Delyea, C. | Search bioRxiv

bioRxiv2021

NOD1 is super-activated through spatially-selective ubiquitination by the Salmonella effector SspH2

As part of its pathogenesis, Salmonella enterica serovar Typhimurium delivers effector proteins into host cells. One effector is SspH2, a member of the novel E3 ubiquitin ligase family, interacts with, and enhances, NOD1 pro-inflammatory signaling, though the underlying mechanisms are unclear. Here, we report the novel discovery that SspH2 interacts with multiple members of the NLRC family to enhance pro-inflammatory signaling that results from targeted ubiquitination. We show that SspH2 modulates host innate immunity by interacting with both NOD1 and NOD2 in mammalian epithelial cell culture. We also show that SspH2 specifically interacts with the NBD and LRR domains of NOD1 and super-activates NOD1- and NOD2-mediated cytokine secretion via the NF-{kappa}B pathway. Mass spectrometry analyses identified lysine residues in NOD1 that were ubiquitinated after interaction with SspH2. Through NOD1 mutational analyses, we identified four key lysine residues that are required for NOD1 super-activation by SspH2, but not its basal activity. These critical lysine residues are positioned in the same region of NOD1 and define a surface on NOD1 that is targeted by SspH2. Overall, this work provides evidence for post-translational modification of NOD1 by ubiquitin, and uncovers a unique mechanism of spatially-selective ubiquitination to enhance the activation of an archetypal NLR. SYNOPSISSspH2 is an E3 ubiquitin ligase injected by Salmonella Typhimurium into host cells that induces pro-inflammatory signaling. The immune receptor, NOD1, is ubiquitinated in the presence of SspH2, resulting in increased pro-inflammatory cytokine secretion. O_LISspH2 super-activates NOD1 and NOD2 to increase pro-inflammatory cytokine secretion, in part, through the NF-{kappa}B pathway C_LIO_LIUbiquitin modification of NOD1 were identified by mass spectrometry C_LIO_LIA specific region of NOD1 is targeted by SspH2 to enhance NOD1 activity. C_LI

microbiology↗

bioRxiv2020

Toll-like receptor 4 is activated by platinum and contributes to cisplatin-induced ototoxicity

Toll-like receptor 4 (TLR4) is famous for recognizing the bacterial endotoxin lipopolysaccharide (LPS) as its canonical ligand. TLR4 is also activated by other classes of agonist including some Group 9/10 transition metals. Roles for these non-canonical ligands in pathobiology mostly remain obscure, though TLR4 interactions with metals can mediate immune hypersensitivity reactions. In this work, we tested whether TLR4 can be activated by the Group 10 transition metal, platinum. We demonstrated that in the presence of TLR4, platinum activates pathways downstream of TLR4 to a similar extent as the known TLR4 agonists LPS and nickel. Platinum is the active moiety in cisplatin, a very potent and invaluable chemotherapeutic used to treat solid tumors in childhood cancer patients. Unfortunately, cisplatin use is limited due to an adverse effect of permanent hearing loss (cisplatin-induced ototoxicity, CIO). Herein, we demonstrated that cisplatin also activates TLR4, prompting the hypothesis that TLR4 mediates aspects of CIO. Cisplatin activation of TLR4 was independent of the TLR4 co-receptors CD14 and MD-2, which is consistent with TLR4 signaling elicited by transition metals. We found that TLR4 is required for cisplatin-induced inflammatory, oxidative and apoptotic responses in an ear outer hair cell line and for hair cell damage in vivo. Thus, TLR4 is a promising therapeutic target to mitigate CIO. We additionally identify a TLR4 small molecule inhibitor able to curtail cisplatin toxicity in vitro. Further work is warranted towards inhibiting TLR4 as a route to mitigating this adverse outcome of childhood cancer treatment.Significance Statement This work identifies platinum, and its derivative cisplatin, as new agonists for TLR4. TLR4 contributes to cisplatin-induced hair cell death in vitro and in vivo. Genetic and small molecule inhibition of TLR4 identify this receptor as a druggable therapeutic target with promise to curtail cisplatin-induced ototoxicity, a devastating side-effect of an otherwise invaluable chemotherapeutic tool.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗