Liu Hongtao, Yu Shujie, Zhang Hua, Xu Jian
Section of Endocrinology, Department of Medicine and Harold Hamm Oklahoma Diabetes Center, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, United States of America.
PLoS One. 2014 May 22;9(5):e98486. doi: 10.1371/journal.pone.0098486. eCollection 2014.
The 26S proteasome plays a fundamental role in almost all eukaryotic cells, including vascular endothelial cells. However, it remains largely unknown how proteasome functionality is regulated in the vasculature. Endothelial nitric oxide (NO) synthase (eNOS)-derived NO is known to be essential to maintain endothelial homeostasis. The aim of the present study was to establish the connection between endothelial NO and 26S proteasome functionality in vascular endothelial cells. The 26S proteasome reporter protein levels, 26S proteasome activity, and the O-GlcNAcylation of Rpt2, a key subunit of the proteasome regulatory complex, were assayed in 26S proteasome reporter cells, human umbilical vein endothelial cells (HUVEC), and mouse aortic tissues isolated from 26S proteasome reporter and eNOS knockout mice. Like the other selective NO donors, NO derived from activated eNOS (by pharmacological and genetic approach) increased O-GlcNAc modification of Rpt2, reduced proteasome chymotrypsin-like activity, and caused 26S proteasome reporter protein accumulation. Conversely, inactivation of eNOS reversed all the effects. SiRNA knockdown of O-GlcNAc transferase (OGT), the key enzyme that catalyzes protein O-GlcNAcylation, abolished NO-induced effects. Consistently, adenoviral overexpression of O-GlcNAcase (OGA), the enzyme catalyzing the removal of the O-GlcNAc group, mimicked the effects of OGT knockdown. Finally, compared to eNOS wild type aortic tissues, 26S proteasome reporter mice lacking eNOS exhibited elevated 26S proteasome functionality in parallel with decreased Rpt2 O-GlcNAcylation, without changing the levels of Rpt2 protein. In conclusion, the eNOS-derived NO functions as a physiological suppressor of the 26S proteasome in vascular endothelial cells.
26S蛋白酶体在几乎所有真核细胞（包括血管内皮细胞）中都发挥着重要作用。然而，蛋白酶体功能在脉管系统中是如何被调节的，在很大程度上仍然未知。内皮型一氧化氮（NO）合酶（eNOS）衍生的NO对于维持内皮细胞稳态至关重要。本研究的目的是建立血管内皮细胞中内皮型NO与26S蛋白酶体功能之间的联系。在26S蛋白酶体报告基因细胞、人脐静脉内皮细胞（HUVEC）以及从26S蛋白酶体报告基因小鼠和eNOS基因敲除小鼠分离得到的小鼠主动脉组织中，检测了26S蛋白酶体报告蛋白水平、26S蛋白酶体活性以及蛋白酶体调节复合物的关键亚基Rpt2的O-连接N-乙酰葡糖胺化修饰。与其他选择性NO供体一样，由活化的eNOS（通过药理学和遗传学方法）产生的NO增加了Rpt2的O-连接N-乙酰葡糖胺修饰，降低了蛋白酶体胰凝乳蛋白酶样活性，并导致26S蛋白酶体报告蛋白积累。相反，eNOS失活则逆转了所有这些效应。催化蛋白质O-连接N-乙酰葡糖胺化修饰的关键酶O-连接N-乙酰葡糖胺转移酶（OGT）的小干扰RNA（siRNA）敲低消除了NO诱导的效应。同样，催化去除O-连接N-乙酰葡糖胺基团的酶O-连接N-乙酰葡糖胺酶（OGA）的腺病毒过表达模拟了OGT敲低的效应。最后，与eNOS野生型主动脉组织相比，缺乏eNOS的26S蛋白酶体报告基因小鼠表现出26S蛋白酶体功能升高，同时Rpt2的O-连接N-乙酰葡糖胺化修饰减少，而Rpt2蛋白水平没有变化。总之，eNOS衍生的NO在血管内皮细胞中作为26S蛋白酶体的生理抑制剂发挥作用。
