Real-time bio-layer interferometry ubiquitination assays as alternatives to western blotting.

Ubiquitination is a crucial cellular pathway enabling normal cellular functions. Abnormalities in the ubiquitination process can lead to cellular dysfunction and cause a range of diseases. Efforts to screen and develop small molecule inhibitors targeting portions of the ubiquitination cascade require rapid and robust methods for detecting ubiquitination. Enormous efforts have been made in the field to detect ubiquitination using various techniques including fluorescence, spectrophotometry, chemiluminescence, NMR, and radioactive tracers. The most common method to detect ubiquitination is western blotting. However, western blotting is time-consuming and difficult to use when seeking fine-grained time course experiments. Here we present the use of bio-layer interferometry to rapidly assay ubiquitination in real-time. An E3 ligase auto-ubiquitination system and a substrate ubiquitination assay have been applied as tests for the newly developed assay. The developed BLI ubiquitination assay provides one-second time resolution and detects the formation of polyubiquitin chains directly on a biosensor-bound target. Results are returned instantaneously, and reagent concentrations are identical to those used by traditional western blot-based ubiquitination assays. The developed BLI ubiquitination assay is a viable alternative to traditional western blot assays to detect ubiquitination in a rapid real-time manner.

Biocatalytic role of cytochrome P450s to produce antibiotics: A review.

Cytochrome P450s belong to a family of heme-binding monooxygenases, which catalyze regio- and stereospecific functionalisation of C-H, C-C, and C-N bonds, including heteroatom oxidation, oxidative C-C bond cleavages, and nitrene transfer. P450s are considered useful biocatalysts for the production of pharmaceutical products, fine chemicals, and bioremediating agents. Despite having tremendous biotechnological potential, being heme-monooxygenases, P450s require either autologous or heterologous redox partner(s) to perform chemical transformations. Randomly distributed P450s throughout a bacterial genome and devoid of particular redox partners in natural products biosynthetic gene clusters (BGCs) showed an extra challenge to reveal their pharmaceutical potential. However, continuous efforts have been made to understand their involvement in antibiotic biosynthesis and their modification, and this review focused on such BGCs. Here, particularly, we have discussed the role of P450s involved in the production of macrolides and aminocoumarin antibiotics, nonribosomal peptide (NRPSs) antibiotics, ribosomally synthesized and post-translationally modified peptide (RiPPs) antibiotics, and others. Several reactions catalyzed by P450s, as well as the role of their redox partners involved in the BGCs of various antibiotics and their derivatives, have been primarily addressed in this review, which would be useful in further exploration of P450s for the biosynthesis of new therapeutics.

H19X-encoded miR-424(322)/-503 cluster: emerging roles in cell differentiation, proliferation, plasticity and metabolism.

miR-424(322)/-503 are mammal-specific members of the extended miR-15/107 microRNA family. They form a co-expression network with the imprinted lncRNA H19 in tetrapods. miR-424(322)/-503 regulate fundamental cellular processes including cell cycle, epithelial-to-mesenchymal transition, hypoxia and other stress response. They control tissue differentiation (cardiomyocyte, skeletal muscle, monocyte) and remodeling (mammary gland involution), and paradoxically participate in tumor initiation and progression. Expression of miR-424(322)/-503 is governed by unique mechanisms involving sex hormones. Here, we summarize current literature and provide a primer for future endeavors.

H19X-encoded miR-322(424)/miR-503 regulates muscle mass by targeting translation initiation factors.

BACKGROUND: Skeletal muscle atrophy is a debilitating complication of many chronic diseases, disuse conditions, and ageing. Genome-wide gene expression analyses have identified that elevated levels of microRNAs encoded by the H19X locus are among the most significant changes in skeletal muscles in a wide scope of human cachectic conditions. We have previously reported that the H19X locus is important for the establishment of striated muscle fate during embryogenesis. However, the role of H19X-encoded microRNAs in regulating skeletal mass in adults is unknown. METHODS: We have created a transgenic mouse strain in which ectopic expression of miR-322/miR-503 is driven by the skeletal muscle-specific muscle creatine kinase promoter. We also used an H19X mutant mouse strain in which transcription from the locus is interrupted by a gene trap. Animal phenotypes were analysed by standard histological methods. Underlying mechanisms were explored by using transcriptome profiling and validated in the two animal models and cultured myotubes. RESULTS: Our results demonstrate that the levels of H19X microRNAs are inversely related to postnatal skeletal muscle growth. Targeted overexpression of miR-322/miR-503 impeded skeletal muscle growth. The weight of gastrocnemius muscles of transgenic mice was only 54.5% of the counterparts of wild-type littermates. By contrast, interruption of transcription from the H19X locus stimulates postnatal muscle growth by 14.4-14.9% and attenuates the loss of skeletal muscle mass in response to starvation by 12.8-21.0%. Impeded muscle growth was not caused by impaired IGF1/AKT/mTOR signalling or a hyperactive ubiquitin-proteasome system, instead accompanied by markedly dropped abundance of translation initiation factors in transgenic mice. miR-322/miR-503 directly targets eIF4E, eIF4G1, eIF4B, eIF2B5, and eIF3M. CONCLUSIONS: Our study illustrates a novel pathway wherein H19X microRNAs regulate skeletal muscle growth and atrophy through regulating the abundance of translation initiation factors, thereby protein synthesis. The study highlights how translation initiation factors lie at the crux of multiple signalling pathways that control skeletal muscle mass.