EDEM1 is a mannosidase-like protein that recruits misfolded glycoproteins from the calnexin/calreticulin folding cycle to downstream endoplasmic reticulum associated degradation (ERAD) pathway. Here, we investigate the role of EDEM1 in the processing of tyrosinase, a tumour antigen overexpressed in melanoma cells. First, we analyzed and modeled EDEM1 major domains. The homology model raised on the crystal structures of human and Saccharomyces cerevisiae ER class I α1,2-mannosidases reveals that the major mannosidase domain located between aminoacids 121–598 fits with high accuracy. We have further identified an N-terminal region located between aminoacids 40–119, predicted to be intrinsically disordered (ID) and susceptible to adopt multiple conformations, hence facilitating protein-protein interactions. To investigate these two domains we have constructed an EDEM1 deletion mutant lacking the ID region and a triple mutant disrupting the glycan-binding domain and analyzed their association with tyrosinase. Tyrosinase is a glycoprotein partly degraded endogenously by ERAD and the ubiquitin proteasomal system. We found that the degradation of wild type and misfolded tyrosinase was enhanced when EDEM1 was overexpressed. Glycosylated and non-glycosylated mutants co-immunoprecipitated with EDEM1 even in the absence of its intact mannosidase-like domain, but not when the ID region was deleted. In contrast, calnexin and SEL 1L associated with the deletion mutant. Our data suggest that the ID region identified in the N-terminal end of EDEM1 is involved in the binding of glycosylated and non-glycosylated misfolded proteins. Accelerating tyrosinase degradation by EDEM1 overexpression may lead to an efficient antigen presentation and enhanced elimination of melanoma cells.

An unglycosylated TRPM8 protein is expressed and is functional in the membrane of PDA cells. Transient receptor potential melastatin 8 inhibits the migration of PDA cells, suggesting a putative role as a biomarker or target for this channel for PDA therapy.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is the main mechanism of targeting ER proteins for degradation to maintain homeostasis, and perturbations of ERAD lead to pathological conditions. ER-degradation enhancing α-mannosidase-like (EDEM1) was proposed to extract terminally misfolded proteins from the calnexin folding cycle and target them for degradation by ERAD. Here, using mass-spectrometry and biochemical methods, we show that EDEM1 is found in auto-regulatory complexes with ERAD components. Moreover, the N-terminal disordered region of EDEM1 mediates protein–protein interaction with misfolded proteins, whilst the absence of this domain significantly impairs their degradation. We also determined that overexpression of EDEM1 can induce degradation, even when proteasomal activity is severely impaired, by promoting the formation of aggregates, which can be further degraded by autophagy. Therefore, we propose that EDEM1 maintains ER homeostasis and mediates ERAD client degradation via autophagy when either dislocation or proteasomal degradation are impaired.

The degradation process of the antigens specific to MHC-I presentation depends mainly on the proteasomal proteases in the cytosol. However, since many antigens are glycoproteins, including tumor antigens or viruses envelope proteins, their glycosylation status could also affect their processing and presentation. Here, we investigate the processing of tyrosinase, a multiple glycosylated tumor antigen overexpressed in human malignant melanoma. By LC-MS/MS analysis of human tyrosinase expressed in a melanoma cell, we show that all seven sites of tyrosinase are at least partially N-glycosylated. Using human CD8+ T-cell clones specific for the tyrosinase epitope YMDGTMSQV (369-377), including an N-glycosylation site, we found that transfectants of single and triple N-glycosylation mutants are recognized by specific T cells. Importantly, single, triple, and the aglycosylated tyrosinase mutants lacking the epitope located N-glycosylation site (N371D) were able to trigger higher CD8+ T-cell activation. The LC/MS analysis showed significant increase of the amount of YMDGTMSQV peptide resulted from accelerated oligomerization and degradation of aglycosylated mutants. The generation of the antigenic peptide by the antigen processing machinery is therefore largely independent of tyrosinase N-glycosylation. However, while distal N-glycans had no effect on the epitope generation, the mutants lacking the N371 glycan generated the antigenic peptide more efficiently. We conclude that epitope located N-glycans limit the ability of human tyrosinase to provide HLA-A2-restricted antigen for recognition by specific CD8+ T cells.

The transient receptor potential ankyrin type 1 (TRPA1) channel belongs to the TRP superfamily of ion channels. TRPA1 is a membrane protein with multiple functions able to respond to noxious stimuli, reactive oxygen species, inflammatory cytokines or pungent substances, and it participates in pain signalling, taste, inflammation and various steps of the tumorigenic process. To date, no reports have addressed the expression and function of TRPA1 in pancreatic ductal adenocarcinoma (PDAC) cells. This work reports the endogenous expression of TRPA1 channels in human pancreatic adenocarcinoma cell lines and provides insights into the function of the TRPA1 protein in the Panc-1 cell line. This study reports that cell lines isolated from PDAC patients had different levels of TRPA1 expression. The channel activity in Panc-1 cells, as assessed with electrophysiological (whole-cell patch clamp) and microfluorimetry methods, showed that non-selective cationic currents were activated by allyl isothiocyanate (AITC) in Panc-1 cells and inhibited by the selective TRPA1 antagonist A-967079. The current elicited by the specific agonist was associated with a robust increase in intracellular Ca2+. Furthermore, siRNA-induced downregulation of TRPA1 enhanced cell migration in the wound healing assay, indicating a possible role of ion channels independent from pore function. Finally, TRPA1 activation changed the cell cycle progression. Taken together, these results support the idea of channel-dependent and independent role for TRPA1 in tumoral processes.

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Since Graphene discovery, their associated derivate nanomaterials, Graphene Oxide (GO) and reduced-GO were in the forefront of continuous developments in bio-nano-technology due to unique physical-chemical properties. Although GO nano-colloids (GON) were proposed as drug release matrix for targeting cancer cells, there is still a concern regarding its cytotoxicity issues. In this study, we report on the fabrication of functional GON bio-coatings by Matrix-Assisted Pulsed Laser Evaporation (MAPLE) to be used as drug carriers for targeting melanoma cells. We first performed a thorough in vitro cytotoxicity assay for comparison between GON and protein functionalized GON coatings. As functionalization protein, Bovine Serum Albumin (BSA) was non-covalently conjugated to GO surface. Safe concentration windows were identified in cytotoxicity tests by live/dead staining and MTS assays for five different human melanoma cell lines as well as for non-transformed melanocytes and human dermal fibroblasts. Hybrid GON-BSA nano-scaled thin coatings incorporating Dabrafenib (DAB) and Trichostatin A (TSA) inhibitors for cells bearing BRAF V600E pathway activating mutation were assembled on solid substrates by MAPLE technique. We further demonstrated the successful immobilization for each drug-containing GON-BSA assembling systems by evaluating cellular BRAF activity inhibition and histone deacetylases activity blocking, respectively. DAB activity was proven by the decreased ERK phosphorylation in primary melanoma cells (SKmel28 BRAF V600E cell line), while TSA effect was evidenced by acetylated histones accumulation in cell's nuclei (SKmel23 BRAF WT cell line). In addition, melanoma cells exposed to GON-BSA coatings with compositional gradient of inhibitors evidenced a dose-dependent effect on target activity. Such functional bio-platforms could present high potential for cell-biomaterial interface engineering to be applied in personalized cancer therapy studies.