14 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
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
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
NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells
Protein-fragment
complementation assays (PCAs) are widely used
for investigating protein interactions. However, the fragments used
are structurally compromised and have not been optimized nor thoroughly
characterized for accurately assessing these interactions. We took
advantage of the small size and bright luminescence of NanoLuc to
engineer a new complementation reporter (NanoBiT). By design, the
NanoBiT subunits (i.e., 1.3 kDa peptide, 18 kDa polypeptide) weakly
associate so that their assembly into a luminescent complex is dictated
by the interaction characteristics of the target proteins onto which
they are appended. To ascertain their general suitability for measuring
interaction affinities and kinetics, we determined that their intrinsic
affinity (<i>K</i><sub>D</sub> = 190 μM) and association
constants (<i>k</i><sub>on</sub> = 500 M<sup>–1</sup> s<sup>–1</sup>, <i>k</i><sub>off</sub> = 0.2 s<sup>–1</sup>) are outside of the ranges typical for protein interactions.
The accuracy of NanoBiT was verified under defined biochemical conditions
using the previously characterized interaction between SME-1 β-lactamase
and a set of inhibitor binding proteins. In cells, NanoBiT fusions
to FRB/FKBP produced luminescence consistent with the linear characteristics
of NanoLuc. Response dynamics, evaluated using both protein kinase
A and β-arrestin-2, were rapid, reversible, and robust to temperature
(21–37 °C). Finally, NanoBiT provided a means to measure
pharmacology of kinase inhibitors known to induce the interaction
between BRAF and CRAF. Our results demonstrate that the intrinsic
properties of NanoBiT allow accurate representation of protein interactions
and that the reporter responds reliably and dynamically in cells
Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate
Bioluminescence methodologies have been extraordinarily
useful
due to their high sensitivity, broad dynamic range, and operational
simplicity. These capabilities have been realized largely through
incremental adaptations of native enzymes and substrates, originating
from luminous organisms of diverse evolutionary lineages. We engineered
both an enzyme and substrate in combination to create a novel bioluminescence
system capable of more efficient light emission with superior biochemical
and physical characteristics. Using a small luciferase subunit (19
kDa) from the deep sea shrimp <i>Oplophorus gracilirostris</i>, we have improved luminescence expression in mammalian cells ∼2.5
million-fold by merging optimization of protein structure with development
of a novel imidazopyrazinone substrate (furimazine). The new luciferase,
NanoLuc, produces glow-type luminescence (signal half-life >2 h)
with
a specific activity ∼150-fold greater than that of either firefly
(<i>Photinus pyralis</i>) or <i>Renilla</i> luciferases
similarly configured for glow-type assays. In mammalian cells, NanoLuc
shows no evidence of post-translational modifications or subcellular
partitioning. The enzyme exhibits high physical stability, retaining
activity with incubation up to 55 °C or in culture medium for
>15 h at 37 °C. As a genetic reporter, NanoLuc may be configured
for high sensitivity or for response dynamics by appending a degradation
sequence to reduce intracellular accumulation. Appending a signal
sequence allows NanoLuc to be exported to the culture medium, where
reporter expression can be measured without cell lysis. Fusion onto
other proteins allows luminescent assays of their metabolism or localization
within cells. Reporter quantitation is achievable even at very low
expression levels to facilitate more reliable coupling with endogenous
cellular processes
Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate
Bioluminescence methodologies have been extraordinarily
useful
due to their high sensitivity, broad dynamic range, and operational
simplicity. These capabilities have been realized largely through
incremental adaptations of native enzymes and substrates, originating
from luminous organisms of diverse evolutionary lineages. We engineered
both an enzyme and substrate in combination to create a novel bioluminescence
system capable of more efficient light emission with superior biochemical
and physical characteristics. Using a small luciferase subunit (19
kDa) from the deep sea shrimp <i>Oplophorus gracilirostris</i>, we have improved luminescence expression in mammalian cells ∼2.5
million-fold by merging optimization of protein structure with development
of a novel imidazopyrazinone substrate (furimazine). The new luciferase,
NanoLuc, produces glow-type luminescence (signal half-life >2 h)
with
a specific activity ∼150-fold greater than that of either firefly
(<i>Photinus pyralis</i>) or <i>Renilla</i> luciferases
similarly configured for glow-type assays. In mammalian cells, NanoLuc
shows no evidence of post-translational modifications or subcellular
partitioning. The enzyme exhibits high physical stability, retaining
activity with incubation up to 55 °C or in culture medium for
>15 h at 37 °C. As a genetic reporter, NanoLuc may be configured
for high sensitivity or for response dynamics by appending a degradation
sequence to reduce intracellular accumulation. Appending a signal
sequence allows NanoLuc to be exported to the culture medium, where
reporter expression can be measured without cell lysis. Fusion onto
other proteins allows luminescent assays of their metabolism or localization
within cells. Reporter quantitation is achievable even at very low
expression levels to facilitate more reliable coupling with endogenous
cellular processes
Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate
Bioluminescence methodologies have been extraordinarily
useful
due to their high sensitivity, broad dynamic range, and operational
simplicity. These capabilities have been realized largely through
incremental adaptations of native enzymes and substrates, originating
from luminous organisms of diverse evolutionary lineages. We engineered
both an enzyme and substrate in combination to create a novel bioluminescence
system capable of more efficient light emission with superior biochemical
and physical characteristics. Using a small luciferase subunit (19
kDa) from the deep sea shrimp <i>Oplophorus gracilirostris</i>, we have improved luminescence expression in mammalian cells ∼2.5
million-fold by merging optimization of protein structure with development
of a novel imidazopyrazinone substrate (furimazine). The new luciferase,
NanoLuc, produces glow-type luminescence (signal half-life >2 h)
with
a specific activity ∼150-fold greater than that of either firefly
(<i>Photinus pyralis</i>) or <i>Renilla</i> luciferases
similarly configured for glow-type assays. In mammalian cells, NanoLuc
shows no evidence of post-translational modifications or subcellular
partitioning. The enzyme exhibits high physical stability, retaining
activity with incubation up to 55 °C or in culture medium for
>15 h at 37 °C. As a genetic reporter, NanoLuc may be configured
for high sensitivity or for response dynamics by appending a degradation
sequence to reduce intracellular accumulation. Appending a signal
sequence allows NanoLuc to be exported to the culture medium, where
reporter expression can be measured without cell lysis. Fusion onto
other proteins allows luminescent assays of their metabolism or localization
within cells. Reporter quantitation is achievable even at very low
expression levels to facilitate more reliable coupling with endogenous
cellular processes
Validation of HTS hits.
<p>A) Bioluminescence activity of cells treated with MNS was measured at 12 hours post treatment and plotted as fold induction. Experiments were performed at least in triplicates (mean ± SEM). B) Representative western blots for Luciferase, cleaved Caspase 3 and PARP or β-Actin as loading control of D54 cells treated with (25 µM) MNS for 12 hrs. C) Bioluminescence activity of cells treated with increasing concentrations of CV3988 at 12 hours post treatment. Data are plotted as fold induction over values obtained from vehicle treated cells. Experiments were performed in triplicates (mean ± SEM). D and E). Bioluminescence activity was measured at various time points using 1833 (D) or D54 (E) cells treated with CV3988 (12.5 µM), Z-VAD (20 µM) or a combination of Z-VAD plus CV3988 for 24 hrs. Data are plotted as fold induction and experiments were performed in triplicates and plotted as mean ± SEM. F) Representative western blots of Luciferase, Caspase 3, PARP or β-actin were performed on lysates obtained from D54 cells. Cells were either treated with CV3988 (12.5 µM), pre-treated with Z-VAD (20 µM) or treated with Z-VAD and CV3988 for 12 hrs.</p