Functional specific roles of FADD: comparative proteomic analyses from knockout cell lines

Abstract

Fas-associated death domain (FADD) is a classical adaptor protein involved in tumor necrosis factor receptor family-mediated apoptosis. Besides being an essential instrument in cell death, it also plays key roles in cell proliferation and survival. The current study shows for the first time that FADD is probably associated with energy metabolism and proteolysis. It has been reported that embryonic death caused by FADD deficiency in mice was not attributable to impaired apoptosis. Furthermore, mice bearing the substitution in FADD of serine 191 to aspartic acid exhibited leaner body size than both wild-type control and serine 191 to alanine mutant mice, indicating metabolic disorders. To study these non-apoptotic effects of FADD, a comprehensive strategy of proteomics identification combined with bioinformatic analyses and further cell biology validation was utilized to identify differentially-expressed proteins in FADD-deficient mouse embryonic fibroblasts (MEFs). A total of 45 unique proteins were determined to be significantly changing due to FADD deficiency. Network analysis of these proteins using MetaCore™ suggested induction of transcriptional factors that are too low to be detected by two-dimensional gels and identified an enriched cluster of changed proteins that are involved in cellular metabolic processes, including lipid metabolism, fatty acids metabolism, glycolysis, tricarboxylic acid (TCA) cycle, and oxidative phosphorylation. Fatty acids β-oxidation was found to be enhanced in FADD-deficient cells. In addition, five proteins related to the ubiquitin–proteasome (UP) pathway were also specifically up-regulated in the FADD^−/− MEFs. Finally, the c-Myc gene represents a convergent hub lying at the center of dysregulated pathways, and was up-regulated in FADD knockout cells. Taken together, these studies show that impaired mitochondrial function and proteolysis may play pivotal roles in the dysfunction associated with FADD deficiency-induced disorders, probably including embryonic lethality. The link between FADD and cell metabolism may provide us new insight for understanding the crosstalk of independent cell signaling pathways.