Fig 2: Protein–protein interactions and association of the PD‐related α‐syn with DJ‐1. (A) Interaction and co‐occurrence protein partners of α‐syn (SNCA) were identified with String and GeneMANIA databases. Circles displayed are indicated by nodes. Predicted functional partners are shown after considering co‐expression, co‐localization, genetic interactions, pathways, and physical interactions. Venn diagram employed to identify the commonly interactive protein partners of α‐syn, including DJ‐1(PARK7), based on GeneMANIA and STRING databases. (B) SK‐MEL‐28 and A375 melanoma cells were transfected with pcDNA3.1 + −wild‐type‐α‐synuclein plasmid and protein levels of α‐syn and DJ‐1 (ng/mL) were assessed in the cell supernatants (secretome) after 24 h of transfection. Mean values with standard deviation (SD) are indicated in the bar graphs. (C) Illustration of α‐syn (receptor, in blue) and DJ‐1 (ligand, in green) interacting protein domains and binding sites using a molecular docking approach. Interacting domain (zoom) that includes the two amino acid residues with the most stable binding affinity (LEU‐75 in the ligand‐DJ‐1 with binding free energy −2.55 kcal/mol and VAL‐52 in the receptor‐α‐syn with −3.74 kcal/mol).

Fig 3: Expression profile of α‐syn and DJ‐1 in melanoma cell lines. (A) Gene expression comparative bar plot for SNCA (encoding α‐syn) and PARK7 (encoding DJ‐1) using the Cancer Cell Line Encyclopedia datasets via the Betastasis platform. (B, C) Validation of selected melanoma cell lines (A375 and SK‐MEL‐28) at the protein level using ELISA immunoassays. α‐syn (B) and DJ‐1 (C) protein levels were measured as ng/mL in the supernatants (secretome, S) and cell extracts (CE) of the selected melanoma cell lines. Mean values with standard deviation (SD) are indicated in the bar graphs.

Fig 4: α‐syn and DJ‐1 protein expression in lymph nodes from metastatic melanoma patients. (A) Immunohistochemistry was performed in lymph nodes of patients with metastatic malignant melanoma with antibodies capturing α‐syn aggregated forms (α‐syn filament and α‐syn‐5G4), the α‐syn‐Ser129 phosphorylated form (α‐syn‐S129), and DJ‐1. Representative images of stained lymph node tissue sections from 2 metastatic malignant melanoma patients (right panel) and a patient with non‐metastatic (N0) carcinoma lymph nodes (control, left panel) are shown. Magnification, 20×. (B) The slides were categorized into three scores according to the percentage of area and intensity of staining. Score 2 represents the highest expression. α‐syn aggregated forms were highly expressed in metastatic malignant melanoma compared to non‐metastatic lymph nodes. Expression of α‐syn phosphorylation form was almost absent in metastatic melanoma lymph nodes, whereas DJ‐1 was expressed in both control and metastatic melanoma lymph nodes.

Fig 5: Expression profiles of SNCA and PARK7 in skin cutaneous melanoma (SKCM). (A, B) RNA sequencing transcriptomic TCGA data from Xena UCSC portal were used to compare Normal (N) (n = 556) with Tumor (T; primary and metastatic) (n = 469) (A), and Normal (N) (n = 556) with Primary Tumor (PT) (n = 102) or Metastatic (M) (n = 367) (B). Both SNCA and PARK7 expression levels are significantly upregulated in SKCM tumor samples (primary and metastatic) compared to normal skin tissues. Wilcoxon rank sum non‐parametric test was applied between groups for statistical significance (***p < 0.001). (C) Positive Spearman correlation was observed between SNCA and PARK7 gene expression in SKCM‐primary (N = 102) (Spearman's ρ: positive correlation (ρ > 0, p < 0.01) whereas not in SKCM‐metastatic tumors.