Temporally controlled targeting of 4-hydroxynonenal to specific proteins in living cells

Article abstract of DOI:10.1021/ja405400k

In-depth chemical understanding of complex biological processes hinges upon the ability to systematically perturb individual systems. However, current approaches to study impacts of biologically relevant reactive small molecules involve bathing of the entire cell or isolated organelle with excess amounts, leading to off-target effects. The resultant lack of biochemical specificity has plagued our understanding of how biological electrophiles mediate signal transduction or regulate responses that confer defense mechanisms to cellular electrophilic stress. Here we introduce a target-specific electrophile delivery platform that will ultimately pave the way to interrogate effects of reactive electrophiles on specific target proteins in cells. The new methodology is demonstrated by photoinducible targeted delivery of 4-hydroxynonenal (HNE) to the proteins Keap1 and PTEN. Covalent conjugation of the HNE-precursor to HaloTag fused to the target proteins enables directed HNE delivery upon photoactivation. The strategy provides proof of concept of selective delivery of reactive electrophiles to individual electrophile-responsive proteins in mammalian cells. It opens a new avenue enabling more precise determination of the pathophysiological consequences of HNE-induced chemical modifications on specific target proteins in cells.

Full text of DOI:10.1021/ja405400k

Communication
pubs.acs.org/JACS
Temporally Controlled Targeting of 4‑Hydroxynonenal to Specific
Proteins in Living Cells
Xinqiang Fang,†,§ Yuan Fu,†,§ Marcus J. C. Long,⊥ Joseph A. Haegele,† Eva J. Ge,† Saba Parvez,†
and Yimon Aye*,†,‡
†Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States
‡Department of Biochemistry, Weill Cornell Medical College, New York, New York 10065, United States
⊥Graduate Program in Biochemistry and Biophysics, Brandeis University, Waltham, Massachusetts 02454, United States
S
* Supporting Information
manner (neither to individual proteins nor subcellular
compartments).5
ABSTRACT: In-depth chemical understanding of com-
plex biological processes hinges upon the ability to
systematically perturb individual systems. However,
current approaches to study impacts of biologically
relevant reactive small molecules involve bathing of the
entire cell or isolated organelle with excess amounts,
leading to off-target effects. The resultant lack of
biochemical specificity has plagued our understanding of
how biological electrophiles mediate signal transduction or
regulate responses that confer defense mechanisms to
cellular electrophilic stress. Here we introduce a target-
specific electrophile delivery platform that will ultimately
pave the way to interrogate effects of reactive electrophiles
on specific target proteins in cells. The new methodology
is demonstrated by photoinducible targeted delivery of 4-
hydroxynonenal (HNE) to the proteins Keap1 and PTEN.
Covalent conjugation of the HNE-precursor to HaloTag
fused to the target proteins enables directed HNE delivery
upon photoactivation. The strategy provides proof of
concept of selective delivery of reactive electrophiles to
individual electrophile-responsive proteins in mammalian
cells. It opens a new avenue enabling more precise
determination of the pathophysiological consequences of
HNE-induced chemical modifications on specific target
proteins in cells.
Current strategy to probe the effects of electrophilic small
molecule modulators, such as HNE, on individual proteins or
pathways of interest involves treatment of the entire cell,1−5
oftentimes with nonphysiological quantities of the reactive
electrophile (Figure 1a). The strategy clearly leads to a large
Figure 1. (a) Current approach to study impacts of reactive
electrophiles on protein of interest (POI) involves treatment of entire
cell/proteome with a reactive entity, unavoidably leading to multiple
off-target effects. Red, HNE. (b) A new approach to interrogate with
biochemical specificity effects of electrophiles on individual proteins.
The strategy focuses on a time-resolved target-specific delivery of
electrophiles to a single POI against the entire cell/proteome. The
synthetic delivery platform “dormant electrophile” shown has three
modules: gray, high affinity/specificity ligand to POI; blue, flexible
linker; pink, inert warheada caged HNE.
his Communication demonstrates a new way to study
Timpacts of reactive small molecules, such as electrophilic
lipids, on specific proteins in living cells. A well-established
example of a reactive lipid is 4-hydroxynonenal (HNE), a key
end-product of polyunsaturated fatty acids (PUFAs) perox-
idation.1 HNE can result either from reactive oxygen species-
initiated peroxidation of PUFAs, or from lipooxygenases-
mediated peroxidation of PUFAs.2 HNE was classified for many
years as a nonspecific, highly reactive entity implicated in
causing neurodegeneration in Alzheimer’s disease.3 However,
mounting evidence has highlighted HNE’s cell signaling
roles.1,4 Although these pioneering studies have opened up
new lines of investigations, progress is hindered because
presently there exists no method to study impact of this
reactive Michael acceptor in a temporally controlled or targeted
amount of noise due to off-target effects. Whole proteome or
isolated organelle incubation with micromolar to millimolar
HNE also precludes rigorous assessments on HNE’s regulatory
capacity where physiological amounts (<0.1−1 μM) are
thought to trigger specific responses.1,6 In fact, there exist
>3400 reports on pathophysiological functions of HNE.7
Received: May 29, 2013
Published: September 9, 2013
© 2013 American Chemical Society
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Myriad regulatory proteins have been implicated to participate
in HNE-induced signaling at submicromolar HNE concen-
trations. However, whether the observed responses stem from
on- vs off-target effects of HNE remains unclear.
synthetic fluorophores.9 (3) Expression vectors encoding
20,000 human and 15,000 mouse genes with HaloTag are
commercially available,10 thus in principle, any desired POI can
be investigated using this platform. In the design of the HNE-
delivery module, we were encouraged by the previously
reported 1-alkoxyanthraquinone-based aldehyde caging strategy
where photoinducible HNE release has been demonstrated in
aqueous methanol.11 We thus aimed to establish our new
approach using this photocaged-HNE system. HaloTag
PreHNE (HtPH, 1) and HaloTag PreHNE-alkyne (HtPHA,
2) (Figure 2) were thus synthesized (Schemes S1 and S2), and
evaluated for the time-dependent HNE liberation upon
photoactivation (Figure S1). Photolysis (4 W, 0.6 mW/cm2,
365 nm) promoted HNE release that reached saturation within
30 min at 37 °C for both HtPH and HtPHA (Figure S1b).
Keap1 (Kelch-like, ECH-associated protein 1), a key
electrophile-sensing regulatory protein in humans, was chosen
to test the idea of selective targeting. Keap1 is a central
regulator of a major antioxidant signaling pathway that
responds to electrophilic stress and promotes cell survival.12
Keap1 is highly susceptible, by virtue of its nucleophilic
cysteines, to post-translational modifications by reactive
electrophiles including HNE.13 These chemical modulations
are thought to in turn influence downstream cell signaling.
Using HaloKeap1 as a model protein, we first showed the
designed HNE-delivery platforms were biocompatible. To
demonstrate cell permeability and affinity of HtPH (1) to
HaloKeap1, we used a commercial chloroalkane ligand-
conjugated TMR dye (HaloTag TMR) that binds selectively
to Halo-tagged proteins labeling them with the TMR red dye.
Initially, treatment of COS-1 cells overexpressing HaloKeap1
with HaloTag TMR resulted in cytosolic localization of the red
dye post wash out (Figure 3a-1). Since Keap1 localizes to the
To overcome this loss in biochemical specificity, we
conceived a biocompatible chemical platform that would for
the first time enable a controlled perturbation of a specific
electrophile-responsive protein of interest (POI) with HNE in
living cells. The platform is designed such that the amount of
reactive electrophile liberated is equivalent to the concentration
of POI, conditions that would mimic endogenous signaling
directed to one target protein. Shown schematically in Figure
1b, the first step involves a high specificity and affinity ligand
(gray triangle) binding the POI. For POIs with no known
ligands, known functionalizable protein tags may be fused to
the POI. The POI or the tag fused to POI is tethered via a
flexible linker (blue line) to a caged HNE (pink sphere). The
cell-permeable small molecule should remain bound to the POI
while the unbound molecule can be washed out, so that at the
point of photoactivated release, HNE is only available in the
cell at concentrations similar to POI. At a precise time, a
nonintrusive stimulation with external light activates a rapid and
localized HNE generation.
Unlike free radicals, longer half-lives of reactive aldehydes
such as HNE render them predisposed to diffusion from
original sites of production.8 However, because the targeted
platform ensures HNE release on or proximal to POI, and
because HNE is released in stoichiometric (or lesser) amounts
relative to POI, we hypothesized that adduction of an
electrophile-responsive POI by the liberated HNE would be
sufficiently rapid so that covalent conjugation would offset
potential diffusion. Effects arising from any leakage of the
liberated HNE to the surroundings are also likely averaged over
the entire proteome.
We first set about synthesis of time-resolved targeted HNE
delivery systems (Figure 2). To target HNE to a specific POI,
Figure 3. (a) Designed ligand is cell-permeable. COS-1 cells
transiently overexpressing cytosolic HaloKeap1 were treated with
either (1) 5 μM HaloTag TMR alone, or (2) 50 μM HtPH (1, Figure
2) for 1 h prior to 5 μM HaloTag TMR. Scale bars, 20 μm. (b)
Designed ligand is non-cytotoxic. Representative data from flow
cytometry dye exclusion assays on viability of HtPHA (2, Figure 2)-
treated (3 h) COS-1 cells. Error bars are SD (N = 3).
Figure 2. Photoinducible HNE-targeting on HaloTag fusion proteins
made possible by HaloTag PreHNE, HtPH (1, R = CH2CH3), and
HaloTag PreHNE-alkyne, HtPHA (2, R = C2H). Also see Figure S1.
Color code refers to the schematic representation of dormant
electrophile in Figure 1b.
HaloTag genetic encoding strategy9 was selected. This enzyme
is an engineered bacterial dehalogenase which is able to react
with chloroalkanes to form a covalent enzyme-bound
intermediate, via SN2 attack of an Asp at an alkyl chloride
function. However, unlike in the wild type dehalogenase, the
intermediate O-alkyl ester is hydrolytically stable.9 We chose
HaloTag because (1) O-alkyl ester bond formation between the
Asp residue of Halo and the chloroalkane ligand is irreversible
and highly specific. (2) A range of sterically demanding groups
are tolerated on the chloroalkane ligand, demonstrated by
cytosol,12 this is consistent with TMR selectively labeling the
functional HaloKeap1 fusion protein. Addition of 50 μM HtPH
(1) for 1 h prior to addition of HaloTag TMR prevented
labeling of the COS-1 cells overexpressing HaloKeap1 (Figure
3a-2). This result showed that HtPH (1) can saturate to the
Halo domain within HaloKeap1 in the cells rendering, to the
limit of detection, all HaloKeap1 unable to be dye-labeled. This
observation also suggests that previously unrecognized strategy
to deliver HNE to a subcellular compartment such as cytosol
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can be possible. In addition, analysis of cell morphology and
flow cytometry dye exclusion assays of COS-1 cells subsequent
to HtPH (1) and HtPHA (2) treatments and propidium iodide
staining, showed both of our compounds had a low effect on
viability over this period (Figure 3b).
Our next step was to test the possibility of targeted delivery
of HNE in cells. To enable quantitation of % targeting
efficiency in cells, we used a simple setup that allows separation
of the recognition domain (Halo) from the fused target protein
(Keap1) (Figure 4a). We made modifications to the
(Figure 4a). Click coupling of Cy5-azide dye to the terminal
alkyne was subsequently performed. The alkyne moiety could
either exist within the unreacted HtPHA (2) conjugated to
Halo, within released HNE-alkyne adducted with Cys-rich
Keap1, or within liberated HNE-alkyne conjugated to other
cellular proteins. The data showed efficient transfer of Cy5
fluorescence signal to a band corresponding to MW of Keap1
(Figure 4c, lane 3). There was negligible fluorescence observed
in the bulk cellular proteins. In controls with either no light
(lane 2) or no Tev (lane 4) treatment, the signal respectively
located to Halo or Halo−Keap1. We also showed that delivery
of HNE to Keap1 required an interaction between HtPHA (2)
and Halo domain by addition of an unsubstituted chloroalkane
ligand (3) (Scheme S1) (i.e., without the pink warhead in
Figure 1b) to the cells prior to addition of HtPHA (2). This
procedure would block the Halo domain binding site similarly
to the experiment shown in Figure 3a and render Halo unable
to bind to HtPHA (2). Under these conditions, no Cy5 signal
was observed (Figure S4b, lane 7). As a corollary, the use of
PreHNE-alkyne (PHA) (4) (Scheme S3), which does not have
a chloroalkane recognition motif, showed no Cy5 fluorescence
(Figure S4b, lane 5).
The data from independent replicates revealed that 55
13% of Cy5 signal was transferred from Halo to Keap1
subsequent to photoactivation (Figures 4c and S4). The fact
that PreHNE-alkyne is present in stoichiometric amounts to
Keap1 prior to light exposure, as suggested by the blocking
experiments (Figure 3a, Figure S4b, lane 7, Figure S5b, lane 4),
further indicates that the 55% 13% of the total POI in the
proteome is HNEylated using the targeted approach. The data
is consistent with directed delivery of HNE from Halo to
Keap1. We note that the observed selective targeting was not
due to artifacts from high levels of overexpression of Halo−
Keap1 since SDS-PAGE analysis (Figure 4b) showed many
bands of equal or greater intensity to Keap1 in the lysate,
underscoring that delivery is specific to Keap1. We also note
that because of the bicistronic expression system (Figure S2)
used in our gene delivery, DsRed protein was simultaneously
overexpressed at either equal or higher concentrations than
Keap1.15 No Cy5 signal was observed around 28 kDa, MW of
DsRed (Figure 4c), consistent with absence of nonspecific
targeting.
We next decided to test the concept on tumor suppressor
protein PTEN (phosphatase and tensin homologue), an
upstream antagonist of Akt-oncogenic signaling.16 This protein
represents a sterner test of our methodology because unlike
Keap1, reports on sensitivity of PTEN to electrophiles such as
HNE are limited.17 HNE-directed targeting to Halo−PTEN
(Figure S3) expressed in HEK cells was found to yield a similar
outcome (Figure S5). As in Keap1, results from control
experiments with Halo−PTEN suggested targeted delivery.
The lower targeting efficiency of 28 10% likely reflects the
lower electrophile sensitivity of PTEN over Keap1. We also
chose to compare our method to conventional approach of
bathing the cells with HNE. HNE-alkyne (HA) (5) (Scheme
S4) was synthesized and added to HEK cells at 25 μM, a
concentration previously used to study PTEN signaling
triggered by HNE.17b Unsurprisingly, global treatment showed
nonspecific targeting by the reactive enal (Figure S5b, lane 5).
In summary, we have been able to demonstrate selective
delivery of a reactive, biologically relevant small molecule to
specific electrophile-responsive proteins of interest. The
strategy was demonstrated in living cells on targeting Keap1
Figure 4. (a) Directed HNE targeting strategy demonstrated by
selective Keap1 targeting in living cells. COS-1 cells transiently
expressing DsRed and Halo−Keap1 (Figure S2) were treated with 25
μM HtPHA (2) for 2.5 h. Subsequent to exposure to a 4 W, 0.6 mW/
cm2, 365 nm lamp (20 min), cells were lysed and lysate was treated
with Tev protease, followed by Cy5-Click assay reagents. This setup
enables quantitation of % targeting efficiency: Cy5 signal intensity on
Halo band prior to light exposure serves as the input, and that on the
Keap1 band post photouncaging and Tev-cleavage serves as the
output. (b) A representative resultant SDS-PAGE with Coomassie
staining. (c) In-gel Cy5-fluorescence analysis of the same SDS-PAGE.
Lane 1, MW ladder; lane 2, cells not exposed to light; lane 3, cells
exposed to light; lane 4, no Tev cleavage in lysate. *, Halo−Keap1
(104 kDa); +, Keap1 (70 kDa); ×, Halo (33 kDa). From independent
replicates, average Cy5 signal intensity transfer from Halo band before
light exposure, to Keap1 band after light exposure, is quantitated to be
55 13%.
HaloKeap1 construct such that a Tev protease recognition
site was placed in between the two domains (“Halo−Keap1”,
Figure S2). The positioning of the Tev site permits selective
Tev-protease-mediated cleavage and separation of Halo and
Keap1 subsequent to cell lysis, thus allowing Keap1-targeted in-
cell delivery of HNE-alkyne from HtPHA (2) to be assessed
using click chemistry.14 The physical separation of the HaloTag
from the POI is necessary because alkyne function reports on
the presence of both HNE-precursor covalently bound to Halo
as well as the liberated HNE (Figure 4a). The Tev-cleavage
setup enables quantitation of % targeting efficiency, where the
Cy5 signal intensity on the Halo band after the Tev-cleavage
from the sample not exposed to light serves as an input signal,
while that on POI (Keap1) band after the Tev-cleavage from
the sample exposed to light is the output.
The cells were treated with 25 μM HtPHA (2) for 2.5 h and
exposed to 4 W, 0.6 mW/cm2, 365 nm lamp for 20 min. Cell
lysis was followed by Tev cleavage to separate Halo and Keap1
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ASSOCIATED CONTENT
* Supporting Information
■
(14) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B.
Angew. Chem., Int. Ed. 2002, 41, 2596.
S
Experimental details; small-molecule synthesis procedure and
characterization data; and additional biochemical data. This
material is available free of charge via the Internet at http://
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AUTHOR INFORMATION
Corresponding Author
ya222@cornell.edu
■
Author Contributions
§X.F. and Y.F. contributed equally.
Notes
The authors declare no competing financial interest.
L.; Gems, D.; Nystrom, T.; Belousov, V.; Schumacker, P. T.;
̈
Winterbourn, C. C. Cell. Metab. 2011, 13, 361.
ACKNOWLEDGMENTS
■
This research was supported by the Cornell University junior
faculty startup funds and the Milstein New Faculty Fellowship
(to Y.A). Y.A. thanks the Howard and Abby Milstein
Foundation. M.J.C.L acknowledges the Howard Hughes
Medical Institute for an international predoctoral student
fellowship. J.A.H. acknowledges the CBI training grant
(T32GM008500, PI - Steven E. Ealick). We thank Dr. Anthony
Condo, Jr., for help with LDI mass characterizations on
compounds 1, 2, and 4 which were carried out at the Cornell
Center for Materials Research Shared Facilities supported
through NSF MRSEC program (DMR-1120296). Cornell
Biotech Resource Center Imaging Facility is acknowledged
for the use of the confocal fluorescence microscope.
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