Protein Derived Radicals

该小组继续实施和验证名为免疫自旋捕获的技术，该技术结合了DMPO(5，5-二甲基-1-吡咯啉N-氧化物)自旋捕获的特定自由基反应性和硝酮抗体的敏感性。抗DMPO抗体已被证明是一种高度特异的抗体，与非DMPO对照很容易识别任何非特异性问题。这种类型的控制在抗体工作中并不常见，但非常有用。与电子自旋共振(ESR)检测自由基中间体不同，抗DMPO免疫自旋捕获不依赖于瞬时自由基中间体，其灵敏度约为ESR的10,000倍和MS的100倍。多年来，我们继续使用免疫自旋捕获方法在体外、细胞和体内研究以蛋白质为中心的自由基的形成。与ESR自旋捕获相比，该免疫分析有许多优势：1)免疫自旋捕获需要的物质(微克蛋白质而不是毫克)要少得多；2)最终氧化产物DMPO-硝酮加合物(一种稳定的非自由基物种)的稳定性比ESR所需的顺磁性DMPO-自由基加合物(T1/2=秒-分钟)大得多，从而大大提高了灵敏度；3)可以使用标准的ELISA法、Western blotting技术和免疫组织学技术进行免疫自旋捕获，使免疫自旋捕获比ESR自旋捕获更容易获得，也更具通用性。这一进展极大地扩展了自旋捕获技术的用途，使其摆脱了ESR光谱的量子力学复杂性。 我们发现，增强型绿色荧光蛋白(EGFP)等荧光蛋白的表达可诱导细胞内的氧化应激。在哺乳动物、细菌细胞和过度表达荧光蛋白的动物中，超氧化物歧化酶(O2)和过氧化氢(H2O2)等活性氧物种(ROS)的增加可以解释先前报道的细胞毒性和组织异常。O2和H2O2都诱导氧化还原信号机制，导致参与细胞增殖、细胞分化和细胞死亡的细胞调控蛋白的基因表达改变。因此，这些发现可能会对许多常规使用荧光蛋白标签的研究结果的解释产生重大影响。 在甘露醇和除草剂百草枯诱导的帕金森病模型中，我们发现细胞色素c在突触核蛋白自由基的形成和寡聚中起着关键作用。大量证据表明，突触核蛋白影响多条生物学途径，最终导致甘露醇和除草剂百草枯诱导的帕金森病小鼠模型神经元死亡。突触核蛋白的存在或不存在(分别在野生型或突触核蛋白基因敲除的小鼠中)决定了对甘露醇和百草枯联合暴露的所有反应的差异。这些结果表明，依赖于清除突触核蛋白自由基或降低突触核蛋白水平的实验方法，可能会导致帕金森病潜在疗法的发展。

The group continued to implement and validate the technique called immuno-spin trapping, which combines the specific free radical reactivity of the DMPO (5,5-dimethyl-1-pyrroline N-oxide) spin trap with nitrone-antibody sensitivity. Anti-DMPO has proven itself to be a highly specific antibody with the no-DMPO control easily identifying any non specificity issues. This type of control is uncommon among antibody work, but is of great utility. Unlike electron spin resonance (ESR) detection of radical intermediates, anti-DMPO immuno-spin trapping is not dependent on transient free radical intermediates and is about 10,000 times more sensitive than ESR and 100 times more sensitive than MS. In years we have continued using the immuno-spin trapping approach to investigate the formation of protein-centered free radicals in vitro, in cells, and in vivo. The immunoassay has many advantages compared to ESR spin-trapping: 1) immuno-spin trapping requires much less material (micrograms of protein rather than milligrams); 2) the stability of the final oxidation product, the DMPO-nitrone adduct (a stable non-radical species), is much greater than that of the paramagnetic DMPO-radical adducts (t1/2 = sec-min) required for ESR, resulting in greatly enhanced sensitivity, and 3) immuno-spin trapping can be performed using standard ELISA, Western blotting techniques, and immuno-histological techniques, making immuno-spin trapping far more accessible and versatile than ESR spin trapping. This advance greatly expanded the utility of the spin-trapping technique, freeing it from the quantum mechanical complexity of ESR spectroscopy. We found that expression of fluorescent proteins such as enhanced green fluorescent protein (eGFP) induces oxidative stress in cells. The increased formation of reactive oxygen species (ROS) such as superoxide (O2) and hydrogen peroxide (H2O2) can explain the cytotoxicity and tissue abnormalities reported previously in mammalian and bacterial cells and animals overexpressing fluorescent proteins. Both O2 and H2O2 induce redox signaling mechanisms, leading to altered gene expression of cell regulatory proteins involved in cell proliferation, cell differentiation, and cell death. Thus, these findings could have a major influence on the interpretation of results obtained from numerous studies that routinely use fluorescent protein tags. In the Maneb and the herbicide paraquat induced Parkinsons disease model, we found that the cytochrome c plays crucial roles in -synuclein radical formation and oligomerization. Extensive evidence were provided that -synuclein affects several biological pathways, which ultimately contribute to neuronal death in the Maneb and the herbicide paraquat induced mouse model of Parkinsons disease. The presence or absence of -synuclein (in wild-type or -synuclein knockout mice, respectively) determined all the difference in response to Maneb and paraquat co-exposure. These results suggest that experimental approaches which rely on scavenging -synuclein radical or decreasing -synuclein levels, can possibly lead to development of potential therapeutics for Parkinsons disease.