Monaco, Vittoria (2024) Study of the molecular mechanisms relevant to the interactions between pathogens and hosts. [Tesi di dottorato]

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Abstract

To date, the study of the impact of pathogens on their hosts has been a hot topic, especially when it concerns viruses. The mechanisms of pathogen invasion into the host, i.e. the identification of the receptors involved, as well as the characterisation of the proliferation mechanisms used by the pathogen to replicate within a host cell, can be addressed using untargeted approaches relying on Omics Sciences, such as proteomics. Each pathogen protein exerts a specific function, such as the promotion of replication, the inhibition of the host response, etc, exploiting molecular relationships with many hosts’ protein complexes. The identification and characterisation of the latter is therefore a fundamental element in the development of potential drugs. The study of physical and functional interactions between pathogen and host proteins and the identification of processes as potential targets of bioactive molecules in the treatment of infections are the two main topics of investigation within this PhD project. The focus, in particular, has been addressed to the elucidation of molecular mechanisms in host cells affected by the Spike protein from SARS-CoV-2. These interactions have been encompassed by using functional proteomics approaches. Spike is structured in two domains, known as S1 and S2, which are involved in two different aspects of viral internalisation: the recognition of receptors on the host cell surface and the process of membrane rearrangement functional for its internalisation, respectively. The identification of the interacting partners of the S1 domain, achieved by an AP-MS approach, allowed the characterisation of novel receptors and/or co-receptors and the description of the intracellular trafficking of viral particles, suggesting alternative roles for this protein never described before. Among these, the identification of LDHB suggests a specific role for Spike in the anaerobic switch of host metabolism, typical of viral infection. We demonstrated that Spike inhibits LDHB by subtracting its cofactor NAD+. The binding site of NAD+ on Spike was also described by using a proteomics-limited mass spectrometry integrated approach. Moreover, the role of the S2 domain in membrane remodelling was also investigated to better characterise viral entry, trafficking and spread in SARS-CoV-2 infections. In particular, the S2 interactome identified by an AP-MS strategy, suggested the routes followed by protein from the ER to the plasma membrane, through the COP shuttles, and the involvement of S2 in a spread mechanism mediated by the formation of tunnelling nanotubes. Finally, the characterisation of the mode of action and the identification of protein targets of specific drugs has been carried out by using different targeted and untargeted methodologies proteomics-based, integrated with biochemical and biophysical approaches. As a targeted approach, we focused our attention on specific SARS-CoV-2 proteins, such as the PLpro protease, in order to describe the molecular mechanism of inhibition of specific drugs, by in vitro methods such as SPR, limited proteolysis and mass spectrometry. Alternatively, the untargeted approaches passed through the identification of physical and molecular targets within entire proteomes. During my period abroad, I set up a method relying on chemical proteomics approaches known as ABPP (Affinity-Based Protein Profiling) for the in vitro and in vivo target identification of a specific antimalarial drug (plasmodione). The mode of action of different antiviral drugs active in the SARS-CoV-2 infections was also investigated using label-free differential proteomics approaches, which allowed the identification of processes targeted by each drug upon the pharmacological treatment. As a final and general consideration, no single method is able to find satisfying answers to all biological questions. However, the integration of different strategies, including cell biology, biochemical and omics approaches, allows definitely a detailed description of biological systems, opening up unexpected avenues also in tailoring new therapeutic treatments against viral, parasitic or bacterial infections.

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