A short binding site in the KPC1 ubiquitin ligase mediates processing of NF-κB1 p105 to p50: A potential for a tumor-suppressive PROTAC

Abstract

Nuclear factor κB (NF-κB) is an important transcriptional regulator that is involved in numerous cellular processes, including cell proliferation, immune response, cell survival, and malignant transformation. It relies on the ubiquitin–proteasome system (UPS) for several of the steps in the concerted cascade of its activation. Previously, we showed that the ubiquitin (Ub) ligase KPC1 is involved in ubiquitination and limited proteasomal processing of the NF-κB1 p105 precursor to generate the p50 active subunit of the “canonical” heterodimeric transcription factor p50–p65. Overexpression of KPC1 with the generation of an excessive amount of p50 was shown to suppress tumors, an effect which is due to multiple mechanisms. Among them are suppression of expression of programmed cell death-ligand 1 (PD-L1), overexpression of a broad array of tumor suppressors, and secretion of cytokines which results in recruitment of suppressive immune cells into the tumor. Here, we show that the site of KPC1 to which p105 binds is exceptionally short and is made up of the seven amino acids WILVRLW. Attachment of this short stretch to a small residual part (∼20%) of the ligase that also contains the essential Really Interesting New Gene (RING)-finger domain was sufficient to bind p105, conjugate to it Ub, and suppress tumor growth in an animal model. Fusion of the seven amino acids to a Von Hippel–Lindau protein (pVHL)-binding ligand (which serves as a “universal” ligase for many proteolysis-targeting chimeras; PROTACs) resulted in a compound that stimulated conjugation of Ub to p105 in a cell-free system and its processing to p50 in cells and restricted cell growth.

The NF-ĸB p50 subunit generated by KPC1-mediated ubiquitination and limited proteasomal processing, suppresses tumor growth

“…To identify the p105-binding domain in KPC1, we deleted systematically stretches of amino acids in KPC1, covering the entire reading frame, and examined the interaction with p105 in a co-immunoprecipitation assay. We found that a short domain consisting of 7 amino acids—968-WILVRLW-974—mediates the interaction between two proteins, which is completely abrogated following its deletion [ 32 ]. Interestingly, a chimera protein consisting of the 7 amino acids attached to a short segment of KPC1 which contains the RING (Really Interesting New Gene) domain required for binding of the ubiquitin-conjugating enzyme, E2 (and therefore is essential for the ubiquitin-ligating activity of the protein), was able to interact with and to ubiquitinate p105.…”

“…Interestingly, a chimera protein consisting of the 7 amino acids attached to a short segment of KPC1 which contains the RING (Really Interesting New Gene) domain required for binding of the ubiquitin-conjugating enzyme, E2 (and therefore is essential for the ubiquitin-ligating activity of the protein), was able to interact with and to ubiquitinate p105. This activity of the mini-KPC1 was dependent on the presence of WILVRLW [ 32 ].…”

“…1 ). Similar to suppressing anchorage independence in cells, the tumor suppressive activity of KPC1 was also dependent on its RING finger domain and on the presence of the 7 amino acids stretch in the ligase to which p105 binds [ 29 , 32 ].

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“…The resulted molecule stimulated the ubiquitination of p105 by purified pVHL ubiquitin ligase complex in a cell-free system. In cells, the PROTAC enhanced processing of p105 to p50 and consequently restricted cell growth [ 32 ]. It is now important to monitor the effect of the PROTAC on tumor models.…”

From classic medicinal chemistry to state‐of‐the‐art interdisciplinary medicine: Recent advances in proteolysis‐targeting chimeras technology

“…14,15 Goldhirsh et al found that the WILVRLW sequence in KPC1 is the key to its interaction with p105 and the ubiquitination process. 17 They developed a PROTAC based on the WILVRLW peptide as the warhead, connecting the VHL E3 ligase with a polyethylene glycol (PEG) linker in an attempt to induce limited processing from p105 to p50. It was confirmed that the ubiquitination of p105 outside the cell and the increase of p50 intracellularly could be achieved.…”