Construction method and application of a cell line for high-throughput screening of AMPK pathway activators

Abstract

This invention relates to the fields of biomedicine and molecular biology, and particularly to the application of a cell line for high-throughput screening of AMPK pathway agonists and its construction method. The method provided by this invention can target the AMPK pathway, maximally mimicking in vivo physiological conditions for rapid high-throughput screening of pathway agonists. The cell line constructed by this invention can accurately and objectively reflect the state of the AMPK pathway, revealing the mechanism of action between prebiotics and the host, enriching research on prebiotic targeting, overcoming the shortcomings of existing technologies, and enabling high-throughput screening through real-time monitoring of pathway status in living cells.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and molecular biology, and in particular to the application of a cell line for high-throughput screening of AMPK pathway agonists and its construction method. Background Technology

[0002] AMPK (AMP-activated protein kinase) is an evolutionarily highly conserved serine / threonine protein kinase widely found in eukaryotic cells. It is hailed as the cell's "energy sensor" and "metabolic regulatory center," activated when cellular energy is insufficient to help restore energy homeostasis. AMPK consists of a catalytic α subunit and two regulatory β and γ subunits. The γ subunit contains an energy state sensing site capable of binding AMP, ADP, and ATP. As a heterotrimeric kinase complex, AMPK senses changes in the AMP / ATP ratio (energy stress) or responds to calcium signals (CaMKK2 pathway) and lysosomal glucose sensing (FBP-aldolase axis) via the CBS domain of its γ subunit. After phosphorylation activation at Thr172, it distributes in a spatiotemporally specific manner to subcellular compartments such as the cytoplasm, mitochondria, lysosomes, and endoplasmic reticulum. It phosphorylates specific substrate networks—acutely inhibiting anabolism (mTORC1, lipid synthesis, glycogen synthesis) and promoting catabolism (glycolysis, fatty acid oxidation, autophagy), and chronically promoting mitochondrial and lysosomal biogenesis, thereby restoring cellular metabolic homeostasis during energy depletion. Its pharmacological activation has become an important strategy for treating diabetes, obesity, and cancer. AMPK activation is a multi-level regulatory process, mainly including four aspects: energy state sensing, phosphorylation regulation of upstream kinases, exogenous factor-mediated activation, and dephosphorylation inactivation. Tumor suppressor kinase LKB1 is the main upstream AMPK kinase. Under low-energy conditions, the LKB1 complex phosphorylates the key threonine site (Thr172 in human α1) in the activation loop of the AMPK α subunit, which is a necessary condition for the complete activation of AMPK.

[0003] Researchers both domestically and internationally have conducted extensive research on the precise detection of AMPK pathway activity, primarily employing methods including traditional biochemical enzyme activity assays and Western blotting (WB). While traditional enzyme activity assays rely on purified proteins for in vitro reactions and offer the advantage of intuitive operation, they are susceptible to interference from endogenous thiols (such as glutathione) and nonspecific background in the sample, leading to increased false-positive signals and failing to reflect the true in situ cellular activity. Immunoassays (such as ELISA), on the other hand, are limited by antibody cross-reactivity, poor thermal stability, and high cost, making them unsuitable for high-throughput and high-precision mechanism analysis.

[0004] While mass spectrometry (MS)-based phosphorylated protein quantification techniques have significantly improved detection accuracy, they are limited by complex sample pretreatment, high instrument costs, and a high barrier to entry for specialized operation, making it difficult to meet the rapid screening needs of routine laboratories. These new methods have high requirements for the precision of equipment and experimental systems and have not yet achieved universal application. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the above-mentioned shortcomings, this invention proposes a novel sensing technology that uses gene-encoded FRET fluorescent probes to achieve high spatiotemporal resolution detection at the live cell level through a signal amplification strategy. It can track conformational changes induced by AMPK activation in real time. However, it suffers from problems such as high probe construction complexity and easy interference of the fluorescence signal by the cellular microenvironment (such as pH and ionic strength).

[0007] Therefore, as one aspect of the present invention, the present invention overcomes the shortcomings of the prior art and provides a cell line for high-throughput screening of AMPK activators.

[0008] To address the aforementioned technical problems, this invention provides the following technical solution: Using long and flexible EV linkers can significantly improve the dynamic range of many FRET biosensors by reducing the baseline FRET signal. Therefore, we designed a highly sensitive FRET biosensor. This technology involves the continuous expression of a fusion protein (ECFP-FHA1-AMPKsub-EYFP) via a promoter. When AMPK is activated and phosphorylates the AMPKsub substrate, the FHA1 domain specifically binds to the phosphorylation site, inducing protein conformational folding. This brings ECFP and EYFP closer together, generating a highly efficient FRET signal. By observing the intensity of the receptor fluorescence using the excitation wavelength of the donor fluorescence, the real-time activity of intracellular AMPK can be accurately quantified, providing an ideal tool for exploring AMPK pathway activators.

[0009] In one specific embodiment, expression can be achieved by integrating recombinant plasmid pairs into host cells.

[0010] The structure of the recombinant plasmid ECFP-FHA1-EVLinker-AMPKsub-EYFP is described as follows: The original lentiviral vector plasmid PLV3-ECFP-EYFP was digested with two restriction enzyme sites, BamH1 and Xho1, to obtain a linearized vector. Then, the EQ IDNO:1 sequence was added with a homologous arm and homologously recombinated with the digested PLV3-ECFP-EYFP vector.

[0011] The integration can be lentiviral system integration. First, the ECFP-FHA1-EVLinker-AMPKsub-EYFP overexpression vector is co-transfected with psPAX2 and pMD2.G packaging plasmids into 293T cells for lentiviral packaging and amplification to obtain ECFP-FHA1-EVLinker-AMPKsub-EYFP virus. The cells are then infected with this virus, and cell selection is performed to obtain a cell line that stably expresses ECFP-FHA1-EVLinker-AMPKsub-EYFP.

[0012] In the previous specific embodiment, the fluorescent protein expression module and the EV plug module can be integrated into the plasmid ECFP-FHA1-EVLinker-AMPKsub-EYFP and stably expressed in the host cell.

[0013] The present invention provides a method for constructing the cell structure described above, comprising introducing a recombinant vector of the fluorescent protein expression module and the EV plug module described above into mammalian cells to obtain the recombinant cells.

[0014] The mammalian cells used in the above-described method are human embryonic kidney cell lines.

[0015] The human embryonic kidney cell line mentioned above is the 293T cell line.

[0016] This invention also provides the application of the method described above in high-throughput screening of AMPK activators.

[0017] This invention also provides a method for high-throughput screening of AMPK activators, comprising the following steps: Culture the recombinant cells described above; When the cell culture density reaches 50%, use low-glucose DMEM (phenol red-free) medium for starvation culture for 12 hours; Add the sample to be tested to the cells described in step 2), and incubate with the cells for 1 hour after adding the sample; Cyan fluorescence images were captured at a wavelength of 430 nm under confocal conditions, and yellow fluorescence images were captured at an excitation wavelength of 470 nm. Bleaching of yellow fluorescence and observation of changes in cyan fluorescence brightness under constant parameters.

[0018] If the cyan fluorescence in the same area increases in brightness after bleaching the yellow fluorescence, it proves that the drug being tested has activated the AMPK signaling pathway.

[0019] The main achievement of this invention is to solve the problem of high-throughput screening of AMPK activators, reducing the time and financial costs. Based on gene-encoded FRET fluorescent probes, a signal amplification strategy enables high spatiotemporal resolution detection at the live-cell level, allowing real-time tracking of conformational changes induced by AMPK activation. This method is more convenient and efficient compared to other detection methods.

[0020] Compared with existing technologies, this technology has the following advantages: Live cell in situ detection without cell lysis: Western blot requires lysis of fixed cells, resulting in loss of spatiotemporal information and disruption of the cellular microenvironment; FRET directly observes intracellular AMPK activation under physiological conditions, without lysis artifacts, phosphorylation degradation, or artificial enrichment errors.

[0021] Millisecond-level real-time dynamic tracking captures instantaneous phosphorylation: AMPK activation and dephosphorylation are rapid and reversible in seconds to minutes. Western blotting can only measure the single-point value of the endpoint cleavage and cannot capture instantaneous fluctuations, oscillation signals, or upstream and downstream time series relationships.

[0022] Single-cell / subcellular spatial localization: WB is a population average signal and cannot distinguish the differences in AMPK between cytoplasm, mitochondria, and lysosomes; FRET fluorescence imaging can locate organelles, single-cell heterogeneity, and local energy stress.

[0023] Higher sensitivity and extremely low background: ECFP-EYFP has good spectral matching and ratio detection, with a signal-to-noise ratio much higher than flow cytometry and ordinary fluorescence, and can be detected with weak AMPK activation.

[0024] Caption for attached image: Figure 1 This is a diagram of the nucleotide coding sequence of the EV plug module in Embodiment 1 of the present invention. Figure 2 This is an agarose gel verification result after amplification of the nucleotide coding sequence of the EV plug module synthesized using AMPK partial specific primers in the plasmid; Figure 3 This is a single colony plate image of PLV3-ECFP-MCS-EYFP-EF1a-Puro plasmid transformed in Example 2 of this invention. Figure 4 This is a diagram showing the sequencing alignment results of the PLV3-ECFP-MCS-EYFP-EF1a-Puro plasmid extracted from bacterial culture in Example 2 of this invention. Figure 5This is a schematic diagram of double enzyme digestion of the PLV3-ECFP-MCS-EYFP-EF1a-Puro plasmid in Example 2 of the present invention; Figure 6 This is a gel image of the PLV3-ECFP-MCS-EYFP-EF1a-Puro plasmid after double enzyme digestion in Example 2 of this invention. Figure 7 This is a single colony plate image of the PLV3-AMPK recombinant plasmid in Example 3 of the present invention; Figure 8 This is a comparison diagram of the sequencing results of single colony extraction plasmids from the PLV3-AMPK recombinant plasmid in Example 3 of the present invention; Figure 9 This is a fluorescence expression diagram of a single clonal cell line stably transfected with the PLV3-AMPK recombinant plasmid in Example 4 of this invention. Figure 10 This is a comparison of fluorescence bleaching before and after the PLV3-AMPK recombinant plasmid stably transfected monoclonal cell lines in Example 5 of the present invention, after the fluorescence pair was replaced.

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are only for explaining the invention, and the scope of protection of this invention is not limited by these embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort fall within the scope of protection of this invention.

[0026] Unless otherwise specified, the technical solutions described in this invention are conventional solutions in the art; the reagents or materials described, unless otherwise specified, are all derived from commercial sources. The invention will be further illustrated below through specific embodiments.

[0027] The vector plasmid pLV3-CMV-ECFP-MCS-EYFP-EF1a-puro used in the following examples was purchased from Miaoling Biotechnology, catalog number P62104.

[0028] The PSPAX2 and PMD2G plasmids in the following examples are all plasmids currently existing in the laboratory.

[0029] The EV plug domain in the following examples is a synthetic plasmid from Sangon Biotech.

[0030] The FHA1 domain is derived from the FHA1 domain of the yeast Rad53 protein, amino acid positions 241–382 (aa 241–382). The EV Linker is derived from the Eevee backbone system of Komatsu et al. (2011), developed by the Matsuda Laboratory at Kyoto University. The AMPK substrate sequence is based on the peptide sequence designed from the natural AMPK substrate. The designed plasmid map was then sent to a biotechnology company for plasmid synthesis. Figure 1 ).

[0031] Take 1 μL of the synthesized AMPK plasmid and add it to 100 μL of DH5α competent cells. Incubate on a shaker (37°C, 220 rpm) for 4 hours.

[0032] Take 50 μL of bacterial culture and spread it on LB solid medium with ampicillin resistance. Incubate at 37°C for 15 h. Pick large and round single colonies and incubate them in 1 mL of LB liquid medium with cannabinoid resistance for 4 h. Then add 15 mL of LB liquid medium with ampicillin resistance and incubate for 15 h.

[0033] After plasmid extraction using a plasmid extraction kit (omega, D6948-01), the AMPK region was amplified using specific primers with homologous arms. The purified product was then recovered using an agarose gel for verification. The verification results are shown below. Figure 2 ).

[0034] Primers with homologous arms: Forward primer: 5′-3′ gctgtacaagggaggaggtGGATCCaagtttttctcaagaacagatcggcgaaaaca Reverse primer: 5′-3′ gctgtacaagggaggaggtGGATCCaagtttttctcaagaacagatcggcgaaaaca

[0035] Take 1 μL of the purchased PLV3-ECFP-MCS-EYFP-EF1a-Puro plasmid and add it to 100 μL of Stbl3 competent cells. Incubate on a shaker (30℃, 220 rpm) for 4 hours.

[0036] Take 50 μL of bacterial suspension and spread it on LB solid medium containing ampicillin resistance. Incubate at 30°C for 15 h, and pick large, round single colonies. Figure 3 The samples were cultured in 1 mL of ampicillin-resistant LB liquid medium for 4 h, and then added to 15 mL of ampicillin-resistant LB liquid medium and cultured for 15 h.

[0037] After plasmid extraction using a plasmid extraction kit (omega, D6948-01), the plasmid was sent to Sangon Biotech for plasmid sequencing. Figure 4 ).

[0038] The vector plasmid PLV3-ECFP-MCS-EYFP-EF1a-Puro was linearized using double enzyme digestion. Figure 5 The enzyme digestion system is shown in the table below (total volume: 50 uL). The enzyme digestion system was validated using 5% agarose gel. Figure 6 The target band size was 8800bp. The purified enzyme digestion product was obtained by gel recovery for homologous recombination.

[0039] The purified EV plug product with homologous arms was subjected to homologous recombination with the enzyme-cleaved purified product. The reaction system is shown in the table below. The operation was performed on ice. React at 50°C for 30 minutes, then immediately place on ice.

[0040] Recombinant product transformation Thaw the chemically competent Stbl3 cells used for cloning on ice. Add 10 μl of the recombinant product to... Mix 100 μl of competent cells by gently tapping the tube wall and incubating on ice for 30 min.

[0041] After heat shock in a 42°C water bath for 30 seconds, immediately place on ice to cool for 2-3 minutes. Add 900 μl of LB liquid culture medium (without antibiotics) and incubate at 37°C for 1 hour (220 rpm).

[0042] Preheat the ampicillin LB solid medium plates in a 37°C incubator.

[0043] Centrifuge at 5,000 rpm (2,500 × g) for 5 min, and discard 900 μl of supernatant. Resuspend the bacterial culture in the remaining culture medium and gently spread it evenly on a plate containing the correct antibiotic using a sterile spreader. Incubate upside down in a 30°C incubator for 15 h.

[0044] Identification of recombinant products: single colonies were picked ( Figure 7 Add the culture to 1 mL of ampicillin-resistant liquid LB medium and shake at 37°C for 1 h (220 rpm). Then transfer the culture to 15 mL of large-system LB liquid medium and shake at 37°C for 12 h (220 rpm).

[0045] Extract plasmids from 10 mL of overnight bacterial culture, and verify the recombinant primers using Sangon Biotech's third-generation sequencing technology. Figure 8 ).

[0046] After 293T cells were stably passaged for 3 generations, they were seeded into 100 mm cell culture dishes. When the cells reached a density of 60%-70%, the PLV3-AMPK recombinant vector was co-transfected into 293T cells with psPAX2 and pMD2.G packaging plasmids for lentiviral packaging and amplification to obtain PLV3-AMPK virus solution.

[0047] Select a suitable HCT116 cell line, seed it into a 6-well cell culture plate, and culture it until the cell density reaches 70%-80%. Infect the cells with PLV3-AMPK virus, and replace the medium with fresh medium containing 1ug / mL Puromycin (concentration depends on the cell line) after 24 h for cell selection.

[0048] After obtaining positive polyclonal cells, they were transferred to 96-well plates for further culture to obtain a monoclonal cell line stably expressing PLV3-AMPK and to observe its fluorescence expression. Figure 9 ).

[0049] Once the obtained stable expression cell lines are cultured in cell culture dishes, they can be used for high-throughput screening of AMPK pathway agonists.

[0050] Due to limitations in instrumentation, this validation used red-green fluorescent pairs instead of cyan-yellow fluorescent pairs, while keeping the rest unchanged. After stable passage of the transfected cell line for three generations, the cells were seeded in cell culture dishes and cultured until the cell density reached 50%. The cells were then cultured in DMEM low-glucose, phenol red-free medium for 12 hours, followed by incubation with 50 μM of A-769662 for 1 hour.

[0051] Observation of changes in green fluorescence before and after red fluorescence bleaching under confocal microscopy ( Figure 10 ).

[0052] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cell line for high-throughput screening of AMPK pathway agonists, characterized in that, The cell line stably expresses the fluorescent plasmid module and the EV plug module.

2. The fluorescent plasmid module and EV plug module according to claim 1, characterized in that, It includes a fluorescent pair capable of fluorescence resonance energy transfer—cyan fluorescent protein (ECFP) and yellow fluorescent protein (EYFP); the EV plug module includes an FHA1 phosphorylation-binding domain, a flexible linker peptide, and an AMPK-specific phosphorylated substrate short peptide.

3. The fluorescent pair capable of fluorescence resonance energy transfer—cyan fluorescent protein (ECFP) and yellow fluorescent protein (EYFP)—according to claim 2, is characterized in that, Its excitation and emission wavelengths are 430 / 470 and 470 / 530, respectively.

4. The EV plug module according to claim 1, characterized in that, The nucleotide coding sequence of the inserted EV plug module is EQ IDNO:

1.

5. As described in claims 1-4, characterized in that, The method for constructing a cell line for high-throughput screening of AMPK pathway agonists involves inserting the EV plug module as claimed in claim 4 between the fluorescent base pairs described in claim 3 to obtain a recombinant vector, which is then introduced into mammalian cells to obtain a stable cell line.

6. The method according to claim 5, characterized in that, The analyte was added to a stable cell line to observe the fluorescence resonance energy transfer phenomenon.

7. The method according to claim 6, characterized in that, If a fluorescence-neutral energy transfer occurs, it proves that the substance can activate the AMPK pathway.

8. The use of the recombinant cells according to any one of claims 1-5 in high-throughput screening of AMPK pathway agonists.

9. The application of the method according to any one of claims 6-7 in high-throughput screening of AMPK pathway agonists.

10. A method for high-throughput screening of AMPK pathway activators, characterized in that... It includes the following steps: Cultivate the recombinant cells according to any one of claims 1-5; Add the sample to be tested to the recombinant cells described in step 1), and incubate with the cells for 1 hour after adding the sample. Observe the fluorescence intensity using the cyan fluorescence excitation wavelength and record it as F0. By observing the yellow fluorescence in the cell sample from bleaching step 2), the fluorescence intensity of the cyan fluorescence is recorded as F1; If yellow fluorescence can be observed using the cyan fluorescence excitation wavelength, and the cyan fluorescence in the same area is enhanced after bleaching the yellow fluorescence, it indicates that fluorescence resonance energy transfer has occurred, thus proving that the sample can activate the AMPK pathway.

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