No abstract

Chemically induced dimerization (CID) has proven to be a powerful tool for modulating protein interactions. However, the traditional dimerizer rapamycin has limitations in certain in vivo applications because of its slow reversibility and its affinity for endogenous proteins. Described herein is a bioorthogonal system for rapidly reversible CID. A novel dimerizer with synthetic ligand of FKBP' (SLF') linked to trimethoprim (TMP). The SLF' moiety binds to the F36V mutant of FK506-binding protein (FKBP) and the TMP moiety binds to E. coli dihydrofolate reductase (eDHFR). SLF'-TMP-induced heterodimerization of FKBP(F36V) and eDHFR with a dissociation constant of 0.12 μM. Addition of TMP alone was sufficient to rapidly disrupt this heterodimerization. Two examples are presented to demonstrate that this system is an invaluable tool, which can be widely used to rapidly and reversibly control protein function in vivo.

In order to elucidate the components of the oxygen sensory complex in HepG2 cells which regulates the production of erythropoietin, we have microinjected recombinant variants of the human small GTP-binding protein hRac1 and measured their effects on the production of reactive oxygen species (ROS) by the dihydrorhodamine-123 technique. The dominant-negative mutant hRac1(T17N) inhibits the NADH-stimulated production of ROS in HepG2 cells, whereas the constitutively activated hRac1(G12V) leads to an increase in intracellular ROS concentration. Reverse transcriptase PCR analysis showed that the hRac1, but not the hRac2, gene is expressed in HepG2 cells. These results demonstrate that hRac1, and not hRac2, is involved in the regulation of ROS production in HepG2 cells and suggest that hRac1 specifically functions in the non-phagocytic NAD(P)H oxidase complex.