Effect of miP-PSTPIP2 overexpression on endothelial gene expression in homeostasis and inflammation. [ext501]

Detecting microproteins (miPs) translated from small open reading frames (smORFs) by mass spectrometry poses major challenges. Here we report a high-throughput proteo-genomic pipeline that provides cumulative evidence for a large number of bona fide miPs in human and murine endothelial cells. Vascular inflammation in vitro and in vivo profoundly altered smORF expression and a large number of miPs was identified in the blood stream of patients following heart tissue damage. Human miPs presented high homology with other primates and their functional perturbation identified miPs that were essential for endothelial cell growth and viability. Moreover, combining gain/loss-of-function with multi-omic approaches demonstrated the involvement of an inflammation-regulated miP in the regulation of gene expression, proliferation and inflammation through interaction with proteins in different subcellular compartments. Thus, large numbers of uncharacterized miPs exist, play a major role in the regulation of cell function and may become biomarkers or therapeutic targets in cardiovascular disease. MiP-PSTPIP2 is upregulated in inflammatory conditions in vitro and in vivo. Therefore this study investigated global transcriptomic alterations following overexpression of FLAG-miP-PSTPIP2 in homeostatic conditions as well as after treatment with interleukin-1beta in human endothelial cells. As the overexpression of FLAG-miP-PSTPIP2 was achieved by adenoviral infection, infection with an adenovirus to express the small protein FLAG-miRFP670nano served as control. The analysis included non infected human endothelial cells under the same experimental conditions to control for infection-dependent effects.