MICA: A molecular dissection of the interplay between diabetes and cancer: an integrated, multidisciplinary approach. II.
Lead Research Organisation:
University of York
Department Name: Chemistry
Abstract
This research programme focuses on the detailed actions of human insulin and Insulin-like Growth Factors-1 and 2 (IGF1/2); these are closely related protein hormones. Through evolution they acquired separate biological functions, with insulin becoming a key regulator of metabolism, while IGF1/2 are major growth factors behind cell growth and differentiation. The levels of activity of these hormones determine how long and how healthy we live in the face of lifestyle, diet and disease. When released into the blood the hormones bind, tightly and specifically, to their receptors: Insulin- (IR) and IGF-1R, respectively, large complex protein molecules on the cell surface. Receptor binding, through which the hormone activity is delivered into cells, involves structural changes in both the hormone and the receptors. Here we aim to understand the key events in the translation of hormone signal from the outside to the inside of the cell.
Despite their fundamental medical importance, such as insulin signaling malfunctions in Type 1 (T1D) and Type 2 Diabetes (T2D), IGF1/2 are major drivers of cancer, it is still not understood how these hormones achieve their specific signals and induce different biological effects via their receptors. The complexity of insulin & IG1/2 molecular actions are further convoluted by the existence of two, very similar forms of the IR, and the ability of these hormones to bind in some way to all receptors. There are two forms of the IR; the IR-B form controls metabolic actions of insulin, while IR-A binds also IGF1/IGF2, and can stimulate cell growth and proliferation.
This very complex, and intertwined molecular activity of insulin and IGF1/2 is the basis of their huge societal and human health impact. ~£25mln/day of the NHS budget is spent on T2D, largely to treat associated complications such as cardiovascular and kidney disorders, cancer, and neurodegeneration. Hence there is an urgent need to understand insulin and IGF1/IGF2 specificity at the hormonal and receptor level. This could be then exploited in the design and delivery of new, safer, forms of insulin (analogues), and new IGF1/2 analogues with anti-cancer and beneficial (e.g. anti-neurodegenerative) selective properties, without side-effects seen in diabetes. This research programme is responding to this challenge by offering a consolidated, multidisciplinary (structural, chemical and cell biology) approach to these problems, addressing all the key aspect of insulin & IGF1/2 biology: hormones, receptors and cells.
Fundamental research is the foundation cornerstone of this programme. However, the advanced expertise of this group in applied biomedical sciences will enable thorough clinical translation for the benefit of patients with different conditions.
This programme will deliver:
- on the receptor level: (i) the description of insulin binding to its receptors IR-A and IR-B, (ii) delineation of the structural signatures in the IR-A and IR-B extracellular, hormone-binding parts, (iii) how the hormone-triggered signal is transduced to the inside of the cell in IR and IGF-1R receptors, (iv) what are the structural determinants of insulin and IGF1/2 specific actions through their receptors
- on the hormone level: (i) description of the metabolic and mitogenic elements of human insulin, (ii) description of IGF1/IGF2 hormonal determinants behind their specificities, (iii) development of highly-metabolic, safe insulin analogues, (iv) development of IGF-1R specific IGF1 and IGF2 analogues, including IGF-1R antagonist with anti-cancer applicability
-on the cell-level: (i) description of the contribution of IR-A, IR-B and IGF-1R to hormone-activated glucose uptake into human muscle and fat tissue, (ii) development of advanced human cell-systems with specific receptors to optimise development of insulin analogues, and for study of T2D, (iii) validation of the available human tissue models used in glucose transport studies.
Despite their fundamental medical importance, such as insulin signaling malfunctions in Type 1 (T1D) and Type 2 Diabetes (T2D), IGF1/2 are major drivers of cancer, it is still not understood how these hormones achieve their specific signals and induce different biological effects via their receptors. The complexity of insulin & IG1/2 molecular actions are further convoluted by the existence of two, very similar forms of the IR, and the ability of these hormones to bind in some way to all receptors. There are two forms of the IR; the IR-B form controls metabolic actions of insulin, while IR-A binds also IGF1/IGF2, and can stimulate cell growth and proliferation.
This very complex, and intertwined molecular activity of insulin and IGF1/2 is the basis of their huge societal and human health impact. ~£25mln/day of the NHS budget is spent on T2D, largely to treat associated complications such as cardiovascular and kidney disorders, cancer, and neurodegeneration. Hence there is an urgent need to understand insulin and IGF1/IGF2 specificity at the hormonal and receptor level. This could be then exploited in the design and delivery of new, safer, forms of insulin (analogues), and new IGF1/2 analogues with anti-cancer and beneficial (e.g. anti-neurodegenerative) selective properties, without side-effects seen in diabetes. This research programme is responding to this challenge by offering a consolidated, multidisciplinary (structural, chemical and cell biology) approach to these problems, addressing all the key aspect of insulin & IGF1/2 biology: hormones, receptors and cells.
Fundamental research is the foundation cornerstone of this programme. However, the advanced expertise of this group in applied biomedical sciences will enable thorough clinical translation for the benefit of patients with different conditions.
This programme will deliver:
- on the receptor level: (i) the description of insulin binding to its receptors IR-A and IR-B, (ii) delineation of the structural signatures in the IR-A and IR-B extracellular, hormone-binding parts, (iii) how the hormone-triggered signal is transduced to the inside of the cell in IR and IGF-1R receptors, (iv) what are the structural determinants of insulin and IGF1/2 specific actions through their receptors
- on the hormone level: (i) description of the metabolic and mitogenic elements of human insulin, (ii) description of IGF1/IGF2 hormonal determinants behind their specificities, (iii) development of highly-metabolic, safe insulin analogues, (iv) development of IGF-1R specific IGF1 and IGF2 analogues, including IGF-1R antagonist with anti-cancer applicability
-on the cell-level: (i) description of the contribution of IR-A, IR-B and IGF-1R to hormone-activated glucose uptake into human muscle and fat tissue, (ii) development of advanced human cell-systems with specific receptors to optimise development of insulin analogues, and for study of T2D, (iii) validation of the available human tissue models used in glucose transport studies.
Technical Summary
The experimental paths are very different, due to diverse methodologies of the structural, chemical and cell biology.
Receptors. The structural studies in York (IR-A,IR-B,IGF-1R) are re-focused due to gains achieved in the current MRC grant. Receptors and their constructs will be expressed only in the baculovirus system (yielding currently more reproducible/better crystals). Complexes with insulin/IGF1/2 will be expanded for highly specific analogues to decipher their structural signatures. Hormone receptor affinities will be evaluated by ITC & Novo Nordisk proprietary assays. TK protein expression/structural work will be carried in Cork, while TK crystal studies will be done with York. Frequent BAG beam access to DLS (Didcot), and remote data collection are well established. The exploitation of the larger constructs (i.e. IR ectodomains) by cryoEM is initiated, and it will be continued. However, when suitable grids:constructs are established, the cryoEM work will form a separate grant application.
Hormones. Analogues of insulin, IGF1/2 will be semi-/fully-synthesised or expressed in E.coli; IOCB has key expertise here. The established wide range of analogue assays (receptor affinities and autophosphorylation, cell viability etc.), will be expanded for IGF-2R receptor essays; unique radio-active (125I, Eu) IGF2 tracers will be produced. All analogues with novel properties will be used in structural studies with the receptors, and optimised further in novel human muscle/fat-tissue cell-based platforms developed here.
Cells. CRISPR-Cas9 will be used to knockout all IR isoforms individually (and IGF-1R), creating a novel insulin unresponsive cell line. This line will be transfected to express specific receptor(s) profile, then the insulin/analogues responsiveness of these cell lines will be characterised with respect to i) glucose transport, ii) the proportion of HA-GLUT4-GFP expressed/present at the cell surface, iii) IR downstream effectors activation.
Receptors. The structural studies in York (IR-A,IR-B,IGF-1R) are re-focused due to gains achieved in the current MRC grant. Receptors and their constructs will be expressed only in the baculovirus system (yielding currently more reproducible/better crystals). Complexes with insulin/IGF1/2 will be expanded for highly specific analogues to decipher their structural signatures. Hormone receptor affinities will be evaluated by ITC & Novo Nordisk proprietary assays. TK protein expression/structural work will be carried in Cork, while TK crystal studies will be done with York. Frequent BAG beam access to DLS (Didcot), and remote data collection are well established. The exploitation of the larger constructs (i.e. IR ectodomains) by cryoEM is initiated, and it will be continued. However, when suitable grids:constructs are established, the cryoEM work will form a separate grant application.
Hormones. Analogues of insulin, IGF1/2 will be semi-/fully-synthesised or expressed in E.coli; IOCB has key expertise here. The established wide range of analogue assays (receptor affinities and autophosphorylation, cell viability etc.), will be expanded for IGF-2R receptor essays; unique radio-active (125I, Eu) IGF2 tracers will be produced. All analogues with novel properties will be used in structural studies with the receptors, and optimised further in novel human muscle/fat-tissue cell-based platforms developed here.
Cells. CRISPR-Cas9 will be used to knockout all IR isoforms individually (and IGF-1R), creating a novel insulin unresponsive cell line. This line will be transfected to express specific receptor(s) profile, then the insulin/analogues responsiveness of these cell lines will be characterised with respect to i) glucose transport, ii) the proportion of HA-GLUT4-GFP expressed/present at the cell surface, iii) IR downstream effectors activation.
Planned Impact
The multi-facetted approach of our proposal maximizes the cohort of researchers on whose work our studies will impact. Our work will be of especial interest to all members of the research community who work on various aspects of insulin action, metabolism and diabetes, as well as those working on cell-signaling pathways. Our research has potential to greatly impact the underexplored research area seeking to define the molecular cross talk between signals emanating from distinct, but closely related cell surface receptors, and how perturbations in such processes underlie patho-physiologies including diabetes and numerous types of cancers.
As a collective, we are well-connected both nationally and internationally to disseminate our findings informally as well as making them publicly available through presentation at scientific meetings and publication in open-access journals. Through the very nature of our programme we have distinct research profiles; an asset that, as well as ensuring our work addresses questions that are important in multiple fields, will facilitate impact of this work.
The cell biology platforms we aim to generate will facilitate a paradigmatic shift in approach to allow characterization of the consequences on cellular physiology of hormone analogues that have hitherto been studies using biophysical and biochemical techniques. By making these platforms available to other researchers our work will increase the impact of the whole field (hormone/receptor structure/function)
The focus of our work on growth factor-signaling means that the work will also have impact on the pharmaceutical industry which has a substantial interest in Type-2 diabetes, insulin-resistance and cancers. Our work will pave the way for development/design of diagnostic and therapeutic strategies for these disease states. Given the substantial problem that these conditions represent in the U.K (and globally) not only in terms of morbidity and mortality, but also from an economic perspective, it is hard to overstate the impact this could have.
Presentation of our work at national and international meetings attended by academics and representatives of major pharmaceutical companies will maximise its impact in these areas, as will the other forms of engagement outlined in our 'Pathways to Impact'. It should also be noted that the Universities of all partners have Communications Departments dedicated to highlighting research outputs, and Commercial Exploitation Units to assist with commercialisation of any research output as need arises.
Finally, all PIs have a track record in engaging the general public, for example by presentation at diabetes support groups, and work on National Diabetes groups. Such meetings are often attended by policy makers, providing opportunity for our research to influence directly the nation's health, wealth and culture. While such developments clearly represent a long-term investment, they are nonetheless important.
As a collective, we are well-connected both nationally and internationally to disseminate our findings informally as well as making them publicly available through presentation at scientific meetings and publication in open-access journals. Through the very nature of our programme we have distinct research profiles; an asset that, as well as ensuring our work addresses questions that are important in multiple fields, will facilitate impact of this work.
The cell biology platforms we aim to generate will facilitate a paradigmatic shift in approach to allow characterization of the consequences on cellular physiology of hormone analogues that have hitherto been studies using biophysical and biochemical techniques. By making these platforms available to other researchers our work will increase the impact of the whole field (hormone/receptor structure/function)
The focus of our work on growth factor-signaling means that the work will also have impact on the pharmaceutical industry which has a substantial interest in Type-2 diabetes, insulin-resistance and cancers. Our work will pave the way for development/design of diagnostic and therapeutic strategies for these disease states. Given the substantial problem that these conditions represent in the U.K (and globally) not only in terms of morbidity and mortality, but also from an economic perspective, it is hard to overstate the impact this could have.
Presentation of our work at national and international meetings attended by academics and representatives of major pharmaceutical companies will maximise its impact in these areas, as will the other forms of engagement outlined in our 'Pathways to Impact'. It should also be noted that the Universities of all partners have Communications Departments dedicated to highlighting research outputs, and Commercial Exploitation Units to assist with commercialisation of any research output as need arises.
Finally, all PIs have a track record in engaging the general public, for example by presentation at diabetes support groups, and work on National Diabetes groups. Such meetings are often attended by policy makers, providing opportunity for our research to influence directly the nation's health, wealth and culture. While such developments clearly represent a long-term investment, they are nonetheless important.
Publications
Asai S
(2022)
Characterization of insulin crystalline form in isolated ß-cell secretory granules.
in Open biology
Asai S
(2021)
A radioligand receptor binding assay for measuring of insulin secreted by MIN6 cells after stimulation with glucose, arginine, ornithine, dopamine, and serotonin.
in Analytical and bioanalytical chemistry
Brezina K
(2018)
Can Arginine Inhibit Insulin Aggregation? A Combined Protein Crystallography, Capillary Electrophoresis, and Molecular Simulation Study.
in The journal of physical chemistry. B
Chrudinová M
(2018)
A versatile insulin analog with high potency for both insulin and insulin-like growth factor 1 receptors: Structural implications for receptor binding.
in The Journal of biological chemistry
Chrudinová M
(2021)
Characterization of viral insulins reveals white adipose tissue-specific effects in mice
in Molecular Metabolism
Dzianová P
(2020)
The efficiency of insulin production and its content in insulin-expressing model ß-cells correlate with their Zn2+ levels.
in Open biology
Fabre B
(2018)
Probing Tripodal Peptide Scaffolds as Insulin and IGF-1 Receptor Ligands
in European Journal of Organic Chemistry
Jirácek J
(2020)
Radiolabeled hormones in insulin research, a minireview.
in Journal of labelled compounds & radiopharmaceuticals
Description | A regional cryoEM facility at the University of York |
Amount | £1,601,545 (GBP) |
Funding ID | 206161/Z/17/Z |
Organisation | Wellcome Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 12/2018 |
End | 07/2023 |
Description | Guy Dodson Fund |
Amount | £150,000 (GBP) |
Organisation | Novo Nordisk |
Sector | Private |
Country | Denmark |
Start | 09/2014 |
End | 09/2018 |
Description | Revealing Molecular Bases of Signal Transduction through the Drosophila Insulin Receptor: cryoEM and Functional Studies. |
Amount | £563,843 (GBP) |
Funding ID | BB/W003783/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 07/2022 |
End | 01/2025 |
Description | University of York - Centre for Future Health Fellowship |
Amount | £90,000 (GBP) |
Organisation | University of York |
Sector | Academic/University |
Country | United Kingdom |
Start | 01/2018 |
End | 12/2019 |
Description | X-ray Diffraction Equipment for Macromolecular Crystallography at York |
Amount | £750,000 (GBP) |
Funding ID | BB/T017805/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 08/2020 |
End | 06/2022 |
Title | STRUCTURE OF [ASP58]-IGF-I ANALOGUE |
Description | |
Type Of Material | Database/Collection of data |
Year Produced | 2019 |
Provided To Others? | Yes |
URL | https://bmrb.io/data_library/summary/?bmrbId=34408 |
Description | Collaboration with Dr Sean Sweeney on in vivo evaluation of insulin analogues |
Organisation | University of York |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We designed and made insulin analogues with different signaling profiles: with different affinities to Insulin Receptor isoforms. |
Collaborator Contribution | Dr. Sweeney's lab applied/used our analogues to their in vivo neurons systems to evaluate the neuroprotective impact of these analogues. This is still ongoing work. |
Impact | to be publsihed |
Start Year | 2019 |
Description | Collaboration with Dr Sean Sweeney on in vivo evaluation of insulin analogues |
Organisation | University of York |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We designed and made insulin analogues with different signaling profiles: with different affinities to Insulin Receptor isoforms. |
Collaborator Contribution | Dr. Sweeney's lab applied/used our analogues to their in vivo neurons systems to evaluate the neuroprotective impact of these analogues. This is still ongoing work. |
Impact | to be publsihed |
Start Year | 2019 |
Description | Collaboration with Novo Nordisk (Copenhagen) |
Organisation | Novo Nordisk |
Department | Diabetes Research Unit |
Country | Denmark |
Sector | Private |
PI Contribution | Novo Nordisk is interested in all York-based IR-related research. Hence all York IR-oriented work is within the scope of this collaboration and future exploitation by both sides. York input spans very broad activity of cloning, protein production and characterisation, and structural work |
Collaborator Contribution | Novo Nordisk discloses its know-how related to production and function of insulin receptor. It also provides human insulin and other insulin-like hormones (insect DILP5) needed for the research in York. Novo Nordisk provides also funds (£140,000) for the employment of York-based research technician for work on insulin receptor. |
Impact | Work in progress. |
Start Year | 2018 |
Description | Collaboration with Prof. Emrah Altindis (Boston College Biology Department, MA, 02467, USA) on viral insulins |
Organisation | Boston College |
Country | United States |
Sector | Academic/University |
PI Contribution | York group is involved in the structural characterization of viral insulin that have been synthesized in the IOCB (Prague) and discovered by Boston group (Biology Department) of Prof. Altindis. This is an ongoing research. |
Collaborator Contribution | Double chain viral insulins (VILPs) were chemically synthesized based on DNA sequences identified in several iridoviridae. Binding affinities to IR-A, IR-B or IGF1R and post-receptor signaling were determined. One of VILPS displays white adipose tissue specific effects in mice. Results show that VIPLs are active members of the insulin superfamily with unique characteristics. Elucidating the mechanism of tissue specificity for GIV dcVILP will help to design new analogs that specifically target the tissues and provide new insights into VILPs potential role in disease. IOCB (LZ, JJ) assisted with binding competition/experiments and analysis of the data. |
Impact | Martina Chrudinová, François Moreau, Hye Lim, Terezie Páníková, Lenka Žáková, Randall H. Friedline, Francisco A. Valenzuela, Jason K. Kim, Jirí Jirácek, C. Ronald Kahn, Emrah Altindis. Characterization of viral insulins reveals white adipose tissue-specific effects in mice. Molecular Metabolism 2020, 44: 101121. doi: 10.1016/j.molmet.2020.101121. |
Start Year | 2020 |
Description | Collaboration with Prof. NIa Bryant (Dept of Biology, University of York) on specific activation of glucose GLUT4 transporter |
Organisation | University of York |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We delivered clones of Insulin receptor isoforms IR-A and IR-B, and IGF-1R to make specific cells where the activation of GLUT4 glucose transporter may be studied in detail. |
Collaborator Contribution | Development of cell-based systems for specific glucose GLUT4 transporter activation - work in progress. |
Impact | Work in progress |
Start Year | 2018 |