• PISA
• photostimulation
• photo-induced degradation
• orthogonal
• optogenetics
• optogenetica
• nanobody
• molecular biology
• LOV
• light
• LED
• intrabody
• gephyrin
• fotostimolazione
• degradazione proteica
• degradation
• biologia sintetica
• biologia molecolare
• AtLOV2
• AsLOV2
• anticorpo intracellulare
• anticorpo
• antibody
• AcH3K9
• protein degradation
• scFv
• selective protein degradation
• single-chain fragment variable
• SIT
• synthetic biology
• synuclein
• tau

The biological research based on selective gene knock-out or knock-down at transcriptomic level has been useful for studies of protein functions. Instead, the functional interference at proteomic level can be useful for targeting and dissecting specific protein properties, such as post-translational modifications as well as conformations, with a rapid kinetic of the process.
Intracellular antibodies (intrabodies) represent a well-established molecular tool for directly interfering and inactivating a protein, without manipulation on genes or mRNAs (such as CRISPR, knock-out, RNAi …).
Indeed, the study of the physiological function of a cellular protein often requires the evaluation of the consequences of its removal or inactivation. Intrabodies can inactivate the target protein through the antibody intrinsic properties of protein function neutralisation, which are not selectable a priori, through a retargeting strategy towards different cell compartments or by exploiting the ubiquitin-proteasome pathway with the suicide intrabody technology (SIT). SIT is based on the ligand-inducible and ubiquitin-dependent degradation of the intrabodies with the resulting redirection of the target protein to the proteolytic machinery, which selectively degrades it. Even though SIT represents a potentially generalizable approach for any protein of interest, it requires TNFα as ligand for the induction of the degradation, thus being not orthogonal to the cellular machineries. Furthermore, the ligand-based inducible mechanisms do not allow a spatial focus on subcellular compartments.
In recent years, different proteins that change conformation upon a light stimulus have been widely exploited as optogenetic tools for molecular and cell biology. In particular, some LOV domains, which are both the light-sensitive and the effector core of photosensitive proteins, have been recently engineered to obtain photoinducible degron modules.
The induction of such modules is based on light, an orthogonal signal with respect to the cellular machineries, which allows in principle a high spatio-temporal resolution, since the stimulus is quickly switchable between the active and inactive state, and easily focusable on subcellular regions. Moreover, these modules exploit ubiquitin-independent mechanisms of degradation without requiring exogenous cofactors.
This master’s thesis project is aimed at exploring the feasibility and setting the conditions for a proof of concept study, combining the features of intrabodies and optogenetic degron domains. To this aim, fusion constructs between intrabodies and LOV-based degron modules will be developed, tested and validated in mammalian cell lines and yeast and the experimental equipment for this new molecular tool will be set up, in order to assess the effectiveness of the intrabody-LOV domain fusions for selective target protein degradation. Intrabodies targeting neurobiologically relevant proteins such as tau, gephyrin, synuclein and the post-translationally modified site of acetylated histone H3, will be used in the format of single chain variable fragments (scFv) or nanobodies (nb). The experimental setup and protocol for the illumination of cells will be designed and developed, along with determining the optimal cell transfection protocol.