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.

Novel Dent disease 1 cellular models reveal biological processes underlying ClC-5 loss-of-function.

Dent disease 1 (DD1) is a rare X-linked renal proximal tubulopathy characterized by low molecular weight proteinuria and variable degree of hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressing to chronic kidney disease. Although mutations in the electrogenic Cl-/H+ antiporter ClC-5, which impair endocytic uptake in proximal tubule cells, cause the disease, there is poor genotype-phenotype correlation and their contribution to proximal tubule dysfunction remains unclear. To further discover the mechanisms linking ClC-5 loss-of-function to proximal tubule dysfunction, we have generated novel DD1 cellular models depleted of ClC-5 and carrying ClC-5 mutants p.(Val523del), p.(Glu527Asp) and p.(Ile524Lys) using the human proximal tubule-derived RPTEC/TERT1 cell line. Our DD1 cellular models exhibit impaired albumin endocytosis, increased substrate adhesion and decreased collective migration, correlating with a less differentiated epithelial phenotype. Despite sharing functional features, these DD1 cell models exhibit different gene expression profiles, being p.(Val523del) ClC-5 the mutation showing the largest differences. Gene set enrichment analysis pointed to kidney development, anion homeostasis, organic acid transport, extracellular matrix organization and cell-migration biological processes as the most likely involved in DD1 pathophysiology. In conclusion, our results revealed the pathways linking ClC-5 mutations with tubular dysfunction and, importantly, provide new cellular models to further study DD1 pathophysiology.

EDEM3 Domains Cooperate to Perform Its Overall Cell Functioning.

EDEM3 recognizes and directs misfolded proteins to the ER-associated protein degradation (ERAD) process. EDEM3 was predicted to act as lectin or as a mannosidase because of its homology with the GH47 catalytic domain of the Man1B1, but the contribution of the other regions remained unresolved. Here, we dissect the molecular determinants governing EDEM3 function and its cellular interactions. LC/MS analysis indicates very few stable ER interactors, suggesting EDEM3 availability for transient substrate interactions. Sequence analysis reveals that EDEM3 consists of four consecutive modules defined as GH47, intermediate (IMD), protease-associated (PA), and intrinsically disordered (IDD) domain. Using an EDEM3 knock-out cell line, we expressed EDEM3 and domain deletion mutants to address EDEM3 function. We find that the mannosidase domain provides substrate binding even in the absence of mannose trimming and requires the IMD domain for folding. The PA and IDD domains deletions do not impair the trimming, but specifically modulate the turnover of two misfolded proteins, NHK and the soluble tyrosinase mutant. Hence, we demonstrate that EDEM3 provides a unique ERAD timing to misfolded glycoproteins, not only by its mannose trimming activity, but also by the positive and negative feedback modulated by the protease-associated and intrinsically disordered domain, respectively.

New factors for protein transport identified by a genome-wide CRISPRi screen in mammalian cells.

Protein and membrane trafficking pathways are critical for cell and tissue homeostasis. Traditional genetic and biochemical approaches have shed light on basic principles underlying these processes. However, the list of factors required for secretory pathway function remains incomplete, and mechanisms involved in their adaptation poorly understood. Here, we present a powerful strategy based on a pooled genome-wide CRISPRi screen that allowed the identification of new factors involved in protein transport. Two newly identified factors, TTC17 and CCDC157, localized along the secretory pathway and were found to interact with resident proteins of ER-Golgi membranes. In addition, we uncovered that upon TTC17 knockdown, the polarized organization of Golgi cisternae was altered, creating glycosylation defects, and that CCDC157 is an important factor for the fusion of transport carriers to Golgi membranes. In conclusion, our work identified and characterized new actors in the mechanisms of protein transport and secretion and opens stimulating perspectives for the use of our platform in physiological and pathological contexts.

Sodium channel TRPM4 and sodium/calcium exchangers (NCX) cooperate in the control of Ca2+-induced mucin secretion from goblet cells.

Regulated mucin secretion is essential for the formation of the mucus layer that protects the underlying epithelial cells from foreign particles. Alterations in the quantity or quality of secreted mucins are therefore detrimental to airway and colon physiology. Based on various biochemical assays in several human cell lines, we report here that Na+/Ca2+ exchanger 2 (NCX2) works in conjunction with transient receptor potential cation channel subfamily M member 4 (TRPM4), and perhaps TRPM5, Na+ channels to control Ca2+-mediated secretion of both mucin 2 (MUC2) and MUC5AC from HT29-18N2 colonic cancer cells. Differentiated normal bronchial epithelial (NHBE) cells and tracheal cells from patients with cystic fibrosis (CFT1-LC3) expressed only TRPM4 and all three isoforms of NCXs. Blocking the activity of TRPM4 or NCX proteins abrogated MUC5AC secretion from NHBE and CFT1-LC3 cells. Altogether, our findings reveal that NCX and TRPM4/TRPM5 are both required for mucin secretion. We therefore propose that these two proteins could be potential pharmacological targets to control mucus-related pathologies such as cystic fibrosis.

KChIP3 coupled to Ca2+ oscillations exerts a tonic brake on baseline mucin release in the colon.

Regulated mucin secretion from specialized goblet cells by exogenous agonist-dependent (stimulated) and -independent (baseline) manner is essential for the function of the epithelial lining. Over extended periods, baseline release of mucin can exceed quantities released by stimulated secretion, yet its regulation remains poorly characterized. We have discovered that ryanodine receptor-dependent intracellular Ca2+ oscillations effect the dissociation of the Ca2+-binding protein, KChIP3, encoded by KCNIP3 gene, from mature mucin-filled secretory granules, allowing for their exocytosis. Increased Ca2+ oscillations, or depleting KChIP3, lead to mucin hypersecretion in a human differentiated colonic cell line, an effect reproduced in the colon of Kcnip3-/- mice. Conversely, overexpressing KChIP3 or abrogating its Ca2+-sensing ability, increases KChIP3 association with granules, and inhibits baseline secretion. KChIP3 therefore emerges as the high-affinity Ca2+ sensor that negatively regulates baseline mucin secretion. We suggest KChIP3 marks mature, primed mucin granules, and functions as a Ca2+ oscillation-dependent brake to control baseline secretion. Editorial note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter).

Inhibition of N-glycan processing modulates the network of EDEM3 interactors.

We present here data on EDEM3 network of ER resident interactors and the changes induced upon this network by perturbing the early ER N-glycan processing with mannosidase and glucosidase inhibitors. By coupling immunoprecipitation with mass spectrometry we identified EDEM3 interactors and assigned statistical significance to those most abundant ER-residents that might form functional complexes with EDEM3. We further show that this ER interaction network changes in both content and abundance upon treatment with kifunensine (kif) and N-butyldeoxynojirimycin (NB-DNJ) which suggests that when interfering with the N-glycan processing pathway, the functional complexes involving EDEM3 adapt to maintain the cellular homeostasis. In order to increase the scope of EDEM3 network contenders, the set of MS identified species was further supplemented with putative interactors derived from in silico simulations performed with STRING. Finally, the most interesting candidates to this network were further validated by immunoprecipitation coupled with Western Blotting, which strengthened the confidence in the inferred interactions. The data corroborated herein suggest that besides ER residents, EDEM3 interacts also with proteins involved in the ERAD cargo recognition and targeting to degradation translocation into the cytosol, including UBA1 and UBA2 ubiquitinating enzymes. In addition, the results indicate that this network of EDEM3 interactors is highly sensitive to interfering with early ER N-glycan processing.