Regulation of Cidea protein stability by the ubiquitin-mediated proteasomal degradation pathway.
Cidea, one of three members of the CIDE (cell-death-inducing DNA-fragmentation-factor-45-like effector) family of proteins, is highly enriched in brown adipose tissue, 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. However, little is known as to how the Cidea protein is regulated. In the present study we show that Cidea is a short-lived protein as measured by cycloheximide-based protein chase experiments in different cell lines or in differentiated brown adipocytes. Proteasome inhibitors specifically increased the stability of both transfected and endogenous Cidea protein. Furthermore, Cidea protein was found to be polyubiquitinated when overexpressed in different culture cells as well as in differentiated mature brown adipocytes. Extensive mutational analysis of individual lysine residues revealed that ubiquitinated lysine residues are located in the N-terminal region of Cidea, as alteration of these lysine residues to alanine (N-5KA mutant) renders Cidea much more stable when compared with wild-type or C-terminal lysine-less mutant (C-5KA). Furthermore, K23 (Lys23) within the N-terminus of the Cidea was identified as the major contributor to its polyubiquitination signal and the protein instability. Taken together, the results of our study demonstrated that the ubiquitin-proteasome system confers an important post-translational modification that controls the protein stability of Cidea.
Chan
Siu Chiu
SC
Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, People's Republic of China.
Lin
Sheng-Cai
SC
Li
Peng
P
D000595:Amino Acid Sequence; D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D061988:Proteasome Inhibitors; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology*; D013816:Thermodynamics; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
D051017:Apoptosis Regulatory Proteins; C112510:Cidea protein, mouse; D011480:Protease Inhibitors; D061988:Proteasome Inhibitors; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex
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