Identification of a Bromodomain-like Region in 15-Lipoxygenase-1 Explains Its Nuclear Localization

Article abstract of DOI:10.1002/anie.202106968

Lipoxygenase (LOX) activity provides oxidative lipid metabolites, which are involved in inflammatory disorders and tumorigenesis. Activity-based probes to detect the activity of LOX enzymes in their cellular context provide opportunities to explore LOX biology and LOX inhibition. Here, we developed Labelox B as a potent covalent LOX inhibitor for one-step activity-based labeling of proteins with LOX activity. Labelox B was used to establish an ELISA-based assay for affinity capture and antibody-based detection of specific LOX isoenzymes. Moreover, Labelox B enabled efficient activity-based labeling of endogenous LOXs in living cells. LOX proved to localize in the nucleus, which was rationalized by identification of a functional bromodomain-like consensus motif in 15-LOX-1. This indicates that 15-LOX-1 is not only involved in oxidative lipid metabolism, but also in chromatin binding, which suggests a potential role in chromatin modifications.

Full text of DOI:10.1002/anie.202106968

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How to cite:
International Edition: doi.org/10.1002/anie.202106968
German Edition: doi.org/10.1002/ange.202106968
Activity-Based Probes
Hot Paper
Identification of a Bromodomain-like Region in 15-Lipoxygenase-
1 Explains Its Nuclear Localization
Deng Chen+, Zhangping Xiao+, Hao Guo, Dea Gogishvili, Rita Setroikromo,
Petra E. van der Wouden, and Frank J. Dekker*
Abstract: Lipoxygenase (LOX) activity provides oxidative
lipid metabolites, which are involved in inflammatory disor-
ders and tumorigenesis. Activity-based probes to detect the
activity of LOX enzymes in their cellular context provide
opportunities to explore LOX biology and LOX inhibition.
Here, we developed Labelox B as a potent covalent LOX
inhibitor for one-step activity-based labeling of proteins with
LOX activity. Labelox B was used to establish an ELISA-
based assay for affinity capture and antibody-based detection
of specific LOX isoenzymes. Moreover, Labelox B enabled
efficient activity-based labeling of endogenous LOXs in living
cells. LOX proved to localize in the nucleus, which was
rationalized by identification of a functional bromodomain-
like consensus motif in 15-LOX-1. This indicates that 15-LOX-
1 is not only involved in oxidative lipid metabolism, but also in
chromatin binding, which suggests a potential role in chroma-
tin modifications.
leukotrienes (LTs). The activity of LOX enzymes is inves-
tigated by changes in the composition of the metabolites
resulting from their activity. The LOX lipid metabolites play
key roles in various physiological processes involved in
inflammatory diseases,[3] cancers and other disease.[4–7] There-
fore, small molecule modulation of LOX activity has been
explored in drug discovery. However, until now, it remained
difficult to investigate the activity and inhibition of specific
endogenous LOX isoenzymes, which limits progress in drug
discovery.
Currently, there are several methods available to study
LOX activity. A common strategy is the identification and
quantification of metabolites that originate from LOX
activity.[8,9] Despite its power, this method does not enable
distinction of the activity of LOX enzymes with similar
substrate preferences, such as 15-LOX-1 and 15-LOX-2.[10]
Moreover, it does not allow to distinguish the subcellular
localization of LOX activity. Nevertheless, the subcellular
localization is important for LOX activity, as exemplified by
the activity of 5-LOX. Upon cell stimulation, 5-LOX traffics
and binds to 5-lipoxygenase-activating protein (FLAP), which
is an integral nuclear envelope protein essential for 5-LOX
mediated leukotriene A4 (LTA4) production.[11] Expounding
the relationship between LOXs intracellular localization and
activity would promote the understanding of LOX functions.
Moreover, the possibility to identify the inhibitory selectivity
among endogenous LOX isoenzymes will facilitate our
understanding of LOX inhibition and will thus facilitate drug
discovery. Therefore, we aim to develop novel tools to
investigate the activity of endogenous LOX isoenzymes to
advance LOX-oriented research and drug discovery.
In past decades, activity-based protein profiling (ABPP)
has been employed as a powerful tool[12] facilitating studies on
the functions of enzymes in their cellular context[13] and
investigations of small molecule inhibitors.[14] Although
ABPP of oxidoreductases gained relatively little attention,
several ABPs have been developed for this group of
enzymes,[15] such as cytochrome P450,[16] monoamine oxidas-
es[17] and flavin monooxygenases.[18] Nevertheless, oxidore-
ductases remain challenging targets for ABPs. Previously, our
group developed the first activity-based probe (ABP) for
enzymes with lipoxygenase activity.[19] The probe N144
enables two-step labeling of lipoxygenase activity by the use
of a bis-alkyne as a lipoxygenase reactive functionality, and an
alkene functionality as the reactive tag for subsequent
biotinylation of alkene labeled proteins. However, the two-
step labeling proved to be inconvenient and not compatible
with cellular imaging studies. Here, we developed a probe for
Introduction
Small molecule modulation of enzyme activity is an
important strategy in drug discovery. However, measurement
of the activity of specific isoenzymes from the relevant
endogenous sources remains challenging. This is particularly
true for Lipoxygenases (LOXs), which is a group of enzymes
known to be involved in oxidative metabolism of polyunsa-
turated fatty acids (PUFAs),[1] such as arachidonic acid (AA),
and linoleic acid (LA). In humans, LOXs are classified
according to the position of O2 insertion in AA as 5-LOX, 12-
LOX or 15-LOX.[2] LOX activity provides a range of
products, such as hydroperoxyl eicosatetraenoic acids
(HPETEs), hydroxy eicosatetraenoic acids (HETEs), and
[*] D. Chen,[+] Z. Xiao,[+] H. Guo, D. Gogishvili, R. Setroikromo,
P. E. van der Wouden, Prof. Dr. F. J. Dekker
Department Chemical and Pharmaceutical Biology
Groningen Research Institute of Pharmacy (GRIP)
University of Groningen
Antonius Deusinglaan 1, 9713 AV Groningen (The Netherlands)
E-mail: f.j.dekker@rug.nl
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202106968.
ꢀ 2021 The Authors. Angewandte Chemie International Edition
published by Wiley-VCH GmbH. This is an open access article under
the terms of the Creative Commons Attribution License, which
permits use, distribution and reproduction in any medium, provided
the original work is properly cited.
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one-step labeling ABP of LOX activity that we employed to
investigate isoenzyme selective inhibition by small molecule
inhibitors and to visualize the subcellular localization of LOX
activity. The observed 15-LOX-1 localization in the nucleus
enabled identification of a bromodomain-like region in 15-
LOX-1 that directly interacts with acetylated histone H3. The
presence of an epigenetic reader domain in 15-LOX-1 could
also play a role in epigenetics and chromatin remodeling.
Results
Labelox B (3) is a potent covalent inhibitor for 15-LOX-1.
We developed a concise two-step synthesis route to prepare
probes for one-step activity-based labeling of LOX, which
contains a bis-alkyne part for LOX labeling and a biotin tag
for detection. In brief, an alkyne-alcohol was linked to
propargyl halide (Cl or Br) using a CuI-catalyzed Sonogashira
cross-coupling reaction followed by biotinylation of the
alcohol using EDCI and HOBt as reagents to provide the
desired biotinylated bis-alkyne probes. The biotinylated bis-
alkynes were subjected to 15-LOX-1 binding studies using
a 15-LOX-1 activity assay based on the UV-absorbance
change for linoleic acid conversion (Figure 1a). The potency
was estimated using inhibiting concentration 50% (IC50)
values determined after fixed pre-incubation times. Com-
pound 1 proved to have an intermediate potency with an IC50
around 40 mM. The LOX inhibitory potency was lost upon
methyl branching of the propargyl methylene linker as found
in compound 2. Also, extending the propargyl methylene
linker to two or three carbon atoms, as found in 4 and 5,
provided a loss of LOX inhibitory potency. Extending the
methylene linker to 4 carbon atoms, as found in compound 7,
provided a regain in potency. Next, we evaluated the effect of
the extension of the terminal ethyl substitution of inhibitor 1.
Extending the terminal ethyl to a pentyl, as found in inhibitor
3 provided a major improvement of the IC50 to reach 0.62 mM
(Figure 1c and Table 1). Combination of the terminal pentyl
with extensions of the propargyl methyl linker as found in 6
and 8 did not improve the potency. To further assess the mode
of binding, we generated Lineweaver–Burk plots for inhibitor
3 (Figure 1d) as well as inhibitors 1, 7, and 8 (Figure S1a),
which indicate non-competitive LOX inhibition in which the
Km is constant and the Vmax decreases with the inhibitor
concentration. These observations are consistent with our
observations for the previously described bis-alkyne probe
N144, which also showed non-competitive inhibition on 15-
LOX-1.[19]
Figure 1. Development and characterization of the activity-based probe
for lipoxygenases. a) Chemical structure of Labelox B (3). b) Schematic
representation of ABPs covalently labeling LOXs. c) IC50 curve for
Labelox B (3). Data are meanÆSME. nꢀ3 independent experiments.
d) Lineweaver–Burk plots for 15-LOX-1 inhibition by Labelox B (3).
Data are mean, n=2 independent experiments.
Subsequently, Kitz-Wilson analysis was performed to
distinguish the equilibrium binding constant (Ki) from the
rate of covalent inactivation (ki). The different bis-alkynes
provided remarkably constant kiꢀs that varied by a factor 3 or
less, whereas the Kiꢀs varied by a factor 200 (Table 1).
Remarkably, inhibitor 3 provided a Ki of 0.5 mM, which is
a 100-fold improvement compared to inhibitor 1. Also
comparing the Ki of 3 to the Ki of the previously reported
probe N144, indicates an improvement by one or two orders
of magnitude. Improved non-covalent binding is an important
characteristic for activity-based labeling because it implies
improved specific recognition properties to the target of
interest. Therefore, inhibitor 3 has been used for further
development of a probe for activity-based labeling of lip-
oxygenase activity with 15-LOX-1 as a reference enzyme. For
convenient discussion, we named inhibitor 3, “Labelox B”.
Labelox B efficiently labels endogenous LOXs. Lipoxy-
genase labeling by Labelox B was investigated using cell-
based assays. As a first step, labeling efficiency was estimated
in intact RAW 264.7 macrophage cells that were treated with
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Table 1: Structure–activity relationship studies for inhibition of 15-LOX-1 activity.
Structure
Probe
R
IC50 [mM]
ki [minÀ1
]
Ki [mM]
Value
38.2
+ Error
À Error
1
2
9.2
7.4
0.0136Æ0.0061
116.44Æ19.48
>100
N/A
N/A
N/A
N/A
Labelox B (3)
0.6
0.2
0.1
0.0191Æ0.0038
0.0093Æ0.0031
0.0066Æ0.0047
N/A
0.50Æ0.14
16.58Æ3.29
38.72Æ6.33
N/A
4
5
6
7
11.1
19.8
>100
>100
2.3
1.9
3.6
3.0
N/A
N/A
N/A
N/A
N/A
N/A
8
>100
>100
>100
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
9
10
Labelox B in concentrations ranging between 1 mM and
100 mM with incubation times between 0.5 h and 6 h. Western
blotting indicated that Labelox B labeled proteins with a size
corresponding to the mass of endogenous LOXs in a dose and
time-dependent manner (Figure 2a,b). This indicates that
Labelox B is sufficiently cell-permeable to label endogenous
LOX in intact cells effectively in half an hour at a concen-
tration of 25 mM. As a next step, a sandwich ELISA assay was
Figure 2. Labelox B labels endogenous LOXs in a dose- and time-dependent manner. a,b) Western blot analysis of Labelox B labeled LOXs in
intact RAW 264.7 for indicated doses and time points. Biotinylation was visualized by western blotting, and b-actin was employed as a loading
control. c) Sandwich ELISA assays to capture lipoxygenase isoenzymes and to monitor biotinylation by Labelox B. Samples excluding Labelox B
were used as negative controls. Data are meanÆs.d., nꢀ3 independent experiments. d) Dose-dependent labeling by Labelox B in living cells.
A549 cells were treated with Labelox B for 1 h at 378C. Then, the cells were fixed and permeabilized with methanol. The labeled LOXs were
visualized with Cy2-conjugated streptavidin. The pictures were captured by a Leica DM4000b fluorescence microscope.
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established in order to find proof that probe Labelox B is
LOX inhibitors. However, metabolite analysis does not
indeed labeling proteins with lipoxygenase enzyme activity.
Towards this aim, A549 cell lysates were treated with sub-
micromolar to micromolar concentrations of Labelox B.
Subsequently, the endogenous LOXs were captured on an
anti-LOX antibody precoated 96-well plate followed by
detection of Labelox B mediated biotinylation using HRP-
conjugated streptavidin. The ELISA data showed that Label-
ox B efficiently labeled four lipoxygenase isoenzymes, namely
5-LOX, 12-LOX, 15-LOX-1, and 15-LOX-2, with an effective
concentration 50% (EC50) around 3.5 mM (Figure 2c), thus
creating an ABP-based ELISA assay for all LOX isoenzymes.
Furthermore, probe Labelox B enabled visualization of
localized LOX activity in living cells (Figure 2c). A 549 cells
were treated with different doses of Labelox B. After fixation
and permeabilization, biotin-tagged LOXs were visualized by
Cy2-conjugated streptavidin. The fluorescence intensity in-
creased with increasing doses of Labelox B and labeling was
observed throughout the cells in the cytoplasm and the
nucleus of A549. Altogether, this indicates that Labelox B
labels cellular proteins in a time and concentration-dependent
manner, that it labels all human lipoxygenase isoenzymes and
that labeling can be visualized cells using fluorescence
microscopy.
enable the distinction of 15-LOX-1 and 15-LOX-2 activity.
Here, we employed the ABP-based ELISA assay to study the
specificity of LOX inhibitors among LOX isoenzymes. Three
well-known LOX inhibitors were employed to gain insight in
the performance of this assay. The first inhibitor is Baicalein,
which is a redox inhibitor for LOXs[20,21] with an IC50 of less
than 4 mM in vitro. The second inhibitor is PD146176, which is
a 15-LOX inhibitor[22] with micromolar potency. The third
inhibitor is Zileuton,[23] which is a 5-LOX inhibitor that is
currently on market for the maintenance treatment of asthma.
The inhibitors were evaluated in the linoleic acid conversion
assay using recombinant 15-LOX-1 produced in E.coli (Fig-
ure 3A). In this assay, PD146176 provided inhibition with IC50
around 4 mM and a Hill-slope of 1, whereas Baicalein
provided a similar IC50 value with a much higher Hill-slope.
We note that Hill-slopes deviating from 1 indicate binding
behavior deviating from a 1:1 non-covalent interaction. For
Baicalein, the deviating Hill-slope might be connected to
redox inhibition of 15-LOX-1 activity. Inhibitors PD147176,
Baicalein, and Zileuton were also evaluated in the ABP-
based ELISA assay conducted with recombinant 15-LOX-
1 (Figure 3B). We found that PD146176 efficiently attenuated
the Labelox B mediated LOXs labeling with an IC50 around
15 mM and a Hill slope around 1. Baicalein also attenuated 15-
LOX-1 activity in the ABP-based ELISA assay with a similar
IC50 value similar to the linoleic acid conversion assay,
Assessment of lipoxygenase inhibitor selectivity by the
ABP-based ELISA. Currently, the analysis of LOX metab-
olites is a common strategy to determine the selectivity of
Figure 3. ABP-based ELISA assay is a fast and convenient method to estimate inhibitory selectivity among LOX isoenzymes. A) Inhibition of
recombinant 15-LOX-1 catalyzed linoleic acid conversion in a UV absorbance assay. B) Inhibition of recombinant 15-LOX-1 ABP-labeling using the
ABP-based ELISA assay. C) ABP-based ELISA assay with A549 cell lysate reveals the distinct specificity of LOX inhibitors among LOX isoenzymes.
A549 cell lysate was treated with a serial dilution of the respective inhibitor. Subsequently, samples were labeled by treatment with 1 mM of
Labelox B for 1 min. EDTA then efficiently stopped the reaction (Figure S5). Data are meanÆs.d., nꢀ3 independent experiments.
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however here the Hill-slope proved to be lower than 1. We
anticipate that 15-LOX-1 inhibition by redox activity rather
than non-covalent interaction causes a dissimilarity between
both assays.
isoenzymes 12-LOX, 15-LOX-1, and 15-LOX-2 were local-
ized in both cytoplasm and nuclei of RAW264.7 and A549
cells, whereas the 5-LOX localization seemed to be restricted
to the nucleus. Some interesting observations were made.
First, in RAW264.7 cells, a relatively large amount of 12-LOX
was observed that could not be colocalized with Labelox B
labeling, whereas other LOX isoenzymes do also not seem to
accumulate in the cytoplasm (Figure 4a). This could indicate
a pool of inactive 12-LOX in the cytoplasm that disappears
upon LPS stimulation. Next, we observed a change in
localization of the LOX isoenzymes that was the clearest in
RAW264.7 macrophages. Upon stimulation, the localization
becomes more pronounced in the nuclear membrane and
remarkably also on distinct locations in the nucleus. This
staining pattern for LOX localization has been observed
before for 5-LOX in macrophages.[11] Importantly, the change
in localization of all LOX isoenzymes colocalized with
a change in activity-based LOX labeling with probe Labelox
B (Figure 4). This indicates that the LOX isoenzymes are
activated at the nuclear membranes as described in literature
before.[11,24] However, LOX enzyme activity at distinct
nuclear localizations have not been described before. We
were intrigued by this finding and set out to rationalize the
LOX localization in the nucleus.
Subsequently, we employed the ABP-based ELISA assay
for cellular lipoxygenase enzymes by the use of isoenzyme
selective antibodies to study the inhibitory potency of lip-
oxygenase inhibitors. The results indicate that PD146176
inhibited 5-LOX, 12-LOX, and 15-LOX-1 with IC50 values of
around 17 mM, while 15-LOX-2 has an IC50 greater than
100 mM (Figure 3C, left). The Hill-slope for inhibition of 5-
LOX, 12-LOX and 15-LOX-1 by PD146176 is one (Table S1),
which suggests that binding between PD146176 and these
isoenzymes is a 1:1 binding event. The IC50 and Hill-slope for
PD146176 mediated inhibition of cellular 15-LOX-1 activity
is similar to that of recombinant 15-LOX-1 (IC50 = 15 mM).
Furthermore, this analysis indicated that PD146176 inhibited
ABP labeling of 5-LOX, 12-LOX, and 15-LOX-1 with equal
potency, whereas the ABP labeling of 15-LOX-2 is much less
affected. This indicates that PD146176 is selective among 15-
LOX-1 and 15-LOX-2 but not among 5-LOX, 12-LOX, and
15-LOX-1. In contrast to PD146176, Baicalein has less
influence on ABP-labeling of cellular 15-LOX-1 compared
to recombinant 15-LOX-1 and no selectivity among the
cellular LOX enzymes was observed (Figure 3C, middle).
This might be caused by the redox inhibitory mechanism,
which might be less effective in a cellular context in
comparison to the recombinant enzyme. For the 5-LOX
inhibitor, Zileuton,[23] we found inhibition of 5-LOX and 12-
LOX at high micromolar concentrations, whereas little or no
effect on 15-LOX-1 and 15-LOX-2 was observed(Figure 3C,
right). Thus the ABP-based assay enables determination of
the potency and selectivity of inhibitors among the endoge-
nous lipoxygenase isoenzymes.
Lipopolysaccharide stimulates nuclear LOX accumula-
tion. The ability of probe Labelox B to perform activity-based
labeling of endogenous LOX isoenzymes in mammalian cells
(Figure 2c) motivated us to investigate the utility to localize
cellular LOX activity using fluorescence microscopy. In this
study we note however, that we can not exclude the possibility
that Labelox B also labels non-LOX proteins and therefore
we refer in this part to putatively active LOX. By comparing
immunostaining of LOX isoenzymes with activity-based
labeling of total LOX activity, we aimed to gain more insight
in cellular localizations of LOX activity. Previously, it has
been shown that LOX isoenzymes are distributed over
cytoplasm and nuclei.[11,24–26] LOX enzyme activity has been
described to occur mainly on the nuclear membrane, where
arachidonic acid is released to act as a substrate for LOX
mediated oxygenation. Here, we investigated how the local-
ization and activation of cellular lipoxygenases changes under
the influence of lipopolysaccharide(LPS) as a stimulus in
RAW 264.7 macrophages and in A549 cells. We employed
confocal microscopy to colocalize active LOXs labeled with
Labelox B and LOX isoenzymes labeled by immunostaining.
The confocal pictures demonstrated that LOX antibody-
and Labelox B labeled LOXs were mainly colocalized,
suggesting that Labelox B was sufficient to visualize active
LOXs in living cells. Under non-stimulated conditions, the
15-LOX-1 has a bromodomain-like region that binds to
acetylated histones. In our search for a physiological explan-
ation for the distinct nuclear localization of LOX isoenzymes,
we focused on 15-LOX-1. The primary sequence of 15-LOX-
1 was analyzed for epigenetic reader domains based on the
presumption that chromatin binding could explain the distinct
nuclear localization of 15-LOX-1. We identified the consen-
sus motifs for
a bromodomain, Nx2or3D(E)x2or3Y(A/
V),[27–29] in the primary structure of 15-LOX-1. This bromo-
domain consensus sequence is also found in other bromodo-
main containing proteins, such as bromodomain and extra
terminal domain (BET) and histone acetyltransferase (HAT)
family members (Figure 5a, lower part). Such consensus
residues are also conserved in 15-LOX-1 across mammalian
species (Figure 5a, upper part). Among these residues,
asparagine is the most essential as it directly interacts with
acetylated lysine residues on histones.[27–29] Based on this
sequence analysis, we hypothesized that 15-LOX-1 binds to
histones depending on their acetylation level. Therefore, we
prepared two histone extractions with different levels of total
acetylation. Highly acetylated histones were extracted from
A549 cells treated with inhibitor CI994, a histone deacetylase
inhibitor, whereas lowly acetylated histones were prepared
from A549 cells treated with A485, a histone acetyltransfer-
ase inhibitor. An ELISA-binding assay demonstrated that
histones with higher acetylation levels had increased 15-LOX-
1 binding compared to histones with lower acetylation levels,
thus supporting the hypothesis that recombinant 15-LOX-
1 binds acetylated histones (Figure 5b). Moreover, confocal
microscope pictures showed that LOX labeling with probe
Labelox B colocalized with both acetylated H3 and H4
(Figure 5c,d). Upon LPS stimulation, more active LOXs were
recruited to acetylated H3 and H4, especially at the inner
nuclear envelope area (Figure 5c,d, white arrows). Further-
more, a microarray with acetylated histone H3 peptides was
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ing to H3K27Ac and
H3K56Ac acetylated pep-
tides with affinities in the
low micromolar range.
Discussion
Lipoxygenases
have
classically been recognized
as a group of enzymes in-
volved in lipid signaling in
inflammatory diseases and
oncology.[3,30,31] More re-
cently, a connection with
ferroptosis has been de-
scribed.[32–35] Despite their
importance in physiology, it
remained difficult to study
the activity of lipoxygenase
isoenzymes in their cellular
context.
Here, we described
a versatile probe, Labelox
B, for one-step ABP-label-
ing of LOX isoenzymes.
Labelox B is a cell mem-
brane permeable ABP-
probe (Figure 3a, b, and d)
that can be employed to
investigate
intracellular
LOX activity and LOX ac-
tivity in in cell lysates (Fig-
ure 3c). Using this proper-
ty, an ABP-based ELISA
was established to deter-
mine Labelox B ABP-label-
ing per LOX isoenzyme
and the inhibitory selectiv-
Figure 4. Colocalization analysis of active LOXs and total LOXs in a) RAW264.7 and b) A549. Most of Labelox
B labeled LOXs are colocalized with the antibody labeled LOXs, indicating that most of the lipoxygenases in
cells are catalytically active. However, in RAW264.7 cells, a large number of 12-LOXs are inactive. LPS
stimulation promotes the change of inactive 12-LOX to a catalytically active state. Pictures are representative
of three independent experiments.
performed to further characterize the binding of 15-LOX-1 to
acetylated histones. This microarray indicated that H3K27Ac
and H3K56Ac are binding to 15-LOX-1, whereas H3K4Ac
does not bind to 15-LOX-1 (Figure 5e). To confirm the
microarray data, a binding study between the respective
lysine-acetylated histone peptides and 15-LOX-1 was per-
formed using an ELISA formatted assay. A clear binding
curve was observed for binding of the peptides H3K27Ac and
H3K56Ac with 15-LOX-1 to provide a Kd of 1.13 and
1.65 mM, respectively. The Hill-slopes of these binding curves
equal 1 (Figure 5 f). For comparison, a binding study to the
corresponding non-acetylated histone H3 peptide showed
a lower binding affinity to 15-LOX-1 with a Kd of 22 mM
(Figure S6). Another comparison was made by determining
the binding affinity of a H3K4Ac peptide towards 15-LOX-
1 (Figure 5 f). The Kd for binding of H3K4Ac to 15-LOX-
1 exceeded the concentration range used in this assay because
no saturation of binding was observed at concentration up to
20 mM. Taken together, this indicates that 15-LOX-1 has
a functional bromodomain-like sequence that provides bind-
ity of LOX inhibitor among these LOX isoenzymes. Interest-
ingly, the 12/15-LOX selective inhibitor PD146176 provided
only selectivity among 15-LOX-1 and 15-LOX-2 but not for 5-
LOX and 12-LOX. The inhibition of 5-LOX and 12-LOX has
not been reported before,[22,26] whereas the lack of 15-LOX-2
inhibition is in line with the literature. The group of Anne
et al. reported that PD146176 did not inhibit the formation of
15-LOXs product formation in neutrophils which express
only 15-LOX-2.[10] This indicates that PD146176 is selective
among 15-LOX-1 and 15-LOX-2. Also, the lack of inhibitory
selectivity of Baicalein among LOX isoenzymes is in line with
the literature. Previous enzymatic assays confirmed that
Baicalein has potent inhibitory effects on both 5-, 12-, and
15-LOX.[21,36] Additionally, the antioxidative activity of
Baicalein was confirmed to contribute to the inhibition of
other oxidases, such as xanthine oxidase (XO).[37] Besides,
Baicalein has potent free radical scavenging effects, especially
À
on superoxide radicals (CO2 ).[37] Such effects attenuate
oxidative stress and protect cells from reactive oxygen species
(ROS).[38] Furthermore, we found that Zileuton inhibits both
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Figure 5. 15-LOX-1 contains a bromodomain-like region that specifically interacts with histone H3K27Ac and H3K56Ac, but not H3K4Ac or
H3K79Ac. a) The consensus motifs of a bromodomain, Nx2or3D(E)x2or3Y(A/V), are conserved in many other bromodomains containing proteins
as well as in 15-LOX-1 across mammalian species. b) ELISA assay for binding of 15-LOX-1 to histone coated plates with various levels of
acetylation. Data are meanÆs.d., *P<0.03, **P<0.003, ****P<0.0001, nꢀ3 independent experiments, Two-way ANOVA. c,d) Confocal
fluorescence microscopy analysis showed that Labelox B-labeled LOXs colocalized with acetylated histone H3 and H4. Pictures are representative
of three independent experiments. e) Acetylated histone H3 peptide array identified specific residues interacting with 15-LOX-1. Data are
meanÆs.d., **P<0.003, ***P<0.0003, n=3 readouts, One-way ANOVA. f) ELISA showed distinct binding affinity between 15-LOX-1 and diverse
H3Ac peptides. Data are meanÆSME, nꢀ3 independent experiments.
5-LOX and 12-LOX, but not 15-LOXs. This also coincides
with the previous findings. Zileuton attenuated the produc-
tion of 5-HETE, 5-HEPE, 12-HETE, but not 15-HETE.[39]
Although we were not able to find studies that directly
determined the binding of Zileuton to 5-LOX, cell-based
assays indicate that Zileuton potently inhibits the biosynthesis
of 5-LOX metabolites, like LTBs and cysteinyl leuko-
trienes.[40] However, this effect might be attributed to
inhibition of arachidonic acid release in addition to direct
effects on 5-LOX.[40] Thus the 5-LOX inhibition observed in
our assay is in line with previous reports on the 5-LOX
inhibitory potency. Taking this together, we conclude that the
ABP-based ELISA assay provides a fast and convenient
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complementary method to assess the selectivity of LOX
inhibitors.
The ABP-based ELISA assay may provide a better
Kd of H4K4Ac against the firstly identified bromodomains of
BRD2 and BRD4 are 4.3 and 3.1 mM, respectively.[45] The Kd
of BAZ2B bromodomain-H3K14Ac pair is 6.3 mM.[45] Even
bromodomain-acetylated peptide interactions with lower
affinity values were reported, such as a H3K36Ac peptide
interaction with the CREBBP bromodomain, which provides
a Kd of merely 122 mM,[45] whereas the crystal structure[43]
clearly demonstrates a specific interaction.
prediction of in vivo potency of LOX inhibitors compared
to activity assay on purified recombinant enzymes. This idea is
supported by observations made for the LOX inhibitor
Baicalein that proved to be much less potent in the ABP-
based ELISA on endogenous lipoxygenase activity (Fig-
ure 3b,c). A similar observation was reported by Lukas et al.,
who observed that Baicalein less potently rescued cells from
ferroptotic cell death compared to PD146176,[32] whereas
Baicalein and PD146176 have a comparable IC50 for LOX
inhibition. Thirdly, the ABP-based ELISA does not depend
on expression and purification of different LOXs isoforms.
This, together, with the straightforward two-step synthetic
procedure to synthesize probe Labelox B and the one-step
labeling procedure for LOX activity provides a convenient
method that can be used complementary to already existing
methods.
A limited number of studies discuss the connection
between the localization and activity of endogenous LOXs.
Christmas et al. reported that only 5-LOX and not 15-LOX
translocated to the nuclear envelope upon stimulation.[11]
Later, Jones et al. identified three nuclear-localization-se-
quence (NLS) in 5-LOX.[41,42] Mutation of each NLS impaired
nuclear import of 5-LOX. Further, Luo et al. observed that
the disruption of all three NLS in 5-LOX eliminated the
production of leukotriene B4 (LTB4) in cells, while the
arachidonic acid conversion activity of such mutants largely
remained as observed in a cell-free enzymatic assay.[25] The
authors argue that this isoenzyme specific translocation plays
a role in the production of specific oxygenation products with
specific functions.[11,25] Our observations are complementary
to previous observations. We confirm that cellular stimula-
tion, by LPS, induced accumulation of LOX isoenzymes on
the nuclear envelope. This is particularly pronounced for 5-
LOX (Figure 4a,b). The change in localizations for all LOX
isoenzymes overlapped with a change in localization of ABP
probe Labelox B, which indicates that a change in localization
is accompanied by a change in the localization of LOX
activity (Figure 4a).
Application of probe Labelox B indicated that LPS
stimulation triggered LOX accumulation at the nuclear
envelope as well as at distinct locations in the nucleus. This
nuclear accumulation of LOX isoenzymes has been observed
before. However, it was expected that LOX was not active in
the nucleus[25] Remarkably, we observed clear colocalization
of probe Labelox B labeling with nuclear localization of LOX
ABP labeling, thus suggesting that LOX is active in the
nucleus. The connection between LOX localization and LOX
ABP labeling at distinct localization in the nucleus could
indicate a functional role for the LOX isoenzymes in the
nucleus. Using sequence comparison and biochemical tools
we were able to identify a bromodomain-like region in 15-
LOX-1 with low micromolar affinity for specific lysine
acetylated histone peptides. These observations are in line
with previous observations for bromodomain-containing
proteins that also provide particular epitope preferences
and binding constants in the low micromolar range.[27,43,44] The
The classical bromodomain contains a continuous four
helices (aZ, aA, aB, aC) with two inter-helical loops (ZA and
BC) of variable length and sequence, in which, the ZA loop is
likely to determine the acetyl-lysine binding.[46] The essential
consensus motifs, Nx2or3D(E)x2or3Y(A/V), are located in
this loop. Crystal structure shows that the acetyl group in
a lysine residue forms a conventional hydrogen bond with an
asparagine residue located in the ZA loop.[27–29] Binding of
histone peptides to bromodomains is attenuated by the lack of
acetylated lysine residues.[47] The observations that lysine
acetylated histone peptides have a higher affinity for the
putative bromodomain-like region in 15-LOX-1 are in line
with these prior observations. Also, identification of the
essential loop with the Nx2or3D(E)x2or3Y(A/V) motif in 15-
LOX-1 (Figure S7) supports the idea that 15-LOX-1 contains
a functional bromodomain-like region.
Taken together, we generated probe Labelox B for
convenient one-step activity-based labeling of lipoxygenase
enzyme activity. This probe enabled distinction and quantifi-
cation of ABP-labeling of LOX isoenzymes in an ELISA
assay, which was utilized to determine inhibitory selectivity
among LOX isoenzymes. Moreover, Labelox B enabled
visualization of putatively active endogenous LOXs in cells,
which, for the first time, offers a possibility to connect the
activity of LOXs to their different intracellular localizations
upon treatment with stimuli. The potential of this approach is
demonstrated by the remarkable finding that LOX isoen-
zymes and ABP-labeling co-localized on distinct locations in
the nucleus. This observation led to the identification of
a bromodomain-like region in 15-LOX-1, which rationalizes
the observed nuclear localization 15-LOX-1. Thus, this study
on the one hand provided a novel chemical tool for ABP-
labeling of LOX isoenzymes and on the other hand indicated
that 15-LOX-1 has a bromodomain-like region, which sug-
gests a role in chromatin biology.
Acknowledgements
D.C. and Z.X. are funded by the China Scholarship Council
(grant no 201907720019 for D.C., grant no. 201706010341 for
Z.X.).
Conflict of Interest
The authors declare no conflict of interest.
Keywords: activity-based probes · bromodomains · inhibitors ·
lipoxygenases · oxidoreductases
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Accepted manuscript online: August 13, 2021
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Research Articles
Activity-Based Probes
D. Chen, Z. Xiao, H. Guo, D. Gogishvili,
R. Setroikromo, P. E. van der Wouden,
F. J. Dekker*
&&&& — &&&&
Identification of a Bromodomain-like
Region in 15-Lipoxygenase-1 Explains Its
Nuclear Localization
Activity-based labeling of lipoxygenase
(LOX) activity using the novel one-step
labeling probe Labelox B enables detec-
tion of isoenzyme inhibitory selectivity in
an ELISA-based assay and visualization of
the localization of LOX activity in living
cells. The observed nuclear localization
was rationalized by identification of
a bromodomain-like region in the 15-
LOX-1 isoenzyme.
Angew. Chem. Int. Ed. 2021, 60, 2 – 11
ꢀ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH
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&&&&
These are not the final page numbers!