A recombinant macrophage cell line, method and use for enhancing endogenous hydrogen peroxide localized accumulation in macrophages

Abstract

The application discloses a kind of recombinant macrophage strains for enhancing endogenous hydrogen peroxide local accumulation in macrophage, method and application.It belongs to the field of cell biology and drug screening technology.The application first finds that D-amino acid oxidase has the problem of catalytic activity loss in the cytoplasm, nucleus, endoplasmic reticulum and Golgi body of macrophage, and based on this problem, it is found that mitochondria is the only compartment that can make DAAO catalytic activity stable.Based on this, the application constructs a recombinant macrophage stably expressing mitochondria-targeted D-amino acid oxidase, a method, and realizes the accumulation of endogenous hydrogen peroxide in macrophage.The application also first finds that FCCP and D-ala can produce significant synergistic effect when used together, greatly improving the level of mitochondrial local H2O2 accumulation.The application provides an effective direction for the study of macrophage mitochondrial oxidative stress and the screening of targeted drugs.

Description

Technical Field

[0001] This invention relates to the fields of cell biology and drug screening technology, and more specifically to a recombinant macrophage cell line, method, and application that enhances the local accumulation of endogenous hydrogen peroxide in macrophages. Background Technology

[0002] Mitochondrial H2O2 is a key signaling molecule regulating macrophage function and is closely related to infection and inflammatory diseases, diabetes, and tumor immunotherapy. Precise induction of mitochondrial H2O2 production through chemogenetic methods allows for the exploration of metabolic reprogramming, regulation of pro-inflammatory cytokines, and high-throughput screening of candidate drugs targeting macrophage mitochondrial metabolism.

[0003] D-amino acid oxidase (DAAO), a chemogenetic tool, is widely used in biological research for its catalytic in situ generation of H2O2 from substrates such as D-alanine. DAAO catalyzes the oxidative deamination of D-amino acids to produce the corresponding imino acids, while simultaneously generating H2O2 as a byproduct. Current techniques typically bind DAAO to a localization sequence and introduce it into cells to induce local H2O2 production. In non-immune cells (such as HEK293T), current techniques have shown that DAAO maintains normal catalytic activity and produces products in various subcellular compartments (cytoplasm, mitochondria, endoplasmic reticulum, etc.).

[0004] APEX2 is an engineered soybean ascorbate peroxidase that converts chemiluminescent or fluorescent substrates into detectable signals in the presence of H2O2. Unlike traditional genetically encoded H2O2 probes based on reversible thiol oxidation, APEX2-mediated H2O2 detection is thiol-independent and therefore unaffected by disulfide reduction systems or thiol-reactive compounds. APEX2 can directly and irreversibly convert H2O2 molecules into fluorescent or bioluminescent signals, enabling the accurate detection of localized H2O2 levels within cells.

[0005] The combined use of APEX2 and DAAO has been reported in existing technologies. For example, the literature "An enzymatic cascade enables sensitive and specific proximity labeling proteomics in challenging biological systems" (hereinafter referred to as Literature 1) reports the use of APEX2 and DAAO expressed in two separate open reading frames (ORFs) to target organelles such as mitochondria in non-immune cells (such as IMCD3) for proximity labeling. This method employs dual ORF (dual plasmid / dual gene) sporadic expression. In in vitro high-throughput drug screening or industrial applications, dual ORF delivery faces serious limitations such as low transfection efficiency, uneven expression ratios of various components, and difficulty in stable co-transformation in macrophages. More importantly, Literature 1 aims to use APEX2 to attach biotinylate tags to surrounding proteins for mass spectrometry analysis, and does not address the technical issues of "local H2O2 pharmacokinetics screening in living cells." The literature "Using the heme peroxidase APEX2 to probe intracellular H2O2 flux and diffusion" (hereinafter referred to as Literature 2) reports the highly sensitive intracellular detection of H2O2 using the APEX2 / AmUR system, and the separate expression of DAAO and APEX2 in the same cell for linked detection. Literature 2 only discloses the underlying biochemical mechanism of APEX2 hydrogen detection and DAAO hydrogen production. The common feature of the above-mentioned prior art is that its verification and functional characterization are all completed in conventional non-immune cell lines (such as HEK293T, HeLa, IMCD3), thus forming a generally accepted technical expectation in the field - that DAAO, as a universal tool enzyme, can stably exert catalytic activity in multiple subcellular compartments of different cell types.

[0006] However, the inventors discovered that when APEX2 and DAAO are expressed in macrophages, DAAO cannot maintain its normal catalytic function as it does in non-immune cells—no effective endogenous H2O2 production signal can be detected after substrate stimulation. This finding implies that the existing paradigm for DAAO tool applications based on non-immune cells cannot be simply transferred to macrophage systems. As professional immune cells, macrophages possess a unique, highly antioxidant microenvironment that constitutes a barrier to the application of DAAO tools that has never been revealed in the prior art.

[0007] Therefore, how to overcome the problem that DAAO cannot maintain its normal catalytic function in non-immune cells when APEX2 and DAAO are expressed in macrophages, and how to further amplify the local catalytic efficiency of DAAO in the mitochondrial compartment within a controllable range to improve the sensitivity and practicality of the system in applications such as drug screening, are technical problems that urgently need to be solved by those skilled in the art. Summary of the Invention

[0008] The main technical problem solved by this invention is the complete loss of catalytic activity of DAAO tools in the non-mitochondrial compartments of macrophages due to microenvironmental clearance. Furthermore, it addresses the issue of amplifying the local catalytic efficiency of DAAO in the mitochondrial compartments within a controllable range.

[0009] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0010] The primary objective of this application is to provide a method for enhancing the local accumulation of endogenous hydrogen peroxide in macrophages, wherein in recombinant macrophages stably expressing mitochondrial-targeting D-amino acid oxidase, a D-amino acid oxidase substrate is administered to initiate the local production of endogenous hydrogen peroxide; wherein the D-amino acid oxidase substrate is D-alanine and / or D-valine.

[0011] As a preferred technical solution, the method further includes administering a mitochondrial uncoupling agent simultaneously with the D-amino acid oxidase substrate in recombinant macrophages that stably express mitochondrial-targeting D-amino acid oxidase, so as to initiate the local production of endogenous hydrogen peroxide.

[0012] As a preferred technical solution, the mitochondrial uncoupling agent is 4-trifluoromethoxyphenylhydrazone.

[0013] As a preferred technical solution, the recombinant macrophages also express the APEX2 detection module, which is co-expressed with the mitochondrial-targeted D-amino acid oxidase in the form of a fusion protein.

[0014] Another object of this application is to provide: a recombinant macrophage cell line expressing a recombinant fusion protein, wherein the recombinant fusion protein comprises, from the N-terminus to the C-terminus, a mitochondrial localization sequence, an APEX2 detection module, and a D-amino acid oxidase catalytic module; wherein, when the cell line is stimulated with a D-amino acid oxidase substrate, there is no detectable endogenous hydrogen peroxide accumulation signal in the non-mitochondrial compartment; the amino acid sequence of the recombinant fusion protein is shown in SEQ ID NO. 1; and the nucleotide sequence is shown in SEQ ID NO. 2.

[0015] As a preferred technical solution, the cell line is macrophages derived from the bone marrow of immortalized mice.

[0016] Another object of this application is to provide the use of the recombinant macrophage cell line in the preparation of a cell model for in vitro screening of drugs targeting macrophage mitochondrial oxidative stress regulation.

[0017] Another object of this application is to provide: the use of a mitochondrial uncoupling agent in the preparation of a regulator for synergistically enhancing the catalytic hydrogen peroxide accumulation efficiency of macrophage mitochondrial-localized D-amino acid oxidase, wherein the mitochondrial uncoupling agent is used in combination with a D-amino acid oxidase substrate; wherein the mitochondrial uncoupling agent is 4-trifluoromethoxyphenylhydrazone.

[0018] Another object of this application is to provide: a composition comprising a mitochondrial uncoupling agent and a D-amino acid oxidase substrate, said composition for synergistically enhancing the accumulation of hydrogen peroxide catalyzed by macrophage mitochondrial-localized D-amino acid oxidase; said mitochondrial uncoupling agent is 4-trifluoromethoxyphenylhydrazone; said D-amino acid oxidase substrate is D-alanine and / or D-valine.

[0019] Another object of this application is to provide: a method for in vitro screening of drugs that target the regulation of mitochondrial oxidative stress in macrophages, characterized by comprising the following steps: (1) Provide the aforementioned recombinant macrophage cell line; (2) Contact the candidate drug with the cell line; (3) D-amino acid oxidase substrate was administered, and changes in the level of local hydrogen peroxide accumulation in mitochondria were detected; (4) Based on the changes in hydrogen peroxide accumulation levels, candidate drugs for regulating mitochondrial oxidative stress in macrophages were screened.

[0020] As can be seen from the above technical solution, the present invention has the following beneficial effects: (1) Overcoming the compartment selectivity limitations of the immune microenvironment, this invention achieves unexpected technical effects. Existing technologies have only validated DAAO tools in non-immune cells, failing to reveal their inactivation mechanism in immune cells. This invention is the first to discover that conventionally positioned DAAO completely loses its effective catalytic activity in most compartments of macrophages (cytoplasm / nucleus / endoplasmic reticulum / Golgi apparatus). This invention breaks the technical bias that "enzyme activity can be exerted in any location," discovering that a mitochondrial targeting strategy can successfully bypass the broad-spectrum clearance barrier of macrophages, becoming the only effective compartment for evaluating DAAO catalytic activity. This single-compartment high signal-to-noise ratio response against the backdrop of complete inactivation in other compartments constitutes the core of the inventiveness of this invention.

[0021] (2) Successfully discovered and verified the drug screening value of FCCP in synergistically enhancing local H2O2 accumulation. Based on the mitochondrial-targeted cell model constructed in this invention, this invention has for the first time discovered and confirmed in practical applications that the mitochondrial uncoupling agent FCCP has an unexpected effect of synergistically enhancing DAAO-mediated local H2O2 signal accumulation. Specifically, in macrophage cell lines stably expressing mitochondrial-targeted fusion proteins, FCCP treatment alone did not induce a significant signal (clean background), D-ala treatment alone could establish a baseline signal, while FCCP and D-ala combined treatment showed a significant synergistic amplification effect on the signal. This result not only directly proves that the system of this invention has extremely high drug evaluation sensitivity, but also unexpectedly reveals new application clues of FCCP in regulating the local mitochondrial microenvironment and synergistically enhancing the catalytic efficiency of DAAO enzymes.

[0022] (3) It provides a reliable platform for in vitro high-throughput drug screening. The macrophage mitochondrial targeting system constructed in this invention can use the substrate D-ala as a trigger switch to quantitatively catalyze the production of H2O2 in the local mitochondria, establish a regulated baseline for endogenous hydrogen peroxide reaction, and is suitable for in vitro high-throughput screening of drugs that target macrophage mitochondrial oxidative stress regulation. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 The graph shows the changes in fluorescence intensity (RFU) at each stage for each treatment group in Example 1.

[0025] Figure 2 The graph shows the changes in fluorescence intensity (RFU) at each stage for each treatment group in Example 2; Mock in the graph represents the control group.

[0026] Figure 3 The graph shows the changes in fluorescence intensity (RFU) at each stage for each treatment group in Example 3; Mock in the graph represents the control group.

[0027] Figure 4 Example 4: Fluorescence intensity (RFU) variation curves of each treatment group at each stage. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Unless otherwise specified, the methods used in this embodiment are conventional methods known to those skilled in the art, and the reagents and materials used are commercially available products.

[0030] Cell lines: HEK293T cells (human embryonic kidney epithelial cell line, derived from Peking Union Medical College Hospital Cell Center, 1101HUM-PUMC000091) were cultured in DMEM medium containing 10% FBS.

[0031] iBMDM macrophages (immortalized mouse bone marrow-derived macrophages, from Nature. 2015 Oct 29;526(7575):660-5.) were cultured in DMEM medium containing 10% FBS.

[0032] pLV-EF1α-BlastR Lentiviral Vector: Derived from Miaoling Biotechnology, Product No.: P41259.

[0033] AmUR probe (Thermo Fisher Scientific, A36006).

[0034] D-alanine (D-ala, MedChemExpress, HY-41700).

[0035] D-norvaline (D-norvaline, MedChemExpress, HY-41309).

[0036] H2O2 solution (Sigma, 88597).

[0037] Carbonyl cyanide-4-trifluoromethoxyphenylhydrazone (FCCP, mitochondrial uncoupling agent, MedChemExpress, HY-100410).

[0038] Lipofectamine 3000 (Thermo Fisher Scientific, L3000).

[0039] Example 1 APEX2 Reporting System Methodology Quality Control This embodiment aims to verify the specificity and sensitivity of the APEX2 / AmUR detection system to H2O2 response and to perform basic quality control of the system.

[0040] The APEX2 / AmUR detection system works as follows: APEX2 is an engineered heme peroxidase that catalyzes the oxidation of non-fluorescent Amplex UltraRed (AmUR) using H2O2 as a substrate, generating Resorufin, which exhibits strong red fluorescence. Therefore, by detecting the fluorescence intensity of Resorufin (excitation / emission wavelength 510 / 590 nm), the level of local H2O2 within living cells can be reflected in real-time and specifically. The APEX2 module acts as a signal converter in this system—converting the chemical signal of H2O2 into a quantitatively detectable fluorescent signal; AmUR acts as a signal reporter substrate, which is itself non-fluorescent and only fluoresces when APEX2 and H2O2 are present simultaneously, thus ensuring high detection specificity. This embodiment uses systematic factorial experiments to perform methodological quality control on the specificity and sensitivity of this detection system.

[0041] (1) Cell seeding and transfection: HEK293T cells were seeded in 12-well plates and allowed to adhere overnight. When the cell confluence reached 60%-70%, plasmid transfection was performed. Two genotype groups were set up for transfection: empty vector control group (transfected with pLV-BlastR, Miaoling Biotechnology, P41259) and APEX2 expression group (transfected with pLV-APEX2). pLV-APEX2 was constructed from the empty vector pLV-BlastR using conventional molecular cloning methods, i.e., BamHI was selected at the multiple cloning site for single enzyme digestion, and then the APEX2 fragment was inserted through homologous recombination. The APEX2 sequence was synthesized by Shanghai Sangon Biotech Co., Ltd. and was consistent with the APEX2 sequence marked in step (1) of Example 3. Using Lipofectamine 3000 transfection reagent, the corresponding plasmid (1 μg per well) was transiently transfected into each group of cells strictly according to the instructions.

[0042] (2) Plating and grouping: 24-36 hours after transfection, cells were digested and seeded into 96-well plates with a black transparent bottom coated with poly-L-lysine (PLL). Cells from each genotype group were seeded into four treatment groups (three replicates per group): ① The double-free group (AmUR-, H2O2-).

[0043] ② The group with only H2O2 added (AmUR-, H2O2+).

[0044] ③ The AmUR group (AmUR+, H2O2-) is added alone.

[0045] ④ Double addition group (AmUR+, H2O2+).

[0046] Add 100 μL of complete DMEM medium to each well and incubate overnight to achieve a cell density of approximately 70% the next day.

[0047] (3) Washing and preparation before detection: Discard the culture medium before detection, gently wash the cells 3 times with PBS, and then add 100 μL of pre-warmed FluoroBrite DMEM to each well.

[0048] (4) Baseline detection: Place the 96-well plate in a multi-functional microplate reader and record the baseline fluorescence signal of each well for 10 min.

[0049] (5) First stage of sample addition and detection (AmUR incubation stage): After the baseline detection is completed, the first stage of sample addition is carried out according to the treatment group design: For the AmUR(+) group (group ③ and group ④), Amplex UltraRed (AmUR) is added to the well to make the final concentration 50 μM, and the fluorescence signal is immediately and continuously detected for 10 min.

[0050] For the AmUR(-) group (group ① and group ②), add an equal volume of FluoroBrite DMEM diluent (Gibco, A1896701) to the wells and immediately and continuously detect the fluorescence signal for 10 min.

[0051] (6) Second stage of sample addition and detection (H2O2 stimulation stage): After the first stage of detection is completed, the second stage of sample addition is carried out immediately: For the H2O2(+) group (group ② and group ④), exogenous H2O2 is added to the same reaction well to make the final concentration 0.2mM, and the fluorescence signal is detected continuously for 10min immediately.

[0052] For the H2O2(-) group (group ① and group ③), add an equal volume of FluoroBrite DMEM diluent to the same reaction well and immediately and continuously detect the fluorescence signal for 10 min.

[0053] (7) Fluorescence detection parameter settings: excitation wavelength 510nm, emission wavelength 590nm.

[0054] Results analysis: such as Figure 1 As shown, a strong increase in fluorescence signal was observed only in the double-addition experimental group (expressing APEX2+, containing AmUR+, and with the addition of exogenous H2O2); while in the control group lacking APEX2 vector, lacking AmUR probe, or without the addition of exogenous H2O2, no significant increase in fluorescence signal was observed. This factorial quality control result demonstrates that the APEX2 / AmUR detection system has high specificity and dependence on H2O2, with no non-specific fluorescence interference, laying a reliable methodological foundation for the subsequent detection of H2O2 in live cells using the fusion protein system of this invention.

[0055] Example 2 Validation of 293T transient conversion of basic fusion protein This embodiment aims to verify the ability of the APEX2-DAAO basic fusion protein without a localization tag to induce and detect hydrogen peroxide in conventional non-immune cells, serving as a technical benchmark for subsequent macrophage models.

[0056] (1) HEK293T cells were seeded in 12-well plates and divided into two groups: a pLV-BlastR transfection control group and a pLV-APEX2-DAAO transfection group (the specific sequence of APEX2-DAAO is described in step (1) of Example 3). The APEX2-DAAO gene fragment was then inserted into the pLV-EF1α-BlastR lentiviral vector backbone to construct the recombinant plasmid pLV-APEX2-DAAO. The transfection steps were the same as in Example 1.

[0057] (2) After transfection, the cells were digested and seeded in 96-well plates coated with poly-L-lysine (PLL) and cultured overnight.

[0058] (3) Discard the culture medium before detection, wash with PBS 3 times, and add 100 μL FluoroBrite DMEM to each well.

[0059] (4) Record the baseline with an enzyme-linked immunosorbent assay (ELISA) reader for 10 min, then add AmUR (final concentration 50 μM) and incubate in the dark for 10 min.

[0060] (5) Perform sequential sample addition stimulation and detection: First stage (substrate stimulation): Add D-ala (final concentration 10mM) and continuously detect fluorescence signal for 10min; Second stage (positive control): Add exogenous H2O2 (final concentration 0.2mM) to the same reaction well and continuously detect fluorescence signal for 10min.

[0061] (6) The fluorescence detection parameters are set in the same way as in Example 1.

[0062] Results analysis: such as Figure 2 As shown, in the D-ala stimulation phase, the experimental group transfected with pLV-APEX2-DAAO showed a significant increase in fluorescence signal after the addition of D-ala, while the control group showed no signal. In the exogenous H2O2 positive control phase, the fluorescence signal further increased after the addition of exogenous H2O2. This embodiment confirms that the APEX2-DAAO fusion protein constructed in this invention has a complete structure, and the functions of the DAAO module in catalyzing the production of H2O2 from D-ala and the APEX2 module in in situ detecting H2O2 are perfectly linked. This indicates that the system can exert its catalytic activity normally in the cytoplasmic environment of conventional non-immune cells, laying the foundation for subsequent translation into macrophages and revealing the phenomenon of compartment-selective inactivation.

[0063] Example 3 Sequential stimulation verification of macrophage multi-compartment stable strains This embodiment aims to explore the spatial limitations of DAAO tools in the harsh microenvironment of macrophages, verify whether mitochondrial-specific compartmentalization targeting can bypass the clearance barrier, and establish a stable evaluation system.

[0064] (1) Construction of fusion protein: 1) Construction of the core mitochondrial-targeting fusion protein (COX8-APEX2-DAAO) The order of COX8-APEX2-DAAO from N to C is as follows: [COX8]x2 - [V5 tag]-[APEX2] - [Flag tag]- [DAAO].

[0065] Structural Description: The N-terminal [COX8]x2 (consisting of two tandem COX8 sequences, providing a very strong and specific mitochondrial localization signal); the mid-terminal APEX2 module; and the C-terminal DAAO module constitute the core dual-function combination of this invention. The DAAO module is responsible for catalyzing the production of endogenous H2O2 in the presence of a substrate (D-alanine) (hydrogen peroxide-induced function); the APEX2 module acts as an in-situ detector for H2O2, catalyzing the conversion of the non-fluorescent AmUR substrate into the highly fluorescent halogen in the presence of H2O2 (hydrogen peroxide detection function). Both are co-expressed in the mitochondrial matrix as a single fusion protein, achieving integrated coupling of "local production-in-situ detection". V5 and Flag are merely standard epitope tags for convenient in vitro immunoassay.

[0066] The amino acid sequence of COX8-APEX2-DAAO: MSVLTPLLLRGLTGSARRRLPVPRAKIHSLGDLSVLTPLLLRGLTGSARLPVPRAK IHSLGDTG GKPI PNPLLGLDST GKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGTFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAVEVTGGPKVPFHPGREDKPEPPPE GRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHTIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDKYAADEDAFFADYAEAHQKLSELGFADAGGGGGS DYKDDDDKMHSQKRVVVLGSGVIGLSSALILARKGYSVHILARDLPEDVSSQTFASPWAGANWTPFMTLTDGPRQAKWEESTFKKWVELVPTGHAMWLKGTRRFAQNEDGLLGHWYKDITPNYRPLPSSECPPGAIGVTYDTLSVHAPKYCQYLARELQKLGATFERRTVTSLEQAFDGADLVVNATGLGAKS IAGIDDQAAEPIRGQTVLVKSPCKRCTMDSSDPASPAYIIPRPGGEVICGGTYGVGDWDLSVNPETVQRILKHCLRLDPTISSDGTIEGIEVLRHNVGLRPARRGGPRVEAERIVLPLDRTKSPLSLGRGSARAAKEKEVTLVHAYGFSSAGYQQSWGAAEDVAQLVDEAFQRYHGAARES, SEQ ID NO: 1.

[0067] [COX8]x2: The first underscore indicates this.

[0068] [V5 tag]: indicated by the second underscore.

[0069] [APEX2]: The first one is shown in bold.

[0070] [Flag]: As indicated by the third underscore.

[0071] [DAAO]: The second one is in bold.

[0072] The nucleotide sequence of COX8-APEX2-DAAO:

[0073] 2) Construction of the basic targetless fusion protein (APEX2-DAAO) The order of APEX2-DAAO from N to C is: [V5 tag] - [APEX2] - [Flag tag] - [DAAO].

[0074] Structural Description: This protein is a basic fusion protein without any organelle localization tags. Its sequence is based on the core sequence SEQ ID NO: 1, except that the N-terminal [COX8]x2 sequence has been removed. All other functional modules (V5-APEX2-Flag-DAAO) are completely identical to the core sequence of SEQ ID NO: 1.

[0075] The amino acid sequence of APEX2-DAAO: , SEQ ID NO: 3.

[0076] The nucleotide sequence of APEX2-DAAO:

[0077] 3) Construction of the cytoplasmic localization fusion protein (APEX2-DAAO-NES) The order of APEX2-DAAO-NES from N end to C end is as follows: [V5 tag] - [APEX2] - [Flag tag] - [DAAO] - [NES].

[0078] Structural description: A conventional NES short peptide tag is attached to the C-terminus of the basic untargeted fusion protein (APEX2-DAAO), and its NES amino acid sequence is: LPPLELTL, SEQ ID NO: 5.

[0079] APEX2-DAAO-NES amino acid sequence: , SEQ ID NO: 6.

[0080] APEX2-DAAO-NES nucleotide sequence:

[0081] 4) Construction of nuclear localization fusion protein (APEX2-DAAO-NLS) The order of APEX2-DAAO-NLS from N end to C end is as follows: [V5 tag] - [APEX2] - [Flag tag] - [DAAO] - [NLS]x3.

[0082] Structural description: A conventional NLS short peptide tag is attached to the C-terminus of the basic untargeted fusion protein (APEX2-DAAO), and its NLS amino acid sequence is: PKKKRKV, SEQ ID NO: 8.

[0083] APEX2-DAAO-NLS amino acid sequence: , SEQ IDNO: 9.

[0084] APEX2-DAAO-NLS nucleotide sequence:

[0085] 5) Construction of the endoplasmic reticulum localization fusion protein (prolactin-APEX2-DAAO-KDEL) The order of prolactin-APEX2-DAAO-KDEL from N-terminus to C-terminus is as follows: [prolactin signal peptide] - [V5 tag] - [APEX2] - [Flag tag] - [DAAO] - [KDEL].

[0086] Structural description: A prolactin signal peptide was attached to the N-terminus of the basic untargeted fusion protein (APEX2-DAAO), with the sequence: MDSKGSSQKGSRLLLLLVVSNLLLCQGVVST, SEQ ID NO: 11; and an endoplasmic reticulum retention signal KDEL was attached to the C-terminus, with the sequence: KDEL, SEQ ID NO: 12.

[0087] prolactin-APEX2-DAAO-KDEL amino acid sequence: MDSKGSSQKGSRLLLLLVVSNLLLCQGVVSTGGKPIPNPLLGLDSTGKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGTFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAVEVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHTIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDKYAADEDAFFADYAEAHQKLSELGFADAGGGGGSDYKDDDDKMHSQKRVVVLGSGVIGLSSALILARKGYSVHILARDLPEDVSSQTFASPWAGANWTPFMTLTDGPRQAKWEESTFKKWVELVPTGHAMWLKGTRRFAQNEDGLLGHWYKDITPNYRPLPSSECPPGAIGVTYDTLSVHAPKYCQYLARELQKLGATFERRTVTSLEQAFDGADLVVNATGLGAKSIAGIDDQAAEPIRGQTVLVKSPCKRCTMDSSDPASPAYIIPRPGGEVICGGTYGVGDWDLSVNPETVQRILKHCLRLDPTISSDGTIEGIEVLRHNVGLRPARRGGPRVEAERIVLPLDRTKSPLSLGRGSARAAKEKEVTLVHAYGFSSAGYQQSWGAAEDVAQLVDEAFQRYHGAARESKDEL, SEQ ID NO: 13.

[0088] Nucleotide sequence of prolactin-APEX2-DAAO-KDEL:

[0089] 6) Construction of Golgi-localized fusion protein (OSBP(PH)-APEX2-DAAO) The order of OSBP(PH)-APEX2-DAAO from N to C is as follows: [OSBP(PH)] - [V5 tag] - [APEX2] - [Flag tag] - [DAAO].

[0090] Structural description: An OSBP(PH) is attached to the N-terminus of the basic untargeted fusion protein (APEX2-DAAO), with the sequence: SGSAREGWLFKWTNYIKGYQRRWFVLSNGLLSYYRSKAEMRHTCRGTINLATANITVEDSCNFIISNGGAQTYHLKASSEVERQRWVTALELAKAKAVK, SEQ ID NO: 15.

[0091] The amino acid sequence of OSBP(PH)-APEX2-DAAO: MSGSAREGWLFKWTNYIKGYQRRWFVLSNGLLSYYRSKAEMRHTCRGTINLATANITVEDSCNFIISNGGAQTYHLKASSEVERQRWVTALELAKAKAVKRSIATGKPIPNPLLGLDSTGKSYPTVSADYQDAVEKAKKKLRGFIAEKRCAPLMLRLAFHSAGTFDKGTKTGGPFGTIKHPAELAHSANNGLDIAVRLLEPLKAEFPILSYADFYQLAGVVAVEVTGGPKVPFHPGREDKPEPPPEGRLPDPTKGSDHLRDVFGKAMGLTDQDIVALSGGHTIGAAHKERSGFEGPWTSNPLIFDNSYFTELLSGEKEGLLQLPSDKALLSDPVFRPLVDKYAADEDAFFADYAEAHQKLSELGFADAGGGGGSDYKDDDDKMHSQKRVVVLGSGVIGLSSALILARKGYSVHILARDLPEDVSSQTFASPWAGANWTPFMTLTDGPRQAKWEESTFKKWVELVPTGHAMWLKGTRRFAQNEDGLLGHWYKDITPNYRPLPSSECPPGAIGVTYDTLSVHAPKYCQYLARELQKLGATFERRTVTSLEQAFDGADLVVNATGLGAKSIAGIDDQAAEPIRGQTVLVKSPCKRCTMDSSDPASPAYIIPRPGGEVICGGTYGVGDWDLSVNPETVQRILKHCLRLDPTISSDGTIEGIEVLRHNVGLRPARRGGPRVEAERIVLPLDRTKSPLSLGRGSARAAKEKEVTLVHAYGFSSAGYQQSWGAAEDVAQLVDEAFQRYHGAARES, SEQ ID NO: 16.

[0092] Nucleotide sequence of OSBP(PH)-APEX2-DAAO:

[0093] (2) Construction of recombinant vector The gene fragments of the fusion protein obtained in step (1) were inserted into the pLV-EF1α-BlastR lentiviral vector backbone to construct recombinant plasmids pLV-COX8-APEX2-DAAO, pLV-APEX2-DAAO-NES, pLV-APEX2-DAAO-NLS, pLV-prolactin-APEX2-DAAO-KDEL, and pLV-OSBP(PH)-APEX2-DAAO.

[0094] (3) Lentiviral packaging and collection Lentiviral expression plasmids pLV-COX8-APEX2-DAAO, pLV-APEX2-DAAO-NES, pLV-APEX2-DAAO-NLS, pLV-prolactin-APEX2-DAAO-KDEL, and pLV-OSBP(PH)-APEX2-DAAO were co-transfected into HEK293T cells with lentiviral packaging plasmids psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259), respectively. Cell supernatants rich in lentiviral particles were collected 48–72 hours after transfection.

[0095] (4) Macrophage infection and screening: iBMDM cells in good growth condition were plated. The collected lentivirus supernatant was added to the culture medium, and Polybrene (Santa Cruz Biotechnology, sc-134220) was added to a final concentration of 8 μg / mL to assist infection. 24-48 hours after infection, the virus solution was removed, and the medium was replaced with complete culture medium containing 10 μg / mL blastomycin (Solarbio, B9300) for pressure screening. Screening continued for 1 week until all cells in the control group (uninfected group) died. The surviving cells were the stable iBMDM cell lines expressing the five corresponding fusion proteins.

[0096] (5) The iBMDM stable cell lines expressing the above 5 fusion proteins were seeded into 96-well plates with black transparent bottoms. Each cell line was set up as an experimental group and a blank control group, and each group was set up with 3 parallel replicates.

[0097] (6) Add 100 μL of complete culture medium to each well and incubate overnight in a 37°C, 5% CO2 incubator. The cell adhesion and confluence should be about 70% by the next day.

[0098] (7) Discard the culture medium before detection, gently wash the cells three times with PBS to remove residual background, and then add 100 μL of pre-warmed FluoroBrite DMEM to each well.

[0099] (8) Place the 96-well plate in the microplate reader and record the baseline fluorescence signal of each well for 10 min.

[0100] (9) Dilute AmUR with FluoroBrite DMEM and add it to each well to make a final concentration of 50 μM, and continue to incubate for 10 min.

[0101] (10) After incubation, sequential stimuli were added and real-time monitoring was performed according to the experimental design: For the experimental group: First stage: Add D-alanine (D-ala) to each well to a final concentration of 10 mM, and immediately use a microplate reader to continuously detect the fluorescence signal for 10 min.

[0102] Second stage: Immediately afterwards, D-norvaline (D-nva) was added directly to the same reaction well to a final concentration of 2 mM, and the fluorescence signal was immediately and continuously detected for 10 min.

[0103] Third stage: Finally, H2O2 was added directly to the same reaction well to make the final concentration 0.2 mM, and the fluorescence signal was immediately and continuously detected for 10 min.

[0104] The purpose of adding D-nva after D-ala: a. Eliminating false negatives due to occasional failure of a single substrate: Given that non-mitochondrial compartments (such as the cytoplasm and nucleus) showed no response to D-ala stimulation, the structurally different substrate D-nva was added in situ, and still no response was observed. This aimed to eliminate false negatives caused by loss of affinity for a specific single substrate or restricted substrate transport by the DAAO enzyme. This design rigorously demonstrated that the absence of H2O2 signaling in non-mitochondrial compartments is caused by the broad-spectrum and potent antioxidant scavenging effect of the macrophage's local microenvironment, rather than the catalytic failure of a single substrate.

[0105] b. Verification of the substrate universality of the mitochondrial targeting model: For the only surviving mitochondrial targeting (COX8) group, the sequential addition of two different D-amino acids could effectively excite fluorescence response, confirming that the cell model has excellent enzymatic catalytic applicability to a variety of DAAO substrates, further supporting the high stability and practical value of the model in the in vitro screening system.

[0106] For the blank control group (drug-free parallel control): First stage: Add an equal volume of FluoroBrite DMEM to each well and immediately use a microplate reader to continuously detect the fluorescence signal for 10 min.

[0107] Second stage: Immediately afterwards, an equal volume of FluoroBrite DMEM was added directly to the same reaction well, and the fluorescence signal was continuously detected for 10 min.

[0108] Third stage: Finally, add an equal volume of FluoroBrite DMEM directly to the same reaction well and immediately and continuously detect the fluorescence signal for 10 min.

[0109] (11) Fluorescence detection parameter settings: excitation wavelength 510nm, emission wavelength 590nm.

[0110] (12) The changes in fluorescence intensity at each stage and time point (RFU dynamic curve) reflect the dynamic changes in the local H2O2 level in the subcellular compartment of the living cell.

[0111] Results analysis: such as Figure 3 As shown, DAAO catalytic activity was severely inactivated in the non-mitochondrial compartments of macrophages: When endogenous stimulation was achieved with the substrates D-ala and D-nva, no significant increase in fluorescence signal was detected in stable transgenic cells localized in the cytoplasm (NES), nucleus (NLS), endoplasmic reticulum (KDEL), and Golgi apparatus (OSBP) (signal flatness). This indicates that in these compartments, the trace amounts of endogenous H2O2 produced by DAAO catalysis were instantaneously cleared by the high-concentration antioxidant system in the local macrophage, resulting in the complete loss of effective catalytic activity.

[0112] Mitochondrial targeting successfully bypassed the scavenging barrier: In contrast, stable transgenic strains of mitochondrial targeting (COX8) showed significant and time-dependent increases in fluorescence signal after substrate addition. This confirms that the specific microenvironment of the mitochondrial matrix successfully avoided the broad-spectrum scavenging effect, allowing DAAO catalytic activity to be stably maintained and endogenous H2O2 accumulation to be achieved.

[0113] In situ positive control to rule out false negatives: Subsequently, exogenous H2O2 was added to the four groups (NES / NLS / KDEL / OSBP) that showed no obvious response. The results showed that the fluorescence signal in each group increased rapidly. This ruled out false negatives caused by protein non-expression or detection module inactivation, confirming that the lack of signal was due to the local microenvironment clearing products.

[0114] This embodiment successfully overcomes the technical bias that "enzyme activity can be achieved at any location," confirming the existence of a stringent clearance barrier in the non-mitochondrial compartments of macrophages. This invention utilizes mitochondrial-specific compartmentalization targeting, becoming the only effective method for stably assessing the in-situ catalytic activity of DAAO in macrophages, and successfully establishing a high signal-to-noise ratio cell screening system.

[0115] Example 4 FCCP synergistic sensitization drug screening application This embodiment aims to verify the feasibility and sensitivity of the above-mentioned mitochondrial assessment system in targeted drug screening, and to confirm the unexpected effect of the mitochondrial uncoupling agent FCCP in synergistically enhancing the catalytic activity of DAAO.

[0116] (1) The iBMDM-COX8-APEX2-DAAO stable cell line obtained in Example 3 was seeded into 96-well plates with black transparent bottoms. Four treatment groups were set up in the experiment, with three replicates in each group: ① Control group (untreated).

[0117] ②Incubation group with FCCP alone (10μM FCCP).

[0118] ③ The group that received D-ala stimulation alone (10mM D-ala).

[0119] ④ Combined treatment group (10 μM FCCP + 10 mM D-ala).

[0120] (2) Add 100 μL of complete culture medium to each well and incubate overnight in a 37°C, 5% CO2 incubator so that the cell adhesion and confluence is about 70% the next day.

[0121] (3) Discard the culture medium before detection, gently wash the cells three times with PBS, and then add 100 μL of pre-warmed FluoroBrite DMEM to each well.

[0122] (4) Place the 96-well plate in the microplate reader and record the baseline fluorescence signal of each well for 10 min.

[0123] (5) Dilute AmUR with FluoroBrite DMEM and add it to each well to make a final concentration of 50 μM. Continue to incubate and detect the fluorescence signal for 10 min.

[0124] (6) After incubation, quickly add the corresponding stimulant to each well according to the group: ① Untreated control group: Add an equal volume of FluoroBrite DMEM diluent (or control solution containing an equal proportion of DMSO).

[0125] ② FCCP incubation group: Add FCCP to the wells to make the final concentration 10 µM.

[0126] ③ Single D-ala stimulation group: D-alanine was added to the wells to a final concentration of 10 mM.

[0127] ④ Combined treatment group: FCCP (final concentration 10 µM) and D-alanine (final concentration 10 mM) were added to the wells at the same time.

[0128] (7) Immediately after adding the drug, place the 96-well plate in the microplate reader and continuously detect the fluorescence signal for 1 hour.

[0129] (8) Fluorescence detection parameter settings: excitation wavelength 510nm, emission wavelength 590nm.

[0130] The regulatory effect of different drug treatments on the endogenous H2O2 level in the mitochondria of living cells was evaluated by the dynamic change curve of fluorescence intensity within 1 h for each treatment group, or by the cumulative fluorescence intensity (RFU) at the end of 1 h.

[0131] Results analysis: such as Figure 4 As shown, both the control group and the FCCP-only group maintained extremely low baseline fluorescence signals within 1 hour. This demonstrates that the model has a clean background and that treatment with the candidate drug FCCP alone does not induce non-specific fluorescence interference.

[0132] In the D-ala group alone, the fluorescence signal showed a significant time-dependent increase. This confirms that the system can stably establish an endogenous H2O2 reaction baseline with a specific substrate.

[0133] The combined treatment group (FCCP+D-ala) showed a significant synergistic enhancement effect in fluorescence signal, with both the signal rise rate and the terminal cumulative fluorescence intensity being significantly higher than the group with D-ala alone.

[0134] The aforementioned drug screening application data demonstrate that the macrophage mitochondrial targeting system constructed in this invention possesses extremely high drug evaluation sensitivity. This invention unexpectedly discovered and confirmed that the mitochondrial uncoupling agent FCCP, as a "catalytic accumulation synergist," can significantly enhance the DAAO-mediated local H2O2 accumulation efficiency when used in combination with D-ala. This directly verifies the practical value of the system of this invention as an in vitro high-throughput screening platform, providing a novel drug combination strategy for targeted regulation of macrophage mitochondrial oxidative stress.

[0135] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0136] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for enhancing the local accumulation of endogenous hydrogen peroxide in macrophages, characterized in that, In recombinant macrophages that stably express mitochondrial-targeting D-amino acid oxidase, administration of D-amino acid oxidase substrates to initiate local production of endogenous hydrogen peroxide; the D-amino acid oxidase substrates are D-alanine and / or D-valine.

2. The method according to claim 1, characterized in that, The method further includes administering a mitochondrial uncoupling agent simultaneously with the D-amino acid oxidase substrate in recombinant macrophages that stably express mitochondrial-targeting D-amino acid oxidase to initiate the local production of endogenous hydrogen peroxide.

3. The method according to claim 2, characterized in that, The mitochondrial uncoupling agent is 4-trifluoromethoxyphenylhydrazone.

4. The method according to claim 1 or 2, characterized in that, The recombinant macrophages also express the APEX2 detection module, which is co-expressed with the mitochondrial-targeted D-amino acid oxidase in the form of a fusion protein.

5. A recombinant macrophage cell line, characterized in that, The recombinant macrophage cell line expresses a recombinant fusion protein, which, from the N-terminus to the C-terminus, comprises: a mitochondrial localization sequence, an APEX2 detection module, and a D-amino acid oxidase catalytic module; when the cell line is stimulated with D-amino acid oxidase substrate, no detectable endogenous hydrogen peroxide accumulation signal is found in the non-mitochondrial compartment. The amino acid sequence of the recombinant fusion protein is shown in SEQ ID NO.1; the nucleotide sequence is shown in SEQ ID NO.

2.

6. The recombinant macrophage cell line according to claim 5, characterized in that, The cell line is macrophage derived from the bone marrow of immortalized mice.

7. The use of the recombinant macrophage cell line according to any one of claims 5-6 in preparing a cell model for in vitro screening of drugs targeting macrophage mitochondrial oxidative stress regulation.

8. The application of mitochondrial uncoupling agents in the preparation of regulators for synergistically enhancing the catalytic efficiency of macrophage mitochondrial-localized D-amino acid oxidase in hydrogen peroxide accumulation, characterized in that, The mitochondrial uncoupling agent is used in combination with a D-amino acid oxidase substrate; the mitochondrial uncoupling agent is 4-trifluoromethoxyphenylhydrazone.

9. A composition, characterized in that, The composition comprises a mitochondrial uncoupling agent and a D-amino acid oxidase substrate, and is used to synergistically enhance the accumulation of hydrogen peroxide catalyzed by macrophage-localized D-amino acid oxidase; the mitochondrial uncoupling agent is 4-trifluoromethoxyphenylhydrazone; and the D-amino acid oxidase substrate is D-alanine and / or D-valine.

10. A method for in vitro screening of drugs targeting the regulation of mitochondrial oxidative stress in macrophages, characterized in that, Includes the following steps: (1) Provide the recombinant macrophage cell line according to any one of claims 5-6; (2) Contact the candidate drug with the cell line; (3) D-amino acid oxidase substrate was administered, and changes in the level of local hydrogen peroxide accumulation in mitochondria were detected; (4) Based on the changes in hydrogen peroxide accumulation levels, candidate drugs for regulating mitochondrial oxidative stress in macrophages were screened.

Images