5 research outputs found
CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide
Intracellular
signaling pathways are mediated by changes in protein
abundance and post-translational modifications. A common approach
for investigating signaling mechanisms and the effects induced by
synthetic compounds is through overexpression of recombinant reporter
genes. Genome editing with CRISPR/Cas9 offers a means to better preserve
native biology by appending reporters directly onto the endogenous
genes. An optimal reporter for this purpose would be small to negligibly
influence intracellular processes, be readily linked to the endogenous
genes with minimal experimental effort, and be sensitive enough to
detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino
acids) capable of producing bright and quantitative luminescence through
high affinity complementation (<i>K</i><sub>D</sub> = 700
pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9,
we demonstrate that HiBiT can be rapidly and efficiently integrated
into the genome to serve as a reporter tag for endogenous proteins.
Without requiring clonal isolation of the edited cells, we were able
to quantify changes in abundance of the hypoxia inducible factor 1A
(HIF1α) and several of its downstream transcriptional targets
in response to various stimuli. In combination with fluorescent antibodies,
we further used HiBiT to directly correlate HIF1α levels with
the hydroxyproline modification that mediates its degradation. These
results demonstrate the ability to efficiently tag endogenous proteins
with a small luminescent peptide, allowing sensitive quantitation
of the response dynamics in their regulated expression and covalent
modifications
CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide
Intracellular
signaling pathways are mediated by changes in protein
abundance and post-translational modifications. A common approach
for investigating signaling mechanisms and the effects induced by
synthetic compounds is through overexpression of recombinant reporter
genes. Genome editing with CRISPR/Cas9 offers a means to better preserve
native biology by appending reporters directly onto the endogenous
genes. An optimal reporter for this purpose would be small to negligibly
influence intracellular processes, be readily linked to the endogenous
genes with minimal experimental effort, and be sensitive enough to
detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino
acids) capable of producing bright and quantitative luminescence through
high affinity complementation (<i>K</i><sub>D</sub> = 700
pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9,
we demonstrate that HiBiT can be rapidly and efficiently integrated
into the genome to serve as a reporter tag for endogenous proteins.
Without requiring clonal isolation of the edited cells, we were able
to quantify changes in abundance of the hypoxia inducible factor 1A
(HIF1α) and several of its downstream transcriptional targets
in response to various stimuli. In combination with fluorescent antibodies,
we further used HiBiT to directly correlate HIF1α levels with
the hydroxyproline modification that mediates its degradation. These
results demonstrate the ability to efficiently tag endogenous proteins
with a small luminescent peptide, allowing sensitive quantitation
of the response dynamics in their regulated expression and covalent
modifications
Deciphering the Cellular Targets of Bioactive Compounds Using a Chloroalkane Capture Tag
Phenotypic screening of compound
libraries is a significant trend in drug discovery, yet success can
be hindered by difficulties in identifying the underlying cellular
targets. Current approaches rely on tethering bioactive compounds
to a capture tag or surface to allow selective enrichment of interacting
proteins for subsequent identification by mass spectrometry. Such
methods are often constrained by ineffective capture of low affinity
and low abundance targets. In addition, these methods are often not
compatible with living cells and therefore cannot be used to verify
the pharmacological activity of the tethered compounds. We have developed
a novel chloroalkane capture tag that minimally affects compound potency
in cultured cells, allowing binding interactions with the targets
to occur under conditions relevant to the desired cellular phenotype.
Subsequent isolation of the interacting targets is achieved through
rapid lysis and capture onto immobilized HaloTag protein. Exchanging
the chloroalkane tag for a fluorophore, the putative targets identified
by mass spectrometry can be verified for direct binding to the compound
through resonance energy transfer. Using the interaction between histone
deacetylases (HDACs) and the inhibitor, Vorinostat (SAHA), as a model
system, we were able to identify and verify all the known HDAC targets
of SAHA as well as two previously undescribed targets, ADO and CPPED1.
The discovery of ADO as a target may provide mechanistic insight into
a reported connection between SAHA and Huntington’s disease
Deciphering the Cellular Targets of Bioactive Compounds Using a Chloroalkane Capture Tag
Phenotypic screening of compound
libraries is a significant trend in drug discovery, yet success can
be hindered by difficulties in identifying the underlying cellular
targets. Current approaches rely on tethering bioactive compounds
to a capture tag or surface to allow selective enrichment of interacting
proteins for subsequent identification by mass spectrometry. Such
methods are often constrained by ineffective capture of low affinity
and low abundance targets. In addition, these methods are often not
compatible with living cells and therefore cannot be used to verify
the pharmacological activity of the tethered compounds. We have developed
a novel chloroalkane capture tag that minimally affects compound potency
in cultured cells, allowing binding interactions with the targets
to occur under conditions relevant to the desired cellular phenotype.
Subsequent isolation of the interacting targets is achieved through
rapid lysis and capture onto immobilized HaloTag protein. Exchanging
the chloroalkane tag for a fluorophore, the putative targets identified
by mass spectrometry can be verified for direct binding to the compound
through resonance energy transfer. Using the interaction between histone
deacetylases (HDACs) and the inhibitor, Vorinostat (SAHA), as a model
system, we were able to identify and verify all the known HDAC targets
of SAHA as well as two previously undescribed targets, ADO and CPPED1.
The discovery of ADO as a target may provide mechanistic insight into
a reported connection between SAHA and Huntington’s disease
Improved Deconvolution of Protein Targets for Bioactive Compounds Using a Palladium Cleavable Chloroalkane Capture Tag
The
benefits provided by phenotypic screening of compound libraries
are often countered by difficulties in identifying the underlying
cellular targets. We recently described a new approach utilizing a
chloroalkane capture tag, which can be chemically attached to bioactive
compounds to facilitate the isolation of their respective targets
for subsequent identification by mass spectrometry. The tag minimally
affects compound potency and membrane permeability, enabling target
engagement inside cells. Effective enrichment of these targets is
achieved through selectivity in both their rapid capture onto immobilized
HaloTag and their subsequent release by competitive elution. Here,
we describe a significant improvement to this method where selective
elution was achieved through palladium-catalyzed cleavage of an allyl-carbamate
linkage incorporated into the chloroalkane capture tag. Selective
tag cleavage provided robust release of captured targets exhibiting
different modes of binding to the bioactive compound, including prolonged
residence time and covalent interactions. Using the kinase inhibitors
ibrutinib and BIRB796 as model compounds, we demonstrated the capability
of this new method to identify both expected targets and “off-targets”
exhibiting a range of binding affinities, cellular abundances, and
binding characteristics