Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the main causes of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). Hypoxia is an important factor related to PAH and can induce the excessive proliferation of PASMCs and inhibit apoptosis. To explore the possible mechanism of hypoxia-related PAH, human PASMCs are exposed to hypoxia for 24 h and tandem mass tag (TMT)-based quantitative proteomic and phosphoproteomic analyses are performed. Proteomic analysis revealed 134 proteins are significantly changed (p < 0.05, |log2 (fold change)| > log2 [1.1]), of which 48 proteins are upregulated and 86 are downregulated. Some of the changed proteins are verified by using qRT-PCR and Western blotting. Phosphoproteomic analysis identified 404 significantly changed (p < 0.05, |log2 (fold change)| > log2 [1.1]) phosphopeptides. Among them, 146 peptides are upregulated while 258 ones are downregulated. The kinase-substrate enrichment analysis revealed kinases such as P21 protein-activated kinase 1/2/4 (PAK1/2/4), protein-kinase cGMP-dependent 1 and 2 (PRKG1/2), and mitogen-activated protein-kinase 4/6/7 (MAP2K4/6/7) are significantly enriched and activated. For all the significantly changed proteins or phosphoproteins, a comprehensive pathway analysis is performed. In general, this study furthers our understanding of the mechanism of hypoxia-induced PAH.
Phenolic acids are the main bioactive compounds in Salvia miltiorrhiza, which can be increased by salicylic acid (SA) elicitation. However, the specific molecular mechanism remains unclear. The nonexpresser of PR genes 1 (NPR1) and its family members are essential components of the SA signaling pathway. Here, we report an NPR protein, SmNPR4, showed strongly expression in hairy root after SA treatment, acting as a negative moderator of SA-induced phenolic acid biosynthesis in Salvia miltiorrhiza (S. miltiorrhiza). Moreover, a basic leucine zipper family transcription factor SmTGA5 was identified and was found to interact with SmNPR4. SmTGA5 activates the expression of phenolic acid biosynthesis gene SmTAT1 through binding to the as-1 element. Finally, a series of biochemical assays and dual gene overexpression analysis demonstrated that the SmNPR4 significantly inhibited the function of SmTGA5, and SA can alleviate the inhibitory effect of SmNPR4 on SmTGA5. Overall, our results reveal the molecular mechanism of salicylic acid regulating phenolic acid biosynthesis in S. miltiorrhiza and provides new insights for SA signaling to regulate secondary metabolic biosynthesis.
Pulmonary arterial hypertension (PAH) is a rare but fatal disease characterized by elevated pulmonary vascular resistance and increased pressure in the distal pulmonary arteries. Systematic analysis of the proteins and pathways involved in the progression of PAH is crucial for understanding the underlying molecular mechanism. In this study, we performed tandem mass tags (TMT)-based relative quantitative proteomic profiling of lung tissues from rats treated with monocrotaline (MCT) for 1, 2, 3 and 4 weeks. A total of 6759 proteins were quantified, among which 2660 proteins exhibited significant changes (p-value < 0.05, fold change < 0.83 or >1.2). Notably, these changes included several known PAH-related proteins, such as Retnla (resistin-like alpha) and arginase-1. Furthermore, the expression of potential PAH-related proteins, including Aurora kinase B and Cyclin-A2, was verified via Western blot analysis. In addition, we performed quantitative phosphoproteomic analysis on the lungs from MCT-induced PAH rats and identified 1412 upregulated phosphopeptides and 390 downregulated phosphopeptides. Pathway enrichment analysis revealed significant involvement of pathways such as complement and coagulation cascades and the signaling pathway of vascular smooth muscle contraction. Overall, this comprehensive analysis of proteins and phosphoproteins involved in the development and progression of PAH in lung tissues provides valuable insights for the development of potential diagnostic and treatment targets for PAH.
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