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Identification of protein targets of 12/15-lipoxygenasederived lipid electrophiles in mouse peritoneal macrophages using omega-alkynyl fatty acid Yosuke Isobe, Yusuke Kawashima, Tomoaki Ishihara, Kenji Watanabe, Osamu Ohara, and Makoto Arita ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b01092 • Publication Date (Web): 20 Feb 2018 Downloaded from http://pubs.acs.org on February 22, 2018

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ACS Chemical Biology

Identification of protein targets of 12/15-lipoxygenase-derived lipid electrophiles in mouse peritoneal macrophages using omega-alkynyl fatty acid

Yosuke Isobe1,3*, Yusuke Kawashima2, Tomoaki Ishihara1,3, Kenji Watanabe1,3, Osamu Ohara2, and Makoto Arita1,3,4*

1

Laboratory for Metabolomics, 2Laboratory for Integrative Genomics, RIKEN Center for Integrative

Medical Sciences (IMS) 1-7-22, Suehiro-cho, Tsurumi, Yokohama, Kanagawa, 230-0045, Japan 3

Graduate School of Medical Life Science, Yokohama City University, 1-7-29, Suehiro-cho, Tsurumi,

Yokohama, Kanagawa, 230-0045, Japan 4

Division of Physiological Chemistry and Metabolism, Keio University Faculty of Pharmacy, 1-5-30,

Shibakoen, Minato-ku, Tokyo, 105-0011, Japan

*Correspondence to Makoto Arita, PhD, and Yosuke Isobe, PhD. E-mail address: [email protected] (M.A.) and [email protected] (Y.I.)

Abstract The 12/15-lipoxygenase (12/15-LOX) enzyme introduces peroxyl groups, in a position-specific manner, into polyunsaturated fatty acids to form various kinds of bioactive lipid metabolites, including lipid-derived electrophiles (LDE). The resident peritoneal macrophage is the site of greatest 12/15-LOX expression in the mouse. However, the role of the enzyme in the regulation of resident macrophages is not fully understood. Here, we describe a chemoproteomic method to identify the targets of enzymatically generated LDE. By treating mouse peritoneal macrophages with omega-alkynyl arachidonic acid (aAA), we identified a series of proteins adducted by LDE generated through a 12/15-LOX catalyzed reaction. Pathway analysis revealed a dramatic enrichment of proteins involved in energy metabolism, and found that glycolytic flux and mitochondrial respiration were significantly affected by the expression of 12/15-LOX. Our findings thus highlight the utility of chemoproteomics using aAA for identifying intracellular targets of enzymatically generated LDE.

Body text The 12/15-lipoxygenase (12/15-LOX) enzyme is a member of the LOX family of enzymes that insert peroxyl groups into double bonds of polyunsaturated fatty acids (PUFAs) such as arachidonic acid (AA). The resident peritoneal macrophage is the site of greatest 12/15-LOX expression in the mouse1. However, the role of the enzyme in the regulation of resident macrophages

1 ACS Paragon Plus Environment

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is not fully understood. Previous studies indicate that 12/15-LOX can contribute to the formation of an array of bioactive lipid mediators such as lipoxins, resolvins, and protectins2. Such lipid mediators, in turn, are identified as autacoids that stimulate the resolution of inflammation. The 12/15-LOX enzyme catalyzes PUFAs to form peroxides and hydroperoxides, as well as secondary electrophilic metabolites that can covalently modify biomolecules3. Therefore, understanding the protein targets of such lipid-derived electrophiles (LDE) could be critical for elucidating the functions of 12/15-LOX in macrophages. Chemoproteomic methods are particularly useful for identifying a large number of proteins that react with LDE in cells. In recent studies, terminal alkynyl analogs of 4-hydroxy-2(E)-nonenal (HNE) and 4-oxo-2(E)-nonenal (ONE) were used to globally profile LDE adduction of proteins4. Besides oxygenated PUFA-derived reactive metabolites, this methodology has widely been applied to related natural products, such as oxygenated polyacetylenes5,6. Wang et al. have reported an alternative chemoproteomic strategy, which maps targets of LDE through the competition of lipid aldehydes against a cysteine-reactive iodoacetamide-alkyne reactivity-based probe7. However, 12/15-LOX can produce a diverse array of LDE which may include previously unknown metabolites. In addition, 12/15-LOX–derived electrophiles are mainly generated in the cytosol, and can react covalently with neighboring proteins. Therefore, previous strategies to identify protein targets of LDE would fail to address the in-situ formation of electrophiles in specific intracellular locations. Clickable lipid analogs, including omega-alkynyl PUFAs, are potentially useful probes for tracking the fate of fatty acid metabolism8,9. Using omega-alkynyl linoleic acid, a recent study has identified protein targets of LDE which are generated by reactive oxygen species in cells10. However, there are no reports focusing on enzymatically generated LDE derived from PUFAs. In this study, we sought to identify protein targets of LDE generated through enzymatic oxidation in peritoneal macrophages by using omega-alkynyl AA (aAA) (Figure 1A). Previous studies have demonstrated that alkynyl PUFAs are metabolized by LOX enzymes to produce profiles similar to those of native PUFAs8,9. However, it has not been demonstrated whether peritoneal macrophages metabolize aAA in a manner similar to AA. Since 12/15-LOX is predominantly expressed in macrophages among the various cell types present in the mouse peritoneum (Figure 1B), we determined whether aAA is a suitable traceable analog of AA for studying its 12/15-LOX–dependent metabolism in resident peritoneal cells. At first, monooxygenated products of aAA were globally detected by liquid chromatography–tandem mass spectrometry (LC-MS/MS)– based lipidomics, using a multiple reaction monitoring (MRM) transition of m/z 315/253. Since daughter ion of MRM transition m/z 315/253 (i.e. 253) comes from neutral loss of H2O and CO2 from aAA, this MRM transition correspond to the addition of one atom of oxygen to aAA. Peritoneal cells from wild-type (WT) mice produced dramatic amounts of aAA monooxygenate, whereas fewer products were detected from the incubation of 12/15-LOX–deficient peritoneal cells (Figure 1C). To


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identify the metabolites depicted in Figure 1C, MS/MS analysis of a product peak at m/z = 315 was performed (Figure 1D). Based on MS/MS spectra, a peak of WT peritoneal cells was identified as alkynyl 12-hydroxyeicosatetraenoic acid (a12-HETE), with corresponding fragments at m/z 315 (M-H), 297 (M-H-H2O), and 253 (M-H-H2O-CO2), and diagnostic ions at m/z 179, 161 (179-H2O), and

135

(179-CO2).

It

is

known

that

mouse

12/15-LOX

mainly

generates

12-hydroperoxyeicosatetraenoic acid (12-HpETE) from AA, which is subsequently converted into secondary products including 12-HETE (Figure 1E)11,12. Therefore, these results indicate that aAA can be metabolized, similar to AA, by 12/15-LOX in peritoneal macrophages. To get an initial impression of whether aAA-derived LDEs might covalently bind to cellular proteins in peritoneal macrophages, we performed gel-based profiling as follows: Peritoneal cells were treated with aAA for 30 min. An Alexa Fluor 647 azide tag was appended to probe-labeled proteins by copper-catalyzed azide–alkyne cycloaddition (CuAAC). The analysis of probe targets by SDS–polyacrylamide gel electrophoresis (PAGE) and in-gel fluorescence scanning revealed that treatment of aAA resulted in a substantial increase in measurable protein adduction in peritoneal macrophages, and that the signal was displaced by AA in a concentration-dependent manner (Figure 2A). In addition, such protein adduction was not observed in 12/15-LOX–deficient peritoneal cells (Figures 2B), containing almost the same percentage of macrophages as WT (Supplementary Figures S1A, S1B). Taken together, these results indicate that aAA is metabolized by 12/15-LOX, thereby generating lipid electrophiles that adduct proteins in peritoneal macrophages. We next sought to identify protein targets of 12/15-LOX–derived electrophiles in peritoneal macrophages. To identify the direct protein targets detected by gel-based methods, we applied LC–MS/MS–based chemoproteomic analysis. Affinity purified, electrophile-adducted proteins from the three biological replicates were analyzed via LC–MS/MS. This analysis identified a total of 221 targets which were significantly enriched in aAA-treated peritoneal cells (Figure 3A, Table S1). Of note, 12/15-LOX (termed Alox15 in Figure 3A and Table S1) itself was included as a potential target. It is known that LOXs are gradually inactivated as a result of the covalent binding of PUFA-derived reactive intermediates, which are generated via LOX-catalyzed oxygenation13. Direct adduction of aAA or its metabolites to 12/15-LOX was further confirmed by using recombinant 12/15-LOX protein (Figure 3B). These results indicate that our strategy is capable of detecting protein targets of LDE generated via enzymatic oxygenation. Next, we conducted a pathway enrichment analysis in WebGestalt using the WikiPathways database, and revealed the enrichment of proteins involved in central metabolic pathways (Table 1). While the relationship between 12/15-LOX and energy metabolism has not been appreciated before, proteins in central metabolic pathways were enriched in a previous study that identified protein targets of LDE generated by reactive oxygen species in Raw264.7 macrophages10. Moreover, a recent chemoproteomic study has shown that



enzymes that play key roles in energy metabolism are enriched as targets of certain reactive environmental chemicals14. Therefore, these target proteins may be particularly sensitive to reactive chemicals and metabolites, including 12/15-LOX–derived electrophiles. To further confirm adduct formation by 12/15-LOX–derived electrophiles, we transfected representative target proteins from central metabolic pathways (ACADL in mitochondrial fatty acid beta-oxidation, and GAPDH in glycolysis and gluconeogenesis), with or without mouse 12/15-LOX, in HEK293 cells and subsequently treated aAA. As shown in Figures 3C and D, the expression of 12/15-LOX alone resulted in a substantial increase in measurable protein adduction in aAA-treated cells as compared to mock-transfected cells. In addition, we show that each target was labeled by aAA only when 12/15-LOX was co-expressed (Figure 3C, D). These results indicate that aAA was metabolized by 12/15-LOX and subsequently labeled target proteins. From the results of the pathway enrichment analysis, we hypothesized that 12/15-LOX may regulate energy metabolism in peritoneal macrophages. To test this hypothesis, we purified peritoneal resident macrophages from WT and 12/15-LOX–deficient mice and measured glycolysis and mitochondrial respiration using an extracellular flux analyzer. The extracellular acidification rate (ECAR), which is predominately from the excretion of lactic acid, was increased by the addition of glucose in WT peritoneal macrophages, highlighting the glycolytic rate of the cells (Figure 3E). The subsequent addition of an ATP synthase inhibitor, oligomycin, which decreases the ATP/ADP ratio and drives glycolysis, had little effect in increasing the ECAR, suggesting peritoneal macrophages had little glycolytic reserve capacity (Figure 3E). A glucose-induced increase in the ECAR was significantly lowered in 12/15-LOX–deficient macrophages, whereas such a difference was abolished by the addition of a hexokinase inhibitor, 2-deoxy-D-glucose (2-DG; Figure 3E). These results indicate that glycolytic flux was significantly impaired in 12/15-LOX–deficient peritoneal macrophages. The oxygen consumption rate (OCR), an indicator of mitochondrial respiration, was also significantly reduced in 12/15-LOX–deficient macrophages (Figure 3F). These results suggest that 12/15-LOX regulates energy metabolism, possibly through the formation of 12/15-LOX–derived lipid electrophiles and their actions on energy metabolism-related proteins identified in this study. In this study, we identified a series of proteins adducted by 12/15-LOX–derived electrophiles in peritoneal macrophages. Pathway enrichment analysis revealed the potential relationship between 12/15-LOX and energy metabolism. Indeed, 12/15-LOX–deficient peritoneal macrophages showed an impaired glycolytic rate and mitochondrial respiration. Although a detailed molecular mechanism has to be further addressed, these results demonstrate, for the first time, that 12/15-LOX activity is functionally linked to the regulation of energy metabolism. Besides defects of energy metabolism, previous studies using 12/15-LOX–deficient peritoneal macrophages have highlighted the involvement of the enzyme in several biological processes. For example, 12/15-LOX–


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deficient peritoneal macrophages show defective phagocytosis linked to altered actin polymerization in mice15. Phagocytosis-related proteins (e.g. Actb, Rab5a, Atp6v1b2, Atp6v1a, Tuba1b, Tubb4b, Tubb5) were significantly enriched in this study (Table S1). Our findings identified the LDE modification of these candidates as a potential mechanism to explain functional changes in 12/15-LOX–deficient peritoneal macrophages. Further analysis of candidate proteins should reveal novel mechanisms of 12/15-LOX that regulate cellular functions.

Methods Chemicals AA and aAA were purchased from Cayman Chemical. All solvents of LC/MS grade were obtained from Sigma-Aldrich. Other chemicals were purchased from Wako Chemicals unless otherwise indicated.

Isolation of mouse peritoneal macrophages Peritoneal lavages from C57BL/6 wild-type (from CLEA Japan, Inc.) and 12/15-LOX KO mice (8–12 weeks) were collected using phosphate buffered saline (PBS). Macrophages were isolated from peritoneal lavages using phycoerythrin (PE)-conjugated anti-mouse F4/80 (BM8; BioLegend) and anti-PE microbeads (Miltenyi Biotec) on an autoMACS cell separator (Miltenyi Biotec). All animal experiments were approved by the Animal Care and Use Committee of RIKEN Yokohama Institute and Yokohama City University.

Cell culture and transfection HEK293 cells were maintained in Dulbecco’s modified Eagle’s medium (D-MEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 100 units/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine (Life Technologies) in 5% CO2 at 37℃. Full length mouse 12/15-LOX cloned into pCAGGS was generated in our previous study12. Similarly, full length cDNAs encoding for Acadl and Gapdh were cloned from cDNA derived from C57BL/6 mouse peritoneal cells into pCAGGS with an N-terminal FLAG tag. Plasmids were transfected into HEK293 cells with ViaFect Transfection Reagent (Promega).

Click chemistry procedures to append analytical handles to aAA-treated cell proteome Protein from aAA-treated HEK293 cells was harvested by brief sonication in PBS. Peritoneal cells were lysed in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific) containing EDTA-Free Complete Protease Inhibitor Cocktail (Roche). The protein concentrations of samples were determined by a bicinchoninic acid assay (Thermo Fisher Scientific). Cell lysates were diluted to 1.0



mg/mL (total protein concentration), and 80 µL of each proteome was subjected to a click reagent mixture containing Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA, 5 µL/sample, 100 mM in H2O, Sigma–Aldrich), CuSO4 (5 µL/sample, 20 mM in H2O, Thermo Fisher Scientific), sodium L-ascorbate (5 µL/sample, 300 mM in H2O, Sigma–Aldrich), and Alexa Fluor 647 azide (5 µL/sample, 5 mM in DMSO, Thermo Fisher Scientific). After incubating for 1 h at room temperature, each reaction was quenched and proteomes were precipitated by adding 300 µL of methanol (MeOH), 75 µL of chloroform, and 200 µL of H2O. Precipitated proteins were washed in MeOH, then resolubilized in phase-transfer surfactant (PTS) buffer (12 mM sodium deoxycholate (SDC), Sigma-Aldrich, 12 mM sodium N-lauroyl sarcosinate, Tokyo Chemical Industry, in 100 mM Tris-HCl, pH 9.0). Samples were resolved using SDS–PAGE (10% acrylamide gel) and detected by in-gel fluorescence scanning on a ChemiDoc imaging system (Bio-Rad).

Chemoproteomics to identify targets of aAA metabolites in resident peritoneal cells After click reactions using biotin-azide (Thermo Fisher Scientific), protein precipitation and resolubilization as above, biotin-labeled proteins were bound to Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific) while rotating 4 h at 4oC. Bead-linked proteins were enriched by washing in wash buffer 1 (2% SDS), wash buffer 2 (0.1% SDC, 1% Triton X-100, 500 mM NaCl, 1 mM EDTA, and 50 mM HEPES, pH 7.4), wash buffer 3 (250 mM LiCl, 0.5% NP-40, 0.5% SDC, 1 mM EDTA, and 10 mM Tris-HCl, pH 8.0), and wash buffer 4 (50 mM Tris-HCl, pH 7.4, and 50 mM NaCl). Samples were then boiled in SDS–PAGE sample buffer with 10 mg/mL of saturated biotin to 95oC for 5 min. Eluted proteins were precipitated with trichloroacetic acid/acetone. The precipitate was dissolved in PTS buffer. The dissolved sample was treated with 10 mM dithiothreitol at 50ºC for 30 min. and then subjected to alkylation with 30 mM iodoacetamide in the dark at room temperature. The mixture was diluted 4-fold with 50 mM ammonium bicarbonate and digested by Lys-C and trypsin (Promega) overnight at 37°C. An equal volume of ethyl acetate was added to the digested samples, and the mixture was acidified with 0.5% trifluoroacetic acid according to PTS protocols16,17. The mixture was shaken and centrifuged at 12,000 × g for 2 min for phase separation, and then the aqueous phase was retrieved. The volume of the digested sample thus recovered was reduced to a half or less of the original volume by a centrifugal evaporator for complete removal of ethyl acetate, and then desalted with C18-StageTips18 followed by drying in a centrifugal evaporator. The dried peptides were finally dissolved in 3% acetonitrile and 0.1% formic acid.

LC–MS/MS analysis of proteolytic peptides Peptides were directly injected onto a 75 µm × 20 cm, PicoFrit emitter (New Objective) packed in-house with 3.0 µm reversed-phase C18 particles (ReproSil-Pur 120 C18-AQ; Dr.Maisch) and then


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separated with a 120-min gradient at a flow rate of 300 nL/min using an Eksigent ekspert nanoLC 400 high performance liquid chromatography (HPLC) system (Sciex). Peptides eluting from the column were analyzed on a TripleTOF 5600+ mass spectrometer (Sciex) for both shotgun-MS and Sequential Windowed Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH-MS) analyses19. For shotgun-MS based experiments, MS1 spectra were collected in the range of 400–1200 m/z for 250 ms. The top 18 precursor ions with charge states of 2+ to 5+ that exceeded 150 counts/s were selected for fragmentation with a rolling collision energy, and MS2 spectra were collected in the range of 100– 1800 m/z for 90 ms. The dynamic exclusion time was set to 16 s. For SWATH–MS–based experiments, the mass spectrometer was operated in a consecutive data-independent acquisition with a 160 ms precursor ion scan followed by MS2 scans for 80 variable SWATH segments, each at 32 ms accumulation time. Precursor ions were fragmented for each MS2 experiment using rolling collision energy. All shotgun-MS files were searched against a mouse UniProt Swiss-Prot database (May 2017 release) using ProteinPilot software v. 4.5 and the Paragon algorithm (Sciex) used for protein identification. The protein confidence threshold was a ProteinPilot unused score 1.3 with at least one peptide with 95% confidence. The global false discovery rate for both peptides and proteins was lower than 1% in this study. The protein/peptide identification files were used as the protein/peptide library. The SWATH-MS data files were then annotated from the protein/peptide library using PeakView v.2.2 (Sciex). Within the PeakView, we allowed up to 5 peptides per protein and 8 transition ions per peptide to be used, and shared and modified peptides were excluded. Protein abundances were estimated from the summed abundances of selected peptides, and peptide abundances were estimated from the summed areas of 8 selected transition peaks. To discover protein targets of 12/15-LOX–derived electrophiles in proteome of the aAA-treated peritoneal cell, we extracted the proteins that had changed by more than 1.5-fold and pass significantly different (p < 0.05) as compared to the vehicle-treated samples.

Sample extraction and LC-MS/MS-based lipidomics Oxidized aAA were purified from samples by solid-phase extraction using a MonoSpin C18 column (GL Sciences) with LTB4-d4 as an internal standard. Subsequent LC-MS/MS–based lipidomic analyses were performed using an HPLC system (UPLC, Waters) with a linear ion trap quadrupole mass spectrometer (QTRAP5500, Sciex) equipped with an Acquity UPLC BEH C18 column (1.0 mm×150 mm×1.7 µm; Waters) as reported previously20.

Expression and purification of recombinant mouse 12/15-LOX



Recombinant mouse 12/15-LOX was cloned into a pCold-TF vector (TaKaRa) and overexpressed as N-terminal His-tagged fusion proteins in E. coli BL21 (DE3) cells in Luria–Bertani medium. Expression was induced by the addition of 1 mM isopropyl β-D-thiogalactopyranoside and cells were incubated at 16°C overnight. The protein was purified using a HisTrap HP column (GE Healthcare) and dialyzed against PBS.

Western blotting Anti–12/15-LOX polyclonal antibody was prepared from a rabbit by immunization with a synthetic peptide of mouse 12/15-LOX (RNHREEELEERRSL; Eurofins Genomics). Anti-DYKDDDDK tag antibody (L5) was purchased from BioLegend. Cells lysates were prepared as described above. Proteins were resolved by SDS–PAGE and transferred to polyvinylidene fluoride membranes using a Transblot apparatus (Bio-Rad). Blots were blocked with 5% nonfat milk in Tris-buffered saline containing 0.05% (v/v) Tween 20 (TBS-T) for 30 min at room temperature, washed in TBS-T, and probed with primary antibody overnight at 4℃. Following washes with TBS-T, the blots were incubated in the dark with a horse radish peroxidase-linked secondary antibody at room temperature for 1 h. Blots were visualized using a Chemidoc.

Immunocytochemistry Cells were fixed with 3.7% formaldehyde in PBS at room temperature for 15 min and permeabilized with 0.5% Triton X-100 in PBS at room temperature for 15 min. After blocking with 3% BSA in PBS, cells were stained with biotin-conjugated anti-mouse F4/80 (BM8; BioLegend) and rabbit anti-mouse 12/15-LOX antibodies, and then with Alexa Fluor 488 streptavidin and secondary antibody labeled with Alexa Fluor 594–conjugated donkey anti-rabbit IgG (Thermo Fisher Scientific).

Extracellular acidification and oxygen consumption rate The ECAR and OCR of murine peritoneal macrophages were analyzed in a Seahorse XFp Extracellular Flux Analyzer (Agilent). Briefly, 1 × 105 cells were seeded in each well of Seahorse XFp Cell Culture Miniplates 1 h prior to the assay. For ECAR measurements, the glycolysis stress test was employed by sequential injections of 10 mM glucose, 2 µM oligomycin, and 50 mM 2-DG. For OCR measurements, a cell mito stress test was employed by sequential injections of 2 µM oligomycin, 2 µM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, and 0.5 µM rotenone/antimycin A.

Statistical analysis Results are expressed as the mean ± standard error of the mean (SEM). Differences between two


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groups were tested by Student’s t-test. A significance level of p < 0.05 was used.

Supporting Information The supporting information is available free of charge via the internet at http://pubs.acs.org.

Acknowledgements This work was funded in part by the Japan Society for the Promotion of Science KAKENHI JP15H05897, 15H05898, 15H04648 (M.A.), and the RIKEN Special Postdoctoral Researcher Program (Y.I.). We thank Yuki Ariyasu for skillful technical assistance in immunocytochemistry.

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Figure legends Figure 1. Formation of aAA metabolites from incubations of mouse peritoneal macrophages. (A) The structures of AA and its aAA. (B) Peritoneal cells from wild-type (WT) and 12/15-LOX knockout (KO) mice were stained for 12/15-lipoxygenase (12/15-LOX) and F4/80, a macrophage marker. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; in blue). Scale bars represent 50 µm. (C) Profile of aAA metabolites in peritoneal cells. Monooxygenated products of aAA were monitored by LC-MS/MS–based lipidomics, using an MRM transition of 315/253 m/z. (D) Structures of monohydroxylated aAA. Dashed lines depict suggested sites of fragmentation. (E) Scheme for biosynthesis of 12-HETE. Mouse 12/15-LOX mainly generates 12-HpETE from AA, which is subsequently converted into secondary products including 12-HETE.

Figure 2. 12/15-LOX–dependent protein adduction with aAA metabolites in peritoneal cells. (A) Purified murine peritoneal macrophages were treated with aAA for 30 min in the presence or absence of AA. Proteins adducted with aAA metabolites were ligated with fluorescent dye via click reaction. The degree of adduction was determined by SDS–PAGE and in-gel fluorescence. (B) Peritoneal cells from WT or 12/15-LOX KO mice were treated with aAA. Proteins adducted with aAA metabolites were ligated with fluorescent dye via click reaction. The degree of adduction was determined by SDS–PAGE and in-gel fluorescence.

Figure 3. Target identification of aAA-derived electrophiles in mouse peritoneal cells. (A) The SWATH ratio for proteins identified from peritoneal cells treated with vehicle versus aAA. (B) Recombinant 12/15-LOX was expressed with trigger factor (TF) as a solubilization tag. After TF cleavage using human rhinovirus 3C protease, 12/15-LOX was incubated with aAA. Proteins adducted with aAA or its metabolites were ligated with fluorescent dye via click reaction. The degree of adduction is determined by SDS–PAGE and in-gel fluorescence. (C and D) Representative targets of aAA were validated by FLAG-tagged recombinant target and 12/15-LOX overexpression in HEK293 cells. Asterisks show targets adducted by aAA metabolites in a 12/15-LOX–dependent manner. (E) ECAR and (F) OCR in resident peritoneal macrophages from WT (blue) or 12/15-LOX KO (red) mice. Glycolytic flux (E) and mitochondrial respiration (F) are impaired in peritoneal macrophages from 12/15-LOX KO mice. n=3, *p < 0.05 compared to WT.

Table 1. WikiPathway Enrichment for the significantly enriched proteins in the pull down experiment.



Figure 1

A

B O OH

Arachidonic Acid (AA)

WT F4/80

12/15-LOX

Merge

F4/80

12/15-LOX

Merge

O

AA alkyne (aAA)

KO

OH

D

C 60

WT

40 20 0 14

18

22

60

KO

40

relative abundance

O

aAA+O (315/253)

Intensity/IS(LTB4-d4)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 16

100

315 161 179

50

179 253

135

200

10

14

18 min

22

300

12-HpETE O

0

OH

m/z

E AA

20

a12-HETE

315 = M-H 297 = M-H-H2O 253 = M-H-H2O-CO2 161 = 179-H2O 135 = 179-CO2

297 0 100

OH

O

O OH

OH

12/15-LOX

ACS Paragon Plus Environment

12-HETE

OOH

OH

OH

Page 13 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2

A

B

AA (µM) 0 aAA (µM)

0

0

10 100 300

10 10 10 10

(kDa)

WT KO

250

(kDa)

150 250

100

150

75

100 50 75 37 50 25 20

37

Coomassie


Coomassie



Page 14 of 16

Page 15 of 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47


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112x68mm (72 x 72 DPI)


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