcl-2 is regulated by the level of thioredoxin and, in part, correlated with cAMP-dependent protein kinase (PKA)-catalyzed phosphorylation of CREB (cyclic AMP-responsive element binding protein) induced by thioredoxin treatment. The PKA activity is elevated by glutaredoxin-catalyzed deglutathionylation of the catalytic subunit of PKA at its low pKa cysteine, C199. This is in agreement with the notion that the glutathionylated form of this catalytic subunit has been shown to facilitate the dephosphorylation of its threonine 197 and converting it to an inactive enzyme. (v) External electric fields affect cellular systems in a multitude of ways ranging from low field effects associated with signaling, wound healing, growth, and transport to relatively large pulsed fields that lead to membrane permeabilization or cell death via the apoptotic pathway. Using relatively large but short pulsed fields, we demonstrated a method for achieving selective membrane permeabilization. Our results revealed permeabilization of a selective population in mixed vesicle populations of similar size distribution but with varied internal resistivities and permeabilization of endocytosed membrane vacuoles with a diameter of 2-3 micrometers in COS-7 cells with limiting effects on the integrity of the outer cell membrane.