Biology subjects

Webb, A. M.

Publications and source records attributed to Webb, A. M..

3 recordsLinked to original sources

bioRxiv2020

Notch Regulates Vascular Collagen IV Basement Membrane Through Modulation of Lysyl Hydroxylase 3 Trafficking

SUMMARYDuring angiogenesis, endothelial cells secrete proteins that make up a planar protein network surrounding blood vessels termed basement membrane (BM). Collagen type IV (Col IV) is a BM protein associated with early blood vessel morphogenesis and is essential for blood vessel stability. To date, little is known about how endothelial cells mediate intracellular transport and selective secretion of Col IV. We have identified the GTPase Rab10 as a major regulator of Col IV vesicular trafficking during vascular development. Knockdown of Rab10 reduced de novo Col IV secretion in vivo and in vitro. Mechanistically, we determined that Rab10 is an indirect mediator of Col IV secretion, partnering with atypical Rab25 to deliver the enzyme lysyl hydroxylase 3 (LH3) to Col IV-containing vesicles staged for secretion. Loss of Rab10 or Rab25 resulted in depletion of LH3 from Col IV-containing vesicles and rapid lysosomal degradation of Col IV. Furthermore, we demonstrated that Rab10 activation is downstream of Notch signaling, indicating a novel connection between permissive Notch-based vessel maturation programs and vesicle trafficking. Overall, our results illustrate both a new trafficking-based component in the regulated secretion of Col IV and how this vesicle trafficking program interfaces with Notch signaling to fine-tune BM secretion during blood vessel development.Competing Interest StatementThe authors have declared no competing interest.View Full Text

developmental biology↗

bioRxiv2020

EHBP1 and EHD2 regulate Dll4 caveolin-mediated endocytosis during blood vessel development

Despite the absolute requirement of Delta/Notch signaling to activate lateral inhibition during early blood vessel development, many mechanisms remain unclear. Here, we identify EHD2 and EHBP1 as novel regulators of Notch activation in endothelial cells through controlling endocytosis of Delta-like ligand 4 (Dll4). Knockout of EHBP1 and EHD2 in zebrafish produced a significant increase in ectopic sprouts in zebrafish intersomitic vessels during development and a reduction in downstream Notch signaling. In vitro, EHBP1 and EHD2 localized to plasma membrane-bound Dll4 and actin independently of clathrin. Disruption of caveolin endocytosis resulted in EHBP1 and EHD2 failing to organize around Dll4 as well as loss of Dll4 internalization in endothelial cells. Overall, we demonstrate that EHBP1 and EHD2 regulate Dll4 endocytosis by anchoring caveolar endocytic pits to the actin cytoskeleton.

cell biology↗

bioRxiv2019

Convergent Hybrid Phase Ligation Strategy for Efficient Total Synthesis of Large Proteins Demonstrated for 212-residue Linker Histone H1.2

Simple and efficient total chemical synthesis of large proteins remains a significant challenge. Here, we report development of a convergent hybrid phase native chemical ligation (CHP-NCL) strategy that should be generally applicable for facile preparation of large proteins. Key to the strategy is the use of sequential ligation on the solid phase for the directed assembly of ~100-residue segments from short, synthetically accessible peptide components. These segments can then be assembled via convergent solution phase ligation, exploiting o-aminoaniline as a chemically flexible cryptic thioester with multiple activation modalitiies on resin and in situ. We demonstrate the feasibility of our approach through the total synthesis of 212-residue linker histone H1.2 in unmodified, phosphorylated, and citrullinated forms, each from eight component peptide segments. We further demonstrate that fully synthetic H1.2 replicates the binding interactions of linker histones to intact mononucleosomes, as a proxy for the essential function of linker histones in the formation and regulation of higher order chromatin structure.

biochemistry↗