title: A bidirectional system for the dynamic small molecule control of intracellular fusion proteins.
abstract: Small molecule control of intracellular protein levels allows temporal and dose-dependent regulation of protein function. Recently, we developed a method to degrade proteins fused to a mutant dehalogenase (HaloTag2) using small molecule hydrophobic tags (HyTs). Here, we introduce a complementary method to stabilize the same HaloTag2 fusion proteins, resulting in a unified system allowing bidirectional control of cellular protein levels in a temporal and dose-dependent manner. From a small molecule screen, we identified N-(3,5-dichloro-2-ethoxybenzyl)-2H-tetrazol-5-amine as a nanomolar HALoTag2 Stabilizer (HALTS1) that reduces the Hsp70:HaloTag2 interaction, thereby preventing HaloTag2 ubiquitination. Finally, we demonstrate the utility of the HyT/HALTS system in probing the physiological role of therapeutic targets by modulating HaloTag2-fused oncogenic H-Ras, which resulted in either the cessation (HyT) or acceleration (HALTS) of cellular transformation. In sum, we present a general platform to study protein function, whereby any protein of interest fused to HaloTag2 can be either degraded 10-fold or stabilized 5-fold using two corresponding compounds.
authors: Department of Molecular Cellular and Developmental Biology, Yale University, 219 Prospect Street, New Haven, CT 06511, USA.
authors: Taavi K
authors: TK
authors: Neklesa
authors: Department of Molecular Cellular and Developmental Biology, Yale University, 219 Prospect Street, New Haven, CT 06511, USA.
authors: Devin J
authors: DJ
authors: Noblin
authors: Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, NY 10027, USA.
authors: Alexander P
authors: AP
authors: Kuzin
authors: Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, NY 10027, USA.
authors: Scott
authors: S
authors: Lew
authors: Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, NY 10027, USA.
authors: Jayaraman
authors: J
authors: Seetharaman
authors: Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
authors: Thomas B
authors: TB
authors: Acton
authors: Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
authors: Gregory J
authors: GJ
authors: Kornhaber
authors: Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
authors: Rong
authors: R
authors: Xiao
authors: Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, and Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.|Department of Biochemistry and Molecular Biology, Robert Wood Johnson Medical School-UMDNJ, Piscataway, NJ 08854, USA.
authors: Gaetano Thomas
authors: GT
authors: Montelione
authors: Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, NY 10027, USA.
authors: Liang
authors: L
authors: Tong
authors: Department of Molecular Cellular and Developmental Biology, Yale University, 219 Prospect Street, New Haven, CT 06511, USA.|Department of Chemistry, Yale University, 225 Prospect Street, New Haven, CT 06511, USA.|Department of Pharmacology, Yale University, 333 Cedar Street, New Haven, CT 06511, USA.
authors: Craig M
authors: CM
authors: Crews
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000378:metabolism*; D013696:Temperature; D054875:Ubiquitination
chemical_list: D029721:Drosophila Proteins; C486141:Halo protein, Drosophila; D011993:Recombinant Fusion Proteins; D054852:Small Molecule Libraries
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