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While the degradation of disease-causing proteins [1] is now possible thanks to targeted protein degradation (TPD) technologies such as PROTACs [2], new targeted protein stabilization (TPS) technologies called DUBTACs [3] have been designed to stabilize and restore proteins that may promote cell proliferation when aberrantly degraded.
Tumor suppressing proteins are proteins which, as their name suggests, eliminate cancer cells by influencing their cell cycle or promoting their apoptosis, that is, their death. However, in many cancers, some tumor suppressing proteins are actively ubiquitinated and degraded. Their degradation by the ubiquitin-proteasome system (UPS) [4] allows cancer cells to proliferate and create a microenvironment conducive to tumor growth and spread.
The p53 protein is a well-known example of a tumor suppressor protein. In most human cancers the TP53 gene is mutated, or changes in its regulatory pathway alter p53 activity. Both of these can have pro-cancerous effects.As a DNA-binding transcription factor, p53 binds to DNA under cellular stress or DNA damage. This activates the transcription of genes involved in cell cycle arrest and promotes the arrest of cell growth. In this way, p53, as ‘guardian of the genome’ [6], plays a role in stopping tumorigenesis (that is the formation of cancer).
However, cancer cells have found ways of escaping this type of regulation. This includes reprogramming the degradation system to excessively and rapidly degrade such regulatory proteins so that the cells can proliferate uncontrollably.
Under regular conditions, the mechanism involved in the degradation of a protein requires two elements: poly-ubiquitination of the target protein and degradation by the proteasome. Ubiquitination involves the addition of ubiquitin to the target protein by an E3 ligase, and this addition can be iterated to obtain poly-ubiquitination on the same protein.. Ubiquitination and polyubiquitination act as degradation signals. Once poly-ubiquitinated, the protein is ready to be degraded by the proteasome, hence the term ubiquitin-proteasome system. The proteasome can be seen as a factory that cuts the protein into small pieces called peptides.
In parallel with this degradation process, there are proteins that cleave the ubiquitin chain to prevent degradation by the proteasome, making the ubiquitination process reversible. These proteins are called DUBs, [7] for deubiquitylating enzymes or deubiquitinases. DUBS are of particular interest to researchers in Prof Daniel K Nomura’s laboratory [8], as they can be used to stabilize proteins that are aberrantly degraded. As mentioned above, these proteins can be tumor suppressing proteins, but also mutated and/or misfolded proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR) membrane protein. One of the most common mutations in CFTR leads to abnormal degradation [9], resulting in cystic fibrosis [10]. This disease is a rare genetic disorder known to affect the lungs and other organs.
The researcher’s idea was to recruit these DUBs specifically to proteins carrying a (poly-)ubiquitination signal. To do this, they used an existing technology that is currently being clinically tested [11]: PROTACs. PROTACs are molecules consisting of two linked ligands with distinct functions. One part is an E3 ligase ligand that recruits the E3 ligase responsible for protein ubiquitination and the other part is a protein-targeting ligand that recruits the protein to be degraded. So, by recruiting the E3 ligase and the protein to be degraded into close proximity, the system can induce the specific degradation of a protein.
DUBTAC technology is also based on a similar technology, with two linked ligands. These are a non-inhibitory DUB ligand and a protein-targeting ligand. To demonstrate that DUBTACs ultimately stabilize proteins and restore their protein level, two proteins were tested for proof-of-concept: mutated ΔF508-CFTR protein and tumor suppressor kinase WEE1 protein.
These two proteins were good candidates for proof-of-concept because both undergo ubiquitin-dependent degradation. ΔF508-CFTRdegradation is responsible for an imbalance in chloride ion traffic in epithelial cells, leading to deregulation of mucus production.WEE1 is a tumor suppressing protein downregulated in cancer tissues [13].
The crucial element in DUBTAC construction is the non-inhibitory DUB ligand. This is a ligand that binds to the DUB enzyme without inhibiting it, so as not to lose the enzymatic capacity of DUB, which involves the cleavage of ubiquitin linkages on a target protein.
By employing DUBTACs targeting the mutated ΔF508-CFTR and WEE1, the expression level of these proteins stabilized.n addition, for CFTR, there was a restoration of its transepithelial chloride conductance function [3] in bronchial epithelial cells—that is, its capacity to regulate chloride ions passage through the membrane of the epithelial cells.
Finally, this proof of concept for DUBTACs technology by targeting two proteins involved in different diseases demonstrates that DUBTACs have a promising therapeutic future. This includes the regulation of tumorigenesis in many cancers. DUBTACs could therefore be used to restore aberrantly degraded proteins in cancers, such as p53. This can restore tumor suppressor protein activity to suppress or even reverse tumor progression.
Together with PROTACs, they constitute a whole new arsenal of drugs that modulate specific proteins.
Edited by Mia Hubert
Works Discussed
[1] Lu, Jing et al. “Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently Target BRD4.” Chemistry & biology vol. 22,6 (2015): 755-63. doi:10.1016/j.chembiol.2015.05.009
[2]Sakamoto, K M et al. “Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation.” Proceedings of the National Academy of Sciences of the United States of America vol. 98,15 (2001): 8554-9. doi:10.1073/pnas.141230798
[3]Henning, Nathaniel J et al. “Deubiquitinase-targeting chimeras for targeted protein stabilization.” Nature chemical biology vol. 18,4 (2022): 412-421. doi:10.1038/s41589-022-00971-2
[4]Sakamoto, K. Protacs for Treatment of Cancer. Pediatr Res 67, 505–508 (2010). https://doi.org/10.1203/PDR.0b013e3181d35017
[5]Vogelstein, B., Lane, D. & Levine, A. Surfing the p53 network. Nature 408, 307–310 (2000). https://doi.org/10.1038/35042675
[6]Lane, D. p53, guardian of the genome. Nature 358, 15–16 (1992). https://doi.org/10.1038/358015a0
[7]Komander, David et al. “Breaking the chains: structure and function of the deubiquitinases.” Nature reviews. Molecular cell biology vol. 10,8 (2009): 550-63. doi:10.1038/nrm2731
[8] Henning, N.J., Boike, L., Spradlin, J.N. et al. Deubiquitinase-targeting chimeras for targeted protein stabilization. Nat Chem Biol 18, 412–421 (2022). https://doi.org/10.1038/s41589-022-00971-2
[9]Ward, C L et al. “Degradation of CFTR by the ubiquitin-proteasome pathway.” Cell vol. 83,1 (1995): 121-7. doi:10.1016/0092-8674(95)90240-6
[10]Riordan, John R. “CFTR function and prospects for therapy.” Annual review of biochemistry vol. 77 (2008): 701-26. doi:10.1146/annurev.biochem.75.103004.142532
[11] ‘OUR SCIENCE’, 2023 Arvinas, https://www.arvinas.com/our-science/
[12]Békés, M., Langley, D.R. & Crews, C.M. PROTAC targeted protein degraders: the past is prologue. Nat Rev Drug Discov 21, 181–200 (2022). https://doi.org/10.1038/s41573-021-00371-6
[13]Esposito, Francesca et al. “Wee1 Kinase: A Potential Target to Overcome Tumor Resistance to Therapy.” International journal of molecular sciences vol. 22,19 10689. 1 Oct. 2021, doi:10.3390/ijms221910689
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