WO2023136472A1 - Composition for ferroptosis regulation comprising marh6 (marchf6) regulator, and method for same - Google Patents

Abstract

The present invention relates to: a composition for ferroptosis regulation, the composition comprising a MARH6 regulator; a method for ferroptosis regulation; and a screening method for screening a substance for ferroptosis regulation, wherein the screening method targets MARH6. The present invention, which is the first invention based on the discovery that MARH6 is involved in ferroptosis regulation, can induce or inhibit ferroptosis by regulating the activity and expression of MARH6. Therefore, the present invention can be diversely used to regulate various physiological and pathological conditions in which ferroptosis is involved, or develop a therapeutic agent for related diseases, wherein the agent targets MARH6 and the ferroptosis mechanism.

Description

Composition and method for regulating ferrotosis, including MARH6 (MARCHF6) modulator

The present invention relates to a composition and method for regulating ferrotosis containing a MARH6 modulator, and a method for screening a material for regulating ferrotosis targeting MARH6.

Ferrotosis, one of the cell death methods, is induced through lipid peroxidation by excessive iron ions. Ferrotosis is basically regulated by iron ions, reactive oxygen species, lipid peroxidation, and a system that neutralizes lipid peroxidation, and changes in the cell environment or cell metabolism can be signals that cause ferrotosis. Recent studies have revealed the connection between ferrotosis and various diseases. Physiological and pathological aspects of ferrotosis have been revealed to the extent that the categories of the diseases include cancer diseases, brain diseases, organ damage, immune and inflammation-related diseases, etc. importance is emerging.

MARH6 (MARCHF6/MARH6/TEB4/RNF176) is one of the E3 ubiquitin ligases located in the endoplasmic reticulum membrane and is involved in protein degradation in the endoplasmic reticulum membrane or cytoplasm. Target proteins degraded by MARH6 include MARH6 itself, squalene monooxygenase (SM) involved in cholesterol synthesis, regulator of G-protein signaling 2 (RGS2), a blood pressure regulator, and Plin2 (Perilipin 2) involved in lipid droplet formation. there is Although recent studies have revealed an association between MARH6 and lipid and cholesterol metabolism, the physiological role of MARH6 has not been clearly defined.

While researching targets and methods for regulating ferrotosis, which have recently been found to be associated with various physiological and pathological symptoms, the present inventors found that MARH6, whose role had not been clearly identified, was involved in the ferrotosis regulatory mechanism. It was confirmed that they were involved and the present invention was completed.

Accordingly, the present invention relates to a ferrotosis regulating composition comprising a MARH6 modulator, a ferrotosis regulating method using the same, and a screening method for a ferrotosis regulating substance targeting MARH6.

Accordingly, the present invention provides a composition for inducing ferrotosis comprising an inhibitor of MARH6 activity or expression.

In addition, the present invention provides a composition for inhibiting ferrotosis comprising MARH6 or an activator thereof.

In addition, the present invention comprises the steps of: 1) processing a candidate material for controlling ferrotosis in a sample; 2) measuring the change in activity or expression of MARH6 after treatment with the candidate substance; It provides a screening method for substances for controlling ferrotosis, comprising a.

In addition, the present invention comprises the steps of regulating the expression or activity of MARH6 in vitro; It provides a method for controlling ferrotosis of cells comprising a.

In addition, the present invention comprises the steps of treating the subject with an inhibitor of MARH6 activity or expression; Ferrotosis induction method comprising a; treating the subject with MARH6 or an activator thereof; It provides a ferrotosis inhibition method comprising a.

The present invention is the first invention to confirm that MARH6 is involved in ferrotosis regulation, and ferrotosis can be induced or inhibited through the regulation of MARH6 activity and expression. Therefore, it can be used in various ways to control various physiological and pathological conditions involved in ferrotosis or to develop therapeutic agents for related diseases by targeting MARH6 and the ferrotosis mechanism.

Figure 1A is a diagram showing the results of confirming lipid ROS levels in HeLa cells treated with MARH6 siRNA and control for 48 hours through three independent experiments. The left side is a diagram showing the percentage of cells exhibiting increased fluorescence. The right side is a diagram showing the mean ± SD of lipid ROS quantitative values measured using C11-BODIPY (Two-tailed t -test. p**** < 0.0001).

1B and C are diagrams showing the results of three independent experiments on lipid ROS levels after treatment/untreatment with erastin (5 μM) for 24 hours in wild-type and MARH6 KO HeLa cells (B) or HEK293T cells (C). . The left side is a diagram showing the percentage of cells exhibiting increased fluorescence. The right side is a diagram showing the mean ± SD of lipid ROS quantitative values measured using C11-BODIPY (one-way ANOVA with Tukey's HSD post-hoc test. **p<0.01 and ****p<0.0001).

FIG. 1D is a diagram showing the results of three independent experiments on lipid ROS levels in MARH6 KO HEK293T cells after 12 hours of treatment/untreatment with wild-type RSL3 (0.1 μM). The left side is a diagram showing the percentage of cells exhibiting increased fluorescence. The right side is a diagram showing the mean ± SD of lipid ROS quantitative values measured using C11-BODIPY (one-way ANOVA with Tukey's HSD post-hoc test. **p<0.01 and ****p<0.0001).

1E and F are diagrams showing the results of three independent experiments on lipid ROS levels after treatment/untreatment with erastin (5 μM) for 24 hours in wild-type and MARH6 KO A549 cells (E) or HCT116 cells (F). . The left side is a diagram showing the percentage of cells exhibiting increased fluorescence. The right side is a diagram showing the mean ± SD of lipid ROS quantitative values measured using C11-BODIPY (Two-tailed t -test. p** < 0.05, p*** < 0.001, p**** < 0.0001 ).

Figure 2 is a diagram showing the result of confirming the accumulation of lipid ROS at the organism level after treating MARH6 ^-/- mice with the ferrotosis inducer elastin. Each result was expressed as mean ± SD of 3 independent experiments (One-way ANOVA with Tukey's HSD post-hoc test: p** <0.01; p*** <0.001; p**** < 0.0001).

3 is a diagram showing the result of confirming the relative viability of wild-type, MARH6 KO HeLa, HEK293T, A549, or HCT116 cells treated with the ferrotosis inducer Erastin or RSL3 at different concentrations.

Figure 4a is a diagram showing the results of confirming the sensitivity to RSL3 and erastin in MARH6 KO HeLa cells expressing blank, MARH63f, and MARH6 _3f ^C9A through the relative viability of MARH6 KO HeLa (two-tailed t -test. p**** < 0.0001).

Figure 4b is a diagram showing the results of confirming the relative cell viability after treating RSL3 with mock, DFO, Fer-1, Z-VAD or Nec-1 in MARH6 KO HeLa or MARH6 KO HEK293T (one-way ANOVA with Tukey's HSD post-hoc test. p* < 0.05, p**** < 0.0001).

FIG. 5 is a view showing the results confirming the effect of stabilizing MARH6 substrate SM according to the treatment of elastin and RSL3, which are ferrotosis inducers (FIG. 5A shows an erastin-treated group, and FIG. 5B shows an RSL3-treated group).

6 is a diagram showing the results of confirming down-regulated genes and their functions in MARH6 KO HeLa cells compared to wild-type control through DEG (differentially expressed genes).

7 is a diagram showing the results of confirming the relative amount of NADP or NADPH in wild-type and MARH6 KO HeLa cells (mean ± SD (three biological replicates), two-tailed t -test. p*** < 0.001).

8 is a diagram showing the structure of MARH6 and the sequence alignment of MARH6 FRR in various species, including humans.

9 is a view showing the results of confirming the cell viability according to the ferrotosis inducer treatment after expressing the full-length or partial sequence-deleted MARH6 in MARH6 KO HeLa cells (mean ± SD (three independent experiments), One-way ANOVA with Tukey's HSD post-hoc test (p* < 0.05, p**** < 0.0001).

10 is a diagram showing the results of confirming and quantifying the degradation rates of MARH6 _3f 1-900 and full-length MARH6 _3f 1-910 through CHX tracking analysis.

11 is a diagram showing the results of a GST-pulldown assay for confirming an interaction site between a ring domain and an FRR domain.

12 is a view showing the result of performing an in vitro ubiquitination assay (a) and the result of confirming the type of linking chain (b) for confirming the interaction between the ring domain and the FRR domain.

13 is a diagram showing the results of 2',5'-adenosine diphosphate (ADP)-agarose affinity chromatography used to confirm the binding between NADPH and FRR.

Figure 14 shows the results of 2',5'-ADP-agarose affinity chromatography for mapping the FRR region recognizing NADPH (A), the results of GST pull-down assay of GST or GST-FRR (B), and the presence of NADPH or NADH It is a diagram showing the ubiquitination analysis result (C) according to.

Figure 15 shows the proteolysis patterns of ferrotosis regulatory proteins ACSL4 (A), ALOX5 (B), TfR1 (C), and p53 (D) according to the presence or absence of the MARH6 gene in wild-type and MARH6 KO HeLa or A549 cells by CHX tracking assay. This is a diagram showing the results obtained through

Figure 16 is a view showing the results of confirming the expression and degradation rate of pro-ferroptosis protein and anti-ferrotosis protein according to MARH6 deletion.

Figure 17: liver tissue of MARH6 ^-/- embryos and wild type isolated from Erastin treated/untreated mothers; and wild-type and MARH6 KO A549 cell-derived xenograft tumors showing pro-ferroptosis protein and anti-ferroptosis protein expression changes.

18 is a schematic view of xenotransplantation experiment (A) for confirming the tumor growth inhibitory effect of MARH6 KO and a diagram showing the result (B) confirming the tumor growth inhibitory effect.

19 is a diagram showing a mating schematic diagram (A) and a result (B) of confirming the survival rate for confirming the survival rate of MARH6 ^-/- baby mice according to the vitamin E diet.

20 is a schematic diagram showing the ferrotosis regulation mechanism of MARH6 according to the present invention.

The present invention relates to a composition and method for regulating ferrotosis including a MARH6 modulator.

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for inducing ferrotosis comprising an inhibitor of MARH6 activity or expression.

Also, the present invention relates to a composition for inhibiting ferrotosis comprising MARH6 or an activator thereof.

According to the present invention, when the activity or expression of MARH6 is inhibited, ferrotosis is induced and death by ferrotosis can be induced, and conversely, when the activity of MARH6 is enhanced through MARH6 or an activator thereof, ferrotosis It is possible to protect the individual from death by ferrotosis by inhibiting.

MARH6 of the present invention is an E3 ubiquitin-protein ligase and is an enzyme encoded by the MARH6 gene. MARH6, also known as MARCHF6, MARCH6, TEB4, or RNF176, is a protein present in the ER membrane. MARH6 contains 14 transmembrane domains, N-terminal and C-terminal domains containing RING sites directed to the cytoplasm (see FIG. 8). MARH6 is found in various animals and its C-terminus is known to be particularly conserved in vertebrates. Human MARH6 (GenBank: AAI36462.1) consists of a total of 910 amino acids.

The term 'Ferrotosis' is a cell death method by iron-mediated lipid peroxidation, in which iron meets reactive oxygen species (ROS) to induce the Fenton reaction. Through this, lipid peroxides accumulate in the cells and the cells die. That is, ferrotosis is iron-dependent cell death, and may refer to cell death caused by generation and accumulation of iron-dependent reactive oxygen species and lipid peroxidation. Recently, a study on a method of killing cancer cells using such ferrotosis has been reported.

In the present invention, the MARH6 activity or expression inhibitor capable of inducing ferrotosis is an antisense nucleotide that complementarily binds to the mRNA of the MARH6 gene, short hairpin RNA (shRNA), small interfering RNA , siRNA), ribozymes, compounds that specifically bind to MARH6 protein, peptides, peptide mimetics, substrate analogs, aptamers, and antibodies.

The activity or expression inhibitor refers to a substance that further promotes ferrotosis by inhibiting the role that MARH6 plays in the ferrotosis step. For example, MARH6 activity or expression inhibitors can increase the sensitivity of an individual to ferrotosis and promote cell death by ferrotosis.

In addition, the activity or expression inhibitor of the present invention may inhibit the binding of MARH6 and NADPH or the binding of MARH6 ring domain and ferroptosis regulatory region (FRR) domain. The ferroptosis regulatory region (FRR) region of the C-terminal tail of MARH6 was identified for the first time, and NADPH binds to the FRR region to promote rapid ubiquitination. Therefore, for the purpose of inducing ferrotosis, a substance that inhibits the binding between the FRR region of MARH6 and NADPH may be used as an inhibitor of MARH6 activity or expression. The MARH6 ring domain may be a region from 1 to 69 amino acids from the N-terminus of MARH6 based on human MARH6 (GenBank: AAI36462.1), and the FRR domain of MARH6 may be a region 870-910 that binds to the ring domain of MARH6. there is. Since FRR activates enzymatic RING of MARH6 by binding to the RING domain, substances that inhibit their binding can be used as MARH6 activity or expression inhibitors.

The MARH6 activity or expression inhibitor of the present invention inhibits degradation of one or more pro-ferroptosis proteins selected from the group consisting of ACSL4, ALOX5, TFR1 and p53; Alternatively, it may down-regulate the expression of at least one anti-ferrotosis factor selected from the group consisting of SLC7A11 (cystine/glutamate antiporter xCT), GPX4, and NRF2.

In the present invention, in the absence of MARH6, it was confirmed that major pro-ferroptosis effectors such as ACSL4, ALOX5 and TfR1 and squalene monooxygenase SM were stabilized by reducing the degradation rate, and the anti-ferrotosis protein SLC7A11 (cystine/glutamate It was confirmed that the levels of antiporter xCT), GPX4, and NRF2 were downregulated. Therefore, inhibitors of MARH6 activity or expression may inhibit the degradation of pro-ferroptosis or down-regulate the expression of anti-ferroptosis factors.

Also, the present invention relates to a composition for inhibiting ferrotosis comprising MARH6 or an activator thereof.

In the present invention, it was confirmed that ferrotosis is promoted when MARH6 is inhibited, and ferrotosis can be inhibited when MARH6 is activated.

In the present invention, the MARH6 activator may be a MARH6 protein or a gene encoding the protein, which can inhibit ferrotosis by restoring the expression of MARH6 due to MARH6 knockout or increasing the amount of MARH in cells there is. The MARH6 activator may include, without limitation, one that promotes the E3 ubiquinine ligase activity of MARH6, and in particular, may be NADPH. In the present invention, it was confirmed that MARH6 can act as a NADPH sensing ferrotosis regulator, NADPH binds to the FRR domain, and the FRR region present at the C-terminus binds to the RING domain present at the N-terminus. It can be increased, and it was confirmed that rapid ubiquitination can be promoted through this.

Meanwhile, in the present invention, the FRR region, FerR ( Fer roptosis Repression ) domain, and FerS ( Fer roptosis Stimulation ) domain of MARH6 were determined through the study of the ferrotosis mechanism of MARH6. More specifically, it was confirmed that residues 870-891 in vertebrates that are evolutionarily conserved, and in particular in human MARH6 (GenBank: AAI36462.1), are the sites that bind to the ring, thereby confirming that they are FerR domains, and the C-terminal 10 of the residues It was confirmed that the residue stretch, residues 901-910, corresponds to the FerS domain.

It was confirmed that the overexpression of full-length MARH6 in the MARH6 knockout model substantially increased the survival rate from ferrotosis induced by a ferrotosis inducer. That is, this is a result showing that MARH6 can protect an individual from ferrotosis stress. Additionally, it was confirmed that over-expression of MARH6 _1-900 in which the 10 C-terminal FerS domains were removed from full-length MARH6 showed a more excellent inhibitory effect on ferrotosis-induced cell death compared to the experimental group overexpressing full-length MARH6. Accordingly, it can be seen that the FerS domain of MARH6 mediates ferrotosis promotion, and conversely, it can be seen that MARH6 in which the 10 residues MARH6 _901-910 region is removed can inhibit ferrotosis.

Accordingly, in the present invention, MARH6 or its activator may be MARH6 _1-900 from which the sequence of MARH6 _901-910 has been removed, as well as full-length MARH6 itself.

On the other hand, in the present invention, it was confirmed that ferrotosis regulated by MARH6 plays an important role in regulating ferrotosis during embryonic development and birth, and that MARH6 ^-/- mice have increased sensitivity to ferrotosis, leading to an increase in the embryonic development stage. It was confirmed that growth was delayed after undergoing ferrotosis or high mortality after birth. However, when a vitamin E-rich diet was supplied to the pregnant mother of these mice, it was confirmed that the survival rate of the object increased remarkably by protecting the object from ferrotosis. Therefore, it can be confirmed that additional supply of vitamin E can inhibit ferrotosis in the process of inhibiting ferrotosis regulated by MARH6, and in the present invention, ferrotosis further comprising vitamin E in addition to MARH6 or its activator An inhibitory composition is provided.

In the present invention, inhibition of ferrotosis includes all protective actions that reduce cell death caused by ferrotosis and protect an individual from stress caused by ferrotosis.

In addition, the present invention comprises the steps of treating the subject with an inhibitor of MARH6 activity or expression; Ferrotosis induction method comprising a; treating the subject with MARH6 or an activator thereof; It provides a ferrotosis inhibition method comprising a.

In addition, the present invention comprises the steps of regulating the expression or activity of MARH6 in vitro; It provides a method for controlling ferrotosis of cells comprising a. In addition, in the present invention, the composition may include a reagent composition and a medium composition without limitation.

The subject may include, without limitation, any subject that has undergone or is likely to undergo ferrotosis, and may include vertebrates, preferably mammals including humans.

The description of the ferrotosis inhibition and induction method may equally apply to all of the descriptions of the composition described above.

In addition, since the present invention newly derives a correlation between MARH6 regulation and ferrotosis regulation, it provides a screening method for ferrotosis regulation substances based on this mechanism.

More specifically, the present invention comprises the steps of 1) treating a candidate for ferrotosis regulation in a sample; 2) measuring the change in activity or expression of MARH6 after treatment with the candidate substance; It provides a screening method for substances for controlling ferrotosis, comprising a.

In the present invention, when the MARH6 activity or expression level in step 2) increases, the step of determining the candidate substance as a ferrotosis inhibitor may be further included, and the MARH6 activity or expression level in step 2) decreases. If it is, the step of determining the candidate substance as a ferrotosis inducer may be further included.

The candidate substance is an unknown substance expected to inhibit or promote the expression or activity of MARH6, and may include compounds, proteins, natural products, extracts, genes, and the like without limitation.

Measurement of the activity or expression change of MARH6 can be measured using any method known in the art without limitation, such as polymerase reaction (PCR), reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction ( Western blotting, such as Competitive RT-PCR), Realtime RT-PCR, RNase protection assay (RPA), northern blotting, or DNA microarray analysis ), ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion method, Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay ( immunoprecipitation assay), complement fixation assay (complete fixation assay), flow cytometry (Fluorescence Activated Cell Sorter, FACS) or protein chip (protein chip) assay, etc. may be used, but is not limited thereto.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited by the following examples.

Example 1. Confirmation of lipid ROS increase according to MARH6 knockout

In order to confirm the relationship between ferrotosis and MARH6, experiments using HEK293T, HeLA, A549 or HCT116 cells were performed. First, lipid ROS levels were compared in MARH6 knockdown cells using siRNA and control cells through flow cytometry using the lipid peroxidation sensor C11-BODIPY ^581/591 . In addition, wild-type or MARH6 knockout cells were treated with the ferrotosis inducer erastin (a cystine/glutamate antiporter inhibitor) or RSL3 (a glutathione peroxidase 4 (GPX4) inhibitor), and changes in lipid ROS levels were measured. The results are shown in Figure 1.

As shown in Figure 1, MARH6 knockout increased lipid ROS levels, consistent with several pairs of wild-type and CRISPR/Cas9 system-based MARH6 KO HeLa, HEK293T, A549 or HCT116 cancer cell lines. In particular, lipid ROS production was shown to be increased in MARH6 KO cells treated with the ferrotosis inducer erastin (a cystine/glutamate antiporter inhibitor) or RSL3 (a glutathione peroxidase 4 (GPX4) inhibitor).

Additionally, using MARH6 ^-/- mice prepared using the CRISPR/Cas9 system, an experiment was performed to confirm whether MARH6 affects lipid ROS accumulation at the organism level. Because MARH6 ^-/- mice showed growth retardation and/or high postnatal lethality, corn oil (control) or elastin (15 mg/kg per body weight) was injected daily intraperitoneally into pregnant mice for 7 days, followed by littermate littermates. Lipid ROS production was measured in the embryonic liver of , and the results are shown in FIG. 2 . The production of lipid ROS was evaluated by malondialdehyde (MDA), a by-product of lipid peroxidation.

As shown in Figure 2, upregulated lipid ROS levels were confirmed in the livers of corn oil-treated E18.5 Marh6 ^-/- embryos compared to livers of Marh6 ^+/+ or Marh6 ^+/- embryos. As in human cell lines, administration of erastin markedly increased lipid ROS levels in Marh6 ^−/− embryos compared to wild-type or heterozygous livers.

Example 2. Confirmation of ferrotosis inhibitory ability of MARH6

Since an increase in lipid ROS levels was confirmed in MARH6 deficient human cell lines and mice through Example 1, dose-dependent responsiveness to ferrotosis inducers was confirmed in wild-type and MARH6 KO cells. The relative viability of wild-type, MARH6 KO HeLa, HEK293T, A549, or HCT116 cells was confirmed after treatment with erastin and RSL3 for 24 hours at each concentration, and the results are shown in FIG. 3 .

As shown in FIG. 3 , it was confirmed that the sensitivity of various MARH6 KO cells to treatment with elastin or RSL3, which is a ferrotosis inducer, was increased, and more apoptosis was induced at the same concentration.

On the other hand, in order to confirm whether the sensitivity of MARH6 KO cells to elastin or RSL3 is improved by the catalytically inactive MARH6 _3f ^C9A mutant, elastin (15 μM) and RSL3 (0.15 μM) were treated for 24 hours. The viability of MARH6 KO cells expressing the empty vector, MARH63f, and MARH6 _3f ^C9A was confirmed. The results are shown in Figure 4a.

As shown in Figure 4a, sensitivity to either erastin or RSL3 was not improved in the catalytically inactive MARH6 _3f ^C9A mutant. Unlike the ectopic expression of the C-terminal triple flag-tagged wild-type MARH6 _3f , this result does not improve in the MARH6 _3f ^C9A mutant, indicating that the E3 Ub ligase activity of MARH6 is required for ferrotosis inhibition.

MARH6 KO HeLa cells were treated with mock, DFO (100 μM), Fer-1 (5 μM), Z-VAD (20 μM), or Nec-1 (40 μM) with RSL3 0.15 μM for 24 hours and cell viability confirmed. In addition, MARH6 KO HEK293T cells were treated with 0.1 μM of RSL3, and cell viability was confirmed by performing the same experiment. The result of confirming the cell viability is shown in FIG. 4B.

As shown in Figure 4b, the viability of MARH6 KO cells against the ferrotosis inducer RSL3 was significantly recovered by DFO (deferoxamine) or Fer-1 (ferrostatin-1), but by the caspase inhibitor Z-VAD-FMK was hardly recovered, and even with nerostatin-1 (Nec-1), a necroptosis inhibitor, it was hardly recovered or only partially recovered to a low degree. These results indicate that MARH6 is not related to other forms of cell death such as apoptosis or necroptosis, but is particularly related to ferrotosis regulation.

Example 3. Stabilization of MARH6 substrate by ferrotosis inducer

A cycloheximide (CHX)-tracking assay of proteolysis was performed to determine whether ferrotosis inducers affect the metabolic turnover of MARH6 substrates. After treatment with mock, erastin (5 μM), or RLS3 (0.15 μM), CHX tracking analysis of SM in HeLa cells was performed for 2 hours, and the results are shown in FIG. 5 .

As shown in Fig. 5, treatment with erastin or RSL3 in HeLa cells strongly stabilized endogenous SM, a degron-bearing MARH6 substrate.

Example 4. Confirmation of NADPH metabolism regulation of MARH6

Whole genome RNA sequencing analysis of wild-type and MARH6 KO HeLa cells was performed to confirm the mechanism of MARH6 regulating fertility. Using WikiPathways enrichment, DEGs (differentially expressed genes) were identified in wild-type and MARH6 KO HeLa cells and their functions were analyzed, and genes in priority among downregulated genes in MARH6 KO HeLa cells were identified. is shown in Figures 6 and 7.

As shown in FIG. 6 , it was confirmed that MARH6 is related to NAD(P), sterols, vitamin metabolism, regulated necrosis, inflammation, etc., suggesting the importance of its relationship with NAD(P) production in particular. As shown in Figure 7, as a result of confirming the relative amount of NADP or NADPH in wild-type and MARH6 KO HeLa cells, it was confirmed that the corresponding metabolites were significantly down-regulated in MARH6 KO HeLa cells.

EXAMPLE 5. MARH6 Ferrotosis Regulatory Region (FRR) for Substrate Degradation

The structure of MARH6 and the sequence alignment of MARH6 FRR in various species including humans are shown in FIG. 8 .

As shown in FIG. 8, MARH6 has a ring domain responsible for E3 activity at the N-terminus and FRR, which is important for ferrotosis regulation, is located at the C-terminus. Human MARH6 contains a highly conserved CTE (residues 877-892) and a Ferroptosis regulatory region (FRR) with a relatively less conserved C-terminal tail.

To investigate the role of FRR in MARH6-mediated fertility inhibition, MARH6 knockout HeLa cells were transfected with full length MARH6 _3f 1-910, FRR deletion (MARH6 _3f 1-869) and MARH6 _3f 1-900 (last 10 C-terminal residues). deletion) was expressed through a vector, and the survival rate was confirmed by treatment with 15 μM of erastin or 0.15 μM of RSL3, which is a ferrotosis inducer, and the results are shown in FIG. 9 . In addition, CHX tracking analysis of full-length and MARH6 _3f 1-900 was performed in MARH6 KO HeLa cells, and the degradation rate was confirmed by quantifying them, and the results are shown in FIG. 10 .

As shown in Figure 9, overexpression of full-length MARH6 _3f 1-910 substantially increased the viability of MARH6 KO HeLa cells treated with Elastin or RSL3, whereas MARH6 _3f 1-869 had no effect on cell viability. confirmed that it is not. On the other hand, it was unexpectedly confirmed that the overexpression of MARH6 _3f 1-900 more efficiently inhibited cell death induced by ferrotosis inducers in MARH6 KO HeLa cells than the full-length MARH6 _3f 1-910.

Also, as shown in FIG. 10, MARH6 _3f 1-900 was decomposed much faster than MARH6 _3f 1-910. These results show that the C-terminal 10 residues potentially mediate ferrotosis promotion by inhibiting MARH6 activity.

Additionally, a GST-pulldown assay was performed to analyze the mechanism of MARH6 activity regulation. Perform GST pulldown assay using GST, GST-MARH6 ^870-910 , GST-MARH6 ^870-900 , GST-MARH6 ^870-891 , or GST-MARH6 ^892-910 (30 μg each) and RINGha (2 μg) and confirmed the specific interaction of MARH6, and the results are shown in FIG. 11.

As shown in FIG. 11, it was confirmed that the N-terminal ring-containing fragment (residues 1-91) specifically interacted with the C-terminal FRR (residues 870-910), and the C-terminal _ha -labeled RING _ha ( MARH6 _ha 1-71) directly binds to GST-MARH6 870-910, GST-MARH6 870-900 and GST, but does not bind to GST and GST-MARH6 892-910. Residues 870-891 were derived. That is, it was confirmed that the ring domain directly binds to the FRR domain, and even within the FRR domain (MARH6 870-910), it was confirmed that, in particular, the MARH6 970-891 portion plays a decisive role in binding to the ring domain.

The binding of GST-MARH6 ^870-900 and GST-MARH6 ^870-891 to RING _ha showed distinctly stronger binding compared to the binding to GST-MARH6 ^870-910 , and the enhancement of the last C-terminal 10-residue stretch showed FRR -RING interaction was inhibited. These results are consistent with the rapid degradation of MARH6 _3f 1-900 previously identified. Therefore, the highly evolutionarily conserved (particularly in vertebrates) RING-linked/CTE-segment (residues 870-891) containing and C-terminal 10-residue stretch (residues 901-910), here ferroptosis expression ( FerR ) ) domain and FerS ( Ferroptosis Stimulation ) domain, respectively, reflecting their rheostatic role in ferrotosis regulation through MARH6 activation. To confirm the interaction between the ring domain and the FRR domain, RINGha-FRR (0.5 μM) and K0-Ub (25 μM), a Ub mutant that does not contain Ub and Lys, and Ub mutants K6 and K11 that contain a single Lys , K27, K29, K33, K48, K63-Ub ubiquitination assay and anti-Ub immunoblot were performed. The results are shown in FIG. 12 .

As shown in Fig. 12a, the purified linear RING _ha -FRR fusion strongly promoted the formation of long poly-Ub chains in the presence of His6-UBE2J2 ^ΔTM and/or His6-UBE2G2, whereas RING _ha only produced short Ub chains. only produced. Through this, it was confirmed that the interaction between the ring domain and the FRR domain contributes to increasing the activity of the ring domain. FRR activates the enzymatic RING of MARH6, which can promote the transfer of Ub by E3 ligase from Ub-charged E2 to free Ub, unattached Ub chains, or Ub chains attached to proteins. As shown in Fig. 12b, it was confirmed that most of the chains consisted of K48 connections.

Example 6. Direct binding of NADPH to FRR

To confirm whether FRR directly interacts with NADPH, 2', _5' -adenosine diphosphate (ADP) _- Agarose affinity chromatography was performed, and the results are shown in FIG. 13 . The stable NADP(H) analog 2',5'-ADP is commonly used to identify NADPH-binding proteins.

As shown in FIG. 13, 2',5'-ADP-agarose affinity chromatography showed that 2',5'-ADP binds to MARH6 _3f 1-910, but not to MARH6 _3f 1-869. . These results indicate the possibility that FRR is a binding site for NADP(H) for regulating MARH6 activity. In addition, the purified GST-FRR fusion directly bound to 2',5'-ADP-agarose, whereas GST alone did not bind to it. In particular, the binding of GST-FRR to 2',5'-ADP was abolished by competitive treatment with 1 mM NADPH, but was partially maintained by treatment with the same concentration of NADP+. These results show that FRR prefers binding to NADPH rather than NADP+.

Example 7. FRR-RING interaction of NADPH and confirmation of continuous MARH6 activity increasing effect

To map the FRR region recognizing NADPH, 2',5'-ADP-agarose affinity chromatography was further performed using the following purified GST-FRR derivatives: GST-MARH6 870-910, GST- MARH6 870-900, GST-MARH6 870-891 and GST-MARH6 892-910. In addition, to confirm the effect of NADPH and NADH on FRR-RING _ha interaction, GST or GST-FRR (40 μg each) in the presence of mock, NADPH, or NADH (0.1 mM each) and RINGha (2 μg) A GST pull-down assay was performed.

As shown in Figure 14A, it was confirmed that 2',5'-ADP binds to GST-MARH6 870-910 and GST-MARH6 870-900, but not to GST-MARH6 870-891 and GST-MARH6 892-910. did This indicates that the FRR domain (MARH6 870-900) sequence is responsible for binding to NADPH. Through this, it was confirmed that NADPH and RING overlap with FRR. In addition, as shown in Figure 14B, it was confirmed through GST pull-down analysis that the FRR-RING _ha interaction significantly increased in the presence of NADPH, but not in NADH.

The in vitro ubiquitination assay in FIG. 14C also showed that MARH63f-His6UBE2J2DTM/His6UBE2G2 mediated polyubiquitylation was significantly enhanced by the presence of NADPH but not by the presence of NADH. These results show that the presence of NADPH can increase the activity of MARH6.

Example 8. Confirmation of ferrotosis effector regulation of MARH6

An experiment was performed to see whether MARH6 regulates the metabolic turnover of specific ferrotosis regulators in response to apoptosis. A subset of previously known ferrotosis-related proteins was investigated by monitoring their degradation patterns in wild-type and MARH6 KO HeLa or A549 cells. More specifically, CHX was used to determine whether MARH6 is involved in the degradation of acyl-CoA synthetase long-chain family member 4 (ACSL4), arachidonate 5-lipoxygenase 5 (ALOX5), transferrin receptor 1 (TfR1), and p53, which are proteins that regulate fertility. A follow-up assay was performed. In the absence of the MARH6 gene, the degradation rates of ACSL4, ALOX5, TFR1, and p53 proteins were also confirmed, and the results are shown in FIGS. 15 and 16 .

As shown in FIG. 15, major pro-ferroptosis effectors, such as ACSL4, ALOX5, and TfR1, were significantly or identifiablely stabilized in the absence of MARH6, such as MARH6 KO, and it was confirmed that the rate of protein degradation was remarkably reduced.

In addition, as shown in FIG. 16, MARH6 deletion effectively stabilized pro-ferroptosis proteins and significantly or detectably lowered the levels of key anti-ferrotosis proteins, SLC7A11 (cystine/glutamate antiporter xCT), GPX4, and NRF2. It was also confirmed that downregulation. Among them, SLC7A11 downregulation was most prominent.

Through the above results, it was confirmed that the ferrotosis inducer can stabilize the pro-ferrotosis protein through NADPH depletion and continuous MARH6 inactivation.

To confirm the effect ^of MARH6 on regulating ferrotosis factors under physiological conditions, p53, SLC7A11, SM and 4-HNE (lipid The level of peroxidation marker 4-hydroxynonenal) was measured. In addition, changes in the expression level of the same factors were confirmed in wild-type and MARH6 KO A549 cell-derived xenograft tumors, and the results are shown in FIG. 17 .

As shown in FIG. 17, the levels of pro- ferrotesis factors p53, SM and 4-HNE were increased in Marh6 ^-/- mice compared to the wild type, whereas the level of SLC7A11 was greatly decreased. These results were similar in wild-type and MARH6 KO A549 cell-derived xenograft tumors.

Summarizing the above results, MARH6 is a protein involved in ferrotosis regulation by sensing NADPH, NADPH directly binds to the FRR domain of MARH6 to increase the activity of the ring domain, and MARH6 is a pro-ferroptosis protein. ACSL4, ALOX5, TFR1, and p53 were degraded to confirm that ferrotosis could be inhibited.

Example 9. Confirmation of anticancer activity through ferrotosis regulation of MARH6

Since it was confirmed that MARH6 can be a ferrotosis regulator, an experiment was performed to determine whether MARH6 can regulate tumor growth through ferrotosis control in cancer cells.

First, xenograft experiments using wild-type and MARH6 KO A549 lung cancer cells were performed to confirm whether the removal of MARH6 had an effect on tumor formation. At the beginning of the xenotransplantation, Fer-1 was injected to minimize the effect of ferrotosis, and then Fer-1 was discontinued to confirm the effect of ferrotosis. The volume of the tumor was measured every 4 days, and the results of confirming the growth rates of the wild-type cancer and the cancer from which the MARH6 gene was removed are shown in FIG. 18 .

As shown in Figure 18, the xenograft test results showed that the tumor size was significantly reduced in MARH6 KO cells compared to wild-type cells. As can be seen in Figure 18B, there was no difference in tumor size until 40 days after Fer-1 was injected, but MARH6 KO A549 cells were transplanted from 40 days after injection of Fer-1, a ferrotosis inhibitor, was stopped. In one mouse, it was confirmed that the tumor size was significantly inhibited.

These results show that the growth rate of cancer can be inhibited by inducing ferrotosis of cancer cells through inhibition of MARH6 in cancer cells.

Example 10. Confirmation of survival rate according to ferrotosis regulation of MARH6

It was confirmed that MARH6 ^-/- mice undergo ferrotosis during embryonic development, exhibit growth retardation and/or high mortality after birth. On the other hand, it is known that the addition of vitamin E, a lipophilic antioxidant, to the diet inhibits the fertosis phenotype in Gpx4 ^-/- mice.

In mice whose ferrotosis was induced by MARH6 ^-/- of the present invention, it was confirmed whether the ferrotosis phenotype could be suppressed and the survival rate increased if vitamin E was added to the diet. MARH6 ^+/- and MARH6 ^+/- were mated, and pregnant mothers were fed normal (45 mg/kg food) and high-dose (567 mg/kg food) vitamin E diets until the 19th day. After that, the survival rate of wild type, MARH6 ^+/- mice and MARH6 ^-/- mice was confirmed. A schematic diagram of the experiment and the results of measuring the survival rate are shown in FIG. 19 .

As shown in FIG. 19, it was confirmed that baby mice without the MARH6 gene could be born (12%) in mothers who were abundantly supplied with vitamin E. On the other hand, it was confirmed that in mothers supplied with normal levels of vitamin E, offspring of the MARH6 ^-/- genotype were hardly born due to ferrotosis stress. These results indicate that MARH6 may play an important role in regulating fertility during embryonic development and birth.

Claims (17)

1. A composition for inducing ferrotosis comprising an inhibitor of MARH6 activity or expression.
2. The method of claim 1, wherein the MARH6 activity or expression inhibitor is an antisense nucleotide that complementarily binds to the mRNA of the MARH6 gene, short hairpin RNA (small hairpin RNA, shRNA), small interfering RNA (small interfering RNA, siRNA), ribozyme (ribozyme), a compound that specifically binds to MARH6 protein, a peptide, a peptide mimetic, a substrate analog, at least one member selected from the group consisting of an aptamer and an antibody, a composition for inducing ferrotosis.
3. The composition for inducing ferrotosis according to claim 1, wherein the MARH6 activity or expression inhibitor increases sensitivity to ferrotosis.
4. The composition for inducing ferrotosis according to claim 1, wherein the MARH6 activity or expression inhibitor inhibits the binding of MARH6 and NADPH or the binding of MARH6 ring domain and FRR (ferroptosis regulatory region) domain.
5. The method of claim 1, wherein the MARH6 activity or expression inhibitor is selected from the group consisting of ACSL4, ALOX5, TFR1 and p53 - inhibits degradation of pro-ferroptosis proteins; or

   SLC7A11 (cystine / glutamate antiporter xCT), GPX4, and one or more selected from the group consisting of NRF2 - to down-regulate the expression of ferrotosis factors, ferrotosis induction composition.
6. A composition for inhibiting ferrotosis comprising MARH6 or an activator thereof.
7. The composition for inhibiting ferrotosis according to claim 6, wherein the MARH6 or its activator is a MARH6 protein or a gene encoding the protein.
8. The composition for inhibiting ferrotosis according to claim 6, wherein the MARH6 activator promotes the E3 ubiquitin ligase activity of MARH6.
9. The composition for inhibiting ferrotosis according to claim 6, wherein the MARH6 is full-length MARH6 or MARH6 _1-900 .
10. The composition for inhibiting ferrotosis according to claim 6, wherein the MARH6 activator is NADPH.
11. The composition for inhibiting ferrotosis according to claim 6, further comprising vitamin E.
12. 1) treating the sample with a ferrotosis-regulating candidate;

    2) measuring the change in activity or expression of MARH6 after treatment with the candidate substance; Comprising, the screening method of the material for controlling ferrotosis.
13. According to claim 12,

    3) if the MARH6 activity or expression level in step 2) is increased, determining the candidate substance as a ferrotosis inhibitor; A screening method further comprising a.
14. According to claim 12,

    3) if the MARH6 activity or expression level in step 2) is reduced, determining the candidate substance as a ferrotosis inducer; A screening method further comprising a.
15. Regulating the expression or activity of MARH6 in vitro; A method for regulating ferrotosis of cells comprising a.
16. administering an inhibitor of MARH6 activity or expression to a subject in need thereof; Ferrotosis induction method comprising a.
17. administering MARH6 or an activator thereof to a subject in need thereof; Ferrotosis inhibition method comprising a.

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