**3. Discussion**

Up to now, the role of non-degradative monoubiquitination in human disease has been relatively understudied, as focus was mostly falling on the role of polyubiquitination in proteasomal degradation. Recent emerging evidences have highlighted the key function of monoubiquitination in a wide range of cellular processes. The findings listed here only represent the most characterized enzymes controlling monoubiquitination. Nonetheless, it reflects the high prevalence of alterations of the monoubiquitin pathway in such a broad array of genetic disorders. This suggests that disruption of the monoubiquitin pathway may be a major force driving the pathogenic phenotypes of such diseases.

The abundance of the ubiquitin-related enzymes mutated in genetic disorders indicates that targeting the ubiquitin pathway might have a utility for a range of genetic diseases. However, at present, we lack detailed knowledge on how monoubiquitin signals are generated and how they are decoded by the cell. This is challenged by the diversity and complexity of the ubiquitin pathway. Moreover, monoubiquitinated proteins might not have been accurately identified, because polyubiquitinated conjugates are recognized more e fficiently by anti-ubiquitin specific antibodies. This leads to underestimation of the pool of monoubiquitinated proteins present in the cell and challenges their characterization. The development of novel tools to purify monoubiquitinated proteins using high-a ffinity ubiquitin-binding domains and synthetic biology approaches to e fficiently generate monoubiquitinated proteins overcoming these issues.

It is also worth noting that when looking at the few drugs that were developed to target the ubiquitin pathway, most are meant to inhibit its functioning. Several inhibitors targeting the ubiquitinating enzymes described in this review have been reported. Ubiquitin variants that block the E2-ubiquitin binding surface of the RING domain of CBL were shown to specifically inhibit the activity of phosphorylated CBL [120,121]. A high-throughput screening to identify ITCH inhibitors discovered that clomipramine, a common antidepressant drug, blocks ITCH autoubiquitination and a ffects the ability of ITCH to ubiquitinate its substrates [122]. Screening for the inhibitors of UBE2T/FANCL identified two compounds that sensitize cells to DNA crosslinking [123]. Pharmacological inhibition of USP8 was shown to e ffectively suppress ACTH synthesis in vitro without causing any significant cytotoxicity, indicating its potential for the managemen<sup>t</sup> of ACTH hypersecretion in Cushing's disease [124]. However, considering that the disease-associated alterations of the ubiquitin ligases and DUBs are mostly loss of function, inhibitors targeting these enzymes would not be beneficial. This indicates that there is a need to develop novel strategies for targeted therapies of genetic diseases [125]. Several screens identified compounds activating PARKIN ubiquitin ligase activity [126] and enhancing mitophagy [127], such as the compound described in patent WO2018023029. While no in vivo validation is available for this compound yet, this demonstrates the feasibility of identification of E3 ligase activators, opening novel therapeutic options for patients with genetic disorders.

This review collected multiple evidences that monoubiquitination is a highly relevant process in the pathogenesis of a wide range of genetic diseases; however, further research is necessary to identify specific entry points for therapeutic intervention of monoubiquitination-dependent signaling pathways.

**Funding:** This work was supported by H2020 European Research Council (Ub-RASDisease); Research Foundation Flanders (FWO) postdoctoral fellowship (MFB).

**Conflicts of Interest:** The authors declare no conflict of interest.
