Ubiquitylation of a Melanosomal Protein by HECT-E3 Ligases Serves as Sorting Signal for Lysosomal Degradation

Abstract

The production of pigment by melanocytic cells of the skin involves a series of enzymatic reactions that take place in specialized organelles called melanosomes. Melan-A/MART-1 is a melanocytic transmembrane protein with no enzymatic activity that accumulates in vesicles at the trans side of the Golgi and in melanosomes. We show here that, in melanoma cells, Melan-A associates with two homologous to E6-AP C-terminus (HECT)-E3 ubiquitin ligases, NEDD4 and Itch, and is ubiquitylated. Both NEDD4 and Itch participate in the degradation of Melan-A. A mutant Melan-A lacking ubiquitinacceptor residues displays increased half-life and, in pigmented cells, accumulates in melanosomes. These results suggest that ubiquitylation regulates the lysosomal sorting and degradation of Melan-A/MART-1 from melanosomes in melanocytic cells.

Melan-A/MART-1-Specific CD4 T Cells in Melanoma Patients: Identification of New Epitopes and Ex Vivo Visualization of Specific T Cells by MHC Class II Tetramers

“…1C). The six other responding patients (LAU 618, LAU 42, LAU 321, LAU 672, LAU 4, and LAU 814) showed responses against three additional regions of Melan-A: Melan-A 1-20 , Melan-A [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] , and Melan-A [41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60] . These results showed that among 16 melanoma patients tested, Ͼ50% of them displayed Melan-A-specific CD4 T cell responses.…”

“…We then focused on the responses to the region Melan-A [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] , because this region contains the immunodominant HLA-A2-restricted epitope Melan-A 26 -35 and defining Th epitopes in the same region is of practical interest. We derived clones using the IFN-␥ capture assay and flow cytometry-assisted cell sorting to select CD4 T cells from the primary cultures that were able to respond to short-term stimulation with Melan-A [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] .…”

“…We selected clones from patients LAU 618, LAU 672, LAU 814, and LAU 42 for additional experiments. First, we determined the HLA restriction of Melan-A [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] peptide recognition using either anti-HLA-DR-, -DP-, or -DQ-specific Abs. The Melan-A 21-40 -specific CD4 T cell clones from LAU 618 and LAU 814 recognized the peptide in an HLA-DQ-restricted manner.…”

“…Thus, in the case of LAU 42 and LAU 672, the CD4 T cell response specific for Melan-A 21-40 appeared to be HLA-DR restricted and in the case of LAU 618 and LAU 814 it appeared to be HLA-DQ restricted. To identify the restricting allele, we used a panel of EBV-B cells to present Melan-A [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40] peptide to the different clones. Using different combinations of presenting EBV-B cells, we could determine that the clones from LAU 618 and LAU 814 recognized the Melan-A 21-40 peptide presented by HLA-DQB1*0602, the clones from LAU 672 recognized the peptide presented by HLA-DRB1*0301 and the clones from LAU 42 recognized the same peptide presented by HLA-DRB1*0101 and HLA-DRB1*0102 ( Fig.…”

“…We also used Melan-A [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] and Melan-A 26 -40 for peptide recognition experiments. Melan-A [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] was recognized by very few clones at a level much lower than Melan-A [21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] and Melan-A 26 -40 was never recognized (data not shown). Clones from LAU 618 gave similar results as clones from LAU 814 (data not shown).…”