A general chemical method to regulate protein stability in the mammalian central nervous system.
The ability to make specific perturbations to biological molecules in a cell or organism is a central experimental strategy in modern research biology. We have developed a general technique in which the stability of a specific protein is regulated by a cell-permeable small molecule. Mutants of the Escherichia coli dihydrofolate reductase (ecDHFR) were engineered to be degraded, and, when this destabilizing domain is fused to a protein of interest, its instability is conferred to the fused protein resulting in rapid degradation of the entire fusion protein. A small-molecule ligand trimethoprim (TMP) stabilizes the destabilizing domain in a rapid, reversible, and dose-dependent manner, and protein levels in the absence of TMP are barely detectable. The ability of TMP to cross the blood-brain barrier enables the tunable regulation of proteins expressed in the mammalian central nervous system.
Iwamoto
Mari
M
Department of Chemical and Systems Biology, Stanford University, CA 94305, USA.
Björklund
Tomas
T
Lundberg
Cecilia
C
Kirik
Deniz
D
Wandless
Thomas J
TJ
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002460:Cell Line; D004926:Escherichia coli / Q000201:enzymology; D005493:Folic Acid Antagonists / Q000737:chemistry* / Q000494:pharmacology; D008024:Ligands; D051379:Mice; D015202:Protein Engineering; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D022021:Tacrolimus Binding Proteins / Q000737:chemistry / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014295:Trimethoprim / Q000737:chemistry* / Q000494:pharmacology
D005493:Folic Acid Antagonists; D008024:Ligands; D011993:Recombinant Fusion Proteins; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D022021:Tacrolimus Binding Proteins