Journal Information
Vol. 97. Issue 6.
Pages 808-814 (01 November 2022)
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...
Vol. 97. Issue 6.
Pages 808-814 (01 November 2022)
Research Letter
Open Access
Proteomic study of facial melasma
Visits
...
Luiza Vasconcelos Schaefera,b,
Corresponding author
luizavasconcelos12@hotmail.com

Corresponding author.
, Leticia Gomes de Pontesc,d, Nayara Rodrigues Vieira Cavassanc,d, Lucilene Delazari dos Santosc,d, Hélio Amante Miote
a Department of Pathology, Faculty of Medicine, Universidade Estadual Paulista, Botucatu, SP, Brazil
b Department of Dermatology, Universidade do Oeste Paulista, São Paulo, SP, Brazil
c Department of Research, Centro de Estudos de Venenos e Animais Peçonhentos, Universidade Estadual Paulista, Botucatu, SP, Brazil
d Postgraduate Program in Tropical Diseases, Faculty of Medicine, Universidade Estadual Paulista, Botucatu, SP, Brazil
e Department of Dermatology, Faculty of Medicine, Universidade Estadual Paulista, Botucatu, SP, Brazil
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Dear Editor,

Melasma is hypermelanosis that affects photoexposed areas, especially in adult women, with a significant impact on quality of life by affecting visible areas and being recurrent, despite treatments. Its pathophysiology is not yet fully understood, but it results from the interaction between exposure factors (e.g., solar radiation and sex hormones) and genetic predisposition. Several dermal stimuli have been identified in the maintenance of melanogenesis in melasma, including the activity of fibroblasts, endothelium and mast cells, which promote elastonization of collagen, structural damage to the basement membrane, the release of growth factors (e.g., sSCF, bFGF, NGF, HGF) and inflammatory mediators (e.g., ET1, IL1, VEGF, TGFb).1–3

This study aimed to explore differentially exposed proteins in melasma skin when compared to adjacent, unaffected, photoexposed skin.

A cross-sectional study was carried out involving 20 women with facial melasma, without specific treatments for 30 days. Two biopsies were performed (by the same researcher), one at the edge of facial melasma and another on unaffected skin, 2 cm away from the first, as previously standardized.1,3 The mechanical extraction of proteins was performed, followed by their enzymatic digestion and mass spectrometry. The project was approved by the institutional ethics committee (n. 1,411,931).

The samples were analyzed in duplicate in the nanoACQUITY-UPLC system coupled to a Xevo-Q-TOF-G2 mass spectrometer, and the results were processed with the ProteinLynx Global Server 3.03v software. The proteins were identified using the ion-counting algorithm, whose spectral patterns were searched in the Homo sapiens database, in the UniProt catalog (https://www.uniprot.org/).

All identified proteins with >95% similarity were included in the analysis. The intensities of the ion peaks were normalized, scaled and compared between topographies by a Bayesian algorithm (Monte Carlo method), which returns a value of p ≤ 0.05 for down-regulated proteins and ≥0.95 for up-regulated proteins, corrected by the Benjamini-Hochberg procedure.4

The main outcome of the study was the difference between the intensities of the ionic peaks of the proteins (Melasma: M, Perilesional: P). The effect size was estimated by the ratio of these amounts between topographies (M/P). Proteins with an M/P ratio of ≤0.5 or ≥2.0 were considered in this study.

The identified proteins and their biological functions were diagrammed in a heat map and grouped using the cluster procedure (Ward method).

The mean age (standard deviation) of the patients was 42.8 (8.9) years old, 70% were phototypes III‒IV and 25% worked in professions in which they were exposed to the sun. The age of melasma onset was 29.3 (7.5) years; 55% of the women reported a family history and 30% used contraceptives.

A total of 256 proteins were validated in the skin samples, and the 29 proteins differentially quantified between the topographies are shown in Table 1. The greatest discrepancies occurred for proteins HBD, EXPH5, KRT1, KRT9, REV3L (M/S > 4,00); and ACAP9, ADGB, CA1 (M/S < 0.33).

Table 1.

Proteins and isoforms identified in samples of facial melasma (M) and adjacent photoexposed (P) skin (n = 40) with the difference between the groups (p ≤ 0.05 or ≥0.95) and M/P ratio ≥2.0 or ≤0.5.

Protein code  Protein  PLGS score  Melasma  Perilesional  Log2 M/P (sd)  M/P ratio  p-Valuea 
P1  Actin Alpha Skeletal Muscle ACTA1  958.81  1.34 (0.04)  0.66 (0.04)  1.04 (0.07)  2.05  1.00 
P2Actin Cytoplasmic 2 ACTG1  1164.96  1.40 (0.06)  0.60 (0.06)  1.23 (0.11)  2.34  1.00 
Actin Cytoplasmic 2 ACTG1  1164.96  1.40 (0.07)  0.60 (0.07)  1.24 (0.12)  2.36  1.00 
P3A-Kinase Anchor Protein 13 AKAP13  87.85  1.53 (0.40)  0.47 (0.40)  1.96 (1.03)  3.90  0.97 
A-Kinase Anchor Protein 13 AKAP13  87.85  1.55 (0.37)  0.45 (0.37)  1.99 (1.13)  3.97  0.95 
P4A-kinase Anchor protein 9 AKAP9  12.43  0.35 (0.34)  1.65 (0.34)  −2.39 (1.00)  0.19  0.03 
A-kinase Anchor protein 9 AKAP9  13.08  0.38 (0.20)  1.62 (0.20)  −2.16 (0.50)  0.22  0.00 
A-kinase Anchor protein 9 AKAP9  16.30  0.40 (0.24)  1.60 (0.24)  −2.06 (0.55)  0.24  0.00 
P5Albumin isoform CRA k ALB  542.99  0.62 (0.05)  1.38 (0.05)  −1.14 (0.08)  0.45  0.00 
Serum albumin ALB  5862.44  0.54 (0.18)  1.46 (0.18)  −1.44 (0.32)  0.37  0.00 
Serum albumin ALB  542.99  0.63 (0.04)  1.37 (0.04)  −1.14 (0.07)  0.45  0.00 
Serum albumin ALB  542.99  0.52 (0.07)  1.48 (0.07)  −1.50 (0.12)  0.35  0.00 
Serum albumin ALB  2923.53  0.62 (0.10)  1.38 (0.10)  −1.17 (0.17)  0.44  0.00 
Serum albumin ALB  542.99  0.63 (0.06)  1.37 (0.06)  −1.11 (0.10)  0.46  0.00 
Serum albumin ALB  486.61  0.64 (0.06)  1.36 (0.06)  −1.07 (0.09)  0.48  0.00 
Serum albumin ALB  534.91  0.66 (0.08)  1.34 (0.08)  −1.04 (0.12)  0.49  0.00 
P6  Alpha-1-antitrypsin SERPINA1  1092.60  1.33 (0.13)  0.67 (0.13)  1.00 (0.21)  2.00  1.00 
P7  Androglobin ADGB  69.19  0.48 (0.21)  1.52 (0.21)  −1.69 (0.40)  0.31  0.05 
P8Annexin ANXA2  117.73  1.33 (0.25)  0.67 (0.25)  1.01 (0.41)  2.01  0.95 
Annexin ANXA2  117.73  1.33 (0.26)  0.67 (0.26)  1.02 (0.44)  2.03  0.97 
Annexin ANXA2  117.73  1.34 (0.26)  0.66 (0.26)  1.05 (0.43)  2.08  0.95 
Annexin ANXA2  117.73  1.35 (0.28)  0.65 (0.28)  1.08 (0.43)  2.12  0.95 
P9  Beta-actin-like protein 2 ACTBL2  101.00  1.43 (0.07)  0.57 (0.07)  1.34 (0.12)  2.53  1.00 
P10  BTB/POZ domain-containing protein KCTD7  53.81  1.57 (0.17)  0.43 (0.17)  1.90 (0.40)  3.74  1.00 
P11Carbonic Anhydrase 1 CA1  1112.39  0.39 (0.17)  1.61 (0.17)  −2.09 (0.45)  0.23  0.00 
Carbonic Anhydrase 1 CA1  1386.45  0.47 (0.13)  1.53 (0.13)  −1.70 (0.27)  0.31  0.00 
P12Ceruloplasmin CP  76.55  1.40 (0.17)  0.60 (0.17)  1.23 (0.30)  2.34  1.00 
Ceruloplasmin CP  85.85  1.37 (0.17)  0.63 (0.17)  1.13 (0.29)  2.18  1.00 
Ceruloplasmin CP  85.85  1.43 (0.15)  0.57 (0.15)  1.34 (0.27)  2.53  1.00 
P13DNA polymerase zeta catalytic subunit REV3L  73.85  1.58 (0.34)  0.42 (0.34)  2.03 (0.86)  4.10  0.95 
DNA polymerase zeta catalytic subunit REV3L  153.25  1.52 (0.06)  0.48 (0.06)  1.66 (0.12)  3.16  1.00 
P14  Exophilin-5 EXPH5  40.12  1.79 (0.12)  0.21 (0.12)  3.16 (0.48)  8.94  1.00 
P15  Fibrinogen Gamma chain FGG  211.06  1.33 (0.16)  0.67 (0.16)  1.01 (0.25)  2.01  1.00 
P16Fibrinogen Gamma chain FGG  211.06  1.34 (0.14)  0.66 (0.14)  1.04 (0.24)  2.05  1.00 
Fructose-bisphosphate Aldolase A ALDOA  153.06  1.46 (0.07)  0.54 (0.07)  1.43 (0.14)  2.69  1.00 
Fructose-bisphosphate aldolase A ALDOA  296.85  1.45 (0.07)  0.55 (0.07)  1.41 (0.13)  2.66  1.00 
Fructose-bisphosphate aldolase ALDOA  295.30  1.46 (0.10)  0.54 (0.10)  1.43 (0.17)  2.69  1.00 
Fructose-bisphosphate aldolase ALDOA  295.30  1.47 (0.08)  0.53 (0.08)  1.46 (0.14)  2.75  1.00 
P17G Patch domain-containing protein 1 GPATCH1  95.48  1.37 (0.14)  0.63 (0.14)  1.13 (0.25)  2.18  1.00 
G patch domain-containing protein 1 GPATCH1  88.85  1.60 (0.28)  0.40 (0.28)  2.15 (0.66)  4.44  1.00 
P18  Heat shock protein 75 kDa mitochondrial TRAP1  124.78  1.43 (0.13)  0.57 (0.13)  1.34 (0.24)  2.53  1.00 
P19  Hemoglobin subunit alpha HBA1  8552.23  1.57 (0.02)  0.43 (0.02)  1.88 (0.04)  3.67  1.00 
P20  Hemoglobin subunit beta HBB  91.85  0.65 (0.05)  1.35 (0.05)  −1.07 (0.08)  0.48  0.00 
P21  Hemoglobin subunit delta HBD  42.06  1.94 (0.02)  0.06 (0.02)  5.05 (0.31)  33.12  1.00 
P22Keratin type I cytoskeletal 9 KRT9  340.12  1.61 (0.13)  0.39 (0.13)  2.05 (0.27)  4.14  1.00 
Keratin type I cytoskeletal 9 KRT9  190.36  1.60 (0.26)  0.40 (0.26)  2.06 (0.62)  4.18  1.00 
P23  Keratin type II cytoskeletal 1 KRT1  55.74  1.62 (0.06)  0.38 (0.06)  2.08 (0.14)  4.22  1.00 
P24  POTE ankyrin domain family member F POTEF  101.00  1.47 (0.07)  0.53 (0.07)  1.49 (0.14)  2.80  1.00 
P25  Putative beta-actin-like protein 3 POTEKP  101.00  1.47 (0.09)  0.53 (0.09)  1.47 (0.16)  2.77  1.00 
P26  RNA-binding protein 25 RBM25  29.17  1.57 (0.12)  0.43 (0.12)  1.86 (0.25)  3.63  1.00 
P27  Splicing Regulatory glutamine/Lysine-rich protein 1 SREK1  79.89  1.41 (0.26)  0.59 (0.26)  1.28 (0.48)  2.44  1.00 
P28Tetratricopeptide repeat protein 37 TTC37  443.53  1.45 (0.21)  0.55 (0.21)  1.44 (0.42)  2.72  1.00 
Tetratricopeptide repeat protein 37 TTC37  449.05  1.46 (0.18)  0.54 (0.18)  1.47 (0.36)  2.77  1.00 
P29Triosephosphate isomerase TPI1  475.34  1.39 (0.25)  0.61 (0.25)  1.23 (0.41)  2.34  0.97 
Triosephosphate isomerase TPI1  475.34  1.41 (0.22)  0.59 (0.22)  1.28 (0.40)  2.44  0.97 
Triosephosphate isomerase TPI1  576.90  1.43 (0.19)  0.57 (0.19)  1.34 (0.37)  2.53  1.00 
a

p-Value corrected by false discovery rate.

The main biological functions of these proteins are shown in Table 2. Fig. 1 represents the interaction between the 29 proteins and their biological functions. Proteins ACTG1, ALB, SERPINA1, HBD, ALDOA, and FGG showed to be co-participants in different biological processes, such as oxygen consumption, glycolysis, gluconeogenesis, and cell transport, suggesting an increase in the metabolic activity of the skin with melasma.

Table 2.

Main functional pathways associated with the 29 proteins identified as differentials between melasma and perilesional skin.

Functions  Involved proteins  n (%)  FDRa 
1. Canonical glycolysis  p16, p29  2 (7)  <0.0001 
2. Gluconeogenesis  p16, p29  2 (7)  <0.0001 
3. Fibrinolysis  p8, p15, p23  2 (10)  <0.0001 
4. Platelet degranulation  p2, p5, p6, p15, p16  5 (17)  <0.0001 
5. Regulation of body fluids  p2, p5, p6, p8, p15, p16, p21, p22, p23  8 (28)  <0.0001 
6. Oxygen transport  p7, p19, p20, p21  4 (14)  <0.0001 
7. Vesicle-mediated transport  p2, p5, p6, p10, p14, p15, p16, p19, p20  9 (31)  <0.0001 
8. Platelet activation  p5, p6, p15, p16  4 (14)  <0.0001 
9. Positive regulation of cell adhesion  p15  1 (3)  0.0001 
10. Hemostasis  p2, p5, p6, p15, p16, p21  6 (20)  0.0001 
11. Platelet aggregation  p2, p15  2 (7)  0.0002 
12. Plasminogen activation  p15  1 (3)  0.0003 
13. Single-organism transport  p2, p5, p6, p7, p8, p10, p11, p12, p14, p15, p16, p19, p20, p21  14 (48)  0.0004 
14. Blood clotting  p2, p5, p6, p15, p16, p21  6 (21)  0.0008 
15. Error-prone translesion synthesis  p13  1 (3)  0.0010 
16. Protein activation cascade  p15, p23  2 (7)  0.0014 
17. Retinal homeostasis  p2, p5, p23, p24  4 (14)  0.0015 
18. Down-regulation of trauma response  p8, p10, p15  3 (10)  0.0015 
19. Up-regulation of exocytosis  p14, p15  2 (7)  0.0016 
20. Regulation of exocytosis  p10, p14, p15  3 (10)  0.0017 
21. Down-regulation of endothelial cell apoptosis process  p15  1 (3)  0.0022 
22. Blood clotting, fibrin clot formation  p15  1 (3)  0.0024 
23. Down-regulation of the extrinsic apoptosis signaling pathway through the receptor death domain  p15  1 (3)  0.0024 
24. Transport  p2, p4, p5, p6, p7, p10, p11, p12, p15, p16, p19, p20, p21  13 (45)  0.0029 
25. Monocarboxylic Acid Metabolic Process  p5, p16, p29  3 (10)  0.0030 
26. Regulation of adhesion-dependent cell spread  p15  1 (3)  0.0033 
27. Wound healing  p2, p5, p6, p15, p16, p21  6 (20)  0.0035 
28. Bicarbonate transport  p11, p19, p20  3 (10)  0.0044 
29. Up-regulation of vasoconstriction  p15  1 (3)  0.0044 
30. Response to calcium ion  p2  1 (3)  0.0075 
31. Regulation of transport by vesicles  p8, p10, p14, p15  4 (14)  0.0083 
32. Secretion  p5, p6, p8, p15, p16  5 (17)  0.0092 
33. Down-regulation by external stimuli  p8, p10, p15  3 (10)  0.0098 
34. Response to stress  p2, p5, p6, p13, p16, p19, p20, p21, p23  9 (31)  0.0100 
a

False discovery rate estimated according to the number of proteins expected for the function.

Figure 1.

Heat map and dendrograms between identified proteins (rows) and biological functions (columns). Green highlights: grouping of proteins with a similar pattern of occurrence according to the functions they perform; and in red: the functions with a similar expression pattern, according to the indicated proteins.

(0.36MB).

Exophyllin-5 (EXPH5) is linked to intracellular vesicle transport. It was up-regulated (M/S = 8.94) in melasma, which may be due to the intense epidermal transfer of melanosomes.1 Thirteen of the proteins differentially identified in melasma have been linked to intracellular transport phenomena, which comprise a series of processes ranging from endocytosis to autophagy and several forms of exocytosis. As autophagy and senescence are melanogenesis-related phenomena, characterization of transport vesicles in the melasma epithelium may prove to be important in the pathophysiology of melasma.5,6

Cytokeratins (such as KRT1) are structural constituents of keratinocytes induced in response to oxidative stress. They were identified in greater proportion in melasma (M/S > 4.10). Hemoglobin-δ (but not the other subunits) showed a high ratio (M/S = 33.12) in melasma, and, in addition to oxygen transport, its non-erythrocytic expression occurs in situations of cell stress.7 Likewise, up-regulation of alpha 1-antitrypsin (SERPINA1) and actin gamma-1 (ACTG1) is also seen in tissue stress conditions.8,9 The higher expressions of HBD, ACTG1, SERPINA1, and KRT1 in melasma may be due to oxidative stress sustained by mast cell tryptase activity and the secretory phenotype of upper dermis fibroblasts.3,6

Carbonic anhydrase (CA1) acidifies the extracellular environment of the dermis, favoring the repair process, being down-regulated (M/S < 0.33) in melasma.10 The senescence of dermal fibroblasts, associated with the activity of MMP1 and MMP9, promotes a pro-inflammatory microenvironment with degradation of the extracellular matrix and the basement membrane zone, the repair deficit of which may be a factor in the maintenance of melanogenesis.1,6

Androglobin (ADGB) has a cysteine-endopeptidase regulatory function, being identified in a lower ratio (M/S < 0.33) in melasma. Endopeptidases participate in the degradation of melanosomes in the epidermis, notably reduced in melasma.

The alpha-kinase anchor proteins (ANCHOR9, ANCHOR13) and the z-catalytic subunit of DNA polymerase (REV3L) showed an imbalance in the skin with melasma. They are important in the regulation of protein kinase-A and the p38-MAP kinase pathway, involved in the activation of the CREB protein, which leads to the expression of MTIF, a promoter of melanogenesis.3

Aldolase-A (ALDOA) ​​has a glycolytic function and is associated with the activity of mast cells, which, in the superficial dermis, promote changes in the basement membrane, solar elastosis, and endothelial dilation, reinforcing the idea that stimuli originating in the dermis play a central role in the melanogenesis of melasma.2,3

Fibrinogen-γ (FFG) is an extracellular matrix protein, and interacts in several biological functions, including fibrinolysis, fibrinogen activation and activation of the ERK pathway, a promoter of melanogenesis.

The main limitations of the study are related to transmembrane, serum and lipid-conjugated proteins, which are not identified by the method. However, it consistently points to a number of proteins with a pathophysiological role and potential therapeutic manipulation of which should be explored in specific assays.

In conclusion, the study identified 29 differentially regulated proteins in melasma, involved in energy metabolism, cell transport phenomena, regulation of melanogenesis pathways, hemostasis/coagulation, repair/healing, and response to oxidative stress. This supports the research of therapeutic strategies aimed at the identified proteins and their functions and shows that melasma does not depend exclusively on the hyperfunction of melanocytes but also on functional alterations involving the epidermal melanin unit, basement membrane zone and upper dermis.

Financial support

FUNADERSP (048/2016).

Authors’ contributions

Luiza Vasconcelos Schaefer: Design and planning of the study; drafting and editing of the manuscript; collection, analysis, and interpretation of data; intellectual participation in the propaedeutic and/or therapeutic conduct of the studied cases; critical review of the literature.

Leticia Gomes de Pontes: Collection, analysis, and interpretation of data.

Nayara Rodrigues Vieira Cavassan: Collection, analysis, and interpretation of data.

Lucilene Delazari dos Santos: Critical review of the literature; critical review of the manuscript; collection, analysis, and interpretation of data.

Hélio Amante Miot: Critical review of the literature; critical review of the manuscript; statistical analysis; approval of the final version of the manuscript; design and planning of the study.

Conflicts of interest

None declared.

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Study conducted at the Department of Dermatology and Radiotherapy, Faculty of Medicine, Universidade Estadual Paulista, Botucatu, SP, Brazil and Centro de Estudos de Venenos e Animais Peçonhentos, Universidade Estadual Paulista, Botucatu, SP, Brazil.

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Anais Brasileiros de Dermatologia

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