Multiscale chemical approaches to map oxidative stress

描述（由申请人提供）：自由基是呼吸和环境不可避免的结果，并被小分子抗氧化剂和重解毒酶紧密缓冲。当活性氧水平与细胞清除活性中间体或修复损伤的能力不平衡时，氧化应激就出现了。异常的氧化信号可能是导致衰老、神经变性、心脏病、糖尿病和癌症的最重要因素之一。为了诱导表型变化，氧化应激必须诱导基因组、蛋白质组和/或代谢组的生化改变。晶体学分析表明，DJ-1含有稳定的亚亚硫酸，这种氧化修饰是抑制线粒体氧化应激所必需的。我们现在已经证明，这种亚亚磺酸可以与亚硝基硫醇反应形成硫代磺酸键，然后可以被细胞硫醇还原。令人惊讶的是，在标准的生理缓冲液中，亚硫酸盐与亚硝基硫醇的反应速度比硫醇更快。这为DJ-1的功能提供了一种潜在的机制，该奖项将对其进行进一步探索。我们将这种方法扩展到开发生物素连接的亚硫酸盐，用于直接检测和富集内源性亚硝基化蛋白。在初步实验中，该方法鉴定了>1500内源性亚硝基化蛋白，并建立了一个强大的新平台，从功能上询问s -亚硝基化动力学。此外，我们描述了一种基于正交烷基化试剂选择性富集亚磺酸的新方法，并提出了在蛋白质组中鉴定新的功能亚磺酸的方法。最后，我们提出了一种新的比率荧光探针，用于活细胞成像和体内蛋白质亚砜化的19F-NMR。尽管氧化应激在人体健康中起着核心作用，但由于缺乏选择性的化学和分析方法，我们研究这种修饰的精确机制的能力受到阻碍。在本提案中，我们提出了一系列创新的化学方法来跨实验尺度研究氧化损伤，从活细胞成像到体内成像，以及氧化翻译后修饰的蛋白质组范围注释。此外，我们提出了与帕金森病相关的氧化还原伴侣j -1的可能机制，并提出了基于机制的新探针，对s -亚硝基化（R-SNO）、s -亚砜化（R-SOH）和s -亚砜化（R-SO2H）进行功能注释和分析。

DESCRIPTION (provided by applicant): Radical species are an unavoidable consequence of respiration and the environment, and are tightly buffered by small molecule antioxidants and redo detoxifying enzymes. Oxidative stress emerges when an imbalance develops between the levels of reactive oxygen species and the cell's ability to readily eliminate the reactive intermediates or to repair the resulting damage. Aberrant oxidative signaling is perhaps one of the most important factors contributing to aging, neurodegeneration, heart disease, diabetes, and cancer. In order to induce a phenotypic change, oxidative stress must induce biochemical alterations to the genome, proteome and/or metabolome. Crystallographic analysis revealed that DJ-1 harbors a stabile sulfinic acid, and this oxidative modification is required for the suppression of mitochondrial oxidative stress. We have now shown that this sulfinic acid can react with nitrosothiols to form a thiosulfonate linkage, which can then be reduced by cellular thiols. Amazingly, sulfinates react with nitrosothiols faster than thiolates in standard physiological buffers. This provides a potential mechanism for DJ-1 function, which will be further explored with this award. We extended this approach to develop biotin-linked sulfinates for the direct detection and enrichment of endogenous nitrosylated proteins. In preliminary experiments, this method led to the identification of >1500 endogenous nitrosylated proteins, and establishes a robust new platform to functionally interrogate the dynamics of S-nitrosylation. In addition, we describe a new methodology for the selective enrichment of sulfinic acids based on orthogonal alkylation reagents, and propose to identify novel functional sulfinates in the proteome. Finally, we present a new class of ratiometric fluorescent probes for live-cell imaging and 19F-NMR of protein sulfenylation in vivo. Despite the central role of oxidative stress in human health, our ability to study the precise mechanisms of such modifications is hampered by a lack of selective chemical and analytical methods. In this proposal, we present a series of innovative chemical approaches to study oxidative damage across experimental scales, from live-cell imaging to in vivo imaging, in addition to proteome-wide annotation of oxidative post-translational modifications. Furthermore, we present a likely mechanism for the Parkinson's disease-linked redox chaperone DJ-1, and present new mechanism-based probes to functionally annotate and profile S-nitrosylation (R-SNO), S-sulfenylation (R-SOH), and S-sulfinylation (R-SO2H).