The receptor interacting serine/threonine kinase 2 (RIPK2) is essential for linking activation of the pattern recognition receptors NOD1 and NOD2 to cellular signaling events. Recently, it was shown that RIPK2 can form higher order molecular structures in vitro. Here, we demonstrate that RIPK2 forms detergent insoluble complexes in the cytosol of host cells upon infection with invasive enteropathogenic bacteria. Formation of these structures occurred after NF-κB activation and depended on the caspase activation and recruitment domain of NOD1 or NOD2. Complex formation upon activation required RIPK2 autophosphorylation at Y474 and was influenced by phosphorylation at S176. We found that the E3 ligase X-linked inhibitor of apoptosis (XIAP) counteracts complex formation of RIPK2, accordingly mutation of the XIAP ubiquitylation sites in RIPK2 enhanced complex formation. Taken together, our work reveals novel roles of XIAP in the regulation of RIPK2 and expands our knowledge on the function of RIPK2 posttranslational modifications in NOD1/2 signaling.

Background Invasive mechanical ventilation (IMV) is a standard therapy for intensive care patients with respiratory failure. With increasing population age and multimorbidity, the number of patients who cannot be weaned from IMV increases, resulting in impaired quality of life and high costs. In addition, human resources are tied up in the care of these patients. Methods The PRiVENT intervention is a prospective, mixed-methods interventional, multicentre study with a parallel comparison group selected from insurance claims data of the health insurer Allgemeine Ortskrankenkasse Baden-Württemberg (AOK-BW) conducted in Baden-Württemberg, Germany, over 24 months. Four weaning centres supervise 40 intensive care units (ICUs), that are responsible for patient recruitment. The primary outcome, successful weaning from IMV, will be evaluated using a mixed logistic regression model. Secondary outcomes will be evaluated using mixed regression models. Discussion The overall objective of the PRiVENT project is the evaluation of strategies to prevent long-term IMV. Additional objectives aim to improve weaning expertise in and cooperation with the adjacent Intensive Care Units. Trial registration This study is registered at ClinicalTrials.gov (NCT05260853).

The receptor interacting serine/threonine kinase 2 (RIPK2) is essential for linking activation of the pattern recognition receptors NOD1 and NOD2 to cellular signaling events.Recently, it was shown that RIPK2 forms higher order molecular structures in vitro, which were proposed to activate signaling. Here, we demonstrate that RIPK2 forms detergent insoluble complexes in the cytosol of host cells upon infection with invasive enteropathogenic bacteria. Formation of these structures occurred after NF-κB activation and depends on the CARD of NOD1 or NOD2. Complex formation upon activation was dependent on RIPK2 autophosphorylation at Y474 and influenced by phosphorylation at S176. Inhibition of activity of the cIAP protein XIAP induced spontaneous complex formation of RIPK2 but blocked NOD1-dependet NF-κB activation. Using immunoprecipitation, we identified 14-3-3 proteins as novel binding partners of non-activated RIPK2, whereas complexed RIPK2 was bound by the prohibitin proteins Erlin-1 and Erlin-2.Taken together, our work reveals novel roles of XIAP, 14-3-3 and Erlin proteins in the regulation of RIPK2 and expands our knowledge on the function of RIPK2 posttranslational modifications in NOD1/2 signaling. AUTHOR CONTRIBUTIONSCA, KE, SB and IK performed the experiments.JP performed MS analysis and data processing.TAK conceived and supervised the study.CA, KE and TAK wrote and edited the manuscript. CONFLICT OF INTERESTThe authors declare that they have no conflict of interest.A: Indirect immunofluorescence micrographs of HeLa EGFP-RIPK2 cells infected for 2 h with S. flexneri M90T. Staining for XIAP, EGFP-RIPK2, and merge with DNA-staining is shown.Co-localization is shown at higher magnification in the inlay. Scale bar = 10 µm. B: Fluorescence micrographs of HeLa EGFP-RIPK2 cells, treated for 48 h with a XIAP siRNA or a non-targeting siRNA, following infection with S. flexneri M90T for 2 h or left uninfected. Cells were treated with 1 µg/ml doxycycline for 24 h prior infection. EGFP-RIPK2 and merge with DNA-staining is shown. Scale bar = 10 µm. C: IL-8 release in the supernatants from HeLa EGFP-RIPK2 cells, treated for 48 h with a XIAP siRNA or a non-targeting siRNA, following infection with S. flexneri M90T for 6 h. Mean of n=2 with S.D. is shown. D: Immunoblot analysis of HeLa EGFP-RIPK2 cells infected with S. flexneri M90T or BS176 for the indicated time. Immunoblot was probed with anti-RIPK2 antibody, anti-XIAP, and anti-β-tubulin as loading control. E: Left panel: Fluorescence micrographs of HeLa EGFP-RIPK2 cells, transfected with 500 ng Ubi-SMAC-fl-pDS-RED, Ubi-SMAC-tr-pDS-RED, or Ubi-SMAC-AVPI-pDS-RED plasmid and treated with 1 µg/ml doxycycline for 24 h. EGFP-RIPK2, SMAC-dsRed, and merge with DNA-staining is shown. Scale bar = 10 µm. Right panel: Immunoblot analysis of cells prepared as in the left panel. Protein levels were detected using an anti-RIPK2 antibody and anti-β-tubulin as loading control.Figure 6: Phosphorylation of RIPK2 at S176 and Y474 contribute to RIPosome formation. A: Fluorescence microg...

The receptor interacting serine/threonine kinase 2 (RIPK2) is essential for signal transduction induced by the pattern-recognition receptors NOD1 and NOD2. Upon NOD1/2 activation RIPK2 forms complexes in the cytoplasm of human cells. Here, we identified the molecular composition of these complexes. Infection with Shigella flexneri to activate NOD1-RIPK2 revealed that RIPK2 formed dynamic interactions with several cellular proteins, including A20, Erlin-1, Erlin-2 and 14-3-3. Whereas interaction of RIPK2 with 14-3-3 proteins was strongly reduced upon infection with Shigella, Erlin-1 and Erlin-2 specifically bound to RIPK2 complexes. The interaction of these proteins with RIPK2 was validated by protein binding assays and immunofluorescence staining. Beside bacterial activation of NOD1/2, depletion of the E3 ligase XIAP and treatment with RIPK2 inhibitors also leads to the formation of RIPK2 cytosolic complexes. Whereas Erlin-1 and Erlin-2 were recruited to RIPK2 complexes following XIAP inhibition, they did not associate with RIPK2 structures induced by RIPK2 inhibitors. While the specific recruitment of Erlin-1/2 to RIPK2 suggests a role for these proteins in innate immune signaling, the biological response regulated by the Erlin-1/2-RIPK2 association remains to be determined.