The concept that a substantial fraction of the encoded proteins are intrinsically disordered (IDPs) or contain intrinsically disordered regions (IDRs), that are nevertheless fully functional, has revolutionized our understanding of protein science. This finding has directly challenged the classic view implying 'sequence determines structure determines function'. Since their discovery, IDPs and IDRs have been implicated in several cellular functions, being recognized and/or post-translationally modified by structured domains of interacting partners. Importantly, a number of pathologies have been linked to IDPs, demanding for a better understanding of the physical-chemical principles underlining their function.
Among IDPs, a particularly interesting role is played by Short Linear Motifs (SLiMs), consisting of short stretches of typically 3 to 12 amino acids that, despite their small size, mediate crucial interaction between protein partners often acting as regulators of entire pathways. SLiMs function has been associated with disease. For instance, both papilloma virus and SARS-CoV-2 hijack endogenous SLiMs to cause cervical cancer and facilitate lung infection, respectively.
Another example of recognition afforded by the presence of an IDR is provided by nucleophosmin (NPM1). This protein is overexpressed and mutated in a number of cancers and used by several viruses, including HIV and HBV, to transport viral proteins to nucleoli. Mutations at the IDR compromise this function and have been described in solid cancers and dyskeratosis congenita.
This project is designed to investigate the interactions of selected SLiMs with SH2, SH3 and PDZ domains from protein partners and how these interactions are deranged in cancer and viral infections. Moreover, the role of NPM1 IDR in mediating interactions with fibrillarin and snoRNAs will be investigated. Finally, our structural studies will be used to search for small lead compounds that interfere with these interactions.
This project is placed in one of the most active research fields in Protein Science: the investigation of the chemical-physical features and functional role of IDPs and IDRs as well as their implication in several human pathologies. Among them, it is necessary to mention the mechanisms of viral infection, that perfectly enter in this specific topic, due to the consequences that the global pandemic caused by SARS-CoV-2 is causing all over the world. In this respect it is important to outline that, even if a large part of the scientific community is focusing on the specific ones that are related to this virus, a wider perspective that is focused on the general interaction patterns between plastic protein partners such as IDPs and IDRs could be necessary and useful to prepare the scientific community to the next global threat.
Research on IDPs and IDRs poses indeed many challenges to the investigators because of i) their plasticity, ii) the possibility to undergo structural transitions from disordered to folded states upon binding interaction partners and iii) the promiscuity that many IDPs have in binding several different partners, also at once, using short aminoacidic stretches within their sequences. Such features also explain why IDPs are often found in different supramolecular complexes with disparate partners, playing many specific, and sometimes opposed, functions within the cell. NPM1 is a good example of this promiscuity since it can bind dozens of different IDR-containing proteins with its folded N-terminal domain and use its central IDR to recognize several other folded proteins. The consequences of altering the large interaction patterns of these highly promiscuous proteins just varying binding affinity for a single possible protein partner, is the next level that is necessary to clarify in the new era of structural biology. Accordingly, an interdisciplinary approach and the use of several state-of-the-art technologies is mandatory. Structural analysis methods such as crystallography and molecular modelling may provide snapshots of the interactions but, to reach a full understanding, have to be flanked by biophysical methods that allow to capture the dynamical features at the basis of recognition. In order to fulfill this aim, the research units will strictly collaborate exchanging samples and sharing expertise, also organizing collaborative research teams to participate at beam time sessions available in the Synchrotron Radiation Facilities.
Moreover, a detailed description of the folding mechanism upon binding if any, the description of conformational modifications, the identification of allosteric sites generated upon binding and so on will be pursued along the project. Such complete understanding is crucial not only in terms of general comprehension of how proteins work but also because, very often, pathogenic mutations act subtly and their effect is not immediately understandable by a static structural description of protein complexes.
We have selected a number of model systems that are implicated both in viral infections and in several human cancers. We aim to show, within this project, that research on IDPs and IDRs may have a social impact in that it may allow a considerable increase in the number of targets available for therapeutic interventions. The project is designed to provide a clear proof of this concept. Starting from the detailed description of the mechanistic and structural basis of an interaction, we will search for interfering molecules, we will assess their potency in disrupting the interaction in vitro and we will finally investigate their effect in cell model systems. If successful, this project will provide to the scientific community new putative targets for the development of novel marketable drugs.