Phosphorylation promoter of p62 protein

Abstract

To promote phosphorization of p62 / SQSTM1 protein by using diacylglycerol PEG adduct.SOLUTION: A phosphorization promoter contains diacylglycerol PEG adduct as an active ingredient, and the diacylglycerol PEG adduct is a phosphorization promoter of p62 / SQSTM1 protein. The diacylglycerol PEG adduct is selected from the group consisting of glycerol dimyristate PEG-12(GDM12), glycerol distearate PEG-12(GDS12), glycerol distearate PEG-23(GDS23), glycerol palmitate PEG-23(GDP23), and glycerol dioleate PEG-12(GDO12).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a substance that promotes phosphorylation of a phosphorylation site of the p62 / SQSTM1 protein.

Background Art

[0002] In vivo, the homeostasis of biological functions is maintained by inherently having the "in vivo antioxidant system", which is a defense mechanism against oxidative stress. This system induces the expression of antioxidant enzymes and antioxidant protein genes that detoxify oxidative stress such as reactive oxygen species (ROS) in cells. One of the main in vivo antioxidant systems is the Nrf2-Keap1 pathway. Nrf2 is a transcription factor that expresses antioxidant genes, and Keap1 is a protein that suppresses and controls Nrf2. Under non-oxidative stress conditions, Keap1 forms a complex with Nrf2 in the cytoplasm and constantly degrades Nrf2 to suppress its activation. When cells are exposed to oxidative stress or electrophilic substances, the thiol group of Keap1 is oxidized, inhibiting its binding to Nrf2, and Nrf2 dissociates from Keap1 and translocates into the nucleus. In the nucleus, Nrf2 binds to the antioxidant response element (ARE) to enhance the expression of antioxidant factors.

[0003] As another in vivo defense system, selective autophagy that degrades abnormal substances in cells is known. The p62 / SQSTM1 protein (hereinafter abbreviated as "p62"), known as an adapter factor for selective autophagy, has a Keap1-interacting region (KIR), which is a domain capable of binding to Keap1 (Patent Document 1, Non-Patent Document 1). However, since the binding affinity of p62 to Keap1 is much lower than that of Nrf2, it usually does not affect the binding of Keap1 and Nrf2.

[0004] p62 has multiple phosphorylation domains, and these domains are phosphorylated during the process of selective autophagy. In recent years, when serine (Ser) 351 in the KIR of p62 (in the case of mice; Ser349 in humans) is phosphorylated, the binding affinity of phosphorylated p62 for Keap1 is enhanced to the same extent as that of Nrf2 for Keap1. As a result, it has been revealed that Keap1 binds to phosphorylated p62, and at the same time, Nrf2 dissociates from Keap1 and is stabilized (Patent Document 2, Non-Patent Documents 2, 3, 4). That is, in addition to the oxidation of its thiol group, the binding of Keap1 to Nrf2 is also inhibited by phosphorylated p62, allowing Nrf2 to dissociate from Keap1. Also in this pathway involving phosphorylated p62, similar to the Nrf2-Keap1 pathway, Nrf2 that has translocated into the nucleus enhances the expression of antioxidant factors. Since p62 is also included among the factors expressed by Nrf2, a positive feedback that replenishes the p62 reduced by the production of phosphorylated p62 works.

[0005] Here, although not directly related to the above, closed vesicles (vesicles) composed of phospholipids and surfactants are known. These are also referred to as liposomes. Patent Document 3 presents a preparation method for spontaneously forming vesicles by using a lipid mainly composed of a diacylglycerol polyethylene glycol adduct (hereinafter sometimes referred to as "diacylglycerol PEG adduct") instead of phospholipids and mixing it with water or a surfactant. Such vesicles are used in a drug delivery system in which target substances such as proteins and antibodies are encapsulated or bound inside or on the surface thereof and delivered to cells in the living body.

[0006] Vesicles with diacylglycerol PEG adduct as the lipid have a form in which their surface is covered with hydrophilic PEG chains, and have good permeability in the living body and stability in the blood. Patent Document 4 describes that by binding a charged element to the surface of vesicles composed of diacylglycerol PEG adduct to make them positively charged, the permeability and retention in the stratum corneum of the epidermis can be improved.

[0007] Furthermore, Patent Document 5 discloses an antioxidant expression enhancer based on the newly discovered in-vivo action of diacylglycerol PEG adducts themselves, rather than as carriers for target substances. According to Patent Document 5, diacylglycerol PEG addition has the effect of enhancing the expression of Nrf2 in cells.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0010] According to the above findings, phosphorylation of p62 causes activation of Nrf2 by the so-called phosphorylated p62-Keap1-Nrf2 pathway, thereby enhancing the expression of antioxidant factors by Nrf2 that has translocated into the nucleus. This activation of Nrf2 is a different pathway from the activation of Nrf2 by the Keap1-Nrf2 pathway under oxidative stress conditions. Phosphorylation of p62 usually occurs by intracellular phosphorylating enzymes (such as mTORC1). Also, such phosphorylation of p62 occurs as part of the selective autophagy pathway for capturing and degrading abnormal protein aggregates and pathogens that have invaded the cell.

[0011] However, at present, no attempt has been made to intentionally promote phosphorylation of p62 by administration of an external substance, nor has such a substance been presented.

[0012] An object of the present invention is to utilize the newly discovered properties of diacylglycerol PEG adducts, and in particular, to promote phosphorylation of p62 protein in vivo, thereby enhancing the expression of Nrf2.

Means for Solving the Problems

[0013] In order to achieve the above object, the present invention provides the following configuration. An aspect of the present invention includes a diacylglycerol PEG adduct as an active ingredient, the diacylglycerol PEG adduct has the following structural formula, the carbon number of R in the long-chain fatty acid is in the range of 11 to 23, and n in the polyethylene glycol chain is in the range of 11 to 46, and it is a phosphorylation promoter for the p62 / SQSTM1 protein. Another aspect of the present invention includes a diacylglycerol PEG adduct as an active ingredient, the diacylglycerol PEG adduct has the following structural formula, the carbon number of R in the long-chain fatty acid is in the range of 11 to 23, and n in the polyethylene glycol chain is in the range of 11 to 46, and it is a method for promoting the phosphorylation of the p62 / SQSTM1 protein.

[0014]

Chemical Formula

[0015] Preferably, the diacylglycerol PEG adduct is selected from the group consisting of glycerol dimyristate PEG-12 (GDM12), glycerol distearate PEG-12 (GDS12), glycerol distearate PEG-23 (GDS23), glycerol dipalmitate PEG-23 (GDP23), and glycerol dioleate PEG-12 (GDO12).

[0016] Preferably, the diacylglycerol PEG adduct penetrates into the epidermis in a solution state. Preferably, the diacylglycerol PEG adduct penetrates into the epidermis in a vesicle state.

[0017] The present invention further provides an Nrf2 expression enhancer using the above phosphorylation promoter for the p62 / SQSTM1 protein.

Effects of the Invention

[0018] According to the present invention, a phosphorylation promoter of p62 protein containing a diacylglycerol PEG adduct as an active ingredient is realized. Further, according to the present invention, an Nrf2 expression enhancer using such a phosphorylation promoter of p62 protein is realized.

Brief Description of the Drawings

[0019]

Figure 1

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Modes for Carrying Out the Invention

[0020] Hereinafter, embodiments of the present invention will be described with reference to the drawings. The present invention is made by utilizing the newly discovered properties of diacylglycerol polyethylene glycol adducts (diacylglycerol PEG adducts). The newly discovered property here is the action of promoting the phosphorylation of p62 / SQSTM1 protein in the human body.

[0021] The structural formula of the diacylglycerol PEG adduct, which is a lipid molecule related to the present invention, is schematically shown.

[0022]

Chemical formula

[0023] The diacylglycerol PEG adduct is composed of a glycerol backbone (CH2CHCH2) having three carbons, a PEG chain which is a linear polyethylene glycol bonded to one of the terminal carbons of the three carbons of the backbone, and the same kind of long-chain fatty acids (COOR) bonded to the other two of the three carbons respectively. The PEG chain part is hydrophilic and the long-chain fatty acid part is hydrophobic.

[0024] In the following description, when representing a specific diacylglycerol PEG adduct, it is referred to as "[di] + [long-chain fatty acid name] + [glycerol] + [PEG-n]" based on the type of long-chain fatty acid and the number n of the PEG chain. For example, when the long-chain fatty acid is stearic acid and n of the PEG chain is 23, it is "distearic acid PEG-23". Also, a specific diacylglycerol PEG adduct may sometimes be shown in a further abbreviated form.

[0025] The number of carbon atoms of R in the long-chain fatty acid can be in the range of 11 to 23. The long-chain fatty acids included in this range are, for example, myristic acid, palmitic acid, stearic acid, or oleic acid, etc. The number n of the PEG chain can be in the range of 11 to 46. As the diacylglycerol PEG adducts related to the present invention, the following can be exemplified. In parentheses, the melting point and the abbreviation are shown. · Dimyristic acid glycerol PEG-12 (25.0 °C: GDM12) · Glycerol distearate PEG-12 (40.0 °C: GDS12) · Glycerol distearate PEG-23 (39.8 °C: GDS23) · Glycerol dipalmitate PEG-23 (31.2 °C: GDP23) · Glycerol dioleate PEG-12 (25.0 °C: GDO12)

[0026] As described above, the Nrf2-Keap1 pathway is well known as a defense mechanism against oxidative stress in vivo. Under non-oxidative stress conditions, Nrf2 and Keap1 form a complex in the cytoplasm, and at the same time, Nrf2 is constantly degraded and inactivated by Keap1. When the living body is subjected to oxidative stress, Keap1 is inactivated by oxidation of its thiol group by reactive oxygen species or electrophilic substances. As a result, Nrf2 dissociates from Keap1 and is activated, translocates into the cell nucleus, and produces antioxidant enzymes.

[0027] On the other hand, p62, known as an adapter factor for selective autophagy, increases its binding affinity to Keap1 by phosphorylation of its Keap1 interaction region. As a result, Keap1 binds to phosphorylated p62 without undergoing thiol group oxidation. As a result, Nrf2 dissociates from Keap1 and is activated, translocates into the cell nucleus, and produces antioxidant enzymes and the like. This is an Nrf2 activation pathway involving phosphorylated p62, which is different from the Nrf2 activation pathway by the above oxidative stress.

[0028] The inventors have found that the phosphorylation of p62 is promoted by applying a diacylglycerol PEG adduct to human epidermal cells. This promotion of p62 phosphorylation in living cells is a newly discovered effect of the diacylglycerol PEG adduct and can be said to be a new property of the diacylglycerol PEG adduct. The relationship between the diacylglycerol PEG adduct and p62 has not been known at all so far. The promotion of p62 phosphorylation means the promotion of the binding between phosphorylated p62 and Keap1. In other words, the diacylglycerol PEG adduct can contribute to the activation pathway of Nrf2 involving phosphorylated p62.

[0029] The present invention provides a p62 phosphorylation promoter containing a diacylglycerol PEG adduct as an active ingredient, utilizing the newly discovered property in this diacylglycerol PEG adduct. This can also be said to be a method for promoting p62 phosphorylation using a diacylglycerol PEG adduct.

[0030] There are multiple types of diacylglycerol PEG adducts. When applied to a living body, only one type may be used, or a combination of multiple types may be used.

[0031] Intracellular p62 is usually maintained at a certain level. According to the present invention, the diacylglycerol PEG adduct that has reached the human epidermis promotes the phosphorylation of p62, thereby promoting the detachment of Nrf2 from Keap1 compared to the case without it, and Nrf2 translocates into the cell nucleus. This process was confirmed by the tests described later. As a result of the activation of Nrf2, antioxidant enzymes and the like are produced, making it possible to reduce or prevent cell damage in the epidermis caused by external oxidative stress, such as ultraviolet rays and air pollutants.

[0032] Therefore, according to the present invention, it is also expected to provide, for example, cosmetics or pharmaceuticals containing a diacylglycerol PEG adduct as an active ingredient.

[0033] As one of the methods for reaching the diacylglycerol PEG adduct into the human epidermis, there is a method of reaching it into the epidermis in a solution state where the diacylglycerol PEG adduct is dissolved in water or a predetermined solvent. For example, a diacylglycerol PEG adduct solution at a predetermined concentration using phosphate buffered saline PBS(-) as a solvent can be prepared and applied to the skin surface to penetrate into the epidermis. The applied solution penetrates into the outermost stratum corneum and further penetrates into the granular layer under the stratum corneum. And the diacylglycerol PEG adduct promotes the phosphorylation of p62 present in the cells of each layer in the penetrated epidermis.

[0034] In a preferred method, the diacylglycerol PEG adduct can be reached into the epidermis in a vesicle state. Such a vesicle is formed as a closed spherical shell composed of a bilayer of the diacylglycerol PEG adduct or a multiple layer in which multiple bilayers overlap, and the hydrophilic PEG chains are arranged on the surface of the outermost layer. A vesicle of the diacylglycerol PEG adduct can be prepared and applied to the skin surface to penetrate into the epidermis. After reaching the epidermis, the vesicle decomposes and separates into individual molecules, so that the diacylglycerol PEG adduct itself can exert its function.

[0035] In the conventional drug delivery system, the diacylglycerol PEG adduct, which is the material of the vesicle, has been considered merely as a carrier of the target substance. However, in the present invention, the diacylglycerol PEG adduct itself is used as an active ingredient. Therefore, in the present invention, the target substance incorporated into the vesicle in the normal drug delivery system is basically unnecessary. In the present invention, by penetrating a vesicle formed by mixing only water and the diacylglycerol PEG adduct into the epidermis, the diacylglycerol PEG adduct itself can function as an accelerator for phosphorylating p62 in cells.

[0036] Some diacylglycerol PEG adducts spontaneously form vesicles by mixing with water at a predetermined temperature (Patent Document 3). For example, by mixing 2% by mass of GDM12 or GDO12 with 98% by mass of deionized water at room temperature and stirring, a suspension of vesicles of GDM12 or GDO12 can be obtained. As another example, after dissolving 2% by mass of GDS12 or GDS23 at 45 to 55°C, it is mixed with 98% by mass of deionized water at 45 to 55°C and stirred to obtain a suspension of vesicles of GDS12 or GDS23. As yet another example, after dissolving 2% by mass of GDP23 at 37°C, it is mixed with 98% by mass of deionized water at 37°C and stirred to obtain a suspension of vesicles of GDP23. Even when the suspension obtained at a temperature higher than room temperature is cooled to room temperature, the vesicles are stable.

[0037] As another example, when vesicles formed by mixing and stirring an aqueous solution of various substances and a diacylglycerol PEG adduct are used in place of water, it is also included in the scope of the present invention. In that case, another function can also be imparted to the substance contained in the aqueous solution.

[0038] The size of the vesicles formed by the diacylglycerol PEG adduct is, for example, about 20 to 300 nm in diameter. In particular, the refined vesicles in the range of 20 to 40 nm in diameter are suitable because they have good permeability to the epidermis. Such refined vesicles can be obtained, for example, by mixing the diacylglycerol PEG adduct with squalane and cholesterol, which are other lipids (Patent Document 5).

[0039] As yet another example, when the surface of the vesicles formed by mixing and stirring water or an aqueous solution and a diacylglycerol PEG adduct is modified with a charged element such as a cationic surfactant and used, it is also included in the scope of the present invention. Patent Document 4 describes that positively charged vesicles are particularly excellent in permeability and retention to the epidermis.

[0040] Cosmetics and pharmaceuticals containing a diacylglycerol PEG adduct as an active ingredient can be provided in various forms such as aqueous solutions, emulsions, gels, creams, etc.

[0041] Hereinafter, test data regarding the promoting effect of the diacylglycerol PEG adduct on the phosphorylation of p62 are shown. (1) Confirmation test of the promoting effect on the phosphorylation of p62 (1-1) Test method Using a 24-well plate with a medium (HuMedia-KG2 medium: manufactured by Kurabo), normal human epidermal keratinocytes (NHEK: manufactured by Kurabo) were seeded at a cell density of 1.0×10 5 cells / well. Subsequently, they were cultured for 24 hours under the conditions of 37°C and 5% CO2. Thereafter, using the medium (HuMedia-KB2: manufactured by Kurabo), samples added with three different types of diacylglycerol PEG adducts were respectively cultured under the conditions of 37°C and 5% CO2. The types, addition amounts, and culture times of the diacylglycerol PEG adducts in each sample are shown in Table 1. The culture time of 0 is a comparative example.

[0042]

Table 1

[0043] For each sample after each time elapsed, the Western blot method was applied to detect the proteins of p62 and phosphorylated p62. First, SDS-PAGE containing 10% mercaptoethanol was performed to extract the proteins. The protein extract was separated by electrophoresis using a polyacrylamide gel, and the proteins were transferred to the membrane by the semi-dry method. Thereafter, using each antibody, the proteins of p62 and phosphorylated p62 (pp62) were quantified. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal standard. Each antibody used, its dilution ratio, origin species, etc. are shown in Table 2.

[0044]

Table 2

[0045] After correcting the amount of each target protein with the value of the amount of GAPDH (control), which is an internal standard in the same sample, the corrected value of each sample was calculated when the corrected value of the control was set to 1 (the same applies to the following tests).

[0046] (1-2) Test results Figures 1 and 2 show the test results for GDS23, Figures 3 and 4 show the test results for GDM12, and Figures 5 and 6 show the test results for GDS12. Figures 1, 3, and 5 are the band images of Western blots at each time for each sample. Figures 2, 4, and 6 are graphs showing the quantitative analysis results of Western blots at each time for each sample. These data are mean ± S.D. (n = 4), *p < 0.05, **p < 0.01, ***p < 0.001 (Dunnet test).

[0047] When each sample of GDS23, GDM12, and GDS12 was added to normal human epidermal keratinocytes and cultured for several hours, in each case, phosphorylated p62 increased, and no change was observed in p62. The increase in phosphorylated p62 is considered to be due to the phosphorylation of p62. On the other hand, the decrease in p62 is considered to be due to the production and replenishment of new p62. As a p62 replenishment mechanism, it is said to include a positive feedback that p62 also exists among the factors expressed by Nrf2 released by the binding of Keap1 to phosphorylated p62. By this test, the promoting effect of phosphorylation of p62 by each of GDS23, GDM12, and GDS12 was confirmed. It was confirmed that phosphorylation of p62 significantly occurred after 12 hours of culture for GDS23 and after 18 hours of culture for GDM12 and GDS12 under these test conditions.

[0048] (2) Confirmation test of Nrf2 activation and nuclear translocation Using immunostaining, a test was conducted to confirm the nuclear translocation of Nrf2 after phosphorylation of p62. In this test, glycerol distearate PEG-23 (GDS23) was used. (2-1) Test method Normal human epidermal keratinocytes (NHEK: manufactured by Kurabo) were seeded on a 25 mmφ polylysine-coated cover glass (manufactured by Matsunami Glass Industry Co., Ltd.) at a cell density of 2×10 4 cells / well. Subsequently, the cells were cultured for 5 days at 37°C under 5% CO2 conditions using a medium (HuMedia-KG2: manufactured by Kurabo). Thereafter, a medium containing 50 μM GDS23 (HuMedia-KB2: manufactured by Kurabo) and the medium alone (HuMedia-KB2: manufactured by Kurabo) as a control were added, and the cells were cultured for 24 hours at 37°C under 5% CO2 conditions.

[0049] After removing the sample, the cover glass was subjected to permeabilization treatment by treating it with a 4% formaldehyde solution (dissolved in PBS(-)) for 10 minutes. Also, the cells were washed by treating them with 50 mM ammonium chloride (dissolved in PBS(-)) for 10 minutes. Thereafter, blocking was performed by treating them with 10% Normal Goat Serum (dissolved in PBS(-) containing 3% BSA) at room temperature for 1 hour. Subsequently, the primary antibody (rabbit Anti-Nrf2 monoclonal antibody, diluted 1 / 100 using PBS(-) containing 3% BSA) was treated at 4°C overnight to perform the primary antibody reaction. Further, after washing with PBS(-), the secondary antibody (goat anti-rabbit IgG (Alexa Fluor 488), diluted 1 / 400 using PBS(-) containing 3% BSA) was treated at room temperature in the dark to perform the secondary antibody reaction. After washing with PBS(-), nuclear staining was performed by treating with 2.0 μg / mL of DAPI (4',6-diamidino-2-phenylindole) at room temperature in the dark for 10 minutes. After staining, the cover glass was observed using a confocal laser microscope. The observed laser conditions are as follows. · Laser intensity: DAPI 45%, Alexa 488 50% · Laser intensity: DAPI 55%, Alexa 488 55%

[0050] (2-2) Test results Figure 7 shows the confocal laser microscope images of Nrf2 immunostaining. Cells were treated with 50 μM GDS23, and Nrf2 immunostaining was observed 12 hours after treatment with GDS23, when phosphorylation of p62 occurred significantly, especially 24 hours after 18 hours and later. It was found that in the GDS23-treated samples, Nrf2 was more highly expressed in the cell nucleus compared to the control not treated with GDS23. From this result, it was confirmed that Nrf2 translocated into the nucleus and was activated.

[0051] (3) Confirmation test of Nrf2 activation by the binding of Keap1 and phosphorylated p62 Using an inhibitor (K67) that inhibits the binding of phosphorylated p62 and Keap1, the inhibitory effect on Nrf2 activation (when the binding of Keap1 and phosphorylated p62 is blocked and Nrf2 cannot be released from Keap1) was confirmed. In this test, glycerol distearate PEG-23 (GDS23) was used.

[0052] (3-1) Test method <Method 1> Normal human epidermal keratinocytes (NHEK: manufactured by Kurabo) were seeded in a 96-well plate at a cell density of 2.0×10 4 cells / well using medium (HuMedia-KG2: manufactured by Kurabo) and cultured for half a day under conditions of 37 °C and 5% CO2. Next, 25 μM K67 (manufactured by Sigma-Aldrich) was prepared using medium (HuMedia-KB2: manufactured by Kurabo) and cultured for 14 hours under conditions of 37 °C and 5% CO2. Subsequently, 50 μM GDS23 was prepared using medium (HuMedia-KB2: manufactured by Kurabo) and cultured for 24 hours or 30 hours under conditions of 37 °C and 5% CO2.

[0053] After removing the sample, it was washed with PBS(-), and RNA was extracted using an RNA purification kit (RNeasy Mini kit: QIAGEN). Furthermore, cDNA was synthesized by performing a reverse transcription reaction using a PCR apparatus (PCR Thermal Cycler Dice: Takara Bio Inc.). Then, PCR reagents (SYBR (registered trademark) Green Master Mix: Thermo Fisher Scientific) were added, and mRNA quantification was performed by the ΔΔCt method using a PCR apparatus (StepOne Real-Time PCR System: Applied Biosystems). The value of the expression level of GAPDH was used as an internal standard (control) for correction.

[0054] <Method 2> Normal human epidermal keratinocytes (NHEK: Kurabo) were seeded at a cell density of 1.0×10 5 cells / well in a 24-well plate using a medium (HuMedia-KG2: Kurabo). A 25 μM solution of K67 (Sigma-Aldrich) was prepared using a medium (HuMedia-KB2: Kurabo) and cultured at 37°C under 5% CO2 for 14 hours. Subsequently, a 50 μM solution of glycerol distearate PEG-23 (GDS23) was prepared using a medium (HuMedia-KB2: Kurabo) and cultured at 37°C under 5% CO2 for 24 hours or 30 hours.

[0055] After each time period, 10% mercaptoethanol-containing SDS-PAGE was performed to extract proteins from the cells. Then, the protein extract was separated by electrophoresis using a polyacrylamide gel, and the proteins were transferred to a membrane by the semi-dry method. Subsequently, the protein amount was quantified using each antibody. The value of the protein amount of GAPDH was used as an internal standard (control) for correction.

[0056] (3-2) Test Results Figure 8 is a graph showing the quantitative analysis results of HO-1 (Heme Oxygenase-1) and GCLC (Glutamate-Cysteine Ligase Catalytic subunit (GCLC): γ-glutamylcysteine synthetase) by <Method 1>. HO-1 is a rate-limiting enzyme for heme degradation and a cytoprotective protein that protects cells from damage caused by oxidative stress. In Figure 8, it is mean ± S.D. (n = 4), **p < 0.01, ***p < 0.0001 (Tukey test).

[0057] Figure 9 is a band image of a Western blot of HO-1 and NQO1 (NAD(P)H Quinone Oxidoreductase-1) by <Method 2>.

[0058] In cells treated with only GDS23, the production levels of antioxidant genes and antioxidant proteins enhanced by the activation of Nrf2 were much greater than those in the control. In contrast, in cells treated with GDS23 after pretreatment with K67, the production levels of antioxidant genes and antioxidant proteins enhanced by the activation of Nrf2 decreased. From these results, it was confirmed that the phosphorylation of p62 promoted by treatment with GDS23 causes Nrf2 to dissociate from Keap1 and become activated, and that activation of Nrf2 does not occur when the binding between p62 and Keap1 is inhibited even though p62 is phosphorylated.

[0059] As described above, the present invention has been described with reference to the examples, but the present invention is not limited to these examples, and obvious modifications from these are also included in the present invention.

Claims

1. A phosphorylation promoter of p62 / SQSTM1 protein, which contains a diacylglycerol PEG adduct as an active ingredient, wherein the diacylglycerol PEG adduct has the following structural formula, the carbon number of R in the long-chain fatty acid is in the range of 11 to 23, and n in the polyethylene glycol chain is in the range of 11 to 46. 【Chemical 1】

2. The phosphorylation promoter of p62 / SQSTM1 protein according to claim 1, wherein the diacylglycerol PEG adduct is selected from the group consisting of glycerol dimyristate PEG-12 (GDM12), glycerol distearate PEG-12 (GDS12), glycerol distearate PEG-23 (GDS23), glycerol dipalmitate PEG-23 (GDP23), and glycerol dioleate PEG-12 (GDO12).

3. The phosphorylation promoter of p62 / SQSTM1 protein according to claim 1 or 2, wherein the diacylglycerol PEG adduct penetrates into the epidermis in a solution state.

4. The phosphorylation promoter of p62 / SQSTM1 protein according to claim 1 or 2, wherein the diacylglycerol PEG adduct penetrates into the epidermis in a vesicle state.

5. An enhancer for enhancing the expression of Nrf2, which uses the phosphorylation promoter of p62 / SQSTM1 protein according to claim 1 or 2.

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