ANIMAL MODEL FOR OXIDATIVE STRESS RESEARCH AND USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to and the benefit of Korean Patent Application No. 10-2021-0113959, filed on Aug. 27, 2021, the disclosure of which is incorporated herein by reference in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (Name: 5142.0010001_Seqlisting_ST26; Size: 13,492 bytes; and Date of Creation: Dec. 20, 2022) is herein incorporated by reference in its entirety.

BACKGROUND
Field of the Invention

The present invention relates to a use of a deletion mutant for an RCAT gene having a regulatory function for an oxidation stress in Caenorhabditis elegans and a human cell line transfected with the RCAT gene as an animal model for oxidative stress research.

Discussion of Related Art

Caenorhabditis elegans lives in soil, has a body size of about 1 mm, and is the first multicellular organism whose DNA sequence has been fully elucidated. Caenorhabditis elegans has about 19,000 genes similar to humans. C. elegans has many unique features that make it an ideal model organism, such as a convenient inbreeding reproductive mode (i.e., hermaphroditic), a short life span (2˜3 weeks), a large brood size (>300 progeny), a variety of readily produced genetic mutants, and ease of cultivation in the laboratory Its body is transparent in which the number of somatic cells and nerve cells is as small as 1,000 and 320, respectively. Oxidative stress or reactive oxygen species (ROS) is produced during energy metabolism or by bacterial infection and heavy metals. Oxidative stress is known to contribute to the progression of various diseases such as metabolic diseases, neurodegenerative diseases, aging, cancer and diabetes. Nrf2/SKN-1, which is well known as a master transcription factor for controlling oxidative stress, carries out cellular defense functions during the detoxification process in animals including humans. Although many studies have been conducted on the regulation of oxidative stress where Nrf2/SKN plays an important role, the transcriptional regulation of Nrf2/SKN-1 is remained elusive in animals including C. elegans. That is, although extensive studies of searching a regulator for Nrf2/SKN1, there still remains largely unknown for the presence of biological regulator of Nrf2/SKN-1.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an animal model for oxidative stress research, including a deletion mutant of regulator of cat (RCAT, R02D3.7) and the mammalian cell lines overexpressing the nematode RCAT gene.

It is another object of the present invention to provide a model animal for use of RCAT.

It is still another object of the present invention to provide a method for screening an antioxidant drug using the animal model.

To achieve these objectives, the present invention provides an animal model for oxidative stress research, including a regulator of cat (RCAT) gene deletion mutant represented by a base sequence of SEQ ID NO: 1.

The present invention also provides an antioxidant composition including a regulator of cat (RCAT) gene represented by a base sequence of SEQ ID NO: 2 or 3, or a protein encoded therefrom.

The present invention also provides a method for screening an antioxidant drug, the method including:

handling of an animal model for oxidative stress research, including a regulator of cat (RCAT) gene deletion mutant represented by a base sequence of SEQ ID NO: 1 with an antioxidant candidate drug; and

measuring whether the generation amount of reactive oxygen species (ROS) is reduced in the animal model.

According to the present invention, an RCAT mutant including the RCAT gene deletion mutant may be used not only as an animal model for oxidative stress research, but also in the screening of an antioxidant drug because the generation amount of reactive oxygen species (ROS) is increased.

According to the present invention, a human HEK293T cell line transfected with RCAT derived from Caenorhabditis elegans and Nrf2 may be used not only as an animal cell model for oxidative stress research, but also in the screening of an antioxidant drug because the transcriptional activity of an antioxidant factor Nrf2 is increased by the action of RCAT originating from Caenorhabditis elegans.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objectives, features and advantages of the present invention will become more apparent to those of ordinary skill in the art by describing in detail exemplary embodiments thereof with reference to the accompanying drawings, in which:

FIG. 1 illustrates the results of from comparison of a gene deletion site in an RCAT mutant (ok1745) with a wild type (left side of FIG. 1: SEQ ID NO: 2) and an RCAT mutant (right side of FIG. 1: SEQ ID NO: 1). The corresponding drawing was adopted from the data of wormbase.org;

FIG. 2 illustrates the results from DCFDA analysis in that a reactive oxidative species (ROS) level is highly elevated in a Caenorhabditis elegans RCAT deletion mutant, meaning that the greener the fluorescence, the higher the ROS level;

FIG. 3 is a graph obtained from qRT-PCR analysis in that the expression levels of detoxification genes activated by oxidative stress, such as SKN-1 and its downstream targets, GCS-1 and GST-4, are decreased in a Caenorhabditis elegans RCAT deletion mutant but increased again in a mutant strain where RCAT is overexpressed. FIG. 3 shows that the levels of SKN-1 and detoxification genes are directly regulated in the presence of RCAT;

FIG. 4 illustrates an HA-RCAT portion, which is a junction part between a plasmid vector and RCAT gene in pcDNA3.1-HA-RCAT that is transfected into a human HEK293T cell line. The corresponding drawing was produced using a Snapgene (GSL Biotech, CA, USA) program;

FIG. 5 shows the results of expressing FLAG-tagged Keap1 and HA-tagged RCAT in a human HEK293T cell line where the two proteins actually bind as assessed by immunoprecipitation (IP). The left side shows the result of IP using an HA-antibody, while the right side displays the result of IP using a FLAG antibody. Since both Keap1 and RCAT are detected in both experiments, it is confirmed that the two proteins are physically interacted;

FIG. 6 shows the results of western blot analysis to show whether free Nrf2 is actually generated by the action of RCAT in a human HEK293T cell line and enters the nucleus; and

FIG. 7 shows the luciferase reporter analysis results for the transactivation activity of Nrf2 by RCAT overexpressed in human HEK293T cell line.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Hereinafter, the configuration of the present invention will be specifically described.

The present invention relates to an animal model for oxidative stress research, including a regulator of cat (RCAT) gene deletion mutant represented by SEQ ID NO: 1.

The regulator of cat (RCAT) is a common name for R02D3.7, which was shown to suppress transcriptional expression of cat-1 and cat-2 (‘cat’ means catecholamine), that are dopamine metabolism-related enzyme genes of Caenorhabditis elegans. However, the biological function, action mechanism and its association with human diseases thereof and the degree of utilization using this gen have not been well elucidated. The present inventors discovered the ability of RCAT to transcriptionally regulate cat-1 or cat-2, and based on this, they named R02D3.7 as regulator of cat (RCAT).

The RCAT gene deletion mutant has a deletion of five exons including the start codon and some promoter regions as shown in the nucleotide sequences of SEQ ID NO: 2, and the aforementioned deletion site may be represented by a base sequence of SEQ ID NO: 4. Preferably, the RCAT gene deletion mutant may be represented by a base sequence of SEQ ID NO: 1.

According to an exemplary embodiment of the present invention, the RCAT mutant generated more ROS in non-dopaminergic cells of Caenorhabditis elegans (FIG. 2). Further, it was found that the expression of oxidative stress-related genes known to be regulated by SKN-1 (homolog of human Nrf2) of Caenorhabditis elegans, which is an oxidative stress-related transcription factor associated with ROS generation, was reduced in an RCAT mutant compared to that in N2 wild type worms, and the reduction in expression was partially recovered in an RCAT overexpressed strain (FIG. 3).

It is known that human Nrf2 binds to Keap1, which is then degraded under the normal physiological conditions. However, when cells are under the oxidative stress condition, Nrf2 is released from the Nrf2-Keap1 complex and then enters the nucleus, thereby suppresses the transcription of oxidative stress-related genes.

Inventors investigated whether RCAT and Nrf2 can interact and work together in a human HEK293T cell line as seen in C. elegans, they found that when RCAT was added, Keap1 competitively interacted with RCAT instead of Nrf2, and accordingly, unbound Nrf2s then freely entered the nucleus (FIGS. 5 and 6).

It was also observed that binding of RCAT to Keap1 causes degradation of Keap1 in the human HEK293T cell line, thereby enhances the transactivation of Nqo-1 by Nrf2 (FIG. 7).

Therefore, RCAT is a novel regulator that controls antioxidant Nrf2/SKN-1 in non-dopaminergic cells and an RCAT deletion mutant has an increased level of ROS compared to the wild type, and thus may be useful for numerous studies as an animal model in studying oxidative stress.

Therefore, the animal model may be a Caenorhabditis elegans mutant including an RCAT gene deletion mutant; or a human HEK293T cell line overexpressing RCAT and Nrf2.

The Caenorhabditis elegans mutant including the RCAT gene deletion mutant may have an increased level of reactive oxygen species (ROS) compared to the wild-type N2 Caenorhabditis elegans. Preferably, the animal model may be Caenorhabditis elegans R02D3.7 (ok1745).

Furthermore, the human HEK293T cell line overexpressing RCAT and Nrf2/SKN-1 may be a human HEK293T cell line transfected with an RCAT gene of Caenorhabditis elegans and an Nrf2/SKN-1 gene. The human HEK293T cell line may increase the transcriptional activity of an antioxidant transcription factor Nrf2/SKN-1 by the action of RCAT originating from Caenorhabditis elegans.

In an exemplary embodiment of the present invention, the RCAT gene may be overexpressed by inserting an RCAT cDNA represented by a base sequence of SEQ ID NO: 3 into an expression vector, and then transfecting the expression vector into a human HEK293T cell line. Specifically, a human HEK293T cell line is transfected with Flag-Keap1 (Addgene, MA, USA), pCDNA3-Myc3-Nrf2 (Addgene, MA, USA), or pcDNA3.1-HA-RCAT, wherein the expression vector pCDNA3.1-HA-RCAT which is constructed by conjugating Caenorhabditis elegans RCAT cDNA with pcDNA3.1-HA (Addgene, MA, USA).

As used herein, the term “animal model” refers to an animal that mimics or simulates the characteristics of human diseases, meaning that the animal model may be used for the disease research in which it is difficult to test and apply therapeutic method.

The present invention also relates to an antioxidant composition including a regulator of cat (RCAT) gene represented by a base sequence of SEQ ID NO: 2 or 3, or a protein encoded therefrom.

Nrf2/SKN-1, which is well known as a master transcription factor, carries out cell defense functions during the detoxification process of oxidative stress in animals including humans. It is known that Nrf2/SKN-1 binds to Keap1 and then this Nrf2/Keap1 complex to be degraded under a normal physiological condition. However, under the oxidative stress condition, RCAT binds Keap1, leaving free Nrf2, that can enter the nucleus, thereby suppressing the transcription of oxidative stress-related genes.

According to exemplary embodiments of the use of the present invention, as a result of expressing RCAT and Nrf2 in an HEK293T cell line, one can examine whether C. elegans RCAT and Nrf2/SKN-1 can interact and exert their anti-oxidation function. It was found that when RCAT was added, Keap1 bound to RCAT instead of Nrf2, and accordingly, more Nrf2s that freely enter the nucleus. In addition, it was observed that RCAT bound to Keap1 caused degradation of Keap1 in the human HEK293T cell line, thereby increasing the transcriptional activity of NQO-1, a mammalian NAD(P)H dehydrogenase, by Nrf2. Therefore, RCAT is a regulatory factor that suppresses the expression of antioxidant Nrf2/SKN-1 in non-dopaminergic cells, and thus may be used as an antioxidant component.

The present invention also relates to a method for screening an antioxidant drug, the method including: microinjection or addition of anti-oxidant drug candidates into the growth media (plate for C. elegans, culture media for human mammalian cells) where an animal model (i.e., C. elegans RCAT mutant strain or RCAT overexpressed HEK293 cell lines) can take up biological for response to drugs. Herein RCAT gene deletion mutant is represented by a base sequence of SEQ ID NO: 1 with an antioxidant candidate drug; and

measuring whether a generation amount of ROS is reduced in the animal model.

The animal model of the present invention is an RCAT gene deletion mutant, and is characterized by an increased generation amount of ROS. Therefore, the antioxidant candidate drug may be brought into contact with the RCAT mutant to select the candidate drug in which the ROS generation amount of the RCAT mutant is reduced as an antioxidant drug. Preferably, the animal model may be Caenorhabditis elegans R02D3.7 (ok1745).

The antioxidant candidate drug may include any molecules such as proteins, peptides, small organic molecules, polysaccharides, polynucleotides and a wide range of compounds. Such candidate materials also include synthetic materials as well as natural products.

Hereinafter, the present invention will be described in more detail through the Examples according to the present invention, but the scope of the present invention is not limited by the Examples as suggested below.

<EXAMPLE 1> IDENTIFICATION OF CHARACTERISTICS OF C. elegans RCAT MUTANT
1. Strain

The species of C. elegans used in the present experiment is as follows: Wild type: Bristol (N2 species); RCAT [R02D3.7 (ok1745)]; RCAT OE [R02D3.7::GFP, rol-6]

Unless otherwise stated, C. elegans was cultured in a solid nematode growth medium (NGM) containing OP50 at 20° C. using a standard method, and a synchronized culture was prepared by treating an adult (gravid adult) with hypochlorite as previously described (Brenner, S. Genetics 77, 71-94, 1974).

2. ROS Analysis

ROS generation levels in wild type and mutants of C. elegans were measured using 2,7-dicholorodihydro-fluorescein-diaceate (DCFDA). The ROS measurement method was performed according to a previously established protocol (Kampkφtter, A. et al. Arch Toxicol 81, 849-858. 2007).

3. Confocal Microscopy Analysis

Fifty mM sodium azide was added to synchronized L3 stage worms, and the worms were cultured for 5 minutes. Worms treated under each condition were placed on 3% agar on a glass slide for microscopic observation. For fluorescence observation, an LSM700 confocal microscope (ZEISS, Jena, Germany) equipped with a FITC filter was used. All images were 8-bit and analyzed using ZEN Black software (ZEISS).

4. RNA Extraction and cDNA Synthesis

Synchronized L3 stage worms were incubated on NGM plates and washed 3 times with S-basal and 1 time with water for collection. TRIzol was added to a pellet and frozen in nitrogen gas. Proteins and other impurities were removed by addition of chloroform, and RNA was precipitated with isopropyl alcohol. RNA concentration was measured with an ND-1000 spectrophotometer. cDNA was synthesized using a GE Healthcare kit. cDNA was additionally purified, and the RNA concentration was measured using the ND-1000 spectrophotometer.

5. Quantitative RT-PCR (qRT-PCR)

Total RNA was isolated from the sample as described in the manual using

RNAspin mini columns (GE Healthcare, UK), and 2 μg of manually provided RNA was transcribed using a transcriptor first standard cDNA synthesis kit (Roche, IN, USA) along with oligoDT priming. qRT-PCR was performed using SYBR Green PCR Master Mix (Qiagen) according to the manufacturer's manual.

<EXAMPLE 2> CHARACTERIZATION OF RCAT IN HUMAN CELL LINE
1. Cell Line

HEK293T cells (Korean Cell Line Bank, 21573) were maintained under conditions of 37° C. and 5% CO₂in a Dulbecco's Modified Eagle Medium (DMEM; Hyclone, HS3243.01) supplemented with 10% fetal bovine serum (Hyclone, SV30087.02) and 1% penicillin-streptomycin (Biowest, L0022). The expression vectors transfected into the cell line are as follows: Flag-Keap1 (Addgene, MA, USA); pCDNA3-Myc3-Nrf2 (Addgene, MA, USA); and pcDNA3.1-HA-RCAT. The expression vector pCDNA3.1-HA-RCAT was constructed by conjugating Caenorhabditis elegans RCAT cDNA with pcDNA3.1-HA (Addgene, MA, USA). The conjugated site of the corresponding expression vector is shown in FIG. 4. The RCAT cDNA sequence inserted into the expression vector is set forth in SEQ ID NO: 3.

2. Immunoblot Analysis

For immunoprecipitation (IP), HEK293T cells were lysed in a lysis buffer (including 50 mM Tris-HCl (pH 7.5), 125 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10% glycerol, 0.3% TritonX-100, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5% Nonidet P-40 (NP-40), 10 mM β-phosphoglyceride, 1 mM Na₃V0₄, 5 mM NaF and 1 mg/mL aprotinin and leupeptin). A cell lysate was centrifuged and IP was applied to the resulting supernatant with an antibody against FLAG HA or Myc using protein G-sepharose beads.

For immunoblotting, sodium dodecyl sulfate polyacrylamide gel electrophoresis was performed on the cell lysates or immunoprecipitates, and an isolated protein was transferred to a polyvinylidene difluoride membrane, incubated with a primary antibody at 4° C. overnight, and then incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for 1 hour. Visualization was performed using an enhanced chemiluminescence lightning solution (Thermo Fisher Scientific).

3. Luciferase Reporter Analysis

Nrf2 activity was measured by dual luciferase after transfection of a reporter plasmid including an antioxidant response element (ARE) and a canonical Nrf2 binding motif HEK293T cells were transfected with an expression vector for RCAT, Keap1 and Nrf2 along with a pNqo1-ARE reporter plasmid (provided by H.-S. Choi) and pRL-TK (endogenous control, Promega) for 24 hours. And then, luciferase activity was analyzed from the cells using a dual luciferase reporter gene analysis system (Promega).

<EXPERIMENTAL EXAMPLE 1> IDENTIFICATION OF CORRELATION OF RCAT (ok1745) STRAIN WITH ROS

FIG. 1 illustrates the results of comparing a gene deletion site of an RCAT mutant (ok1745) with a wild type (left side of FIG. 1: SEQ ID NO: 2) and an RCAT mutant (right side of FIG. 1: SEQ ID NO: 1). The corresponding drawing was adopted from the data of wormbase.org.

Further, as a result of measuring ROS generation amounts using DCFDA assay in a normal wild type and an RCAT mutant in order to identify the antioxidant effect of RCAT, as illustrated in FIG. 2, it was confirmed that more ROS were generated in the RCAT mutant.

In addition, the expression level of oxidative stress-related genes known to be regulated by SKN-1 was measured using QRT-PCR in order to identify whether such ROS generation is associated with the oxidative stress-related transcriptional factor Nrf2/SKN-1.

As illustrated in FIG. 3, it was found that the expression of oxidative stress-related genes known to be regulated by SKN-1 was reduced in the RCAT mutant and recovered in the overexpressed species.

Furthermore, it is known that human Nrf2 binds to Keap1 to be degraded in a normal situation, and when oxidative stress is applied, free Nrf2 enters the nucleus while the bond between the two is broken, thereby regulating the transcription of oxidative stress-related genes. In order to identify whether the action of RCAT and Nrf2/SKN-1 occurs not only in Caenorhabditis elegans, but also in a human cell line, the interaction was observed by expressing RCAT and Nrf2 in the HEK239T cell line.

As illustrated in FIG. 5, it was found that when RCAT was added, Keap1 bound to RCAT instead of Nrf2, and accordingly, a lot of Nrf2 freely entering the nucleus was demonstrated.

It was confirmed through western blot whether free Nrf2 is actually generated by the action of RCAT in a human HEK293T cell line and enters the nucleus.

As illustrated in FIG. 6, it can be seen that the detection amount of Keap1 decreases as the amount of injected RCAT increases, meaning that a Keap1-RCAT complex is degraded by binding to the injected RCAT. Further, through the fact that the larger the injection amount of RCAT, more Nrf2 in the nucleus is detected, it is confirmed that RCAT binds to Keap1 instead of Nrf2, and accordingly, Nrf2 freely enters the nucleus.

In addition, in order to identify whether the transactivation activity of Nrf2 is actually generated by RCAT in the human HEK293T cell line, M-Nrf2, F-Keap1 and H-RCAT were expressed in the HEK293T cell line with the luciferase reporter plasmid pNqo-1 ARE.

As illustrated in FIG. 7, the transactivation activity of Nqo-1 is increased by Nrf2, and is decreased by Keap1 known as a suppressor of Nrf2.

Furthermore, it was observed that RCAT bound to Keap1 to degrade Keap1, thereby increasing the transactivation activity of Nqo-1 by Nrf2.

ANIMAL MODEL FOR OXIDATIVE STRESS RESEARCH AND USE THEREOF

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