Background and Aim. Microproteins (miPs) are short peptides encoded by small open reading frames (smORFs - shorter than 100 codons) that have been implicated in the regulation of biological processes ranging from cell growth to cellular signalling and metabolism. Endothelial cells, as the inner lining of blood vessels, are situated in a strategic position to respond to cues derived from the circulating blood as well as from the tissues. The aim of this study was to identify and characterize endothelial smORF-encoded miPs.

Methods and Results. We developed a novel proteogenomic strategy consisting of RiboTag and bulk next generation RNA-seq (NGS) as well as mass spectrometry-based proteomics to identify endothelial smORF-encoded miPs. This yielded ~2.2k miPs encoded by previously non-annotated smORFs in primary human endothelial cells. NGS of ribosome associated RNAs from endothelial cell-specific RiboTag mice combined with proteomic analyses revealed the organotypic expression of murine endothelial miPs under homeostatic conditions: 907 in the thoracic aorta, 271 in the aortic arch, ~1k in the heart and ~3k in the lung. Approximately 2.4k endothelial miPs were detected in at least two organs. Endothelial cell activation by interleukin-1β was associated with profound alterations in endothelial miP expression in vitro (1.6k differentially expressed miPs). Similarly, ~1.2k endothelial miPs were differentially expressed in response to inflammation and endothelial dysfunction in mice (AAV-PCSK9 injection combined with partial carotid artery ligation). Endothelial FLAG-tagged miPs (FLAG immunofluorescence) displayed a diverse cellular localization, including the cytosol, plasma membrane, the endoplasmic reticulum or the nucleus. Functional studies with selected miPs revealed their involvement in the regulation of proliferation, cell growth and the response to oxidative stress- as well as cytotoxic drug-induced cell death. The study of selected miP interactomes by mass spectrometry-based proteomics as well as transcriptomes by NGS upon overexpression of selected miPs was performed to functionally characterize these novel molecular players.

Conclusions. Proteogenomic studies enabled the discovery of previously non-annotated endothelial smORFs and their encoded miPs under homeostatic conditions. The profound alterations in the expression of miPs in response to endothelial cell activation suggest a potential involvement in the development of cardiovascular disease.