Abstract：
The selective removal of dysfunctional mitochondria, a process termed mitophagy, is critical for cellular health and impairments have been linked to aging, Parkinson disease, and other neurodegenerative conditions. A central mitophagy pathway is orchestrated by the ubiquitin (Ub) kinase PINK1 together with the E3 Ub ligase PRKN/Parkin. The decoration of damaged mitochondrial domains with phosphorylated Ub (p-S65-Ub) mediates their elimination though the autophagy system. As such p-S65-Ub has emerged as a highly specific and quantitative marker of mitochondrial damage with significant disease relevance. Existing p-S65-Ub antibodies have been successfully employed as research tools in a range of applications including western blot, immunocytochemistry, immunohistochemistry, and enzyme-linked immunosorbent assay. However, physiological levels of p-S65-Ub in the absence of exogenous stress are very low, therefore difficult to detect and require reliable and ultrasensitive methods. Here we generated and characterized a collection of novel recombinant, rabbit monoclonal p-S65-Ub antibodies with high specificity and affinity in certain applications that allow the field to better understand the molecular mechanisms and disease relevance of PINK1-PRKN signaling. These antibodies may also serve as novel diagnostic or prognostic tools to monitor mitochondrial damage in various clinical and pathological specimens.Abbreviations: AD: Alzheimer disease; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ELISA: enzyme-linked immunosorbent assay; HEK293E cell: human embryonic kidney E cell; ICC: immunocytochemistry; IHC: immunohistochemistry: KO: knockout; LoB: limit of blank; LoD: limit of detection; LoQ: limit of quantification; MEF: mouse embryonic fibroblast; MSD: Meso Scale Discovery; n.s.: non-significant; nonTg: non-transgenic; PBMC: peripheral blood mononuclear cell; PD: Parkinson disease; p-S65-PRKN: phosphorylated PRKN at serine 65; p-S65-Ub: phosphorylated Ub at serine 65; Ub: ubiquitin; WT: wild-type.
摘要：
选择性去除功能失调的线粒体，一个叫做线粒体自噬的过程,对细胞健康至关重要，损伤与衰老有关，帕金森病,和其他神经退行性疾病。中枢线粒体自噬途径由泛素（Ub）激酶PINK1与E3Ub连接酶PRKN/Parkin协调。磷酸化Ub（p-S65-Ub）修饰受损的线粒体结构域通过自噬系统介导其消除。因此,p-S65-Ub已经作为具有显著疾病相关性的线粒体损伤的高度特异性和定量标记物出现。现有的p-S65-Ub抗体已被成功地用作一系列应用的研究工具，包括蛋白质印迹，免疫细胞化学,免疫组织化学，和酶联免疫吸附测定。然而,在没有外源胁迫的情况下，p-S65-Ub的生理水平非常低，因此很难检测，需要可靠和超灵敏的方法。在这里，我们产生并表征了一组新的重组体，兔单克隆p-S65-Ub抗体在某些应用中具有高特异性和亲和力，允许该领域更好地了解PINK1-PRKN信号传导的分子机制和疾病相关。这些抗体还可以作为新的诊断或预后工具来监测各种临床和病理标本中的线粒体损伤。缩写：AD：阿尔茨海默病；CCCP：羰基氰化物3-氯苯腙；ELISA：酶联免疫吸附测定；HEK293E细胞：人胚胎肾E细胞；ICC：免疫细胞化学；IHC：免疫组织化学：KO：敲除；LoB：空白极限；LoD：检测极限；LoQ：定量极限；MEF：小鼠胚胎BMBP-NPC-65。非磷酸化UMSOF：NSD-65
