The role of ISG15 and ISGylation in the senescence-associated secretory phenotype

细胞衰老是一种细胞状态，影响与年龄相关的疾病，如炎症，癌症， 发展和癌症进展。进入衰老期的细胞具有衰老相关的分泌性 表型（SASP），对周围的其他细胞具有主要且通常有害的影响 微环境ISG15是一种泛素样蛋白，可以共价修饰宿主和致病蛋白， 改变其稳定性和功能。ISG15也可以分泌并作为细胞因子发挥作用。虽然ISG15 通常被认为是在病原体感染期间诱导的干扰素刺激基因，它也可以 会被DNA损伤和压力上调。最近的研究表明，ISG 15是最重要的 在衰老的啮齿类动物的组织中上调基因。我们和其他人也表明，ISG15是 在人成纤维细胞的复制性衰老期间上调。我们的初步数据进一步表明， ISG15在来自先天性角化不良患者的皮肤成纤维细胞中显著上调， 端粒酶功能失调和端粒缩短的模型。端粒酶诱导DC细胞永生化的研究 激活使ISG15水平恢复正常。关于ISG15上调如何有助于 以及衰老细胞中哪些蛋白质被ISGylated。此外，ISGylation如何改变是未知的。 在细胞永生化之后。我们假设ISG15的上调和伴随的ISG1的ISG2化可能是由于ISG15的表达增加而引起的。 DNA损伤引起的蛋白质对SASP很重要。在本提案中，我们将确定 在衰老和永生化中，分泌的ISG15和ISG化之间存在相互作用。利用我们独特的病人 细胞和细胞系，我们将使用创新的蛋白质组学策略绘制衰老相关的ISGylome 在我们的实验室里开创。我们将比较被诱导衰老的细胞之间的ISGylomes 通过不同的机制，从人类和小鼠，我们将确定分泌ISG15如何影响 周围的细胞。此外，我们还将评估端粒酶或病毒癌基因的永生化如何改变细胞的增殖。 ISGylome。最后，我们将具体确定ISG15和ISGylation如何通过遗传途径影响SASP。 操作细胞以具有降低和增强的ISG化。这些研究将提供关键的洞察力， ISG15的上调和特定细胞蛋白的ISG化在细胞衰老中起作用， 特别是SASP。

Cellular senescence is a cellular state that affects age-related diseases such as inflammation, cancer development, and cancer progression. Cells that enter senescence have a senescence-associated secretory phenotype (SASP) that has major, and usually detrimental, effects on other cells in the surrounding microenvironment. ISG15 is a ubiquitin-like protein that can covalently modify host and pathogenic proteins, changing their stability and function. ISG15 can also be secreted and function as a cytokine. While ISG15 is classically regarded as an interferon-stimulated gene that is induced during infection by pathogens, it can also be upregulated by DNA damage and stress. Recent studies have demonstrated that ISG15 is among the most upregulated genes across tissues in aging rodents. It has also been shown by us and others that ISG15 is upregulated during replicative senescence of human fibroblasts. Our preliminary data further indicates that ISG15 is significantly upregulated in skin fibroblasts derived from patients with dyskeratosis congenita, a human model of dysfunctional telomerase and shortened telomeres. Immortalization of DC cells by telomerase activation restores ISG15 levels to normal. Little is known about how ISG15 upregulation contributes to senescence and what proteins are ISGylated in senescent cells. Further, it is unknown how ISGylation changes upon cell immortalization. We hypothesize that upregulation of ISG15 and concomitant ISGylation of proteins caused by DNA damage is important for the SASP. In this proposal, we will determine what the interplay is between secreted ISG15 and ISGylation in senescence and immortalization. Using our unique patient cells and cell lines, we will map the senescence-associated ISGylome using innovative proteomic strategies pioneered in our laboratories. We will compare the ISGylomes between cells that have been induced to senesce by different mechanisms and from human and mouse, and we will determine how secreted ISG15 affects surrounding cells. Further, we will assess how immortalization by telomerase or viral oncogenes changes the ISGylome. Finally, we will specifically determine how ISG15 and ISGylation affects the SASP through genetic manipulation of cells to have reduced and enhanced ISGylation. These studies will provide key insight into how upregulation of ISG15 and ISGylation of specific cellular proteins play a role in cellular senescence and, in particular, the SASP.