Drug-regulable protease-based modules [electronic resource] : a novel strategy for interrogating newly synthesized pools of synaptic proteins and controlling biotherapeutic tools
Keywords: Biology.

The development of novel drug-regulable tools that allow manipulation or control of target proteins has the potential to meet many unmet needs in basic and applied science. In 2008, with the TimeSTAMP technique, a new class of genetically encoded, drug-regulable protein tags was introduced. This original TimeSTAMP tag allowed for drug-dependent epitope labeling of a protein of interest, allowing the researcher to selectively visualize only the protein copies synthesized within a drug-specified time window. One novel feature of this new class of drug-regulable module was its property of self-cleavage: in addition to a distal epitope tag, TimeSTAMP contained the hepatitis C virus (HCV) NS3/4A protease, and was covalently linked to the target protein via a protease substrate. Thus, the tag underwent autocleavage and removed itself by default. A second novel feature of the system was that it relied on a clinically approved class of small molecule drugs (HCV NS3/4A serine protease inhibitors) to enforce preservation of the tag. Since the tag is only retained on new protein copies synthesized in the presence of drug, this allows selective tagging of the newly synthesized pool of the protein of interest. The ability of drug-regulable protease-based tags to selectively interrogate only the newly synthesized pool of a protein of interest makes it an especially attractive technique for probing the role of de novo protein synthesis in biological processes known to rely on carefully choreographed new protein expression -- for instance, synaptic plasticity. While de novo activity-dependent protein synthesis is known to be necessary for synaptic plasticity and memory consolidation, the question of which specific proteins' new synthesis is required for these processes has proven refractory to investigation, due to the lack of tools with which to selectively interrogate the newly synthesized protein pool for a given protein. Likewise, the properties of drug-regulable protease-based tags offer advantages for biotherapeutic applications. The fact that these protease-based modules can be controlled by clinically relevant small molecules makes them attractive for possible gene therapy applications using protein- or cell-based approaches. While interest in gene therapies, particularly those whose activity or persistence is regulable by small molecules as a safety feature, is currently experiencing a resurgence, the lack of clinically approved bioinert drugs with which to regulate them complicates translation to the clinic. This dissertation details efforts to expand the toolbox of drug-regulable genetically encoded protease-based modules, and leverage them towards answering outstanding questions in neuroscience, and also towards developing drug-activatable proteins relevant to gene therapy. By adapting the TimeSTAMP tag such that it contained a degradation-promoting sequence, a new protease-based tag, SMASh, was developed that can mediate drug-enforced shutoff of the further expression of a protein of interest. The SMASh tool was characterized and subsequently deployed to shut off the synaptic protein PSD95, which is known to be relevant to synaptic plasticity and memory. To apply this tool towards the study of endogenous PSD95, PSD95-SMASh knock-in mice were generated in order to test the hypothesis that de novo hippocampal PSD95 synthesis is necessary for normal expression of fear memory in mice and for normal expression of long-term potentiation (LTP) in hippocampal slices. While an effect on fear memory was not observed, new PSD95 shutoff in hippocampal slices attenuated the rate of passive LTP decay after LTP induction. TimeSTAMP knock-in mouse strains for PSD95 and the synaptic protein Arc were also developed as additional tools for interrogating the newly synthesized pools of key synaptic proteins. Finally, a generalizable NS3 protease-based module called StaPL was developed in order to exert drug control over therapeutically relevant proteins. An inserted StaPL module cleaves a protein into nonfunctional pieces, but application of NS3 protease inhibitor preserves linkage, and thus function. The NS3 protease was also mutagenized in order to produce two orthogonal StaPL modules that are able to be controlled independently by two different NS3 protease inhibitors. StaPL regulation was shown to be extensible to controlling zinc finger-based bidirectional transcriptional effectors, dCas9-based transcriptional effectors, and a caspase-9 suicide switch.