Bhatia Vikram, Maghsoudi Saeid, Hinton Martha, Bhagirath Anjali Y, Singh Nisha, Jaggupilli Appalaraju, Chelikani Prashen, Dakshinamurti Shyamala
Biology of Breathing Theme, Children's Hospital Research Institute of Manitoba, Winnipeg, MB R3E 3P4, Canada.
Department of Oral Biology, University of Manitoba, Winnipeg, MB R3E 0W2, Canada.
Biology (Basel). 2023 Apr 10;12(4):572. doi: 10.3390/biology12040572.
The adenylyl cyclase (AC) pathway, crucial for pulmonary vasodilation, is inhibited by hypoxia. Forskolin (FSK) binds allosterically to AC, stimulating ATP catalysis. As AC6 is the primary AC isoform in the pulmonary artery, selective reactivation of AC6 could provide targeted reinstatement of hypoxic AC activity. This requires elucidation of the FSK binding site in AC6.
HEK293T cells stably overexpressing AC 5, 6, or 7 were incubated in normoxia (21% O) or hypoxia (10% O) or exposed to s-nitrosocysteine (CSNO). AC activity was measured using terbium norfloxacin assay; AC6 structure built by homology modeling; ligand docking to examine FSK-interacting amino acids; roles of selected residues determined by site-directed mutagenesis; FSK-dependent cAMP generation measured in wild-type and FSK-site mutants by biosensor-based live cell assay.
Only AC6 is inhibited by hypoxia and nitrosylation. Homology modeling and docking revealed residues T500, N503, and S1035 interacting with FSK. Mutation of T500, N503, or S1035 decreased FSK-stimulated AC activity. FSK site mutants were not further inhibited by hypoxia or CSNO; however, mutation of any of these residues prevented AC6 activation by FSK following hypoxia or CSNO treatment.
FSK-interacting amino acids are not involved in the hypoxic inhibition mechanism. This study provides direction to design FSK derivatives for selective activation of hypoxic AC6.
腺苷酸环化酶（AC）途径对肺血管舒张至关重要，但会受到缺氧抑制。福斯高林（FSK）与AC变构结合，刺激ATP催化。由于AC6是肺动脉中的主要AC亚型，选择性重新激活AC6可靶向恢复缺氧时的AC活性。这需要阐明AC6中的FSK结合位点。
将稳定过表达AC 5、6或7的HEK293T细胞置于常氧（21% O）或缺氧（10% O）环境中，或暴露于亚硝基半胱氨酸（CSNO）。使用铽诺氟沙星测定法测量AC活性；通过同源建模构建AC6结构；进行配体对接以检查与FSK相互作用的氨基酸；通过定点诱变确定所选残基的作用；通过基于生物传感器的活细胞测定法测量野生型和FSK位点突变体中FSK依赖性环磷酸腺苷（cAMP）的生成。
只有AC6受到缺氧和亚硝基化抑制。同源建模和对接显示残基T500、N503和S1035与FSK相互作用。T500、N503或S1035突变会降低FSK刺激的AC活性。FSK位点突变体不再受缺氧或CSNO进一步抑制；然而，这些残基中的任何一个发生突变都会阻止缺氧或CSNO处理后FSK对AC6的激活。
与FSK相互作用的氨基酸不参与缺氧抑制机制。本研究为设计用于选择性激活缺氧AC6的FSK衍生物提供了方向。
