科研之友
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摘要
Adipogenesis involves a complex signaling network requiring strict temporal and spatial organization of effector molecules. Molecular scaffolds, such as 14-3-3 proteins, facilitate such organization, and we have previously identified 14-3-3 as an essential scaffold in adipocyte differentiation. The interactome of 14-3-3 is large and diverse, and it is possible that novel adipogenic factors may be present within it, but this possibility has not yet been tested. Herein, we generated mouse embryonic fibroblasts from mice overexpressing a tandem affinity purification (TAP) epitope-tagged 14-3-3 molecule. After inducing adipogenesis, TAP-14-3-3 complexes were purified, followed by MS analysis to determine the 14-3-3 interactome. We observed more than 100 proteins that were unique to adipocyte differentiation, 56 of which were novel interacting partners. Among these, we were able to identify previously established regulators of adipogenesis (i.e. Ptrf/Cavin1) within the 14-3-3 interactome, confirming the utility of this approach to detect adipogenic factors. We found that proteins related to RNA metabolism, processing, and splicing were enriched in the interactome. Analysis of transcriptomic data revealed that 14-3-3 depletion in 3T3-L1 cells affected alternative splicing of mRNA during adipocyte differentiation. siRNA-mediated depletion of RNA-splicing factors within the 14-3-3 interactome, that is, of Hnrpf, Hnrpk, Ddx6, and Sfpq, revealed that they have essential roles in adipogenesis and in the alternative splicing of Pparg and the adipogenesis-associated gene Lpin1. In summary, we have identified novel adipogenic factors within the 14-3-3 interactome. Further characterization of additional proteins within the 14-3-3 interactome may help identify novel targets to block obesity-associated expansion of adipose tissues.
- 出版日期2018-5-4
