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: Proximity proteomics identifies endothelial cell septins and PAK2 as decisive regulators of actomyosin mediated expulsion of von Willebrand factor.

ABSTRACT: A Rab27a targeted APEX2 construct was transfected into primary human umbilical endothelial cells (HUVEC) and the proximal proteins surrounding endothelial storage organelles (Weibel Palade bodies) identified by proximity proteomics in unstimulated and stimulated conditions. The procedure was carried out in the presence and absence of two different stimuli (10 minutes-phorbol myristate acetate or a combination of Histamine, adrenalin and 3-isobutyl-1-methylxanthine) and the data presented represents 4 independent experiments each with 2 technical replicates.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Vein Endothelial Cell, Umbilical Vein

SUBMITTER: Vinothini Rajeeve

LAB HEAD: Professor Pedro R Cutillas

PROVIDER: PXD036983 | Pride | 2023-01-11

REPOSITORIES: Pride

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Proximity proteomics identifies septins and PAK2 as decisive regulators of actomyosin-mediated expulsion of von Willebrand factor.

El-Mansi Sammy S Robinson Christopher L CL Kostelnik Katja B KB McCormack Jessica J JJ Mitchell Tom P TP Lobato-Márquez Damián D Rajeeve Vinothini V Cutillas Pedro P Cutler Daniel F DF Mostowy Serge S Nightingale Thomas D TD

Blood 20230201 8

In response to tissue injury, within seconds the ultra-large glycoprotein von Willebrand factor (VWF) is released from endothelial storage organelles (Weibel-Palade bodies) into the lumen of the blood vasculature, where it leads to the recruitment of platelets. The marked size of VWF multimers represents an unprecedented burden on the secretory machinery of endothelial cells (ECs). ECs have evolved mechanisms to overcome this, most notably an actomyosin ring that forms, contracts, and squeezes o ...[more]

PMID: 36564030

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Syntaxin 5 determines Weibel-Palade body size and Von Willebrand factor secretion by controlling Golgi architecture

Project description:Abstract Von Willebrand factor (VWF) is a multimeric hemostatic protein primarily synthesized in endothelial cells (ECs). VWF is stored in endothelial storage organelles, the Weibel-Palade bodies (WPBs), whose biogenesis strongly depends on VWF anterograde trafficking and Golgi architecture. Elongated WPB morphology is correlated to longer VWF strings with better adhesive properties. We previously identified the SNARE SEC22B, which is involved in anterograde ER-to-Golgi transport, as a novel regulator of WPB elongation. To elucidate novel determinants of WPB morphology we explored endothelial SEC22B interaction partners in a mass spectrometry-based approach, identifying the Golgi SNARE Syntaxin 5 (STX5). We established STX5 knockdown in ECs using shRNA-dependent silencing and analyzed WPB and Golgi morphology, using confocal and electron microscopy. STX5-depleted ECs exhibited extensive Golgi fragmentation and decreased WPB length, which was associated with reduced intracellular VWF levels, and impaired stimulated VWF secretion. However, the secretion incompetent organelles in shSTX5 cells maintained WPB markers such as Angiopoietin 2, P-selectin, Rab27A, and CD63. Taken together, our study has identified SNARE protein STX5 as a novel regulator of WPB biogenesis

2022-01-27 | PXD027516 | Pride

Syntaxin-3 is a Weibel-Palade body localized SNARE protein that regulates Von Willebrand factor secretion from endothelial cells

Project description:Endothelial cells store von Willebrand factor (VWF) in rod-shaped secretory organelles, called Weibel-Palade bodies (WPBs). WPB exocytosis is coordinated by a complex network of Rab GTPases, Rab-effectors and SNARE proteins. We have previously identified STXBP1 as the link between the Rab27A-Slp4-a complex on WPBs and the SNARE proteins syntaxin-2 and -3. In this study we investigate the function of syntaxin-3 in VWF secretion. In human umbilical vein endothelial cells (HUVECs) and in blood outgrowth endothelial cells (BOECs) from healthy controls endogenous syntaxin-3 immunolocalized to WPBs. A detailed analysis of BOECs isolated from a patient with variant microvillus inclusion disease (MVID), carrying a homozygous mutation in STX3 (STX3-/-), showed a loss of syntaxin-3 protein and absence of WPB-associated syntaxin-3 immunoreactivity. Ultrastructural analysis revealed no detectable differences in morphology or prevalence of immature or mature WPBs in control versus STX3-/- BOECs. VWF multimer analysis showed normal patterns in plasma of the MVID patient, and media from STX3-/- BOECs, together indicating WPB formation and maturation are unaffected by absence of syntaxin-3. However, a clear defect in Ca2+ and cAMP-mediated VWF secretion was found in the STX3-/- BOECs. Co-immunoprecipitation studies showed that syntaxin-3 associates with the WPB SNAREs SNAP23 and VAMP8. Our data reveal syntaxin-3 as a novel WPB-associated SNARE controlling VWF secretion and highlight the complex regulation of WPB exocytosis by multiple SNARE complexes.

2018-07-04 | PXD006176 | Pride

Proximity proteomics of endothelial Weibel-Palade bodies identifies novel regulator of VWF secretion

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2019-07-11 | PXD013194 | Pride

A CRISPR/Cas9 engineered Von Willebrand factor deficient endothelial cell model shows alternative trafficking of Weibel-Palade body proteins

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2019-11-11 | PXD013857 | Pride

Effect of von Willebrand factor on gene expression in HUVECs

2010-11-10 | GSE19816 | GEO

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2010-11-10 | E-GEOD-19816 | biostudies-arrayexpress