Bioluminescent RIPoptosome Assay for FADD/RIPK1 Interaction Based on Split Luciferase Assay in a Human Neuroblastoma Cell Line SH-SY5Y.

Different programed cell death (PCD) modalities involve protein-protein interactions in large complexes. Tumor necrosis factor α (TNFα) stimulated assembly of receptor-interacting protein kinase 1 (RIPK1)/Fas-associated death domain (FADD) interaction forms Ripoptosome complex that may cause either apoptosis or necroptosis. The present study addresses the interaction of RIPK1 and FADD in TNFα signaling by fusion of C-terminal (CLuc) and N-terminal (NLuc) luciferase fragments to RIPK1-CLuc (R1C) or FADD-NLuc (FN) in a caspase 8 negative neuroblastic SH-SY5Y cell line, respectively. In addition, based on our findings, an RIPK1 mutant (R1C K612R) had less interaction with FN, resulting in increasing cell viability. Moreover, presence of a caspase inhibitor (zVAD.fmk) increases luciferase activity compared to Smac mimetic BV6 (B), TNFα -induced (T) and non-induced cell. Furthermore, etoposide decreased luciferase activity, but dexamethasone was not effective in SH-SY5Y. This reporter assay might be used to evaluate basic aspects of this interaction as well as for screening of necroptosis and apoptosis targeting drugs with potential therapeutic application.

Effect of mutation at positively charged residues (K329 and R330) in a flexible region of firefly luciferase on structure and kinetic properties.

Firefly luciferase as a bioluminescent enzyme has many applications in various fields from scientific research to commercial goals. This enzyme is relatively unstable with low functional capacity due to rapid inactivation in physiological temperature, low in vitro stability and high susceptibility to proteolytic degradation. Based on previous studies, two regions 206-220 and 329-341 on N-domain of Photinus pyralis luciferase are known accessible and flexible. Flexible regions may lead to protein instability. Here, the effect of mutation at positively charged residues Lys(K)329 and Arg(R)330 on the stability of luciferase was studied. Furthermore, the role of these mutations on the structure and function was evaluated. Introducing of these point mutations did not affect the orientation of critical residues in bioluminescence color determination. The kinetic studies showed that thermostability and Km value for luciferin in both mutants were decreased as compared to wild type. However, optimum pH and optimum temperature showed no significant changes in both mutants. Moreover, the structural data revealed an increase in tryptophan fluorescence intensity and secondary structure content for R330Q in compared with wild type, while intrinsic fluorescence and far-UV CD intensity in K329I mutant was decreased.