Structural mechanisms of regulation and assembly in the nephronophthisis INVS-NPHP3-NEK8-ANKS6 module

Cystic kidney diseases represent a major health burden for which there are few effective treatments. Yet we are far from understanding the cell biology behind these diseases. Here, we propose to study the regulation and function of proteins that are mutated in nephronophthisis, an inherited childhood cystic kidney disease. These proteins coordinate signals received by developing kidney cells at antenna-like structures on the cell surface, called primary cilia. Defective signalling leads to loss of cell division control and cyst formation. Our aim is to provide new insights into cystic kidney disease mechanisms that will ultimately lead to new therapies.

We will concentrate our efforts principally on two proteins, NEK8 and INVS. NEK8 belongs to a family of enzymes called protein kinases. These modify other proteins to modulate their functions, and are often found in signalling pathways that pass information from the cell surface to the cell nucleus so that cells may respond to their environment. The activity of protein kinases, that is to say the efficiency with which they modify other proteins, is strictly controlled. Protein kinases and other proteins that control them are very commonly mutated in diseases. The mutations result in uncontrolled kinase activity so that signalling pathways malfunction: they might pass signals to the nucleus when they should not, or they might block signals that ought to be passed on. We discovered that INVS controls the activity of NEK8, and we mapped the part of INVS responsible down to a region of the protein that is usually disrupted in patients that have INVS mutations. In many patients, it seems that disease is caused by missing just this region of INVS.

In the first part of this project, we will investigate how INVS controls the activity of NEK8. To simplify matters, we will study just the two proteins in isolation. Crystallography will be used to produce atomic-resolution models of NEK8, both alone and associated with INVS, to show how its activity is controlled in the maximum possible detail. Crystallography produces detailed but static models. So we will apply advanced biochemical methods that enable us to track the activity of NEK8 over time under a number of different experimental conditions. This will provide us with a detailed understanding of what effects INVS has on NEK8, and what is therefore lacking in patients that have mutations in INVS.

In the second part of this project, we will broaden our study to encompass two further proteins that are associated with NEK8 and INVS and that are also mutated in cystic kidney disease. We will investigate whether and how these proteins associate and how these interactions relate to the activity of NEK8. Finally, we will examine mutations in these proteins that were found in cystic kidney disease patients, to discover their effects on the interactions between proteins.

Although many of the genes involved in cystic kidney disease are known, we have very little insight into how mutations in these genes cause disease. In this project, we will explore the idea that these mutations disrupt the interactions between a group of proteins, leading to loss of control of NEK8 activity. These insights will influence the way we think about signalling in the cilia, and how this goes awry in a range of associated diseases. We will produce a detailed model to explain how NEK8 activity is controlled by INVS and generate the first chemical compounds that block NEK8 activity. Our work will provide the basis for strategies to rescue ciliary signalling in cells with mutations in NEK8, INVS and interacting proteins. If successful, this might eventually lead to new therapies for patients.

There is an urgent need to characterize the molecular pathways that underlie ciliopathies, such as the cystic kidney disease nephronophthisis (NPHP), in order to identify new therapeutic targets. Proteins that are mutated in NPHP can be grouped into modules based on their localization, interactions and functional relationships. One module is formed from the protein kinase NEK8, with INVS, ANKS6 and NPHP3. Disease-associated mutations in NEK8 disrupt its localization, which is dependent on INVS. We have now shown that INVS induces NEK8 to self-activate, and found that the region responsible is disrupted in INVS mutations that cause disease.
Here, through structural, molecular and cell biology approaches, we will investigate the hypothesis that the INVS-NPHP3-NEK8-ANKS6 module acts as a protein complex that localizes NEK8 and modulates its activity and that disease mutations in this module lead to dysregulation of NEK8.

Work Package 1 Regulation of NEK8 catalytic activity by INVS
The mechanism of NEK8 autophosphorylation will be assigned using a kinetic test using an automated kinase assay system. The structures of NEK8 alone and bound to INVS will be determined using X-ray crystallography. Based on the structures, the mechanisms that regulate NEK8 will be probed using site-specific mutagenesis, kinase assays and cell-based assays that address their function in ciliary signalling.

Work Package 2 Molecular interactions within the INVS-NPHP3-NEK8-ANKS6 module
We will characterize the molecular interactions within the module with respect to binding and regulation of NEK8. The four module proteins will be co-expressed in mammalian cells and complex formation assessed using co-immunoprecipitation, co-purification and SEC-MALLS, and NEK8 activity measure using kinase assays. The interactions within NEK8-containing complexes will be dissected by co-expression of module proteins lacking individual domains, having only single domains or harbouring disease mutations.

Research career development. Training the next generation of biomedical research scientists is an important component of our work that has lasting impact. Our aim is that the PDRA who works on this project will be in a strong position to apply for Fellowships either directly afterwards, or after gaining further experience. This approach is highlighted by one of our previous PDRAs, Charlotte Dodson who has been awarded a Research Fellowship from Imperial College to set up her own group. At least in terms of her publications, this award was based largely on her 3 first author papers from her 3 years in the group, the most prominent of which was published in Science Signaling.

Economic and Societal Impact. There are currently no therapeutic options for autosomal recessive cystic kidney disease patients, 30% of whom die in their first month, and many others die in childhood. The path to discovering treatments for these diseases is long and challenging. It requires accurate models that generate ideas for therapeutic development, as well as reagents and assays to support drug discovery. Pharmaceutical companies or charitable/academic drug discovery groups will incorporate our work into drug discovery projects. This will be done through our existing network of collaborators or, with the help of the Enterprise and Business Development Office in Leicester, with new partners. As our track record on Aurora-A and NEK2 kinases shows, we will play a direct role in developing these therapeutics. Some aspects of our work will be of particular significance in this regard:

Mechanisms through which mutations lead to polycystic kidney disease. A key goal of our research is to understand the mechanisms through which mutations found in these patients result in disease. This will form the basis for future work towards treatments for these patients, perhaps through rescue of the defective signalling pathway.

NEK8 structures and inhibitors. We aim to discover the first ATP-competitive inhibitors of NEK8. These will be developed further for use in cell biological or pharmacological contexts. The structures of NEK8 that we determine will enable virtual screening approaches to identify ligands that bind the kinase, and will be essential for efficient optimization of inhibitors. We would like to stress that therapeutically-relevant NEK8 inhibitors will not necessarily be of the ATP-competitive class, and the structures will identify allosteric sites such as the INVS binding site. We are working with MRC-Technology to target allosteric sites such as this, and so we would be strongly placed to drive forward drug discovery on targets in cystic kidney diseases.

Recombinant protein and assays. There are currently no commercial sources of recombinant NEK8 protein, and no assays suitable for supporting a drug discovery campaign on this kinase. Our work will enable companies to develop these products for sale.

Kidney disease groups and patients. Just knowing that relevant research is being done on the disease is helpful to kidney disease patients and their supporters because it shows that scientists are taking an interest. This gives them increased hope for future treatments. We will work with local hospitals and patient groups, and national charities to explain our research.

Junior scientists. Modern biomedical research is an inspirational topic for exciting children and young adults about science. We give talks in schools about our own work, which have to date been focused on cancer, and this project will enable us to broaden our scope to kidney diseases.