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Abstract Background & aims Metallothionein-3 (hMT3) is a structurally unique member of the metallothioneins family of low-mass cysteine-rich proteins. hMT3 has poorly characterized functions, and its importance for hepatocellular carcinoma (HCC) cells has not yet been elucidated. Therefore, we investigated the molecular mechanisms driven by hMT3 with a special emphasis on susceptibility to sorafenib. Methods Intrinsically sorafenib-resistant (BCLC-3) and sensitive (Huh7) cells with or without up-regulated hMT3 were examined using cDNA microarray and methods aimed at mitochondrial flux, oxidative status, cell death, and cell cycle. In addition, in ovo/ex ovo chick chorioallantoic membrane (CAM) assays were conducted to determine a role of hMT3 in resistance to sorafenib and associated cancer hallmarks, such as angiogenesis and metastastic spread. Molecular aspects of hMT3-mediated induction of sorafenib-resistant phenotype were delineated using mass-spectrometry-based proteomics. Results The phenotype of sensitive HCC cells can be remodeled into sorafenib-resistant one via up-regulation of hMT3. hMT3 has a profound effect on mitochondrial respiration, glycolysis, and redox homeostasis. Proteomic analyses revealed a number of hMT3-affected biological pathways, including exocytosis, glycolysis, apoptosis, angiogenesis, and cellular stress, which drive resistance to sorafenib. Conclusions hMT3 acts as a multifunctional driver capable of inducing sorafenib-resistant phenotype of HCC cells. Our data suggest that hMT3 and related pathways could serve as possible druggable targets to improve therapeutic outcomes in patients with sorafenib-resistant HCC.

Leuconostoc lactis is found in vegetables, fruits, and meat and is used by the food industry in the preparation of dairy products, wines, and sugars. We have previously demonstrated that the dextransucrase of Lc. lactis (DsrLL) AV1n produces a high-molecular-weight dextran from sucrose, indicating its potential use as a dextran-forming starter culture. We have also shown that this bacterium was able to produce 10-fold higher levels of dextran at 20°C than at 37°C, at the former temperature accompanied by an increase in dsrLL gene expression. However, the general physiological response of Lc. lactis AV1n to cold temperature in the presence of sucrose, leading to increased production of dextran, has not been yet investigated. Therefore, we have used a quantitative proteomics approach to investigate the cold temperature-induced changes in the proteomic profile of this strain in comparison to its proteomic response at 37°C. In total, 337 proteins were found to be differentially expressed at the applied significance criteria (adjusted p-value ≤ 0.05, FDR 5%, and with a fold-change ≥ 1.5 or ≤ 0.67) with 204 proteins overexpressed, among which 13% were involved in protein as well as cell wall, and envelope component biosynthesis including DsrLL. Proteins implicated in cold stress were expressed at a high level at 20°C and possibly play a role in the upregulation of DsrLL, allowing the efficient synthesis of the protein essential for its adaptation to cold. Post-transcriptional regulation of DsrLL expression also seems to take place through the interplay of exonucleases and endonucleases overexpressed at 20°C, which would influence the half-life of the dsrLL transcript. Furthermore, the mechanism of cold resistance of Lc. lactis AV1n seems to be also based on energy saving through a decrease in growth rate mediated by a decrease in carbohydrate metabolism and its orientation toward the production pathways for storage molecules. Thus, this better understanding of the responses to low temperature and mechanisms for environmental adaptation of Lc. lactis could be exploited for industrial use of strains belonging to this species.

Abstract Metallothionein-3 has poorly characterized functions in neuroblastoma. Cisplatin-based chemotherapy is a major regimen to treat neuroblastoma, but its clinical efficacy is limited by chemoresistance. We investigated the impact of human metallothionein-3 (hMT3) up-regulation in neuroblastoma cells and the mechanisms underlying the cisplatin-resistance. We confirmed the cisplatin-metallothionein complex formation using mass spectrometry. Overexpression of hMT3 decreased the sensitivity of neuroblastoma UKF-NB-4 cells to cisplatin. We report, for the first time, cisplatin-sensitive human UKF-NB-4 cells remodelled into cisplatin-resistant cells via high and constitutive hMT3 expression in an in vivo model using chick chorioallantoic membrane assay. Comparative proteomic analysis demonstrated that several biological pathways related to apoptosis, transport, proteasome, and cellular stress were involved in cisplatin-resistance in hMT3 overexpressing UKF-NB-4 cells. Overall, our data confirmed that up-regulation of hMT3 positively correlated with increased cisplatin-chemoresistance in neuroblastoma, and a high level of hMT3 could be one of the causes of frequent tumour relapses.

Colorectal cancer (CRC) is the second leading cause of cancer-related death worldwide. The five-year survival rate of CRC patients depends on the stage at diagnosis, being higher than 80% when CRC is diagnosed in the early stages but lower than 10% when CRC is diagnosed in advanced stages. Autoantibodies against specific CRC autoantigens (tumor-associated antigens (TAAs)) in the sera of patients have been widely demonstrated to aid in early diagnosis. Thus, we herein aim to identify autoantigens target of autoantibodies specific to CRC that possess a significant ability to discriminate between CRC patients and healthy individuals by means of liquid biopsy. To that end, we examined the protein content of the exosomes released by five CRC cell lines and tissue samples from CRC patients by means of immunoprecipitation coupled with mass spectrometry analysis. A total of 103 proteins were identified as potential autoantigens specific to CRC. After bioinformatics and meta-analysis, we selected 15 proteins that are more likely to be actual CRC autoantigens in order to evaluate their role in CRC prognosis by Western blot (WB) and immunohistochemistry (IHC). We found dysregulation at the protein level for 11 of these proteins in both tissue and plasma exosome samples from patients, along with an association of nine of these proteins with CRC prognosis. After validation, all but one showed a statistically significant high diagnostic ability to distinguish CRC patients and individuals with premalignant lesions from healthy individuals, either by luminescence Halotag-based beads, or by a multiplexed biosensing platform involving the use of magnetic microcarriers as solid support modified with covalently immobilized Halotag fusion proteins constructed for CRC detection. Taken together, our results highlight the usefulness of the approach defined here to identify the TAAs specific to chronic diseases; they also demonstrate that the measurement of autoantibody levels in plasma against the TAAs identified here could be integrated into a point-of-care (POC) device for CRC detection with high diagnostic ability.

The presence of a worn surface in the implanted material, as in the case of a replacement of a damaged osteoarticular joint, is the normal condition after implantation. This manuscript focuses precisely on the comparative study of the cellular behavior on worn CoCr surfaces, analyzing the effect of different surface modifications on macrophages’ responses. CoCr surfaces were modified by the deposition of electrochemically reduced graphene oxide (CoCrErGO), followed by additional surface functionalization with hyaluronic acid (CoCrErGOHA). After the wear corrosion processes, the macrophage response was studied. In addition, macrophage supernatants exposed to the surfaces, before and after wear, were also evaluated for osteoblast response through the analysis of the metabolic activity, plasma membrane damage, and phosphatase alkaline activity (ALP). The proteomic analysis and the quantitative TNF-α/IL-10 ratios of the J774A.1 macrophages exposed to the surfaces under study showed a polarization shift from M0 (basal state) to M1, associated with the pro-inflammatory response of all surfaces. A lower M1 polarization was observed upon exposure to the surface modification with ErGO, whereas posterior HA functionalization attenuated, even more, the M1 polarization. The wear corrosion process contributed to inflammation and exacerbated the M1 polarization response on macrophages to CoCr, which was diminished for the ErGO and attenuated the most for the ErGOHA surfaces. Comparative proteomics showed that the pathways related to M1 polarization were downregulated on the surfaces of CoCrErGOHA, which suggests mechanisms for the observed attenuation of M1 polarization. The suitable immuno-modulatory potential induced by the ErGOHA surface, with and without wear, together with the stimulation of ALP activity in osteoblasts induced by macrophage supernatants, promotes the mineralization processes necessary for bone repair. This makes it feasible to consider the adsorption of ErGOHA on CoCr as a recommended surface treatment for the use of biomaterials in osseous joint applications.

ABSTRACT RepA is a bacterial protein that builds intracellular amyloid oligomers acting as inhibitory complexes of plasmid DNA replication. When carrying a mutation enhancing its amyloidogenesis (A31V), the N-terminal domain (WH1) generates cytosolic amyloid particles that are inheritable within a bacterial lineage. Such amyloids trigger in bacteria a lethal cascade reminiscent of mitochondrial impairment in human cells affected by neurodegeneration. To fulfill all the criteria to qualify as a prion-like protein, horizontal (intercellular) transmissibility remains to be demonstrated for RepA-WH1. Since this is experimentally intractable in bacteria, here we transiently expressed in a murine neuroblastoma cell line the soluble, barely cytotoxic RepA-WH1 wild type [RepA-WH1(WT)] and assayed its response to exposure to in vitro-assembled RepA-WH1(A31V) amyloid fibers. In parallel, murine cells releasing RepA-WH1(A31V) aggregates were cocultured with human neuroblastoma cells expressing RepA-WH1(WT). Both the assembled fibers and donor-derived RepA-WH1(A31V) aggregates induced, in the cytosol of recipient cells, the formation of cytotoxic amyloid particles. Mass spectrometry analyses of the proteomes of both types of injured cells pointed to alterations in mitochondria, protein quality triage, signaling, and intracellular traffic. Thus, a synthetic prion-like protein can be propagated to, and become cytotoxic to, cells of organisms placed at such distant branches of the tree of life as bacteria and mammalia, suggesting that mechanisms of protein aggregate spreading and toxicity follow default pathways. IMPORTANCE Proteotoxic amyloid seeds can be transmitted between mammalian cells, arguing that the intercellular exchange of prion-like protein aggregates can be a common phenomenon. RepA-WH1 is derived from a bacterial intracellular functional amyloid protein, engineered to become cytotoxic in Escherichia coli. Here, we have studied if such bacterial aggregates can also be transmitted to, and become cytotoxic to, mammalian cells. We demonstrate that RepA-WH1 is capable of entering naive cells, thereby inducing the cytotoxic aggregation of a soluble RepA-WH1 variant expressed in the cytosol, following the same trend that had been described in bacteria. These findings highlight the universality of one of the central principles underlying prion biology: No matter the biological origin of a given prion-like protein, it can be transmitted to a phylogenetically unrelated recipient cell, provided that the latter expresses a soluble protein onto which the incoming protein can readily template its amyloid conformation.

Metastasis is the primary cause of colorectal cancer (CRC) death. The liver and lung, besides adjacent lymph nodes, are the most common sites of metastasis. Here, we aimed to study the lymph nodes, liver, and lung CRC metastasis by quantitative spatial proteomics analysis using CRC cell-based models that recapitulate these metastases. The isogenic KM12 cell system composed of the non-metastatic KM12C cells, liver metastatic KM12SM cells, and liver and lung metastatic KM12L4a cells, and the isogenic non-metastatic SW480 and lymph nodes metastatic SW620 cells, were used. Cells were fractionated to study by proteomics five subcellular fractions corresponding to cytoplasm, membrane, nucleus, chromatin-bound proteins, and cytoskeletal proteins, and the secretome. Trypsin digested extracts were labeled with TMT 11-plex and fractionated prior to proteomics analysis on a Q Exactive. We provide data on protein abundance and localization of 4710 proteins in their different subcellular fractions, depicting dysregulation of proteins in abundance and/or localization in the most common sites of CRC metastasis. After bioinformatics, alterations in abundance and localization for selected proteins from diverse subcellular localizations were validated via WB, IF, IHC, and ELISA using CRC cells, patient tissues, and plasma samples. Results supported the relevance of the proteomics results in an actual CRC scenario. It was particularly relevant that the measurement of GLG1 in plasma showed diagnostic ability of advanced stages of the disease, and that the mislocalization of MUC5AC and BAIAP2 in the nucleus and membrane, respectively, was significantly associated with poor prognosis of CRC patients. Our results demonstrate that the analysis of cell extracts dilutes protein alterations in abundance in specific localizations that might only be observed studying specific subcellular fractions, as here observed for BAIAP2, GLG1, PHYHIPL, TNFRSF10A, or CDKN2AIP, which are interesting proteins that should be further analyzed in CRC metastasis.

TRAnsport Protein Particle complexes (TRAPPs) are ubiquitous regulators of membrane traffic mediating nucleotide exchange on the Golgi regulatory GTPases RAB1 and RAB11. In S. cerevisiae and metazoans TRAPPs consist of two large oligomeric complexes: RAB11-activating TRAPPII and RAB1-activating TRAPPIII. These share a common core TRAPPI hetero-heptamer, absent in metazoans but detected in minor proportions in yeast, likely originating from in vitro-destabilized TRAPPII/III. Despite overall TRAPP conservation, the budding yeast genome has undergone extensive loss of genes, and lacks homologues of some metazoan TRAPP subunits. With nearly twice the total number of genes of S. cerevisiae, another ascomycete Aspergillus nidulans has also been used for studies on TRAPPs. We combined size-fractionation chromatography with single-step purification coupled to mass-spectrometry and negative-stain electron microscopy to establish the relative abundance, composition and architecture of Aspergillus TRAPPs, which consist of TRAPPII and TRAPPIII in a 2:1 proportion, plus a minor amount of TRAPPI. We show that Aspergillus TRAPPIII contains homologues of metazoan TRAPPC11, TRAPPC12 and TRAPPC13 subunits, absent in S. cerevisiae, and establish that these subunits are recruited to the complex by Tca17/TRAPPC2L, which itself binds to the 'Trs33 side' of the complex. Thus Aspergillus TRAPPs compositionally resemble mammalian TRAPPs to a greater extent than those in budding yeast. Exploiting the ability of constitutively-active (GEF-independent, due to accelerated GDP release) RAB1* and RAB11* alleles to rescue viability of null mutants lacking essential TRAPP subunits, we establish that the only essential role of TRAPPs is activating RAB1 and RAB11, and genetically classify each essential subunit according to their role(s) in TRAPPII (TRAPPII-specific subunits) or TRAPPII and TRAPPIII (core TRAPP subunits). Constitutively-active RAB mutant combinations allowed examination of TRAPP composition in mutants lacking essential subunits, which led to the discovery of a stable Trs120/Trs130/Trs65/Tca17 TRAPPII-specific subcomplex whose Trs20- and Trs33-dependent assembly onto core TRAPP generates TRAPPII.

Intracellular traffic in Aspergillus nidulans hyphae must cope with the challenges that the high rates of apical extension (1μm/min) and the long intracellular distances (>100 μm) impose. Understanding the ways in which the hyphal tip cell coordinates traffic to meet these challenges is of basic importance, but is also of considerable applied interest, as fungal invasiveness of animals and plants depends critically upon maintaining these high rates of growth. Rapid apical extension requires localization of cell-wall-modifying enzymes to hyphal tips. By combining genetic blocks in different trafficking steps with multidimensional epifluorescence microscopy and quantitative image analyses we demonstrate that polarization of the essential chitin-synthase ChsB occurs by indirect endocytic recycling, involving delivery/exocytosis to apices followed by internalization by the sub-apical endocytic collar of actin patches and subsequent trafficking to TGN cisternae, where it accumulates for ~1 min before being re-delivered to the apex by a RAB11/TRAPPII-dependent pathway. Accordingly, ChsB is stranded at the TGN by Sec7 inactivation but re-polarizes to the apical dome if the block is bypassed by a mutation in geaAgea1 that restores growth in the absence of Sec7. That polarization is independent of RAB5, that ChsB predominates at apex-proximal cisternae, and that upon dynein impairment ChsB is stalled at the tips in an aggregated endosome indicate that endocytosed ChsB traffics to the TGN via sorting endosomes functionally located upstream of the RAB5 domain and that this step requires dynein-mediated basipetal transport. It also requires RAB6 and its effector GARP (Vps51/Vps52/Vps53/Vps54), whose composition we determined by MS/MS following affinity chromatography purification. Ablation of any GARP component diverts ChsB to vacuoles and impairs growth and morphology markedly, emphasizing the important physiological role played by this pathway that, we propose, is central to the hyphal mode of growth.

Oenococcus oeni is the main lactic acid bacterium that carries out the malolactic fermentation in virtually all red wines and in some white and sparkling wines. Oenococcus oeni possesses an array of metabolic activities that can modify the taste and aromatic properties of wine. There is, therefore, industrial interest in the proteins involved in these metabolic pathways and related transport systems of this bacterium. In this work, we report the characterization of the O. oeni ATCC BAA-1163 proteome. Total and membrane protein preparations from O. oeni were standardized and analysed by two-dimensional gel electrophoresis. Using tandem mass spectrometry, we identified 224 different spots corresponding to 152 unique proteins, which have been classified by their putative function and subjected to bioinformatics analysis.

Aspergillus nidulans asexual differentiation is induced by Upstream Developmental Activators (UDAs) that include the bZIP-type Transcription Factor (TF) FlbB. A 2D-PAGE/MS-MS-coupled screen for proteins differentially expressed in the presence and absence of FlbB identified 18 candidates. Most candidates belong to GO term classes involved in osmotic and/or oxidative stress response. Among these, we focused on GmcA, a putative glucose-methanol-choline oxidoreductase which is upregulated in a ΔflbB background. GmcA is not required for growth since no differences were detected in the radial extension upon deletion of gmcA. However, its activity is required to induce conidiation under specific culture conditions. A ΔgmcA strain conidiates profusely under acid conditions but displays a characteristic fluffy aconidial phenotype in alkaline medium. The absence of asexual development in a ΔgmcA strain can be suppressed, on one hand, using high concentrations of non-fermentable carbon sources like glycerol, and on the other hand, when the cMyb-type UDA TF flbD is overexpressed. Overall, the results obtained in this work support a role for GmcA at early stages of conidiophore initiation.