Hybrid bio-nanoporous peptide loaded-polymer platforms with anticancer and antibacterial activities.

In this study, hybrid bio-nanoporous peptides loaded onto poly(N-isopropylacrylamide-co-butylacrylate) (pNIPAM-co-BA) coatings were designed and obtained via matrix-assisted pulsed laser evaporation (MAPLE) technique. The incorporation of cationic peptides magainin (MG) and melittin (Mel) and their combination was tailored to target synergistic anticancer and antibacterial activities with low toxicity on normal mammalian cells. Atomic force microscopy, scanning electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy as well as contact angle and surface energy measurements revealed the successful and functional incorporation of both the peptides within porous polymeric nanolayers as well as surface modifications (i.e. variation in the pore size diameter, surface roughness, and wettability) after Mel, MG or Mel-MG incorporation compared to pNIPAM-co-BA. In vitro testing revealed the impairment of biofilm formation on all the hybrid coatings while testing with S. aureus, E. coli and P. aeruginosa. Moreover, MG was shown to modulate the effect of Mel in the combined Mel-MG extract formulation released via pNIPAM-platforms, thus significantly reducing cancer cell proliferation through apoptosis/necrosis as revealed by flow cytometry analysis performed in vitro on HEK293T, A375, B16F1 and B16F10 cells. To the best of our knowledge, Mel-MG combination entrapped in the pNIPAM-co-BA copolymer has not yet been reported as a new promising candidate with anticancer and antibacterial properties for improved utility in the biomedical field. Mel-MG incorporation compared to pNIPAM-co-BA in in vitro testing revealed the impairment of biofilm formation in all the hybrid formulations.

Methionine oxidation selectively enhances T cell reactivity against a melanoma antigen.

The impact of the peptide amino acids side-chain modifications on the immunological recognition has been scarcely explored. We investigate here the effect of methionine oxidation on the antigenicity of the melanoma immunodominant peptide 369-YMDGTMSQV-377 (YMD). Using CD8+ T cell activation assays, we found that the antigenicity of the sulfoxide form is higher when compared to the YMD peptide. This is consistent with free energy computations performed on HLA-A∗02:01/YMD/TCR complex showing that this is lowered upon oxidation, paired with a steep increase in order at atomic level. Oxidized YMD forms were identified at the melanoma cell surface by LC-MS/MS analysis. These results demonstrate that methionine oxidation in the antigenic peptides may generate altered peptide ligands with increased antigenicity, and that this oxidation may occur in vivo, opening up the possibility that high-affinity CD8+ T cells might be naturally primed in the course of melanoma progression, as a result of immunosurveillance.

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.

Structural Investigation of Betulinic Acid Plasma Metabolites by Tandem Mass Spectrometry.

Betulinic acid (BA) has been extensively studied in recent years mainly for its antiproliferative and antitumor effect in various types of cancers. Limited data are available regarding the pharmacokinetic profile of BA, particularly its metabolic transformation in vivo. In this study, we present the screening and structural investigations by ESI Orbitrap MS in the negative ion mode and CID MS/MS of phase I and phase II metabolites detected in mouse plasma after the intraperitoneal administration of a nanoemulsion containing BA in SKH 1 female mice. Obtained results indicate that the main phase I metabolic reactions that BA undergoes are monohydroxylation, dihydroxylation, oxidation and hydrogenation, while phase II reactions involved sulfation, glucuronidation and methylation. The fragmentation pathway for BA and its plasma metabolites were elucidated by sequencing of the precursor ions by CID MS MS experiments.

A Study on Repositioning Nalidixic Acid via Lanthanide Complexation: Synthesis, Characterization, Cytotoxicity and DNA/Protein Binding Studies.

"Drug repositioning" is a modern strategy used to uncover new applications for out-of-date drugs. In this context, nalidixic acid, the first member of the quinolone class with limited use today, has been selected to obtain nine new metal complexes with lanthanide cations (La3+, Sm3+, Eu3+, Gd3+, Tb3+); the experimental data suggest that the quinolone acts as a bidentate ligand, binding to the metal ion via the keto and carboxylate oxygen atoms, findings that are supported by DFT calculations. The cytotoxic activity of the complexes has been studied using the tumoral cell lines, MDA-MB-231 and LoVo, and a normal cell line, HUVEC. The most active compounds of the series display selective activity against LoVo. Their affinity for DNA and the manner of binding have been tested using UV-Vis spectroscopy and competitive binding studies; our results indicate that major and minor groove binding play a significant role in these interactions. The affinity towards serum proteins has also been evaluated, the complexes displaying higher affinity towards albumin than apotransferrin.

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.

New panel of biomarkers to discriminate between amelanotic and melanotic metastatic melanoma.

Melanoma is a form of skin cancer that can rapidly invade distant organs. A distinctive feature of melanomas is their pigmentation status, as melanin is present in most skin melanomas, whilst many metastatic tumors could become amelanotic. Besides the obvious malfunction of the key genes of the melanin pathway, the amelanotic tumors could bear a characteristic molecular signature accounting for their aggressivity. Using mass spectrometry-based proteomics we report here a distinctive panel of biomarkers for amelanotic aggressive melanoma that differ from the less invasive pigmented cells. The developed method allows the label-free quantification of proteins identified by LC-MS/MS analysis. We found a set of proteins comprising AHNAK, MYOF, ANXA1, CAPN2, ASPH, EPHA2, THBS1, TGM2, ACTN4 along with proteins involved in cell adhesion/migration (integrins, PLEC, FSCN1, FN1) that are highly expressed in amelanotic melanoma. Accompanying the down regulation of pigmentation specific proteins such as tyrosinase and TYRP1, these biomarkers are highly specific for a type of highly invasive melanoma. Interestingly, the LC-MS/MS proteomics analysis in hypoxia revealed that the abundance of this specific set of proteins found in normoxia was rather unaltered in these conditions. These biomarkers could therefore predict a metastatic behaviour for the amelanotic cells in the early stages of the tumor development and thus serve in melanoma prognostic. Applying this algorithm to related databases including melanoma samples published by independent laboratories/public databases we confirm the specificity of the newly found signatures. Overall, we begin to unravel the molecular alterations in the amelanotic melanoma and how basic proteomics offers insights into how to assess the clinical, pathological and misdiagnosis differences between the main subtypes of melanoma.

Dataset of human EDEM2 melanoma cells proteomics, affinity proteomics and deglycoproteomics.

EDEM2 (Endoplasmic reticulum Degradation-Enhancing alpha-Mannosidase-like protein 2) is one of the key-proteins suggested to be involved in the selection and degradation of misfolded proteins from the endoplasmic reticulum. The datasets discussed in this article are related to experiments covering affinity proteomics, label-free quantitative proteomics, deglycoproteomics and SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture) proteomics data of A375 melanoma cells with modified expression of EDEM2. Our first aim was to affinity-enrich EDEM2 alongside its potential interaction partners and analyse the obtained samples by nanoLC-MS/MS to identify novel EDEM2 associated proteins. The dataset was substantiated by SDF (Sucrose Density Fractionation)-nanoLC-MS/MS experiments, in an integrated workflow to validate EDEM2 identified partners and corroborate these with previous data. Our second aim was to delineate novel EDEM2 substrate candidates using a two-step strategy. The first one refers to the deglycoproteomics dataset, which covers nanoLC-MS/MS analysis of Concanavalin A enriched glycopeptides released by endoglycosidase digestion from A375 melanoma cell lysates. This allowed us to map the fraction of glycoproteins with non-matured N-glycans from A375 melanoma cells and find or validate N-glycosylation sites of proteins from the secretory pathway. The same dataset was also used to define glycoproteins altered by the down-regulation of endogenous EDEM2, which should contain its candidate-substrates. In a second step we delineate the degradation kinetics of some of these proteins using a pulse SILAC strategy (pSILAC) thus complementing our initial findings with a fourth dataset. Beside nanoLC-MS/MS analysis our findings were also validated by various biochemical experiments. All the data described are associated with a research article published in Molecular and Cellular Proteomics [1].

Y2O3 Nanoparticles and X-ray Radiation-Induced Effects in Melanoma Cells.

The innovative strategy of using nanoparticles in radiotherapy has become an exciting topic due to the possibility of simultaneously improving local efficiency of radiation in tumors and real-time monitoring of the delivered doses. Yttrium oxide (Y2O3) nanoparticles (NPs) are used in material science to prepare phosphors for various applications including X-ray induced photodynamic therapy and in situ nano-dosimetry, but few available reports only addressed the effect induced in cells by combined exposure to different doses of superficial X-ray radiation and nanoparticles. Herein, we analyzed changes induced in melanoma cells by exposure to different doses of X-ray radiation and various concentrations of Y2O3 NPs. By evaluation of cell mitochondrial activity and production of intracellular reactive oxygen species (ROS), we estimated that 2, 4, and 6 Gy X-ray radiation doses are visibly altering the cells by inducing ROS production with increasing the dose while at 6 Gy the mitochondrial activity is also affected. Separately, high-concentrated solutions of 25, 50, and 100 µg/mL Y2O3 NPs were also found to affect the cells by inducing ROS production with the increase of concentration. Additionally, the colony-forming units assay evidenced a rather synergic effect of NPs and radiation. By adding the NPs to cells before irradiation, a decrease of the number of proliferating cell colonies was observed with increase of X-ray dose. DNA damage was evidenced by quantifying the γ-H2AX foci for cells treated with Y2O3 NPs and exposed to superficial X-ray radiation. Proteomic profile confirmed that a combined effect of 50 µg/mL Y2O3 NPs and 6 Gy X-ray dose induced mitochondria alterations and DNA changes in melanoma cells.

Affinity Proteomics and Deglycoproteomics Uncover Novel EDEM2 Endogenous Substrates and an Integrative ERAD Network.

Various pathologies result from disruptions to or stress of endoplasmic reticulum (ER) homeostasis, such as Parkinson's disease and most neurodegenerative illnesses, diabetes, pulmonary fibrosis, viral infections, and cancers. A critical process in maintaining ER homeostasis is the selection of misfolded proteins by the ER quality-control system for destruction via ER-associated degradation (ERAD). One key protein proposed to act during the first steps of misfolded glycoprotein degradation is the ER degradation-enhancing α-mannosidase-like protein 2 (EDEM2). Therefore, characterization of the EDEM2-associated proteome is of great interest. We took advantage of using melanoma cells overexpressing EDEM2 as a cancer model system, to start documenting at the deglycoproteome level (N-glycosites identification) the emerging link between ER homeostasis and cancer progression. The dataset created for identifying the EDEM2 glyco clients carrying high mannose/hybrid N-glycans provides a comprehensive N-glycosite analysis mapping over 1000 N-glycosites on more than 600 melanoma glycoproteins. To identify EDEM2-associated proteins, we used affinity proteomics and proteome-wide analysis of sucrose density fractionation in an integrative workflow. Using intensity and spectral count-based quantification, we identify seven new EDEM2 partners, all of which are involved in ER quality-control system and ERAD. Moreover, we defined novel endogenous candidates for EDEM2-dependent ERAD by combining deglycoproteomics, stable isotope labeling with amino acids in cell culture-based proteomics, and biochemical methods. These included tumor antigens and several ER-transiting endogenous melanoma proteins, including integrin alpha-1 and protocadherin 2, the expression of which was negatively correlated with that of EDEM2. Tumor antigens are key in the antigen presentation process, whereas integrin alpha-1 and protocadherin 2 are involved in melanoma metastasis and invasion. EDEM2 could therefore have a regulatory role in melanoma through the modulation of degradation and trafficking in these glycoproteins. The data presented herein suggest that EDEM2 is involved in ER homeostasis to a greater extent than previously suggested.

Rare-Earth Metal Complexes of the Antibacterial Drug Oxolinic Acid: Synthesis, Characterization, DNA/Protein Binding and Cytotoxicity Studies.

"Drug repositioning" is a current trend which proved useful in the search for new applications for existing, failed, no longer in use or abandoned drugs, particularly when addressing issues such as bacterial or cancer cells resistance to current therapeutic approaches. In this context, six new complexes of the first-generation quinolone oxolinic acid with rare-earth metal cations (Y3+, La3+, Sm3+, Eu3+, Gd3+, Tb3+) have been synthesized and characterized. The experimental data suggest that the quinolone acts as a bidentate ligand, binding to the metal ion via the keto and carboxylate oxygen atoms; these findings are supported by DFT (density functional theory) calculations for the Sm3+ complex. The cytotoxic activity of the complexes, as well as the ligand, has been studied on MDA-MB 231 (human breast adenocarcinoma), LoVo (human colon adenocarcinoma) and HUVEC (normal human umbilical vein endothelial cells) cell lines. UV-Vis spectroscopy and competitive binding studies show that the complexes display binding affinities (Kb) towards double stranded DNA in the range of 9.33 × 104 - 10.72 × 105. Major and minor groove-binding most likely play a significant role in the interactions of the complexes with DNA. Moreover, the complexes bind human serum albumin more avidly than apo-transferrin.

High resolution mass spectrometry provides novel insights into the ganglioside pattern of brain cavernous hemangioma.

In this study we have optimized nanoelectrospray ionization (nanoESI) high resolution mass spectrometry (HR MS) performed on Orbitrap instrument in the negative ion mode for the determination of the composition and structure of gangliosides extracted from human brain cavernous hemangioma. The optimized HR MS platform, allowed the discrimination of 62 ions, corresponding to 52 different ganglioside species, which represents roughly twice the number of species existing in the current inventory of human brain hemangioma-associated gangliosides. The experiments revealed a ganglioside pattern dominated by GD-type of structures as well as an elevated incidence of species characterized by a low degree of sialylation and short glycan chains, including asialo GA1 (d18:1/18:0), which offer a new perspective upon the ganglioside composition in this benign tumor. Many of the structures are characteristic for this type of tumor only and are to be considered in further investigations for their potential use in early brain hemangioma diagnosis based on molecular markers. The detailed fragmentation analysis performed by collision-induced dissociation (CID) tandem MS provided information of structural elements related to the glycan core and ceramide moiety, which confirmed the molecular configuration of GD3 (d18:1/24:1) and GD3 (d18:1/24:2) species with potential biomarker role.

Functionalized Graphene Oxide Thin Films for Anti-tumor Drug Delivery to Melanoma Cells.

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 BRAFV600E 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 BRAFV600E 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.

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.

Cross-talk between Dopachrome Tautomerase and Caveolin-1 Is Melanoma Cell Phenotype-specific and Potentially Involved in Tumor Progression.

l-Dopachrome tautomerase (l-DCT), also called tyrosinase-related protein-2 (TRP-2), is a melanoma antigen overexpressed in most chemo-/radiotherapeutic stress-resistant tumor clones, and caveolin-1 (CAV1) is a main regulator of numerous signaling processes. A structural and functional relationship between DCT and CAV1 is first presented here in two human amelanotic melanoma cell lines, derived from vertical growth phase (MelJuSo) and metastatic (SKMel28) melanomas. DCT co-localizes at the plasma membrane with CAV1 and Cavin-1, another molecular marker for caveolae in both cell phenotypes. Our novel structural model proposed for the DCT-CAV1 complex, in addition to co-immunoprecipitation and mass spectrometry data, indicates a possible direct interaction between DCT and CAV1. The CAV1 control on DCT gene expression, DCT post-translational processing, and subcellular distribution is cell phenotype-dependent. DCT is a modulator of CAV1 stability and supramolecular assembly in both cell phenotypes. During autocrine stimulation, the expressions of DCT and CAV1 are oppositely regulated; DCT increases while CAV1 decreases. Sub-confluent MelJuSo clones DCT(high)/CAV1(low) are proliferating and acquire fibroblast-like morphology, forming massive, confluent clusters as demonstrated by immunofluorescent staining and TissueFAXS quantitative image cytometry analysis. CAV1 down-regulation directly contributes to the expansion of MelJuSo DCT(high) subtype. CAV1 involved in the perpetuation of cell phenotype-overexpressing anti-stress DCT molecule supports the concept that CAV1 functions as a tumor suppressor in early stages of melanoma. DCT is a regulator of the CAV1-associated structures and is possibly a new molecular player in CAV1-mediated processes in melanoma.

Epitope located N-glycans impair the MHC-I epitope generation and presentation.

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.