Role of autophagy-dependent Pellino3a downregulation during TLR4 signaling in macrophages

• Applicant: Professor Dr. Andreas von Knethen
• Subject Area: Public Health, Healthcare Research, Social and Occupational Medicine
• Term: from 2015 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 284401149

Lipopolysaccharide (LPS)-induced activation of toll-like receptor 4 (TLR4) signaling provokes a proinflammatory response in macrophages. This inflammatory reaction is terminated by autophagy. We found that Pellino3a, which is an E3-ubiquitin-ligase and scaffold protein in TLR4-signaling, is regulated by autophagy in macrophages after LPS-stimulation. Pellino3a protein gets stabilized 6 h after LPS-treatment and subsequently is degraded 24 h after LPS-stimulation. This depletion is caused by binding to the autophagy adaptor protein p62/SQSTM1, initiating its degradation. It remains obscure, how Pellino3a short time protein stabilization is achieved and whether its blockade might improve sepsis outcome. Because Pellino3a is essential in macrophages for LPS-TLR4-mediated expression of proinflammatory cytokines such as IL-1beta, TNFalpha, IL-6, and IFNgamma, we hypothesize that inhibiting the early increase of Pellino3a expression blocks the hyperinflammatory response. Since no mechanistic insights are available for regulation of Pellino3a expression in inflammation, we characterize first by mass spectrometry (MS) and a fluorescence polarization assay which modification(s) of Pellino3a and/or p62/SQSTM1 cause their interaction. Next we check, whether induction of autophagy and Pellino3a degradation can be enhanced by overexpression of p62/SQSTM1 or pharmacologically by Torin2 treatment, thus decreasing proinflammatory cytokine expression. Considering that Keap1 binds to p62/SQSTM1 as well, we determine by FACS-FRET-analysis whether this interaction blocks Pellino3 binding, thereby stabilizing Pellino3a. We clarify by MS whether Pellino3a-dependent ubiquitination of other proteins contributes to its degradation. Moreover, we analyze whether Pellino3a protein expression is transcriptionally modulated during sepsis and whether this can be used to inhibit Pellino3a expression. These experiments will be performed in J774A.1 and RAW264.7 macrophages, following endogenous gene tagging by CRSIPR/Cas9. We aim at identifying, how the early stabilization of Pellino3a protein can be blocked and explore to directly translate this mechanism to an in vivo setup in the mouse, the endotoxin model. We act on the assumption that inhibition of Pellino3a or p62/SQSTM1 expression in macrophages decreases the hyperinflammatory response in vivo.

• DFG Programme: Research Grants
• Cooperation Partners: Dr. Phillip Grote; Professor Dr. Eugen Proschak