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Severe junctional epidermolysis bullosa is a rare genetic, postpartum lethal skin disease, predominantly caused by nonsense/premature termination codon (PTC) sequence variants in LAMB3 gene. LAMB3 encodes LAMB3, the β subunit of epidermal–dermal skin anchor laminin 332. Most translational reads of a PTC mRNA deliver truncated, nonfunctional proteins, whereas an endogenous PTC readthrough mechanism produces full-length protein at minimal and insufficient levels. Conventional translational readthrough-inducing drugs amplify endogenous PTC readthrough; however, translational readthrough-inducing drugs are either proteotoxic or nonselective. Ribosome editing is a more selective and less toxic strategy. This technique identified ribosomal protein L35/uL29 (ie, RpL35) and RpL35-ligands repurposable drugs artesunate and atazanavir as molecular tools to increase production levels of full-length LAMB3. To evaluate ligand activity in living cells, we monitored artesunate and atazanavir treatment by dual luciferase reporter assays. Production levels of full-length LAMB3 increased up to 200% upon artesunate treatment, up to 150% upon atazanavir treatment, and up to 170% upon combinatorial treatment of RpL35 ligands at reduced drug dosage, with an unrelated PTC reporter being nonresponsive. Proof of bioactivity of RpL35 ligands in selective increase of full-length LAMB3 provides the basis for an alternative, targeted therapeutic route to replenish LAMB3 in severe junctional epidermolysis bullosa.

Background: Dystrophic Epidermolysis bullosa (DEB) is a rare inherited mechanobullous disease characterised by the hyperfragility of the skin and mucous membranes. It is (typically) caused by (loss-of-function) mutations in the COL7A1 gene that impair the formation of collagen type VII, which represents the major constituent of anchoring fibrils within the basement membrane zone of epithelialised tissues. In a 4-year-old patient diagnosed with the clinical features of recessive DEB, genotyping via Next-Generation EB Panel Sequencing initially revealed the homozygosity of the maternal c.425A>G mutation, while the paternal heterozygosity in exon 3 was lacking. This genetic profile suggested incongruent gene transmission due to uniparental isodisomy (UPD) or the occurrence of a hemizygous deletion of unknown size. Methods: Thus, the EB panel sequencing of genomic DNA, followed by a paternity test and analysis of microsatellite markers, as well as multiplex ligation-dependent probe amplification (MLPA) copy number analysis using patient and parental DNA, were performed. Results: This approach revealed a paternally derived hemizygous deletion spanning from exon 3 to exon 118. Linear amplification-mediated PCR (LAM-PCR) determined the breaking points within intron 2 of the COL7A1 gene, comprising a 40kb segment within intron 1 of the adjacent PFKFB4 gene. Conclusion: This report highlights the relevance of advanced molecular profiling to determine new/exceptional/unusual genotypes and the accurate mode of genetic transmission in DEB.

NADPH oxidases of human cells are not only functional in defense against invading microorganisms and for oxidative reactions needed for specialized biosynthetic pathways but also during the past few years have been established as signaling modules. It has been shown that human Nox4 is expressed in most somatic cell types and produces hydrogen peroxide, which signals to remodel the actin cytoskeleton. This correlates well with the function of Yno1, the only NADPH oxidase of yeast cells. Using two established tumor cell lines, which are derived from hepatic and neuroblastoma tumors, respectively, we are showing here that in both tumor models Nox4 is expressed in the ER (like the yeast NADPH oxidase), where according to published literature, it produces hydrogen peroxide. Reducing this biochemical activity by downregulating Nox4 transcription leads to loss of F-actin stress fibers. This phenotype is reversible by adding hydrogen peroxide to the cells. The effect of the Nox4 silencer RNA is specific for this gene as it does not influence the expression of Nox2. In the case of the SH-SY5Y neuronal cell line, Nox4 inhibition leads to loss of cell mobility as measured in scratch assays. We propose that inhibition of Nox4 (which is known to be strongly expressed in many tumors) could be studied as a new target for cancer treatment, in particular for inhibition of metastasis.

Life requires the maintenance of molecular function in the face of stochastic processes that tend to adversely affect macromolecular integrity. This is particularly relevant during ageing, as many cellular functions decline with age, including growth, mitochondrial function and energy metabolism. Protein synthesis must deliver functional proteins at all times, implying that the effects of protein synthesis errors like amino acid misincorporation and stop-codon read-through must be minimized during ageing. Here we show that loss of translational accuracy accelerates the loss of viability in stationary phase yeast. Since reduced translational accuracy also reduces the folding competence of at least some proteins, we hypothesize that negative interactions between translational errors and age-related protein damage together overwhelm the cellular chaperone network. We further show that multiple cellular signalling networks control basal error rates in yeast cells, including a ROS signal controlled by mitochondrial activity, and the Ras pathway. Together, our findings indicate that signalling pathways regulating growth, protein homeostasis and energy metabolism may jointly safeguard accurate protein synthesis during healthy ageing.