Role of hyperplastic discs in ubiquitin-mediated control of intercellular communication

Lead Research Organisation: University of Edinburgh
Department Name: Royal (Dick) School of Veterinary Scienc

Abstract

Summary: Organismal development and subsequent maintenance of the adult animal requires the co-ordinated action of cell growth, proliferation, differentiation and cell death. Through these concerted actions cells are able to form and maintain a diverse array of different tissues and organs. Extracellular signalling molecules, such as morphogens, establish temporal and spatial gradients that help determine cellular fate decisions. The restriction of morphogen expression to a small subset of specific cells, such as those in stem cell niches, is crucial to allow correct orchestration of cellular fate within a tissue or organ. However, very little is known about the intracellular signalling pathways and molecular mechanisms that regulate morphogen expression. This proposal's principal aim is to uncover a new upstream regulatory pathway governing morphogen expression. Its central hypothesis concerns addressing whether a unique ubiquitin-protein ligase called 'Hyperplastic Discs' (Hyd) uses the addition of a small protein, called ubiquitin, to control morphogen expression and influence intercellular communication. Addition of ubiquitin to other proteins, in a process called ubiquitylation, provides the cell with a powerful 'molecular handle' to control the modified protein's behaviour. Hyd represents a particularly intriguing and important ubiquitin-protein ligase and this work aims to comprehensively uncover its ubiquitin-associated molecular mechanism. Taken together the proposal's findings will not only advance our understanding of the mechanisms governing animal development and cellular homeostasis, but also expand our knowledge of the versatility and regulatory potential of ubiquitylation as modification system. Potential applications and benefits of this work are manifold. One direct benefit would help us understand a possible cause of a number of human diseases that demonstrate aberrant morphogen expression, including cancer and many congenital disorders. Similarly, as a process ubiquitylation is central to numerous biological processes that when disrupted can lead to a wide array of human diseases. Therefore, elucidation of novel modes of ubiquitylation action will both expand our knowledge of its signalling potential and guide our understanding of its role in both this as well as several other important areas of biology. Furthermore, the identification of a Hyd-associated molecular mode of action will allow the development of small-molecule therapeutic compounds. Therapeutic intervention with such compounds could then correct the aberrant, disease-associated morphogen expression. Finally, Hyd-associated therapeutic intervention could also provide huge advances in regenerative medicine by promoting morphogen expression and stimulating dormant stem cells into dividing and promoting tissue regeneration. Design and development of small molecule compounds requires detailed insights in a protein's molecular mechanism. Only a comprehensive approach will allow accurate dissection and elucidation of the molecular events controlled by Hyd and its downstream effectors. Hence, the proposal's three individual projects use a combination of cutting-edge proteomic, biochemical and genetic techniques to address the specific objectives of: (1) Identification of Hyd-mediated ubiquitylated targets, (2) the type of ubiquitylation signal made on them and (3) the proteins responsible for translating and propagating the ubiquitin signal into a molecular event. Overall this work is both important and timely and will provide important insights into extremely important areas of biology and molecular biology. Whose findings will impact upon our understanding of numerous biological processes and potentially lead to the generation of treatments for human disease.

Technical Summary

Technical Summary: This proposal's objectives concern exploring the hypothesis that Hyperplastic Discs (Hyd) uses ubiquitylation to control morphogen expression and influence intercellular communication. Long term this work aims to comprehensively uncover Hyd's ubiquitin-associated molecular mechanism in a biologically relevant setting. These objectives will be addressed using a combination of proteomic, genetic and biochemical techniques: Proteomic techniques: For the purification and identification of Hyd binding partners and ubiquitylated substrates: Stable Isotope Labelling with Amino Acids in Cell Culture (SILAC); Fourier Transform Ion Cyclotron Resonance and Orbitrap Mass Spectrometry coupled with Electron Coupled Dissociation and 2D liquid chromatography; Haemagglutinin-Strep-Tactin tandem-affinity purification under native conditions (protein binding studies) with inhibition of deubiquitylase and phosphatase activity with Chloracetamide and Sodium Orthovanadate / Sodium Fluoride, respectively; V5-Histidine6 tandem affinity purification for isolation of ubiquitylated material initially under denaturing conditions (Ni2+-affinity purification) with 6M Guanidine HCl and 8M Urea denaturing affinity purification followed by native conditions (V5-affinity purification). Drosophila Genetic techniques: Generation of transgenic flies: P-element-mediated (pUAST) transgenesis (BestGene). Purification of ubiquitylated material: pUAS-V5-His6-ubiquitin and K>R and I44A mutant variants. Clonal analysis and Temporal and Spatial expression of transgenes: Mosaic Analysis with a Repressible Cell Marker (MARCM) analysis; Gal4-UAS system; inverted-repeat-mediated RNAi; EGUF/Cell-lethal-mediated clonal selection. Confocal and Electron-Microscopic (Scanning and Transmission) Imaging of Drosophila Cell-based Techniques: LacZ- and Luciferase-based reporter assays and quantitative RT-PCR dsRNAi Western Blotting and Immunoprecipitations

Planned Impact

Impact Summary: As described in the Beneficiaries section this work will have a significant impact upon academic and corporate investigators working in a diverse range of fields. Enhancing both technical and biological knowledge of the extremely important and timely fields of ubiquitylation and morphogen expression. Particular impact could be felt in the rapidly advancing areas of regenerative medicine and stem cell biology, which ultimately could be used to benefit the lives of many people suffering from diseases related to defects in stem-cell behavior and/or morphogen expression. Additionally, this work could also help patients who would benefit from advances in stem cell based therapies. While researchers in the ubiquitylation field will also benefit from the knowledge and tools generated from these studies. Advances in our knowledge of morphogen expression may provide a powerful tool to regulate the number and type of cells in a human body. Such control would directly benefit the nation's health by correcting numerous human disease and developmental defects. Accordingly, such improvements would indirectly increase the nation's wealth through increasing the pool of working adults. Additionally, development of therapeutic compounds / research tools could also provide a marketable opportunity for pharmaceutical and biotech involvement. Greater elucidation of morphogen regulatory pathways would allow the identification of new genetic markers to detect and optimise personalised treatment plans. Although the timescale for such developments extend beyond the length of the BBSRC New Investigator Award, it is my intention to pursue and develop these initial findings and follow a translational path into the development of therapeutic compounds. Edinburgh Research and Innovation will govern and facilitate the exploitation, application and commercialisation of scientific advances, discoveries and inventions. In addition to aiding the exploitation of the proposal's findings, Edinburgh University also provides excellent career development opportunities. As a renowned centre of excellence for investing in people, Edinburgh University provides training in both scientific and transferable skills including: Project and Time Management, communication, presentation and interpersonal skill courses. As a signature of the recent Concordat to support the career development of researchers, Edinburgh University clearly supports and invests in the comprehensive training of it staff. I have already benefited enormously from these courses and will continue to attend them. Accordingly I will encourage the Post Doc to also attend them to promote their personal and career development. Importantly, the Post Doc will also aid in the preparation and writing of presentations and manuscripts for publication.
 
Description This work identified which protein are bound by Hyd to regulate the activity of a signalling pathway involved in important normal and disease processes. The work in the Drosophila fruit fly has helped direct our murine and human research efforts.
Exploitation Route Our findings have guided our own translational work into mammalian systems and we believe the same will be true for other research groups interested in Hyd and Hedgehog signalling. Recent publication and our own indicate clear role for mammalian Hyd in human disease-related processes such as cancer and osteoarthritis.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Our identification of a link between UBR5 and Hedgehog signalling prompted a collaboration to whole genome sequence family members with early onset osteoarthritis.While it did not identify an obvious UBR5 coding mutation, it did identify a mutation in a decoy TNF receptor. This information will help inform family members wishing to genetically screen other existing family members for disease susceptibility as well as potential genetic screening of potential family members.
First Year Of Impact 2015
Sector Healthcare
Impact Types Societal

 
Description Investigating Skeletal Homeostasis 
Organisation Medical Research Council (MRC)
Department MRC Human Genetics Unit
Country United Kingdom 
Sector Academic/University 
PI Contribution Directed murine crosses to address specific research questions. Analysed embryonic and adult tissue for histological and molecular changes.
Collaborator Contribution Housed and maintained murine lines
Impact Publication covering the generation and validation of the novel mouse model.
Start Year 2011
 
Description UBR5 in intestinal homeostasis and cancer 
Organisation Beatson Institute for Cancer Research
Country United Kingdom 
Sector Academic/University 
PI Contribution We generated Drosophila-based findings in the intestine that prompted generation of murine model. We analysed the Hedgehog profile of material generated by the collaborators
Collaborator Contribution They generated and housed the various mice from which we received material
Impact Meeting abstracts and poster presentations.
Start Year 2011