Autophagy-based Anti-aging and senile disease regulator screening system using ATG4b cleavage site, and method for producing cell line and animal model using same

Abstract

The present invention relates to a reporter system capable of more accurately and sensitively analyzing autophagy activation on the basis of an autophagy reporter including an ATG4B cleavage site than existing systems, a method for screening an anti-aging or senile disease regulator using same, and a cell line and an animal model expressing the system.

Description

Autophagy-based screening system for anti-aging and age-related disease regulatory substances using an ATG4B cleavage site and method for preparing cell lines and animal models using the same

[0001] The present invention relates to a reporter system capable of analyzing autophagy activation more accurately and sensitively than existing systems based on an autophagy reporter comprising an ATG4B cleavage site, a method for screening autophagy-related disease-regulating substances using the same, and cell lines and animal models expressing the system.

[0002] Autophagy is a crucial cellular process for maintaining cellular homeostasis by removing damaged organelles or unnecessary proteins within the cell. This process is activated by various intracellular stressors closely associated with aging and geriatric diseases, such as infection, starvation, energy stress (including ischemia), mechanical stress, hypoxia, ER stress, and oxidative stress (including organelle damage). Autophagy plays a role in the degradation and removal of proteins and organelles, as well as regulating adaptation to stress, immunity, inflammatory responses, and apoptosis. Therefore, abnormal regulation of autophagy is known to be highly associated with various physiological and pathological conditions, including not only aging but also geriatric diseases (Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS)).

[0003] As such, since autophagy is associated with various diseases, a reporter system capable of accurately monitoring the autophagy state is required to identify substances that regulate it.

[0004] Existing autophagy reporter systems (GFP-LC3, mCherry-GFP-LC3) have primarily used a method of visually confirming the formation of autophagosomes by fusing a fluorescent protein to an autophagy-related protein such as LC3.

[0005] The GFP-LC3 reporter is one of the most widely used autophagy markers, and LC3 is closely associated with the formation of autophagosomes. Since GFP-LC3 binds to the membrane of autophagosomes, it has the advantages of enabling visual monitoring of the autophagy process, allowing for high-resolution imaging, and facilitating the easy tracking of autophagy activation through changes in the localization of LC3 within the cell. However, because GFP can be degraded inside lysosomes, it may be difficult to accurately monitor the autophagy flux (the entire process), and there are limitations in studying the late stages of autophagy as it is difficult to observe the autophagy process after the point where GFP degrades.

[0006] Another autophagy reporter system, the mCherry-GFP-LC3 reporter, utilizes two fluorescent proteins, GFP and mCherry, to monitor the autophagy process more precisely. Since GFP degenerates in lysosomes while mCherry is relatively stable, it has the advantage of being able to distinguish between the formation of autophagosomes and their transition to autolysosomes, thereby allowing for more accurate measurement of autophagy flux. However, expressing both fluorescent proteins can be complex, fluorescence interference may occur, and there may be technical limitations in simultaneously detecting both fluorescent signals depending on the equipment.

[0007] In addition, these existing reporter systems have limitations in providing accurate information about specific stages of autophagy due to variability depending on the intensity of the fluorescence signal and the accumulation of fluorescent proteins within autophagosomes.

[0008] Therefore, there is a need for a means to monitor the autophagy process more accurately and sensitively.

[0009]

[0010] The objective of the present invention is to provide a reporter system capable of specifically measuring the cleavage of the LC3B protein by the ATG4B enzyme by developing a reporter system using an ATG4B cleavage site.

[0011] Another objective of the present invention is to provide a method for screening substances that regulate autophagy-related diseases using such a reporter system.

[0012] Another objective of the present invention is to provide cell lines and animal models that express such a reporter system.

[0013]

[0014] To achieve the above objective, the present invention provides a reporter system for measuring ATG4B activity, having a structure comprising an enhanced green fluorescent protein (EGFP), an ATG4B cleavage site targeting LC3B, and then a destabilized sequence and a ubiquitination sequence, and cell lines and animal models utilizing the same.

[0015] In addition, the present invention relates to a method for screening substances that regulate autophagy-related diseases, wherein

[0016] 1) a step of adding a reporter system according to the present invention to cells expressing the ATG4B enzyme and measuring the EGFP fluorescence intensity;

[0017] 2) a step of treating the above cells with a test substance and measuring the EGFP fluorescence intensity; and

[0018] 3) A step of comparing the fluorescence intensity of Step 1) and the fluorescence intensity of Step 2)

[0019] Provides a method including

[0020] In addition, the present invention provides a cell line and an animal model expressing the reporter system.

[0021] The reporter system according to the present invention can observe the process of specific protein cleavage by ATG4B in real time by directly detecting the activity of the ATG4B enzyme, and thus can be used to analyze autophagy activity with greater specificity and precision than existing systems. Furthermore, it can be used to screen autophagy-regulating substances, and in particular, to effectively screen therapeutic substances for autophagy-related diseases, specifically anti-aging and age-related disease-regulating substances.

[0022]

[0023] Figure 1 schematically shows the structure of a reporter system according to the present invention.

[0024] Figure 2 illustrates the mechanism related to the components of the reporter system according to the present invention. Figure 2a explains the mechanism by which the ATG4B enzyme is involved in autophagy, and Figure 2b schematically shows that when enhanced green fluorescent protein (EGFP) is fused with a protein destabilization sequence and a ubiquitination sequence, fluorescence is not exhibited.

[0025] Figure 3 shows the map (Figure 3a) and sequencing results (Figure 3b) of an ATG4B active reporter vector designed to be inserted into a safe-harbor (AAVS1 locus) within the gene.

[0026] Figure 4 shows the genotyping PCR results (bottom) and primer positions and sequences for detection (top) when an ATG4B active reporter vector is inserted into the AAVS1 gene of 293 or U2OS cells at a safe-harbor (AAVS1 locus).

[0027] Figure 5 shows the results confirming that the expression of green fluorescence increased when cells in a 293 ATG4B reporter cell line constructed using a fluorescence microscope were starved (cultured in EBSS medium) to activate autophagy. It was found that green fluorescence was not expressed under relatively complete media (DMEM+FBS) conditions.

[0028] Figure 6 shows the results confirming that the expression of green fluorescence increased when cells in a U2OS ATG4B reporter cell line constructed using a fluorescence microscope were starved (cultured in EBSS medium) to activate autophagy. It was found that green fluorescence was not expressed under relatively complete media conditions.

[0029] Figure 7 shows the results of confirming that the expression of green fluorescence increased when cells in 293 ATG4B reporter cell lines were starved (cultured in EBSS medium) at 4-hour intervals for 72 hours using real-time fluorescence microscopy to activate autophagy. It was found that green fluorescence was not expressed under relatively complete media conditions.

[0030] Figure 8 shows the results of confirming that the expression of green fluorescence increased when cells in the U2OS ATG4B reporter cell line were starved (cultured in EBSS medium) at 4-hour intervals for 72 hours using real-time fluorescence microscopy to activate autophagy. It was found that green fluorescence was not expressed under relatively complete media conditions.

[0031] Figure 9 shows the results of confirming that when siRNA for ATG4B was introduced into 293 ATG4B reporter cell lines using a fluorescence microscope to reduce ATG4B expression, the expression of green fluorescent protein (EGFP), which is enhanced by cell starvation (culture in EBSS medium), did not appear. This demonstrates that the ATG4B reporter reflects the state of autophagy activity through a reduction in fluorescence signal under conditions where ATG4B activity is inhibited.

[0032] Figure 10 shows the results of confirming that when siRNA for ATG4B was introduced into U2OS ATG4B reporter cell lines using a fluorescence microscope to reduce ATG4B expression, the expression of green fluorescent protein (EGFP), which is enhanced by cell starvation (culture in EBSS medium), did not appear. This demonstrates that the ATG4B reporter reflects the state of autophagy activity through a reduction in fluorescence signal under conditions where ATG4B activity is inhibited.

[0033] Figure 11 shows the results of confirming that green fluorescent protein (GFP) expression decreased due to cell starvation (culture in EBSS medium) when siRNA targeting ATG4B was introduced into 293 ATG4B reporter cell lines via the Western blot method to reduce ATG4B expression. At this time, to prove that siRNA targeting ATG4B was induced, ATG5 was confirmed to have no change in expression as a control, and while the expression of ATG4B increased when cell starvation was induced in the group treated with control siRNA, ATG4B was knocked down in the group treated with ATG4B siRNA, verifying that the siRNA experiment was successfully conducted.

[0034] Figure 12 shows the results confirming that the expression of green fluorescent protein increased after treating four U2OS ATG4B reporter cell lines constructed using a fluorescence microscope with 10 μM of 1,10-phenanthroline for 24 hours to activate autophagy.

[0035] Figure 13 shows the results of confirming that the expression of green fluorescent protein (GFP) enhanced by 1,10-phenanthroline increased when autophagy was induced in U2OS ATG4B reporter cell lines using the Western blot method. At this time, ATG5 was used as a control to confirm that there was no change in expression to prove that autophagy was induced targeting ATG4B, and LC3B was used to verify that autophagy was successfully induced.

[0036] Figure 14 shows the results of confirming the expression of green fluorescent protein after treating U2OS ATG4B reporter cell lines constructed using fluorescence microscopy with various autophagy-inducing substances. It shows the results confirming a significant increase in green fluorescence with 1,10-phenanthroline, MG132, bafilomycin A1, and molibresib. Here, blue fluorescence is from nuclei stained with DAPI.

[0037] Figure 15 shows the results confirmed by Western blot after treating U2OS ATG4B reporter cell lines with four different autophagy-inducing substances for 24 hours. It was confirmed that green fluorescent protein (GFP) expression increased in the treatment groups excluding Torin1. At this time, ATG5 was used as a control to confirm that there was no change in expression to prove that it induced autophagy targeting ATG4B, and LC3B was used to verify that autophagy was successfully induced.

[0038] Figure 16 shows the results of confirming that the expression of green fluorescent protein enhanced by autophagy-inducing substances was reduced when siRNA against ATG4B was introduced into U2OS ATG4B reporter cell lines using a fluorescence microscope to reduce ATG4B expression (siATG4B #2 experimental group). This demonstrates that the ATG4B reporter reflects the state of autophagy activity through a reduction in fluorescence signal under conditions where ATG4B activity is inhibited.

[0039] Figure 17 shows the results confirming that the expression of green fluorescent protein increased after treating two HeLa ATG4B reporter cell lines constructed using a fluorescence microscope with 10 μM of 1,10-phenanthroline for 24 hours to activate autophagy.

[0040] Figure 18 shows the results of confirming that the expression of green fluorescent protein (GFP) was increased by 1,10-phenanthroline when autophagy was induced in HeLa ATG4B reporter cell lines using the Western blot method. At this time, ATG5 was used as a control to confirm that there was no change in expression to prove that autophagy was induced targeting ATG4B, and LC3B was used to verify that autophagy was successfully induced.

[0041] FIGS. 19a and 19b schematically show the structure of a vector for producing a mouse in which an ATG4B active reporter according to the present invention is precisely inserted into a safe location (mROSA26 locus) of the mouse.

[0042] Figure 20 shows the result of confirming the insertion of the ATG4B active reporter vector using the Sanger sequencing method, which shows that the transformation vector was accurately and precisely inserted into a safe site (mROSA26 locus) of the transformation mouse.

[0043] Figure 21 shows the PCR results of offspring genotyping obtained by crossing heterozygous Tg mice using the corresponding primers, through which transgenic mice introduced with an ATG4B activity monitoring vector were selected.

[0044] Figure 22 shows the results of confirming that the expression of green fluorescent protein increased for each somatic cell line after treating somatic cell lines constructed from each organ of ATG4B reporter transgenic mice (ATG4B activity monitoring Tg) with 50 μM of 1,10-phenanthroline for 24 hours to induce autophagy using a fluorescence microscope.

[0045] Figure 23 shows the results of confirming that the expression of green fluorescent protein increased in the group treated with an autophagy-inducing substance (1,10-phenanthroline) in somatic cell lines constructed from heart, liver, and kidney tissues of ATG4B reporter transgenic mice (ATG4B activity monitoring Tg) via the Western blot method. At this time, to prove that ATG5 induced autophagy targeting ATG4B, no change in expression was confirmed as a control, and LC3B was used to verify that autophagy was successfully induced.

[0046]

[0047] One embodiment of the present invention relates to a reporter system capable of analyzing autophagy activation more accurately and sensitively than existing systems, based on an autophagy reporter comprising an ATG4B cleavage site.

[0048] Specifically, the reporter system according to the present invention is a protein having a structure in which an ATG4B cleavage site targeting LC3B is inserted after an enhanced green fluorescent protein (EGFP), followed by a destabilized sequence and a ubiquitination sequence (see FIG. 1). By adopting this structure, the green fluorescent protein is not expressed when autophagy is inactive, and when autophagy is activated, ATG4B cleaves the cleavage site, thereby removing the destabilized sequence and the ubiquitination sequence, and EGFP is stably expressed, resulting in green fluorescence.

[0049] This reporter system, by including the cleavage site of the ATG4B enzyme, can measure ATG4B activity in real time and monitor specific stages of autophagy, and has the following features and advantages, particularly compared to existing autophagy reporter systems.

[0050] 1) ATG4B Specificity: While existing reporter systems focus on autophagosome formation, this system directly detects the activity of the ATG4B enzyme using an ATG4B cleavage site. This cleavage site plays a crucial role in the conversion phase of LC3 and similar proteins during autophagy, allowing for real-time observation of specific protein cleavage processes by ATG4B. In particular, specific stages of autophagy can be monitored specifically through the ATG4B cleavage reaction occurring in the early stages of autophagy, which provides higher specificity than existing LC3-based reporter systems.

[0051] 2) Precise stage analysis: The reporter system of the present invention can intensively analyze activity during the stage involving ATG4B among the various stages of autophagy, and can provide more specific and precise data than existing reporter systems.

[0052] 3) Increased sensitivity of fluorescence signals: When the reporter protein is cleaved by the activity of ATG4B, the separation of the two fluorescence signals can be clearly observed, allowing for the detection of autophagy more sensitively than existing reporter systems. While existing systems display fluorescence signals merely through changes in intracellular location at various stages of the autophagy process, the present invention provides a form in which the fluorescence signal is clearly turned ON / OFF according to autophagy activity. This allows for more intuitive confirmation of the presence or absence of autophagy activity, thereby significantly enhancing its usefulness as a research and diagnostic tool.

[0053] 4) Wide range of applications: The reporter system of the present invention can be applied to study not only autophagy but also other ATG4B-related pathways, and can be utilized to screen autophagy-related disease-regulating substances, particularly anti-aging substances and substances for the treatment or prevention of geriatric diseases.

[0054] Due to these features and advantages, the reporter system according to the present invention can provide a more sophisticated monitoring system for the autophagy pathway through the combination of an ATG4B enzyme-specific cleavage site, an instability sequence, and a ubiquitination sequence compared to existing reporter systems such as the GFP-LC3 reporter and the mCherry-GFP-LC3 reporter, and additionally, this structural improvement causes a clear difference in fluorescent signals depending on whether autophagy is activated, thereby enabling more accurate measurement of autophagy.

[0055] The reporter system according to the present invention has the following features:

[0056] 1. Introduction of ATG4B-specific cutting sites

[0057] Existing reporter systems primarily use methods to detect autophagy activation that directly monitor changes in autophagy-related proteins such as LC3 or indirectly track the formation of autophagosomes.

[0058] In comparison, the reporter system of the present invention proposes a system that responds more directly to the activity of the ATG4B enzyme during the autophagy pathway by introducing an ATG4B-specific cleavage site, which enables more precise detection of specific degradation processes of the autophagy pathway.

[0059] 2. Introduction of destabilizing and ubiquitizing sequences

[0060] It has a structure in which a dominant destabilization sequence and a ubiquitination sequence are placed behind EGFP (enhanced green fluorescent protein), and such a structure does not exist in existing reporter systems.

[0061] By suppressing EGFP expression when autophagy is inactive, the background signal in the inactive state can be significantly reduced, thereby enabling increased signal sensitivity and clarity.

[0062] In addition, when autophagy is activated, ATG4B specifically performs cleavage to remove destabilized and ubiquitinated sequences, allowing EGFP to be stably expressed and produce a signal, which enables a system to visualize the activation of autophagy at high resolution.

[0063] 3. Regulation of EGFP expression

[0064] The characteristic structure of the present invention, in which EGFP is not expressed when autophagy is inactive but is promoted by the action of ATG4B when autophagy is activated, represents a mechanism not observed in existing reporter systems. In particular, since the fluorescence signal changes clearly according to the activation of autophagy, experimental observation and interpretation become easier.

[0065] 4. Direct visualization of autophagy activation

[0066] While existing systems indirectly measure various steps of the autophagy pathway, the reporter system of the present invention specifically responds to the activity of ATG4B, a key enzyme involved in autophagy, thereby enabling more direct observation of specific mechanisms and activities of autophagy. This feature can serve as a highly useful tool for autophagy research and related drug development.

[0067] Another embodiment of the present invention relates to a method for screening substances that regulate autophagy-related diseases.

[0068] Specifically, the present invention

[0069] 1) a step of adding a reporter system according to the present invention to cells expressing the ATG4B enzyme and measuring the EGFP fluorescence intensity;

[0070] 2) a step of treating the above cells with a test substance and measuring the EGFP fluorescence intensity; and

[0071] 3) A step of comparing the fluorescence intensity of Step 1) and the fluorescence intensity of Step 2)

[0072] A method for screening substances that regulate autophagy-related diseases, comprising: a test substance, wherein if the fluorescence intensity of step 2) is higher than that of step 1), the test substance can be determined to be a substance for the treatment or prevention of autophagy-related diseases, particularly anti-aging substances or geriatric diseases.

[0073] In the present invention, autophagy-related diseases are particularly geriatric diseases, and include, but are not limited to, cancer, neurodegenerative diseases, diabetes, cardiovascular diseases, infectious diseases, and inflammatory diseases.

[0074] In the present invention, cancer includes, but is not limited to, pituitary adenoma, glioma, brain tumor, nasopharyngeal cancer, laryngeal cancer, thymoma, mesothelioma, breast cancer, lung cancer, stomach cancer, esophageal cancer, colorectal cancer, liver cancer, pancreatic cancer, pancreatic endocrine tumor, gallbladder cancer, penile cancer, ureteral cancer, renal cell carcinoma, prostate cancer, bladder cancer, non-Hodgkin lymphoma, myelodysplastic syndrome, multiple myeloma, plasma cell tumor, leukemia, pediatric cancer, skin cancer, ovarian cancer, and cervical cancer.

[0075] In the present invention, degenerative neurological diseases include, but are not limited to, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, and amyotrophic lateral sclerosis (ALS).

[0076] In the present invention, cardiovascular diseases include, but are not limited to, photoarterial heart disease, cardiomyopathy, hypertensive heart disease, heart failure, cor pulmonary heart, cardiomyopathy, endocarditis, inflammatory cardiomegaly, myocarditis, valvular heart disease, cerebrovascular disease, peripheral artery disease, congenital heart disease and cardiac rheumatism.

[0077] In the present invention, the infectious disease is a virus (e.g., adenovirus, herpesvirus (e.g., HSV-I, HSV-II, CMV, or VZV), poxvirus (e.g., variola, vaccinia, or orthopoxvirus such as molluscum contagiosum, picornavirus (e.g., rhinovirus or enterovirus)), orthomyxovirus (e.g., influenza virus), paramyxovirus (e.g., 5-parainfluenza virus, mumps virus, measles virus, and respiratory syncytial virus), coronavirus (e.g., SARS), papovavirus (e.g., papillomavirus causing genital warts, common warts, or plantar warts), hepardnavirus (e.g., hepatitis B virus), flavivirus (e.g., hepatitis C virus or dengue virus) or retrovirus (e.g., HIV, etc.) Infectious diseases caused by lentiviruses, infectious diseases caused by bacteria (e.g., Escherichia, Enterobacter, Salmonella, Staphylococcus, Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium, Campylobacter, Vibrio, Serratia, Providencia, Cromobacter, Brucella, Yersinia, Haemophilus, or Bordetella genera) and other infectious diseases (e.g., fungal diseases including Chlamydia and candidiasis, aspergillus, histomaplasmosis, and cryptococcal meningitis, and malaria, pneumoniae pneumoniae, leishmaniasis, ryptosporidiosis, toxoplasmosis, and trypasonoma infections) Includes, but is not limited to, parasitic diseases.

[0078] In the present invention, inflammatory diseases include, but are not limited to, osteoarthritis, rheumatoid arthritis, gout, ankylosing spondylitis, tendinitis, tenosynovitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, asthma, atopy, Crohn's disease, and ulcerative colitis.

[0079] Another embodiment of the present invention relates to a cell line expressing a reporter system according to the present invention.

[0080] The reporter system according to the present invention consists of a vector (Sequence No. 1) expressing a reporter protein containing an ATG4B cleavage site and a vector (Sequence No. 2) inserted into a safe-harbor locus within the gene and used to produce a cell line. When this reporter vector is inserted, an antibiotic resistance gene (puromycin resistance) is expressed, allowing only cell lines into which the ATG4B reporter vector is inserted to be selected, and whether the cell line production was accurately performed can be confirmed through a genotyping PCR method.

[0081] - ATG4 activity monitoring vector sequence

[0082]

[0083]

[0084]

[0085]

[0086] - ATG4 activity monitoring mouse production vector sequence

[0087]

[0088]

[0089]

[0090]

[0091]

[0092] When autophagy is activated in the cell line prepared in this way, the reporter protein expressed in the cell line is cleaved by ATG4B, and consequently, a change in the fluorescence or luminescence signal occurs, making it possible to measure the activity of ATG4B.

[0093] Another embodiment of the present invention relates to an animal model expressing a reporter system according to the present invention.

[0094] In the present invention, usable animals include, but are not limited to, rats, mice, guinea pigs, hamsters, dogs, cats, rabbits, cattle, sheep, pigs, guinea pigs, and monkeys.

[0095]

[0096] Hereinafter, preferred embodiments of the present invention will be described in detail. However, these embodiments are intended solely to illustrate the present invention, and the scope of the present invention should not be interpreted as being limited by these embodiments.

[0097]

[0098] Example 1: Preparation of ATG4B active reporter vector

[0099] In this example, a reporter vector (ATG4 activity monitoring vector) capable of monitoring changes in the activity of ATG4B protein was designed on the VectorBuilder online platform (https: / www.vectorbuilder.kr) and then produced through custom synthesis and QC (Fig. 1).

[0100] Specifically, the reporter synthesized a DNA fragment containing a specific cleavage sequence of human LC3B (ATG4B substrate) (e.g., Gly-Gly cleavage motif), fused EGFP and MODC (mouse ornithine decarboxylase) PEST (aa 422–461)-based destabilization sequences to both sides of the sequence, and additionally fused HIF-1α's ODDD (aa 338–608) to induce VHL-dependent ubiquitination and proteasome degradation to minimize background signals and configured the stability and expression level of EGFP to change upon changes in ATG4B activity (Fig. 2).

[0101] The above components were sequentially assembled into a 9,385 bp vector along with essential regulatory elements of the vector, such as the IRES sequence, EGFP, Kozak sequence, CAG promoter, CMV enhancer, chimeric intron, bGH poly-A signal, AmpR, and pUC ori. The entire sequence is a circular plasmid, and the entire cassette was assembled using de novo gene synthesis and standard cloning. The structural and sequence accuracy was verified through quality control (QC), which included retransformation, restriction enzyme digestion, and Sanger sequencing. Additionally, the vector was selectively selected and propagated in E. coli based on AmpR antibiotic resistance (Fig. 3).

[0102]

[0103] Example 2: Preparation of a cell line inserted with an ATG4B active reporter vector

[0104] Transformed cell lines were constructed by introducing the ATG4B activity monitoring vector prepared in Example 1 into HEK 293 cells derived from human embryonic kidney and U2OS cells derived from human osteosarcoma (Fig. 4).

[0105] First, HEK 293 and U2OS cells are seeded into 100 mm culture dishes, and when the cells are cultured to about 70% to 80%, the 4D-Nucleofector TMUsing the equipment, 5 μg of the ATG4B activity monitoring vector was introduced into the cells according to the SF kit, CM1-130 program, and the manufacturer's manual. After 3 days, when the cells were cultured to about 90%, 3 μl of puromycin (#A11138-03, Gibco) was added to 10 ml of culture medium to perform puromycin screening. After 14 days, when the cells were cultured to about 70% to 80% again, cells that did not express green fluorescent protein were secondarily screened using a flow cytometer (Fluorescence-activated Cell Sorting, FACSAria™ III), and the screened single cells were inoculated into 96-well plates and cultured, then subcultured into 24-well plates to establish a transformed cell line.

[0106] To confirm whether the ATG4B activity monitoring vector was accurately inserted into the safe-harbor (AAVS1 locus) of HEK 293 and U2OS cells, genomic DNA was extracted from the acquired transformed cell lines and genotyping PCR was performed to determine whether the gene was inserted.

[0107] Specifically, for genotyping PCR, genomic DNA was extracted from cells using the DNeasy Blood & Tissue kit (#69506, Qiagen) according to the manufacturer's manual. Genotyping PCR was performed using the extracted genomic DNA and ExPrime Taq Premix (2X, 8-strip) (#G-6000, GeNet Bio) under the conditions shown in Table 1 below.

[0108]

[0109] Samples for genotyping PCR were subjected to electrophoresis for 30 minutes at 120V on a 0.8% agarose gel prepared using Agarose, LE, Analytical Grade (#V3125, Promega) and 0.5X TAE (50X TAE; TR2002-100-00, Biosesang 3rd order diluted to 0.5X). Subsequently, wild-type, hetero-type, and homo-type cells were distinguished by comparing the band sizes of the samples. PCR primers for genomic DNA amplification were designed in silico using Primer3 software (https: / bioinfo.ut.ee / primer3-0.4.0 / ) and are shown in Table 2 below.

[0110]

[0111] Figure 4 shows the electrophoresis results. As shown in Figure 4, in HEK293 and U2OS cells (WT) without the ATG4B activity monitoring vector inserted (knock-in; KI), a PCR product of 339 bp was observed. In the case of heterogenomes inserted into one allele (HEK 293 #1 to #2, U2OS #1 to #6), PCR products of 339 bp and 658 bp were observed, and in the case of homogenomes (HEK 293 #8, U2OS #2 to #6), only a PCR product of 658 bp was observed.

[0112] Example 3: Evaluation of Green Fluorescent Protein Expression in ATG4B Reporter Cell Lines Under Condition

[0113] Among the HEK293 and U2OS cells with the ATG4B activity monitoring vector inserted verified in Example 2, homogenomic #8 (HEK 293) and #6 (U2OS) cells were treated with EBSS (Earle's Balanced Salt Solution) for 72 hours to induce starvation, and then changes in fluorescence were confirmed through fluorescence microscopy and real-time fluorescence analysis, which are shown in Figs. 5, 6, 7, and 8, respectively.

[0114] As a result, fluorescence microscopy confirmed that fluorescence increased in both HEK293 and U2OS when EBSS was treated for 72 hours in the homo genotype inserted into both alleles (Figs. 5, 6).

[0115] In addition, as shown in Figures 7 and 8, real-time fluorescence analysis confirmed that fluorescence increased in real time when EBSS was treated for 72 hours in the homogenome inserted into both alleles.

[0116] Through this, it was possible to observe fluorescence changes caused by autophagy using HEK293 and U2OS cells inserted with the ATG4B activity monitoring vector.

[0117]

[0118] Example 4: Evaluation of green fluorescent protein expression under conditions in ATG4B reporter cell lines with reduced ATG4B expression

[0119] Changes in the expression of green fluorescent protein through the reduction of ATG4B expression were confirmed using HEK293 and U2OS cells inserted with the ATG4B activity monitoring vector verified in Example 3. #8 (HEK 293) and #6 (U2OS) cells were treated with siRNA targeting ATG4B and starvation was induced by treating them with EBSS for 72 hours. The changes in fluorescence were then confirmed using a fluorescence microscope and real-time fluorescence analysis, as shown in Figures 9 and 10, respectively.

[0120] As a result, fluorescence microscopy confirmed that in the group treated with control siRNA, fluorescence increased upon treatment with EBSS for 72 hours in both HEK293 and U2OS cells, whereas in the group with reduced ATG4B expression, green fluorescence expression decreased in both HEK293 and U2OS cells (Figs. 9, 10).

[0121] In addition, the expression of green fluorescent protein through the inhibition of ATG4B expression was confirmed via Western blot.

[0122] Specifically, Western blot was performed through the following process. 30 to 60 μg of protein lysates were separated from an 8 to 12% sodium dodecyl sulfate-polyacrylamide gel by SDS-PAGE and then transferred to 0.45 μm nitrocellulose membranes (#HAWP04700, EMD Millipore, Burlington, MA, USA).

[0123] The above membranes were immersed in a solution containing 10 mM Tris pH 7.4, 150 mM NaCl, and 0.02% Tween-20 (TBST), blocked with 5% skim milk (#232100, BD Biosciences) or bovine serum albumin (#A9647, Sigma-Aldrich), and then incubated overnight at 4°C with primary antibodies (GFP, #ab290, abcam, 1:2000; ATG4B, #13507S, Cell signaling technology, 1:1000; ATG5, #8540S, Cell signaling technology, 1:1000; GAPDH, #LF-MA0038, AB Frontier, 1:2000).

[0124] Subsequently, the membrane was washed three times with TBST and blocked again with 5% skim milk, then incubated at room temperature for 1 hour with a secondary antibody bound to horseradish peroxidase. After incubation, the blot was washed with TBST, and antibody binding was confirmed using SuperSignal West Pico PLUS Chemiluminescent Substrate (#34580, Thermo Fisher Scientific) according to the manufacturer's instructions.

[0125] As a result, it was confirmed that the expression of green fluorescent protein decreased in ATG4B activity monitoring #8 HEK 293 cells as ATG4B expression was inhibited (Fig. 11).

[0126]

[0127] Example 5: Evaluation of Green Fluorescent Protein Expression Following Treatment with Autophagy-Inducing Substances in ATG4B Reporter Cell Lines

[0128] U2OS cells inserted with the ATG4B activity monitoring vector prepared in Example 3 were treated with 1,10-phenanthroline, a substance that induces autophagy, for 24 hours, and the expression of green fluorescence was confirmed through fluorescence microscopy and the degree of protein expression was confirmed through Western blot.

[0129] As a result, using U2OS cell lines (#5, #9, #10, and #17) inserted with four types of ATG4B activity monitoring vectors, it was confirmed that the expression of green fluorescence increased when cells were treated with 1,10-phenanthroline for 24 hours, and through analysis at the protein level, it was also confirmed that the expression of green fluorescence increased through the induction of autophagy by 1,10-phenanthroline (Figs. 12, 13).

[0130] Additionally, the versatility of the ATG4B reporter system was confirmed by treating with various substances that induce autophagy (1,10-phenanthroline, Tonrin1, AP15). Three substances that strongly act on the ATG4B reporter system were selected by confirming an increase in green fluorescence expression after treating with nine substances that affect autophagy (Fig. 14).

[0131] Monitoring of 4 types of ATG4B activity: U2OS cell lines were treated with three types of autophagy-inducing substances and EBSS, which induces starvation, and the expression of green fluorescent protein was confirmed by Western blot. Although there were differences in the expression levels of green fluorescent protein among the substances, it was confirmed that the expression of green fluorescent protein increased in all substances (Fig. 15).

[0132] In addition, the effect on the expression of green fluorescent protein through an autophagy inducer was confirmed after inhibiting the expression of ATG4B using the siRNA used in Example 3. As a result, it was confirmed that the green fluorescence, which was increased by 1,10-phenanthroline in the control group, decreased under conditions where ATG4B was inhibited (Fig. 16).

[0133] Additionally, a cell line was constructed by introducing an ATG4B activity monitoring vector into HeLa cells derived from human cervical cancer. After inducing autophagy with 1,10-phenanthroline, an increase in green fluorescence was confirmed via fluorescence microscopy and an increase in the expression of green fluorescent protein was confirmed via Western blot (Figs. 17, 18).

[0134] Through this, it was confirmed that the expression of green fluorescence increases not only through starvation but also through substances that induce autophagy. Furthermore, analysis at the protein level also confirmed that the expression of green fluorescence increases through the induction of autophagy by various substances.

[0135]

[0136] Example 6: Production of transgenic mice introduced with the ATG4B reporter system and evaluation of GFP expression following autophagy in mouse somatic cell lines

[0137] To produce a transgenic mouse in which the ATG4B reporter vector of Example 1 was inserted into a safe place (Safe-harbor) (ROSA26 locus) of the mouse, it was designed on the VectorBuilder online platform (https: / www.vectorbuilder.kr) and then produced through custom synthesis and QC (Fig. 19).

[0138] Specifically, the mROSA26 AT4B activity monitoring vector was designed by placing a left homology arm (1083 bp) and a right homology arm (805 bp) derived from intron 1 of the mouse ROSA26 gene on both sides of the vector so that the target gene sequence could be inserted into the ROSA26 locus in a homologous recombination manner.

[0139] To confirm whether the mROSA26 AT4B activity monitoring vector prepared above was accurately inserted into the mouse ROSA26 site, genomic DNA was extracted from transgenic mice and Sanger sequencing and genotyping PCR were performed to determine whether the gene was inserted (Figs. 20, 21).

[0140] Specifically, for genotyping PCR, genomic DNA was extracted from transgenic mouse offspring using the DNeasy Blood & Tissue kit (#69506, Qiagen) according to the manufacturer's manual. Genotyping PCR was performed using the extracted genomic DNA and ExpPrime Taq Premix (2X, 8-strip) (#G-6000, Gent Bio) under the conditions shown in Table 3 below.

[0141]

[0142] Samples for genotyping PCR were subjected to electrophoresis at 120V for 30 minutes on a 0.8% agarose gel prepared using Agarose, LE, Analytical Grade (#V3125, Promega) and 0.5X TAE (50X TAE; TR2002-100-00, Biosesang 3rd order diluted to 0.5X). Subsequently, wild-type, hetero-type, and homo-type cells were distinguished by comparing the band sizes of the samples. PCR primers for genomic DNA amplification were designed in silico using Primer3 software (https: / bioinfo.ut.ee / primer3-0.4.0 / ) and are shown in Table 4 below.

[0143]

[0144] The electrophoresis results are shown in Fig. 21. As shown in Fig. 21, a PCR product of 412 bp was observed in transgenic mice (WT) without the ATG4B activity monitoring vector inserted (knock-in; KI); PCR products of 241 bp and 412 bp were observed in heterogenomes with the vector inserted into one allele; and only a 241 bp PCR product was observed in homogenomes. We intended to confirm the expression of green fluorescent protein after inducing autophagy with 1,10-phenanthroline in somatic cell lines constructed from each organ of the selected transgenic mice. As a result of confirming the expression of green fluorescent protein after inducing autophagy in somatic cell lines constructed from the liver, kidney, heart, lung, and spleen using a fluorescence microscope, it was confirmed that the expression of green fluorescent protein increased (Fig. 22). As a result of confirming the expression of green fluorescent protein after inducing autophagy in somatic cell lines constructed from heart, liver, and kidney using the Western blot method, it was confirmed that the expression of green fluorescent protein increased (Fig. 23).

[0145]

[0146] The reporter system according to the present invention can observe the process of specific protein cleavage by ATG4B in real time by directly detecting the activity of the ATG4B enzyme, and thus can be used to analyze autophagy activity with greater specificity and precision than existing systems. Furthermore, it can be used to screen autophagy-regulating substances, and in particular, to effectively screen therapeutic substances for autophagy-related diseases, specifically anti-aging and age-related disease-regulating substances.

Claims

1. A reporter system for measuring ATG4B activity, having a structure comprising enhanced green fluorescent protein (EGFP), then an ATG4B cleavage site targeting LC3B, then a destabilized sequence and a ubiquitination sequence.

2. The reporter system according to claim 1, wherein when autophagy is activated, the ATG4B cleavage site is cleaved by ATG4B, and a fluorescent protein is expressed to generate a visual signal.

3. In paragraph 1, the reporter system is a reporter system capable of precisely analyzing the initial stage of autophagy.

4. In claim 1, the reporter system is a reporter system capable of detecting a specific protein cleavage process by an ATG4B enzyme in real time.

5. The reporter system according to claim 1, wherein the reporter system comprises a vector expressing a reporter protein comprising the ATG4B cleavage site of SEQ ID NO. 1, and a vector inserted into a safe-harbor locus within the gene of SEQ ID NO. 2 and used to construct a cell line.

6. As a method for screening substances that regulate autophagy-related diseases, 1) A step of adding a reporter system according to any one of claims 1 to 5 to a cell expressing the ATG4B enzyme and measuring the EGFP fluorescence intensity; 2) a step of treating the above cells with a test substance and measuring the EGFP fluorescence intensity; and 3) A step of comparing the fluorescence intensity of Step 1) and the fluorescence intensity of Step 2) A method including 7. In paragraph 6, the method wherein the above-mentioned autophagy-related disease-regulating substance is an anti-aging substance or a substance for the treatment or prevention of geriatric diseases.

8. The method according to claim 7, wherein the above-mentioned geriatric disease is one or more selected from the group consisting of cancer, degenerative neurological disease, diabetes, cardiovascular disease, infectious disease and inflammatory disease.

9. A method according to claim 8, wherein the above-mentioned geriatric disease is one or more selected from the group consisting of Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis.

10. In claim 6, the method determines that the test substance is a substance that controls autophagy-related diseases when the fluorescence intensity of step 2) is higher than that of step 1).

11. A cell line expressing the reporter system of any one of paragraphs 1 to 5.

12. An animal model expressing the reporter system of any one of paragraphs 1 to 5.

13. An animal model according to claim 12, wherein the animal is one or more species selected from the group consisting of rats, mice, guinea pigs, hamsters, dogs, cats, rabbits, cattle, sheep, pigs, guinea pigs, and monkeys.

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