Selection of Intracellular Antibodies against genetically encoded PTMs in target proteins

The complexity of the proteome outnumbers that of the transcriptome by orders of magnitude, due to the diverse post-translational modifications (PTMs) of proteins. PTMs (like acetylation, phosphorylation) have a central role in physiological and pathological intracellular processes. Therefore, they represent a very rich source of targets of biological and therapeutic interest, that, however, is not deeply exploited. As a matter of fact, the functional study and validation of individual PTM targets present formidable challenges and can only be indirect. Intracellular antibodies (intrabodies) represent, in principle, a class of molecules that could be exploited to selectively target PTMs. Recent results provided a robust selection platform to isolate intrabodies against native PTM-proteins: the Post-translational Intracellular Silencing Antibodies (PISA) technology. PISA permits in vivo isolation of the required intrabody from a library of antibody domains using i) yeast two hybrid system (Y2H) and ii) Tethered Catalysis, by which it is possible to create two-hybrid system baits that present a constitutive post-translational modification. As first proof of concept, PISA yielded a specific intrabody (scFv-58F) that selectively binds acetylated lysine 9 of histone H3 (H3K9ac), which was subsequently used to bind the Acetylated Histone H3 in cell, and to obtain a functional in cell interference of the acetylated h3 Histone pool. In this thesis, the scFv-58F was exploited to achieve a PTM-directed interference, testing the hypothesis that targeting H3K9ac yields more specific effects on gene expression than inhibiting the corresponding modifying enzyme (HAT) that installs that PTM. The results collected here prospect the H3K9ac-specific intrabody as the founder of a new class of molecules to directly target histone PTMs, inverting the paradigm from inhibiting the enzymes to acting on the PTM mark. Furthermore, in this thesis work, an improved version of the PISA platform, referred to as PISA 2.0, was also developed. The technology in its 1.0 form represents a significant breakthrough in fact, but tethered catalysis-based selection may present some limitations. To name but one the impossibility of a priori predicting the site of the single PTMs. Accordingly, here I implemented the PISA platform, exploiting the Genetic Code Expansion (GCE) methodology that allows to genetically encode the post translational (PT-) modified amino acid in a desired position in the bait protein. Specifically, new Y2H strains were generated by integration of different orthogonal aminoacyl tRNA synthetase/tRNA (aaRS/tRNA) pairs to incorporate PT-modified amino acids (namely Acetyl-Lysine and Phospho-serine) in a specific residue (TAG codon) of bait proteins of interest, stably expressed and ready to be screened against binding domain libraries. Eventually, since all the collected results highlighted poor incorporation efficiency in S.cerevisiae, I took advantage from the well stated bacterial-two-hybrid (B2H) system as a intrabody selection platform and the wide range of functional and orthogonal aaRS/tRNA pairs available in E.coli to set up the new 2.0 version of PISA in this model organism. In conclusion, my PhD work is part of a wider project aimed at addressing the complexity of the proteome by the selection of intracellularly working binders of functional epitopes. In particular my independent work major advances have been reached in i) in demonstrating the specific interfering activity of one of our anti PTM-intrabodies (namely the scFv58) compared to currently used indirect methods to study PTMs and notably ii) in improving the current PISA platform by exploiting Expanded Genetic Code to genetically encode the desired target PTM, in order to facilitate, accelerate and automatize the isolation of anti-individual PTM intrabodies, specifically anti-Acetyl-Lysine binders.