Identification of Nitric Oxide as an Endogenous Inhibitor of 26S Proteasomes in Vascular Endothelial Cells

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（26S proteasome）在几乎所有真核细胞（包括血管内皮细胞）中发挥核心生理功能。然而，目前学界对脉管系统中蛋白酶体功能的调控机制仍知之甚少。已知内皮型一氧化氮合酶（endothelial nitric oxide synthase, eNOS）产生的一氧化氮（NO）对维持内皮细胞稳态至关重要。本研究旨在明确血管内皮细胞中内皮源性一氧化氮与26S蛋白酶体功能之间的关联。

研究分别检测了26S蛋白酶体报告细胞、人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVEC）以及从26S蛋白酶体报告小鼠和eNOS基因敲除小鼠中分离的小鼠主动脉组织中的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关键亚基Rpt2的O-连接N-乙酰葡糖胺糖基化（O-GlcNAcylation）水平。

结果显示，与其他选择性一氧化氮供体类似，通过药理学和遗传学手段激活eNOS所产生的一氧化氮，可增强Rpt2的O-GlcNAc修饰、降低蛋白酶体的胰凝乳蛋白酶样活性，并导致26S蛋白酶体报告蛋白积累。反之，抑制eNOS活性则可逆转上述所有效应。

小干扰RNA（siRNA）敲低催化O-连接N-乙酰葡糖胺修饰的关键酶O-连接N-乙酰葡糖胺转移酶（O-GlcNAc transferase, OGT），可消除一氧化氮诱导的上述效应。与之一致的是，腺病毒介导的过表达负责去除O-连接N-乙酰葡糖胺基团的酶O-糖基化酶（O-GlcNAcase, OGA），可模拟OGT敲低产生的效果。

最终实验结果表明，与eNOS野生型小鼠的主动脉组织相比，缺失eNOS的26S蛋白酶体报告小鼠的主动脉组织中，26S蛋白酶体功能显著增强，同时Rpt2的O-连接N-乙酰葡糖胺糖基化水平降低，而Rpt2蛋白的表达水平并未发生改变。综上，内皮源性一氧化氮可作为生理性抑制剂调控血管内皮细胞中的26S蛋白酶体功能。