Kinetics, Regulation, And Mechanisms Of Biochemical Reac

生化反应的动力学、调控和机制

• 批准号: 7154186
• 负责人: P. BOON Chock
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7154186
• 关键词: RNA interference; actins; apoptosis; biological signal transduction; cell line; cell senescence; cysteine endopeptidases; enzyme activity; enzyme mechanism; free radical oxygen; glutathione; glycation; laboratory mouse; mass tissue /cell culture; membrane permeability; nitric oxide; oxidative stress; phosphorylation; posttranslational modifications; protein kinase A; superoxide dismutase; thioredoxin; tissue /cell culture; ubiquitin

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.

自由基和活性氧物种(ROS)与许多疾病的病因和/或进展、衰老以及正常的生物功能有关。代谢调节科的研究人员进行了研究，以阐明自由基和ROS的产生机制和发挥其生物效应。在本财年，我们的研究集中在：(I)蛋白质相加已被证明在氧化应激中发挥作用。利用蛋白质组学和分子生物学方法，我们已经证明了SUMO-1过表达并没有显著改变HEK 293细胞的生长，并且，像对照细胞一样，修饰后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，在pTRE2hyg2-Myc载体中构建了编码Myc-His标记的SUMO-1、SUMO-2和SUMO-3及其非结合形式SUMO-1/2/3 DeltaGG的质粒，并将其导入HEK 293 Tet-on细胞系。通过潮霉素筛选获得稳定的转染系。经多西环素诱导后，用抗Myc抗体进行Western印迹分析，发现Myc-His标记的SUMO-1/2/3和SUMO-1/2/3 DeltaGG高表达。我们观察到，相扑-2和相扑-3的全局修饰模式非常相似，但它们与相扑-1的修饰模式有显著不同。通过这种方法，14个蛋白质被确定为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，高表达SUMO-2或SUMO-3的细胞表现为早衰表型。这与p21的升高有关。Eitehr P53或pRb基因的敲除显着减少了高表达SUMO-2/3的早衰表型，表明过度表达SUMO-2/3诱导的衰老是P53和pRb依赖的。类似的方法也被用来鉴定另一种泛素样蛋白NEDD8的目标蛋白。(Ii)我们已经建立了一种稳定和可控的RNAi技术，可以在细胞过程或动物生命的所需阶段击倒蛋白质。该方法已被证明在细胞水平上是成功的。我们目前正在进行老鼠模型的研究。我们正处于筛选同时含有Tet-on控制元件和敲除目标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)SOD1基因编码区的错义突变与家族性肌萎缩侧索硬化症(FALS)有关。人们普遍认为，fals SOD1突变体是通过获得细胞毒功能来发挥作用的(S)，其确切性质尚有争议。目前的数据表明，由fals突变体引起的过程导致了含有SOD1的聚集体的形成。我们之前的研究表明，两个fals突变体增强了产生自由基的催化活性，从而促进了导致蛋白质聚集的反应。我们目前正在研究纯化的野生型和fals突变体在体外对蛋白质聚集体形成的影响。为此，杆状病毒中适当折叠的突变蛋白的生产受到了超氧化物歧化酶(CCS)铜伴侣相对较低的表达的阻碍。我们改进了过表达方法，并对感染的昆虫细胞进行了大规模培养。用纯化的突变酶，我们计划在明确的条件下研究功能获得和聚集形成的性质。(4)自由基介导的抗神经母细胞瘤SH-SY5Y细胞凋亡的研究表明，ROS诱导NO升高，进而激活PKG介导的硫氧还蛋白、MnSOD和Bcl-2的表达。进一步的研究表明，Bcl2的表达增加受硫氧还蛋白水平的调节，部分与硫氧还蛋白诱导的cAMP依赖的蛋白激酶(PKA)催化的CREB(环磷酸腺苷反应元件结合蛋白)的磷酸化有关。通过谷氧还蛋白催化的PKA催化亚基在其低PKA半胱氨酸C199上的去谷胱甘肽基化来提高PKA的活性。这与这种催化亚基的谷胱甘肽形式被证明有助于其苏氨酸197的去磷酸化并将其转化为失活酶的概念是一致的。(V)外电场以多种方式影响细胞系统，从与信号传递、伤口愈合、生长和运输相关的低场效应到相对较大的脉冲场，这些脉冲场通过凋亡途径导致膜通透性或细胞死亡。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通透性的方法。我们的结果显示，在COS-7细胞中，大小分布相似但内阻不同的混合囊泡种群的选择性群体通透性和直径为2-3微米的内吞膜空泡的通透性，限制了外细胞膜的完整性。