Bennewitz, K. | Search bioRxiv

bioRxiv2026

Global mapping of VEGF-A driven changes in the RNA bound phospho-proteome reveals a molecular switch to coordinate angiogenesis in endothelial cells

RNA binding proteins (RBPs) serve as upstream master regulators of cellular fate transitions at the post-transcriptional level. RBP-RNA interactions are dynamically regulated by post-translational modifications to coordinate cellular responses. We hypothesised that changes in RBP phosphorylation will play a key role in coordinating angiogenesis, the formation of new blood vessels, in endothelial cells (ECs). Therefore, we used phospho-proteomic analysis of the mRNA bound proteome to identify global phosphorylation changes in mouse cardiac ECs during acute and prolonged VEGF-A stimulation. Of the differentially phosphorylated RBPs, we found that the phosphorylation of serine 199 within mRNA bound Phf6 (PHD finger protein 6) was consistently increased following VEGF-A stimulation. Phf6 phosphorylation at this specific site was sufficient and required to induce a pro-angiogenic phenotype, facilitating a change in Phf6 conformation promoting interaction with distinct RNAs. We show here that Phf6 phosphorylation at serine 199, downstream of VEGF-A, acts as a molecular switch to differentially coordinate a decrease of anti-angiogenic RNAs (i.e. extracellular matrix genes) and an increase in pro-angiogenesis related RNAs, in part by differentially regulating their stability, to promote angiogenesis.

cell biology↗

bioRxiv2021

Deep metabolic profiling assessment of tissue extraction protocols for three model organisms

Metabolic profiling harbors the potential to better understand various disease entities such as cancer, diabetes, Alzheimers, Parkinsons disease or COVID-19. Deciphering these intricate pathways in human studies requires large sample sizes as a means of reducing variability. While such broad human studies have discovered new associations between a given disease and certain affected metabolites, i.e. biomarkers, they often provide limited functional insights. To design more standardized experiments, reduce variability in the measurements and better resolve the functional component of such dynamic metabolic profiles, model organisms are frequently used. Standardized rearing conditions and uniform sampling strategies are prerequisites towards a successful metabolomic study. However, further aspects such as the choice of extraction protocol and analytical technique can influence the outcome drastically. Here, we employed a highly standardized metabolic profiling assay analyzing 630 metabolites across three commonly used model organisms (Drosophila, mouse and Zebrafish) to find the optimal extraction protocols for various matrices. Focusing on parameters such as metabolite coverage, metabolite yield and variance between replicates we compared seven extraction protocols. We found that the application of a combination of 75% ethanol and methyl tertiary-butyl ether (MTBE), while not producing the broadest coverage and highest yields, was the most reproducible extraction protocol. We were able to determine up to 530 metabolites in mouse kidney samples, 509 in mouse liver, 422 in Zebrafish and 388 in Drosophila and discovered a core overlap of 261 metabolites in these four matrices. To enable other scientists to search for the most suitable extraction protocol in their experimental context and interact with this comprehensive data, we have integrated our data set in the open-source shiny app "MetaboExtract". This will enable scientists to search for their metabolite or metabolite class of interest, compare it across the different tested extraction protocols and sample types as well as find reference concentrations.

biochemistry↗