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Fusobacterium nucleatum is an oral pathogen associated with periodontal disease, preterm births and the exacerbation of colorectal, esophageal, pancreatic, and breast cancers. F. nucleatum was previously found to inhibit killing of cancer cells by natural killer (NK) cells and tumor infiltrating T cells by inducing the NK cells killing-suppressing receptors TIGIT and CEACAM1. In this study, we show that the incubation of F. nucleatum with primary human natural killer (NK) cells causes the cleavage of the activating receptors CD16, NKp44 and NKp46 by fusolisin. Fusolisin is a fusobacterial outer-membrane, auto-transporter, serine protease and to date the only functional protease found in F. nucleatum. We previously showed that fusolisin is essential for fusobacterial growth in culture. High expression of a functional recombinant fusolisin in E. coli was enabled using codon optimization and replacement of the fusolisin's signal peptide with that of the E. coli OmpA. Recombinant fusolisin was found to cleave the same NK-activating receptors degraded by F. nucleatum. Cleavage of these activating receptors by fusolisin inhibited NK cells activity including killing of tumor cells in-vitro and in-vivo. Our results provide a new bacterial protease-dependent mechanism in which tumors colonized by F. nucleatum are protected from NK cells attack by exploiting fusolisin proteolytic activity. Importantly, our previous and current results suggest that fusolisin might serve to target for tumor-colonized fusobacteria. Citation Format: Amjad Shhadeh, Liat Dassa, Jamal Fahoum, Nicole Haj, Reuven Wiener, Ofer Mandelboim, Gilad Bachrach. Suppression of anti-tumor immunity by the Fusobacterium nucleatum protease fusolisin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5893.

Recent studies on the oral, anaerobic, gram-negative bacterium Fusobacterium nucleatum revealed its presence and involvement in colorectal, esophageal and breast cancer. We previously demonstrated that F. nucleatum binds and activates the human inhibitory receptors TIGIT and CEACAM1 leading to inhibition of T and NK cell anti-tumor immunity. CEACAM1 was found to be bound and activated by the fusobacterial trimeric autotransporter adhesin CbpF. Here we report the generation of a recombinant E. coli expressing full-length CbpF that efficiently binds and activates CEACAM1.

We describe a molecular characterization of the interaction between the cancer-related proteins WWOX and p73. This interaction is mediated by the first of two WW domains (WW1) of WWOX and a PPXY-motif-containing region in p73. While phosphorylation of Tyr33 of WWOX and association with p73 are known to affect apoptotic activity, the quantitative effect of phosphorylation on this specific interaction is determined here for the first time. Using ITC and fluorescence anisotropy, we measured the binding affinity between WWOX domains and a p73 derived peptide, and showed that this interaction is regulated by Tyr phosphorylation of WW1. Chemical synthesis of the phosphorylated domains of WWOX revealed that the binding affinity of WWOX to p73 is decreased when WWOX is phosphorylated. This result suggests a fine-tuning of binding affinity in a differential, ligand-specific manner: the decrease in binding affinity of WWOX to p73 can free both partners to form new interactions.

Ufmylation is a post-translational modification essential for regulating key cellular processes. A three-enzyme cascade involving E1, E2 and E3 is required for UFM1 attachment to target proteins. How UBA5 (E1) and UFC1 (E2) cooperatively activate and transfer UFM1 is still unclear. Here, we present the crystal structure of UFC1 bound to the C-terminus of UBA5, revealing how UBA5 interacts with UFC1 via a short linear sequence, not observed in other E1-E2 complexes. We find that UBA5 has a region outside the adenylation domain that is dispensable for UFC1 binding but critical for UFM1 transfer. This region moves next to UFC1’s active site Cys and compensates for a missing loop in UFC1, which exists in other E2s and is needed for the transfer. Overall, our findings advance the understanding of UFM1’s conjugation machinery and may serve as a basis for the development of ufmylation inhibitors., Ufmylation is a well-established ubiquitin-like protein modification, but its mechanism is largely unclear. Here, the authors present a crystal structure of the ufmylation-specific E1-E2 complex, revealing differences to the ubiquitination machinery and mechanistic details of the ufmylation process.