U.S. patent application number 14/675321 was filed with the patent office on 2015-10-08 for modulation of melanogenesis by modification of tyrosinase by palmitoylation.
The applicant listed for this patent is ELC Management LLC. Invention is credited to Lieve DECLERCQ, Yoko Niki, Naoaki Saito, Daniel B. Yarosh.
Application Number | 20150283054 14/675321 |
Document ID | / |
Family ID | 50974888 |
Filed Date | 2015-10-08 |
United States Patent
Application |
20150283054 |
Kind Code |
A1 |
Yarosh; Daniel B. ; et
al. |
October 8, 2015 |
MODULATION OF MELANOGENESIS BY MODIFICATION OF TYROSINASE BY
PALMITOYLATION
Abstract
Compositions and methods for modulating melanogenesis by
modifying tyrosinase palmitoylation are provided.
Inventors: |
Yarosh; Daniel B.; (Merrick,
NY) ; DECLERCQ; Lieve; (Ekeren, BE) ; Niki;
Yoko; (Kakogawa, JP) ; Saito; Naoaki;
(Kakogawa, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ELC Management LLC |
Melville |
NY |
US |
|
|
Family ID: |
50974888 |
Appl. No.: |
14/675321 |
Filed: |
March 31, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14104412 |
Dec 12, 2013 |
|
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14675321 |
|
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61740048 |
Dec 20, 2012 |
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Current U.S.
Class: |
424/59 ;
424/62 |
Current CPC
Class: |
A61P 43/00 20180101;
A61P 17/00 20180101; A61K 8/66 20130101; A61K 38/08 20130101; A61Q
5/08 20130101; A61Q 17/04 20130101; A61K 38/005 20130101; A61K
45/06 20130101; A61K 2800/782 20130101; A61K 8/361 20130101; A61K
31/20 20130101; A61Q 5/10 20130101; A61Q 19/02 20130101; A61K 8/64
20130101; A61K 38/06 20130101 |
International
Class: |
A61K 8/66 20060101
A61K008/66; A61Q 5/08 20060101 A61Q005/08; A61Q 19/02 20060101
A61Q019/02; A61K 8/36 20060101 A61K008/36; A61Q 17/04 20060101
A61Q017/04 |
Claims
1. A method for reducing the appearance of melanin in mammalian
skin or hair in need of such reduction by treating the skin or hair
with a melanin-reducing effective amount at least one inhibitor of
PPT.
2. The method of claim 1 wherein the at least one inhibitor of PPT
is at least one peptide containing at least one palmitoylated amino
acid.
3. The method of claim 2 wherein the at least one inhibitor
comprises PP1.
4. The method of claim 2 wherein the at least one inhibitor
comprises PP2.
5. The method of claim 1, which comprises treating the skin or hair
with a composition comprising a melanin-reducing effective amount
at least one inhibitor of PPT in combination with at least one of a
further melanin-reducing ingredient, a sunscreen agent, or a
combination thereof.
6. A method for reducing the appearance of melanin in mammalian
skin or hair in need of such reduction by treating the skin or hair
with a melanin reducing-effective amount of at least one
palmitoylated peptide.
7. The method of claim 6 wherein the at least one palmitoylated
peptide is at least one peptide containing at least one
palmitoylated amino acid.
8. The method of claim 7 wherein the at least one palmitoylated
peptide comprises PP1.
9. The method of claim 7 wherein the at least one palmitoylated
peptide comprises PP2.
10. The method of claim 6, which comprises treating the skin or
hair with a composition comprising with a melanin-reducing
effective amount of at least one palmitoylated peptide in
combination with at least one of a further melanin-reducing
ingredient, a sunscreen agent, or a combination thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/104,412, filed Dec. 12, 2013, which claims priority to
U.S. Provisional Patent Application No. 61/740,048, filed Dec. 20,
2012.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to the regulation of melanin
synthesis. More specifically, the invention is concerned with
compositions and methods which modify the activity of
tyrosinase.
[0004] 2. Description of the Prior Art
[0005] Pigmentation of human skin and hair is a concern for people
around in the world. In some cases the desire is to have an overall
lighter complexion. Most generally people wish to have even skin
tone regardless of the underlying color. All people wish to delay
the appearance of grey hair, which is a sign of aging.
[0006] Pigmentation of skin and hair is controlled by the
specialized cell called a melanocyte. The melanocyte contains
within it an organelle called a melanosome which manufactures the
forms of melanin (including black eumelanin and red pheomelanin)
and then the melanocyte distributes it within the skin or hair. The
most important enzyme in the biosynthesis of melanin is tyrosinase,
and its inhibition has been the focus of most commercial products
that are designed to lighten skin color.
[0007] Tyrosinase is a metalloprotein that is synthesized at
ribosomes, trafficked through the Golgi apparatus to a
pre-melanosome, and then loaded into it. As the pre-melanosome
matures, copper molecules are also loaded into it as co-factors for
the tyrosinase, and the pH is increased to reach the optimum for
the enzyme. The mature melanosome then begins synthesizing
melanin.
[0008] The trafficking of tyrosinase is not well understood.
Recently, palmitoylation of proteins has received attention as a
mechanism of subcellular localization of mammalian proteins
(reviewed in T. Iwanaga, R. Tsutsumi, J. Noritake, Y. Fukata, M.
Fukata "Dynamic protein palmitoylation in cellular signaling,
Progress in Lipid Research 48:117-127, 2009; C. Salaun, J. Greaves,
L. Chamberlain "The intracellular dynamic of protein
palmitoylation" J. Cell Biol. 191:1229-1238, 2010; C. Aicart-Ramos,
R. Valero, I. Rodrigues-Crespo "Protein palmitoylation and
subcellular trafficking" Bioch. Biophys. Acta 1808:2981-2994.
2011). Tyrosinase is not disclosed as a target of palmitoylation in
any of these reviews.
BRIEF SUMMARY OF THE INVENTION
[0009] In accordance with one aspect of the invention, a method for
increasing the appearance of melanin in mammalian skin or hair in
need of such increase, comprising treating the skin or hair with
one or more inhibitors of DHHC 2, 3, 7, 15, 6, 11, 17, 21, or 22 is
provided.
[0010] In accordance with a second aspect of the invention, a
method for reducing the appearance of melanin in mammalian skin or
hair in need of such reduction by treating the skin or hair with
one or more inhibitors of PPT-type enzymes is provided.
[0011] In accordance with a third aspect of the invention, a method
is provided for reducing the appearance of melanin in mammalian
skin or hair in need of such reduction by treating the skin or hair
with at least one palmitoylated peptide.
[0012] In accordance with a fourth aspect of the invention, a
cosmetic or dermatological composition for increasing the
appearance of melanin in mammalian skin or hair, comprising a
melanin increasing-effective amount of one or more inhibitors of
DHHC 2, 3, 7, 15, 6, 11, 17, 21, or 22, in a cosmetically or
dermatologically acceptable vehicle is provided.
[0013] In accordance with a fifth aspect of the invention, there is
provided a cosmetic or dermatological composition for reducing the
appearance of melanin in mammalian skin or hair, comprising a
melanin reducing-effective amount of one or more inhibitors of
PPT-type enzymes in a cosmetically or dermatologically acceptable
vehicle.
[0014] In accordance with a sixth aspect of the invention, a
cosmetic or dermatological composition for reducing the appearance
of melanin in mammalian skin or hair comprising a melanin
reducing-effective amount of at least one palmitoylated peptide in
a cosmetically or dermatologically acceptable vehicle is
provided.
[0015] Other aspects and objectives of the present invention will
become more apparent from the ensuing description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1 is a schematic representation of the cycle of protein
palmitoylation.
[0017] FIGS. 2A and 2B depict the effect of palmitoylation
inhibitor (2-BP) on palmitoylation of tyrosinase.
[0018] FIGS. 3A and 3B represent the effect of 2-BP on melanin
synthesis in NHEM and reconstructed human skin model.
[0019] FIGS. 4A and 4B illustrate the effect of 2-BP on mRNA
expression and glycosylation of tyrosinase.
[0020] FIGS. 5A and 5B represent the effect of 2-BP on degradation
of tyrosinase.
[0021] FIG. 6 depicts the effect of 2-BP on ubiquitination of
tyrosinase.
[0022] FIG. 7 represents the effect of 2-BP on tyrosinase
palmitoylation.
[0023] FIG. 8 is an illustration of the DHHC (Palmitoylation)
family of proteins.
[0024] FIG. 9 represents the results of screening for tyrosinase
specific DHHC.
[0025] FIGS. 10A and 10B illustrate the effect of DHHC 2, 3, 7 and
15 silencing on tyrosinase palmitoylation in MNT-1 cell.
[0026] FIGS. 11A and 11B represent analysis of the intracellular
localizations of DHHC 2, 3, 7 and 15-myc in NHEM.
[0027] FIGS. 12A and 12B illustrate the Palmitoylation site in
human tyrosinase.
[0028] FIGS. 13A and 13B illustrate the Palmitoylation site in
human tyrosinase.
[0029] FIGS. 14A-14D depict the effect of PP-2 and KA on melanin
synthesis in NHEM.
[0030] FIG. 15 represents the effect of PP-2 and KA on human
tyrosinase (DOPA oxidation) activity in vitro.
[0031] FIG. 16 depicts the effect of PP-2 on tyrosinase protein in
NHEM.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
[0032] Protein palmitoylation or S-acylation is a reversible
post-translational lipid modification that affects the localization
and activity of many proteins. As indicated schematically in FIG.
1, palmitoylation is an enzymatic process of attaching a palmitic
acid group to a cysteine residue of a protein accomplished by a
palmitoyl acyl transferase (PAT). PATs share a DHHC domain
(aspartate-histidine-histidine-cysteine signature motif) and there
are 23 or 24 separate DHHC-containing proteins in mammalian cells,
each with its own substrate specificity. The consensus sequence for
the target of the DHHC-containing proteins is not known, other than
it contains a cysteine. Dozens of proteins are palmitoylated and
the list is not yet complete. DHHC-containing proteins are mainly
localized in the Golgi membrane where they cause relocation of
target proteins by palmitoylation shortly after their
synthesis.
[0033] Palmitoylation is enzymatically reversible by acyl-protein
thioesterases (APTs) and palmitoyl-protein thioesterases (PPTs),
which are ubiquitously located in the cytosol. There appear to be
several APTs but only two PPTs in mammalian cells. Palmitoylated
proteins are trafficked away from Golgi and attach to endosomes,
lysozomes or plasma membranes. As a consequence of relocation they
may be degraded (e.g. in lysozomes) or they can become substrates
for PPT, change their localization and/or return to the Golgi for
re-palmitoylation. In this way, the relative activity of a DHHC PAT
and the APT/PPTs determine the localization of a protein, whether
more concentrated near the Golgi or dispersed to endosome or plasma
membrane. The system is dynamic, with proteins constantly shuttled
back and forth and being degraded.
[0034] Inhibitors of Palmitoyl:protein thioesterase (PPT1) are
described by Dawson et al. (Dawson, G., Schroeder, C., Dawson, P.
Palmitoyl:protein thioesterase (PPT1) inhibitors can act as
pharmacological chaperones in infantile Batten Disease. Bioch.
Biophys. Res. Comm. 395:66-69, 2010.) Inhibitors with the
N-terminal addition of the lipophilic fluorophore NBD, CS38
(NBD-.beta.AGDap(Pal)VKIKK), was a 3-fold better inhibitor than
Dap1 (AcGDap(Pal)VKIKK). CS38 was the most potent peptide inhibitor
with an IC.sub.50 of 2 .mu.M. The NBD form of the authentic thiol
(CS8: GGC(Pal) VKIKK) had comparable inhibitory activity to CS38.
Additional peptides with TAT like polyarginine (R.sub.7) tails
(AcGDap(Palm)GGR.sub.7) designed to promote cellular uptake, were
also strong PPT1 inhibitors (for example AcGDap(Pal)GG)R)7). Any
truncation of the VKIKK eliminated inhibitory activity. Inhibition
of APTs is much less effective in blocking melanogenesis.
[0035] Virtually nothing is known about the role of palmitoylation
in melanogenesis. In a genome-wide screen of genes that influence
melanogenesis, the gene ZDHHC9 was identified as one in which
inhibition by siRNA reduced melanogenesis (A. Ganesan, H. Ho, B.
Bodemann, S. Petersen, J. Aruri, S. Koshy, Z. Richardson, L. Le, T.
Krasieva, M. Roth, P. Farmer, M. White "Genome-wide siRNA
functional genomics of pigmentation identifies novel genes and
pathways that impact melanogenesis in human cells". PLOS Genetics
4(12):e1000298, doi:10.1371/journal.pgen.1000298, 2008). The siRNA
to this PAT inhibited MITF (microthalmia-associated transcription
factor) RNA and tyrosinase protein accumulation. The siRNA
inhibition of tyrosinase protein was reversed by incubation with
bafilomycin, which raises the pH of lysozomes and inhibits protein
degradation. They suggest that their screening method identifies
novel genes that impact melanosome trafficking/sorting of
melanosome protein cargo. However, they imply that inhibition of
palmitoylation should reduce melanogenesis. In fact, as uncovered
in the present invention, general inhibition of palmitoylation
increases melanogenesis, and PAT DHHC9 is not a specific modifier
of tyrosinase.
[0036] Wu et al. studied palmitoylation of melanoregulin, a protein
that, in its palmitoylated form, prevents the transfer of melanin
from melanocytes to keratinocytes, (X. Wu, J. Martina, J. Hammer
"Melanoregulin is stably targeted to the melanosome membrane by
palmitoylation" Bioch. Biophys. Res. Comm. 426:209-214.)
Palmitoylation localizes the melanoregulin protein to melanosomes
thereby inhibiting the transfer of melanin from melanocytes. It was
observed that inhibition of palmitoylation of melanoregulin reduces
its accumulation at lysozomes and, by inference, at melanosomes. Wu
et al. do not present any evidence regarding melanogenesis or
tyrosinase.
[0037] The present invention resides in the surprising discovery by
the inventors that palmitoylation/depalmitoylation of tyrosinase
plays a role in melanogenesis. More specifically, the inventors
have discovered that the palmitoylation of tyrosinase in
melanocytes, by PATs (Palmitoyl-acyl transferases or DHHCs), leads
to trafficking of tyrosinase away from melanosomes, resulting in a
decrease in the appearance of melanin in skin and hair. The
inventors theorize that the altered localization of tyrosinase away
from the melanosome leads to its degradation. Furthermore, the
inhibition of the palmitoylation of tyrosinase, that is, the
inhibition of PATs, presumably results in the retention of
tyrosinase in the melanosomes, and in the increased appearance of
melanin in skin and hair. Compounds which inhibit PATs include
2-bromopalimitate (2-BP). As shown in the examples, below, the
inventors have determined that the PATs responsible for the
tyrosinase palmitoylation are DHHC 2, 3, 7 and 15, while DHHC 6,
11, 17, 21, and 22 show weak affinity, and DHHC 9 does not appear
to palmitoylate tyrosinase. It was further ascertained that
tyrosinase is palmitoylated on cysteine 500, since mutating this
amino acid to alanine blocks palmitoylation of the enzyme.
[0038] The inventors have further discovered, as shown in the
examples below, that inhibiting PPTs (palmitoyl-protein
thioesterases), which are responsible for the de-palmitoylation of
tyrosinase, leads to increased degradation of tyrosinase and a
decrease in the appearance of melanin in skin and hair. PPTs are
inhibited by palmitoylated peptides; that is, peptides containing a
palmitoylated amino acid. Exemplary peptides useful for the
purposes of the present invention include Palmitoylated peptide-1
or PP1 (Glyceryl Polymethacrylate (and) PEG-8 (and) Palmitoyl
Oligopeptide, containing 100 ppm of the palmitoylated hexapeptide
Palmitoyl-val-gly-val-ala-pro-glu), available as Biopeptide EL from
Sederma, and Palmitoylated peptide-2 or PP2 (water (and) Butylene
Glycol (and) Dextran (and) Palmitoyl Tripeptide-8, containing 500
ppm of a palmitoylated tri-peptide consisting of arginine,
histidine, and phenylalanine), available as Neutrazen from Lucas
Meyer Cosmetics.
[0039] The inhibitors of DHHC, PPT and/or APT may be used either
alone or in combination with other inhibitors or promoters of
pigmentation, such as hydroquinone, kojic acid and the like. These
inhibitors can be combined with other cosmetically or
dermatologically useful compounds such as sunscreens,
anti-inflammatories, vitamin A and its derivatives, vitamin C and
its derivatives and other vitamins, trace minerals and nutrients.
The inhibitors can be mixed with common delivery forms for skin,
such as emulsions, lotions, creams, serums, hydrogels, polymers,
silicones, and the like. They can be combined with fragrances,
anti-microbial compounds, preservatives and antioxidants.
EXAMPLES
Example 1
Effect Of Palmitoylation Inhibitor 2-BP On Palmitoylation Of
Tyrosinase
[0040] Normal human epidermal melanocytes (NHEM) were incubated
with 2-bromopalmitate (2-BP) for 24 hours, and total tyrosinase was
determined by Western-blot (WB) using the mouse monoclonal
anti-tyrosinase IgG antibody T311 (1:120 dilution). Total
tyrosinase protein level was normalized by tubulin. Palmitoylated
tyrosinase was evaluated by acyl-RAC and WB. The results shown in
FIGS. 2A and 2B, are the average of three determinations SE.
*p<0.05 vs. DMSO.
[0041] Incubation of NHEM with various concentrations of 2-BP, an
inhibitor of palmitoylation, compared with a DMSO control, resulted
in a statistically significant increase in the total amount of
tyrosinase protein per cell. Additionally, while the amount of
total protein increased, the amount of palmitoylated tyrosinase
decreased.
Example 2
Effect Of 2-BP On Melanin Synthesis In NHEM And Reconstructed Human
Skin Model
[0042] NHEM were treated with indicated concentrations of 2-BP for
48 hours. Melanin content (closed bar) and cell number (open bar)
were measured. Values are the average of three
determinations.+-.SE. *p<0.05 vs. DMSO. Macroscopic views of
cell pellet were taken by digital camera (FIG. 3A). Reconstructed
human skin models (Asian type) were incubated with 25 .mu.M 2-BP
for 17 days. Eumelanin (EM) and pheomelanin (PM) contents were
determined and calculated by following formula. EM=PTCA*25,
PM=4-AHP*9. Values are the average of three determinations.+-.SE.
*p<0.05 vs. DMSO. Macroscopic views of a human skin model after
17 days were taken by digital camera (FIG. 3B).
[0043] Incubation of NHEM with 2-BP at various concentrations,
compared with a DMSO control, resulted in a statistically
significant increase in melanin content per cell. Furthermore,
incubating human skin models with 2-BP also resulted in a
statistically significant increase in total melanin: eumelanin (EM)
and pheomelanin (PM) content of the tissue.
Example 3
[0044] Effect Of 2-BP On mRNA Expression And Glycosylation Of
Tyrosinase
[0045] NHEM were cultured with 2-BP for 6 or 24 hours. Tyrosinase
mRNA levels were analyzed by real time quantitative PCR. The
results shown are the average of three determinations.+-.SE.
*p<0.05 vs. DMSO (FIG. 4A). NHEM were incubated with 5 .mu.M
2-BP for 24 hours. Glycosidase digestion treatment by endo H and WB
analysis on mature and immature form of tyrosinase were conducted
by WB (FIG. 4B). It was observed that incubation of NHEM with 2-BP
resulted in a decrease of tyrosinase mRNA expression, but had no
effect on the glycosylation of tyrosinase.
Example 4
Effect Of 2-BP On Degradation Of Tyrosinase
[0046] NHEM were treated with 1 .mu.g/ml cycloheximide, protein
synthesis inhibitor, and with (.box-solid.) or without (.diamond.)
10 .mu.M 2-BP for 4 hours. Tyrosinase levels were analyzed by WB
using the mouse monoclonal anti-tyrosinase IgG antibody T311 (1:120
dilution). The band intensities of tyrosinase were normalized by
the band intensities of Glyceraldehyde 3-phosphate dehydrogenase
(GAPDH) as internal control for each condition. The results shown
are the average of three determinations.+-.SE. *p<0.05 vs. DMSO
(FIGS. 5A and 5B).
[0047] As shown in the figures, when NHEM are treated with
cycloheximide, a protein synthesis inhibitor, tyrosinase levels are
decreased. However, the additional presence of 2-BP in the
incubation medium suppresses the effect of the cycloheximide,
indicating that 2-BP suppresses the degradation of tyrosinase.
Example 5
Effect Of 2-BP On Ubiquitination Of Tyrosinase
[0048] NHEM were treated with 5 .mu.M 2-BP for 24 hours. Equal
protein amounts of cell lysate were used for IP by tyrosinase, and
the ubiquitinated (UB) level of tyrosinase was analyzed by WB using
anti-UB antibody.
[0049] The results, shown in FIG. 6 indicate that 2-BP has no
effect on ubiquitination of tyrosinase. Thus, it was observed, as
indicated in the schematic in FIG. 7, that while 2-BP suppresses
tyrosinase degradation and results in activation of melanin
synthesis, the ubiquitin proteasomal system does not participate in
this phenomenon.
Example 6
Screening Of Tyrosinase-Specific DHHC
[0050] As indicated hereinabove, PATs share a DHHC-rich domain and
can be clustered into genetically conserved DHHC family proteins
(Tsutsumi R, Fukata Y, Fukata M "Discovery of
protein-palmitoylating enzymes", Eur J Physiol. 2008 September;
456(6):1199-206.). This is shown schematically in FIG. 8. To
elucidate which of these DHHCs is involved in tyrosinase
palmitoylation, HEK293T cells were transfected with tyrosinase and
individual DHHCs. After metabolic labeling with [3H]palmitate,
proteins were separated by SDS-PAGE (FIG. 9). The upper panel shows
WB by anti-tyrosinase antibody; the lower panel shows an
autoradiograph. DHHC 3, 7, 15 each show a strong effect, and DHHC
2, 6, 11, 17, 21, 22 each show a weak effect, on tyrosinase
palmitoylation.
Example 7
Effect Of DHHC 2, 3, 7, And 15 Silencing On Tyrosinase
Palmitoylation In MNT-1 Cells
[0051] To assess whether DHHC 2, 3, 7 and/or 15 participate in
tyrosinase palmitoylation, the inventors assessed the effect of
knocking down DHHC 2, 3, 7 or 15 on tyrosinase. MNT-1 cells were
transfected with siRNA against DHHC 2, 3, 7 and 15 or control
siRNA. After 3 days, siRNA transfection was repeated. At 6 days
after the initial transfection, tyrosinase palmitoylation was
analyzed by acyl-RAC and WB. The results (FIG. 10) show the average
of six determinations.+-.SE. *p<0.05 vs. DMSO. Results indicate
that DHHC 2, 3, 7 and 15 may participate in tyrosinase
palmitoylation. The reductions observed in the amounts of
palmitoylated tyrosinase using siRNA to DHHC 2 and 15 are
statistically significant.
Example 8
Analysis Of The Intracellular Localizations Of DHHC 2-, 3-, 7- And
15-myc In NHEM
[0052] NHEM (Asian) transfected with DHHC 2, 3, 7 and 15 with myc
tags were stained with anti-myc antibody and also labeled with
antibodies against Vtilb (Golgi body) or HMB45 (melanosome).
Results are shown in FIGS. 11A and 11B. DHHC 2 was found to be
localized in the melanosome, the plasma membrane, and in the Golgi
body. Also found to be localized in the Golgi body were DHHC 7 and
15. DHHC 3 was found to be localized in the endoplasmic reticulum
(ER).
Example 9
Palmitoylation Site In Human Tyrosinase
[0053] MNT-1 cells were transfected with a wild-type tyrosinase or
one of three tyrosinases, mutated at one of three key cysteine
codons, which resulted in the replacement of cysteine with alanine,
as follows: CBA, C35A or C500A. After 48 hours, palmitoylation of
tyrosinase was analyzed by acyl-RAC and WB. Results shown in FIG.
12A indicate that mutation in the codon for Cys500 results in a
loss of tyrosinase palmitoylation, indicating that Cys500 is a
palmitoylation site in human tyrosinase. Prediction of the most
likely S-palmitoylation sites in human tyrosinase by software
CSS-Palm 3.0, as shown in FIG. 12B, supports the finding.
Example 10
Palmitoylation Site In Human Tyrosinase
[0054] HEK293T cells were transfected with tyrosinase and
individual DHHC. After 4 hour metabolic labeling with
[3H]palmitate, proteins were separated by SDS-PAGE. In each of
FIGS. 13A and 13B, the upper panel is an autoradiograph and the
lower panel shows a WB analysis using anti-tyrosinase antibody. The
results indicate that tyrosinase with mutation of C500A was not
palmitoylated by DHHC 2, 3, 7 or 15 (white asterisks), further
supporting the observation in Example 9, above, that the
palmitoylation site of tyrosinase is Cys500.
Example 11
Effect Of PP2 On Melanin Synthesis In NHEM
[0055] NHEM (Asian) were incubated for 120 hours in the presence of
PP2, with or without 2-BP. PP2 inhibited melanin synthesis both in
2-BP-treated cells and in cells not treated with 2-BP without
cytotoxicity (FIGS. 14A and 14B). Mean.+-.S.E. (N=3)*p<0.05, vs.
control.
[0056] Kojic Acid (KA) was used as a positive control. As shown in
FIGS. 14C and 14D, KA mildly inhibited melanin synthesis both in
2-BP treated cells and cells not treated with 2-BP.
Example 12
Mechanism Of Action Of PP2 On Melanogenesis: Effect Of PP2 On
Melanin Synthesis In NHEM
[0057] As shown in FIG. 15, PP2 did not inhibit human tyrosinase
(DOPA oxidation) activity in vitro, while Kojic acid (positive
control) inhibited tyrosinase activity in a dose dependent manner.
Mean .+-.S.E. (N=4) * p<0.05, vs. control.
Example 13
Mechanism Of Action Of PP2 On Melanogenesis: Effect Of PP2 On
Tyrosinase Protein Level In NHEM
[0058] NHEM were incubated for 48 hours in the presence of PP2 with
or without 2-BP. As shown in FIG. 16, PP2 decreased tyrosinase
protein level in 2-BP-treated human NHEM (Asian). Mean.+-.S.E.
(N=3) * p<0.05, vs. control.
[0059] While the present invention has been described hereinabove
with reference to specific embodiments, features and aspects, it
will be recognized that the invention is not thus limited, but
rather extends in utility to other modifications, variations,
applications, and embodiments, and accordingly all such other
modifications, variations, applications, and embodiments are to be
regarded as being within the spirit and scope of the present
invention.
* * * * *