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bioRxiv · 10.1101/2025.11.07.687176

Microprotein miP-PSTPIP2 affects cytoskeleton dynamics to modulate endothelial cell endocytosis, barrier function and migration

Gueven, B.·Bezuneh, B.·Raheja, M.·Szymanski, W.·Brandes, R. P.·Graumann, J.·Fleming, I.·Siragusa, M.
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Abstract

BackgroundA large number of microproteins (miPs) encoded by small open reading frames (smORFs) are expressed in endothelial cells, yet their function remains largely unknown. In this study, we characterized a novel 46-amino-acid miP encoded by a smORF within the proline-serine-threonine phosphatase interacting protein 2 (PSTPIP2) transcript that was upregulated under inflammatory conditions and we refer to as miP-PSTPIP2. MethodsImmunoprecipitation coupled with mass spectrometry-based proteomics, immunoblotting, immunofluorescence and proximity ligation assays were used to identify and validate miP-PSTPIP2 interacting proteins in human endothelial cells. The impact of adenovirus-mediated overexpression of miP-PSTPIP2 on endocytosis, cytoskeleton dynamics and abundance of proteins involved in these processes was investigated by confocal microscopy and immunoblotting. Live cell imaging was used to assess endothelial cell migration and vascular permeability. ResultsmiP-PSTPIP2 physically associated with caveolar proteins, proteins involved in the regulation of cytoskeleton dynamics, intracellular transport, clathrin adaptor activity, as well as nuclear proteins. Human endothelial cells overexpressing miP-PSTPIP2 demonstrated enhanced endocytosis and transcytosis of transferrin as well as low-density lipoprotein. Mechanistically, miP-PSTPIP2 modulated Arp2/3-mediated actin nucleation and branching, which are required for dynamic cytoskeleton rearrangements. Moreover, altered cytoskeleton dynamics in miP-PSTPIP2-expressing endothelial cells resulted in impaired cell migration as well as increased permeability and monocyte trans-endothelial migration. ConclusionsmiP-PSTPIP2 is an inflammation-induced endothelial miP that regulates Arp2/3-dependent actin dynamics, thereby enhancing lipid uptake and leukocyte permeability. Its upregulation under inflammatory conditions suggests a contributory role in endothelial dysfunction and vascular inflammation.

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Gueven, B., Bezuneh, B., Raheja, M., Szymanski, W., Brandes, R. P., Graumann, J., Fleming, I., Siragusa, M.. 2025-11-08. Microprotein miP-PSTPIP2 affects cytoskeleton dynamics to modulate endothelial cell endocytosis, barrier function and migration. https://doi.org/10.1101/2025.11.07.687176

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