**10. Alternative or Synergistic Approaches for Treating Diseases Caused by Destabilizing Missense Mutations**

### *10.1. Screening Specific DUBs for PAH and FAH Proteins*

The protein misfolding and mis-assembly in PKU and HT1 cause rapid protein degradation, which it is important to preempt. The UPS is one major pathway for intracellular protein degradation. DUBs play a central role in ubiquitin signaling and protein homeostasis. The development of DUB inhibitors has been showcased as a promising strategy for treating cancers and other diseases by enhancing the degradation of DUB substrates. No one has reported on the role of DUBs in misfolding diseases, in which treatments need to stabilize and increase the concentration of mutated proteins. DUBs can recognize di fferent chain linkages that are specific for particular functions, such as the K6, K11, and K48 chains for proteasomal degradation and K63 chains for lysosomal targeting, DNA repair, and NF-κB activation. Thus, DUBs are highly specific in their action [106].

Any misfolded protein is tagged with mono-/poly-ubiquitination chains that mark them for proteasomal degradation. Those degradation tags can be recognized and removed by DUBs, thus aborting the protein degradation cycle. Therefore, we hypothesize that DUBs could play a decisive role in rescuing functional but misfolded PAH and FAH proteins from degradation. Instead of DUB inhibitors, drugs to enhance the function of DUBs that specifically regulate PAH and FAH protein degradation need to be identified. Various DUBs might be suitable for interacting with PAH and FAH proteins to dissociate ubiquitin molecules and maintain the relatively small amount of functional protein needed in the correct subcellular destination to prevent disease phenotypes.

Ernst et al. proposed a highly active viral DUB derived from the protease domain of Epstein-Barr virus, the BPLF1 gene called EBV-DUB, which removed ubiquitin chains and stabilized misfolded proteins in 293T cells. EBV-DUB was less toxic to cells than proteasome inhibitors, because no ubiquitylated proteins accumulated. Because the ubiquitylated proteins did not accumulate, the pool of free Ub was also maintained in the cells. Proteasomal inhibitors reduce protein synthesis with even a short exposure, whereas EBV-DUB did not immediately block the translation, again giving it an advantage over proteasomal inhibitors [132]. Inhibiting proteasomal degradation can derange many other cellular processes. Of all the attempts to stabilize the PAH and FAH mutant proteins, this approach should be an important focus of research and surveillance.

### *10.2. Modified PROTAC Technology to Use DUBs to Stabilize Partially Functional PAH and FAH Proteins*

Small-molecule induced protein degradation using proteolysis-targeting chimeras (PROTACs) is an emerging technology targeting a broad range of proteins. This technology is based on event-driven pharmacology in the sense that it degrades a target protein as soon as the drug transiently binds to the target. After binding and degrading a protein, the PROTACs can again serve their function for multiple rounds of activity. PROTACs are bifunctional molecules that bind to the protein of interest with one end and to an E3 ligase with the other. The bound E3 ligase then attracts the E2 enzyme to transfer Ub to the protein of interest, targeting it for proteasomal degradation (Figure 2). Thus, this technology uses proximity-induced ubiquitination and degradation and has minimal off-target effects at a low concentration. PROTACs have already proved successful in an acute myeloid leukemia xenograft model and a disseminated lymphoma mouse model [133,134]. Therefore, they might also be used to remove null mutant PAH and FAH proteins from cells.

The recent success of small-molecule PROTACs has opened the door to a wide range of applications. This technique to induce protein degradation might also rescue proteins from degradation with some modifications. One end of the PROTAC could be designed to bind to the protein of interest, while the other, instead of binding to the E3 ligase, could be designed to bind to specific DUBs to regulate the target protein. Binding to the PROTAC would thus bring a ubiquitin-tagged misfolded protein and its DUB into close proximity, allowing the DUB to recognize and remove the ubiquitin tag and aborting the degradation (Figure 2). DUBs are usually high-molecular-weight proteins, which might give the modified PROTAC less permeability. Therefore, small molecular ligands that can recruit a specific DUB could be used instead. Due to their high specificity and low toxicity, PROTACs can be modified into selective rescuers of misfolded proteins that would otherwise be rapidly degraded. Therefore, efforts should focus on identifying a specific DUB candidate or small molecule ligand to attract DUBs that regulate PAH and FAH proteins and designing a PROTAC suitable for freezing the degradation process.
