Endothelial nitric oxide synthase dimerization is regulated by heat shock protein 90 rather than by phosphorylation.
Endothelial nitric oxide synthase dimerization is regulated by heat shock protein 90 rather than by phosphorylation.
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DOI：
10.1371/journal.pone.0105479
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Ye M
中科院分区：
文献类型：
--
作者：
Chen W;Xiao H;Rizzo AN;Zhang W;Mai Y;Ye M
内皮一氧化氮合酶 (eNOS) 是一种多功能酶，在多种细胞过程中发挥作用，包括血管生成、组织重塑和血管张力的维持。 eNOS 的单体和二聚体形式存在于多种组织中。 eNOS 的二聚体形式被认为是活性形式，单体形式被认为是无活性的。 eNOS 的活性还受到许多其他机制的调节，包括氨基酸磷酸化和与其他蛋白质的相互作用。然而，调节 eNOS 二聚化、磷酸化和活性的精确机制仍未完全确定。我们利用纯化的 eNOS 和牛主动脉内皮细胞 (BAEC) 来研究调节 eNOS 降解的机制。纯化的牛 eNOS 中同时存在 eNOS 单体和二聚体。将纯化的牛 eNOS 与蛋白磷酸酶 2A (PP2A) 一起孵育，会导致二聚体和单体的丝氨酸 1179 (Ser1179) 去磷酸化，并导致 eNOS 活性降低。然而，eNOS 二聚体：单体比率没有变化。同样，蛋白磷酸酶 1 (PP1) 诱导 eNOS 在苏氨酸 497 (Thr497) 处去磷酸化，但不改变 eNOS 二聚体与单体的比例。与纯化的 eNOS 不同，在培养的 BAEC 中，eNOS 主要以二聚体形式存在。然而，用蛋白酶体抑制剂乳胞素处理后，eNOS 单体会积累。此外，用血管内皮生长因子 (VEGF) 处理 BAEC 会导致 eNOS 二聚体中 Ser1179 的磷酸化，而不会改变任一形式中 Thr497 的磷酸化状态。热休克蛋白 90 (Hsp90) 的抑制或 Hsp90 沉默使 eNOS 二聚体不稳定，并伴随 Ser1179 和 Thr497 的去磷酸化。总之，我们的研究表明，eNOS 单体（而非 eNOS 二聚体）会被泛素化降解。此外，二聚体 eNOS 结构是 eNOS 氨基酸修饰和活性调节的主要条件。最后，eNOS 二聚体的不稳定不仅导致 eNOS 降解，还会导致 eNOS 氨基酸修饰发生变化，从而进一步影响 eNOS 活性。
Endothelial nitric oxide synthase (eNOS) is a multifunctional enzyme with roles in diverse cellular processes including angiogenesis, tissue remodeling, and the maintenance of vascular tone. Monomeric and dimeric forms of eNOS exist in various tissues. The dimeric form of eNOS is considered the active form and the monomeric form is considered inactive. The activity of eNOS is also regulated by many other mechanisms, including amino acid phosphorylation and interactions with other proteins. However, the precise mechanisms regulating eNOS dimerization, phosphorylation, and activity remain incompletely characterized. We utilized purified eNOS and bovine aorta endothelial cells (BAECs) to investigate the mechanisms regulating eNOS degradation. Both eNOS monomer and dimer existed in purified bovine eNOS. Incubation of purified bovine eNOS with protein phosphatase 2A (PP2A) resulted in dephosphorylation at Serine 1179 (Ser1179) in both dimer and monomer and decrease in eNOS activity. However, the eNOS dimer∶monomer ratio was unchanged. Similarly, protein phosphatase 1 (PP1) induced dephosphorylation of eNOS at Threonine 497 (Thr497), without altering the eNOS dimer∶monomer ratio. Different from purified eNOS, in cultured BAECs eNOS existed predominantly as dimers. However, eNOS monomers accumulated following treatment with the proteasome inhibitor lactacystin. Additionally, treatment of BAECs with vascular endothelial growth factor (VEGF) resulted in phosphorylation of Ser1179 in eNOS dimers without altering the phosphorylation status of Thr497 in either form. Inhibition of heat shock protein 90 (Hsp90) or Hsp90 silencing destabilized eNOS dimers and was accompanied by dephosphorylation both of Ser1179 and Thr497. In conclusion, our study demonstrates that eNOS monomers, but not eNOS dimers, are degraded by ubiquitination. Additionally, the dimeric eNOS structure is the predominant condition for eNOS amino acid modification and activity regulation. Finally, destabilization of eNOS dimers not only results in eNOS degradation, but also causes changes in eNOS amino acid modifications that further affect eNOS activity.
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DOI：
10.1158/1940-6207.capr-13-0209
发表时间：
2013-10
期刊：
Cancer prevention research (Philadelphia, Pa.)
影响因子：
--
作者：
Bhat TA;Nambiar D;Tailor D;Pal A;Agarwal R;Singh RP
通讯作者：
Singh RP
影响因子：
7.3
作者：
Gélinas, DS;Bernatchez, PN;Sirois, MG
通讯作者：
Sirois, MG
DOI：
10.1073/pnas.192345199
发表时间：
2002-09-17
影响因子：
11.1
作者：
Kolodziejski, PJ;Musial, A;Eissa, NT
通讯作者：
Eissa, NT
影响因子：
7.4
作者：
Komarov, AM;Wink, DA;Schmidt, HHHW
通讯作者：
Schmidt, HHHW
影响因子：
2.9
作者：
Chen W;Druhan LJ;Chen CA;Hemann C;Chen YR;Berka V;Tsai AL;Zweier JL
通讯作者：
Zweier JL