Cidea is a highly enriched protein found in brown adipose tissue (BAT) in which it plays a critical role in adaptive thermogenesis and fat accumulation. Cidea-null mice have increased energy expenditure with resistance to high fat diet-induced obesity and diabetes. Although Cidea clearly plays an important role in the energy homeostasis, its regulatory processes have not yet been elucidated. In this study, we showed that Cidea is a short-lived protein as measured by cycloheximide (CHX)-based protein chase experiments in different cell lines or in differentiated brown adipocytes. In addition, we demonstrated that proteasome inhibitors specifically increase the stability of both transfected and endogenous Cidea protein. Furthermore, our results indicated that Cidea is polyubiquitinated wh...[ Read more ]

Cidea is a highly enriched protein found in brown adipose tissue (BAT) in which it plays a critical role in adaptive thermogenesis and fat accumulation. Cidea-null mice have increased energy expenditure with resistance to high fat diet-induced obesity and diabetes. Although Cidea clearly plays an important role in the energy homeostasis, its regulatory processes have not yet been elucidated. In this study, we showed that Cidea is a short-lived protein as measured by cycloheximide (CHX)-based protein chase experiments in different cell lines or in differentiated brown adipocytes. In addition, we demonstrated that proteasome inhibitors specifically increase the stability of both transfected and endogenous Cidea protein. Furthermore, our results indicated that Cidea is polyubiquitinated when over-expressed in different culture cells as well as in differentiated mature brown adipocytes. Extensive analysis of individual lysine residue mutations revealed that ubiquitinated lysine residues are located in the N-terminal region of Cidea, as alteration of these lysine residues to alanine (N-5KA) rendered Cidea much more stable when compared with the wild-type protein or a C-terminal lysine-less mutant (C-5KA). Furthermore, we identified lysine-23 (K23) within the N-terminus of Cidea as the major contributor to Cidea’s protein instability. Taken together, our results demonstrated that the ubiquitin-proteasome system confers an important post-translational modification that controls the protein stability of Cidea.

Sarcosin was identified as a strong Cidea-interacting protein by yeast two-hybrid screen using Cidea’s first 145 amino acids as bait. Their interaction was further confirmed by in vivo coimmunoprecipitation experiments, domain-mapping experiments and subcellular localization studies. Interestingly, Sarcosin coexpression stabilized Cidea in our CHX-based protein chase experiments by decreasing the amount of polyubiquitinated species for proteasomal degradation. This observation implies that Cidea protein stability could be modulated by interacting with other proteins.

The SB156 protein shares high sequence homology with a hydroxypyruvate isomerase in prokaryotes and was isolated as a binding protein to Cidea’s N-terminus in the same yeast two-hybrid screen. We identified SB156 as a novel promyelocytic leukemia (PML) nuclear body-associated protein that is colocalized with Cidea in Cos-7 cells. More importantly, SB156 is sequestered with Cidea in the cytosol without targeting to PML nuclear bodies. Strikingly, we used RT-PCR to determine the tissue distribution of SB156. We found that its mRNA is at higher levels in BAT, liver and white adipose tissue, which are coincidental with the tissue-specific expression of Cidea, Cideb and Fsp27, respectively. With in vivo coimmunoprecipitation experiments, we demonstrated that SB156 can associate with Cideb, and Fsp27 as well. This suggests that SB156 may be a unique Cide-N domain interacting protein for all Cide family proteins for metabolic regulation, transcription control and apoptosis.