Optobody: an intrabody-based optogenetic tool for inducible and reversible targeted protein degradation

Protein homeostasis, or proteostasis, refers to the network of cellular processes that maintains the proper balance of protein concentration, localization, conformation, and interactions within the cell. Many human diseases display proteostasis alterations, hence regulating proteostasis is a crucial objective for target validation and drug discovery. While regulation of protein synthesis and folding is essential for maintaining appropriate protein levels, another critical component of proteostasis is proteolysis, the controlled degradation of proteins. Thus, removing aged, misfolded, mislocalized, or aberrant proteins is often necessary to preserve cellular function. In this context, targeted protein degradation has emerged as a groundbreaking strategy in drug discovery, enabling direct intervention at the protein level to modulate the activity of specific proteins. To date, the most common targeted protein degradation (TPD) strategy employs proteolysis targeting chimeras, which are hybrid molecules, usually of chemical synthesis, also called heterobifunctional, for their capacity of binding the target protein and connecting it to the ubiquitin-dependent degradative pathway. The bifunctional nature of such tools remains the bottleneck in the discovery processes, because the target binding moiety of the heterobifunctional degrader requires an ad hoc chemical development process for each target. To overcome the issue of the chemical nature of the target binding moiety capacity, a genetically encoded target-binding moiety would be desirable, being more general. To this aim, we harnessed the broad diversity of antibody binding repertoires and exploited the Intracellular Antibody Capture Technology (IACT) selection platform, to select antigen binders directly from the target cDNA sequence. The selected antibody domains are then equipped with a degradation signal capable of redirecting to the degradative pathway the intracellular target protein. Beyond developing a new genetically encoded heterobifunctional tool based on intracellular antibodies (the primary objective), my second goal was to create a stimulusactivated targeted degradation system. To achieve this, I fused the intrabody domain with a Light-Oxygen-Voltage (LOV) domain and incorporated an ornithine decarboxylase (ODC) degron sequence, enabling conditional engagement of the degradative pathway. [...] Indeed, as for the TDP-43 and phospho-Tau protein the intrabody-based optogenetic tools can become tools to investigate the misfolding and the aggregation process in cellular models for ALS, FTLS and AD respectively, the anti-Arc-optobody can pave a way for investigating, with a dendritic subcellular resolution, the mechanism of the synaptic plasticity subserving behind the memory processes.