E3泛素连接酶FBXW17降解PRMT6对COPD炎症基因的转录调控研究

Chronic obstructive pulmonary disease (COPD) is a major cause of chronic morbidity and mortality throughout the world. Chronic inflammation plays the pivotal role in pathogenesis of COPD. The mechanism of the inflammatory gene expression in this disease is yet illusive. Epigenetic control of inflammatory gene expresion in COPD has been reported recently. A decreased deacetylase activity was observed in patients indicating that cross-talk among histone acetlylation, deacetlylation, and/or other modifications regulates gene expression in COPD. Albeit, the contribuition of histone methylation in inflammatory gene expression in COPD is largely unknown. Our preliminary study proved a decreased protein level of a histone methylation enzyme protein arginine methyltransferase (PRMT6) in COPD patient lung tissues. PRMT6 is an epigenetic code inhibiting gene expression by methylation of histone H3R2. We observed that PRMT6 directly involved in inflammatory gene expression. Proinflammatory gene expression was repressed by overexpression of PRMT6, while knockdown of PRMT6 unleashed proinflammatory gene expression in cultured lung epithelial cells. Of note, we found that PRMT6 is a short life protein with a half-life of approximately 4 h. We identified that PRMT6 is specifically degraded by ubiquitin-proteasomal machinery, proteasome inhibitor MG132 treatment stops PRMT6 degradation and results in PRMT6 accumulation. Furthermore, we discovered a novel E3 ubiquitin ligase FBXW17 that degrades PRMT6 in a dose dependent manner by screening the E3 ubiquitin ligase. Overexpression of FBXW17 accelerates PRMT6 degradation as compared to control E3 ubiquitin ligase FBXW14 that does not degrade PRMT6. Therefore, we hypothesis that histone methylation enzyme PRMT6 is ubiquitin-proteasomal degraded by FBXW17, PRMT6 degradation leads to inflammatory gene upregulation in COPD. To prove this hypothesis, we will 1) investigate the mRNA and protein levels of PRMT6 and FBXW17 in CSE treated lung epthelial cells, COPD model animal lung tissues and human COPD lung tissues, and their levels correlation to proinflammatory gene upregulations; 2) determine the molecular mechanism of ubiquitin-proteasomal degradation of PRMT6 by FBXW17; and 3) interfer inflammatory gene expression in the pathogenesis of COPD by change the protein level of PRMT6 and FboxL18. Thus, we have uncovered a new E3 ubiquitin ligase that epigenetically controls inflammatory gene expression by degradation of histone methylase PRMT6 in COPD. Execution of these studies will lay the groundwork for a potentially significant conceptual advance with regard to a novel E3 ubiquitin lagse involved in the pathogenesis of COPD and the epigenetic control of genes during inflammation in COPD. Completion of this study may represent a new therapeutic option in the prevention and treatment of COPD.

慢性炎症在慢性阻塞性肺疾病（COPD）的发病机制中扮演着重要角色，但对该疾病中炎症基因转录调控机制仍知之甚少。蛋白精氨酸甲基转移酶6（PRMT6）通过对组蛋白H3R2非对称双甲基化抑制基因转录。我们前期研究显示COPD中PRMT6蛋白表达下降并直接参与调控炎症基因表达。吸烟所致泛素-蛋白酶体系统异常活化参与了COPD病程，我们已证实PRMT6半衰期短，蛋白酶体特异性抑制剂能抑制其降解，同时E3泛素连接酶FBXW17能特异性降解PRMT6蛋白。但COPD中是否存在FBXW17降解PRMT6增加，进而影响组蛋白甲基化并激活肺内炎症基因转录仍有待进一步求证。本研究拟从COPD患者、动物和细胞水平中观察FBXW17、PRMT6、组蛋白甲基化和炎症基因的表达，探讨PRMT6通过组蛋白甲基化调控COPD炎症基因转录的机制，并进一步研究FBXW17调控PRMT6降解的机制，为COPD治疗探索新的途径。

慢性炎症在慢性阻塞性肺疾病（COPD）的发病机制中扮演着重要角色，组蛋白甲基化修饰介导的表观遗传学调控基因转录在肺气肿/COPD 慢性炎症机制中起着重要的作用。蛋白精氨酸甲基转移酶6（PRMT6）通过对组蛋白H3R2 非对称双甲基化（H3R2me2a）并拮抗H3K4位点上的三甲基化（H3K4me3）修饰抑制基因转录。本课题通过慢性阻塞性肺疾病（COPD）病人、动物模型和细胞三个层面对PRMT6在COPD中的作用及其调控机制进行了研究。我们的研究显示在COPD患者中PRMT6 表达下调并伴随组蛋白H3R2me2a水平下调而H3K4me3水平上调，并直接参与调控炎症基因的转录表达。本次实验采用腹腔注射香烟提取物（CSE）的方法成功建立了COPD小鼠模型。在动物实验中我们同样发现COPD小鼠模型肺组织PRMT6的蛋白表达明显下调并伴随组蛋白H3R2me2a信号水平下调而H3K4me3信号水平上调，而进一步采用PRMT6过表达慢病毒载体干预COPD小鼠模型则发现，过表达PRMT6后肺组织H3R2me2a表达上调并H3K4me3表达下调从而下调炎症因子、氧化应激和细胞凋亡等基因的转录从而减轻小鼠肺气肿改变并改善小鼠肺功能。采用CSE并PRMT6过表达载体干预细胞，我们同样发现PRMT6介导CSE引起的炎症、凋亡和氧化应激过程。我们研究发现PRMT6蛋白通过泛素蛋白酶体途径降解；通过过表达质粒筛选，我们发现E3 泛素连接酶FBXW17特异性介导PRMT6蛋白的降解。荧光定位及免疫共沉淀显示FBXW17 和PRMT6 相互结合并共存于细胞核内。过表达FBXW17可加速PRMT6的降解，进而影响组蛋白甲基化并抑制抗氧化基因转录，CSE可提高FBXW17的表达，而抑制FBXW17则可部分挽救PRMT6的降解。同时本项目还发现HDAC2依赖的MORF4L1去乙酰化增强MORF4L1的同源二聚体化，进而抑制细胞增殖过程；而MORF4L1促进肺炎内皮细胞损伤并可能成为肺炎非抗生素治疗的潜在靶标。另外，采用气管内移植原代培养EPC干预吸烟COPD小鼠模型，发现EPC可减轻COPD模型炎症细胞浸润、凋亡过程、抑制基质酶的活性和提高抗氧化活性从而减轻肺气肿；而临床方面的研究则发现认知干预的戒烟措施是吸烟COPD患者戒烟的有效方法。

内容获取失败，请点击重试