Rapid quantification of nuclear citrullination in human neutrophils

项目总结 染色质结构和基因表达受组蛋白翻译后修饰控制 核小体上的(PTMS)，染色质的基本重复单位。染色质解缩在一定程度上受到控制 通过依赖PAD4的精氨酸在组蛋白上转化为瓜氨酸(即瓜氨酸化)。值得注意的是，PAD4 中性粒细胞的激活启动了一种不同于凋亡或坏死的细胞死亡程序，其中染色质是 在中性粒细胞胞外陷阱(即NET)中，细胞内出现高瓜氨酸化、去凝聚和排出。这个 瓜氨酸染色质释放到血液中被认为是PAD4相关病理的直接因素。 因此，瓜氨酸核小体(Cit-nucs)是一种很有前途的血液可及生物标记物 自身免疫性疾病，包括类风湿性关节炎(RA)、癌症血栓形成和败血症。然而，有一些 没有能够可靠地从血清或血浆中定量测定核小体瓜氨酸化的分析方法。 目前旨在定量Cit-Nucs的检测方法使用瓜氨酸组蛋白作为检测标准 开发和校准。这些类型的标准有两个主要原因：第一， 瓜氨酸组蛋白不能提供准确的核小体定量，特别是在低浓度时； 第二，游离组蛋白是高带电的，很容易在血浆中聚集，这显著影响了它们的线性 从血浆样本中回收。相比之下，我们(和其他人)发现，核小体在 这表明这些底物可以为定量检测提供更好的标准。EpiCypher® 正在开创携带生理功能的重组设计核小体(DNucs)的商业化 用于下一代表观遗传学分析的组蛋白PTMS。在这里，我们正在开发CitNuc™，这是第一个 准确量化用于蚊虫病研究和临床前生物标记物开发的Cit-Nucs。我们的创新 用重组瓜氨酸设计核小体(Cit-dNucs)作为抗体的定量标准 配对选择/验证和可靠的检测定量。重要的是，与组蛋白不同，Cit-dNucs可以 从血浆样本中准确回收，使开发出高灵敏度的ELISA成为可能。 在第一阶段，我们成功地使用Cit-dNucs鉴定了高度特异的抗体并建立了可靠的 血浆中硫氰菊酯的定量标准。我们还检查了关键的生物分析参数并验证了 CitNuc-EL ISA检测正常人与RA患者Cit-nucs差异的能力 证明了我们的检测方法在临床环境中的实用性。在第二阶段，我们将完成生物分析测试 并为CitNuc ELISA试剂盒(目标1)确定可靠的批次释放策略，为商业化铺平道路。在……里面 目标2和3，我们将开发该方法的临床前应用，以发现生物标记物，重点是RA 和癌症血栓形成，这两种疾病与高水平的依赖PAD4的核小体瓜氨酸化有关。 总之，这些目标将导致第一代CitNuc酶联免疫吸附试验的商业推出，它将 用于蚊虫病研究和生物标记物开发应用。

PROJECT SUMMARY Chromatin structure and gene expression are controlled by histone post-translational modifications (PTMs) on nucleosomes, the basic repeating unit of chromatin. Chromatin decondensation is controlled in part by the PAD4-dependent conversion of arginine to citrulline on histones (i.e. citrullination). Significantly, PAD4 activation in neutrophils initiates a cell death program distinct from apoptosis or necrosis, wherein chromatin is hypercitrullinated, decondensed, and extruded from the cell in neutrophil extracellular traps (i.e. NETosis). The release of citrullinated chromatin into the blood is thought to contribute directly to PAD4-related pathologies. Thus, citrullinated nucleosomes (Cit-Nucs) are a promising blood-accessible biomarker for a range of autoimmune diseases, including rheumatoid arthritis (RA), cancer thrombosis, and sepsis. However, there are no assays capable of reliably quantifying nucleosome citrullination from serum or plasma. Current assays that aim to quantify Cit-Nucs use citrullinated histones as standards for assay development and calibration. These type of standards are problematic for two major reasons; First, citrullinated histones fail to provide accurate quantification of nucleosomes, especially at low concentrations; second, free histones are highly charged and readily aggregate in plasma, which significantly impacts their linear recovery from plasma samples. By contrast, we (and others) have found that nucleosomes are highly stable in plasma, suggesting that these substrates may provide superior standards for assay quantification. EpiCypher® is pioneering the commercialization of recombinant designer nucleosomes (dNucs) carrying physiological histone PTMs for next-generation epigenetics assays. Here, we are developing CitNuc™, the first ELISA to accurately quantify Cit-Nucs for NETosis research and preclinical biomarker development. Our innovative assay uses recombinant citrullinated designer nucleosomes (Cit-dNucs) as quantification standards for antibody pair selection / validation and reliable assay quantification. Importantly, unlike histones, Cit-dNucs can be faithfully recovered from plasma samples, enabling development of a highly sensitive ELISA. In Phase I, we successfully used Cit-dNucs to identify highly specific antibodies and establish reliable standards for quantification of Cit-Nucs in plasma. We also examined key bioanalytical parameters and validated the ability of CitNuc ELISA to detect differences in Cit-Nucs between healthy and RA patient samples, demonstrating the utility of our assay in a clinical environment. In Phase II, we will complete bioanalytical testing and define reliable lot-release strategies for CitNuc ELISA kits (Aim 1), paving the way for commercialization. In Aims 2 and 3, we will develop the preclinical application of this assay for biomarker discovery, focusing on RA and cancer thrombosis, two diseases associated with high levels of PAD4-dependent nucleosome citrullination. Together, these Aims will result in the commercial launch of a first-generation CitNuc ELISA assay, which will be marketed for NETosis research and biomarker development applications.