Multiscale analysis of extracellular regulation of BMP signalling

Lead Research Organisation: The University of Manchester
Department Name: School of Biological Sciences

Abstract

The Bone Morphogenetic Proteins (BMPs) are powerful "growth factors" which are essential for development of nearly all organs and tissues, as well as subsequently maintaining normal tissue structure and function. During development, cells sense signals from their environment that are converted into cellular responses, resulting in a change in their behaviour and often the type of cell they will become. The research proposed here focuses on one major type of signal, the BMP signal, which is critically important in early embryos, where it controls cell fates along the front to back axis.

The action of BMPs is controlled by regulator proteins that can inhibit or enhance the BMP signal, although how they do this is not yet understood. The inhibitors are large proteins that bind to the BMPs, thereby preventing them from interacting with their receptors and sending a message to the cell. We are interested in two BMP regulators, Chordin and Tsg, which are necessary for correct embryonic development. However, there are currently few details of these protein complexes and their interactions which presents a major obstacle to understanding BMP regulation. The main aim of our research therefore is to understand the mechanisms by which these regulators modulate BMP signals. We will achieve this by visualising the location of Chordin and Tsg using microscopy and analysing their structures to determine how these regulator proteins interact with each other and BMPs.

Chordin and Tsg control BMP signalling during development of embryos from simple organisms, such as fruitflies, up to humans. Therefore, we will use the fruitfly Drosophila as a model for this research as it has many advantages as a research organism, including a rapid life cycle, ease of maintenance and amenability to genetics and genome editing. Given that Chordin and Tsg regulate BMP signalling in both fruitflies and humans, our findings will be directly relevant to human biology.

Our results will allow us to gain a better understanding of how BMP regulation occurs and how these interactions underpin the important roles that Chordin and Tsg play in tissue assembly and embryo development. Results from this study will not only be important for understanding embryonic development, but will ultimately allow the design of new therapeutics modulating BMPs, as many human diseases arise when cells receive excessive or insufficient BMP signal. Moreover, a major goal in the stem cell field is to be able to efficiently differentiate stem cells into a particular cell type. Often this process is achieved by adding or inhibiting BMPs. Therefore, by increasing understanding of precisely how to manipulate BMP activity, our results will also be of major benefit to the stem cell field.

Technical Summary

Bone morphogenetic proteins (BMPs) are powerful signalling molecules that are essential for embryonic dorsal-ventral axis patterning, and development and homeostasis of nearly all organs and tissues. Additionally, BMP regulation is of therapeutic interest for a broad range of pathologies including cancer, vascular disease and arthritis. BMP activity is modulated by extracellular proteins including Chordin (called Sog in Drosophila) and Tsg. Together these extracellular regulators bind BMPs and inhibit signalling, moreover Tsg is unusual in that it can also promote BMP signalling. Currently we lack an understanding of how these regulators control BMP activity and the dynamics of their localisation in vivo. Therefore, the overall aim of this proposal is to determine the molecular mechanisms that modulate extracellular BMP signalling by combining synergistic structural, molecular, developmental and imaging approaches. Using cryoEM, we will determine the structure of Chordin/Sog alone and in complex with its binding partner Tsg. In parallel, we will use live and super-resolution imaging to visualise the localisation of Sog and Tsg in the early embryo. We will then use mutational analysis and Affimers, small non-antibody binding proteins, to determine the effect of disabling particular protein interactions on extracellular function in vivo. The Drosophila embryo is the ideal model for this study, due to its rapid development, amenability to manipulation and genome engineering, and extremely well characterised BMP responses that will allow signalling levels to be sensitively assessed. The findings from this study will not only demonstrate how a signalling ligand is regulated extracellularly, shifting the emphasis away from events downstream of receptor activation, but also reveal new strategies for enhancing and inhibiting BMP signalling, both of which represent major goals therapeutically.
 
Description We have generated a knockout fly stock of the BMP antagonist sog, which allows simple reintegration of altered versions of the sog coding sequence. As proof-of-principle, we tested the requirement for two cysteine residues that were previously identified as targets for palmitoylation, which has been proposed to enhance Sog secretion. However, we show that the sogC27,28S mutant is viable with only very mild phenotypes, indicating that these residues and their potential modification are not critical for Sog secretion in vivo.
We show that the N-terminal region of the BMP-regulator Twisted gastrulation binds to BMPs in both humans and flies and a twisted gastrulation mutant that cannot bind BMPs fails to mediate BMP gradient formation required for dorsal-ventral axis patterning of the early Drosophila embryo.
Exploitation Route The sog knockout fly model will form a useful tool for the community.
Knowledge of the BMP-twisted gastrulation binding site offers potential avenues or manipulating BMP function for therapeutic intervention.
Sectors Healthcare

 
Title Cryo-EM structure of the C terminal region of PTX3 with a section of coiled-coil 
Description Cryo-EM structure of the C terminal region of PTX3 with a section of coiled-coil 
Type Of Material Database/Collection of data 
Year Produced 2024 
Provided To Others? Yes  
Impact Publication of paper in Matrix Biology 
URL https://www.ebi.ac.uk/emdb/EMD-19717
 
Title Cryo-EM structure of the C terminal region of PTX3 with a section of coiled-coil 
Description Cryo-EM structure of the C terminal region of PTX3 with a section of coiled-coil 
Type Of Material Database/Collection of data 
Year Produced 2024 
Provided To Others? Yes  
Impact Paper published in Matrix Biology 
URL https://www1.rcsb.org/structure/8S50
 
Title Structure of Human PTX3 C-terminal domain 
Description Crystal Structure of Human PTX3 C-terminal domain 
Type Of Material Database/Collection of data 
Year Produced 2024 
Provided To Others? Yes  
Impact Publication of paper in Matrix Biology 
URL https://doi.org/10.2210/pdb8PVQ/pdb
 
Description Biology Open First Person Interview 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact First Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Sophie Frampton was the postdoc on the grant and first author on our publication ' Modelling the structure of Short Gastrulation and generation of a toolkit for studying its function in Drosophila', published in BiO. Sophie was solicited to do the in person interview after publication of her manuscript.
Year(s) Of Engagement Activity 2022
URL https://journals.biologists.com/bio/article/11/6/bio059463/275706/First-person-Sophie-Frampton
 
Description British Science Week 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact A video showcasing electron microscopy was made aimed at school pupils and general public and was hosted on the University of Manchester's website for British Science Week in 2021 and 2022.
Year(s) Of Engagement Activity 2021,2022
URL https://express.adobe.com/page/qb9O9OjRPk930/
 
Description Interview for the Node 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact The Ashe lab was featured as lab of the week in the Node, an online community site for stem cell and developmental biologists, which increased awareness of the lab's research in the international research community.
Year(s) Of Engagement Activity 2023
URL https://thenode.biologists.com/lab-meeting-with-the-ashe-lab/lablife/
 
Description University Community Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact 200 pupils and parents attended our lab stand at the annual University Community Festival.
Educational activities were demonstrated to hundreds of children and parents over the weekend, illustrating the stretchy nature of human tissues.
Year(s) Of Engagement Activity 2019,2022,2024