PB0817 - Proximity biotinylation proteomics to identify novel regulators of Weibel-Palade body morphology and von Willebrand factor secretion

Background: Defects in von Willebrand factor (VWF) that impair its synthesis and secretion in endothelial cells (ECs) can cause quantitative von Willebrand Disease (VWD) in patients with bleeding tendencies. When synthesized, VWF is processed through the secretory pathway and stored in secretory organelles called Weibel-Palade bodies (WPBs). The regulatory network that controls trafficking and secretion of VWF involves many dynamic, transient and/or weak interactions that can be identified using novel proteomic strategies.

Aims: To map the composition of the regulatory network of the EC secretory pathway using proximity biotinylation proteomics and determine which of these proteins influence VWF secretion.

Methods: Proximity-dependent biotinylation is performed using turbo BirA biotin ligase (TurboID) tagged to the longin SNARE SEC22B, a known regulator of WPB morphology which resides on the interface between the endoplasmic reticulum (ER) and the Golgi. The TurboID-SEC22B fusion is nucleofected into human umbilical vein endothelial cells (HUVECs). After 60 minutes of labeling with biotin, biotinylated proteins within cell lysates are precipitated using streptavidin beads. Label free quantitative DIA mass spectrometry-based proteomics will be used for identification and quantitation of target proteins.

Results: Expression of the TurboID-SEC22B fusion constructs in ECs is localized to the ER and Golgi. Proteins in its direct environment are biotinylated through the SEC22B recruited BirA ligase, as visualized using cellular staining with fluorescently-tagged streptavidin. Labeling of intracellular proteins can be increased by prolonging biotin incubation time. The known SEC22B interactor and WPB regulator syntaxin-5 (STX5) is successfully enriched from the HUVEC lysate, indicating that our biotinylation strategy is capable of picking up specific and relevant components of the endothelial secretory pathway.

Conclusion(s): Our preliminary results show that TurboID biotin labeling is a promising tool to identify novel regulators of VWF trafficking in ECs.