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 homo-dimers, 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 homo-dimer. 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. 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.

Utilizing the Estrogen Receptor Ligand-Binding Domain for Controlled Protein Translocation to the Insoluble Fraction

“…To test the ability of ERLBD to control a protein’s localization, and therefore function, we subcloned a mutated coiled-coil (CCmut3) motif from Bcr-Abl previously developed in our lab (8). When introduced into Bcr-Abl positive CML cells, this coiled-coil disrupts signaling by Bcr-Abl and induces apoptosis.…”

“…The utility of this controlled localization was demonstrated by fusing the ERLBD protein to a functional peptide (CCmut3) that had previously been shown to bind to and disrupt Bcr-Abl signaling in K562 cells and induce apoptosis (8). We hypothesized that after fulvestrant addition, ERLBD-CCmut3 protein would translocate away from Bcr-Abl to insoluble clusters, and would relieve the Bcr-Abl signaling inhibition.…”

“…In addition, the EGFP-ERLBD-CCmut3 was constructed by amplifying the DNA encoding ERLBD by polymerase chain reaction, using the primers 5′-GGATCACTCTC GGCATGG-3′ and 5′-GCGCGCGCGCTCCGGAGCTA GTGGGCGCATGTAGG-3′ followed with BspEI digestion. This was subcloned into the BspEI restriction enzyme site in our published EGFP-CCmut3 plasmid (8). …”

“…Cover slips were mounted to glass slides with Fluoromount G (Electron Microscopy Sciences, Hatfield, PA). Fixed slides were viewed with an Olympus IX81 FV1000-XY confocal microscope (Imaging Core Facility, University of Utah) with a 60X objective and 3.0x digital zoom as previously described (8). Excitation and emission filters for EGFP and Texas Red were 488 nm excitation, 500–530 nm emission filter, and 543 excitation, 555–655 nm emission filter, respectively.…”

“…This protein switch utilizes a version of the estrogen receptor ligand-binding domain (ERLBD) that is exclusively responsive to fulvestrant, a non-natural ligand that can be used for exquisite regulation of the protein location in the cell. We provide proof of concept of dysregulation of an active peptide (8) whose function is altered when triggered to the cytoskeletal fraction upon addition of ligand.…”