KR-20260062843-A - Screening system for anti-aging and geriatric disease-modulating substances based on autophagy using ATG4B cleavage site and method for producing cell lines and animal model using the same

Abstract

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 substances that regulate anti-aging or age-related diseases using the same, and cell lines and animal models expressing the system.

Inventors

• 박영호
• 김선욱
• 채운빈
• 이동길
• 양해준
• 송인성
• 조두신

Assignees

• 한국생명공학연구원

Dates

Publication Date

20260507

Application Date

20251020

Priority Date

20241025

Claims (13)

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. In claim 1, the reporter system is a reporter system in which, 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 claim 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 an ATG4B cleavage site of SEQ ID NO. 1, and a vector inserted into a safe-harbor locus within a gene of SEQ ID NO. 2 and used to produce 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 claim 6, 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. A 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. In paragraph 12, the animal model 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.

Description

Screening system for anti-aging and geriatric disease-modulating substances based on autophagy using ATG4B cleavage site and method for producing cell lines and animal model using the same 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. 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)). 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. 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. 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. 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. 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. Therefore, there is a need for a means to monitor the autophagy process more accurately and sensitively. Figure 1 schematically shows the structure of a reporter system according to the present invention. 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. 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. 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). 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. 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 fl