Elfin UI:a graphical interface for protein design with modular building blocks

Molecular models have enabled understanding of biological structures and functions and allowed design of novel macro-molecules. Graphical user interfaces (GUIs) in molecular modeling are generally focused on atomic representations, but, especially for proteins, do not usually address designs of complex and large architectures, from nanometers to microns. Therefore, we have developed Elfin UI as a Blender add-on for the interactive design of large protein architectures with custom shapes. Elfin UI relies on compatible building blocks to design single- and multiple-chain protein structures. The software can be used: (1) as an interactive environment to explore building blocks combinations; and (2) as a computer aided design (CAD) tool to define target shapes that guide automated design. Elfin UI allows users to rapidly build new protein shapes, without the need to focus on amino acid sequence, and aims to make design of proteins and protein-based materials intuitive and accessible to researchers and members of the general public with limited expertise in protein engineering

A versatile Halo- and SNAP-tagged BMP/TGFβ receptor library for quantification of cell surface ligand binding

TGFβs, BMPs and Activins regulate numerous developmental and homeostatic processes and signal through hetero-tetrameric receptor complexes composed of two types of serine/threonine kinase receptors. Each of the 33 different ligands possesses unique affinities towards specific receptor types. However, the lack of specific tools hampered simultaneous testing of ligand binding towards all BMP/TGFβ receptors. Here we present a N-terminally Halo- and SNAP-tagged TGFβ/BMP receptor library to visualize receptor complexes in dual color. In combination with fluorescently labeled ligands, we established a Ligand Surface Binding Assay (LSBA) for optical quantification of receptor-dependent ligand binding in a cellular context. We highlight that LSBA is generally applicable to test (i) binding of different ligands such as Activin A, TGFβ1 and BMP9, (ii) for mutant screens and (iii) evolutionary comparisons. This experimental set-up opens opportunities for visualizing ligand-receptor binding dynamics, essential to determine signaling specificity and is easily adaptable for other receptor signaling pathways

Atheroprone fluid shear stress-regulated ALK1-Endoglin-SMAD signaling originates from early endosomes

Background Fluid shear stress enhances endothelial SMAD1/5 signaling via the BMP9-bound ALK1 receptor complex supported by the co-receptor Endoglin. While moderate SMAD1/5 activation is required to maintain endothelial quiescence, excessive SMAD1/5 signaling promotes endothelial dysfunction. Increased BMP signaling participates in endothelial-to-mesenchymal transition and inflammation culminating in vascular diseases such as atherosclerosis. While the function of Endoglin has so far been described under picomolar concentrations of BMP9 and short-term shear application, we investigated Endoglin under physiological BMP9 and long-term pathophysiological shear conditions. Results We report here that knock-down of Endoglin leads to exacerbated SMAD1/5 phosphorylation and atheroprone gene expression profile in HUVECs sheared for 24 h. Making use of the ligand-trap ALK1-Fc, we furthermore show that this increase is dependent on BMP9/10. Mechanistically, we reveal that long-term exposure of ECs to low laminar shear stress leads to enhanced Endoglin expression and endocytosis of Endoglin in Caveolin-1-positive early endosomes. In these endosomes, we could localize the ALK1-Endoglin complex, labeled BMP9 as well as SMAD1, highlighting Caveolin-1 vesicles as a SMAD signaling compartment in cells exposed to low atheroprone laminar shear stress. Conclusions We identified Endoglin to be essential in preventing excessive activation of SMAD1/5 under physiological flow conditions and Caveolin-1-positive early endosomes as a new flow-regulated signaling compartment for BMP9-ALK1-Endoglin signaling axis in atheroprone flow conditions

A versatile Halo- and SNAP-tagged BMP/TGFβ receptor library for quantification of cell surface ligand binding.

Funder: Morbus Osler Society Germany Einstein Center for Regenerative Therapies, BerlinTGFβs, BMPs and Activins regulate numerous developmental and homeostatic processes and signal through hetero-tetrameric receptor complexes composed of two types of serine/threonine kinase receptors. Each of the 33 different ligands possesses unique affinities towards specific receptor types. However, the lack of specific tools hampered simultaneous testing of ligand binding towards all BMP/TGFβ receptors. Here we present a N-terminally Halo- and SNAP-tagged TGFβ/BMP receptor library to visualize receptor complexes in dual color. In combination with fluorescently labeled ligands, we established a Ligand Surface Binding Assay (LSBA) for optical quantification of receptor-dependent ligand binding in a cellular context. We highlight that LSBA is generally applicable to test (i) binding of different ligands such as Activin A, TGFβ1 and BMP9, (ii) for mutant screens and (iii) evolutionary comparisons. This experimental set-up opens opportunities for visualizing ligand-receptor binding dynamics, essential to determine signaling specificity and is easily adaptable for other receptor signaling pathways

Additional file 2 of Atheroprone fluid shear stress-regulated ALK1-Endoglin-SMAD signaling originates from early endosomes

Additional file 2. RNAseq csv file: RPKM values

Additional file 1 of Atheroprone fluid shear stress-regulated ALK1-Endoglin-SMAD signaling originates from early endosomes

Additional file 1: Supplementary Figs. S1-S5. and primer sequences used in qPCR experiments. Figure S1. RNAseq analysis and HAoEC flow marker validation. Figure S2. Validation of used flow set up by comparison with existing data. Fig. S3. Human Serum experiment and additional flow regulated genes. Figure S4. Expression data of selected genes in Endoglin knock-down. Figure S5. Caveolin-1 positive early endosomes are signaling hotspots for FSS induced BMP signaling in HAoECs. Table S1. Primers used for quantitative PCR analysis

Additional file 3 of Atheroprone fluid shear stress-regulated ALK1-Endoglin-SMAD signaling originates from early endosomes

Additional file 3. RNAseq csv file: Differential expression values of LSS vs. HSS

Additional file 4 of Atheroprone fluid shear stress-regulated ALK1-Endoglin-SMAD signaling originates from early endosomes

Additional file 4. Uncropped Western Blot images