KR20070070300A - Method to Inhibit Melanosome Movement Using SiRNA of Prohibitin Protein - Google Patents

Abstract

A method for inhibiting melanosome migration by using prohibitin siRNA(small interference RNA) is provided to inhibit expression of prohibitin in a melanin synthesis cell and uniformed distribution of melanosome in the melanin synthesis cell, so that the skin is whitened. The melanosome migration to the end of melanin synthesis cell is inhibited by administering prohibitin siRNA into the melanin synthesis cell, melan-a, thereby inhibiting binding of prohibitin to melanophilin as a melanosome vehicle and uniformed distribution of melanosome in the melanin synthesis cell.

Description

Method of inhibiting melanosome migration using prohibition siRNA using probitin protein siRNA

FIG. 1 shows the results of Western blot whether prohibitin is expressed in melan-a, melan-producing cells.

Figure 2 is the result of confirming the position where the prohibitin (Prohibitin) is expressed in melanocytes melan-a cells by performing an immunofluorescence assay (immunoflourescence assay).

3 shows the results of mass spectrometry for prohibitin peptides.

Figure 4 shows the combination of prohibitin (melanophilin) with prohibitin (melanophilin) is seen through co-immunoprecipitation (co-immunoprecipitation).

Figure 5a shows the results of treatment with prohibitin siRNA to Melan-a melanocytes, the immunoblot method to see whether the inhibition of the protein expression of PHB actually occurs, Figure 5b is a prohibitin protein over time It is a graph which shows the expression amount of.

6 is a result of observing the cell morphology of melan-a melanogenesis cells treated with prohibitin siRNA under an optical microscope.

The present invention relates to a method for inhibiting melanosomal migration using siRNA of prohibitin protein present in melanocytes. The prohibitin is a protein that binds to melanophilin, one of the melanosome carriers, and when the prohibitin siRNA that inhibits the expression of the prohibitin is injected into melanocytes, the melanosomes in the melanocytes The uniform distribution is inhibited and the movement of the melanosomes toward the cell ends is inhibited, and the melanoma movement of the melanocytes is inhibited.

Knowledge of the chemical and enzymatic basis of melanogenesis as a mechanism for regulating the color of the skin is well known. Melanocytes migrate from the embryonic neural crest to the skin to produce melanosomes that produce melanin, the particles they secrete. The key enzyme in melanogenesis is tyrosinase, which leads to a reaction cascade that converts tyrosine into raw polymer melanin. Two tyrosinase-related proteins (TRP's) are known, TRP-1 and TRP-2, which share about 40% homology with tyrosinase and have a catalytic activity as well as a regulatory role in melanogenesis. . The melanin occurs in the organelle called melanosome (melanosome) in which the enzymes are distributed, and the melanosome containing melanin is later introduced into keratinocytes (keratinocytes, keratinocytes) through melanocyte producing cells dendrites. Delivered. This melanin delivered to keratinocytes is actually an important factor in determining the skin color.

As the main enzyme involved in the production of melanin is tyrosinase, there are also major constructs for transferring the produced melanosomes to melanocyte dendrites. They are called Melanosome Transport Complex (MTC) and are composed of three proteins, Melanoophilin, Rab27a, and Myosin 5a. Rab27a is a protein that binds to the surface of the melanosome and acts as a carrier that recognizes and transports the melanosome. In order to move to the end, Rab27a must meet the myosin 5a protein, a cytoskeleton, and recognize the two proteins in the middle. It's a protein called melanophilin that acts as an adapter to connect and connect. When these three constructs meet to form a complex, the melanosomes can be normally transferred to the cell ends, and ultimately, they can be delivered to keratinocytes.

Phenotypes of various genetic disorders showing abnormalities in skin color are generally related to the well-known genes related to the key enzymes of melanin production, but recently, the skin color becomes bright due to abnormal genes related to the transfer of melanosomes. Diseases that show up are being introduced. These diseases show that melanosomes are not normally delivered to keratinocytes, and that melanosomes are not deposited on keratinocytes. As a result, the skin color becomes brighter than normal. Anatomically looking at the melanocytes present in the skin of these genetic diseases, unlike melanocytes containing melanin in melanocytes are distributed evenly throughout the cell, the melanocytes are only around the nucleus. In the aggregated cell terminal dendrites, there is a characteristic that the distribution of melanosomes is significantly reduced. These melanogenesis cells have no difference in melanin synthesis from normal, and even in melanocytes, melanocytes do not migrate to the ends of the cells, resulting in over-aggregation around the nucleus.

Therefore, the transfer of melanosomes and delivery to keratinocytes is considered to be one of the mechanisms that can affect the skin whitening and inhibit melanin biosynthesis, and also regulate the mechanism of melanin migration in melanocytes. Could be a new target.

Accordingly, the present inventors have studied the mechanism of inhibiting the migration of melanocytes of melanocytes, and found that siRNA of prohibitin protein present in melanocytes can inhibit the movement of melanosomes. The present invention has been completed.

Accordingly, an object of the present invention is to provide a method for inhibiting melanosomal migration using siRNA of prohibitin protein present in melanocytes.

In order to achieve the above object, the method for inhibiting melanosomal migration of the present invention is inhibited by uniform distribution of melanosomes in melanocytes when prohibitin siRNA that inhibits the expression of prohibitin is injected into melanocytes. It is characterized by appearing by inhibiting the movement of the melanosomes received.

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

The inventors first discovered that the prohibitin protein is expressed in melan-a cells, which are melanocytes, and binds to the melanophilin protein, which regulates the movement of melanosomes. Injecting the siRNA to inhibit the melanogenesis cells can be found to inhibit the melanocyte migration of the melanogenesis cells to provide a method for inhibiting the melanosomal movement of the prohibitin protein. Therefore, the prohibitin protein may have a whitening effect through the action of inhibiting the melanosome migration.

Melanosomes of the melanogenesis cells are intracellular organelles that produce melanin that has a function of blocking the destruction of cells by ultraviolet light, and these organelles must be delivered to keratinocytes to minimize damage to the cells caused by ultraviolet light in the skin. It is an important factor.

Hereinafter, although an Example is given and this invention is demonstrated in detail, this invention is not limited only to these examples.

Example 1 Expression of Prohibitin Protein in Melanocytes

[Step 1] Cell Line and Cell Culture

Melan-a cells, mouse immortalized melanin-producing cells, were replaced with RPMI 1640 medium containing 10% fetal bovine serum every 10 days until medium was exchanged every 3 days until 90% confluency was obtained. Incubated in a CO ₂ incubator.

[Step 2] Prohibition of prohibitn protein expressed in melanocytes

The melanocytes obtained in step 1 were washed with PBS, treated with RIPA buffer solution, and left for 30 minutes on ice to destroy the cells. Next, the supernatant was collected by centrifugation for 10 minutes at a gravity acceleration of 15,000 g, and the protein was quantified using BIO-RAD protein Dye Reagent ^™ .

[Step 3] Identification of prohibitn protein from protein obtained from melanocytes

50 ug of the protein obtained in step 2 was electrophoresed using a 10% SDS-PAGE gel, and then blotted onto PVDF (BIO-RAD) membrane at 250 m for 90 minutes. The blot was blocked with PBS containing 5% nonfat milk for 1 hour, followed by 1 hour with Prohibitin Primary Antibody (RDI), followed by anti-HRP (horse radish peroxidase) binding. Rabbit (anti-rabbit) IgG (Amersham) was used as a secondary antibody, and antibody reaction was carried out using an enhanced chemilumicescence (ECL) kit from Amersham. The reacted membrane was developed after being exposed to an x-ray film to confirm protein expression. At this time, the mouse adipose tissue (mouse adipose tissue) was used as a positive control group, the results are shown in Figure 1 below.

As can be seen in Figure 1, it was confirmed that the prohibitin protein is expressed in melan-a cells, which are melanogenesis cells near 36kD.

Example 2 Distribution of Prohibitin Protein Expressed in Melanocytes

[Step 1] Culture of Melanin-producing Cells

 The culture was carried out in the same manner as in Step 1 of Example 1.

[Step 2] Identification of Prohibitn in Melanin-producing Cells by Immunofluorescence Assay

The cells cultured in step 1 were fixed with 3.5% paraformaldehyde for 15 minutes and then washed three times with PBS. Next, the cell membrane was opened by treating PBS solution containing 0.1% Triton X-100 for 5 minutes, and then anti-prohibitin (anti-prohibitin) and mitotracker (primary antibody) as a primary antibody, and FITC as a secondary antibody. (Or Rhodamine) bound IgG (molecular probe) was used, the nucleus was stained with 0.1% DAPI solution. After mounting with an anti-fade solution (mounting), it was observed using a confocal laser scanning microscope. The results are shown in FIG. 2.

In FIG. 2, the green stained mitochondria and the red stained prohibitin were prohibitin, and the mitochondria and the expression site were overlapped and expressed in the nucleus and cytoplasm.

Example 3 Confirmation of Prohibitin and Melanophilin in Melanosite

[Step 1] Melanogenic Cell Culture

The culture was carried out in the same manner as in Step 1 of Example 1.

[Step 2] Search for protein that binds melanophilin

Using the melanin-producing cells cultured in step 1, cell lysates were obtained using the same method as in step 2 of Example 1. The thus prepared fusion was poured into a column cross-linked with recombinant melanophilin protein. After reacting at 4 ° C. for 3 hours, those which did not bind to the column were filtered out, the column was washed well with RIPA buffer, and the proteins bound to the column were separated by SDS electrophoresis. Next, SDS electrophoresis, each protein band seen in the gel was cut and reacted with trypsin for 16 hours to obtain a peptide, and the protein was found by mass spectrometry (MASS spectrometry). The results are shown in FIG. 3.

As shown in FIG. 3 below, a prohibitin protein was found in the binding protein, and a peptide shown in bold among all the prohibitin amino acid sequences described below was identified by mass spectrometry.

Prohibitin amino acid sequence

MAAKVFESIG K FGLALAVAG GVVNSALYNV DAGHR AVIFD RFRGVQDIVV

GEGTHFLIPW VQKPIIFDCR SRPRNVPVIT GSKDLQNVNI TLRILFRPVA

SQLPRIYTSI GEDYDERVLP SITTEILKSV VARFDAGELI TQRELVSRQV

SDDLTERAAT FGLILDDVSL THLTFGKEFT EAVEAKQVAQ QEAERARFVV

EKAEQQKKAA IISAEGDSKA AELIANSLAT AGDGLIELRK LEAAEDIAYQ

LSRSRNITYL PAGQSVLLQL PQ

[Step 3] Co-immunoprecipitation between prohibitin and melanophylline

In the same manner as in step 2, B16 melanocyte producing cell lysate, which is a cancer cell of a mouse, was obtained, and then, using a column in which antibodies of melanoophilin and prohibitin were combined, The binding protein was isolated in the same manner as in step. The protein thus obtained was electrophoresed using a 10% SDS-PAGE gel, and then blotted onto PVDF (BIO-RAD) membrane at 250 m for 90 minutes. The blot was blocked with PBS containing 5% nonfat milk for 1 hour, followed by 1 hour with prohibitin primary antibody (neomarker) and melanophylline primary antibody (self-made), and then horse radish (HRP) Anti-rabbit IgG (Amersham) and anti-mouse IgG (Amersham) bound to peroxidase were used as secondary antibodies, and antibodies were obtained using Amersham's enhanced chemilumicescence kit. Reacted. The reacted membrane was then exposed to an x-ray film and developed to confirm the binding of each protein (see FIG. 4).

Example 4 Inhibitory Effects of Melanosomes by Inhibiting Prohibitin Expression

[Step 1] Securing Prohibitin siRNA Sequences

The siRNA sequence for inhibiting the expression of prohibitin was obtained by purchasing validated siRNA sequences of Ambion and Invitrogen, and the sequences are as follows.

PHB1 siRNA sense template:

5'-GGG ACU CAU UUC CUC AUC C (dTdT) -3 '

PHB1 siRNA antisense template:

5'-GGA UGA GGA AAU GAG UCC C (dTdT) -3 '

PHB2 siRNA sense template:

5'-GCC AAA GUG UUU GAG UCC AUU GGA A-3 '

PHB2 siRNA antisense template:

5'-UUC CAA UGG ACU CAA ACA CUU UGG C-3 '

[Step 2] Confirmation of Prohibitin Expression Inhibition by Prohibitin siRNA

The prohibitin siRNA obtained in step 1 was transfected into lipofectamine 2000 (invitirogen) in melan-a cells cultured by the method described in step 1 of Example 1 above. Thereafter, melan-a cells were collected every 24, 48 and 96 hours, and the same method as described in step 2 and 3 of Example 1 was performed to confirm whether the expression of prohibitin protein was inhibited. The results are shown in FIG. 5.

As can be seen from the expression level of prohibitin protein in Figure 5b it was confirmed that the P1 sequence is well suppressed to show a protein expression amount of 40% or less within 24 hours.

[Step 3] Inhibitory Effects of Melanosomes on Melanogenic Cells by Inhibiting Prohibitin Expression

The melan-a cells obtained in step 2 were observed for the shape of the cells using an optical microscope. The results are shown in FIG. 6.

As can be seen in FIG. 6, the melanosomes are not evenly distributed throughout the cells in the cells treated with the prohibitin siRNA compared to the control group that did not receive the prohibitin siRNA with the melanosome, It was confirmed that melanosomes gathered around the nucleus.

As described above, the present invention revealed that the prohibitin protein is expressed in Melan-a cells, which are melanogenesis cells, and that the protein is expressed not only in the nucleus and mitochondria but also in the cytoplasm. Prohibitin protein is a protein that binds to melanophilin, which plays a major role in melanosomal migration, and when treated with siRNA that inhibits its expression, the melanosomal movement is disturbed and is driven around the nucleus. This provides the use of prohibitin as a target with whitening efficacy as a modulator to regulate the migration of melanosomes.

Claims (3)

A method for inhibiting melanosomal migration, characterized by inhibiting the movement of intracellular melanosomes by injecting prohibitin siRNA that inhibits the expression of prohibitin into melanocytes. A method for inhibiting melanosomal migration characterized by inhibiting the binding of prohibitin and melanocytes protein by injecting prohibitin siRNA that inhibits the expression of prohibitin into melanocytes. A skin whitening method through the method of claim 1 or 2.

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