Chemical Development of Intracellular Protein Heterodimerizers

Abstract

Cell activation initiated by receptor ligands or oncogenes triggers complex and convoluted intracellular signaling. Techniques initiating signals at defined starting points and cellular locations are attractive to elucidate the output of selected pathways. Here, we present the development and validation of a protein heterodimerization system based on small molecules cross-linking fusion proteins derived from HaloTags and SNAP-tags. Chemical dimerizers of HaloTag and SNAP-tag (HaXS) show excellent selectivity and have been optimized for intracellular reactivity. HaXS force protein-protein interactions and can translocate proteins to various cellular compartments. Due to the covalent nature of the HaloTag-HaXS-SNAP-tag complex, intracellular dimerization can be easily monitored. First applications include protein targeting to cytoskeleton, to the plasma membrane, to lysosomes, the initiation of the PI3K/mTOR pathway, and multiplexed protein complex formation in combination with the rapamycin dimerization system.

Localized light-induced protein dimerization in living cells using a photocaged dimerizer

“…For our non-caged covalent ligand/receptor pair, we chose the Halotag system, which consists of a bacterial alkyldehalogenase enzyme mutated to irreversibly form a covalent bond with chloroalkyl-containing substrates 12 . This system offers rapid covalent protein labelling that is bioorthogonal in human cells, and it has been successfully utilized for protein heterodimerization 13,14 . For clarity, we will refer to the Halotag protein as Haloenzyme and the chlorohexane group as Haloligand.…”

“…Alternative photocages could be used to allow uncaging at longer wavelengths, or the protein-binding ligands could be replaced to allow the use of a different receptor. For example, a completely orthogonal dimerizer could be designed by replacing the Halotag ligand with a SNAP-tag ligand 13 , TMP with dexamethasone 18 and NVOC with a redshifted coumarin-based photocage 23 , which would allow for two different proteins of interest to be selectively recruited using different wavelengths of light. An expanding toolkit for light-induced protein dimerization will enable an exciting new era of experimental cell biology, in which researchers can optically control protein localization and interactions.…”

Molecular characterization of a eukaryotic-like phenol hydroxylase from <i>Corynebacterium glutamicum</i>

“…Hence, according to the reports that some protein would be interconvertible between monomer and dimer when binds to ligands (Nan et al, 2006(Nan et al, , 2010 and the indication of previous researches that protein-protein interactions between the components is essential for the shift of FAD such as alkanesulfonate monooxygenase from E. coli (Abdurachim and Ellis, 2006) and arylamine oxygenase from Pseudomonas fluorescens (Lee and Zhao, 2007), we considered that chemically induced dimerization (CID) of Phe protein mediated by phenol would take place while induced with the presence of phenol. As we know, CID system has been regarded as a powerful tool for inducible manipulation and specifically control protein homo-and heterodimerization (Corson et al, 2008;Erhart et al, 2013). So we consider it feasible that phenol-induced protein complex formation can be exploited to accelerate protein translocation to perform different functions and to activate degradation signaling pathways.…”

Molecular tools for acute spatiotemporal manipulation of signal transduction

“…In addition to dimerization systems based on plant hormones, other synthetic heterodimerizers have been created by covalently linking two orthogonal ligands, enzyme substrates, or protein-targeting tags [19]. For example, Erhart et al developed a dimerizer of HaloTag and SNAP tag by integrating HaloTag- and SNAP-tag-binding moieties into a single molecule called HaXS [20]. HaXS-mediated dimerization occurs on the order of 10s of minutes and, unlike the other systems described, results in the two proteins being covalently bound.…”