Immuno-Competitive Capture Mass Spectrometry, a novel unbiased approach to study endogenous protein-protein interactions

Protein-protein interactions (PPIs) are controlling the majority of biological functions and are the main driver of cellular processes observed in normal as well as pathological conditions. Such a level of controlling is only possible via a high degree of complexity; i.e. a massive number of protein-protein interactions (in the range of couple hundreds of thousands), a variety of physical and structural properties and their reversibility. Moreover, binding affinities can span from micro-molar to high pico-molar level and some proteins are acting as “hubs” by having multiple partners. This sophisticated organization and regulation of PPIs explains why their study is so challenging. No single approaches can capture the full picture and there is an urgent need for innovative platforms to study and analyze PPIs. In this thesis, a novel platform named Immuno-Competitive Capture Mass Spectrometry (ICC-MS) was developed to screen in an unbiased fashion intracellular PPIs. ICC-MS was designed to reach higher specificity compared to classical affinity purification mass spectrometry by introducing a competition step between free and capturing antibody prior to immunoprecipitation. This antibody-based label-free quantitative approach was then combined with a rigorous statistical analysis to extract the cellular interactome of proteins of interest while filtering out non-specifically binding proteins. ICC-MS was first applied to elucidate hepatitis C viral non-structural protein 5A interactome in human hepatoma cells revealing LATS kinases as potential important regulators of viral infection. The study of Glypican-2 and HtrA1 interacting partners further confirmed the ability of ICC-MS to deliver a limited number of highly confident interacting proteins being promising candidates for functional validation. Interestingly, ICC-MS can also be adapted to study interactions formed between proteins and oligonucleotides (Oligo-Competitive Capture Mass Spectrometry or OCC-MS). While it contributed to a better understanding of the mode of action of an SMN2 splicing modifier, the approach could not elucidate the role of protein interactions in antisense oligonucleotides toxicity. Taken together, this innovative approach is suitable to improve the comprehensiveness and accuracy of current protein-protein interactions databases in term of true biological interactome representation

Alternative splicing liberates a cryptic cytoplasmic isoform of mitochondrial MECR that antagonizes influenza virus.

Viruses must balance their reliance on host cell machinery for replication while avoiding host defense. Influenza A viruses are zoonotic agents that frequently switch hosts, causing localized outbreaks with the potential for larger pandemics. The host range of influenza virus is limited by the need for successful interactions between the virus and cellular partners. Here we used immunocompetitive capture-mass spectrometry to identify cellular proteins that interact with human- and avian-style viral polymerases. We focused on the proviral activity of heterogenous nuclear ribonuclear protein U-like 1 (hnRNP UL1) and the antiviral activity of mitochondrial enoyl CoA-reductase (MECR). MECR is localized to mitochondria where it functions in mitochondrial fatty acid synthesis (mtFAS). While a small fraction of the polymerase subunit PB2 localizes to the mitochondria, PB2 did not interact with full-length MECR. By contrast, a minor splice variant produces cytoplasmic MECR (cMECR). Ectopic expression of cMECR shows that it binds the viral polymerase and suppresses viral replication by blocking assembly of viral ribonucleoprotein complexes (RNPs). MECR ablation through genome editing or drug treatment is detrimental for cell health, creating a generic block to virus replication. Using the yeast homolog Etr1 to supply the metabolic functions of MECR in MECR-null cells, we showed that specific antiviral activity is independent of mtFAS and is reconstituted by expressing cMECR. Thus, we propose a strategy where alternative splicing produces a cryptic antiviral protein that is embedded within a key metabolic enzyme