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

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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

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.