Roles of RIPK1/RIPK3 homo- and hetero-dimerization in cell death and pro-inflammatory signaling
Yang, Xuzhong.
2017
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Abstract: Necroptosis, a form of regulated necrosis, has been reported to be involved in numerous inflammatory diseases including multiple sclerosis, ischemia reperfusion injury, atherosclerosis, inflammatory bowel syndrome, and pancreatitis. Two homologous Ser/Thr kinases, receptor-interacting protein 1 (RIPK1) and RIPK3, play an important role in mediating necroptosis. RIPK1 and RIPk3 form heter... read moreodimeric amyloid scaffold through their RIP homotypic interaction motif (RHIM) domains, which serves as the platform for the downstream mixed lineage kinase domain-like (MLKL) mediated cell death and Erk1/2 mediated pro-inflammatory signaling induced by LPS. It is reported that RIPK3 homo-dimerization is sufficient to induce cell death in L929 and MEF cell lines. But it is still unclear how RIPK1/RIPK3 homo- or hetero-dimerization work on the pro-inflammatory signaling. In this study, we use FKBP-FRB-AP21976 inducible dimer system to artificially control the formations of different dimers between RIPK1 (RHIM domain mutated to avoid interaction with endogenous RIPK1/3) and RIPK3 (RHIM domain mutated) in L929 and RAW264.7 cells. We confirmed that RIPK3 homo-dimerization, rather than RIPK1 homo- or RIPK1/RIPK3 hetero-dimerization, could cause 40% cell death in L929 cell line. In the first several test sets, RIPK1 homo-dimerization could induce the expression of a number of pro-inflammatory mRNAs, including CSF2, CCL5, CXCL2 and this induction would be abolished by adding Nec-1s, the inhibitor of RIPK1 or SCH772984, the inhibitor of Erk1/2. However, this response was eventually lost, likely due to FKBP-RIPK1/FRB-RIPK1 aggregation without dimerizer AP21976. In the RAW264.7 cells, there is no cell death or inflammatory signaling induction after dimerization, which may indicate the requirement for RHIM in these cells. The establishment of cell lines expressing inducible FKBP-FRB RIPK1 (wild type RHIM) is ongoing.
Thesis (M.S.)--Tufts University, 2017.
Submitted to the Dept. of Pharmacology & Experimental Therapeutics.
Advisor: Alexei Degterev.
Keyword: Pharmacology.read less - ID:
- 5712mk493
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