Ubiquitination is a reversible post-translational modification process during which the highly conserved 76-aminoacid protein ubiquitin is conjugated to target proteins. Ubiquitin can be conjugated to a protein substrate via distinct mechanisms. Monoubiquitination is the attachment of a single ubiquitin molecule to a single lysine residue on a substrate protein, whereas multi-monoubiquitination is the conjugation of a single ubiquitin molecule to multiple lysine residues. Polyubiquitination occurs when ubiquitin molecules are attached end-to-end to a lysine residue on a substrate protein to form a poly-ubiquitin chain. In this case, ubiquitin molecules are conjugated through one of the seven lysine residues present on the ubiquitin itself (K6, K11, K27, K29, K33, K48, and 63) or the N-terminal methionine (M1). While most of the studies have described the role of specific polyubiquitination, such as K48-linked polyubiquitination for proteasomal degradation [1,2] or K63-linked polyubiquitination for vesicular trafficking [3], emerging evidences implicate monoubiquitination and multi-monoubiquitination in controlling numerous aspects of protein function, such as degradation, subcellular localization, and protein–protein interaction. In this review, we focus on the role of monoubiquitin conjugation in normal physiology and genetic disease.

Comparing to the role of polyubiquitination in proteasomal degradation, the function of non-degradative monoubiquitination in human disease has been relatively understudied so far. 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 be of therapeutic use for a range of genetic diseases. However, at present, we lack a 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 efficiently by anti-ubiquitin specific antibodies. This leads to the 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-affinity ubiquitin-binding domains and synthetic biology approaches to efficiently generate monoubiquitinated proteins can overcome 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 only 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 CBL [120,121]. A high-throughput screening to identify ITCH inhibitors discovered that clomipramine, a common antidepressant drug, blocks ITCH autoubiquitination and affects 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 effectively suppress ACTH synthesis in vitro without causing any significant cytotoxicity, indicating its potential for the management of ACTH hypersecretion in Cushing’s disease [124]. However, considering that the disease-associated alterations of the ubiquitin ligases and hydrolases 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.

The analysis of the alterations of the ubiquitin system associated to genetic diseases generated evidence that monoubiquitination is a key process underlying the development of such diseases; highlighting the need for further research to identify new monoubiquitination-dependent signaling pathways as novel targets suitable for therapeutic approach of genetic diseases.

This entry is adapted from the peer-reviewed paper 10.3390/ijms21093036