Intrabodies are fragments of antibodies that can be expressed within a cell, therefore bringing the binding specificity of antibodies to the intracellular compartment. Using intracellular antibody capture (which is based on yeast two-hybrid screening), Rabbitts and colleagues isolated a single domain intrabody that binds to oncogenic HRAS-G12V, called iDab#6. Using COS-7 cells, they showed that iDab#6 binds to oncogenic HRAS mutants 10 times more efficiently than wild-type HRAS, and the intrabody also recognized other oncogenic Ras family members such as NRAS mutants. Furthermore, the authors showed that retroviral infection of vectors that encode iDab#6-memb, which contains a membrane localization signal, could revert the tumorigenic phenotype of NIH3T3-EJ (which express HRAS-G12V), HT1080 (which express NRAS-Q61K) or DLD-1 (which express both KRAS-G13D and p53-S241F) cancer cell lines. Moreover, tumours or lung metastases did not form when these cells, which expressed iDab#6-memb, were subcutaneously injected into immunocompromised mice, demonstrating that iDab#6 can inhibit Ras-dependent tumorigenesis and metastasis.
So, how does iDab#6 prevent oncogenic Ras tumorigenicity? The authors determined the X-ray crystallographic structures of bacterially purified oncogenic HRAS with iDab#6, and showed that the intrabody binds to the Switch I and II regions that undergo significant conformational changes when Ras exchanges GDP for GTP. Moreover, they showed that the GDP bound conformation was unable to make the necessary H-bonds with iDab#6, indicating the intrabody recognizes the active form of Ras. The downstream Ras-effector molecules, Raf, Ral guanine nucleotide-dissociation stimulator (RALGDS) and phosphatidylinositol 3-kinase (PI3K) also bind to the Switch I domain of Ras–GTP, and further analysis indicates that these effector binding sites overlap significantly with that of iDab#6. The dissociation constant of iDab#6 is lower than that for the downstream effectors, indicating that iDab#6 functions to prevent Ras–GTP signal transduction by inhibiting the interaction of Ras with its downstream effectors, which was also confirmed with competition experiments in vitro.
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