cGMP interacts with tropomyosin and downregulates actin-tropomyosin-myosin complex interaction
Lihui Zou1, Junhua Zhang1, Jingli Han1
1The MOH Key Laboratory of Geriatrics, Beijing Hospital, National Center of Gerontology, Beijing, 100730, People's Republic of China.
Insights
Cyclic guanosine monophosphate (cGMP) directly interacts with tropomyosin, reducing its affinity for actin. This finding clarifies a downstream signaling pathway for pulmonary arterial hypertension (PAH) and suggests new therapeutic targets.
Area of Science:
- Molecular Biology
- Biochemistry
- Cardiovascular Research
Background:
- The nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) pathway is crucial in pulmonary arterial hypertension (PAH) pathogenesis.
- The precise molecular mechanisms underlying this pathway's role in PAH are not fully understood.
Purpose of the Study:
- To identify proteins regulated by cGMP.
- To elucidate the downstream signaling pathway of NO-sGC-cGMP in the context of PAH.
Main Methods:
- Biotin-cGMP pull-down assays and in vivo antibody-dependent pull-down assays were used to detect cGMP-protein interactions.
- Real-time PCR, Western blot, immunofluorescence, and isothermal titration calorimetry (ITC) were employed to analyze tropomyosin expression, localization, and binding affinities.
Main Results:
- cGMP was found to interact with tropomyosin, specifically with the 68-208 amino acid region.
- TPM1 gene, encoding tropomyosin isoform 4, is the primary isoform in human pulmonary artery smooth muscle cells (HPASMCs).
- cGMP-tropomyosin interaction was shown to decrease the affinity of tropomyosin for actin, without altering tropomyosin expression or localization.
Conclusions:
- This study elucidates a novel downstream signaling pathway for NO-sGC-cGMP involving tropomyosin.
- The findings offer insights into the molecular mechanisms of PAH and highlight potential targets for innovative therapeutic agents.
Background:
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); however, its exact molecular mechanism remains undefined.
Methods:
Biotin-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.
Results:
cGMP 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.
Conclusions:
We 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.
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