student profile: Miss Camilla Faoro


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Thesis work

Thesis title: The Signal Recognition Particle : new functions for drug discovery

Supervisors: Sandro FERNANDES ATAIDE , Jacqueline MATTHEWS

Thesis abstract:

�div style="text-align: justify;"�The Signal Recognition Particle (SRP) is an essential ribonucleoprotein complex (RNP) that is conserved across all kingdoms of life and responsible for the co-translational delivery of membrane and secretory proteins to the plasma membrane in prokaryotes and to the endoplasmic reticulum in eukaryotes. In the simpler version found in prokaryotes, SRP comprises one protein (Ffh) and the small 4.5S RNA. In eukaryotes the SRP consists of six proteins (the heterodimer SRP 9/14, SRP 19, SRP 54, and the heterodimer SRP 68/72) along with a large RNA moiety, the 7S RNA. There are hints that SRP components might also have non-canonical functions that have not been fully characterized. SRP 72 has been identified as the only SRP S domain component to possess a phosphorylation site and be involved in cleavage signaling during apoptosis. SRP68 and SRP72 were identified to interact with the tail of histone H4 and further characterize to regulate several gene expression indicating their role and interaction with other cellular components outside the protein synthesis cycle. Moreover, antibodies against SRP components have been detected in the serum of patients with severe myopathies and Lupus-type autoimmune diseases. In this project, I present proteomics and genomics approaches to carry out a systematic search for the SRP network, focusing on the S domain components. The identification of new partners will allow a broader comprehension of the non-canonical functions of SRP that will shed light on its full range of activities in mammalian cells. RIP-seq method was used to capture the SRP transcriptome and to identify coding and non-coding RNA that interact with each SRP subunits of the S domain, while LC-MS/MS analysis was used to characterize the proteomic profile of SRP54 and the heterodimer SRP68/72. Understanding the differences between the SRPs from eukaryotes and prokaryotes, specifically which elements are responsible for triggering GTP hydrolysis, is essential for unlocking the potential of the SRP system as a drug target. Truncations or mutations on any of the bacterial components of the SRP system have proven to be either lethal or severely impact cell viability, indicating that disruption of this system could be a suitable target for drug discovery. An interesting point identified in previous crystal structures of the bacterial SRP complex with SR (FtsY) indicated new and specific interactions of SR with SRP RNA that are essential for early complex formation. In order to investigate how cofactors such as GTP modulate FtsY structure and its interactions with RNA, we have analyzed the binding of FtsY to GTP analogues, such as GMPPNP and GCPPNP as well as to GDP and 4.5S RNA using Nuclear Magnetic Resonance (NMR) spectroscopy and crystallography. Using a Fragment-Based Drug Design (FBDD) approach coupled with NMR we have discovered three novel compounds that bind to FtsY. The x-ray crystallography structures of FtsY with these compounds represents a starting point for the rational targeting of the SRP receptor in bacteria. �/div� �br /� �br /�

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