title: Improved coiled-coil design enhances interaction with Bcr-Abl and induces apoptosis.
abstract: The oncoprotein Bcr-Abl drives aberrant downstream activity through trans-autophosphorylation of homo-oligomers in chronic myelogenous leukemia (CML).(1, 2) The formation of Bcr-Abl oligomers is achieved through the coiled-coil domain at the N-terminus of Bcr.(3, 4) We have previously reported a modified version of this coiled-coil domain, CCmut2, which exhibits disruption of Bcr-Abl oligomeric complexes and results in decreased proliferation of CML cells and induction of apoptosis.(5) A major contributing factor to these enhanced capabilities is the destabilization of the CCmut2 homodimers, increasing the availability to interact with and inhibit Bcr-Abl. Here, we included an additional mutation (K39E) that could in turn further destabilize the mutant homodimer. Incorporation of this modification into CCmut2 (C38A, S41R, L45D, E48R, Q60E) generated what we termed CCmut3, and resulted in further improvements in the binding properties with the wild-type coiled-coil domain representative of Bcr-Abl [corrected]. A separate construct containing one revert mutation, CCmut4, did not demonstrate improved oligomeric properties and indicated the importance of the L45D mutation. CCmut3 demonstrated improved oligomerization via a two-hybrid assay as well as through colocalization studies, in addition to showing similar biologic activity as CCmut2. The improved binding between CCmut3 and the Bcr-Abl coiled-coil may be used to redirect Bcr-Abl to alternative subcellular locations with interesting therapeutic implications.
authors.affiliation: Department of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, University of Utah, Salt Lake City, Utah 84108, United States.
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Dixon
authors.forename: Geoffrey D
authors.initials: GD
authors.lastname: Miller
authors.forename: Benjamin J
authors.initials: BJ
authors.lastname: Bruno
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Constance
authors.forename: David W
authors.initials: DW
authors.lastname: Woessner
authors.forename: Trevor P
authors.initials: TP
authors.lastname: Fidler
authors.forename: James C
authors.initials: JC
authors.lastname: Robertson
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Cheatham
authors.forename: Carol S
authors.initials: CS
authors.lastname: Lim
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D017209:Apoptosis; D019556:COS Cells; D049109:Cell Proliferation; D002522:Chlorocebus aethiops; D016044:Fusion Proteins, bcr-abl / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015316:Genetic Therapy; D006801:Humans; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D050505:Mutant Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D016044:Fusion Proteins, bcr-abl