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Discovering the signalling pathways and physiology of active rhomboid proteases in the brain

Periodic Reporting for period 1 - NeuroRhomboid (Discovering the signalling pathways and physiology of active rhomboid proteases in the brain)

Reporting period: 2015-04-07 to 2017-04-06

Protease enzymes catalyse irreversible post-translational modifications that control many distinct cellular processes. An important class are intramembrane proteases, which have catalytic activity that is confined within the lipid bilayer. Intramembrane proteases function to cleave their specific transmembrane substrates. To our knowledge, the human genome encodes fourteen intramembrane proteases, of which five are rhomboid proteases. Rhomboid proteases are defined and characterised by their distinct mode of catalysis (a catalytic dyad of serine and histidine residues) and their evolutionarily conserved six-transmembrane domain core structure. Despite their intensive study, we do not know the physiological roles for mammalian rhomboid proteases; in particular few roles have been ascribed for the four rhomboids that are localised within the secretory pathway. This is mainly due to a lack of identified substrates. The core aim of this project is to identify novel substrates for rhomboid proteases, to enable us to expose their physiological and medical significance.

Several intramembrane proteases have been linked to human pathologies such as neurodegeneration and cancer. Perhaps the most well known example is that of gamma-secretase and its pivotal role in the formation and accumulation of beta-amyloid, which characterises Alzheimer’s Disease. Interestingly, rhomboid proteases have also been linked to Alzheimer’s Disease; RHBDL4 has been shown to process APP but in a distinct manner to that of gamma-secretase. Furthermore, PARL, the mitochondrially targeted rhomboid protease, cleaves PINK1, a mitochondrial kinase mutated in Parkinson’s Disease. Overall, these examples illustrate the potential impact that can be made by the discovery of a protease-substrate relationship. Rhomboid proteases are present in all kingdoms of life. Such conservation throughout evolution suggests these enzymes have important functions in human biology, which are only now being illuminated, and can only be fully comprehended through substrate discovery.

The overall objective of the action is to discover the physiological and medical significance of active rhomboid proteases in mammals. Two largely uncharacterised rhomboids are enriched in the brain, and are the main focus of our investigations. To achieve our objective, we work toward three goals: 1) to identify novel substrates for the rhomboid proteases, 2) to validate the identified substrates biologically and 3) to uncover their physiological significance through the screening of mutant cells and model organisms.
In accordance with the proposals outlined in fellowship proposal, we successfully designed and performed screens for novel interacting proteins and potential substrates for the rhomboid proteases. This took the form of mass spectrometry-based screens and novel cell biological assays designed in-house. Furthermore, we have generated loss-of-function model systems for the analysis of the hits from the screen, which are currently being used to validate the putative substrates identified by the screen. This research has been communicated at various regional and international meetings and conferences. Overall, the main objective of the project has been fully met and the results are likely to be the foundation for further in-depth characterisation and subsequent publications.

During the period of the fellowship funding, a review on the rhomboid family of intramembrane proteases has been published in an open-access peer-reviewed journal, which has helped to increase awareness of the significance of fundamental research on rhomboid proteases, and the importance of substrate discovery:

Lastun, V.L. Grieve, A.G. & Freeman, M. (2016) Substrates and physiological functions of secretase rhomboid proteases. Semin Cell Dev Biol, doi: 10.1016/j.semcdb.2016.07.033

Further to this, a methodology paper is currently in press, describing cell biological assays for rhomboid protease function. This will help others in the field to efficiently screen for rhomboid protease substrates:
Moncada-Pazos, A. & Grieve, A.G. (2017) A simple cell-based assay for the detection of surface protein shedding by rhomboid proteases. Methods in Molecular Biology, In Press.

Outside of the scope of our initial plan, we investigated the function of rhomboid family member 2, RHBDF2 (also known as iRhom2), and its role in inflammation through regulation of the metalloprotease, TACE. This work represents the discovery of key mechanisms that control the cleavage and release of the primary inflammatory cytokine, TNF. This research on RHBDF2 and inflammation has been published in an internationally recognised open-access journal, and widely communicated through social networking:

Grieve, A.G. et al. (2017) Phosphorylation of iRhom2 at the plasma membrane controls mammalian TACE-dependent inflammatory and growth factor signalling. Elife, 6, doi: 10.7554/eLife.23968.

In addition to many talks and conferences where this research has been presented, we wrote a description of our work in the journal using lay terms, so that the impact of fundamental biological research is communicated as widely as possible (known as an ‘eLife digest’). The aim of which was to narrow the gap between active scientists and the general public with particular interest in the biosciences.
To date, there are few substrates known for the rhomboid proteases, and their physiological roles have not been elucidated. Our screens have provided strong evidence of rhomboid substrates that are currently under further investigation. In addition, we have also employed cutting-edge approaches to identify the interactome of the rhomboid proteases, such as BioID. This has provided us with both substrates and regulators of the protease-substrate relationship. Overall these findings have led to a manuscript that is currently in preparation for an open-access journal, and is the seed for many further projects. Moreover, we believe that the discovery of rhomboid substrates is likely to have medical significance.
The findings from the screens undertaken during the period covered by the fellowship will also form the basis of applications for further funding and recruitment of staff, internationally, which will increase the number of trained scientists, promote the dissemination of skills and knowledge and further enhance European competitiveness in biological science. Last, all our publications are published in an open-access journals, available to any interested party, and has already attracted citations in other peer-reviewed journals. Overall, these are major enhancements in the field.

Last, we have discovered a key mechanism that underpins the control of inflammation in mammals: phosphorylation of the rhomboid related protein, RHBDF2. This work has been published in a high-impact journal, and has wide societal implications. Excessive inflammation, often triggered by the uncontrolled release of TNF, can lead to rheumatoid arthritis, cancer and many other diseases. Therefore, RHBDF2 phosphorylation could be a promising new target for anti-inflammatory drugs that may help to treat these conditions.
iRhom2 phosphorylation controls TACE-dependent growth factor and inflammatory signalling