Composition for regulating ferroptosis containing marchf6 protein-derived domain

Abstract

The present invention relates to a C5 domain derived from a MARCHF6 protein. The C5 domain can specifically recognize a degron (Ac / N-degron) formed by N-terminal acetylation of a target protein and mediate degradation of the target protein to regulate intracellular ferroptosis. Therefore, the present invention can be widely applied to a therapeutic composition, a functional food, and a quasi-drug for inducing or inhibiting ferroptosis, and further to a screening technique for searching for a ferroptosis regulator.

Description

A composition for regulating ferroptosis, comprising a MARCHF6 protein-derived domain

[0001] The present invention relates to a composition for regulating ferroptosis, comprising a MARCHF6 protein-derived domain.

[0002] Maintaining intracellular protein homeostasis is essential for diverse physiological processes, including cell survival, differentiation, and apoptosis. This quality control and regulation of proteins is primarily accomplished through the ubiquitin-proteasome system (UPS). This system precisely regulates protein levels by conjugating ubiquitin to target proteins and then degrading them via the proteasome.

[0003] Ubiquitination is carried out by substrate-specific E3 ligases, which recognize specific degron sequences and selectively degrade the corresponding proteins. Recently, various degrons have been discovered, and it has been reported that one of them, a degron formed by N-terminal acetylation (Ac / N-degron), is recognized by a specific E3 ligase when acetylation occurs at the N-terminus of a target protein, leading to its degradation.

[0004] Ligases that act on the existing Ac / N-degron pathway, such as Doa10, Not4, and TEB4 (MARCHF6), have been identified; however, most of them have not yet demonstrated clear functions or structures in humans, and the molecular mechanisms that recognize Ac / N-degrons in human proteins and are directly involved in their degradation have not yet been fully elucidated.

[0005] Meanwhile, recent studies have reported that an E3 ligase called MARCHF6 (Membrane-Associated RING-CH Finger Protein 6) may be involved in regulating ferroptosis, an iron-dependent cell death, by recognizing Ac / N-degrons and mediating the degradation of various substrate proteins, such as RGS2 and PLIN2.

[0006] However, systematic research on the specific Ac / N-degron-recognizing domain within the MARCHF6 protein, its structural characteristics, and the techniques for artificially modulating this interaction remains insufficient. Therefore, there is a pressing need to secure a structural unit that specifically recognizes Ac / N-degrons and develop a ferroptosis-modulating tool based on this unit.

[0007] The purpose of the present invention is to provide a C5 domain derived from MARCHF6 protein.

[0008] In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease by inhibiting ferroptosis.

[0009] In addition, another object of the present invention is to provide a pharmaceutical product for improving a disease by inhibiting ferroptosis.

[0010] In addition, another object of the present invention is to provide an anticancer adjuvant.

[0011] In addition, another object of the present invention is to provide a gene therapy composition for increasing intracellular ferroptosis sensitivity.

[0012] In addition, another object of the present invention is to provide a method for providing information necessary for diagnosing a functional abnormality or ferroptosis state of a C5 domain derived from MARCHF6 protein in a sample.

[0013] In addition, another object of the present invention is to provide a method for screening a substance for controlling ferroptosis.

[0014] In addition, another object of the present invention is to provide a method for inducing degradation of a protein comprising a degron formed by N-terminal acetylation in a sample.

[0015] In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease caused by excessive ferroptosis.

[0016] In addition, another object of the present invention is to provide a pharmaceutical product for improving a disease caused by excessive ferroptosis.

[0017] In addition, another object of the present invention is to provide a peptide in which at least one amino acid in the amino acid sequence of the C5 domain derived from human MARCHF6 protein is modified.

[0018] In addition, another object of the present invention is to provide a method for producing a peptide that has lost the binding function to a degron (Ac / N-degron) formed by N-terminal acetylation of a protein.

[0019] In addition, another object of the present invention is to provide an N-terminally acetylated peptide that induces or inhibits ferroptosis in a MARCHF6-dependent manner.

[0020] In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease caused by excessive ferroptosis, which comprises the peptide.

[0021] In addition, another object of the present invention is to provide a pharmaceutical product for improving a disease caused by excessive ferroptosis, which includes the peptide.

[0022] In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating a disease by inhibiting ferroptosis, which comprises the peptide.

[0023] In addition, another object of the present invention is to provide a pharmaceutical product for improving a disease by inhibiting ferroptosis, which includes the peptide.

[0024] In addition, another object of the present invention is to provide a method for preventing or treating a disease caused by inhibition of ferroptosis.

[0025] In addition, another object of the present invention is to provide a method for preventing or treating a disease caused by excessive ferroptosis.

[0026] In addition, another object of the present invention is to provide a method for increasing the intracellular ferroptosis sensitivity of an individual.

[0027] To achieve the above purpose, the present invention provides a C5 domain derived from MARCHF6 protein that specifically recognizes a degron (Ac / N-degron) formed by N-terminal acetylation of a target protein.

[0028] In addition, to achieve the above another object, the present invention provides a pharmaceutical composition for preventing or treating a disease by inhibition of ferroptosis, comprising an activator of the C5 domain or the Ac / N domain derived from the MARCHF6 protein.

[0029] In addition, in order to achieve the above-mentioned another object, the present invention provides a pharmaceutical product for improving a disease by inhibiting ferroptosis, comprising an activator of the C5 domain or the Ac / N domain derived from the MARCHF6 protein.

[0030] In addition, to achieve the above another object, the present invention provides an anticancer adjuvant comprising an activator of the C5 domain or the Ac / N domain derived from the MARCHF6 protein.

[0031] In addition, to achieve the above another object, the present invention provides a gene therapy composition for increasing intracellular ferroptosis sensitivity, comprising a vector expressing a C5 domain or an Ac / N domain derived from a MARCHF6 protein.

[0032] In addition, in order to achieve the above another object, the present invention provides a method for providing information necessary for diagnosing a functional abnormality or ferroptosis state of a C5 domain derived from MARCHF6 protein in a sample, comprising the step of measuring the presence or decomposition of a protein containing Ac / N-degron in a sample isolated from an individual.

[0033] In addition, in order to achieve the above another object, the present invention provides a method for screening a substance for regulating ferroptosis, comprising: 1) treating a sample containing a protein formed by N-terminal acetylation, separated from an organism, with a candidate substance for regulating ferroptosis; and 2) measuring a change in the activity of a C5 domain derived from MARCHF6 protein, a change in the activity of an Ac / N domain, or a binding ability to an Ac / N-degron after treating with the candidate substance.

[0034] In addition, in order to achieve the above another object, the present invention provides a method for inducing degradation of a protein including a degron formed by N-terminal acetylation in a sample, the method comprising the step of treating a MARCHF6 protein-derived C5 domain, an Ac / N domain, a C5 domain analogue that maintains the function of recognizing an Ac / N-degron, an Ac / N domain analogue that maintains the function of recognizing an Ac / N-degron, or an activator thereof in a sample in vitro.

[0035] In addition, to achieve the above another object, the present invention provides a pharmaceutical composition for preventing or treating a disease caused by excessive ferroptosis, comprising a MARCHF6 protein-derived C5 domain, an Ac / N domain, an inhibitor of the MARCHF6 protein-derived C5 domain, or an inhibitor of the Ac / N domain.

[0036] In addition, in order to achieve the above another object, the present invention provides a pharmaceutical product for improving a disease caused by excessive ferroptosis, comprising a MARCHF6 protein-derived C5 domain, an Ac / N domain, an inhibitor of the MARCHF6 protein-derived C5 domain, or an inhibitor of the Ac / N domain.

[0037] In addition, in order to achieve the above another object, the present invention provides a peptide in which at least one amino acid in the amino acid sequence of the C5 domain derived from human MARCHF6 protein is modified, wherein the peptide has lost the binding function to a degron (Ac / N-degron) formed by N-terminal acetylation of the protein.

[0038] In addition, in order to achieve the above another object, the present invention provides a method for producing a peptide having lost the binding function to a degron (Ac / N-degron) formed by N-terminal acetylation of a target protein, the method comprising the step of substituting any one or more amino acids selected from the group consisting of positions 566 and 571 of the amino acid sequence of a C5 domain derived from human MARCHF6 protein with alanine.

[0039] In addition, to achieve the above another object, the present invention provides an N-terminal acetylated peptide represented by the following general formula and which induces or inhibits ferroptosis dependently on MARCHF6:

[0040] [General Formula 1]

[0041] Ac-X₁-X₂-R

[0042] In the above general formula 1, Ac means an N-terminal acetyl group,

[0043] When the above X₁ is Ala, X₂ is Ser, Gly, Asp, Val, Thr, Pro, Gln, Tyr, Ala, Leu or Ile,

[0044] When the above X₂ is Ser, X₁ is Ala, Gly, Asp, Val, Thr, Phe, Ser, Pro or Leu,

[0045] The above R is -OH or -NH₂.

[0046] In addition, in order to achieve the above another object, the present invention provides a pharmaceutical composition for preventing or treating a disease caused by excessive ferroptosis, comprising the peptide.

[0047] In addition, in order to achieve the above another object, the present invention provides a pharmaceutical composition for improving a disease caused by excessive ferroptosis, comprising the peptide.

[0048] In addition, in order to achieve the above another purpose, the present invention provides a pharmaceutical composition for preventing or treating a disease by inhibition of ferroptosis, comprising the peptide.

[0049] In addition, in order to achieve the above-mentioned another object, the present invention provides a pharmaceutical composition for improving a disease by inhibiting ferroptosis, comprising the peptide.

[0050] In addition, to achieve the above-mentioned another purpose, the present invention provides a method for preventing or treating a disease caused by inhibition of ferroptosis, comprising the step of administering to a subject an activator of the C5 domain or the activator of the Ac / N domain derived from the MARCHF6 protein.

[0051] In addition, the present invention provides a method for preventing or treating a disease caused by inhibition of ferroptosis, comprising the step of administering to a subject an N-terminally acetylated peptide that induces or inhibits ferroptosis in a MARCHF6-dependent manner, represented by the following general formula:

[0052] [General Formula 1]

[0053] Ac-X₁-X₂-R

[0054] In the above general formula 1, Ac means an N-terminal acetyl group,

[0055] When the above X₁ is Ala, X₂ is Pro or Asp,

[0056] When the above X₂ is Ser, X₁ is Pro or Asp,

[0057] The above R is -OH or -NH₂

[0058] In addition, to achieve the above-mentioned another purpose, the present invention provides a method for preventing or treating a disease caused by excessive ferroptosis, comprising the step of administering to a subject a MARCHF6 protein-derived C5 domain, an Ac / N domain, an inhibitor of the MARCHF6 protein-derived C5 domain, or an inhibitor of the Ac / N domain.

[0059] In addition, the present invention provides a method for preventing or treating a disease caused by excessive ferroptosis, comprising the step of administering to a subject an N-terminally acetylated peptide that is represented by the following general formula and induces or inhibits ferroptosis in a MARCHF6-dependent manner:

[0060] [General Formula 1]

[0061] Ac-X₁-X₂-R

[0062] In the above general formula 1, Ac means an N-terminal acetyl group,

[0063] When the above X₁ is Ala, X₂ is Ser, Ala, Gly, Ile or Leu,

[0064] When the above X₂ is Ser, X₁ is Ala, Gly, Ser, Thr, Val or Leu,

[0065] The above R is -OH or -NH₂.

[0066] In addition, to achieve the above another object, the present invention provides a method for increasing intracellular ferroptosis sensitivity of an individual, comprising the step of administering to the individual a vector expressing a C5 domain or an Ac / N domain derived from a MARCHF6 protein.

[0067] According to the present invention, the C5 domain derived from the MARCHF6 protein can specifically recognize a degron (Ac / N-degron) formed by N-terminal acetylation of the protein, thereby mediating the degradation of the corresponding substrate protein, thereby regulating intracellular ferroptosis. Therefore, the present invention can be utilized in various ways, such as a therapeutic composition for inducing or inhibiting ferroptosis, a functional food and a quasi-drug, and further, a screening technology for searching for a ferroptosis regulating substance.

[0068] Figures 1 to 5 show the results of confirming the structure essential for Ac / N-degron recognition by introducing alanine substitutions into the cytoplasmic exposure region (C1 to C8) of MARCHF6. The above Fig. 1 is a structural diagram that searches for the region involved in Ac / N-degron recognition by designing 25 mutants in which evolutionarily conserved residues are substituted with alanine by comparing the cytoplasmic-directed sequences of human and mouse MARCHF6 and its yeast ortholog, Doa10. Fig. 2 is a result of evaluating whether there is interaction with M-RGS2 through immunoprecipitation to confirm mutations that affect Ac / N-degron recognition among the 25 designed MARCHF6 Ala-stretch mutants. Fig. 3 is a result of analyzing in detail the effect of a specific mutation in the C5 region on binding to M-RGS2. Fig. 4 is a result of confirming the binding ability of a specific mutation in the C5 region to A-PLIN2. Fig. 5 is a result of confirming whether a mutation in the C5 region of MARCHF6 specifically affects only the Ac / N-degron substrate.

[0069] Figures 6 to 9 show the results of analyzing various C-terminal truncations of MARCHF6 using the split-ubiquitin system to confirm the minimum protein length that can interact with the Ac / N-degron substrate. Figure 6 is a schematic diagram of the operating principle of the split-ubiquitin reporter system for quantitatively analyzing the substrate recognition ability of MARCHF6, Figure 7 is a result of defining the minimum region required for substrate recognition by confirming which range of the C-terminal truncations of MARCHF6 can interact with the Nt-acetylated substrate, Figure 8 is a result of confirming that Nt-acetylation of the substrate is essential for recognition by confirming that MARCHF6 fragments do not bind to non-acetylated substrates, and Figure 9 is a result of reconfirming the acetylation dependence of substrate binding by evaluating the interaction with MARCHF6 using P-PLIN2.

[0070] Figures 10 to 15 show the results of evaluating whether each cytoplasmic region of MARCHF6 directly binds to the Ac / N-degron substrate. The above FIG. 10 is a diagram illustrating a split-ubiquitin experimental system that can independently evaluate whether each cytoplasmic domain (C1 to C8) of MARCHF6 can bind to an Ac / N-degron substrate by fixing it to the cell membrane, FIG. 11 shows the results of selecting which domain among the eight cytoplasmic domains (C1 to C8) is limited to the Ac / N-degron recognition function, FIG. 12 shows the results of confirming whether the C5 domain is involved in direct binding to the Ac / N-degron substrate, FIG. 13 shows the results of confirming whether the C5 domain specifically recognizes and binds to the Ac / N-degron substrate in the cell, FIG. 14 shows the results of confirming whether the C5 domain (541-632) actually physically binds to the Ac / N-degron substrate in the cell, and FIG. 15 shows the results of confirming which of the sub-fragments of the C5 region can interact with the substrate.

[0071] Figures 16 to 23 show the results of identifying key residues that cause loss of substrate binding among mutants in which evolutionarily conserved amino acids in the MARCHF6 Ac / N domain were substituted with alanine. The above Figure 16 shows the results of selecting evolutionarily conserved amino acid residues by comparing the Ac / N domain sequences between human MARCHF6 and MARCHF6 orthologs of various biological species (mouse, chicken, yeast, etc.), and Figure 17 shows the results of confirming the binding of mutants in which the conserved residues in the Ac / N domain were substituted with alanine to the Nt-acetylated substrate (RGS2), Figure 18 shows the results of confirming the binding of mutants in which the conserved residues in the Ac / N domain were substituted with alanine to the Nt-acetylated substrate (A-PLIN2), Figure 19 shows the results of confirming the binding of an Ac / N recognition defective mutant to a non-Ac / N substrate (SM), Figure 20 shows the results of confirming the binding of an Ac / N recognition defective mutant to a non-Ac / N-degron substrate (p53), and Figure 21 shows the results of confirming the binding of an Ac / N recognition defective mutant to a non-Ac / N substrate (ACSL4) to distinguish the multi-substrate recognition mechanism, and 22 is the result of verifying whether the Ac / N domain is essential for the degradation function by comparing the RGS2 protein degradation ability after introducing the wild-type MARCHF6 or L571A mutant into cells, and FIG. 23 is the result of verifying the effect of the Ac / N domain defect on substrate stability by comparing the PLIN2 protein degradation ability after introducing the wild-type MARCHF6 or L571A mutant into cells.

[0072] Figures 24 to 29 show the results of examining the effects of overexpression of Nt-acetylated and non-acetylated substrates on intracellular lipid peroxide (ROS) accumulation and cell survival. The above Figure 24 shows the results of confirming the expression of Ac / N-degron substrate (M-RGS2) and its non-acetylated version (P-RGS2) in HeLa cells and A549 cells, and Figure 25 shows the results of verifying the expression of Ac / N-degron substrate (A-PLIN2) and its non-acetylated version (P-RGS2, P-PLIN2) in cells and A549 cells, thereby verifying the expression status of substrate proteins in cell lines to be used in subsequent experiments, and Figure 26 shows the results of analyzing the effect of Ac / N-degron substrates on ferroptosis sensitivity by comparing the levels of lipid peroxides (oxidized lipid ROS) after treating HeLa cells and 549 cells expressing each substrate with a ferroptosis inducer, and Figure 27 shows the results of confirming the universality of the substrate effect through the expression of A-PLIN2 and P-PLIN2 in the same experiment as Figure 5C, and Figure 28 shows the results of verifying the expression status of each substrate in cells expressing each substrate. This is the result of quantitatively confirming the difference in resistance (or sensitivity) to ferroptosis depending on the presence of Ac / N-degron by checking the survival rate after treating with a ferroptosis inducer, and Fig. 29 is the result of confirming the reliability and reproducibility of the results by comparing the effect of Ac / N-degron substrate expression on ferroptosis sensitivity between substrates through the expression of A-PLIN2 and P-PLIN2 in the same experiment as Fig. 5E.

[0073] Figures 30 to 33 show the results of measuring changes in cell viability depending on the presence or absence of Ac / N-degron under ferroptosis induction conditions. The above Figure 30 shows the results of evaluating the effect of substrate stability on intracellular oxidative stress by comparing the effects of Nt-acetylated (M-RGS2) and non-acetylated (P-RGS2) substrate expression on lipid ROS production in HeLa and A549 cells without ferroptosis inducer treatment, and Figure 31 shows the results of verifying consistency between cell lines and substrate effects by comparing the differences in the effects of A-PLIN2 and P-PLIN2 expression on lipid ROS levels, and Figure 32 shows the results of verifying the effect of Nt-acetylation of the substrate on ferroptosis resistance by measuring the difference in the viability of HeLa cells depending on the treatment conditions after treating HeLa and A549 cells with a ferroptosis inducer, and Figure 33 shows the results of verifying the effects of A-PLIN2 and P-PLIN2 expression on ferroptosis resistance and verifying the differences in responses and reproducibility between cell types and substrates.

[0074] Figures 34 to 36 show the results of analyzing how substrate degradation and ferroptosis induction change when the MARCHF6 L571A mutant fails to recognize the Ac / N-degron substrate. The above FIG. 34 shows the results of analyzing the changes in the expression levels of Ac / N-degron substrates, ferroptosis-inducing substrates, and ferroptosis-inhibiting substrates when wild-type MARCHF6 or an Ac / N recognition defective mutant (L571A) was expressed in MARCHF6 KO HeLa and A549 cells, and FIG. 35 shows the results of evaluating the effect of Ac / N recognition function on intracellular oxidative stress by measuring the difference in lipid peroxide (ROS) levels after treating MARCHF6 KO HeLa and A549 cells expressing MARCHF6 with a ferroptosis-inducing agent, and FIG. 36 shows the results of quantitatively analyzing the effect of Ac / N recognition function on regulating ferroptosis sensitivity by comparing the survival rates of the same cells as in FIG. 7B.

[0075] Figure 37 is an image showing a three-dimensional binding model between the Ac / N domain and acetylated peptides based on AlphaFold3 and selective recognition and degradation of Ac / N-degron substrates in the C5 region of MARCHF6, a three-dimensional structure of the Ac / N domain of MARCHF6 (dark gray) and a binding model with Nt-acetylated peptide (Ac-ASVAVD, yellow), and Figure 38 is an image explaining that MARCHF6 has a dual function in regulating ferroptosis.

[0076] Figures 39 to 44 show the results of producing a peptide (Ac-AS-NH₂) composed of two amino acids, with the N-terminus acetylated and the C-terminus amidated, capable of binding to the Ac / N domain of MARCHF6, and confirming its effect. The above FIG. 39 is a drawing showing the structure of the Ac-AS-NH₂, FIG. 40 is a result of confirming cell viability after treating wild-type and MARCHF6-KO HeLa cells with Ac-AS-NH₂ and treating a ferroptosis inducer, FIG. 41 is a result of confirming cell viability after treating wild-type and Marchf6-KO A549 cells with Ac-AS-NH₂ and treating a ferroptosis inducer, FIG. 42 is a result of analyzing the change in the expression level of specific proteins after treating WT and KO A549 cells with Ac-AS-NH₂, FIG. 43 is a result of confirming cell viability after overexpressing wild-type MARCHF6 or the L571A mutant form in MARCHF6-KO cells to confirm whether the effect of Ac-AS-NH₂ on inhibiting ferroptosis is dependent on the Ac / N domain function of MARCHF6, and FIG. 44 is a result of confirming lipid peroxide accumulation.

[0077] Figures 45 and 46 show the results of producing a peptide consisting of two amino acids and having an acetylated N-terminus that can bind to the Ac / N domain of MARCHF6 and confirming its effect. The above Figure 45 shows the results of confirming the cell viability when the N-terminally acetylated X (20 amino acids; alanine, glycine, aspartate, valine, threonine, serine, glutamate, proline, lysine, methionine, tyrosine, tryptophan, histidine, glutamine, asparagine, cysteine / cysteine, isoleucine, leucine, phenylalanine, and arginine)-serine peptide was treated in wild-type or MARCHF6 KO A549 cells and then treated with a ferroptosis inducer, and Figure 46 shows the results of confirming the cell viability when the N-terminally acetylated alanine-X (0 amino acids; alanine, glycine, aspartate, valine, threonine, serine, glutamate, proline, lysine, methionine, tyrosine, tryptophan, histidine, glutamine, asparagine, This is the result of confirming cell viability when wild-type or MARCHF6 KO A549 cells were treated with peptides (cysteine / cysteine, isoleucine, leucine, phenylalanine, and arginine) and then treated with a ferroptosis inducer.

[0078] Hereinafter, the present invention will be described in more detail.

[0079] The present invention provides a C5 domain derived from MARCHF6 protein that specifically recognizes a degron (Ac / N-degron) formed by N-terminal acetylation of a target protein.

[0080] MARCHF6 (Membrane-Associated RING-CH Finger protein 6) is a membrane-associated E3 ubiquitin ligase located in the endoplasmic reticulum membrane, has multiple transmembrane structures, and cooperates with other proteins (E1, E2) to attach ubiquitin to specific substrates and induce their degradation by the proteasome.

[0081] In the present invention, it was confirmed that among the various domains of MARCHF6, the C5 domain can specifically recognize a degron (Ac / N-degron) formed by N-terminal acetylation of the protein.

[0082] In the present invention, the C5 domain is characterized by including an amino acid sequence from positions 541 to 632 of the amino acid sequence of the human MARCHF6 protein. In addition, the C5 domain may include an amino acid sequence represented by the following SEQ ID NO: 1, but is not limited thereto.

[0083] EQGHTRQWLKGLVRAWTVTAGYLLDLHSYLLGDQEENENSANQQVNNNQHARNNNAIPVVGEGLHAAHQAILQQGGPVGFQPYRRPLNFPLR (SEQ ID NO: 1).

[0084] In the present invention, the Ac / N-degron may be recognized by the Ac / N domain within the C5 domain.

[0085] The Ac / N domain is characterized by including an amino acid sequence from positions 552 to 600 of the amino acid sequence of the human MARCHF6 protein. In addition, the Ac / N domain may include, but is not limited to, an amino acid sequence represented by SEQ ID NO: 2 below.

[0086] LVRAWTVTAGYLLDLHSYLLGDQEENENSANQQVNNNQHARNNNAIPVVG (SEQ ID NO: 2).

[0087] In addition, the Ac / N domain may have a helix-loop-helix structure, and in the present invention, the C5 domain is characterized by inducing ferroptosis by mediating the degradation of a target protein containing an Ac / N-degron.

[0088] In addition, when any one or more amino acids selected from the group consisting of the 566th and 571st amino acids of the C5 domain are modified, the C5 domain is characterized in that it does not recognize a degron (Ac / N-degron) formed by N-terminal acetylation of a target protein. The 566th amino acid of the C5 domain is L, and the 571st amino acid is also L, and Leu566 and Leu571 are evolutionarily conserved residues that form a hinge loop and play an important role in maintaining the overall shape of the tweezers structure.

[0089] It will be apparent to those skilled in the art that biological functional equivalents that may be included within the scope of the C5 domain and Ac / N domain of the present invention will be limited to those that include mutations in the amino acid sequence that exhibit biological activity equivalent to that of the C5 domain and Ac / N domain of the present invention.

[0090] These amino acid mutations are based on the relative similarity of the amino acid side-chain substituents, such as hydrophobicity, hydrophilicity, charge, and size. Analysis of the size, shape, and type of amino acid side-chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine are similar in size; and phenylalanine, tryptophan, and tyrosine are similar in shape. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0091] When introducing mutations, the hydrophobicity index of an amino acid can be considered. Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0092] The hydrophobic amino acid index is crucial for imparting interactive biological functions to peptides. It is well known that amino acids with similar hydrophobic indices must be substituted to retain similar biological activity. When introducing mutations based on hydrophobic indices, substitutions are preferably made between amino acids with a difference in hydrophobic indices of ±2, more preferably ±1, or even more preferably ±0.5.

[0093] Meanwhile, it is also well known that substitutions between amino acids having similar hydrophilicity values ​​result in peptides with equivalent biological activity. In the art, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); Tryptophan (-3.4).

[0094] When introducing mutations with reference to hydrophilicity values, substitutions are made between amino acids that exhibit a difference in hydrophilicity values ​​of preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

[0095] Amino acid exchanges in peptides that do not alter the overall activity of the molecule are known in the art, the most common being exchanges between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.

[0096] Considering the mutations having the above-described biological equivalent activity, the C5 domain and Ac / N domain of the present invention are interpreted to also include sequences that show substantial identity with the sequences listed in the sequence listing. The substantial identity means a sequence that shows at least 80% homology, more preferably 90% homology, when the C5 domain and Ac / N domain sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Any method known in the art can be used without limitation as an alignment method for sequence comparison.

[0097] The C5 domain and Ac / N domain according to the present invention can be prepared by chemical synthesis known in the art. Representative methods include, but are not necessarily limited to, liquid or solid phase synthesis, fragment condensation, and F-MOC (9-fluorenylmethoxycarbonyl) or T-BOC (tert-butyloxycarbonyl) chemistry. In addition, the C5 domain and Ac / N domain of the present invention can be prepared by genetic engineering methods. First, a DNA sequence encoding the C5 domain and Ac / N domain is constructed according to a conventional method. The DNA sequence can be constructed by PCR amplification using appropriate primers. Alternatively, the DNA sequence can be synthesized by a standard method known in the art, for example, using an automated DNA synthesizer (e.g., one sold by Biosearch or Applied Biosystems). The produced DNA sequence is inserted into a vector containing one or more expression control sequences (e.g., promoter, enhancer, etc.) that are operatively linked to the DNA sequence and control the expression of the DNA sequence, and a host cell is transformed with the recombinant expression vector formed thereby. The produced transformant is cultured in an appropriate medium and conditions to allow the DNA sequence to be expressed, and a substantially pure peptide encoded by the DNA sequence is recovered from the culture. The recovery can be performed using a method known in the art (e.g., chromatography). The term "substantially pure peptide" as used herein means that the C5 domain and Ac / N domain according to the present invention do not substantially contain any other protein derived from the host.In addition, the C5 domain and Ac / N domain according to the present invention may be modified at the N-terminus or C-terminus or protected with various organic groups in order to protect against protein cleavage enzymes in the body and increase stability. That is, the C-terminus of the C5 domain and the Ac / N domain may be modified with a hydroxyl group (-OH) or an amino group (-NH2) without any particular limitation as long as it can be modified to increase stability. In addition, the N-terminus of the C5 domain and the Ac / N domain may be modified with a group selected from the group consisting of an acetyl group, a fluorenyl methoxycarbonyl (Fmoc) group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG) without any particular limitation as long as it can be modified to increase stability.

[0098] The present invention also provides a pharmaceutical composition for preventing or treating a disease by inhibition of ferroptosis, comprising an activator of the C5 domain or an activator of the Ac / N domain derived from the MARCHF6 protein.

[0099] In the pharmaceutical composition of the present invention, the activator of the C5 domain or the activator of the Ac / N domain is characterized in that it enhances Ac / N-degron recognition of the C5 domain through a mechanism that mediates at least one selected from the group consisting of enhancing substrate recognition ability of the domain, stabilizing the structure of the substrate binding site, and enhancing functional interaction of the domain.

[0100] Therefore, the pharmaceutical composition is characterized in that it can induce degradation of a target protein by promoting the ubiquitination activity of MARCHF6 endogenous to the target protein containing a degron (Ac / N-degron) formed by N-terminal acetylation.

[0101] Examples of the above activator include, but are not limited to, small-molecule compounds that bind to the three-dimensional structure or substrate binding pocket of the C5 domain or Ac / N domain and enhance recognition ability for Ac / N-degron substrates (small-molecule activator), short peptides that interact with adjacent regions of the C5 domain or Ac / N domain to favorably control the spatial arrangement of the core recognition domain, proteins / accessory proteins that cooperate with the C5 domain or Ac / N domain of MARCHF6 to enhance substrate accessibility, small molecules or proteins that stabilize the folding or membrane positioning of the C5 domain to enhance functional activity (structural stabilization substance (Folding chaperone)), enzymes or catalyst derivatives that promote functional transition by inducing phosphorylation / acetylation, etc., at specific residues of the C5 domain or Ac / N domain (post-translational activator), etc.

[0102] The term "prevention" in the present invention means any act of inhibiting or delaying the onset of a disease by inhibiting ferroptosis by administering a composition according to the present invention.

[0103] The term "treatment" in the present invention means any act of improving or beneficially changing the symptoms of the disease by administering the pharmaceutical composition.

[0104] The "pharmaceutical composition" of the present invention is intended for use in the prevention and / or treatment of diseases caused by inhibition of ferroptosis. For prophylactic use, the pharmaceutical composition of the present invention is administered to a subject suspected of having or at risk of developing a disease, disorder, or condition described herein. That is, it can be administered to a subject at risk of developing a disease caused by inhibition of ferroptosis and its related diseases. For therapeutic use, the pharmaceutical composition of the present invention is administered to a subject, such as a patient already suffering from a disease described herein, in an amount sufficient to treat or at least partially arrest the symptoms of a disease, disorder, or condition described herein. An amount effective for such use will depend on the severity and course of the disease, disorder, or condition, previous treatment, the subject's health status and responsiveness to the drug, and the judgment of a physician or veterinarian.

[0105] In the present invention, the disease caused by ferroptosis inhibition may be at least one selected from the group consisting of cancer, drug-resistant cancer, and fibrotic disease, but is not limited thereto. Induction of ferroptosis is being studied as a potential anticancer strategy for selectively removing cancer cells, and ferroptosis provides a new death pathway for cancer cells resistant to existing anticancer drugs. Fibrosis is induced by abnormal cell accumulation and inflammation, and thus inducing ferroptosis in fibrotic cells is attracting attention as an antifibrotic strategy.

[0106] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined according to factors including the type and severity of the individual, age, sex, activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field. For example, the activator of the C5 domain or the activator of the Ac / N domain can be administered at a dose of 0.01 to 5000 mg / kg per day, specifically 10 to 1000 mg / kg, and the administration can be administered once a day or in several divided doses.

[0107] The above pharmaceutical composition can be administered as an individual treatment or in combination with other treatments, and can be administered sequentially or simultaneously with conventional treatments. It can be administered singly or in multiple doses. Taking all of the above factors into account, it is important to administer the amount that achieves maximum effect with the minimum amount possible without causing side effects, a decision that can be readily made by those skilled in the art.

[0108] In addition, the pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage may vary depending on the patient's condition and weight, the degree of the disease, the drug form, the route of administration, and the time, but may be appropriately selected by a person skilled in the art.

[0109] The term "administration" in the present invention refers to the act of introducing an activator of the C5 domain or an activator of the Ac / N domain into a subject by an appropriate method. The term "subject" in the present invention refers to any animal, including rats, mice, and livestock, including humans, that has developed or may develop a disease caused by inhibition of ferroptosis. A specific example may be a mammal, including humans.

[0110] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions, and the carrier may comprise a non-naturally occurring carrier. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0111] In addition, the pharmaceutical composition may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, oral solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizers, and suppositories, and may be various oral or parenteral dosage forms. When formulated, it may be prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0112] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives.

[0113] Formulations for parenteral administration may be in the form of injections, infusions, sprays, liquids, or patches. These formulations include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cocoa butter, laurin butter, and glycerogelatin.

[0114] The present invention may preferably be in the form of an injection such as an injectable ampoule, an injectable such as an infusion bag, and a spray such as an aerosol preparation. The injectable ampoule may be mixed and prepared with an injection solution immediately before use, and the injection solution may be physiological saline solution, glucose, Ringer's solution, etc. In addition, the infusion bag may be made of polyvinyl chloride or polyethylene. In the present invention, administration means providing a predetermined composition of the present invention to a subject by any suitable method.

[0115] Meanwhile, the present invention can be provided as a protein degradation complex comprising a MARCHF6 protein-derived C5 domain; a linker, and a substance acting on a target cell.

[0116] The substance acting on the target cells may be, but is not limited to, an anticancer agent, a cytotoxic substance, etc.

[0117] The above complex can be used to simultaneously induce protein degradation and drug delivery effects by recognizing a protein containing an Ac / N-degron, inducing ubiquitination of the target protein, and delivering the bound substance to the target cell through the linker.

[0118] In addition, the present invention provides a pharmaceutical product for improving a disease by inhibiting ferroptosis, comprising an activator of the C5 domain or an activator of the Ac / N domain derived from the MARCHF6 protein.

[0119] In the present invention, the above "quasi-drug" means a fiber, rubber product or similar product used for the purpose of treating, alleviating, managing or preventing a disease of humans or animals; a product that has a weak effect on the human body or does not directly affect the human body, is not an apparatus or machine and similar product; and a product corresponding to one of the preparations used for sterilization, insecticide and similar purposes for preventing infection. It means a product used for the purpose of diagnosing, treating, alleviating, managing or preventing a disease of humans or animals, excluding products that are not apparatuses, machines or devices; and products used for the purpose of exerting a pharmacological effect on the structure and function of humans or animals, excluding products that are not apparatuses, machines or devices; and also includes external skin preparations and personal hygiene products.

[0120] When the activator of the C5 domain or the activator of the Ac / N domain of the present invention is included in an over-the-counter drug for the purpose of preventing or improving a disease by inhibiting ferroptosis, the activator of the C5 domain or the activator of the Ac / N domain may be used as is or together with other over-the-counter drug ingredients, and may be used appropriately according to a conventional method. The mixing amount of the active ingredients may be appropriately determined depending on the intended use.

[0121] The over-the-counter drug of the present invention is not particularly limited thereto, but may be manufactured and used in the form of, for example, an injection, infusion, spray, liquid, or patch, a cream, lotion, aerosol, gel, or pack.

[0122] In the case of creams, ointments, gels or packs, bases such as white petrolatum, yellow petrolatum, lanolin, bleached beeswax, cetanol, stearyl alcohol, stearic acid, hydrogenated oils, gelling hydrocarbons, polyethylene glycol, liquid paraffin, squalane, etc.; solvents and solubilizers such as oleic acid, isopropyl myristate, glycerin triisooctanoate, crotamiton, diethyl sebacate, diisopropyl adipate, hexyl laurate, fatty acids, fatty acid esters, aliphatic alcohols, vegetable oils, etc.; antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, etc.; preservatives such as parahydroxybenzoate esters, etc.; moisturizers such as glycerin, propylene glycol, sodium hyaluronate, etc. Surfactants such as polyoxyethylene derivatives, glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, and lecithin; thickeners such as carboxyvinyl polymers, xanthan gum, carboxymethylcellulose, carboxymethylcellulose sodium salts, hydroxypropylcellulose, and hydroxypropylmethylcellulose.

[0123] In the case of aerosols, bases such as white petrolatum, yellow petrolatum, lanolin, bleached beeswax, cetanol, stearyl alcohol, stearic acid, hydrogenated oils, gelling hydrocarbons, polyethylene glycol, liquid paraffin, and squalane used in the preparation of ointments, creams, gels, suspensions, emulsions, solutions, and lotions; solvents and solubilizing agents such as oleic acid, isopropyl myristate, diisopropyl adipate, isopropyl sebacate, glycerin triisooctanoate, crotamiton, diethyl sebacate, hexyl laurate, fatty acids, fatty acid esters, aliphatic alcohols, and vegetable oils; antioxidants such as tocopherol derivatives, L-ascorbic acid, dibutylhydroxytoluene, and butylhydroxyanisole; preservatives such as parahydroxybenzoic acid esters; Humectants such as glycerin, propylene glycol, and sodium hyaluronate; surfactants such as polyoxyethylene derivatives, glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, and lecithin; thickeners such as carboxyvinyl polymers, xanthan gum, carboxymethylcellulose, carboxymethylcellulose sodium salts, hydroxypropylcellulose, and hydroxypropylmethylcellulose; In addition, various stabilizers, buffers, coagulants, suspending agents, emulsifiers, fragrances, preservatives, solubilizers, and other suitable additives can be blended. In addition, stabilizers, preservatives, absorption promoters, pH adjusters, and other suitable additives can be blended as needed.

[0124] For the pharmaceutical product of the present invention, a more specific description is the same as the description for the above pharmaceutical composition.

[0125] In addition, the present invention provides an anticancer adjuvant comprising an activator of the C5 domain or the Ac / N domain derived from the MARCHF6 protein.

[0126] In the present invention, the “anticancer adjuvant” refers to a preparation that can improve, enhance or increase the anticancer effect of an anticancer agent.

[0127] In the present invention, the anticancer adjuvant can be used as an anticancer agent or an anticancer adjuvant depending on the treatment concentration, and can enhance the sensitivity of anticancer agents. In the present invention, the anticancer adjuvant can be administered in combination with a known compound that has the effect of preventing, improving, or treating cancer.

[0128] The above known compound may be at least one anticancer agent selected from the group consisting of 5-FU (5-fluorouracil), Doxorubicin, Oxaliplatin, Irinotecan, Carboplatin, Paclitaxel, Gemcitabine, and Bortezomib, but is not limited thereto.

[0129] In addition, the present invention provides a gene therapy composition for increasing intracellular ferroptosis sensitivity, comprising a vector expressing a C5 domain or an Ac / N domain derived from the MARCHF6 protein.

[0130] The above composition may contain one or more active ingredients capable of inducing ferroptosis, and the above composition may be manufactured by additionally containing one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers may include saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and mixtures of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets, and target organ-specific antibodies or other ligands may be combined with the above carriers to specifically act on target organs. Furthermore, it can be formulated preferably according to the disease or ingredient using an appropriate method in the relevant technical field.

[0131] The composition can be delivered into the body by injection via routes such as intravenous, intraperitoneal, intramuscular, subcutaneous, intradermal, nasal, mucosal, inhalation, and oral. The dosage range varies depending on the subject's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. The daily dosage is about 0.01 to 100 mg / kg for the compound, preferably 0.5 to 10 mg / kg, and it is more preferable to administer once or several times a day in divided doses.

[0132] The present invention also provides a method for providing information necessary for diagnosing a functional abnormality or ferroptosis state of a C5 domain derived from MARCHF6 protein in a sample, comprising the step of measuring the presence or degradation of a protein containing an Ac / N-degron in a sample isolated from an object.

[0133] In addition, the present invention provides a method for screening a substance for regulating ferroptosis, comprising: 1) treating a sample containing a protein formed by N-terminal acetylation, separated from an organism, with a candidate substance for regulating ferroptosis; and 2) measuring a change in the activity of a MARCHF6 protein-derived C5 domain, a change in the activity of an Ac / N domain, or binding ability to an Ac / N-degron after treating with the candidate substance.

[0134] In the screening method of the present invention, 3) a step of determining the candidate substance as a ferroptosis inducer when the activity level of the C5 domain, the activity level of the Ac / N domain, or the binding ability to Ac / N-degron of step 2) is increased may be further included.

[0135] In addition, in the screening method of the present invention, 3) a step of determining the candidate substance as a ferroptosis inhibitor when the activity level of the C5 domain, the activity level of the Ac / N domain, or the binding ability to Ac / N-degron of step 2) is reduced may be further included.

[0136] In addition, the present invention provides a method for inducing degradation of a protein comprising a degron formed by N-terminal acetylation in a sample, the method comprising the step of treating a MARCHF6 protein-derived C5 domain, an Ac / N domain, a C5 domain analogue that maintains the function of recognizing an Ac / N-degron, an Ac / N domain analogue that maintains the function of recognizing an Ac / N-degron, or an activator thereof in a sample in vitro.

[0137] The present invention also provides a pharmaceutical composition for preventing or treating a disease caused by excessive ferroptosis, comprising a MARCHF6 protein-derived C5 domain, an Ac / N domain, an inhibitor of the MARCHF6 protein-derived C5 domain, or an inhibitor of the Ac / N domain.

[0138] The MARCHF6 protein-derived C5 domain or Ac / N domain exhibits competitive inhibition (decoy) with the endogenous MARCHF6 protein-derived C5 domain or Ac / N domain and Ac / N-degron, thereby blocking ubiquitination and degradation of a protein containing Ac / N-degron, thereby inhibiting ferroptosis.

[0139] In addition, the C5 domain inhibitor or Ac / N domain inhibitor is characterized by inhibiting Ac / N-degron recognition of the C5 domain through a mechanism that mediates at least one selected from the group consisting of inhibition of substrate recognition ability of the domain, structural change of the substrate binding site, and inhibition of functional interaction of the domain.

[0140] Therefore, the pharmaceutical composition of the present invention is characterized in that it can inhibit the degradation of a target protein by reducing the ubiquitination activity of MARCHF6 inherent to the target protein, which includes a degron (Ac / N-degron) formed by N-terminal acetylation.

[0141] Examples of the above inhibitors include, but are not limited to, small-molecule activators that directly bind to the substrate binding site or recognition pocket and prevent the approach of Ac / N-degron, decoy peptides that exhibit competitive binding with the domain by using peptides with a structure similar to Ac / N-degron, protein structure destabilizing compounds (chaotropic agent-like compounds) such as urea, guanidinium chloride, perchlorate, and thiocyanate that inhibit the folding of the C5 domain or Ac / N domain of MARCHF6 or collapse the functional stereostructure by inducing partial denaturation, or gene expression inhibitors such as siRNA, shRNA, and antisense oligonucleotides that inhibit the expression of the C5 domain or Ac / N domain itself.

[0142] In addition, diseases caused by excessive ferroptosis may be one or more selected from the group consisting of, but not limited to, neurodegenerative diseases, cardiovascular diseases, acute kidney injury, chronic kidney injury, liver injury, lung injury, pulmonary inflammatory diseases, and diabetic neuropathy. Excessive ferroptosis contributes to the death of nerve cells, and increased ferroptosis activity has been reported in Alzheimer's disease, Parkinson's disease, etc. In addition, lipid ROS and iron imbalance are observed in the process of cardiomyocyte damage and heart failure, which can induce ferroptosis. Renal tubular cells are sensitive to iron-dependent damage, and it has been reported that inhibition of ferroptosis alleviates tissue damage in acute kidney disease (AKI) and chronic kidney disease (CKD). Ferroptosis is a major pathway of hepatocyte death. Alveolar epithelial cells undergo ferroptosis in response to high oxygen concentrations, toxic substances, and infections. A link between ferroptosis and acute lung injury (ALI), pulmonary fibrosis, and chronic obstructive pulmonary disease (COPD) has been reported. Furthermore, hyperglycemic environments have been reported to increase lipid peroxidation in peripheral nerves due to oxidative stress and disruption of iron homeostasis, thereby increasing the incidence of ferroptosis.

[0143] Excessive ferroptosis has been reported to contribute to various tissue damages and the progression of chronic diseases, and inhibition of Ac / N-degron recognition by the C5 domain or Ac / N domain can suppress excessive ferroptosis.

[0144] The description of the pharmaceutical composition for preventing or treating a disease caused by excessive ferroptosis of the present invention is the same as the description of the pharmaceutical composition for preventing or treating a disease caused by inhibition of ferroptosis described above.

[0145] In addition, the present invention provides a pharmaceutical product for improving a disease caused by excessive ferroptosis, comprising a MARCHF6 protein-derived C5 domain, an Ac / N domain, an inhibitor of the MARCHF6 protein-derived C5 domain, or an inhibitor of the Ac / N domain.

[0146] The description of the quasi-drug for improving a disease caused by excessive ferroptosis of the present invention is the same as the description of the quasi-drug for improving a disease caused by inhibition of ferroptosis described above.

[0147] In addition, the present invention provides a peptide in which at least one amino acid in the amino acid sequence of the C5 domain derived from human MARCHF6 protein is modified, wherein the peptide has lost the binding function to a degron (Ac / N-degron) formed by N-terminal acetylation of the protein.

[0148] The above peptide may be a peptide in which at least one amino acid selected from the group consisting of the 566th and 571st amino acids of the C5 domain is modified, and the 566th amino acid of the C5 domain is L and the 571st amino acid is L.

[0149] Preferably, at least one amino acid selected from the group consisting of positions 566 and 571 of the C5 domain may be substituted with alanine.

[0150] When the 566th amino acid of the C5 domain derived from human MARCHF6 protein is substituted with alanine, it may include an amino acid sequence represented by SEQ ID NO: 3 below. Based on the amino acid sequence represented by SEQ ID NO: 3 of the present invention, the 566th amino acid is the 26th amino acid. In addition, when the 571st amino acid of the C5 domain derived from human MARCHF6 protein is substituted with alanine, it may include an amino acid sequence represented by SEQ ID NO: 4 below. Based on the amino acid sequence represented by SEQ ID NO: 4 of the present invention, the 571st amino acid is the 31st amino acid.

[0151] EQGHTRQWLKGLVRAWTVTAGYLLDAHSYLLGDQEENENSANQQVNNNQHARNNNAIPVVGEGLHAAHQAILQQGGPVGFQPYRRPLNFPLR (SEQ ID NO: 3).

[0152] EQGHTRQWLKGLVRAWTVTAGYLLDLHSYLAGDQEENENSANQQVNNNQHARNNNAIPVVGEGLHAAHQAILQQGGPVGFQPYRRPLNFPLR (SEQ ID NO: 4)

[0153] The present invention also provides an N-terminally acetylated peptide represented by the following general formula, which induces or inhibits ferroptosis in a MARCHF6-dependent manner:

[0154] [General Formula 1]

[0155] Ac-X1-X₂-R

[0156] In the above general formula 1, Ac means an N-terminal acetyl group,

[0157] When the above X1 is Ala, X2 is Ser, Gly, Asp, Val, Thr, Pro, Gln, Tyr, Ala, Leu or Ile,

[0158] When the above X₂ is Ser, X1 is Ala, Gly, Asp, Val, Thr, Phe, Ser, Pro or Leu,

[0159] The above R is -OH or -NH₂.

[0160] In the present invention, the peptide may be Ac-AS-NH2, Ac-GS, Ac-SS, Ac-TS, Ac-VS, Ac-LS, Ac-AA, Ac-AG, Ac-AI, Ac-AL, Ac-PS, Ac-DS, Ac-AP or Ac-AD.

[0161] The above Ac-AS-NH2, Ac-GS, Ac-SS, Ac-TS, Ac-VS, Ac-LS, Ac-AA, Ac-AG, Ac-AI or Ac-AL peptide may inhibit ferroptosis.

[0162] In addition, the above Ac-PS, Ac-DS, Ac-AP or Ac-AD peptide may induce ferroptosis.

[0163] According to an embodiment of the present invention, the Ac-AS-NH2 peptide decreased the expression of one or more pro-ferroptotic proteins selected from the group consisting of SM, p53, and ACSL4, and increased the expression of one or more anti-ferroptotic proteins selected from the group consisting of SLC7A11, GPX4, and NRF2.

[0164] In addition, the above Ac-GS, Ac-SS, Ac-TS, Ac-VS, Ac-LS, Ac-AA, Ac-AG, Ac-AI or Ac-AL peptides increased the survival rate of cells treated with a ferroptosis inducer.

[0165] In contrast, the Ac-PS, Ac-DS, Ac-AP or Ac-AD peptides further reduced the viability of cells treated with ferroptosis inducers.

[0166] It will be apparent to those skilled in the art that biologically functional equivalents that may be included within the scope of the peptides of the present invention will be limited to those that include variations in the amino acid sequence that exhibit equivalent biological activity to the peptides of the present invention.

[0167] These amino acid mutations are based on the relative similarity of the amino acid side-chain substituents, such as hydrophobicity, hydrophilicity, charge, and size. Analysis of the size, shape, and type of amino acid side-chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine are similar in size; and phenylalanine, tryptophan, and tyrosine are similar in shape. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0168] When introducing mutations, the hydrophobicity index of an amino acid can be considered. Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0169] The hydrophobic amino acid index is crucial for imparting interactive biological functions to peptides. It is well known that amino acids with similar hydrophobic indices must be substituted to retain similar biological activity. When introducing mutations based on hydrophobic indices, substitutions are preferably made between amino acids with a difference in hydrophobic indices of ±2, more preferably ±1, or even more preferably ±0.5.

[0170] Meanwhile, it is also well known that substitutions between amino acids having similar hydrophilicity values ​​result in peptides with equivalent biological activity. In the art, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); Tryptophan (-3.4).

[0171] When introducing mutations with reference to hydrophilicity values, substitutions are made between amino acids that exhibit a difference in hydrophilicity values ​​of preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

[0172] Amino acid exchanges in peptides that do not alter the overall activity of the molecule are known in the art, the most common being exchanges between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.

[0173] Considering the mutations having biologically equivalent activity described above, the peptides of the present invention are interpreted to also include sequences that exhibit substantial identity with the sequences listed in the sequence listing. The substantial identity refers to a sequence that exhibits at least 80% homology, more preferably 90% homology, when the peptide sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequences are analyzed using an algorithm commonly used in the art. Any method known in the art for sequence comparison can be used without limitation.

[0174] The peptide according to the present invention can be prepared by chemical synthesis known in the art. Representative methods include, but are not necessarily limited to, liquid or solid phase synthesis, fragment condensation, and F-MOC (9-fluorenylmethoxycarbonyl) or T-BOC (tert-butyloxycarbonyl) chemistry. In addition, the peptide of the present invention can be prepared by genetic engineering methods. First, a DNA sequence encoding the peptide is constructed according to a conventional method. The DNA sequence can be constructed by PCR amplification using appropriate primers. Alternatively, the DNA sequence can be synthesized by a standard method known in the art, for example, using an automated DNA synthesizer (e.g., sold by Biosearch or Applied Biosystems). The constructed DNA sequence is inserted into a vector containing one or more expression control sequences (e.g., promoter, enhancer, etc.) that are operatively linked to the DNA sequence and control the expression of the DNA sequence, and a host cell is transformed with the recombinant expression vector formed thereby. The resulting transformant is cultured under appropriate media and conditions to allow the expression of the DNA sequence, and a substantially pure peptide encoded by the DNA sequence is recovered from the culture. The recovery can be performed using a method known in the art (e.g., chromatography). The term "substantially pure peptide" as used herein means that the C5 domain and Ac / N domain according to the present invention do not substantially contain any other proteins derived from the host. In addition, the peptide according to the present invention may be in a form in which the N-terminus or C-terminus is modified or protected with various organic groups in order to protect it from protein cleavage enzymes in the body and increase its stability.That is, the C-terminus of the peptide may be modified with a hydroxyl group (-OH) or an amino group (-NH2) without any particular limitation, as long as it is in a form that can be modified to increase stability. In addition, the N-terminus of the peptide may be modified with a group selected from the group consisting of an acetyl group, a fluorenyl methoxycarbonyl (Fmoc) group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG), without any particular limitation, as long as it is in a form that can be modified to increase stability.

[0175] In addition, the present invention provides a method for producing a peptide having lost the binding function to a degron (Ac / N-degron) formed by N-terminal acetylation of a protein, comprising the step of substituting any one or more amino acids selected from the group consisting of positions 566 and 571 of the amino acid sequence of the C5 domain derived from human MARCHF6 protein with alanine.

[0176] The present invention also provides a pharmaceutical composition for preventing or treating a disease caused by excessive ferroptosis, comprising an N-terminally acetylated peptide that induces or inhibits ferroptosis in a MARCHF6-dependent manner, represented by the following general formula:

[0177] [General Formula 1]

[0178] Ac-X1-X₂-R

[0179] In the above general formula 1, Ac means an N-terminal acetyl group,

[0180] When the above X1 is Ala, X₂ is Ser, Ala, Gly, Ile or Leu,

[0181] When the above X₂ is Ser, X₁ is Ala, Gly, Ser, Thr, Val or Leu,

[0182] The above R is -OH or -NH₂.

[0183] In addition, the present invention provides a pharmaceutical composition for improving a disease caused by excessive ferroptosis, which comprises an N-terminal acetylated peptide that induces or inhibits ferroptosis in a MARCHF6-dependent manner, and is represented by the following general formula:

[0184] [General Formula 1]

[0185] Ac-X1-X₂-R

[0186] In the above general formula 1, Ac means an N-terminal acetyl group,

[0187] When the above X1 is Ala, X₂ is Ser, Ala, Gly, Ile or Leu,

[0188] When the above X₂ is Ser, X₁ is Ala, Gly, Ser, Thr, Val or Leu,

[0189] The above R is -OH or -NH₂.

[0190] The above peptide may be an Ac-AS-NH2, Ac-GS, Ac-SS, Ac-TS, Ac-VS, Ac-LS, Ac-AA, Ac-AG, Ac-AI or Ac-AL peptide.

[0191] The present invention also provides a pharmaceutical composition for preventing or treating a disease by inhibition of ferroptosis, comprising an N-terminally acetylated peptide that induces or inhibits ferroptosis in a MARCHF6-dependent manner, represented by the following general formula:

[0192] [General Formula 1]

[0193] Ac-X1-X₂-R

[0194] In the above general formula 1, Ac means an N-terminal acetyl group,

[0195] When the above X1 is Ala, X₂ is Pro or Asp,

[0196] When the above X₂ is Ser, X₁ is Pro or Asp,

[0197] The above R is -OH or -NH₂.

[0198] In addition, the present invention provides a pharmaceutical composition for improving a disease by inhibiting ferroptosis, which comprises an N-terminal acetylated peptide that induces or inhibits ferroptosis in a MARCHF6-dependent manner, and is represented by the following general formula:

[0199] [General Formula 1]

[0200] Ac-X1-X₂-R

[0201] In the above general formula 1, Ac means an N-terminal acetyl group,

[0202] When the above X1 is Ala, X₂ is Pro or Asp,

[0203] When the above X₂ is Ser, X₁ is Pro or Asp,

[0204] The above R is -OH or -NH₂.

[0205] The above peptide may be an Ac-PS, Ac-DS, Ac-AP or Ac-AD peptide.

[0206] In the present invention, the specific description of the pharmaceutical composition or quasi-drug composition is the same as the description of the above pharmaceutical composition or quasi-drug composition.

[0207] The present invention also provides a method for preventing or treating a disease caused by inhibition of ferroptosis, comprising the step of administering to a subject an activator of the C5 domain or an activator of the Ac / N domain derived from the MARCHF6 protein.

[0208] In addition, the present invention provides a method for preventing or treating a disease caused by inhibition of ferroptosis, comprising the step of administering to a subject an N-terminally acetylated peptide that induces or inhibits ferroptosis in a MARCHF6-dependent manner, represented by the following general formula:

[0209] [General Formula 1]

[0210] Ac-X₁-X₂-R

[0211] In the above general formula 1, Ac means an N-terminal acetyl group,

[0212] When the above X₁ is Ala, X₂ is Pro or Asp,

[0213] When the above X₂ is Ser, X₁ is Pro or Asp,

[0214] The above R is -OH or -NH₂

[0215] In addition, the present invention provides a method for preventing or treating a disease caused by excessive ferroptosis, comprising the step of administering to a subject a MARCHF6 protein-derived C5 domain, an Ac / N domain, an inhibitor of the MARCHF6 protein-derived C5 domain, or an inhibitor of the Ac / N domain.

[0216] In addition, the present invention provides a method for preventing or treating a disease caused by excessive ferroptosis, comprising the step of administering to a subject an N-terminally acetylated peptide that is represented by the following general formula and induces or inhibits ferroptosis in a MARCHF6-dependent manner:

[0217] [General Formula 1]

[0218] Ac-X₁-X₂-R

[0219] In the above general formula 1, Ac means an N-terminal acetyl group,

[0220] When the above X₁ is Ala, X₂ is Ser, Ala, Gly, Ile or Leu,

[0221] When the above X₂ is Ser, X₁ is Ala, Gly, Ser, Thr, Val or Leu,

[0222] The above R is -OH or -NH₂.

[0223] In addition, the present invention provides a method for increasing intracellular ferroptosis sensitivity of a subject, comprising the step of administering to the subject a vector expressing a C5 domain or an Ac / N domain derived from a MARCHF6 protein.

[0224] The above-mentioned subject is preferably a mammal, including a human, and includes all patients who are being treated, have been treated, or need to be treated for a disease caused by ferroptosis inhibition, a disease caused by excessive ferroptosis, or a patient requiring an increase in the ferroptosis sensitivity within the cells of the subject. Patients who have undergone a surgical operation for a disease caused by ferroptosis inhibition, a disease caused by excessive ferroptosis, or an increase in the ferroptosis sensitivity within the cells of the subject may also be included.

[0225]

[0226] Throughout this specification, '%' used to indicate the concentration of a particular substance means (w / w)% for solid / solid, (w / v)% for solid / liquid, and (v / v)% for liquid / liquid, unless otherwise stated.

[0227] The terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the customs of the field to which the present invention pertains. Therefore, the definitions of these terms should be determined based on the contents of this specification as a whole. Throughout this specification, when a part is said to "include" a certain component, unless specifically stated otherwise, this does not mean that other components are excluded, but rather that other components may be included.

[0228] Unless otherwise defined, all technical terms used in this invention have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention. The contents of all publications cited as references herein are incorporated herein by reference.

[0229] Hereinafter, the present invention will be described in detail with reference to the attached drawings, using exemplary embodiments. However, the following exemplary embodiments are provided as illustrative examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0230]

[0231] Preparation Example 1. Reagents and Antibodies

[0232] Cycloheximide (CHX; 01810), dimethyl sulfoxide (DMSO; D2650), L-Glutathione (reduced; G4251), lysozyme (L1667), Nonidet P40 substitute (11754599001), phenylmethylsulphonyl fluoride (PMSF; 93482), and protease inhibitor cocktail tablets (4693132001) were purchased from Sigma-Aldrich. Isopropyl-beta-D-thiogalactoside (IPTG; I2481C5) was purchased from GoldBio. 1,4-Dithiothereitol (DTT; DB0058) was purchased from BioBasic. 2-Mercaptoethanol (161-0710), Clarity Western ECL substrate (1705062), and protein assay dye (5000006) were purchased from Bio-Rad. Bovine serum albumin (BSA; A9647) and Triton X-100 (X100) were purchased from Merck. Dithiobis(succinimidylpropionate) (DSP; 22585), Dynabeads protein G (10004D), Opti-MEM (3185070), Lipofectamine 2000 (11668019), and Pierce RIPA buffer (D3861) were purchased from Thermo Fisher Scientific.C11-BODIPY 581-591 (D3861), (1S,3R)-RSL3 (19288), and MG-132 (474790) were purchased from In vitrogen, Cayman Chemical, and CalbioChem, respectively. Pfu-X DNA polymerase was purchased from SolGent (Daejeon, Korea), and T4 DNA ligase was purchased from Enzynomics. Glutathione Sepharose 4B (17-0756-05), Dulbecco's modified Eagle's medium (DMEM; SH30243.01), and fetal bovine serum (FBS; SH30919.03) were purchased from Cytiva. Cell Titer-Glo 2.0 Cell Viability Assay (G9242) was purchased from Promega.

[0233] In addition, the following antibodies were used for immunoblotting and immunoprecipitation: Anti-flag (F3165 or F7425) and anti-ha (H9658 or HSP908) antibodies were purchased from Sigma-Aldrich. Anti-PLIN2 (ab108323) and anti-GPX4 (ab125066) antibodies were purchased from Abcam. Anti-p53 (SC-126), anti-ACSL4 (SC-365230), and anti-NRF2 (SC-365949) antibodies were purchased from Santa Cruz Biotechnology. Anti-SLC711A (12691S), anti-α-tubulin (T5168), and anti-RGS2 (AT3628a) antibodies were purchased from Cell Signaling Technology, Merck, and Abgent, respectively.

[0234]

[0235] Preparation Example 2. Yeast strain and human cell line

[0236] All yeast strains and cell lines used in the present invention are shown in Table 1 below. Yeast cells were cultured in nutrient-rich yeast peptone-dextrose medium (1% yeast extract, 2% peptone, 2% glucose) or synthetic complete (SC) medium (0.67% yeast nitrogen base containing ammonium sulfate, 2% glucose, and supplements necessary for auxotrophic yeast cell growth). Yeast cells were cultured in selective SC medium based on the plasmid carried by each strain, and DNA transformation was performed into Saccharomyces cerevisiae CHY712 cells using standard yeast techniques. Yeast cell growth (A600) was measured using a spectrophotometer (OPTIGEN POP; KLAB, Daejeon, Korea).

[0237] NameDescriptionSourceCatalog #CHY712doa10D ubr1Din NMY51(20)N / AHeLaHuman breast cancer cellATCCCCL-2MARCHF6-KO HeLaCRISPR / Cas9-based MARCHF6-KO HeLa(20)N / AA549Human lung adenocarcinomaATCCCCL-185MARCHF6-KO A549CRISPR / Cas9-based MARCHF6-KO A549(20)N / A

[0238] HeLa and A549 human cells were cultured in DMEM (SH30243.01; Cytiva) supplemented with 10% fetal bovine serum (FBS) (SH30919.03; HyClone), penicillin (100 units / ml), and streptomycin (100 mg / ml) (SV30010; HyClone). Cells were transfected with pcDNA3 or the appropriate plasmid using Lipofectamine 2000 (11668019; Thermo Fisher Scientific) for 24 h and then cultured at 37°C in a 5% CO2 incubator. Mycoplasma contamination was periodically tested using the e-Myco Valid kit (25239; iNtRON Bio, Korea).

[0239]

[0240] Experimental Example 1. Analysis Method

[0241] 1-1. Plasmid construction

[0242] The plasmids and primers used in this study are listed in Tables 2 and 3 below. pCH6045 (MARCHF6 3f I64A, Y65A, S66A) were constructed by overlapping polymerase chain reaction (PCR) using pCH879 as a template and primer pairs OCH6063 / OCH6064 and OCH5502 / OCH5508. The resulting PCR product was digested with EcoRI / KpnI and inserted into EcoRI / KpnI-digested pCH879.

[0243] pCH6046 (MARCHF6 3f D68A;M69A;P70A3f), pCH6047 (MARCHF6 3f L73A;P74A;I75A), pCH6048 (MARCHF6 3f L82A;V83A;T84A;S85A;I86A), pCH6049 (MARCHF6 3f G187A;H188A;H189A;Q190A), pCH6050 (MARCHF6 3fP194A;G196A), pCH6051 (MARCHF6 3f D227A;Q228A), pCH6052 (MARCHF6 3f E231A;E232A;E233A), pCH6053 (MARCHF6 3f E234A;D235A;N236A), pCH6054 (MARCHF6 3f Q255A;D256A;D257A;M258A;N259A), pCH6055 (MARCHF6 3f M287A;L288A;G289A), pCH6056 (MARCHF6 3f G367A;V368A;C369A), pCH6057 (MARCHF6 3f G449A;V450A;L451A), pCH6058 (MARCHF6 3f W452A;F453A;L454A), pCH6059 (MARCHF6 3f D459A;P460A;D461A), pCH6060 (MARCHF6 3f V465A;Q466A;E467A), pCH6061 (MARCHF6 3f 6W548A;L549A;K550A), pCH6062 (MARCHF6 3f W556A;T557A;V558A), pCH6063 (MARCHF6 3f S568A;Y569A;L570A;L571A;G572A), pCH6064 (MARCHF6 3f Q583A;Q584A;V585A), pCH6065(MARCHF6 3f F707A;Q708A;K709A), pCH6066 (MARCHF6 3f M716A;I717A;M718A), pCH6067 (MARCHF6 3f L802A;H803A;Y804A;I805A), pCH6068 (MARCHF6 3f R807A;K808A;L809A), and pCH6069(MARCHF6 3fK878A;N879A;D880A) were generated in a similar manner to pCH6045, but using the following primer pairs: OCH6065 / OCH6066 and OCH5502 / OCH5508, OCH6067 / OCH6068 and OCH5502 / OCH5508, OCH6069 / OCH6070 and OCH5502 / OCH5508, OCH6071 / OCH6072 and OCH5502 / OCH5508, OCH6073 / OCH6074 and OCH5502 / OCH5508, OCH6075 / OCH6076 and OCH5502 / OCH5508, OCH6077 / OCH6078 and OCH5502 / OCH5508. OCH6079 / OCH6080 and OCH5502 / OCH5508, OCH6081 / OCH6082 and OCH5502 / OCH5508, OCH6083 / OCH6084 and OCH5502 / OCH5508, OCH6085 / OCH6086 and OCH5502 / OCH5508, OCH6087 / OCH6088 and OCH5502 / OCH5508, OCH6089 / OCH6090 and OCH5502 / OCH5508, OCH6091 / OCH6092 and OCH5502 / OCH5508, OCH6093 / OCH6094 and OCH5502 / OCH5508, OCH6095 / OCH6096 and OCH5502 / OCH5508, OCH6097 / OCH6098 and OCH5502 / OCH5508, OCH6099 / OCH6100 and OCH5502 / OCH5508, OCH6101 / OCH6102 andOCH5502 / OCH5508, OCH6103 / OCH6104 and OCH5502 / OCH5508, OCH6105 / OCH6106 and OCH5502 / OCH5508, OCH6107 / OCH6108 and OCH5502 / OCH5508, OCH6109 / OCH6110 and OCH5502 / OCH5508, and OCH6111 / OCH6112 and OCH5502 / OCH5508.

[0244] pCH6201 (MARCHF6 3f W556A), pCH6202 (MARCHF6 3f L566A ), pCH6203 (MARCHF6 3f S568A ), pCH6204 (MARCHF6 3f L571A ), and pCH6205 (MARCHF6 3f P598A ) was amplified by overlapping PCR using pCH879 as a template using primer pairs OCH5502 / 5508 and OCH6362 / 6363, OCH5502 / 5508 and OCH6364 / 6365, OCH5502 / 5508 and OCH6366 / 6367, OCH5502 / 5508 and OCH6368 / 6369, and OCH5502 / 5508 and OCH6370 / 6371, respectively. The resulting PCR products were digested with EcoRI / KpnI and inserted into EcoRI / KpnI digested pCH879. pCH837(M-RGS2 ha ) and pCH6198(P-RGS2 ha ) was PCR amplified using pCH766 as a template and primer pairs OCH1220 / 1223 and OCH7127 / 7128, respectively. The resulting PCR product was digested with Spe I / Cla I and inserted into Spe I / Cla I digested pCH346.

[0245] pCH6106(MARCHF6 552-600 ), pCH6199(MARCHF6 541-579 ), and pCH6200(MARCHF6 580-632 ) was constructed by PCR using pCH879 as a template and primer pairs OCH6208 / OCH6209, OCH1776 / OCH6360, and OCH6361 / OCH1777, respectively. The resulting PCR products were digested with SfiI and inserted into SfiI-digested pCH1722.

[0246] pCH6037(MARCHF6 1~188 ), pCH6038(MARCHF6 1~376), pCH6039(MARCHF6 1~467 ), pCH6040(MARCHF6 1~565 ), pCH6041(MARCHF6 1~721 ), pCH6200(MARCHF6 1~810 ) were also prepared in a similar manner using different primer pairs OCH1767 / OCH6113, OCH1767 / OCH6114, OCH1767 / OCH6115, OCH1767 / OCH6116, OCH1767 / OCH6117, and OCH1767 / OCH6118.

[0247] pCH6087(MARCHF6 3f 541-632 ) was amplified by PCR using pCH879 as a template and primers OCH6154 / 6156. The generated PCR product was digested with EcoRI / XhoI and inserted into pCH4577, which was also digested with EcoRI / XhoI. pCH6728(ACSL4 ha ) and pCH6728(p53 ha ) was prepared by PCR using human cDNA extracted from HeLa cells as a template and the primer pairs OCH8624 / OCH8626 and OCH8573 / OCH8574. The resulting PCR product was digested with KpnI / XhoI and inserted into pCH61 digested with KpnI / XhoI. All final plasmids were verified by DNA sequencing. The plasmids and primer sets used in the present invention are shown in Tables 2 and 3 below.

[0248] PlasmidDescriptionpCH61pcDNA3.1 (+) with ha2pCH346pDL2-Alg5pCH766M-RGS2 ha in pcDNA3(+)pCH821P-RGS2 ha in pcDNA3(+)pCH837M-RGS2 in pDL2pCH879MARCHF6 3f in pcDNA3.1pCH1722MARCHF6 1-91 in pDHB1pCH1723MARCHF6164-283 in pDHB1pCH1724MARCHF6 358-376 in pDHB1pCH1725MARCHF6 443-480 in pDHB1pCH1726MARCHF6 541-632 in pDHB1pCH1727MARCHF6 700-721 in pDHB1pCH1728MARCHF6 786-815 in pDHB1pCH1729MARCHF6 870-910 in pDHB1pCH4677pGEX4T-3pCH6037MARCHF6 1-188 in pDHB1pCH6038MARCHF6 1-376 in pDHB1pCH6039MARCHF6 1-467 in pDHB1pCH6040MARCHF6 1-565 in pDHB1pCH6041MARCHF6 1-721 in pDHB1pCH6042MARCHF6 1-810 in pDHB1pCH6051MARCHF6 3f (C2-7 in pcDNA3.1pCH6052MARCHF6 3f (C2-8) in pcDNA3.1pCH6053MARCHF6 3f (C2-9) in pcDNA3.1pCH6054MARCHF6 3f (C2-10) in pcDNA3.1pCH6055MARCHF6 3f (C2-11) in pcDNA3.1pCH6056MARCHF6 3f (C3-12) in pcDNA3.1pCH6057MARCHF6 3f (C4-13) in pcDNA3.1pCH6058MARCHF6 3f (C4-14) in pcDNA3.1pCH6059MARCHF6 3f (C4-15) in pcDNA3.1pCH6060MARCHF6 3f (C4-16) in pcDNA3.1pCH6061MARCHF6 3f (C5-17) in pcDNA3.1pCH6062MARCHF6 3f(C5-18) in pcDNA3.1pCH6063MARCHF6 3f (C5-19) in pcDNA3.1pCH6064MARCHF6 3f (C5-20) in pcDNA3.1pCH6065MARCHF6 3f (C6-21) in pcDNA3.1pCH6066MARCHF6 3f (C6-22) in pcDNA3.1pCH6067MARCHF6 3f (C7-23) in pcDNA3.1pCH6068MARCHF6 3f (C7-24) in pcDNA3.1pCH6069MARCHF6 3f (C8-25) in pcDNA3.1pCH6087MARCHF6 3f 541-632 in pcDNA3.1pCH6106MARCHF6 552-600 in pDHB1pCH6198P-RGS2 in pDL2pCH6199MARCHF6 541-579 in pDHB1pCH6200MARCHF6 580-632 in pDHB1pCH6201MARCHF 3f W556A in pcDNA3.1pCH6202MARCHF 3F L566A in pcDNA3.1pCH6203MARCHF 3f L568A in pcDNA3.1pCH6204MARCHF 3f L571A in pcDNA3.1pCH6205MARCHF 3f L598A in pcDNA3.1pCH6567A-PLIN2 ha in pcDNA3.1(+)pCH6568P-PLIN2 ha in pcDNA3.1(+)pCH6728ACLS4 ha in pcDNA3.1(+)pCH7041p53 hain pcDNA3.1(+)pCH7059A-PLIN2 in pDL2pCH7060P-PLIN2 in pDL2

[0249]

[0250]

[0251] 1-2. Yeast split-Ub assay

[0252] For split-Ub analysis, the following bait vectors were used: pCH7059 (A-PLIN2), pCH7060 (P-PLIN2), pCH837 (M-RGS2), pCH6198 (P-RGS2) and prey vectors: pCH1722 (MARCHF6 1-91 ), pCH1723 (MARCHF6 164-283 ), pCH1724 (MARCHF6 358-376 ), pCH1725 (MARCHF6 443-480 ), pCH1726 (MARCHF6 541-632 ), pCH1727 (MARCHF6 700-721 ), pCH1728 (MARCHF6 786-810 ), pCH1729 (MARCHF6 870-910 ), pCH6037 (MARCHF6 1-188 ), pCH6038 (MARCHF6 1-376 ), pCH6039 (MARCHF6 1-476 ), pCH6040 (MARCHF6 1-565 ), pCH6041 (MARCHF6 1-721 ), pCH6042 (MARCHF6 1-810 ), pCH6106 (MARCHF6 552-600 ), pCH6199 (MARCHF6 541-579 ), pCH6200 (MARCHF6 580-632 ) were co-transformed into CHY712 cells.

[0253] The selected transformants were cultured in SC (-Leu / -Trp) medium and A 600 The cells were grown until the value reached approximately 1.0. Afterwards, the same amount of cells was taken from each strain, serially diluted 4-fold, and spotted onto SC(-Leu / -Trp) or SC(-Leu / -Trp / -His) medium, and cultured at 30°C for 3 days.

[0254]

[0255] 1-3. Lipid peroxidation analysis

[0256] Wild-type and MARCHF6-KO HeLa and A549 cells were seeded in 6-well culture plates for 24 h, treated with DMSO or 0.15 μM RSL3, and incubated at 37°C for 30 min. The medium was then replaced with medium containing 2 mM C11-BODIPY581 / 591 (D3861; Thermo Fisher Scientific). After an additional 30 min of incubation at 37°C, cells labeled with the fluorescent probe were collected by trypsinization into 15 ml Falcon tubes. The collected cells were washed three times with 1 ml of ice-cold phosphate-buffered saline (PBS) containing 1% BSA and then transferred to ice-cold round-bottom polystyrene tubes (352235; Corning, NY, USA). Lipid peroxidation levels in approximately 10,000 cells were measured using a FACSymphony A1 flow cytometer (BD Biosciences). This flow cytometer used a PE-CF594 filter (for measuring non-oxidized BODIPY-C11) and a fluorescein isothiocyanate (FITC) filter (for measuring oxidized BODIPY-C11). The resulting data were analyzed using FlowJo v10.10 (BD Biosciences).

[0257]

[0258] 1-4. Cell viability assay

[0259] The following plasmids were expressed in the wild-type and MARCHF6-deficient (KO) HeLa and A549 cells at the indicated amounts: pCH60 (empty vector), pCH879 (MARCHF6 3f ), pCH6204 (MARCHF6 3fL571A mutant), pCH6567 (A-PLIN2 ha ), pCH6268 (P-PLIN2 ha ), pCH766 (M-RGS2 ha ), pCH822 (P-RGS2 ha ) These cells were seeded at a density of 10,000 cells per well in Corning black flat-bottom 96-well culture plates (model number 3603).

[0260] After culturing for 24 h, cells were treated with DMSO or 0.15 μM RSL3. After another 24 h, cell viability was analyzed using the CellTiter-Glo 2.0 luminescence-based cell viability assay kit (G9241, Promega) and a GloMax Navigator microplate luminometer (Promega).

[0261]

[0262] 1-5. CHX-chase Analysis

[0263] MARCHF6-KO HeLa cells were seeded at 1 × 10 in 12-well cell culture plates containing DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin. 5Cells were seeded at a density of 10 cells / well. After 24 h of culture, the cells were transfected with the designated plasmids and cultured for another 24 h. The cells were treated with CHX to a final concentration of 0.1 mg / ml and harvested at the designated time points. The cultured cells were lysed on ice for 20 min using lysis buffer (89900; Thermo Fisher Scientific) containing a protease inhibitor cocktail (4693132001; Sigma-Aldrich). The supernatant was collected by centrifugation at 11,200 g for 20 min at 4°C, and the total protein concentration of the supernatant was measured using the Bradford assay (5000006; Bio-Rad). Equal amounts of total protein were analyzed by immunoblotting using anti-flag (1:1000), anti-ha (1:1000), or anti-tubulin (1:2000) antibodies. The intensities of immunoblotting bands were quantified using ImageJ software (https: / / imagej.nih.gov / ij / ) and normalized to the α-tubulin band. Quantification was performed using data from at least three independent CHX-chase experiments.

[0264]

[0265] 1-6. Purification of GST-C5

[0266] Escherichia coli BL21 (DE3) cells containing pCH6087 (GST-C5) were inoculated into 1000 mL of LB (Luria broth) medium containing ampicillin (final concentration 100 mg / mL) in 10 mL of overnight culture. The cells were incubated at 37°C for 1 h. 600This was cultured until the confluency reached approximately 0.7. Expression of GST-C5 was induced by treatment with 1 mM IPTG at 37°C for 2 h. IPTG-induced E. coli cells were harvested by centrifugation at 500 × g for 25 min at 4°C and stored at -80°C. The cell pellet was thawed and resuspended in STE buffer (0.1 M NaCl, 1 mM Na-EDTA, 10 mM Tris-HCl, pH 8.0) containing 1 mM DTT, 1 mM PMSF, 1 mg / ml chicken egg white lysozyme, and 1% Triton X-100. The cell suspension was incubated on ice for 20 min and then sonicated for 1 min three times at 1-min intervals. After centrifugation at 11,200 g for 20 min at 4°C, the supernatant (approximately 25 ml) was incubated with 1 ml of Glutathione Sepharose resin (17-0756-05; Cytiva) at 4°C for 2 h. The beads were washed twice with 25 ml of STE buffer. The GST-C5 protein was eluted with 1 ml of STE buffer containing 10 mM glutathione (GSH) and dialyzed overnight against storage buffer (10% glycerol, 0.15 M NaCl, 10 mM β-mercaptothion, 50 mM HEPES, pH 7.5).

[0267]

[0268] 1-7. Chemical crosslinking and co-IP (co-immunoprecipitation) analysis

[0269] HeLa and A549 cells were transiently transfected with the indicated plasmids and cultured for 36 hours. The cells were then treated with 10 μM MG-132 (474790; In vitrogen) for 4 hours. The cells were then washed twice with PBS and cross-linked with 0.5 mM DSP (22585; Thermo Fisher Scientific), an amino group-specific crosslinking reagent, for 2 hours on ice. The cross-linking reaction was stopped by quenching unreacted DSP with 10 mM Tris-HCl (pH 7.5) at 25°C for 15 minutes. The cross-linked cells were washed twice with 1x PBS, carefully scraped from the plates, and pelleted by low-speed centrifugation at 500 g for 3 minutes at 4°C. Pelleted cells were lysed in 0.8 ml lysis buffer [1% Nonidet P-40 (11754599001; Thermo Fisher Scientific), 0.15 M NaCl, 1 mM Na-EDTA, 50 mM Tris-HCl, pH 7.5] containing protease inhibitor cocktail (4693132001; Sigma-Aldrich) and incubated on ice for 20 min. The cell lysate was centrifuged at 11,200 g for 20 min at 4°C, and the supernatant was transferred to a cold tube, and total protein concentration was determined using the Bradford assay (5000006; Bio-Rad).

[0270] Cell extracts with a protein concentration of 20 mg / ml were diluted 10-fold with lysis buffer and then reacted with 1 μl of IgG Dynabeads G (10004D; Thermo Fisher Scientific) conjugated with anti-FLAG antibody (approximately 0.8 mg / ml) or anti-HA antibody (approximately 0.8 mg / ml) at 4°C for 6 hours.

[0271] The magnetic beads were then washed three times with washing buffer (0.2% Nonidet P-40, 137 mM NaCl, 2 mM Na-EDTA, 10% glycerol, 20 mM Tris-HCl, pH 7.5). The proteins bound to the beads were eluted by adding them to 45 μl of 2x SDS-PAGE sample buffer and reacting at 37°C for 20 minutes.

[0272] The eluted proteins were electrophoresed on Tris-glycine SDS-10% PAGE, and then immunoblot analysis was performed using anti-HA antibody (1:1000 dilution) or anti-FLAG antibody (1:1000 dilution).

[0273]

[0274] 1-8. Isothermal titration calorimetry (ITC) analysis

[0275] All calorimetric experiments were performed at 12°C using a Nano ITC (model: 601000.901, TA Instruments). The syringe contained 1 mM ligand, and the measuring cell contained 100 μM protein in buffer (5% glycerol, 0.15 M NaCl, 10 mM β-mercaptoethanol, 50 mM HEPES, pH 7.5).

[0276] The binding affinity of Ac-ASVAVD, Ac-MQSAMD, ASVAVD, and MQSAMD (Anygen, Gwangju, Korea) to GST-C5 was measured by adding 1 mM of each ligand dropwise to a cell containing 100 μM of GST-C5 with stirring at 200 rpm.

[0277] Raw titration data were integrated and fitted to a single-site binding model using NanoAnalyze v3.11.0 (TA Instruments). From this, the dissociation constant (Kd), enthalpy change (ΔH), and stoichiometry (N) were calculated.

[0278]

[0279] 1-9. Screening of evolutionarily conserved Ac / N domain residues across eukaryotic species

[0280] To identify functionally conserved residues in the Ac / N domain of MARCHF6 across eukaryotic species, the amino acid sequence of the human MARCHF6 Ac / N domain was aligned with those of mouse (Mus musculus), chicken (Gallus gallus), fish (Danio rerio), fruit fly (Drosophila melanogaster), frog (Xenopus laevis), Caenorhabditis elegans, plant (Arabidopsis thaliana), and yeast (S. cerevisiae) using Clustal Omega Sequence alignments.

[0281]

[0282] 1-10. AlphaFold structural prediction and Nt-acetyl peptide docking simulation

[0283] A three-dimensional structural model of the Ac / N domain was generated using AlphaFold3, and peptide docking simulations were performed using AutoDock Vina. The amino acid sequence of human MARCHF6 (UniProt accession: O60337) was downloaded and used to generate a three-dimensional structural model with AlphaFold3. The resulting structure was then refined to generate a putative Ac / N domain while maintaining a rigid helix-turn structure. Docking simulations were performed using the MARCHF6 541-592 structural model as the receptor and the N-terminally acetylated Ac-ASVAVD peptide generated from SMILES strings as the ligand. The docking grid box (40 × 40 × 40 Å) 3 ) was set to include the entire structure of the Ac / N domain model. The highest-ranking docking model showing Nt-acetylation-dependent binding was selected and visualized using Chimera.

[0284]

[0285] 1-11. Quantification and Statistical Analysis

[0286] Data are presented as the mean ± standard deviation (SD) from at least three independent biological experiments or samples. Pairwise comparisons were performed using a two-tailed t test, and multiple comparisons were analyzed using one-way or two-way analysis of variance (ANOVA) using Prism 9.5 (GraphPad). Statistical significance was set at p < 0.05.

[0287]

[0288] Example 1. Identification of Ac / N-degron recognition domains in MARCHF6 through systematic mutational screening.

[0289] Previous studies have shown that MARCHF6 specifically recognizes M-RGS2 and A-PLIN2, which are N-terminally acetylatable (starting with Met), but not P-RGS2 and P-PLIN2, which are N-terminally non-acetylatable (starting with Pro).

[0290] In this context, A-PLIN2, P-RGS2, and P-PLIN2 are proteins generated through Nt-Met cleavage by ribosome-associated Met-aminopeptidase through co-translational cleavage of [M]A-PLIN2, [M]P-RGS2, and [M]P-PLIN2 (parentheses indicate initial Met) encoded in nuclear DNA, respectively. This is because Met-aminopeptidase can efficiently cleave Nt-Met when the amino acid at the second position is alanine (Ala) or proline (Pro). On the other hand, when proline (Nt-Pro) is located at the N-terminus, it is rarely acetylated.

[0291] Given that Nt-acetylation occurs primarily in the cytoplasm, where most Nt-acetylases are located, we hypothesized that one or more of the eight cytosol-facing regions (C1-C8 regions, where Cn represents the order of the cytosol-facing regions from the N-terminus) of MARCHF6 may be involved in the specific recognition of Ac / N-degrons (Fig. 1). To verify this, we compared the cytosol-facing sequences of human and mouse MARCHF6 and their yeast ortholog Doa10, and mutated three to five evolutionarily conserved residues to alanine to generate 25 C-terminal triple-flag-tagged MARCHF6. 3f A mutant was produced (Fig. 1).

[0292] To systematically investigate the mutants with impaired Ac / N-degron recognition among the 25 Ala-stretch MARCHF6 mutants, chemical cross-linking experiments and reciprocal immunoprecipitation-immunoblot analysis were performed using HeLa cells, a human cervical cancer cell line. In this experiment, the HeLa cells were transfected with a series of Ala-stretch MARCHF6 mutants. 3f N-terminally acetylatable M-RGS2 with a mutant and C-terminally ha-tagged construct ha Cells co-expressing MARCHF6 were used. As a result, wild-type MARCHF6 3f and most Ala-stretch mutants are M-RGS2 ha and immunoprecipitated (Fig. 2).

[0293] To further evaluate the impact of specific mutations in the C5 domain on the recognition of MARCHF6 substrates bearing Ac / N-degrons, Nt-acetylatable M-RGS2 ha or Nt-acetylatable A-PLIN2 ha With wild-type, C5-17, C5-18, C5-19, or C5-20 MARCHF6 3f Additional chemical cross-linking experiments and reciprocal immunoprecipitation-immunoblot analysis were performed using HeLa cells co-expressing (Figs. 3 and 4).

[0294] As a result, wild type, C5-17, C5-20 MARCHF6 3f The mutation is M-RGS2 ha or A-PLIN2 ha , whereas the C5-18 and C5-19 mutants did not (Figs. 3 and 4).

[0295] Squalene monooxygenase (SM) is a well-known substrate of MARCHF6, and it is likely that SM interacts with SM through the Nt-transmembrane helices and the central cavity rather than the C5 domain. Wild-type SM (also called M-SM) begins with a Met-Leu sequence at the N-terminus and could therefore be a potential substrate for the NatC Nt-acetylase, but there is currently no direct evidence for N-terminal acetylation of M-SM.

[0296] To rule out this possibility in vivo, M-SM ha P-SM by replacing the N-terminal Met with Pro ha was created. M-RGS2 ha and A-PLIN2 ha Unlike , M-SM is capable of N-terminal acetylation ha and P-SM, which cannot be acetylated ha are all MARCHF6, regardless of whether they have C5-18 or C5-19 mutations. 3f interacted with (Fig. 5).

[0297] Therefore, these results confirmed that a limited region within the C5 domain contributes to the specific recognition of Ac / N-degron by MARCHF6.

[0298]

[0299] Example 2. Identification of the MARCHF6 Ac / N-degron Recognition Mechanism Centered on the C5 Domain

[0300] To identify the putative Ac / N-degron recognition region (Ac / N domain) of ER-transmembrane MARCHF6 using a method independent of chemical cross-linking and co-immunoprecipitation-immunoblotting analysis, a modified split-Ub assay was performed (Fig. 6).

[0301] Specifically, the C-terminal fragment of Ub (Cub) located upstream of the LexA-VP16 transcription factor was ligated to various C-terminally truncated variants of MARCHF6 (MARCHF6 1-X , where 1-X includes residues 1 to X) was directly fused to the peptide (Fig. 7).

[0302] Additionally, Nt-acetylatable M-RGS2 and A-PLIN2, or Nt-acetylatable P-RGS2 and P-PLIN2, were conjugated to the N-terminal fragment of mutated Ub (NubG) (Figures 7-9). Upon interaction, Cub and NubG form a nearly native ubiquitin structure, thereby enabling deubiquitylating enzymes (DUBs) to cleave polypeptides located downstream of Cub.

[0303] Therefore, the LexA-VP16 transcription factor, which was anchored to the endoplasmic reticulum membrane by the transmembrane domain of MARCHF6, is released, inducing the expression of LexA-responsive reporter genes (Fig. 6).

[0304] Split-Ub analysis results showed that wild-type MARCHF6 1-910 , C-terminally truncated MARCHF6 1-810 and MARCHF6 1-721 interacted with Nt-acetylatable M-RGS2 and A-PLIN2, but not with Nt-acetylatable P-RGS2 and P-PLIN2. In contrast, shorter truncated MARCHF6 1-188 , MARCHF6 1-376 , MARCHF6 1-467 , MARCHF6 1-565 did not interact with any substrate, regardless of whether the substrate was N-terminally acetylated (Figs. 7 to 9).

[0305] These results confirmed that the C5 region (residues 541-632 of MARCHF6) is involved in the process by which MARCHF6 specifically recognizes Ac / N-degrons.

[0306] LexA-VP16 transcription factor is a C-terminally truncated MARCHF6 1-X The mutants (where 1-X represents the residues from the first to the last residue of the C-terminally truncated MARCHF6) were directly bound to the Nt-segment (NubG) of the mutated Ub (Fig. 7). In addition, Nt-acetylatable M-RGS2 and A-PLIN2, or Nt-non-acetylatable P-RGS2 and P-PLIN2, were bound to the Nt-segment (NubG) of the mutated Ub (Figs. 7 to 9). Cub and NubG interact to form a nearly native Ub, allowing deubiquitylating enzymes (DUBs) to cleave the newly formed polypeptide downstream of Cub. Consequently, LexA-VP16 released from the endoplasmic reticulum membrane bound through the MARCHF6 transmembrane domain activated the expression of a LexA-responsive reporter (Fig. 6). Split-Ub analysis revealed that wild-type MARCHF6 1-910 and C-terminally truncated MARCHF6 1-810 , and MARCHF6 1-721 It was shown to interact with Nt-acetylatable M-RGS2 and A-PLIN2, but not with Nt-acetylatable P-RGS2 and P-PLIN2 (Fig. 2B to 2D). In contrast, a shorter, C-terminally truncated MARCHF6 1-188 , MARCHF6 1-376 , MARCHF6 1-467 , and MARCHF6 1-565 did not interact with either prey, regardless of Nt-acetylation status (Figs. 7-9). These results also suggest that specific recognition of Ac / N-degron by MARCHF6 is mediated by the C5 domain (MARCHF6 541-632 ) suggests that it is related to.

[0307]

[0308] Example 3. Confirmation that the Ac / N domain is located within the C5 region of MARCHF6.

[0309] To determine whether Ac / N-degron recognition is mediated solely by the C5 domain, without the cooperation of other cytoplasmic or transmembrane domains, we used a complementary split-Ub system that tethers individual cytoplasmic MARCHF6 fragments (Cn) to the endoplasmic reticulum (ER) membrane (Fig. 10). This split-Ub analysis revealed that the C5 domain interacted with the N-terminally acetylatable A-PLIN2 but not with the N-terminally non-acetylatable P-PLIN2. In contrast, the other cytoplasmically exposed domains (except the C8 domain) did not interact with these proteins, and the C8 domain interacted with both A-PLIN2 and P-PLIN2 (Fig. 11).

[0310] Additionally, chemical cross-linking and reciprocal immunoprecipitation-immunoblotting experiments in HeLa cells revealed that the C5 fragment (MARCHF 3f 541-632 ) is an N-terminal acetylation capable M-RGS2 ha and A-PLIN2 ha P-PLIN2 interacts with but is incapable of N-terminal acetylation ha and P-RGS2 ha did not interact with (Figs. 12 and 13).

[0311] In particular, ITC analysis results showed that purified GST-C5 had significantly lower binding affinity for non-acetylated MQSAMD (Kd = 137.5 μM) or ASVAVD (Kd = 789.6 μM) peptides than for N-terminally acetylated Ac-MQSAMD (Kd = approximately 6.2 μM) or Ac-ASVAVD (Kd = 3.5 μM) peptides (Fig. 14). Among these, the ASVAVD peptide was derived from the six-amino acid N-terminal sequence of PLIN2 after Nt-Met removal, and the MQSAMD peptide was derived from the five-amino acid N-terminal sequence of RGS2 with the addition of Asp for solubility. All amino acids of these peptides were represented by single-letter abbreviations [A: Ala, S: Ser, V: Val, D: Asp, Q: Gln].

[0312] To further elucidate the minimal Ac / N domain region required for MARCHF6 substrate recognition by the Ac / N-degron, cleaved C5 fragments (MARCHF6 541-579 , MARCHF6 552-600 , MARCHF6 580-632 ) was used to perform additional split-Ub analysis (Fig. 15). Among them, MARCHF6 552-600 Only A-PLIN2, which is capable of N-terminal acetylation, interacted with MARCHF6 541-579 and MARCHF6 580-632 did not interact (Fig. 15).

[0313] All these results are from MARCHF6 552-600 This suggests that the domain acts as the minimum functional unit of the Ac / N domain, allowing MARCHF6 to specifically recognize the N-terminal acetyl group of substrates bearing the Ac / N-degron.

[0314]

[0315] Example 4. Ac / N degron-specific targeting through Ac / N domain evolution of MARCHF6.

[0316] Sequence alignment of MARCHF6 orthologs across species revealed several amino acid residues conserved within the Ac / N domain from yeast to humans (Fig. 16). Some of these residues, including Trp556, Leu566, Ser568, Leu571, and Pro599, were mutated to alanine (Fig. 16). Chemical cross-linking and co-immunoprecipitation-immunoblotting experiments revealed that MARCH6 3f L566A and L571A mutants, which substituted Leu566 or Leu571 with alanine, were capable of N-terminal acetylation in HeLa cells. ha and A-PLIN2 ha In contrast, mutations in other residues did not show this binding loss effect (Figs. 17 and 18). Surprisingly, MARCHF6 3f L566A Wow MARCHF6 3f L571A Go RGS2 ha Wow PLIN2 ha Despite not being clearly bound to (Figs. 17 and 18), wild-type MARCHF6 3f Similarly, SM, a previously known ferroptosis-related substrate ha ,p53 ha , ACSL4 ha maintained a similar level of binding (Figs. 19 to 21). These results indicate that MARCHF6 binds SM through a substrate binding site other than the Ac / N domain. ha ,p53 ha , ACSL4 ha This suggests that there is a possibility of recognizing .

[0317] Recent molecular structural and functional studies have demonstrated that MARCHF6 and its yeast homolog, Doa10, can target diverse substrates through multiple substrate-binding sites. Furthermore, cycloheximide-chase (CHX-chase) experiments for protein degradation have revealed that Ac / Ndegron-inaccessible MARCHF6 3f L571A Expression of wild-type MARCHF6 did not induce degradation of endogenous RGS2 and PLIN2 in MARCHF6-KOHeLa cells, whereas expression of 3f The expression of showed that it accelerated their decomposition (Figs. 22 and 23).

[0318] These results indicate that MARCHF6 has evolved an Ac / N domain to selectively target substrate proteins bearing Ac / N-degrons for degradation.

[0319]

[0320] Example 5. Effect of RGS2 and PLIN2 on increasing ferroptosis resistance

[0321] To explore the functional and mechanistic link between the Ac / N-degron pathway and ferroptosis, C11-BODIPY, a lipid peroxidation sensor, was used. 581 / 591 HeLa or A549 (lung cancer) cells overexpressing only the empty vector using M-RGS2, an Ac / N-degron substrate ha Wow A-PLIN2 ha , or their non-Ac / N-degron counterpart, P-RGS2 ha Wow P-PLIN2 ha The lipid reactive oxygen species (ROS) levels were evaluated (Figs. 24 to 27; Figs. 30 and 31).

[0322] M-RGS2 ha Wow A-PLIN2 haHeLa and A549 cells overexpressing P-RGS2 showed a marked decrease in lipid ROS levels compared to cells expressing only the empty vector, even in the mock condition without ferroptosis inducer treatment (Figs. 30 and 31). Notably, P-RGS2 ha Wow P-PLIN2 ha In the case of M-RGS2, lipid ROS levels were reduced more significantly, as they are not degraded by MARCHF6. ha and A-PLIN2 ha This appears to be due to a higher steady-state protein concentration (Figs. 30 and 31; P-RGS2 ha and P-PLIN2 ha ) is resistant to MARCHF6-mediated degradation due to the absence of Ac / N-degron.

[0323] Meanwhile, treatment with RSL3, a ferroptosis inducer, resulted in an overall increase in lipid ROS levels in all experimental groups (compare Figs. 26 and 27 vs. Figs. 30 and 31). Nevertheless, M-RGS2 with Ac / N-degron ha Wow A-PLIN2 ha , and P-RGS2 which does not have this ha Wow P-PLIN2 ha In all cases of overexpression, lipid ROS levels were significantly reduced, especially in the case of a protein without Ac / N-degron (P-RGS2 ha , P-PLIN2 ha ) the reduction effect was more pronounced (Figs. 24 to 27; Figs. 30 and 31).

[0324] Also, M-RGS2 ha and A-PLIN2 ha Overexpression of P-RGS2 significantly increased the survival of HeLa and A549 cells upon RSL3 treatment (Figs. 28, 29; 32 and 33). ha and P-PLIN2 haIn this case, this effect was more pronounced (Figs. 28, 29; 32 and 33). This is also interpreted as being because P-proteins are more stable than M-proteins and thus exist in greater amounts within cells (Figs. 24 and 25).

[0325] Consistent with these results, PLIN2 is known to inhibit ferroptosis in several cancer cells. Specifically, PLIN2 appears to function by sequestering polyunsaturated fatty acids (PUFAs), which are vulnerable to lipid peroxidation, from membrane phospholipids and storing them in lipid droplets (LDs), thereby preventing ferroptosis. However, it has also been reported that, under certain circumstances, PLIN2 and LDs may actually increase ferroptosis sensitivity.

[0326] Unlike PLIN2, the direct role of RGS2 in ferroptosis has not yet been clearly elucidated, but its increased expression has been observed to inhibit ferroptosis (Figs. 26 and 28). Intracellular Ca 2+ Considering that over-influx and excessive ER stress can induce ferroptosis, RGS2 may be involved in Ca 2+ activation through inhibition of G-protein signaling. 2+ It is possible that ferroptosis could be inhibited by reducing influx or by alleviating ER stress through translational regulation.

[0327]

[0328] Example 6. Confirmation of the effect of increasing ferroptosis resistance by blocking Ac / N-degron recognition by MARCHF6.

[0329] Based on the observation that substrates with Ac / N-degrons inhibit ferroptosis, we investigated whether MARCHF6 specifically affects ferroptosis through the Ac / N-degron pathway. To this end, empty vector, wild-type MARCHF6 3f, or MARCHF6 mutants that do not recognize Ac / N-degron were expressed in MARCHF6 knockout (KO) HeLa and A549 cells (Fig. 34).

[0330] As expected, wild-type MARCHF6 3f When expressed, the steady-state concentrations of RGS2 and PLIN2, endogenous substrates with Ac / N-degron, were significantly reduced in MARCHF6-KO HeLa and A549 cells (Fig. 34), which was attributed to the promotion of degradation of RGS2 and PLIN2 through the Ac / N-degron pathway (Figs. 22, 23).

[0331] However, despite the reduced expression of these substrates, wild-type MARCHF6 3f Expression of decreased lipid ROS levels in MARCHF6-KO HeLa and A549 cells (Fig. 35) and showed an effect of increasing cell viability upon RSL3 treatment (Fig. 36).

[0332] It is noteworthy that this anti-ferroptosis effect was even stronger when a MARCHF6 mutant that does not recognize the Ac / N-degron was expressed (Figs. 35 and 36), and the mutant did not significantly affect the expression of RGS2 and PLIN2, whereas the wild-type MARCHF6 3f strongly suppressed their expression (Fig. 34).

[0333] In conclusion, our results suggest that inhibiting the degradation of Ac / N-degron substrates by MARCHF6 is a promising strategy to enhance ferroptosis resistance.

[0334]

[0335] Example 7. In silico modeling of Ac / N domains bound to Nt-terminal acetylated peptides.

[0336] Based on the identification of the Ac / N domain in MARCHF6, a three-dimensional complex model of the Ac / N domain and the N-terminally acetylated Ac-ASVAVD peptide (derived from the N-terminal six amino acids of A-PLIN2) was generated using AlphaFold3. The predicted Ac / N domain has a helix-loop-helix (tweezer-like) structure, in which Arg554 and Asn579 interact with the Nt-acetyl group and the first and third amide groups of the Ac-ASVAVD peptide, respectively ( Figure 37 ). In addition, Leu566 and Leu571 are evolutionarily conserved residues that form a hinge loop, which is important for maintaining the overall conformation of the tweezer structure ( Figure 37 ).

[0337] Recent structural, computational, and functional analyses of Doa10 / MARCHF6 have revealed a unique C-shaped (horseshoe-shaped) structure with a lipid-filled central cavity within the membrane (important for E3 ligase activity). This structure consists of an N-terminal RING domain, a rigid scaffold, a flexible gate helix, and a C-terminal regulatory region. Notably, the structural and functional properties of Doa10 / MARCHF6 are consistent with reports that MARCHF6 is activated through a direct interaction between the N-terminal RING domain and the C-terminal regulatory region.

[0338] Moreover, the Ac / N domain (residues 552–600) largely overlaps with the helix-loop-helix region (residues 564–628) in MARCHF6, which corresponds to the L8 / 9 loop (or Buoy-Cap domain, 837–960) in Doa10.

[0339] In particular, the interaction of the L8 / 9 loop of Doa10 with a reporter containing Deg1 (the N-terminal 67 residues of the transcriptional repressor Matα2) has been confirmed. This result suggests that the L8 / 9 loop likely contains an Ac / N domain, since the N-terminus of the Deg1 fusion protein is specifically recognized by Doa10 for degradation via the Ac / N-degron pathway when acetylated. Furthermore, Doa10 targets an amphiphilic degron within Deg1 in addition to the Ac / N-degron, and Deg1 fusion proteins without acetylated N-terminus are degraded via the Arg / N-degron pathway (unmodified N-terminal recognition), complicating the interpretation of substrate recognition of Doa10 for Deg1.

[0340] More intriguingly, Doa10 interacts closely with various cytosolic or cytosol-misoriented substrates via a cytosol-exposed lateral tunnel located next to the L8 / 9 loop. This may aid in directing substrates from the cytosolic side to the lipid-filled central cavity for efficient polyubiquitylation.

[0341] Based on this structure, we hypothesized that Doa10 first recognizes the Ac / N-degron of the Deg1 fusion through its Ac / N domain within the L8 / 9 loop on the cytoplasmic face, and then the lateral tunnel binds to the amphipathic degron within Deg1, accelerating proper positioning of the substrate within the hydrophobic central cavity, thereby inducing efficient ubiquitination.

[0342] Similarly, MARCHF6 was postulated to first recognize Ac / N-degron substrates via its cytoplasmic-facing Ac / N domain, followed by ubiquitination by directing them into a lipid-filled central cavity through a cytoplasmically exposed soluble lateral tunnel. It was also postulated that MARCHF6 could also accommodate substrates without Ac / N-degrons (e.g., pro-ferroptosis effectors such as SM, p53, and ACSL4) into the central cavity either directly via the flexible gate, or indirectly via the cytoplasmically exposed lateral tunnel or other cytoplasmically facing substrate recognition sites.

[0343] Moreover, MARCHF6 plays a variable role in the regulation of ferroptosis. This is because MARCHF6 can target both Ac / N-degron-bearing (antiferroptotic) and Ac / N-degron-less (primarily proferroptotic) substrates via at least two different substrate binding sites (Fig. 38). Therefore, posttranslational modifications of MARCHF6 induced by cell signaling or stress, or competitive binding of small molecules to the Ac / N domain within MARCHF6, would dynamically shift the balance between the antiferroptotic function of the E3 Ub ligase (by promoting the degradation of key proferroptotic effectors) and the proferroptotic function (by promoting the degradation of Ac / N-degron-bearing substrates) (Fig. 38).

[0344]

[0345] Example 8. Method for preparing and analyzing a peptide that binds to the acetylated N-terminal recognition domain of MARCHF6

[0346] 8-1. Preparation of Ac-AS-NH2 peptide

[0347] Ac-AS-NH2 was prepared as a peptide that binds to the acetylated N-terminal recognition domain of MARCHF6, as shown in Figure 39.

[0348] The Ac-AS-NH2 peptide used in the present invention is a peptide composed of an N-terminal acetyl group-alanine-serine-C-terminal amide group, and was synthesized by request to Peptron, Inc. Peptron synthesis was performed using an automatic synthesizer (ASP48S, Peptron, Inc.) according to the Fmoc (9-Fluorenyl methyl oxycarbonyl) solid phase synthesis method (Fmoc-SPPS, solid phase peptide synthesis). All amino acid monomers used in the synthesis had the N-terminus protected with Fmoc, and each residue used an amino acid protected with trityl (Trt), t-butyloxycarbonyl (Boc), t-butyl (tBu), etc. The coupling reaction was carried out using HBTU (2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), HOBt (Hydroxybenzotriazole), and NMM (N-methylmorpholine) as coupling agents. Specifically, the protected amino acid (8 equivalents), HBTU (8 equivalents), and NMM (16 equivalents) were dissolved in DMF (Dimethylformamide), added, and reacted at room temperature for 2 hours. The Fmoc protecting group was removed twice for 5 minutes at room temperature using a 20% (v / v) Piperidine / DMF solution, and the desired peptide sequence was synthesized by repeating this coupling and Fmoc removal process. An acetyl group was attached to the N-terminus of the synthesized peptide.

[0349] The synthesized peptide was separated on a resin using a solution containing TFA (Trifluoroacetic acid), EDT (1,2-ethanedithiol), Thioanisole, TIS (Triisopropylsilane), and H2O in a weight ratio of 90:2.5:2.5:2.5:2.5. The resulting mixed solution was treated with an excess of refrigerated diethyl ether to generate a precipitate, which was then recovered by centrifugation, and excess TFA, thianisole, and ethanedithiol were removed. This process was repeated twice to obtain a solidified precipitate. Thereafter, the obtained precipitate was purified using high-performance liquid chromatography (Nexera Lite HPLC, Shimadzu Prominence, Japan) equipped with a C18 column (4.6 mm × 50 mm, 5 μm, Shiseido Co., Japan) under conditions of a water-acetonitrile liner gradient (acetonitrile concentration 10–50% (v / v)) containing 0.1% (v / v) TFA. The molecular weight of the purified peptide was confirmed using LC / MS (LCMS-2020 system, Shimadzu Prominence, Japan)), and finally, it was obtained as a TFA (Trifluoroacetic acid) salt in the form of a white powder by lyophilization.

[0350]

[0351] 8-2. Confirmation of cell viability after treating cells with Ac-AS-NH2 peptide

[0352] Wild-type and Marchf6-KO HeLa and A549 cells were seeded at a density of 3,000 cells / well in 96-well culture plates and cultured for 24 hours. The existing culture medium was replaced with a culture medium containing Ac-AS-NH2 dissolved at a concentration of 5 mM or 10 mM, and cultured for an additional 24 hours. Cells were treated with 0.15 μM RSL3, cultured for an additional 24 hours, and cell viability was confirmed using the same method as in Examples 1-4.

[0353]

[0354] 8-3. Analysis of intracellular protein expression after treatment with Ac-AS-NH2 peptide

[0355] Wild-type and Marchf6-KO A549 cells were cultured at 0.5 × 10 6 Cells were seeded into 6-well culture plates at a density of 10 cells / well and cultured for 12 hours. The existing culture medium of the cells was replaced with fresh medium or medium containing Ac-AS-NH2 at a final concentration of 10 mM, and the cells were cultured for an additional 24 hours and collected. The collected cells were lysed in lysis buffer containing a protease inhibitor cocktail on ice for 20 minutes. The supernatant was collected by centrifugation at 11,200 × g for 20 minutes at 4°C, and the total protein concentration was measured using the Bradford assay. Equal amounts of total protein were analyzed by immunoblotting with anti-PLIN2 (1:1000), anti-RGS2 (1:1000), anti-SM (1:1000), anti-p53 (1:1000), anti-ACSL4 (1:1000), anti-SLC7A11 (1:1000), anti-GPX4 (1:1000), anti-NRF2 (1:1000), and anti-tubulin (1:2000) antibodies.

[0356]

[0357] 8-4. Cell viability analysis after treatment with Ac-AS-NH2 in cells overexpressing wild-type or acetylated N-terminal recognition domain mutant Marchf6.

[0358] pCH879 (MARCHF6 3f ) or pCH6204 (MARCHF6 3f A549 cells were transformed with the MARCHF6-KO A549 plasmid (L571A mutant). The transformed MARCHF6-KO A549 cells were seeded at a density of 10,000 cells / well in a 96-well culture plate and cultured for 24 hours. The existing culture medium was replaced with a culture medium containing the indicated concentration of Ac-AS-NH2 and cultured for an additional 24 hours. The cells were treated with 0.15 μM RSL3, cultured for 24 hours, and cell viability was confirmed using the same method as in Example 1-4.

[0359]

[0360] 8-5. Lipid peroxidation analysis after Ac-AS-NH2 treatment in cells overexpressing wild-type or acetylated N-terminal recognition domain mutant Marchf6.

[0361] pCH879 (MARCHF6 3f ) or pCH6204 (MARCHF6 3f A549 cells were transformed with the MARCHF6-KO (L571A mutant) plasmid. The transformed MARCHF6-KO A549 cells were seeded at 0.5 × 10 in 6-well culture plates. 6 Cells were seeded at a density of 10 cells / well and cultured for 24 hours. The existing culture medium was replaced with fresh medium or a culture medium containing a final concentration of 10 mM Ac-AS-NH2, and cultured for an additional 24 hours. Cells were then treated with 0.15 μM RSL3, cultured for 24 hours, and lipid peroxidation was analyzed according to Example 1-3.

[0362]

[0363] Example 9. Confirmation of the effect of Ac-AS-NH2 peptide binding to the acetylated N-terminal recognition domain of MARCHF6.

[0364] The peptide Ac-AS-NH2, which binds to the acetylated N-terminal recognition domain of MARCHF6 manufactured through the above Example 8, was output using a molecular visualization program, and it was confirmed that it was a short synthetic peptide containing an N-terminal acetyl group (Ac-) and a C-terminal amide group (-NH2) at the end. When HeLa cells were treated with Ac-AS-NH2 and the survival rate was checked, as shown in Figure 40, in WT cells, the survival rate significantly increased as the concentration of Ac-AS-NH2 increased, but in MARCHF6 KO cells, the survival rate was not affected by Ac-AS-NH2 treatment. Therefore, it was confirmed that the protective effect of Ac-AS-NH2 is dependent on the presence of MARCHF6 protein. In addition, when the survival rate was checked in A549 cells after treatment with Ac-AS-NH2, as shown in Figure 41, in WT cells, the survival rate significantly increased as the concentration of Ac-AS-NH2 increased, but in MARCHF6 KO cells, the survival rate was not affected by Ac-AS-NH2 treatment. Therefore, the effect of Ac-AS-NH₂ required MARCHF6 regardless of the cell line, and was presumed to be based on the interaction with a specific domain.

[0365] In addition, WT and KO A549 cells were treated with Ac-AS-NH₂, and the expression levels of Ac / N-degrons substrates PLIN2 and RGS2, pro-ferroptotic proteins SM, p53, ACSL4, and anti-ferroptotic proteins SLC7A11, GPX4, and NRF2 were analyzed and shown in Fig. 42. As a result of treating WT cells with Ac-AS-NH₂, the expression of PLIN2 and RGS2 increased, the expression of SM, p53, and ACSL4 decreased, and the expression of SLC7A11, GPX4, and NRF2 increased. On the other hand, Ac-AS-NH₂ treatment had no effect on KO cells.

[0366] Therefore, it was confirmed that Ac-AS-NH₂ can regulate the ferroptosis pathway by mediating the degradation or stabilization of substrate proteins by regulating the function of MARCHF6.

[0367] In addition, MARCHF6 wild type and L571 mutant (L571A MARCHF6) were transfected into Marchf6-KO A549 cells, and the survival rates were compared after treatment with Ac-AS-NH₂. As a result, as shown in Figure 43, the survival rate of WT MARCHF6 expressing cells significantly increased by Ac-AS-NH₂, but the survival rate of L571A mutant expressing cells did not change even after Ac-AS-NH₂ treatment. Therefore, it was confirmed that the L571A mutant lost the ability to bind to or mediate function with Ac-AS-NH₂, and it was confirmed once again that L571A is a key amino acid within the functional domain of MARCHF6.

[0368] In addition, MARCHF6 wild type and L571 mutant (L571A MARCHF6) were transfected into Marchf6-KO A549 cells, and lipid peroxidation was analyzed after treatment with Ac-AS-NH₂. As a result, as shown in Figure 44, WT MARCHF6 expressing cells showed a decrease in lipid peroxidation upon Ac-AS-NH₂ treatment, but L571A mutant expressing cells showed no change in lipid peroxidation.

[0369] Therefore, Ac-AS-NH₂ induced lipid oxidation inhibition only in WT MARCHF6, and this effect was blocked by the L571A mutant, confirming a defect in binding or signal mediation function.

[0370]

[0371] Example 10. Method for producing and analyzing a MARCHF6-dependent dipeptide.

[0372] 10-1. Preparation of N-terminal acetylated dipeptide (Ac-XX)

[0373] In addition, a short peptide fragment (dipeptide) whose N-terminus was acetylated but whose C-terminus was not amidated was synthesized by requesting Peptron Co., Ltd. in the same manner as in Example 9-1R. Specifically, by excluding the step of binding an acetyl group to the N-terminus of the peptide during the above process, an N-terminus acetylated X (20 amino acids; alanine, glycine, aspartate, valine, threonine, serine, glutamate, proline, lysine, methionine, tyrosine, tryptophan, histidine, glutamine, asparagine, cysteine / cysteine, isoleucine, leucine, phenylalanine, and arginine)-serine peptide was prepared.

[0374] That is, as shown in Table 4 below, a dipeptide having the structure of Ac-X-Ser-NH₂ or Ac-Ala-X-NH₂ was prepared.

[0375] Structural DipeptideStructural DipeptideAc-X-Ser-NH₂Ac-AS-NH₂Ac-Ala- ₂Ac-VS-NH₂Ac-AI-NH₂Ac-LS-NH₂Ac-AL-NH₂Ac-IS-NH₂Ac-AV-NH₂Ac-CS-NH₂Ac-AC-NH₂Ac-PS-NH₂Ac-AP-NH₂Ac-DS-NH₂Ac-AD-NH₂

[0376]

[0377] 10-2. Analysis of cell viability after treatment with N-terminal acetylated dipeptide (Ac-XX) in wild-type or MARCHF6-KO cells.

[0378] Cell viability was measured using the same method as in Example 8-4 and analyzed using the same method as in Example 1-4. The analysis results were expressed in a heat map format.

[0379]

[0380] Example 11. Confirmation of the effect of a MARCHF6-dependent dipeptide.

[0381] To analyze whether a total of 20 N-terminal acetylated dipeptides have a MARCHF6-dependent inhibitory or inductive effect on ferroptosis, cell viability was examined after treatment with a ferroptosis inducer in A549 wild-type and MARCHF6-KO cells.

[0382] As a result, the following dipeptides significantly increased survival only in wild-type cells, while the same effect was not observed in MARCHF6-KO cells. This was interpreted as a result of the dipeptides selectively binding to the acetylated N-terminal recognition domain (Ac / N domain) of MARCHF6 and inhibiting ferroptosis.

[0383] Ac-X-Ser: Ac-GS, Ac-SS, Ac-TS, Ac-VS, Ac-LS (Figure 45);

[0384] Ac-Ala-X: Ac-AA, Ac-AG, Ac-AI, Ac-AL (Figure 46).

[0385] What these dipeptides have in common is the presence of a small, neutral or hydrophilic amino acid (Gly, Ser, Thr, Val, Leu, Ala, Ile) at the N-terminus. This suggests that the Ac / N domain of MARCHF6 favors specific physicochemical properties (small, flexible, and polar or neutral).

[0386] In contrast, the following dipeptides either had no effect on restoring viability in wild-type cells or even reduced it. This suggests that they either failed to bind to MARCHF6 or failed to induce ferroptosis inhibition by interfering with its binding.

[0387] Ac-X-Ser: Ac-PS, Ac-DS, Ac-CS (Figure 45)

[0388] Ac-Ala-X: Ac-AP, Ac-AD, Ac-AC (Figure 46).

[0389] In particular, Ac-PS, Ac-DS, Ac-AP, and Ac-AD reduced survival in a MARCHF6-dependent manner.

[0390] The failure to bind to MARCHF6 was hypothesized to be due to the presence of structural elements that inhibit binding. For example, Pro (Proline) restricts binding flexibility due to its rigid ring structure, Asp (Aspartate) carries a negative charge and can disrupt the charge balance of the domain, and Cys (Cysteine) is detrimental to binding stability due to its oxidative reactivity.

[0391] Therefore, the amino acid properties (size, polarity, charge, etc.) within the sequence of the dipeptide according to the present invention were closely linked to the binding effect with MARCHF6, and it was expected that this could provide key structure-activity correlation (SAR) information for the development of peptide-based ferroptosis regulators or anticancer agents in the future.

[0392]

[0393] As described above, specific embodiments of the present invention have been described in detail. However, those skilled in the art who understand the spirit of the present invention will be able to easily suggest other inventions that are backwards or other embodiments included within the scope of the spirit of the present invention by adding, changing, or deleting other components within the scope of the same spirit. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention is indicated by the scope of the claims described below rather than the detailed description described above, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. C5 domain derived from MARCHF6 protein that specifically recognizes degron (Ac / N-degron) formed by N-terminal acetylation of target protein.

2. In paragraph 1, The C5 domain comprises an amino acid sequence from positions 541 to 632 of the amino acid sequence of the human MARCHF6 protein.

3. In paragraph 1, A C5 domain comprising an amino acid sequence represented by sequence number 1.

4. In paragraph 1, The above Ac / N-degron is recognized by the Ac / N domain within the C5 domain, the C5 domain.

5. In paragraph 4, The above Ac / N domain is a C5 domain comprising amino acid sequences from positions 552 to 600 of the amino acid sequence of the human MARCHF6 protein.

6. In paragraph 5, The above Ac / N domain is a C5 domain comprising an amino acid sequence represented by sequence number 2.

7. In paragraph 4, The above Ac / N domain is a C5 domain having a helix-loop-helix structure.

8. In paragraph 1, The C5 domain induces ferroptosis by mediating the degradation of a target protein containing an Ac / N-degron.

9. In paragraph 1, A C5 domain characterized in that, when any one or more amino acids selected from the group consisting of the 566th and 571st amino acids of the C5 domain are modified, the C5 domain does not recognize a degron (Ac / N-degron) formed by N-terminal acetylation of a target protein.

10. In paragraph 9, A C5 domain wherein the 566th amino acid of the C5 domain is L and the 571st amino acid is L.

11. A pharmaceutical composition for preventing or treating a disease caused by inhibition of ferroptosis, comprising an activator of the C5 domain or the Ac / N domain derived from the MARCHF6 protein.

12. In paragraph 11, A pharmaceutical composition wherein the activator of the C5 domain or the activator of the Ac / N domain enhances Ac / N-degron recognition of the C5 domain through a mechanism that mediates at least one selected from the group consisting of enhancing substrate recognition ability of the domain, stabilizing the structure of the substrate binding site, and enhancing functional interaction of the domain.

13. In paragraph 11, The pharmaceutical composition above is capable of inducing degradation of a target protein by promoting the ubiquitination activity of MARCHF6 endogenous to the target protein containing a degron (Ac / N-degron) formed by N-terminal acetylation.

14. In paragraph 11, A pharmaceutical composition, wherein the disease caused by the above ferroptosis inhibition is at least one selected from the group consisting of cancer, drug-resistant cancer, and fibrotic disease.

15. A quasi-drug for improving a disease by inhibiting ferroptosis, comprising an activator of the C5 domain or the Ac / N domain derived from the MARCHF6 protein. 16.An anticancer adjuvant comprising an activator of the C5 domain or the Ac / N domain derived from the MARCHF6 protein.

17. A composition for gene therapy for increasing intracellular ferroptosis sensitivity, comprising a vector expressing a C5 domain or an Ac / N domain derived from the MARCHF6 protein.

18. A method for providing information necessary for diagnosing a functional abnormality or ferroptosis state of a C5 domain derived from MARCHF6 protein in a sample, comprising the step of measuring the presence or decomposition of a protein containing an Ac / N-degron in a sample isolated from an object. 19.1) A step of treating a sample containing a protein containing a degron (Ac / N-degron) formed by N-terminal acetylation, separated from an object, with a candidate substance for modulating ferroptosis; and 2) A method for screening a substance for regulating ferroptosis, comprising: a step of measuring a change in the activity of a C5 domain derived from MARCHF6 protein, a change in the activity of an Ac / N domain, or a binding ability to an Ac / N-degron after treatment with a candidate substance.

20. In paragraph 19, 3) A screening method further comprising a step of determining the candidate substance as a ferroptosis inducer when the activity level of the C5 domain of step 2), the activity level of the Ac / N domain, or the binding ability to Ac / N-degron is increased.

21. In paragraph 19, 3) A screening method further comprising a step of determining the candidate substance as a ferroptosis inhibitor when the activity level of the C5 domain, the activity level of the Ac / N domain, or the binding ability to Ac / N-degron of step 2) above is reduced; 22. A method for inducing degradation of a protein comprising a degron formed by N-terminal acetylation in a sample, comprising the step of treating a sample in vitro with an activator of a C5 domain or an activator of an Ac / N domain derived from MARCHF6 protein.

23. A pharmaceutical composition for preventing or treating a disease caused by excessive ferroptosis, comprising a C5 domain derived from MARCHF6 protein, an Ac / N domain, an inhibitor of the C5 domain derived from MARCHF6 protein, or an inhibitor of the Ac / N domain.

24. In paragraph 23, A pharmaceutical composition wherein the C5 domain inhibitor or Ac / N domain inhibitor inhibits Ac / N-degron recognition of the C5 domain through a mechanism that mediates at least one selected from the group consisting of inhibition of substrate recognition ability of the domain, structural change of the substrate binding site, and inhibition of functional interaction of the domain.

25. In paragraph 23, A pharmaceutical composition wherein the MARCHF6 protein-derived C5 domain or Ac / N domain exhibits decoy competition against the endogenous MARCHF6 protein-derived C5 domain or Ac / N domain and Ac / N-degron.

26. In paragraph 23, The pharmaceutical composition can inhibit the degradation of a target protein by reducing the ubiquitination activity of MARCHF6 inherent to the target protein, which contains a degron (Ac / N-degron) formed by N-terminal acetylation.

27. In paragraph 23, A pharmaceutical composition, wherein the disease caused by the above excessive ferroptosis is at least one selected from the group consisting of neurodegenerative diseases, cardiac diseases, acute kidney injury, chronic kidney injury, liver injury, lung damage, pulmonary inflammatory disease, and diabetic neuropathy.

28. A quasi-drug for improving a disease caused by excessive ferroptosis, comprising a C5 domain derived from MARCHF6 protein, an Ac / N domain, an inhibitor of the C5 domain derived from MARCHF6 protein, or an inhibitor of the Ac / N domain.

29. A peptide in which at least one amino acid in the amino acid sequence of the C5 domain derived from human MARCHF6 protein is modified, wherein the peptide has lost the binding function to a degron (Ac / N-degron) formed by N-terminal acetylation of a target protein.

30. In paragraph 29, The above peptide is a peptide in which at least one amino acid selected from the group consisting of the 566th and 571st amino acids of the C5 domain is modified.

31. In paragraph 30, A peptide wherein the 566th amino acid of the C5 domain is L and the 571st amino acid is L.

32. In paragraph 29, A peptide wherein the above amino acid is substituted with alanine.

33. In paragraph 30, A peptide comprising an amino acid sequence represented by SEQ ID NO: 3, wherein the 566th amino acid of the C5 domain derived from human MARCHF6 protein is substituted with alanine.

34. In paragraph 30, A peptide comprising an amino acid sequence represented by SEQ ID NO: 4, wherein the 571st amino acid of the C5 domain derived from human MARCHF6 protein is substituted with alanine.

35. A method for producing a peptide having lost the binding function to a degron (Ac / N-degron) formed by N-terminal acetylation of a protein, comprising the step of replacing any one or more amino acids selected from the group consisting of positions 566 and 571 of the amino acid sequence of the C5 domain derived from human MARCHF6 protein with alanine.

36. An N-terminally acetylated peptide that induces or inhibits ferroptosis in a MARCHF6-dependent manner, represented by the following general formula: [General Formula 1] Ac-X₁-X₂-R In the above general formula 1, Ac means an N-terminal acetyl group, When the above X₁ is Ala, X₂ is Ser, Gly, Asp, Val, Thr, Pro, Gln, Tyr, Ala, Leu or Ile, When the above X₂ is Ser, X₁ is Ala, Gly, Asp, Val, Thr, Phe, Ser, Pro or Leu, The above R is -OH or -NH₂.

37. In paragraph 36, A peptide wherein the peptide is Ac-AS-NH2, Ac-GS, Ac-SS, Ac-TS, Ac-VS, Ac-LS, Ac-AA, Ac-AG, Ac-AI, Ac-AL, Ac-PS, Ac-DS, Ac-AP or Ac-AD.

38. In paragraph 37, The above Ac-AS-NH2, Ac-GS, Ac-SS, Ac-TS, Ac-VS, Ac-LS, Ac-AA, Ac-AG, Ac-AI or Ac-AL peptide is a peptide that inhibits ferroptosis.

39. In paragraph 37, The above Ac-PS, Ac-DS, Ac-AP or Ac-AD peptide is a peptide that induces ferroptosis.

40. A pharmaceutical composition for preventing or treating a disease caused by excessive ferroptosis, comprising an N-terminal acetylated peptide that induces or inhibits ferroptosis in a MARCHF6-dependent manner, and is represented by the following general formula: [General Formula 1] Ac-X₁-X₂-R In the above general formula 1, Ac means an N-terminal acetyl group, When the above X₁ is Ala, X₂ is Ser, Ala, Gly, Ile or Leu, When the above X₂ is Ser, X₁ is Ala, Gly, Ser, Thr, Val or Leu, The above R is -OH or -NH₂.

41. A pharmaceutical composition for improving a disease caused by excessive ferroptosis, comprising an N-terminal acetylated peptide that induces or inhibits ferroptosis in a MARCHF6-dependent manner, and is represented by the following general formula: [General Formula 1] Ac-X₁-X₂-R In the above general formula 1, Ac means an N-terminal acetyl group, When the above X₁ is Ala, X₂ is Ser, Ala, Gly, Ile or Leu, When the above X₂ is Ser, X₁ is Ala, Gly, Ser, Thr, Val or Leu, The above R is -OH or -NH₂.

42. A pharmaceutical composition for preventing or treating a disease by inhibition of ferroptosis, comprising an N-terminal acetylated peptide that induces or inhibits ferroptosis in a MARCHF6-dependent manner, and is represented by the following general formula: [General Formula 1] Ac-X₁-X₂-R In the above general formula 1, Ac means an N-terminal acetyl group, When the above X₁ is Ala, X₂ is Pro or Asp, When the above X₂ is Ser, X₁ is Pro or Asp, The above R is -OH or -NH₂.

43. A pharmaceutical composition for improving a disease by inhibiting ferroptosis, comprising an N-terminal acetylated peptide that induces or inhibits ferroptosis in a MARCHF6-dependent manner, and is represented by the following general formula: [General Formula 1] Ac-X₁-X₂-R In the above general formula 1, Ac means an N-terminal acetyl group, When the above X₁ is Ala, X₂ is Pro or Asp, When the above X₂ is Ser, X₁ is Pro or Asp, The above R is -OH or -NH₂.

44. A method for preventing or treating a disease caused by inhibition of ferroptosis, comprising the step of administering to a subject an activator of the C5 domain or an activator of the Ac / N domain derived from the MARCHF6 protein.

45. A method for preventing or treating a disease caused by inhibition of ferroptosis, comprising administering to a subject an N-terminally acetylated peptide that is expressed by the following general formula and induces or inhibits ferroptosis in a MARCHF6-dependent manner; [General Formula 1] Ac-X₁-X₂-R In the above general formula 1, Ac means an N-terminal acetyl group, When the above X₁ is Ala, X₂ is Pro or Asp, When the above X₂ is Ser, X₁ is Pro or Asp, The above R is -OH or -NH₂.

46. ​​A method for preventing or treating a disease caused by excessive ferroptosis, comprising the step of administering to a subject a C5 domain derived from MARCHF6 protein, an Ac / N domain, an inhibitor of the C5 domain derived from MARCHF6 protein, or an inhibitor of the Ac / N domain.

47. A method for preventing or treating a disease caused by excessive ferroptosis, comprising administering to a subject an N-terminally acetylated peptide that is expressed by the following general formula and induces or inhibits ferroptosis in a MARCHF6-dependent manner; [General Formula 1] Ac-X₁-X₂-R In the above general formula 1, Ac means an N-terminal acetyl group, When the above X₁ is Ala, X₂ is Ser, Ala, Gly, Ile or Leu, When the above X₂ is Ser, X₁ is Ala, Gly, Ser, Thr, Val or Leu, The above R is -OH or -NH₂.

48. A method for increasing intracellular ferroptosis sensitivity of an individual, comprising the step of administering to the individual a vector expressing a C5 domain or an Ac / N domain derived from a MARCHF6 protein.

Images