Stability Studies of New Caged bis-deoxy-coelenterazine Derivatives and Their Potential Use as Cellular pH Probes.

The synthesis of new bis-deoxy-coelenterazine (1) derivatives bearing ester protective groups (acetate, propionate and butyrate esters) was accomplished. Moreover, their hydrolytic stability at room temperature was evaluated in dimethylsulfoxide (DMSO) as solvent, using the nuclear magnetic resonance (NMR) spectra of the key products at different time intervals. The results showed an increasing hydrolysis rate according to longest aliphatic chain, with a half-life of 24 days of the more stable acetate derivative (4a). Furthermore, the analysis of the experimental data revealed the greater stability of the enol tautomer in this aprotic polar solvent. This result was confirmed by theoretical calculations using the density functional theory (DFT) approach, which gave us the opportunity to propose a detailed decomposition mechanism. Additionally, the derivatives obtained were tested by bioluminescence luciferase assays to evaluate their potential use as extracellular pH-sensitive reporter substrates of luciferase. The biological data support the idea that further structural modifications of these molecules may open promising perspectives in this field of research.

Engineering REST-Specific Synthetic PUF Proteins to Control Neuronal Gene Expression: A Combined Experimental and Computational Study.

Regulation of gene transcription is an essential mechanism for differentiation and adaptation of organisms. A key actor in this regulation process is the repressor element 1 (RE1)-silencing transcription factor (REST), a transcriptional repressor that controls more than 2000 putative target genes, most of which are neuron-specific. With the purpose of modulating REST expression, we exploited synthetic, ad hoc designed, RNA binding proteins (RBPs) able to specifically target and dock to REST mRNA. Among the various families of RBPs, we focused on the Pumilio and FBF (PUF) proteins, present in all eukaryotic organisms and controlling a variety of cellular functions. Here, a combined experimental and computational approach was used to design and test 8- and 16-repeat PUF proteins specific for REST mRNA. We explored the conformational properties and atomic features of the PUF-RNA recognition code by Molecular Dynamics simulations. Biochemical assays revealed that the 8- and 16-repeat PUF-based variants specifically bind the endogenous REST mRNA without affecting its translational regulation. The data also indicate a key role of stacking residues in determining the binding specificity. The newly characterized REST-specific PUF-based constructs act as excellent RNA-binding modules and represent a versatile and functional platform to specifically target REST mRNA and modulate its endogenous expression.

Activation of Autophagy, Observed in Liver Tissues From Patients With Wilson Disease and From ATP7B-Deficient Animals, Protects Hepatocytes From Copper-Induced Apoptosis.

BACKGROUND & AIMS: Wilson disease (WD) is an inherited disorder of copper metabolism that leads to copper accumulation and toxicity in the liver and brain. It is caused by mutations in the adenosine triphosphatase copper transporting ß gene (ATP7B), which encodes a protein that transports copper from hepatocytes into the bile. We studied ATP7B-deficient cells and animals to identify strategies to decrease copper toxicity in patients with WD. METHODS: We used RNA-seq to compare gene expression patterns between wild-type and ATP7B-knockout HepG2 cells exposed to copper. We collected blood and liver tissues from Atp7b-/- and Atp7b+/- (control) rats (LPP) and mice; some mice were given 5 daily injections of an autophagy inhibitor (spautin-1) or vehicle. We obtained liver biopsies from 2 patients with WD in Italy and liver tissues from patients without WD (control). Liver tissues were analyzed by immunohistochemistry, immunofluorescence, cell viability, apoptosis assays, and electron and confocal microscopy. Proteins were knocked down in cell lines using small interfering RNAs. Levels of copper were measured in cell lysates, blood samples, liver homogenates, and subcellular fractions by spectroscopy. RESULTS: After exposure to copper, ATP7B-knockout cells had significant increases in the expression of 103 genes that regulate autophagy (including MAP1LC3A, known as LC3) compared with wild-type cells. Electron and confocal microscopy visualized more autophagic structures in the cytoplasm of ATP7B-knockout cells than wild-type cells after copper exposure. Hepatocytes in liver tissues from patients with WD and from Atp7b-/- mice and rats (but not controls) had multiple autophagosomes. In ATP7B-knockout cells, mammalian target of rapamycin (mTOR) had decreased activity and was dissociated from lysosomes; this resulted in translocation of the mTOR substrate transcription factor EB to the nucleus and activation of autophagy-related genes. In wild-type HepG2 cells (but not ATP7B-knockout cells), exposure to copper and amino acids induced recruitment of mTOR to lysosomes. Pharmacologic inhibitors of autophagy or knockdown of autophagy proteins ATG7 and ATG13 induced and accelerated the death of ATP7B-knockout HepG2 cells compared with wild-type cells. Autophagy protected ATP7B-knockout cells from copper-induced death. CONCLUSION: ATP7B-deficient hepatocytes, such as in those in patients with WD, activate autophagy in response to copper overload to prevent copper-induced apoptosis. Agents designed to activate this autophagic pathway might decrease copper toxicity in patients with WD.