Friday, May 16, 2025
5/16/2025
Novel functions of plasminogen and its diverse cargo in blood - Plasminogen (Plg), well-known for its fibrinolytic role in clotting, has emerged as a multi-functional protein that transports lipids and small non-coding RNAs (sRNA) as a lipoprotein-like particle in circulation. Extracellular sRNAs have been demonstrated to be transported by classical lipoproteins and extracellular vesicles; however, a large fraction of extracellular sRNAs are likely carried by currently unknown small lipid-bearing ribonucleoproteins. In preliminary studies, size-exclusion chromatography and lipid-binding agents were used to isolate lipid-based ribonucleoprotein complexes for their identification by mass spectrometry proteomics. Strikingly, Plg was identified and subsequently confirmed as ribonucleoprotein with moderate binding affinity towards single-stranded microRNAs and viral sRNA fragments. Preliminary studies based on sRNA-sequencing demonstrated that the Plg-sRNA profile a) includes both host and non-host sRNAs, b) is distinct from other lipoproteins, and c) is altered in hypercholesterolemia. We also observed that Plg has the capacity to delivery sRNAs to recipient macrophages and potentially facilitate intercellular communication between myeloid cells. Plg treatment of immune cells elicits strong gene expression and cytokine changes, and we hypothesize that Plg regulates macrophage gene expression, functions, and sub-phenotypes through extracellular sRNA transfer and ligand-receptor activation. To test this hypothesis, we aim to define the mechanisms and impact of Plg-sRNA cargo on macrophage phenotype and intercellular communication. We will confirm Plg-sRNA uptake across myeloid cell types and assess selectivity in Plg-sRNA transfer to macrophages. We will define the role of receptor-mediated endocytosis for Plg-sRNA uptake and determine the necessity of Plg/Pla protease activity for sRNA delivery to macrophages. We will assess the roles of known Plg receptors, as well as identify novel Plg- sRNA receptors and binding proteins, for macrophage sRNA uptake and storage. We will assess the impact of sRNA cargo on classic Plg functions, including inflammation through quantifying changes to cytokine gene expression and secretion from recipient macrophages. We aim to establish macrophage mediated Plg-sRNA intercellular communication between myeloid cells. Furthermore, we aim to define the impact of hypercholesterolemia and post-transcriptional modifications (PTxM) on Plg-sRNAs and their regulation of macrophage polarization and functions through ARM-seq, LC-MS/MS, and Nanopore-based direct sRNA- sequencing. Successful completion of these aims and objectives will open an entirely new direction for hematology research and advance the status quo for multiple fields.
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