NPC1 plays a role in the trafficking of specific cargo to melanosomes.

Niemann-Pick type C1 (NPC1) protein is a multimembrane spanning protein of the lysosome limiting membrane that facilitates intracellular cholesterol and sphingolipid transport. Loss-of-function mutations in the NPC1 protein cause Niemann-Pick disease type C1, a lysosomal storage disorder characterized by the accumulation of cholesterol and sphingolipids within lysosomes. To investigate whether the NPC1 protein could also play a role in the maturation of the endolysosomal pathway, here, we have investigated its role in a lysosome-related organelle, the melanosome. Using a NPC1-KO melanoma cell model, we found that the cellular phenotype of Niemann-Pick disease type C1 is associated with a decreased pigmentation accompanied by low expression of the melanogenic enzyme tyrosinase. We propose that the defective processing and localization of tyrosinase, occurring in the absence of NPC1, is a major determinant of the pigmentation impairment in NPC1-KO cells. Along with tyrosinase, two other pigmentation genes, tyrosinase-related protein 1 and Dopachrome-tautomerase have lower protein levels in NPC1 deficient cells. In contrast with the decrease in pigmentation-related protein expression, we also found a significant intracellular accumulation of mature PMEL17, the structural protein of melanosomes. As opposed to the normal dendritic localization of melanosomes, the disruption of melanosome matrix generation in NPC1 deficient cells causes an accumulation of immature melanosomes adjacent to the plasma membrane. Together with the melanosomal localization of NPC1 in WT cells, these findings suggest that NPC1 is directly involved in tyrosinase transport from the trans-Golgi network to melanosomes and melanosome maturation, indicating a novel function for NPC1.

Defining the altered glycoproteomic space of the early secretory pathway by class I mannosidase pharmacological inhibition.

N-glycosylation is a key process for various biological functions like protein folding, maturation and sorting for the conventional secretory compartment, cell-cell communication and immune response. This is usually accomplished by a complex system of mannosidases in which those from class I have an outstanding role, commonly involved in the early protein sorting associated to the Endoplasmic Reticulum (ER) in the N-glycan dependent quality control (ERQC) and ER-associated degradation (ERAD). Although these are vital processes in maintaining cellular homeostasis, large-scale analysis studies for this pool of molecules, further denoted as proteins from the early secretory pathway (ESP), were limited addressed. Here, using a custom workflow employing a combination of glycomics and deglycoproteomics analyses, using lectin affinity and selective Endoglycosidase H (Endo H) digestion, we scrutinize the steady-state oligomannosidic glycoprotein load and delineate ESP fraction in melanoma cells. All of these were assessed by applying our workflow for glycosite relative quantification of both the peptide chain and carbohydrate structure in cells with inhibited activity of class I mannosidases after kifunensine treatment. We found that most of the ESP are transient clients involved in cell communication via extracellular matrix, particularly integrin-mediated communication which adopt Man9 N-glycans in kifunensine-treated cells. Moreover, our results reveal that core-fucosylation is decreased subsequent inhibition of class I mannosidases and this could be explained by a general lower protein level of FUT8, the enzyme responsible for fucosylation. By comparing our data with results obtained following downregulation of a key mannosidase in misfolded protein degradation, we mapped both novel and previously suggested endogenous substrate candidates like PCDH2, HLA-B, LAMB2 or members of the integrin family of proteins such as ITGA1 and ITGA4, thus validating the findings obtained using our workflow regarding accumulation and characterization of ESP transitory members following mannosidase class I inhibition. This workflow and the associated dataset not only allowed us to investigate the oligomannosidic glycoprotein fraction but also to delineate differences mediated at glycosite-level upon kifunensine treatment and outline the potential associated cellular responses.

Profiling Optimal Conditions for Capturing EDEM Proteins Complexes in Melanoma Using Mass Spectrometry.

Endoplasmic reticulum (ER) resident and secretory proteins that fail to reach their native conformation are selected for degradation through the ER-Associated Degradation (ERAD) pathway. The ER degradation-enhancing alpha-mannosidase-like proteins (EDEMs) were shown to be involved in this pathway but their precise role is still under investigation. Mass spectrometry analysis has contributed significantly to the characterization of protein complexes in the last years. The recent advancements in instrumentation, especially within resolution and speed can provide unique insights concerning the molecular architecture of protein-protein interactions in systems biology. Previous reports have suggested that several protein complexes in ERAD are sensitive to the extraction conditions. Indeed, whilst EDEM proteins can be recovered in most detergents, some of their partners are not solubilized, which further emphasizes the importance of the experimental setup. Here, we define such dynamic interactions of EDEM proteins by employing offline protein fractionation, nanoLC-MS/MS and describe how mass spectrometry can contribute to the characterization of such complexes, particularly within a disease context like melanoma.