Integrative Proteomic and Phosphoproteomic Analyses of Hypoxia-Treated Pulmonary Artery Smooth Muscle Cells

Abstract

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.

Sexual dimorphism and the multi-omic response to exercise training in rat subcutaneous white adipose tissue

“…Notably, Grb14 , a negative modulator of insulin signaling, was downregulated with ExT while Olah , a thioesterase involved in medium chain fatty acid synthesis, and Hmgcs2 , the rate-limiting enzyme of cholesterol synthesis, were increased at all training timepoints in females (Supplementary Table 4). The only transcript that displayed differential expression at all training timepoints in males was the hypoxia-inducible carbonic anhydrase ( Ca12 ) 40 , which was downregulated. FGSEA further highlighted sexual dimorphism in the progressive transcriptomic response to ExT (Figs.…”

Oxygen‐regulated post‐translation modifications as master signalling pathway in cells

“…In addition to HIF‐dependent pathways, several studies have employed unbiased whole proteome phosphoproteomics to study global changes to protein phosphorylation in response to hypoxic stress. Phosphoproteomic analyses of hypoxia‐treated pulmonary artery smooth muscle cells revealed large‐scale changes to the phosphoproteome identifying 331 significantly changed phosphoproteins, from a total of 2,347 identified phosphoproteins, representing a change of phosphorylation of approximately 14% of all identified phosphosites (Luo et al , 2022). If this data is extrapolated to consider the 72,000 high confidence Ser/Thr/Tyr phosphosites identified in the human proteome (Kalyuzhnyy et al , 2022), there may be as many as 10,000 hypoxia‐induced changes to the phosphoproteome.…”

“…If this data is extrapolated to consider the 72,000 high confidence Ser/Thr/Tyr phosphosites identified in the human proteome (Kalyuzhnyy et al , 2022), there may be as many as 10,000 hypoxia‐induced changes to the phosphoproteome. The limited hypoxia‐dependent phosphoproteomic experiments performed to date have revealed changes in phosphorylation of proteins in pathways as diverse as mitogen‐activated protein kinase pathways (ERK1 and ERK2), regulation of TGF (tumour growth factor)‐β receptor signalling, changes to Rho GTPase signalling and key factors involved in cytoskeletal organisation (Nilsson et al , 2010; Datta et al , 2021; Luo et al , 2022). These suggest that multiple kinase/phosphorylation‐dependent signalling pathways exist in tandem to modulate the cellular response in low oxygen, to tailor the response to different cellular contexts.…”

“…Ubiquitination is clearly essential for the HIF‐mediated responses to hypoxia, but it also has additional HIF‐independent roles in the hypoxic response. Pathway analysis of hypoxia‐dependent signalling pathways in smooth muscle cells reveals changes in phosphoproteins in the ubiquitin proteasome pathway, indicating major global changes to the hypoxia‐dependent proteome (Luo et al , 2022). In addition, global mRNA levels are altered in hypoxia due to hypoxia‐dependent linear ubiquitination of Argonaute RISC Catalytic Component 2 (AGO2) by the linear ubiquitin chain assembly (LUBAC) complex.…”

“…Ubiquitinated AGO2 restrains miRNA‐mediated gene silencing to have global effects on the global accumulation of mRNAs, likely having great impact on many cell signalling pathways (Zhang et al , 2021). The impact of hypoxia‐mediated ubiquitination on the hypoxic response is likely to affect multiple aspects of cell signalling, with the true extent of its control only revealed by multi‐omics‐based experiments (reviewed in (Luo et al , 2022)), which are still emerging.…”