Protein Derived Radicals

该小组继续实施和验证称为免疫自旋捕获的技术，该技术将DMPO（5，5-二甲基-1-吡咯啉N-氧化物）自旋捕获器的特异性自由基反应性与硝酮抗体敏感性相结合。 抗DMPO已被证明是一种高度特异性的抗体，无DMPO对照品可轻松识别任何非特异性问题。这种类型的控制在抗体工作中是不常见的，但却非常有用。 与自由基中间体的电子自旋共振（ESR）检测不同，抗DMPO免疫自旋捕获不依赖于瞬时自由基中间体，并且比ESR灵敏约10，000倍，比MS灵敏100倍。多年来，我们一直使用免疫自旋捕获方法来研究体外、细胞和体内以蛋白质为中心的自由基的形成。 与ESR自旋捕获法相比，免疫自旋捕获法具有许多优点：1）免疫自旋捕获法需要的材料少得多（蛋白质的微克而不是毫克）; 2）最终氧化产物DMPO-硝酮加合物的稳定性（一种稳定的非自由基物种），是远远大于顺磁性DMPO自由基加合物（t1/2 = sec-min），导致灵敏度大大提高，和3）免疫自旋捕获可以使用标准ELISA、蛋白质印迹技术和免疫组织学技术进行，使得免疫自旋捕获比ESR自旋捕获更容易和通用。 这一进展极大地扩展了自旋捕获技术的实用性，使其摆脱了ESR光谱的量子力学复杂性。 我们发现荧光蛋白如增强型绿色荧光蛋白（eGFP）的表达诱导细胞中的氧化应激。活性氧（ROS）如超氧化物（O2）和过氧化氢（H2 O2）的形成增加可以解释先前在哺乳动物和细菌细胞以及过表达荧光蛋白的动物中报道的细胞毒性和组织异常。O2和H2 O2均诱导氧化还原信号传导机制，导致参与细胞增殖、细胞分化和细胞死亡的细胞调节蛋白的基因表达改变。因此，这些发现可能对解释常规使用荧光蛋白标签的许多研究结果产生重大影响。 在代森锰和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化中起着至关重要的作用。大量证据表明，突触核蛋白影响几种生物途径，最终导致Maneb和除草剂百草枯诱导的帕金森病小鼠模型中的神经元死亡。突触核蛋白的存在或不存在（在野生型或突触核蛋白基因敲除小鼠，分别）决定了所有的差异，在响应代森锰和百草枯共同曝光。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导致开发帕金森病的潜在治疗方法。

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.