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Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists
Published on: November 15, 2013
Novel polymorphisms of nuclear receptor SHP associated with functional and structural changes
Taofeng Zhou1, Yuxia Zhang, Antonio Macchiarulo
1Department of Medicine and Oncological Sciences, Huntsman Cancer Institute, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA.
The Journal of Biological Chemistry
|June 3, 2010
Summary
Genetic variations in the small heterodimer partner (SHP) gene are common. The Lys-170 residue is crucial for SHP protein stability and function, impacting ubiquitination and acetylation.
Area of Science:
- Genetics
- Molecular Biology
- Biochemistry
Background:
- The small heterodimer partner (SHP, NROB2) gene plays a critical role in regulating various metabolic pathways.
- Genetic variations in SHP may contribute to disease susceptibility, including conditions like CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy).
Purpose of the Study:
- To identify and characterize genetic variations in the SHP gene in human subjects.
- To investigate the functional consequences of identified SHP mutations on protein stability, nuclear translocation, and transcriptional repression.
Main Methods:
- Sequencing of the SHP gene to identify single nucleotide polymorphisms (SNPs) and mutations.
- In vitro assays to assess protein nuclear translocation, ubiquitination, and acetylation.
- Analysis of SHP interactions with nuclear receptors (e.g., ERRgamma, HNF4alpha, LRH-1) and co-regulators.
- Molecular dynamics simulations to predict the structural impact of mutations.
Main Results:
- Three heterozygous nonsynonymous SNPs (p.R38H, p.K170N, p.G171A) and intronic/UTR/promoter mutations were identified in SHP.
- The K170N mutation impaired SHP nuclear translocation, increased susceptibility to degradation, and abolished repressive activity on ERRgamma and HNF4alpha.
- The G171A polymorphism enhanced SHP expression and maintained normal function.
- Mutations altered SHP interactions with LRH-1 and EID1, and K170N impaired co-activator recruitment to the apoCIII promoter.
Conclusions:
- Genetic variations in the SHP gene are prevalent in the human population.
- The Lys-170 residue is critical for SHP protein stability, ubiquitination, acetylation, and repressive function.
- SHP mutations can disrupt its regulatory activities, potentially contributing to disease pathogenesis.
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