RESUMO
<p><b>OBJECTIVE</b>To construct certain chimeric E3s expression plasmids targetting oncoprotein Ras by harnessing the theory of protein knockdown.</p><p><b>METHODS</b>We chose the binding domain of Raf-1, PI3K, RalGDS, and the function domain of F-Box as well as the U-Box to construct the plasmids. Then used the double enzyme, PCR, and sequence to test the validity and integrity of the cloned nucleotide fragments. The expression efficiency of the plasmids in eukaryotic cells was detected by Western blot analysis.</p><p><b>RESULTS</b>Five of 6 plasmids in this study expressed the corresponding fusion proteins in HEK293T cells, and (RBD+CRD)(Raf-1)- U-Box-pcDNA3.1 can knocked down the protein level of Ras in PANC-1 cells.</p><p><b>CONCLUSIONS</b>We successfully constructed the chimeric E3 expression plasmids, which provides a solid basis for further research on protein knockdown.</p>
Assuntos
Humanos , Clonagem Molecular , Vetores Genéticos , Células HEK293 , Fosfatidilinositol 3-Quinases , Genética , Plasmídeos , Proteínas Recombinantes de Fusão , Genética , Metabolismo , Transfecção , Ubiquitina-Proteína Ligases , Genética , Fator ral de Troca do Nucleotídeo Guanina , Genética , Proteínas ras , GenéticaRESUMO
The guanine-nucleotide exchange factor (GEF) RalGPS1a activates small GTPase Ral proteins such as RalA and RalB by stimulating the exchange of Ral bound GDP to GTP, thus regulating various downstream cellular processes. RalGPS1a is composed of an Nterminal Cdc25-like catalytic domain, followed by a PXXP motif and a C-terminal pleckstrin homology (PH) domain. The Cdc25 domain of RalGPS1a, which shares about 30% sequence identity with other Cdc25-domain proteins, is thought to be directly engaged in binding and activating the substrate Ral protein. Here we report the crystal structure of the Cdc25 domain of RalGPS1a. The bowl shaped structure is homologous to the Cdc25 domains of SOS and RasGRF1. The most remarkable difference between these three Cdc25 domains lies in their active sites, referred to as the helical hairpin region. Consistent with previous enzymological studies, the helical hairpin of RalGPS1a adopts a conformation favorable for substrate binding. A modeled RalGPS1a-RalA complex structure reveals an extensive binding surface similar to that of the SOS-Ras complex. However, analysis of the electrostatic surface potential suggests an interaction mode between the RalGPS1a active site helical hairpin and the switch 1 region of substrate RalA distinct from that of the SOS-Ras complex.