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Structural characterization of a unique interface between carbohydrate response element-binding protein (ChREBP) and 14-3-3? protein.

ABSTRACT: Carbohydrate response element-binding protein (ChREBP) is an insulin-independent, glucose-responsive transcription factor that is expressed at high levels in liver hepatocytes where it plays a critical role in converting excess carbohydrates to fat for storage. In response to fluctuating glucose levels, hepatic ChREBP activity is regulated in large part by nucleocytoplasmic shuttling of ChREBP protein via interactions with 14-3-3 proteins. The N-terminal ChREBP regulatory region is necessary and sufficient for glucose-responsive ChREBP nuclear import and export. Here, we report the crystal structure of a complex of 14-3-3? bound to the N-terminal regulatory region of ChREBP at 2.4 Å resolution. The crystal structure revealed that the ?2 helix of ChREBP (residues 117-137) adopts a well defined ?-helical conformation and binds 14-3-3 in a phosphorylation-independent manner that is different from all previously characterized 14-3-3 and target protein-binding modes. ChREBP ?2 interacts with 14-3-3 through both electrostatic and van der Waals interactions, and the binding is partially mediated by a free sulfate or phosphate. Structure-based mutagenesis and binding assays indicated that disrupting the observed 14-3-3 and ChREBP ?2 interface resulted in a loss of complex formation, thus validating the novel protein interaction mode in the 14-3-3?·ChREBP ?2 complex.

SUBMITTER: Ge Q

PROVIDER: S-EPMC3516738 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Structural characterization of a unique interface between carbohydrate response element-binding protein (ChREBP) and 14-3-3β protein.

Ge Qiang Q Huang Nian N Wynn R Max RM Li Yang Y Du Xinlin X Miller Bonnie B Zhang Hong H Uyeda Kosaku K

The Journal of biological chemistry 20121018 50

Carbohydrate response element-binding protein (ChREBP) is an insulin-independent, glucose-responsive transcription factor that is expressed at high levels in liver hepatocytes where it plays a critical role in converting excess carbohydrates to fat for storage. In response to fluctuating glucose levels, hepatic ChREBP activity is regulated in large part by nucleocytoplasmic shuttling of ChREBP protein via interactions with 14-3-3 proteins. The N-terminal ChREBP regulatory region is necessary and ...[more]

PMID: 23086940

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Project description:ObjectiveCarbohydrate response element binding protein (ChREBP) is a transcription factor that responds to glucose and activates genes involved in the glycolytic and lipogenic pathways. Recent studies have linked adipose ChREBP to insulin sensitivity in mice. However, while ChREBP is most highly expressed in the liver, the effect of hepatic ChREBP on insulin sensitivity remains unknown. To clarify the importance of hepatic ChREBP on glucose homeostasis, we have generated a knockout mouse model that lacks this protein specifically in the liver (Liver-ChREBP KO).MethodsUsing Liver-ChREBP KO mice, we investigated whether hepatic ChREBP deletion influences insulin sensitivity, glucose homeostasis and the development of hepatic steatosis utilizing various dietary stressors. Furthermore, we determined gene expression changes in response to fasted and fed states in liver, white, and brown adipose tissues.ResultsLiver-ChREBP KO mice had impaired insulin sensitivity as indicated by reduced glucose infusion to maintain euglycemia during hyperinsulinemic-euglycemic clamps on both chow (25% lower) and high-fat diet (33% lower) (p < 0.05). This corresponded with attenuated suppression of hepatic glucose production. Although Liver-ChREBP KO mice were protected against carbohydrate-induced hepatic steatosis, they displayed worsened glucose tolerance. Liver-ChREBP KO mice did not show the expected gene expression changes in liver in response to fasted and fed states. Interestingly, hepatic ChREBP deletion also resulted in gene expression changes in white and brown adipose tissues, suggesting inter-tissue communication. This included an almost complete abolition of BAT ChREBPβ induction in the fed state (0.15-fold) (p = 0.015) along with reduced lipogenic genes. In contrast, WAT showed inappropriate increases in lipogenic genes in the fasted state along with increased PEPCK1 in both fasted (3.4-fold) and fed (5.1-fold) states (p < 0.0001).ConclusionsOverall, hepatic ChREBP is protective in regards to hepatic insulin sensitivity and whole body glucose homeostasis. Hepatic ChREBP action can influence other peripheral tissues and is likely essential in coordinating the body's response to different feeding states.

Project description:The intracellular lipids in muscle cells of farm animals play a crucial role in determining the overall intramuscular fat (IMF) content, which has a positive impact on meat quality. However, the mechanisms underlying the deposition of lipids in muscle cells of farm animals are not yet fully understood. The purpose of this study was to determine the roles of carbohydrate-response element binding protein (ChREBP) and fructose in IMF deposition of chickens. For virus-mediated ChREBP overexpression in tibialis anterior (TA) muscle of chickens, seven 5-d-old male yellow-feather chickens were used. At 10 d after virus injection, the chickens were slaughtered to obtain TA muscles for analysis. For fructose administration trial, sixty 9-wk-old male yellow-feather chickens were randomly divided into 2 groups, with 6 replicates per group and 5 chickens per replicate. The chickens were fed either a basal diet or a basal diet supplemented with 10% fructose (purity ≥ 99%). At 4 wk later, the chickens were slaughtered, and breast and thigh muscles were collected for analysis. The results showed that the skeletal ChREBP mRNA levels were positively associated with IMF content in multiple species, including the chickens, pigs, and mice (P < 0.05). ChREBP overexpression increased lipid accumulation in both muscle cells in vitro and the TA muscles of mice and chickens in vivo (P < 0.05), by activation of the de novo lipogenesis (DNL) pathway. Moreover, activation of ChREBP by dietary fructose administration also resulted in increased IMF content in mice and notably chickens (P < 0.05). Furthermore, the lipidomics analysis revealed that ChREBP activation altered the lipid composition of chicken IMF and tented to improve the flavor profile of the meat. In conclusion, this study found that ChREBP plays a pivotal role in mediating the deposition of fat in chicken muscles in response to fructose-rich diets, which provides a novel strategy for improving meat quality in the livestock industry.

Dataset Information

Structural characterization of a unique interface between carbohydrate response element-binding protein (ChREBP) and 14-3-3? protein.

Publications

Structural characterization of a unique interface between carbohydrate response element-binding protein (ChREBP) and 14-3-3β protein.

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