Characterisation of a new mechanism of regulation for HIF1 and the hypoxic response

Lead Research Organisation: University of York
Department Name: Biology

Abstract

I believe our track record with the BBSRC and our publications demonstrate we can achieve the goals of this exciting project and successfully return this grant with novel and significant insights and advances to LIMD1 biology and the HIF signalling pathway.
Optimum levels of oxygen are required throughout the cells and tissues of our body for survival. Referred to as 'oxygen homeostasis', this is tightly regulated in animals and indeed all multicellular organisms to ensure that tissues are sufficiently supplied with oxygen. Whilst oxygen levels range within the human body from 21% in the upper airway to an average of 5% in most organs, each tissue type has a requirement for a certain concentration of oxygen, below which essential cellular processes such as energy production, protein synthesis and cell growth and division become impaired. Rapid reaction and adaptation of a cell or tissue to low oxygen concentrations ('hypoxia') can enable cells to remain viable, thus reducing potential damage to the organism. This is known as the 'hypoxic response'.

Deregulation of the hypoxic response is a key characteristic in cancer development and also large tumour growth and its spread throughout the body. Furthermore, deregulation of this key cellular control process is also linked to many non-cancerous diseases such as neurological disease, myocardial infarction (heart attacks), stokes, and many ischemic (low oxygen) related diseases. Therefore, a complete understanding of the molecular biology of this critical cellular control process remains an important focus for basic cell and molecular biology research worldwide.

To this end we have recently identified a protein called LIMD1 that is a critical to regulating the normal function of the hypoxic response. Furthermore, we have shown that loss of this protein creates a pseudo-hypoxic environment within the cell, causing it to react as if it is in one of the hypoxic diseased states mentioned above. Our discovery therefore represents a new unknown level of molecular biology critical for the hypoxic response. Moreover, we are currently the only group in the world to be researching this specific area of hypoxic regulation, and hopefully with this BBSRC award, we can continue to investigate this exciting new molecular biology. Furthermore, such information will represent a major new avenue of investigation to basic and clinical molecular biological researchers in this field and represent a new set of protein targets for future development of hypoxic disease-related drug therapies and treatments.

Technical Summary

Hypoxia-inducible factor (HIF) is a transcription factor that is active under low oxygen (hypoxic) conditions. HIF is a heterodimer comprised of alpha and beta subunits; under normal oxygen (normoxic) conditions, the HIF-alpha subunit is degraded in an oxygen-dependent process. HIF-a is hydroxylated by prolyl hydroxylase domain proteins (PHD1/2/3) using available molecular oxygen, which facilitates engagement of the von Hippel-Lindau protein (VHL), the recognition component of an E3 ubiquitin ligase, leading to subsequent ubiquitylation/degradation. We have recently discovered that LIMD1 (LIM domains-containing protein 1) acts as a molecular scaffold by simultaneously binding the PHDs and VHL, creating a PHD-LIMD1-VHL protein complex and enzymatic niche that enables efficient HIF-1a degradation. Depletion of endogenous LIMD1 increases HIF1-a levels and transcriptional activity in both normoxia and hypoxia. LIMD1 and family member proteins Ajuba and WTIP (LAW) also bind to PHDs and VHL, indicating that these LIM domain-containing proteins represent a previously unrecognised family (and therefore level) of hypoxic response regulators.
Specifically aims:
(A) Molecular, biochemical and biophysical characterisation of molecular mechanism of action of PHD-LAW-VHL complexes in vitro and in vivo Here we will significantly advance our mechanistic understanding of how the LAW family of proteins contribute additional levels of complexity to the control of hypoxic signalling through binding and regulation of key proteins involved in this pathway.
(B) What is the biological significance of PHD-LAW-VHL complexes in hypoxic regulation? Here we will correlate this new molecular biology with functional significance and use structure-function analysis in a cell biology context to further validate and scrutinise our molecular observations, culminating in what will be an unprecedented and unparalleled systems biology analysis of the LAW family mem

Planned Impact

The beneficiaries from this research can be clearly divided into three groups. Firstly, there are those in the academic arena, where HIF and hypoxic signalling has become an important cellular pathway for both clinical and non-clinical research. When one considers the degree and variety of cellular processes and disease pathologies shown to be regulated by HIF, we can begin to understand the profound impact such work will have. Continuing on with disease pathogenesis, the second group of potential beneficiaries of this research in the long term are the patients suffering from diseases that are now clearly linked to dysfunction of the HIF pathway, such as neurological dysfunction, myocardial infarction and many ischemic related diseases. The more we understand about the mechanism of HIF regulation by LIMD1 and family members, the better we can design novel treatments and therapies to combat these pathologies. Thirdly, the commercial sector will also benefit. We have shown that regulation of LIMD1 can control stem cell differentiation. We now have obtained additional evidence to suggest that its critical role in hypoxic regulation and subsequent mitochondrial function (unpublished data) significantly contributes to this aspect of stem cell biology. This specific discovery has resulted in the filing of a patent for the use of LIMD1 depletion as a novel and highly efficient new methodology/tool for creating induced pluripotent stem cells. This could help realise the potential for reprogramming autologous somatic cells for patient therapy in the near future (Patent PCT/GB09/19773.2). Our recent discovery of LIMD1 as a key regulator of HIF and thus the hypoxic response has also resulted in the filing of a patent (GB1200743.1) for the use of LIMD1 depletion as a novel and highly efficient new methodology/tool for combating hypoxia-related disease states. We wish to licence these patents to the health care industry, the benefits of which will be in the fields of cancer biology, regenerative medicine and tissue engineering. Again, this will result in significant benefits to long term patient health. Once the pharmaceutical or health care industries become involved, engaging and communicating this research can almost be guaranteed. With the endpoint being to make available related therapies or treatments, such large industries have tremendous resources which can be quickly mobilised to maximise advertisement, public engagement and exploitation of the impacts of our research.

Publications

10 25 50
 
Description Our research has focused on the molecular structure of different functional regions of the protein LIMD1. We have shown that three conserved regions (called lim domains) do not associate with each other. We had hypothesized that these domains may associate to form a molecular surface for other proteins to dock on to. Our data suggests this is not the case. We are exploring whether lim/lim interactions occur in the presence of a binding partner. We have demonstrated that each of the lim domais are entities that can independently fold to form 3D structures. We are currently pursuing the 3D structure of each lim domain using NMR spectroscopy. We anticipate elucidating the 3D structure of each of the 3D lim domains before the termination of the project.
Exploitation Route Knowledge of the molecular structure of a protein (and sub-regions of proteins) is key to understanding its biological function. We are interested in determining how the protein LIMD1 co-ordinates different cellular processes and how interactions with other proteins mediate this function. Our ongoing structural studies of LIMD1 (and those of our collaborators at Bart's Cancer Institute) will be invaluable for other researchers who study this protein and/or these pathways, as well as more broadly for cellular and structural biologists who study how proteins like LIMD1 co-ordinate and integrate signaling and regulatory pathways. In the remaining months of this project we are collating our structural data for dissemination via peer-reviewed articles in the established scientific press, which will facilitate the use of our data and findings by other researchers.
Sectors Pharmaceuticals and Medical Biotechnology

 
Description 4-year iCASE PhD funding
Amount £120,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2018 
End 09/2022
 
Description DL 
Organisation Hull York Medical School
Country United Kingdom 
Sector Academic/University 
PI Contribution a
Collaborator Contribution a
Impact 1. Conserved asymmetry underpins homodimerization of Dicer-associated double-stranded RNA-binding proteins. Heyam A, Coupland CE, Dégut C, Haley RA, Baxter NJ, Jakob L, Aguiar PM, Meister G, Williamson MP, Lagos D, Plevin MJ. Nucleic Acids Res. 2017 Dec 1;45(21):12577-12584. PMID: 29045748 2. Argonaute Utilization for miRNA Silencing Is Determined by Phosphorylation-Dependent Recruitment of LIM-Domain-Containing Proteins. Bridge KS, Shah KM, Li Y, Foxler DE, Wong SCK, Miller DC, Davidson KM, Foster JG, Rose R, Hodgkinson MR, Ribeiro PS, Aboobaker AA, Yashiro K, Wang X, Graves PR, Plevin MJ, Lagos D, Sharp TV. Cell Rep. 2017 Jul 5;20(1):173-187. doi: 10.1016/j.celrep.2017.06.027. PMID: 28683311 3. S6K2-mediated regulation of TRBP as a determinant of miRNA expression in human primary lymphatic endothelial cells. Warner MJ, Bridge KS, Hewitson JP, Hodgkinson MR, Heyam A, Massa BC, Haslam JC, Chatzifrangkeskou M, Evans GJ, Plevin MJ, Sharp TV, Lagos D. Nucleic Acids Res. 2016 Nov 16;44(20):9942-9955. Epub 2016 Jul 12. PMID: 27407113 4. Dissecting the roles of TRBP and PACT in double-stranded RNA recognition and processing of noncoding RNAs. Heyam A, Lagos D, Plevin M. Wiley Interdiscip Rev RNA. 2015 May-Jun;6(3):271-89. PMID: 25630541
Start Year 2012
 
Description TVS 
Organisation Queen Mary University of London
Department Barts Cancer Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution The award was a 2-site project with BCI being the lead institute. Data from structural biology and protein biochemistry research conducted by the PI and research technician in York were communicated to the BCI team. These data were used in the design of experiments at the BCI. The York team also sent construct DNA and protein samples for experiments at BCI.
Collaborator Contribution Dr Tyson Sharp at BCI is a the principal investigator on this funding award. Dr Katharine Bridge at BCI was the PDRA funded by this project. The BCI team generated data concerning LIMD1 interactions and binding partners that were communicated to York. The BCI team also sent construct DNA and protein samples for experiments in York.
Impact Publication - Bridge et al (2017) Cell Reports. 20, 173-187
Start Year 2014