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

Abstract 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.


Supporting Information
. Characterization of ABPs against 15-LOX-1 recombinant Figure S2. Uncropped western blot for Fig. 3a,b Figure S3. Labelox B labeled endogenous LOXs are immunoprecipitated by streptavidin beads. Figure S4. IC50 of EDTA on recombinant 15-LOX-1 crude lysate Figure S5. Acetylation on lysine 27 residue is essential for H3 and 15-LOX-1 binding Figure S6. Putative acetyl-histone binding region of 15-LOX-1 based on the theoretical model Table S1. The hillslope and IC50 of PD146176 on LOXs Table S2. The hillslope and IC50 of Baicalein on LOXs Table S3. The hillslope and IC50 of Zileuton on LOXs 3. The NMR spectra and HRMS of probes

Synthesis
General. All the reagents and solvents were purchased from Sigma-Aldrich, AK Scientific, Fluorochem or Acros and were used without further purification. Reactions were monitored by thin-layer chromatography (TLC) method, in which Merck silica gel 60 F254 plates were used, and spots were detected with potassium permanganate staining. MP Ecochrom silica 32-63, 60 Å was used for column chromatography. Nuclear magnetic resonance spectra, 1 H NMR (500 MHz) and 13 C NMR (126 MHz), were recorded on a Bruker Avance 500 spectrometer. Chemical shifts were reported in ppm. Chemical shifts were referenced to the residual proton and carbon signals of the deuterated solvent, CDCl3: δ = 7.26 ppm ( 1 H) and 77.05 ppm ( 13 C). The following abbreviations were used for spin multiplicity: s (singlet), d (doublet), t (triplet), q (quartet), dd (double of doublets), and m (multiplet). Coupling constants were reported in Hertz (Hz). High-resolution mass spectra were recorded using Fourier Transform Mass Spectrometry (FTMS) and electrospray ionization (ESI) on an Applied Biosystems/SCIEX API3000triple quadrupole mass spectrometer.

Synthesis of control probes
Scheme 2. Synthesis route of control probes.
Subsequently, the organic layer was dried over MgSO4, filtered and concentrated under reduced pressure.

Preparation of recombinant human 15-LOX-1
The 15-LOX-1 protein was produced in E. coli BL21(DE3) as described elsewhere [1] . Briefly, a single colony was used to inoculate 2YT medium with 50 µg/ml kanamycin (Duchefa, Haarlem, The Netherlands). After overnight culturing at 37°C, 200 rpm, 10 ml of such culture was used to inoculate 1 L 2YT medium with 0.2% glycerol. The cells were allowed to grow at 37°C, 200 rpm for around 1.5-2.5 h to reach 0.6 of OD600. The culture was equilibrated to 20°C, followed by adding kanamycin and IPTG to a final concentration of 50 µg/ml and 0.1 mM, respectively. The 15-LOX-1 was produced at 20°C for overnight with 200 rpm shaking. Subsequently, cells were pelleted through centrifugation at 3750 g for 20 min, after which the pellet (around 5 grams) was resuspended in 10 ml LOX assay buffer (25 mM HEPES , pH 7.5) supplemented with 1X protease inhibitor cocktail (PIC, Roche, Mannheim, Germany).
Later, cells were disrupted by sonication at 50% duty cycle, 70% output for 5 cycles (45 seconds sonicating and 3 minutes cooling down for each cycle) on an ice water bath. Cell debris was pelleted through centrifugation at 4°C, 20000 g for 1 h. The aliquot of supernatant containing 15-LOX-1 enzyme was snap-frozen in liquid nitrogen and stored at -80°C for further use. BL21(DE3) transformed with empty plasmid was proceed with the same procedure, which lysate was used as the negative control (referred as BL21 control later) in 15-LOX-1 recombinant enzyme inhibition studies.

15-LOX-1 recombinant enzyme inhibition assay
The 15-LOX-1 inhibition assay has been detailed previously [2] . Briefly, the conversion of linoleic acid to hydroperoxy-(9Z,11E)-octadecadienoic acid (λmax of 234 nm) was used to determine the activity of the 15-LOX-1 enzyme. The conversion rate was the slope of UV-absorbance at 234 nm overtime at the linear increase period.
To determine the IC50 of probes, the cell lysate was pre-diluted in LOX assay buffer at a ratio of 1:160. The inhibition assay was done in an F-bottom UV-STAR 96-well plate, in which 50 µl of the diluted enzyme was incubated with 140 µl of a probe diluted in LOX assay buffer for 10 min at room temperature (RT). Later 10 µl of 500 µM linoleic acid dissolved in ethanol was added to the plate to reach a final concentration of 25 µM. The UV-absorbance at 234 nm was determined by a BioTek Synergy H1 plate reader (BioTek, Winooski, VT, USA). The slope of the 15-LOX-1 sample untreated with the probe was set as 100%, while the slope of the BL21 control sample was set as 0%.
For the determination of enzyme kinetics, a series concentration of linoleic acid was used. Fifty µl of pre-diluted 15-LOX-1 enzyme was incubated with 140 µl of probe solution at a fixed concentration for 10 min at RT. Subsequently, 10 µl of a series concentration of linoleic acid was added to a final concentration ranging from 10 µM to 100 µM. The conversion rate was calculated as the same as described in the IC50 determination assay. Michaelis-Menten plots, Lineweaver-Burk plots, Km, and Vmax, are generated using GraphPad Prism 8.3.0.

Kits-Wilson kinetic analysis
The probes inhibit the 15-LOX-1 enzyme with a two-step reaction. First, a probe binds to an enzyme to get a reversible complex, E*I. The dissociation constant of inhibitor (Ki) is from the formation of E*I. Then, the probe forms a covalent bond with the enzyme, which generates an irreversible complex, E-I. The inactivation rate constant (ki) is to describe the rate of E-I generation. To determine these inactivation parameters, Kitz-Wilson analysis was employed. The procedure has been detailed previously by our group [3] . Following the protocol, the ki and Ki of all probes with IC50 values less than 50 µM were determined.

Immunoblotting Analysis
Raw 264.7 cells were seeded in a 6-well plate at a density of 5x10 5 cells per well in high glucose DMEM media supplemented with 1% penicillin/streptomycin, 10% fetal bovine serum (FBS) (Costar Europe, Badhoevedrop, The Netherlands) at 37°C and cultured overnight. Afterward, cells were treated with the desired concentration of Labelox B at the expected time. Then, the cells were harvested and lysed with RIPA buffer containing 1X PIC. The insoluble cell debris was removed by centrifugation.
Later, 30 μl of each sample was used for immunoblotting analysis. The membrane was blocked with 5 % skimmed milk (w/v) in PBST and incubated with HRP conjugated streptavidin (150 ng/ml) overnight at 4°C. After washing, the blotting bands were detected with enhanced chemiluminescence (ECL) solution (GE Healthcare, Amersham, UK). (1:500 dilution in PBS) for overnight at 4°C. After washing triply with 0.05%PBST, the plate was blocked with 2% (w/v) BSA in PBST. Subsequently, 100 µl of each reaction mixture was added to the LOXantibody-coated wells. There are four LOX antibodies used for coating. Therefore, in total, 400 µl mixture is needed for each reaction. After one hour of incubation, LOXs were pulled down by antibodies accordingly. Next, 100 µl of HRP-conjugated streptavidin (1:2000 dilution in PBS, Thermo Fisher Scientific, USA) was added to each well to detect the D04 labeled LOXs among precipitated LOXs. After washing with PBST, 100 µl of mixed HRP substrate reagent (R&D Systems, Minneapolis, USA) was added, and the colorizing reaction was stopped by adding 100 µl of 1M sulfuric acid. The absorbance at 450 nM was determined via a Synergy H1 plate reader.

Confocal microscopy
To visualize the intracellular location of LOXs, immunofluorescence staining was applied to cells.
A549 or RAW264.7 macrophage cells were seeded on glass coverslips in a 6-well plate at a density of 1x10^5 cells per well. After overnight incubation, cells were treated with 1 µg/ml LPS for 24 h.
Subsequently, cells were fixed with 100% methanol (pre-cooled in -20ºC) for 5 min in a freezer. After triple washes with PBS to remove methanol, cells were treated with 50 µM Labelox B for 1 h at 37°C.

Acetylated histone H3 peptide array
The array kit was purchased from Abcam (ab233494), and the assay was performed following the manufacturer's instructions.

15-LOX-1/Histone peptide-binding ELISA
Fifty μl of purified 15-LOX-1 at a concentration of 100 nM was coated on a high-binding 96-well plate. After blocking with 2% PBSTB, 50 ul of biotinylated-peptides (Anaspec, Fremont, USA) (in 0.05% PBST) with desired concentrations were applied on the plate followed by incubating at RT for 1 h. After washing triply with 0.05% PBST, 50 ul of HRP-conjugated streptavidin (500 ng/ml) was added to each well to detect the histone peptides associated with 15-LOX-1. Later, 100 µl of mixed HRP substrate reagent was added, and the colorizing reaction was stopped by adding 100 µl of 1M sulfuric acid. The absorbance at 450 nM was determined via a Synergy H1 plate reader.     Figure S7. Putative acetyl-histone binding region of 15-LOX-1 based on the theoretical model [4]