Targeting of Ubiquitin E3 Ligase RNF5 as a Novel Therapeutic Strategy in Neuroectodermal Tumors

RNF5, an endoplasmic reticulum (ER) E3 ubiquitin ligase, participates to the ER-associated protein degradation guaranteeing the protein homeostasis. Depending on tumor model tested, RNF5 exerts pro-or anti-tumor activity. The aim of this study was to elucidate the controversial role of RNF5 in neuroblastoma and melanoma, two neuroectodermal tumors of infancy and adulthood, respectively. RNF5 gene levels are evaluated in publicly available datasets reporting the gene expression profile of melanoma and neuroblastoma primary tumors at diagnosis. The therapeutic effect of Analog-1, an RNF5 pharmacological activator, was investigated on in vitro and in vivo neuroblastoma and melanoma models. In both neuroblastoma and melanoma patients the high expression of RNF5 correlated with a better prognostic outcome. Treatment of neuroblastoma and melanoma cell lines with Analog-1 reduced cell viability by impairing the glutamine availability and energy metabolism through inhibition of F1Fo ATP-synthase activity. This latter event led to a marked increase in oxidative stress, which, in turn, caused cell death. Similarly, neuroblastoma-and melanoma-bearing mice treated with Analog-1 showed a significant delay of tumor growth in comparison to those treated with vehicle only. These findings validate RNF5 as an innovative drug target and support the development of Analog-1 in early phase clinical trials for neuroblastoma and melanoma patients

The Autophagy Inhibitor Spautin-1 Antagonizes Rescue of Mutant CFTR Through an Autophagy-Independent and USP13-Mediated Mechanism

The mutation F508del, responsible for a majority of cystic fibrosis cases, provokes the instability and misfolding of the CFTR chloride channel. Pharmacological recovery of F508del-CFTR may be obtained with small molecules called correctors. However, treatment with a single corrector in vivo and in vitro only leads to a partial rescue, a consequence of cell quality control systems that still detect F508del-CFTR as a defective protein causing its degradation. We tested the effect of spautin-1 on F508del-CFTR since it is an inhibitor of USP10 deubiquitinase and of autophagy, a target and a biological process that have been associated with cystic fibrosis and mutant CFTR. We found that short-term treatment of cells with spautin-1 downregulates the function and expression of F508del-CFTR despite the presence of corrector VX-809, a finding obtained in multiple cell models and assays. In contrast, spautin-1 was ineffective on wild type CFTR. Silencing and upregulation of USP13 (another target of spautin-1) but not of USP10, had opposite effects on F508del-CFTR expression/function. In contrast, modulation of autophagy with known activators or inhibitors did not affect F508del-CFTR. Our results identify spautin-1 as a novel chemical probe to investigate the molecular mechanisms that prevent full rescue of mutant CFTR

Genetic Inhibition of the Ubiquitin Ligase Rnf5 Attenuates Phenotypes Associated to F508del Cystic Fibrosis Mutation

Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs CFTR trafficking and gating. F508del-CFTR mistrafficking may be corrected by acting directly on mutant CFTR itself or by modulating expression/activity of CFTR-interacting proteins, that may thus represent potential drug targets. To evaluate possible candidates for F508del-CFTR rescue, we screened a siRNA library targeting known CFTR interactors. Our analysis identified RNF5 as a protein whose inhibition promoted significant F508del-CFTR rescue and displayed an additive effect with the investigational drug VX-809. Significantly, RNF5 loss in F508del-CFTR transgenic animals ameliorated intestinal malabsorption and concomitantly led to an increase in CFTR activity in intestinal epithelial cells. In addition, we found that RNF5 is differentially expressed in human bronchial epithelia from CF vs. control patients. Our results identify RNF5 as a target for therapeutic modalities to antagonize mutant CFTR proteins

Plant Growth-Promoting Microbes from Herbal Vermicompost

Overreliance on chemical pesticides and fertilizers has resulted in problems including safety risks, outbreaks of secondary pests normally held in check by natural enemies, insecticide resistance, environmental contamination, and decrease in biodiversity. The increasing costs and negative effects of pesticides and fertilizers necessitate the idea of biological options of crop protection and production. This includes the use of animal manure, crop residues, microbial inoculum, and composts. They provide natural nutrition, reduce the use of inorganic fertilizers, develop biodiversity, increase soil biological activity, maintain soil physical properties, and improve environmental health

Influence of cell background on pharmacological rescue of mutant CFTR

Cystic fibrosis (CF) is caused by mutations in the CFTR chloride channel. Deletion of phenylalanine 508 (F508del), the most frequent CF mutation, impairs the maturation and gating of the CFTR protein. Such defects may be corrected in vitro by pharmacological modulators named as correctors and potentiators, respectively. We have evaluated a panel of correctors and potentiators derived from various sources to assess potency, efficacy, and mechanism of action. For this purpose, we have used functional and biochemical assays on two different cell expression systems, Fischer rat thyroid (FRT) and A549 cells. The order of potency and efficacy of potentiators was similar in the two cell types considered, with phenylglycine PG-01 and isoxazole UCCF-152 being the most potent and least potent, respectively. Most potentiators were also effective on two mutations, G551D and G1349D, that cause a purely gating defect. In contrast, corrector effect was strongly affected by cell background, with the extreme case of many compounds working in one cell type only. Our findings are in favor of a direct action of potentiators on CFTR, possibly at a common binding site. In contrast, most correctors seem to work indirectly with various mechanisms of action. Combinations of correctors acting at different levels may lead to additive F508del-CFTR rescue

Rescue of the mutant CFTR chloride channel by pharmacological correctors and low temperature analyzed by gene expression profiling

The F508del mutation, the most frequent in cystic fibrosis (CF), impairs the maturation of the CFTR chloride channel. The F508del defect can be partially overcome at low temperature (27°C) or with pharmacological correctors. However, the efficacy of correctors on the mutant protein appears to be dependent on the cell expression system. We have used a bronchial epithelial cell line, CFBE41o-, to determine the efficacy of various known treatments and to discover new correctors. Compared with other cell types, CFBE41o- shows the largest response to low temperature and the lowest one to correctors such as corr-4a and VRT-325. A screening of a small-molecule library identified 9-aminoacridine and ciclopirox, which were significantly more effective than corr-4a and VRT-325. Analysis with microarrays revealed that 9-aminoacridine, ciclopirox, and low temperature, in contrast to corr-4a, cause a profound change in cell transcriptome. These data suggest that 9-aminoacridine and ciclopirox act on F508del-CFTR maturation as proteostasis regulators, a mechanism already proposed for the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). However, we found that 9-aminoacridine, ciclopirox, and SAHA, in contrast to corr-4a, VRT-325, and low temperature, do not increase chloride secretion in primary bronchial epithelial cells from CF patients. These conflicting data appeared to be correlated with different gene expression signatures generated by these treatments in the cell line and in primary bronchial epithelial cells. Our results suggest that F508del-CFTR correctors acting by altering the cell transcriptome may be particularly active in heterologous expression systems but markedly less effective in native epithelial cells