A light-controlled NAMPT enzyme, nucleic acid and its application

Abstract

The present invention relates to the field of biomedicine technology, and in particular to a light-controlled NAMPT enzyme, nucleic acid and its application. The light-controlled NAMPT enzyme includes a NAMPT enzyme and a cpLov2 photosensitive protein domain, the cpLov2 photosensitive protein domain is embedded in the NAMPT enzyme, and the amino acid sequence of the light-controlled NAMPT enzyme is shown in SEQ ID NO.1. After the light-controlled NAMPT enzyme is stably expressed in cells and tissues, the laser irradiation with high temporal and spatial resolution can be used to achieve real-time regulation of the NAMPT enzyme activity in specific cells and specific tissue parts. The present invention also illustrates the role of the light-controlled NAMPT enzyme in tumor immunity research by specifically optically regulating the NAMPT enzyme activity of tumors and immune cells in a tumor and immune cell co-culture system.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a light-controlled NAMPT enzyme, nucleic acid and applications thereof. Background Art

[0002] Nicotinamide adenine dinucleotide (NAD+) is an important coenzyme widely present in cells and is involved in regulating a variety of biological processes. Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the biosynthesis of NAD+ in mammalian cells. NAMPT enzyme plays a key role in the NAD+ biosynthesis pathway by catalyzing the synthesis of nicotinamide (NAM) from nicotinamide (NAM). NAMPT enzyme plays an important role in the occurrence and treatment of a variety of diseases. For example, overexpression of NAMPT enzyme in tumor cells is associated with tumor proliferation, invasion and metastasis. Inhibiting the activity of NAMPT enzyme can block the dependence of tumor cells on NAD+ and inhibit tumor growth. In addition, NAMPT enzyme is also closely related to metabolic diseases, neurodegenerative diseases, etc. NAMPT enzyme can regulate the activity and function of immune cells by regulating NAD+ levels, affecting the degree and duration of inflammatory response. Therefore, the activity regulation of NAMPT enzyme is of great significance for the development of tumor drugs and the analysis of disease mechanisms.

[0003] The regulation of NAMPT enzyme activity mainly relies on small molecule inhibitors, small molecule agonists, small interfering RNA (siRNA) or short hairpin RNA (shRNA), etc.

[0004] The activity of NAMPT enzyme can be interfered with by designing and synthesizing small molecule compounds. These small molecule compounds (i.e. small molecule inhibitors or small molecule agonists) can bind to the active site of NAMPT enzyme and regulate its catalytic action, thereby reducing or increasing the synthesis efficiency of nicotinamide adenine dinucleotide (NAD+). The development of NAMPT small molecule inhibitors is of great significance in the field of tumor treatment. Since tumor cells are highly dependent on the synthesis of NAD+, inhibiting the activity of NAMPT enzyme can interfere with the energy metabolism and survival mechanism of tumor cells, thereby inhibiting the growth and spread of tumors.

[0005] By using siRNA or shRNA technology, the expression level of NAMPT enzyme can be reduced by targeting the mRNA sequence of NAMPT enzyme, and the synthesis of NAMPT enzyme can be inhibited through RNA interference mechanism, thereby reducing the intracellular NAD+ level. However, siRNA and shRNA strategies have not yet been widely used in the field of clinical drugs.

[0006] Although strategies such as small molecule inhibitors or small molecule agonists, siRNA or shRNA play an important role in regulating NAMPT enzyme activity, these strategies have the following limitations:

[0007] First, these strategies mainly rely on systemic administration, so it is usually difficult to precisely regulate the NAD+ metabolism of specific types of cells and tissues. Such strategies usually affect the NAD+ metabolism of different cells at the same time and lack cell and tissue targeting.

[0008] Second, these strategies are difficult to achieve real-time regulation in model tissues and animals. Existing small molecule drugs, siRNA or shRNA strategies are difficult to achieve precise inhibition of NAMPT enzyme activity in specific cells within a few minutes.

[0009] Third, due to the above limitations, the existing NAMPT enzyme activity regulation strategy shows obvious limitations in the scenario of studying the regulatory mechanisms of tumor cells and immune cells. The interaction between tumor cells and immune cells is crucial for immunotherapy and the regulation of the tumor microenvironment. However, the interaction mechanism between NAD+ metabolism in tumor cells and immune cells is not fully understood. It is difficult for drugs to inhibit NAD+ metabolism in immune cells or tumor cells alone, which makes it difficult to analyze the role of NAD+ metabolism in immune cells and tumor cells separately.

[0010] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0011] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a light-controlled NAMPT enzyme, nucleic acid and application thereof, aiming to solve the problem that the existing NAMPT enzyme activity regulation strategy lacks cell and tissue targeting and is difficult to achieve real-time regulation.

[0012] The technical solution of the present invention is as follows:

[0013] A light-controlled NAMPT enzyme, wherein the light-controlled NAMPT enzyme comprises a NAMPT enzyme and a cpLov2 photosensitive protein domain, the cpLov2 photosensitive protein domain is embedded in the NAMPT enzyme, and the amino acid sequence of the light-controlled NAMPT enzyme is shown in SEQ ID NO.1.

[0014] A nucleic acid, wherein the nucleic acid contains a nucleotide sequence encoding the light-controlled NAMPT enzyme of the present invention.

[0015] A vector, wherein the vector carries the nucleic acid described in the present invention.

[0016] A cell, wherein the cell expresses the light-controlled NAMPT enzyme of the present invention, or contains the nucleic acid of the present invention, or contains the vector of the present invention.

[0017] A use of the light-controlled NAMPT enzyme of the present invention in catalyzing the reaction of nicotinamide synthesizing nicotinamide adenine mononucleotide.

[0018] Optionally, the catalytic activity of the light-controlled NAMPT enzyme is regulated by light.

[0019] A use of the light-controlled NAMPT enzyme of the present invention in the preparation of a drug for regulating the metabolism of nicotinamide adenine dinucleotide in cells or tissues.

[0020] Optionally, the catalytic activity of the light-controlled NAMPT enzyme is regulated by light.

[0021] A use of the light-controlled NAMPT enzyme of the present invention in preparing a drug for treating tumors.

[0022] A drug for treating tumors, comprising the light-controlled NAMPT enzyme of the present invention.

[0023] Beneficial effects: The present invention uses the principle of protein engineering to develop a light-controlled NAMPT enzyme (a NAMPT enzyme mutant) whose enzyme activity is controlled by blue light. After the light-controlled NAMPT enzyme is stably expressed in cells and tissues, the NAMPT enzyme activity of specific cells and specific tissue parts can be regulated in real time by laser irradiation with high temporal and spatial resolution. The present invention also specifically optically regulates the NAMPT enzyme activity of tumors and immune cells in a tumor and immune cell co-culture system, illustrating the role of the light-controlled NAMPT enzyme in tumor immunity research.

[0024] The activity of the light-controlled NAMPT enzyme provided by the present invention can achieve reversible inhibition under 420 nm laser irradiation. Compared with the traditional NAMPT small molecule inhibition, and siRNA or shRNA inhibition strategy, the inhibition strategy of the present invention can achieve NAD+ metabolic regulation for specific cells and tissue regions, and can also achieve NAD+ metabolic regulation with significantly improved temporal and spatial resolution, and can also achieve rapid and reversible activity regulation of NAMPT enzyme activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A in the figure shows the main loop structure and site of the NAMPT dimer (chain A and chain B) exposed to the solvent; B shows the schematic diagram of the cpLov2 protein domain after being embedded in the NAMPT enzyme; C shows the schematic diagram of the light-controlled activity regulation of the NAMPT-cpLov2 chimeric protein.

[0026] Figure 2This is a graph showing the test results of the NMN synthesis catalytic activity of different NAMPT-cpLov2 chimeric protein sequences.

[0027] Figure 3 This figure shows the results of optical regulation verification of the NMN synthesis catalytic activity of different NAMPT-cpLov2 chimeric protein sequences.

[0028] Figure 4 This is a diagram showing the results of optical inhibition verification of the NMN synthesis catalytic activity of the NAMPT (N182-L183)-cpLov2 chimeric protein.

[0029] Figure 5 Figure A is the test result of NAMPT (N182-L183)-cpLov2 chimeric protein regulating the NMN content in HEK293T cells; Figure B is the result of measuring the NAD+ level in the cell lysate by HPLC-MS / MS.

[0030] Figure 6 A is a schematic diagram of the differential regulation of NAD+ metabolism of cancer cells and T cells by NAMPT (N182-L183)-cpLov2 chimeric protein; B is a schematic diagram of the cancer cell killing of mixed culture of LLC tumor cells expressing NAMPT (N182-L183)-cpLov2 and empty plasmid T cells (immune cells) before and after illumination; C is a schematic diagram of the cancer cell killing of mixed culture of LLC tumor cells expressing empty plasmid and T cells expressing NAMPT (N182-L183)-cpLov2 before and after illumination; D is a schematic diagram of the regulation of NAD+ metabolism of LLC tumor cells and T cells in mice using the NAMPT small molecule inhibitor FK866. DETAILED DESCRIPTION

[0031] The present invention provides a light-controlled NAMPT enzyme, nucleic acid and application thereof. To make the purpose, technical scheme and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] An embodiment of the present invention provides a light-controlled NAMPT enzyme, wherein the light-controlled NAMPT enzyme includes a NAMPT enzyme and a cpLov2 photosensitive protein domain, the cpLov2 photosensitive protein domain is embedded in the NAMPT enzyme, the light-controlled NAMPT enzyme is recorded as NAMPT-cpLov2 chimeric protein, the NAMPT-cpLov2 chimeric protein is NAMPT (N182-L183) -cpLov2, and the amino acid sequence of NAMPT (N182-L183) -cpLov2 is shown in SEQ ID NO.1.

[0033] The light-controlled NAMPT enzyme (belonging to a NAMPT enzyme mutant) provided in an embodiment of the present invention is NAMPT (N182-L183) -cpLov2 (amino acid sequence as shown in SEQ ID NO.1), and the light-controlled NAMPT enzyme is an enzyme whose NAMPT enzyme activity is optically controlled. The rate at which this light-controlled NAMPT enzyme catalyzes nicotinamide to synthesize nicotinamide adenine mononucleotide is regulated by 420 nm irradiation light. When irradiated with 420 nm blue light, the catalytic activity of the light-controlled NAMPT enzyme drops to about 1% of that of the wild-type NAMPT enzyme, but the light-controlled NAMPT enzyme activity recovers after the irradiation ends. Therefore, the embodiment of the present invention provides an effective method for rapidly and accurately regulating the NAMPT enzyme activity and NAD+ bioavailability in specific cells.

[0034] Specifically, the present invention uses protein engineering principles to design a series of chimeric proteins of NAMPT enzyme and cpLov2 photosensitive protein domain. cpLov2 is a photosensitive protein domain that can be embedded in the appropriate region of NAMPT enzyme to achieve regulation of NAMPT enzyme activity.

[0035] After obtaining a series of preliminary NAMPT-cpLov2 chimeric proteins, the activity screening of NAMPT-cpLov2 chimeric proteins was achieved using NMN fluorescent probes. The NAMPT-cpLov2 chimeric protein was used to catalyze the synthesis of NMN, and its activity was characterized by the synthesis rate of NMN. The activity ratio before and after illumination was used to determine the NAMPT-cpLov2 chimeric protein sequence whose catalytic activity was obviously controlled by illumination, thereby obtaining the light-controlled NAMPT enzyme.

[0036] The following is an overview of the process of determining the sequence of the NAMPT-cpLov2 chimeric protein whose catalytic activity is clearly optically controlled.

[0037] 1. Design and expression of NAMPT-cpLov2 chimeric protein

[0038] Using the principle of protein engineering, the cpLov2 photosensitive protein domain was designed to be embedded in the appropriate position of the NAMPT enzyme to construct the NAMPT-cpLov2 chimeric protein. The main selected cpLov2 embedding site is the relatively flexible ring structure (loop structure) in the NAMPT enzyme structure that is exposed to the solvent side, see Figure 1 As shown in A and B. Based on the above principles, a series of NAMPT-cpLov2 chimeric proteins were designed and their DNA sequences were synthesized.

[0039] Through genetic engineering technology, the designed DNA sequence of NAMPT-cpLov2 chimeric protein is introduced into Escherichia coli for chimeric protein expression to express the chimeric protein. Then, the purified NAMPT-cpLov2 chimeric protein is obtained through protein extraction and purification methods.

[0040] The amino acid sequence of the designed NAMPT-cpLov2 chimeric protein includes:

[0041] NAMPT(N182-L183)-cpLov2, the amino acid sequence of which is shown in SEQ ID NO.1;

[0042] NAMPT (D235-P236)-cpLov2, the amino acid sequence of which is shown in SEQ ID NO. 2;

[0043] NAMPT(S303-T304)-cpLov2, the amino acid sequence of which is shown in SEQ ID NO.3;

[0044] NAMPT(N337-S338)-cpLov2, the amino acid sequence of which is shown in SEQ ID NO.4.

[0045] Screening and characterization of the enzymatic activity of NAMPT-cpLov2 chimeric protein

[0046] The NAMPT enzyme reaction system was constructed, and the real-time measurement of NMN concentration in the reaction system was achieved using an automated bioluminescent microplate reader and a specific bioluminescent NMN fluorescent probe. 10 mM phosphoribosyl pyrophosphate (PRPP), 50 nM NAMPT-cpLov2 chimeric protein, and 1 nM NMN fluorescent probe were added to the reaction buffer (50 mM 4-hydroxyethylpiperazineethanesulfonic acid HEPES, pH 7.2, 50 mM NaCl, 2.5 mM adenine nucleoside triphosphate ATP) in sequence and incubated at 37°C for 10 minutes. 10 mM NAM and 1000-fold diluted bioluminescent protein substrate Furimazine (Furimazine is a luminescent substrate of imidazopyrazinone) were added to start the reaction. The bioluminescent light intensity values ​​at 440 nm and 590 nm were read using an automated bioluminescent microplate reader, and the 590 nm / 440 nm light intensity ratio was used to indicate the NMN concentration. The synthesis rate of NMN in the reaction system was used to characterize the enzymatic activity of the NAMPT-cpLov2 chimeric protein. Based on the above method, the regulatory ability of 420 nm light irradiation on the enzymatic reaction (i.e., the enzymatic activity of the NAMPT-cpLov2 chimeric protein) was compared. Figure 1 As shown in C. NAMPT-cpLov2 chimeric proteins whose NMN synthesis rate was significantly inhibited before and after illumination were selected for subsequent experiments.

[0047] The detection found that the NAMPT (N182-L183)-cpLov2 chimeric protein showed the most significant light inhibition, and this chimeric protein was determined to be the dominant protein for priority use.

[0048] 3. Optical regulation of NAMPT-cpLov2 chimeric protease activity

[0049] The optical response ability of the chimeric protein obtained by screening was verified in the NAMPT enzyme reaction system. 10 mM PRPP, 50 nM NAMPT(N182-L183)-cpLov2 chimeric protein, and 1 nM NMN fluorescent probe were added to the reaction buffer (50 mM HEPES, pH 7.2, 50 mM NaCl, 2.5 mM ATP) in sequence and incubated at 37°C for 10 minutes. 10 mM NAM and 1000-fold diluted bioluminescent protein substrate Furimazine (Furimazine is a luminescent substrate of imidazopyrazinone) were added to start the reaction. The bioluminescent light intensity values ​​at 440 nm and 590 nm were read by an automated bioluminescent microplate reader, and the 590 nm / 440 nm light intensity ratio was used to indicate the NMN concentration. During the reaction, a laser with a wavelength of 420 nm was used to irradiate the reaction system, and the NMN synthesis rate before and after laser irradiation was observed.

[0050] 4. Live cell testing and application of NAMPT-cpLov2 chimeric protein

[0051] A HEK293T cell line stably expressing NAMPT(N182-L183)-cpLov2 and NMN fluorescent probe was constructed. NAMPT(N182-L183)-cpLov2 was used to regulate cellular NAD+ metabolism, and the NMN fluorescent probe was used to detect the NMN concentration in living cells. After the real-time monitoring, the cells were collected and the NAD+ level in the cell lysate was measured by HPLC-MS to verify the regulatory ability of NAMPT(N182-L183)-cpLov2.

[0052] NAMPT(N182-L183)-cpLov2 chimeric protein differentially regulates NAD+ metabolism in cancer cells and immune cells, indicating that NAMPT(N182-L183)-cpLov2 achieves light-controlled differential cell regulation. Mouse LLC lung cancer cell lines stably expressing NAMPT(N182-L183)-cpLov2 and normal mouse mononuclear cells (PBMC cells) expressing NAMPT(N182-L183)-cpLov2 were prepared by lentiviral transfection.

[0053] Under the condition of shielding blue light, the LLC lung cancer cell line expressing NAMPT (N182-L183)-cpLov2 and normal mouse PBMC cells were co-cultured. 420 nm wavelength optical fiber irradiation was introduced to compare the killing of cancer cells in the co-culture system before and after 420 nm light irradiation. In a parallel experiment, the killing of cancer cells with the addition of NAMPT small molecule inhibitors was compared.

[0054] Under the condition of shielding blue light, normal mouse LLC lung cancer cell line and mouse PBMC cells expressing NAMPT (N182-L183)-cpLov2 were co-cultured. 420 nm wavelength optical fiber irradiation was introduced to compare the killing of cancer cells in the co-culture system before and after 420 nm light irradiation. In a parallel experiment, the killing of cancer cells with the addition of NAMPT small molecule inhibitors was compared.

[0055] Experiments show that NAMPT(N182-L183)-cpLov2 can selectively and specifically regulate NAD+ metabolism in specific cells.

[0056] The present invention will be further described below by means of specific examples.

[0057] 1. Screening and characterization of enzyme activity of NAMPT-cpLov2 chimeric protein

[0058] The DNA sequence of the NAMPT-cpLov2 chimeric protein was synthesized by Wuhan Miaoling Biotechnology Co., Ltd. and connected to the pET28a expression plasmid. The expression plasmid was prepared into a plasmid solution with a concentration of 100 ng / μL in ultrapure water, and 2 μL of the plasmid solution was transformed into the expression strain E. coli BL21 (DE3) by chemical competent transformation to express the NAMPT-cpLov2 chimeric protein. Then, the purified NAMPT-cpLov2 chimeric protein was obtained by protein extraction and purification.

[0059] 1.1. Expression of NAMPT-cpLov2 chimeric protein

[0060] 1) Pick a single colony from a petri dish (LB plate) containing solid agar medium of the expression strain E. coli BL21 (DE3), inoculate it into a 5 mL liquid LB medium tube containing 50 μg / μL kanamycin, and culture it overnight at 37°C and 220 rpm.

[0061] 2) According to the 1% inoculation volume, add 1 mL of overnight cultured bacteria into a conical flask containing 200 mL of liquid LB medium, add kanamycin at a final concentration of 50 μg / μL, and culture in a shaker at 37°C, 220 rpm for about 3 h until the OD value of the bacterial solution reaches 0.5.

[0062] 3) Lower the shaker temperature to 16°C, add isopropylthiogalactoside (IPTG) to a final concentration of 0.1 mM to the culture medium to induce protein expression, and continue culturing for 16 h.

[0063] 4) After the culture is completed, pour the bacterial solution into a 500 mL centrifuge bucket and centrifuge at 5000 rpm for 10 min to collect the bacteria.

[0064] 1.2. Purification of NAMPT-cpLov2 chimeric protein

[0065] 1) Bacterial disruption: Use lysis buffer (20 mM Tris, 0.5 M NaCl, 20 mM imidazole, pH 8.0) to suspend the bacteria collected in the centrifuge bucket. Precool the high-pressure homogenizer to 4°C in advance, remove the 20% ethanol used for storage, wash the machine twice with deionized water, and then wash the machine twice with lysis buffer. Increase the machine pressure to about 600 bar to disrupt the bacteria.

[0066] 2) Bacterial precipitation: put the collected bacterial fragments into a centrifuge tube, centrifuge at 12000 rpm for 20 min, and collect the supernatant.

[0067] 3) Column loading and purification: Balance the column with lysis buffer. Pour the supernatant directly into the nickel column and use gravity to make the supernatant flow through the nickel column filler, thereby binding the protein to the nickel column filler. Then use lysis buffer to wash off the impurities that do not specifically bind to the nickel column filler. Finally, elute the NAMPT-cpLov2 chimeric protein with elution buffer (lysis buffer containing 150 mM imidazole).

[0068] 4) Protein concentration: The collected NAMPT-cpLov2 chimeric protein eluate was placed in an ultrafiltration concentration tube and the storage buffer (50 mM HEPES, 100 mM NaCl, 2 mM dithiothreitol DTT) was used as the diluent to finally remove the imidazole from the target protein and concentrate the NAMPT-cpLov2 chimeric protein to a concentration higher than 1 mg / mL.

[0069] 1.3. Activity detection of NAMPT-cpLov2 chimeric protein

[0070] Prepare reaction buffer (50 mM HEPES, pH 7.2, 50 mM NaCl, 2 mM MgCl2, 2.5 mM ATP, 1 mM tris(2-carboxyethyl)phosphine TCEP), add 10 mM PRPP, 50 nM NAMPT-cpLov2 chimeric protein, 1 nM NMN fluorescent probe to the reaction buffer, and incubate at 37°C for 10 minutes. Add 10 mM NAM and 1000-fold diluted Furimazine bioluminescent protein substrate to start the reaction. Use an automated bioluminescence microplate reader to continuously read the bioluminescence intensity values ​​at 440 nm and 590 nm, and use the 590 nm / 440 nm intensity ratio to indicate the NMN concentration. Use the titration curve of the NMN fluorescent probe to react to the concentration of NMN in the system.

[0071] The catalytic activities of the purified different NAMPT-cpLov2 chimeric proteins were compared. The results showed that the four selected NAMPT-cpLov2 chimeric proteins (i.e., NAMPT(N182-L183)-cpLov2, NAMPT(D235-P236)-cpLov2, NAMPT(S303-T304)-cpLov2, and NAMPT(N337-S338)-cpLov2 mentioned above) all showed certain catalytic activities, see Figure 2 Among them, the NAMPT (N182-L183) -cpLov2 chimeric protein has the best photosensitivity, and the catalytic activity of the NAMPT (N182-L183) -cpLov2 enzyme is most regulated by light. The activity decreases the most after 420 nm illumination, and the difference in activity before and after illumination reaches 18.9 times, as shown in Figure 2. Figure 3 shown.

[0072] 4. Optical regulation of the enzymatic activity of the NAMPT-cpLov2 chimeric protein

[0073] Based on the above NAMPT enzyme activity measurement system, the optical regulation of the catalytic activity of the NAMPT(N182-L183)-cpLov2 enzyme was characterized. During the reaction, the reaction solution was irradiated with a laser at a wavelength of 420 nm to observe the generation rate of NMN before and after irradiation.

[0074] Figure 4 The results showed that NAMPT(N182-L183)-cpLov2 exhibited good NMN synthase catalytic activity in the baseline (light-avoiding) state. However, under 420 nm laser irradiation, the enzyme catalytic activity decreased significantly.

[0075] 5. Live cell testing and application of NAMPT-cpLov2 chimeric protein

[0076] Based on the HEK293T cell line that stably expresses the NMN fluorescent probe, the NAMPT (N182-L183)-cpLov2 chimeric protein was stably expressed. HEK293T cells were cultured in DMEM medium containing 10wt% fetal bovine serum FBS at 37°C and 5% CO2. Plasmids pMD2G, psPAX2, and pCDH-CMV-NMORI-EF1-neo were transiently transfected into HEK293T for lentiviral packaging production. The lentiviral supernatant was filtered through a 0.45 μm filter and directly used to infect the target cell line. After 6 hours of infection of the target cell line, fresh culture medium was replaced. After 48 hours, highly transfected cells were sorted out by flow cytometry as stable cell lines. After the cell line was stably cultured in the dark, the cells were irradiated with a 420 nm light source, and the fluorescence intensity ratio of the NMN fluorescent probe was observed using a fluorescence microscope. The light intensity ratio is used to indicate the NMN content in living cells. The FRET fluorescence imaging analysis of NMN fluorescent probe was performed using an Olympus inverted fluorescence microscope (IX83), with a 100x / 1.50 oil objective lens and a Laser 488 / 561 spectroscope. Fluorescence intensities at EX 488 nm, EM 520 nm and EX 488 nm, EM 595 nm were collected simultaneously. The FRET ratio was calculated using ImageJ v1.53t for dual-channel fluorescence images. Cells cultured under the same conditions were collected and lysed, and the NAD+ content of the cell lysate was measured by high-performance liquid chromatography-mass spectrometry.

[0077] NMN fluorescent probe signal was used to characterize the NMN content in living cells. Figure 5 The NMN fluorescent probe in A clearly indicates that the NMN level of HEK293T cells is at a normal level in the initial (light-avoiding) state. Under 420 nm illumination, the NMN level of HEK293T cells decreased significantly. At the same time, HPLC-MS measurement of cell lysate found that the NAD+ level in HEK293T cells decreased significantly after 420 nm illumination, as shown in Figure 1. Figure 5 As shown in B. Since NMN is the main precursor for NAD+ synthesis, the experimental results show that NAMPT(N182-L183)-cpLov2 can significantly regulate NMN in cells, thereby regulating the level of NAD+ in cells.

[0078] NAMPT-cpLov2 chimeric protein differentially regulates NAD+ metabolism in cancer cells and immune cells. The LLC lung cancer cell line containing no plasmid and LLC lung cancer cell line expressing NAMPT(N182-L183)-cpLov2 were prepared by lentiviral method. 8Cells were seeded in cell culture dishes at a concentration of 100 cells / L and cultured in DMEM / F12 medium (Gibco) containing 1 mg / mL penicillin and streptomycin (Invitrogen) at 37°C in a 5% CO2 incubator. Mouse mononuclear cells (PBMCs) were extracted, and the PBMC cell fractions were separated from peripheral blood by Ficoll-Paque density gradient separation and frozen until later use. PBMC cells transfected with empty plasmids and expressing NAMPT(N182-L183)-cpLov2 were prepared using a lentiviral method.

[0079] Tumor cell and lymphocyte coculture: PBMCs were cultured in a medium containing 2 mM Ultraglutamine I, 1:100 penicillin / streptomycin, 10% male AB serum (Sigma-Aldrich), and RPMI 1640 (Gibco) (“T cell medium”). One day before coculture, PBMCs were thawed in prewarmed (37°C) T cell medium (human serum was replaced with FCS upon thawing) and incubated with 25 U / mL benzonase (Merck) for 15 min. After washing, cells were resuspended. 150 U / mL IL-2 was added to T cell medium and cultured overnight at 37°C. The bottom of the cell culture plate was coated with anti-CD28 antibody (clone CD28.2, eBioscience) overnight at 4°C. The anti-CD28 coated plates were washed twice with phosphate buffered saline (PBS), and tumor cells were seeded in the culture plates, and co-cultured tumor cells and PBMC cells were inoculated at a cell ratio of 20:1, and 150 U / mL IL-2 and 20 μg / mL anti-PD-1 blocking antibody were added. All co-cultures were supplemented with 5 μg / mL anti-CD28 antibody and 50 μM β-mercaptoethanol. Subsequent experiments were performed after 72 hours of co-culture.

[0080] 1) The LLC lung cancer cell line expressing NAMPT(N182-L183)-cpLov2 was co-cultured with mouse PBMC cells with no plasmid and irradiated with 420 nm light source for 30 min. 2) The LLC lung cancer cell line with no plasmid was co-cultured with mouse PBMC cells expressing NAMPT(N182-L183)-cpLov2 and irradiated with 420 nm light source for 30 min. 3) The LLC lung cancer cell line with no plasmid was co-cultured with mouse PBMC cells with no plasmid and 1 nM NAMPT inhibitor was added for 30 min.

[0081] Flow cytometry was used to separate viable cancer cells and immune cells and count them separately to determine the differential effects of differential regulation of NAMPT-cpLov2 chimeric proteins on cancer cell and immune cell survival. Single-cell suspensions were incubated with fixable Live / Dead Green (Invitrogen) or Zombie Aqua (Invitrogen) to distinguish viable cells according to the manufacturer's instructions. Cells were washed with 2% FCS-PBS, blocked with anti-CD16 / CD32 FCγ II / III (WEHI Antibody Facility), and incubated with extracellular antibodies for 30 minutes at 4°C. Cells were fixed and permeabilized with the Foxp3 / Transcription Factor Staining Kit (eBiosciences) and stained with intracellular antibodies for 30 minutes at 4°C. Samples were acquired using an Aurora Spectral Unmixing Flow Cytometer (CyTEK) and analyzed by Flowjo and Cytobank.

[0082] Figure 6 Figure A is a schematic diagram of the differential regulation of NAD+ metabolism of cancer cells and immune cells by NAMPT(N182-L183)-cpLov2 chimeric protein. Differential regulation of NAD+ in two types of cells can be achieved by differentially expressing NAMPT(N182-L183)-cpLov2 in mouse LLC tumor cells and immune cells. The NAMPT small molecule inhibitor Dapoline (FK866, purchased from MedChemExpress LLC, located in Building 3, Lane 1999, Zhangheng Road, Pudong New District, Shanghai) cannot achieve differential cell regulation. Figure B is a mixed culture of mouse LLC tumor cells expressing NAMPT(N182-L183)-cpLov2 and immune cells with empty plasmids. It can be seen that light significantly reduces the survival of LLC tumor cells. Immune cells are not significantly affected. Figure C is a mixed culture of empty plasmid mouse LLC tumor cells and immune cells expressing NAMPT(N182-L183)-cpLov2. It can be seen that light significantly reduces the survival of LLC tumor cells. T cells are not significantly affected. D is the use of NAMPT small molecule inhibitor FK866 to regulate the NAD+ metabolism of mouse LLC tumor cells and immune cells. It can be seen that FK866 does not differentiate between the two types of cells and cannot achieve differentiated NAMPT enzyme activity regulation.

[0083] In summary, the present invention uses protein engineering principles to provide a light-controlled NAMPT enzyme whose enzyme activity is controlled by blue light. After the light-controlled NAMPT enzyme is stably expressed in cells and tissues, the NAMPT enzyme activity of specific cells and specific tissue parts can be regulated in real time by laser irradiation with strong temporal and spatial resolution. The present invention also illustrates the role of the light-controlled NAMPT enzyme in tumor immunity research by specifically optically regulating the NAMPT enzyme activity of tumors and immune cells in a tumor and immune cell co-culture system. The activity of the light-controlled NAMPT enzyme provided by the present invention can achieve reversible inhibition under 420 nm laser irradiation. Compared with traditional NAMPT small molecule inhibition, and siRNA or shRNA inhibition strategies, the inhibition strategy of the present invention can achieve NAD+ metabolic regulation for specific cells and tissue regions, and can also achieve NAD+ metabolic regulation with significantly improved temporal and spatial resolution, and can also achieve rapid and reversible activity regulation of NAMPT enzyme activity.

[0084] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A light-controlled NAMPT enzyme, characterized in that: The light-controlled NAMPT enzyme includes NAMPT enzyme and cpLov2 photosensitive protein domain, the cpLov2 photosensitive protein domain is embedded in the NAMPT enzyme, and the amino acid sequence of the light-controlled NAMPT enzyme is shown in SEQ ID NO.

1.

2. A nucleic acid, characterized in that The nucleic acid contains a nucleotide sequence encoding the light-controlled NAMPT enzyme according to claim 1.

3. A carrier, characterized in that The vector carries the nucleic acid according to claim 2.

4. A cell, characterized in that The cell expresses the light-controlled NAMPT enzyme according to claim 1, or contains the nucleic acid according to claim 2, or contains the vector according to claim 3.

5. Use of the light-controlled NAMPT enzyme according to claim 1 in inhibiting the reaction of nicotinamide synthesizing nicotinamide adenine mononucleotide, characterized in that: The catalytic activity of the light-controlled NAMPT enzyme is inhibited by light, thereby inhibiting the reaction of nicotinamide synthesizing nicotinamide adenine mononucleotide.

6. Use of the light-controlled NAMPT enzyme according to claim 1 in inhibiting the reaction of nicotinamide synthesizing nicotinamide adenine dinucleotide, characterized in that: The catalytic activity of the light-controlled NAMPT enzyme is inhibited by light, thereby inhibiting the reaction of nicotinamide synthesizing nicotinamide adenine dinucleotide.

7. Use of the light-controlled NAMPT enzyme according to claim 1 in the preparation of a drug for treating lung cancer.

8. A drug for treating lung cancer, characterized in that: Comprising the light-controlled NAMPT enzyme according to claim 1.

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