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Molecular mechanisms of rhomboid-like proteins in human immunity

Periodic Reporting for period 1 - RhomBioID (Molecular mechanisms of rhomboid-like proteins in human immunity)

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

What is the problem/issue being addressed?
The immune system coordinates responses against plethora of pathogens thanks to messenger molecules and cell surface proteins that serve as antenna. These proteins rely on the secretory pathway to be traffic them around and out of the cell. How cells exert this crucial activity is largely unclear, and a major problem in understanding immunity.

The advances in this field of research is hampered by a major problem represented by the lack of knowledge on the molecular interactions occurring with and around protein traffic in the cell. Rhomboid-like proteins are critical fate determinants of protein traffic, however we lack fundamental understanding of their interactions at molecular level. This project aims to tackle this problem by investigating with two paradigmatic proteins in this superfamily, namely RHBDL4 and iRhom2, at fundamental mechanistic cell biological level and from the physiological-immunological point of view. Any discovery in such context would enormously expand our knowledge of the processes where these two proteins are involved, including the immune system. Therefore, I aim in this project to expand our understanding of molecular interactions of RHBDL4 and iRhom2 by using a biochemical screen to identify potential interactor proteins. This should allow large expansion of mechanistic studies over the function of these two members of the rhomboid-like proteins. Importantly, the same approach could be used for other members of the rhomboid-like proteins, thus initiate a new intense expansion of our knowledge of the processes that involve rhomboid-like proteins.

Why is it important for society?
Rhomboid-like proteins are important regulators of protein traffic in the cell. Most diseases show abnormal protein traffic, and understanding how these regulators function could open the doors to the cure of a vast number of diseases. For example, iRhom2 regulates the traffic of an enzyme critical for the secretion of TNFalpha, a potent immune and inflammatory factor released chronically in rheumatoid diseases. Currently available cure is expensive injections with antibodies capable of neutralising TNFalpha. A design of a drug interfering with molecular interaction and thus inhibiting the release of this factor could reduce enormously the costs of treatment of a common disease in the European Union.
Moreover, RHBDL4 and iRhom2 are paradigms of two types of rhomboid-like proteins: the small enzymatically active rhomboids and the large catalytically inactive pseudo enzymes. Comparing these very different proteins can shed light on conceptual aspects of how this superfamily of proteins function. This becomes important in understanding other members of the superfamily, for example of rhomboid-like proteins of pathogens such as Toxoplasma gondi, Trypanosoma brucei, Mycobacterium tuberculosis. Finally, a number of virus such as Human Immune deficiency virus (HIV) causing acquired immune deficiency syndrome (AIDS), is known to highjack protein traffic within cells using the processes where RHBDL4 and iRhom2 and involved.

What are the overall objectives?
The main objective of the project is to identify molecular interactions of two proteins belonging to the superfamily of rhomboid-like proteins, RHBDL4 and iRhom2, and understand the physiological and cell biological relevance of these interactions. Finally the project aims to dissect at the mechanistic level the role of these proteins in the immune system.
Work performed.
A forward biochemical screen on RHBDL4 and iRhom2 were performed and over hundreds of potential interactions have been identified. Further work has been done on RHBDL4, and a potential regulator protein has been identified in addition to other types of interactors. I combined conventional biochemical methodologies to genetic engineering and enzymological approach to analyse the type of interaction between proteins. Results are consistent with the view that RHBDL4 is the substrate of the regulator protein. We are currently working to dissect the physiological function of these previously unknown molecular interactions and setting up an in vitro system to investigate mechanistically the role of the putative regulator in the physiological function of RHBDL4.

There are no fruitful exploitation currently, as the project is concerned with the understanding of the fundamental cell biological processes. Potentially the delivered discoveries could be instrumental for the development of novel disease treatment strategies, for example, drug discoveries.

These results have been disseminated in occasion of three international conferences, ASCB 2015, BSCB annual spring meeting 2016 and 2017 in the form of posters. In addition, the work has been presented to regular department seminars on cell and developmental biology and at the departmental postdoc symposia of 2016 and 2017.
Prior to this work, Very few proteins were known to be interactors of RHBDL4 and iRhom2. Indeed, only 3 proteins were known as interactions of RHBDL4. During the work period of this project, two more proteins were shown to interact with RHBDL4. In this project, about 100 experimentally reproducible potential interactors were identified. Of those, two appear to have more or less direct interaction with RHBDL4, and more are currently being studied. The proteins described in the literature as interactors of RHBDL4 were also identified in this project. This is promising and encouraging as it indicates that there is the likelihood that many of the proteins in the screen are real interactors, thus are worth pursuing. Similarly, this project identified a previously described interactor of iRhom2. In addition, we have identified about 100 new potential interactors.
In the current literature these is no information weather RHBDL4 or iRhom2 can be substrate of other enzyme. In this work we identified a enzyme-substrate type of interaction where the rhomboid-like proteins are the substrates of a protein that was identified in this project. This is a novel and ground breaking discovery, as it would be first time that a protein is identified as modifier of rhomboid-like protein. We are currently working to investigate the function of this interaction.
This project concerns fundamental cell biological processes, and typically discoveries in this type of project show benefits after many years since the discovery to impact the society. Nonetheless, this project deals with two proteins are known to be involved in a process affected in many diseases, such a cystic fibrosis, rheumatoid diseases, and AIDS.
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