[14-3-3/HIP-55 complex increases the stability of HIP-55].

OBJECTIVE: To further demonstrate the interaction of a new 14-3-3 interaction protein hematopoietic progenitor kinase 1[HPK1]-interacting protein (HIP-55) and 14-3-3 proteins and its potential biological function in HEK293 cells. METHODS: PDEST-N-Venus-HIP-55WT (wild type),PDEST-N-Venus-HIP-55AA (mutants, S269A/T291A, abolishing the binding of HIP-55 to 14-3-3),PDEST-GST-HIP-55WT and PDEST-C-Venus-14-3-3τ plasmids were constructed by gateway system. Their expressions were demonstrated by Western blotting method. Then we used Bimolecular Fluorescence Complementation (BiFC) and co-immunoprecipitation (co-IP) methods to demonstrate the interaction of HIP-55 and 14-3-3 in HEK293 cells. Moreover, the 14-3-3 antagonist peptide, R18 and HIP-55 protein mutant plasmid HIP-55AA were used to detect the protein synthesis of HIP-55 at different time points induced by puromycin, an inhibitor of protein production. RESULTS: The HEK293 cells expressed HIP-55 protein respectively, after being transected with PDEST-N-Venus-HIP-55WT,PDEST-N-Venus-HIP-55AA,PDEST-GST-HIP-55WT plasmids and expressed 14-3-3 protein after being transected with PDEST-C-Venus-14-3-3τ plasmids. We could detect venus fluorescence of venus-HIP-55 protein via confocal microscopy in HEK 293 cells transfected with N-Venus-HIP-55 and C-14-3-3τ plasmids by BiFC, but not in HEK 293 cells transfected with N-Venus-HIP-55 AA (mutants S269A/T291A) and C-14-3-3τ plasmids. The results of BiFC suggested that 14-3-3 interacted with HIP-55 through HIP-55 S269/T291 sites. At the same time, the data of co-IP showed that there were endogenous interactions between 14-3-3 and HIP-55. Furthermore, puromycin had no influence in HIP-55 protein synthesis at hours 0, 4, or 8 in HEK 293 cells expressing GST-HIP-55WT and 14-3-3 plasmids, while puromycin blocked HIP-55 protein synthesis in HEK 293 cells transfected with N-Venus-HIP-55AA (mutants S269A/T291A) and C-14-3-3τ plasmids. The results indicated that the 14-3-3/HIP-55 complex could contributed to the stability of HIP-55. CONCLUSION: HIP-55 forms a complex with 14-3-3 and 14-3-3/HIP-55 interaction increases the stability of HIP-55.

[The Regulatory Proteins of ß-Adrenergic Receptor and Their Functions].

Vascular diseases has become a top killer of human health, and cardiovascular receptors are pivotal in the occurrence, development, prevention and treatment of cardiovascular diseases. As for the important member of G protein-coupled receptor, ß-adrenergic receptor is undoubtedly a most important target of cardiovascular drugs. Being the hot spot in the cardiovascular research and application, ß- adrenergic receptor blocker has been considered as the greatest breakthrough for the prevention and cure of cardiovascular disease after digitalis. The 2012 Nobel Prize in chemistry was awarded again to the researchers on ß-adrenergic receptors. Extensive researchs show that ß-adrenergic receptors are precisely regulated by different regulatory proteins in cells in the transduction of different physiological and pathological signaling pathways. Based on these findings, function-selective ligands recently arise in the receptor research and will be the new chance of drug discovery. In this article we reviewed the related signal pathways and functions of ß-adrenergic receptor regulatory proteins.