• AcH3K9
• antibodies
• anticorpi
• anticorpi bifunzionali
• anticorpi intracellulari
• AsLOV2
• AtLOV2
• bifunctional antibodies
• biologia sintetica
• degradazione proteica
• domini LOV
• fotostimolazione
• intrabodies
• intracellular antibodies
• led
• lievito
• LOV domains
• optogenetica
• optogenetics
• orthogonality
• ortogonalità
• photostimulation
• protein degradation
• sinucleina
• synthetic biology
• synuclein
• yeast

In recent years, intracellular antibodies (intrabodies) have been widely exploited as molecular tools both for basic research and potentially towards therapeutic applications. The general idea is to select target-specific recombinant antibodies, typically in the format of single chain variable fragments (scFvs) or nanobodies (Nb), stable in the intracellular reducing environment, and engineer them to interfere with the physiological or pathological activity of the target protein.
Particularly, intrabodies are emerging as useful tools for knock-down/knock-out strategies at the protein level in order to manipulate the proteome of a cell with high rapidity, flexibility and specificity, without acting on genes or mRNAs (such as CRISPR, RNAi etc.). Moreover, since proteins inside the cells can exist in very different conformations and versions due the presence of post-translational modifications (PTM) and each form can play a very different role inside the cell, sometimes it is desirable to have a tool to eliminate just a particular conformation or PTM version of a given protein.
In this scenario, the current screening strategies allow the selection of target-specific intrabodies, but one cannot be sure a priori to have selected intrinsically neutralizing antibodies. In this sense, the development of intrabodies able to guide the degradation of their target protein would be the solution as shown by the Suicide Intrabody Technology (SIT). However, SIT do not offer a sufficient orthogonal tool for these purposes, due to the fact that here the degradation of the target, through the ubiquitin-dependent pathway, works upon administration of the TNFα, a chemical inductor with different functions inside the cell.
The goal of this master thesis was the development of a light-dependent intrabody-based tool (optobodies) for a rapid, reversible, selective and orthogonal regulation of the proteostasis inside a cell, with high temporal and, potentially, spatial resolution. The idea of the project was to establish a proof-of-concept about the possibility to control the lifetime of potentially every protein simply through a light stimulus, avoiding non-specific effects.
For these purposes, a fusion protein was created, in which an intrabody was bound to a photoinducible degron module, composed by a degron unit and a light-sensitive domain, called LOV domain: this last unit masks the degron, that can be activated through a conformational switch induced by light.
The tool was tasted and validated in yeast, as a robust and simple eukaryotic cellular model, optimal for such a type of proof-of-principle experiments. The effectiveness of the intrabody-LOV domain construct to control the light-induced selective target protein degradation was evaluated for three different intrabodies previously selected: the single-chain intrabody scFv58F, selected against an acetylated lysine on the histone H3 (H3K9Ac), and two nanobodies NbSyn2 and NbSyn87, able to target the monomeric form of the protein α-synuclein.
The optimal illumination conditions were set up and the efficiency of the system was evaluated by western blots, to quantify the degradation of the targets, and trying to induct visible phenotypes in living yeast cells.
The data obtained clearly show that light-induced protein degradation was achieved for two of the three intrabodies tested in this proof-of-concept.