Abstract
BackgroundThe nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling pathway, plays a critical role in the pathogenesis of pulmonary arterial hypertension (PAH); however, its exact molecular mechanism remains undefined.MethodsBiotin-cGMP pull-down assay was performed to search for proteins regulated by cGMP. The interaction between cGMP and tropomyosin was analyzed with antibody dependent pull-down in vivo. Tropomyosin fragments were constructed to explore the tropomyosin-cGMP binding sites. The expression level and subcellular localization of tropomyosin were detected with Real-time PCR, Western blot and immunofluorescence assay after the 8-Br-cGMP treatment. Finally, isothermal titration calorimetry (ITC) was utilized to detect the binding affinity of actin-tropomyosin-myosin in the existence of cGMP-tropomyosin interaction.ResultscGMP interacted with tropomyosin. Isoform 4 of TPM1 gene was identified as the only isoform expressed in the human pulmonary artery smooth muscle cells (HPASMCs). The region of 68-208aa of tropomyosin was necessary for the interaction between tropomyosin and cGMP. The expression level and subcellular localization of tropomyosin showed no change after the stimulation of NO-sGC-cGMP pathway. However, cGMP-tropomyosin interaction decreased the affinity of tropomyosin to actin.ConclusionsWe elucidate the downstream signal pathway of NO-sGC-cGMP. This work will contribute to the detection of innovative targeted agents and provide novel insights into the development of new therapies for PAH.
Highlights
The nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling pathway, plays a critical role in the pathogenesis of pulmonary arterial hypertension (PAH); its exact molecular mechanism remains undefined
The result showed that 5 peptides of the band 1 belonged to the protein tropomyosin, which was involved in the regulatory system of actin-myosin interaction
By performing Biotin-cGMP pull-down assay and isothermal titration calorimetry (ITC) analysis, we found that cGMP interacted with tropomyosin, which lead to the structural and functional alterations in the actin-tropomyosin-myosin complex
Summary
The nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling pathway, plays a critical role in the pathogenesis of pulmonary arterial hypertension (PAH); its exact molecular mechanism remains undefined. PAH is a serious yet poorly understood disease characterized by elevated pulmonary artery pressure and vascular resistance and may result in right heart failure and death. The burden of small pulmonary arteries increases due to the contraction of vascular smooth muscle cells, which plays a crucial role in regulating of pulmonary vascular resistance and in the development of PAH [1]. Malfunctions in NO production, sGC activity, and cGMP degradation cause pulmonary arterial vasodilatation through abnormal vascular smooth muscle cell proliferation and platelet aggregation [2]. We have demonstrated that SERPINB2, known as PAI-2, suppresses the proliferation and
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